KR20070086038A - Antiinfective lipopeptides - Google Patents

Abstract

The present invention relates to novel depsipeptide compounds. The invention also relates to pharmaceutical compositions of these compounds and methods of using these compounds as antibacterial compounds. The invention also relates to methods of producing these novel depsipeptide compounds and intermediates used in producing these compounds.

Description

Anti-infective lipopeptides {ANTIINFECTIVE LIPOPEPTIDES}

Cross Reference of Related Application

This application claims priority to US Provisional Application No. 60 / 710,705, filed August 23, 2005, and US Provisional Application No. 60 / 627,056, filed November 12, 2004, which is incorporated by reference in its entirety.

Government support

Some of the studies disclosed herein were governmentally supported under Small Business Innovation Research (SBIR) Grant No. 5R44GM068173-03 and Grant No. 1R43A156858-1. The government may have some rights in this study.

Field of invention

The present invention relates to novel depsipeptides compounds. The present invention also relates to pharmaceutical compositions of these compounds and methods of using these compounds as antibacterial agents.

The rapid increase in the incidence of Gram-positive infections, including those caused by resistant bacteria, has given new attention to the development of new classes of antibiotics. A class of compounds that have demonstrated potential as useful antibiotics is cyclic depsipeptides. Notable members of the cyclic depsipeptides are described, for example, in US Patent RE 32,333; RE 32,455; RE 32,311; RE 32,310; 4,482,487; 4,537,717; 5,912,226; 6,911,525; And 6,794,490 and International Patent Application WO 01/44272; WO 01/44274; And A21978C lipopeptides disclosed in WO 01/44271. See also US Pat. No. 4,994,270; 5,039,789; And the A54145 compound class disclosed in US Pat.

Daptomycin, also known as LY146032, consists of three amino acid chains linked to a cyclic 10-amino acid peptide and an n-decanoyl side chain linked to the N-terminal tryptophan of the amino acid chain. Daptomycin exhibits effective bactericidal activity in vitro and in vivo against clinically relevant Gram-positive bacteria that cause serious and life-threatening diseases. These bacteria are resistant pathogens, such as vancomycin-resistant enterococci (VRE: vancomycin-resistant enterococci) , methicillin-resistant Staphylococcus aureus (MRSA: methicillin-resistant Staphylococcus aureus ), glycopeptide intermediate susceptible Staphylococcus aureus (GISA: glycopeptide intermediate susceptible Staphylococcus aureus ), Vancomycin-resistant Staphylococcus aureus (VRSA), coagulase-negative staphylococci (CNS), and penicillin-resistant Streptococcus pneumoniae (PRSP), There are few treatment alternatives for these resistant pathogens. See, eg, Tally et al., 1999, Exp. Opin. Invest. Drugs 8: 1223-1238.

There are existing antibacterial agents that have been identified as promising, but new antibiotics are still required. Many pathogens are repeatedly exposed to commonly used antibiotics. This exposure results in the selection of antimicrobial strains that are resistant to a wide range of antibiotics. Loss of efficacy and effectiveness of antibiotics caused by the mechanism of resistance can render antibiotics ineffective, resulting in some life-threatening infections that cannot be cured. When new antibiotics become available, pathogens develop resistance or moderate resistance to new drugs, which actually requires a new series of antibiotics against emerging strains. In addition, compounds that exhibit bactericidal activity offer advantageous advantages over the bacteriostatic compounds present. Thus, new antibacterial agents are expected to be useful in treating intermediate and drug resistant pathogens as well as "natural" pathogens, since such pathogens have never been exposed to new antibiotics. The novel antibacterial agents can show differential effectiveness against different kinds of pathogens.

Summary of the Invention

The present invention provides novel compounds that possess antimicrobial activity against a wide range of bacteria, including drug resistant bacteria, and methods of making these compounds.

In one aspect, the invention provides compounds of formula I and salts thereof:

Where

이고;a) R ² is an amino acid side chain,

ego;

b) R ^{2 *} is either H, or alternatively R ² forms a 5-or 6-membered heterocyclic ring together with R ² and ^*;

또는 비단백질원성(non-proteinogenic) 아미노산 측쇄이고;c) R ³ is

Or a non-proteinogenic amino acid side chain;

d) R ⁵ is H or methyl;

e) R ^{5 *} is an amino acid side chain derived from H or N- methyl-amino acids, or alternatively R ⁵ forms a 5 or 6-membered heterocyclic ring together with R ^{5 *} and;

이고;f) R ⁶ is methyl or

ego;

이며;g) R ⁸ is amino acid side chain, methyl,

Is;

h) R ^{8 *} is H or, or alternatively R ⁸ form a 5 or 6-membered heterocyclic ring together with R ^{8 *,} and;

또는 하나 이상의 카르복실산으로 치환된 아미노산 측쇄이고;i) R ⁹ is

Or an amino acid side chain substituted with one or more carboxylic acids;

이며;j) R ¹¹ is amino acid side chain, methyl,

Is;

k) R ^{11 *} is either H, or alternatively R ¹¹ forms a 5-or 6-membered heterocyclic ring together with R ^{11 *;}

l) R ¹² is H or CH ₃ ;

이며;m) R ¹³ is CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

Is;

n) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of Formula F1 and salts thereof:

Where

이고;a) R ⁸ is hydrogen,

ego;

이며;b) R ¹¹ is methyl,

Is;

c) R ¹² is H or CH ₃ ;

이며;d) R ¹³ is CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

Is;

e) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another aspect, the present invention provides a compound of formula F2 and salts thereof:

Where

이고;a) R ⁸ is hydrogen, methyl,

ego;

b) R ¹² is H or CH ₃ ;

이고;c) R ¹³ is CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

ego;

d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of formula F3 and salts thereof:

Where

이고;a) R ⁸ is hydrogen,

ego;

이며;b) R ¹¹ is methyl,

Is;

c) R ¹² is H or CH ₃ ;

d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of formula F4 and salts thereof:

Where

이고;a) R ⁸ is hydrogen, methyl,

ego;

이며;b) R ¹¹ is methyl, or

Is;

c) R ¹² is H or CH ₃ ;

d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of formula F5 and salts thereof:

Where

이고;a) R ⁸ is hydrogen, methyl,

ego;

이며;b) R ¹¹ is methyl,

Is;

c) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting groups, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of formula F6 and salts thereof:

Where

이고;a) R ⁸ is

ego;

이며;b) R ⁹ is

Is;

이고;c) R ¹¹ is methyl,

ego;

d) R ¹² is H or CH ₃ ;

e) R ¹ is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, imino Amino, or phosphonamino.

In another aspect, the present invention provides a compound of formula F7 and salts thereof:

Where

이고;a) R ⁸ is methyl,

ego;

이며;b) R ⁹ is

Is;

c) R ¹² is H or CH ₃ ;

d) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of Formula F8 and salts thereof:

Where

a) R ^{3 **} is hydroxyl or hydrogen;

이고;b) R ⁸ is methyl,

ego;

이며;c) R ¹¹ is amino acid side chain, methyl,

Is;

d) R ¹² is H or CH ₃ ;

e) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of formula F9 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In another aspect, the invention provides compounds of Formula F10 and salts thereof:

Where

a) R ^{13 *} is H or CH ₃ ;

b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another aspect, the present invention provides a compound of Formula F11 and salts thereof:

Where

a) R ^{13 *} is H or CH ₃ ;

b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another aspect, the present invention provides a compound of formula F12 and salts thereof:

Where

이고;a) R ¹³ is CH (CH ₂ CH ₃ ) CH ₃ or

ego;

b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another aspect, the present invention provides a compound of Formula F13 and salts thereof:

Wherein R, R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of Formula F14 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another aspect, the present invention provides a compound of Formula F15 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In another aspect, the invention provides compounds of Formula F16 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In another aspect, the invention provides compounds of Formula F17 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, imino Amino, or phosphonamino.

In another aspect, the invention provides compounds of Formula F18 and salts thereof:

Where

R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thi Oureido is also iminoamino or phosphonamino.

In another aspect, the present invention provides a compound of formula F19 and salts thereof:

Where

이고;a) R ² is

ego;

이며;b) R ⁶ is methyl or

Is;

이고;c) R ⁸ is methyl or

ego;

d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of formula F20 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{8 **} are amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thi Oureido is also iminoamino or phosphonamino.

In another aspect, the present invention provides a compound of Formula F21 and salts thereof:

Where

이고; a) R ¹ is

ego;

b) R ¹² is H or CH ₃ ;

c) R ^{8 **} is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, Iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of Formula F22 and salts thereof:

                                                     F (22)

Where

R ^{6 *} is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, iminoamino Or phosphonamino.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) and a compound of formula (F1-F22) and a method of using the same.

In another aspect, the invention provides an antimicrobial composition comprising a compound of Formula I and a compound of Formula F1-F22 and a method of using the same.

In a further aspect, the present invention provides a process for preparing a compound of formula (I) and a compound of formula (F1-F22).

Brief description of the drawings

1 depicts biosynthetic gene clusters for daptomycin, A54145, and CDA. Numbers in parentheses indicate amino acid numbers. The following abbreviations were used: Trp: tryptophan; Asn: asparagine; Asp: aspartic acid; Thr: threonine; Gly: glycine; Orn: ornithine; Ala: alanine; Ser: serine; MeGlu: 3-methylglutamic acid; Kyn: kynurerine; Glu: glutamic acid; hAsn: 3-hydroxyasparagine; Sar: sacosin; Lys: lysine; OMeAsp: 3-methoxyaspartic acid; Ile: isoleucine; Val: valine; D-HPG: D-hydroxyphenyl glycine.

Figure 2 shows dptA-H deletion in S. roseosporus , where the dptA-H locus is exchanged for tsr ( thiostrepton resistance) and cat (chloramphenicol) for chromosomes of S. roseosporose. The deletion of dptA-H in S. roseosporose was established by constructing a deletion in the cell .

3 shows a general method for "red-mediated" gene substitution in the daptomycin NRPS pathway. The bacteriophage λ-induced "super recombination" state (ie, "red" system or red mediated recombination) was used to construct a deletion in dptBC and clone the replacement module with a technique called "gap-repair". Abbreviation “C”, condensation domain; "A _ser ", the adenylation domain for serine; "T", thiolated domain; "E", epimerase domain.

4 depicts constructs from S. Roseosporus combinatorial library.

Figure 5 illustrates a module-terminal amino acid organization module (Kinugawa rerin) in dptD related (the interior of the module for the amino acid in the D- dptBC) and thio esterase in dptBC. C is a condensation domain. Circles comprising the amino acid triletter code are adenylated domains specific for amino acids: Asn: asparagine; Ala: alanine; Asp: aspartic acid; 3MGlu: 3-methylglutamic acid; Kyn: Kinurerin. T is a thiolated domain. E is an epimerized domain. TE is a thioesterase domain.

Detailed description of the invention

Justice

The term "acyl" refers to, for example, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, n-decanoyl, 8-methylnonanoyl, dodecanoyl, undecanoyl, acetyl And carbonyl radicals bonded to alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl groups, including, but not limited to, radicals such as benzoyl. In one embodiment of the invention, the acyl group is an "alkanoyl" group defined as a carbonyl radical bound to an alkyl group. In another embodiment of the invention, the alkanoyl group is a "C ₁ -C ₂₀ -alkanoyl" group defined as an alkanoyl group containing a total of 1 to 20 carbon atoms, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoyl group is a "C ₁ -C ₁₅ -alkanoyl" group defined as an alkanoyl group containing a total of 1 to 15 carbon atoms, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoyl group is a "C ₁ -C ₁₃ -alkanoyl" group defined as an alkanoyl group containing from 1 to 13 carbon atoms in total, including carbonyl carbon. The carbon may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoyl group is a "C ₅ -C ₂₀ -alkanoyl" group defined as an alkanoyl group containing a total of 5 to 20 carbon atoms, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoyl group is a "C ₁₀ -C ₂₀ -alkanoyl" group defined as an alkanoyl group containing a total of 10 to 20 carbon atoms, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoyl group is a "C ₁₀ -C ₁₃ -alkanoyl" group defined as an alkanoyl group containing from 1 to 13 carbon atoms in total, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoyl group

to be. In another embodiment of the present invention, a subset of the term acyl is defined as (1) "unsubstituted alkanoyl" defined as a carbonyl radical bound to an unsubstituted alkyl group and (2) a carbonyl radical bound to an unsubstituted alkenyl group. It is defined as "unsubstituted alkenoyl".

The term "acylamino" is defined as a nitrogen radical conjugated to an acyl group. In one embodiment of the invention, the acylamino group is a "alkanoylamino" group defined as a nitrogen radical bound to an alkanoyl group. In another embodiment of the invention, the alkanoylamino group is a "C ₁ -C ₂₀ -alkanoylamino" group defined as an alkanoylamino group containing from 1 to 20 carbon atoms in total, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoylamino group is a "C ₁ -C ₁₅ -alkanoylamino" group defined as an alkanoylamino group containing from 1 to 15 carbon atoms in total, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoylamino group is a "C ₁ -C ₁₃ -alkanoylamino" group defined as an alkanoylamino group containing from 1 to 13 carbon atoms in total, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoylamino group is a "C ₅ -C ₂₀ -alkanoylamino" group defined as an alkanoylamino group containing a total of 5 to 20 carbon atoms, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoylamino group is a "C ₁₀ -C ₂₀ -alkanoylamino" group defined as an alkanoylamino group containing a total of 10 to 20 carbon atoms, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoylamino group is a "C ₁₀ -C ₁₃ -alkanoylamino" group defined as an alkanoylamino group containing from 1 to 13 carbon atoms in total, including carbonyl carbon. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkanoylamino group is

to be.

The term "acyloxy" means an oxygen radical conjugated to an acyl group.

The term "alkenyl" is defined as straight or branched lidacal containing 2 to about 20 carbon atoms, preferably 3 to about 10 carbon atoms and containing at least one carbon-carbon double bond. do. One or more hydrogen atoms are also acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxamido, carboxyamino, sia Furnace, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio , Thioacylamino, thioureido or ureido. The double bond portion (s) of the unsubstituted hydrocarbon chain may be in cis or trans configuration. Examples of alkenyl groups include, but are not limited to, ethyleneyl or phenyl ethyleneyl. The term alkenyl subset is an "unsubstituted alkenyl" which is defined as an alkenyl group containing no substituents.

The term "alkoxy" means an oxygen radical substituted by an alkyl, cycloalkyl or heterocyclyl group. Examples include, but are not limited to, methoxy, tert -butoxy, benzyloxy and cyclohexyloxy.

The term "alkyl" is defined as a straight or branched chain saturated radical containing 1 to about 20 carbon atoms, unless otherwise specified. The term "lower alkyl" is defined as an alkyl group containing 1-4 carbon atoms. One or more hydrogen atoms are also acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxamido, carboxyamino, sia Furnace, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio , Thioacylamino, thioureido or ureido. Examples of alkyl groups include methyl, butyl, tert -butyl, isopropyl, trifluoromethyl, nonyl, undecyl, octyl, dodecyl, methoxymethyl, 2- (2'-aminophenacyl), 3-indolylmethyl , Benzyl, and carboxymethyl, but are not limited thereto. The term alkyl is defined as (1) "unsubstituted alkyl" defined as an alkyl group containing no substituents, and (2) at least one hydrogen atom is acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, Amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, already Meaning an alkyl radical substituted by a substituent selected from noamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido or ureido Substituted alkyl ". In another embodiment of the invention, the alkyl group is a “C ₁ -C ₂₀ -alkyl” group defined as an alkyl group containing a total of 1 to 20 carbon atoms. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkyl group is a "C ₁ -C ₁₅ -alkyl" group defined as an alkyl group containing a total of 1 to 15 carbon atoms. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkyl group is a “C ₁ -C ₁₃ -alkyl” group defined as an alkyl group containing a total of 1 to 13 carbon atoms. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkyl group is a "C ₅ -C ₂₀ -alkanoyl" group defined as an alkyl group containing a total of 5 to 20 carbon atoms. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkyl group is a "C ₁₀ -C ₂₀ -alkyl" group defined as an alkyl group containing a total of 10 to 20 carbon atoms. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkyl group is a “C ₁₀ -C ₁₃ -alkyl” group defined as an alkyl group containing a total of 10 to 13 carbon atoms. In another embodiment of the invention, the alkyl group is a “C ₉ -C ₁₂ -alkyl” group defined as an alkyl group containing a total of 9 to 12 carbon atoms. The carbon atoms may be arranged in a straight or branched chain. In another embodiment of the invention, the alkyl group is nonyl, 7-methyloctyl, 7-methylnonyl, n-decyl, 9-methylundecyl, 9-methyldecyl, n-undecyl.

The term "alkylidedenyl" is defined as a carbon radical of the formula:

.

.

In the formula, R ^x and R ^x1 is also Hi laundry or C ₇ -C ₁₇ are independently selected from unsubstituted alkyl, wherein the total carbon number of R ^x and R ^x1 is no more than 17.

The term "alkynyl" means a straight or branched chain radical containing 2 to about 10 carbon atoms and comprising at least one carbon-carbon triple bond. One or more hydrogen atoms are also acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxamido, carboxyamino, sia Furnace, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio , Thioacylamino, thioureido or ureido. Examples of alkynyl groups include, but are not limited to, propynyl.

The term "amino" is defined as the NH ₂ radical.

The term "amino acid" means a compound of the formula:

.

.

In the above formula, R ^aa is an amino acid side chain. A "naturally occurring amino acid" is an amino acid found in nature. “Essential Amino Acids” include 20 common amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenalanine, proline, serine, threonine, tryptophan, tyrosine and One of valine. A "nonproteinaceous amino acid" is any amino acid other than an essential amino acid. In this specification, specific amino acids are described using the following abbreviations:

Abbreviation (s) amino acid (MeO) Asp or (m) Asp or mAsp or moAsp or mo (Asp) 3-methoxy-aspartic acid (OH) Asn or h (Asn) or hAsn or h-Asn 3-hydroxy-asparagine (OH) Asp or h (Asp) or hAsp or h-Asp 3-hydroxy-aspartic acid 3-MG 3-methylglutamic acid D-HPG D-hydroxyphenyl glycine Ala Alanine Asn Asparagine Asp Aspartic acid Glu Glutamic acid Gly Glycine Ile Isoleucine Kyn Kynurinine Lys Lee Sin Orn Ornithine Sar Sarcosine Ser Serine Thr Threonine Trp Tryptophan Val Valine

In one embodiment of the invention, the amino acids are 3-methoxy-aspartic acid, 3-hydroxy-asparagine, 3-hydroxy-aspartic acid, 3-methylglutamic acid, alanine, asparagine, aspartic acid, glutamic acid, glycine, isoleucine, Kinulinnin, lysine, ornithine, sarcosine, serine, threonine, tryptophan and valine.

Those skilled in the art will understand that the peptides are described by combining the three letter codes. For example, Asp-Asn-Trp means the following compound:

.

.

As an alternative, the compound may also be described as Asp-Asn-Trp-NH ₂ . Those skilled in the art will also understand that the peptides of the present invention may contain protecting groups (see below). If an amino acid contains a protecting group, three letter codes are applied to represent the protecting group. For example, Thr-Asp (OtBu) -Asn (NHTrt) -Trp-NH ₂ means the following compounds:

.

.

Common protecting groups for the amino acids of the present invention include tert -butoxy (tBu), trityl (Trt) and tert -butoxy carbonyl (BOC) protecting groups.

는 하기 구조와 동일하다:Those skilled in the art will also appreciate that cyclic peptides can also be described by three letter codes. For example, three character structures

Is identical to the following structure:

Those skilled in the art will also appreciate that amino acids may be present in the L or D configuration. If it is necessary to indicate an amino acid sequence, the D or L mark is specified before the three letter code.

The term "amino acid residue" means a compound of the formula:

.

.

In the above formula, R ^aa is an amino acid side chain. In one embodiment of the invention, the amino acid residues are derived from natural amino acids. In another embodiment of the invention, the amino acid residue is amino acid 3-methoxy-aspartic acid, 3-hydroxy-asparagine, 3-hydroxy-aspartic acid, 3-methylglutamic acid, alanine, asparagine, aspartic acid, glutamic acid, Derived from glycine, isoleucine, kynurinine, lysine, ornithine, sarcosine, serine, threonine, tryptophan and valine.

The term "amino acid side chain" means any side chain (R group) of naturally occurring or synthetic amino acids. For example, 3-indolylmethyl can also be referred to as tryptophan side chain. Examples of amino acid side chains

히드리도 및 메틸을 들 수 있으나, 이에 한정되는 것은 아니며, 상기 식 중, R^aa1 및 R^aa2는 각각 독립적으로 아미노, 일치환 아미노, 이치환 아미노, 아실아미노, 우레이도, 구아니디노, 카르바모일, 설폰아미노, 티오아실아미노, 티오우레이도, 이미노아미노 또는 포스폰아미노이다. "비단백질성 아미노산 측쇄"는 비단백질성 아미노산으로부터 유도되는 아미노산 측쇄이다(하기 참조). 비단백질성 아미노산 측쇄의 예로는

Hydrido and methyl, but are not limited thereto, wherein R ^aa1 and R ^aa2 are each independently amino, monosubstituted amino, disubstituted amino, acylamino, ureido, guanidino, carba Moyl, sulfonamino, thioacylamino, thioureido, iminoamino or phosphonamino. A "nonproteinaceous amino acid side chain" is an amino acid side chain derived from a nonproteinaceous amino acid (see below). Examples of nonproteinaceous amino acid side chains include

It may include, but is not limited thereto.

In one embodiment of the invention, the amino acid side chains are derived from natural amino acids. In another embodiment of the invention, the amino acid side chains comprise amino acids 3-methoxy-aspartic acid, 3-hydroxy-asparagine, 3-hydroxy-aspartic acid, 3-methylglutamic acid, alanine, asparagine, aspartic acid, glutamic acid, glycine , Isoleucine, kynurinine, lysine, ornithine, sarcosine, serine, threonine, tryptophan and valine.

The term "2- (2'-aminophenacyl)" means a radical of the formula:

.

.

The term "aryl" or "aryl ring" is defined as an aromatic radical in a single or fused carbocyclic ring system containing 5 to 14 membered rings. In a preferred embodiment, the ring system contains 6 to 10 membered rings. One or more hydrogen atoms are also acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, azido, carbamoyl, carboalkoxy, carboxy, carboxamido, carboxy Amino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulf It may be substituted by a substituent selected from fonyl, thio, thioacylamino, thioureido or ureido. Examples of the aryl group include, but are not limited to, phenyl, naphthyl, biphenyl, terphenyl.

The term "aryloxy" means an oxy-containing radical substituted by an aryl or heteroaryl group. Examples include, but are not limited to, phenoxy.

The term "carbamoyl" means a nitrogen radical of the formula:

.

.

^Wherein R ^x2 is selected from hydrido, alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl, and R ^x3 is selected from alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl.

The term "carboalkoxy" is defined as a carbonyl radical conjugated to an alkoxy or aryloxy group.

The term "carboxy" means a COOH radical.

The term "carboxyamino" refers to the CONH ₂ radical.

The term "carboxamido" is defined as a carbonyl radical conjugated to a mono- or di-substituted amino group.

The term "α-carboxy amino acid side chain" is defined as a carbon radical of the formula:

In the above formula, R ^x4 is defined as the amino acid side chain.

The term "carboxymethyl" refers to a CH ₂ CO ₂ H radical.

The term "cycloalkyl" or "cycloalkyl ring" means a saturated or partially saturated carbocyclic ring in a single or fused carbocyclic ring system containing 3 to 12 membered rings. In a preferred embodiment, cycloalkyl is a ring system containing 3 to 7 membered rings. One or more hydrogen atoms are also acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxamido, carboxyamino, sia Furnace, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio , Thioacylamino, thioureido or ureido. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclohexyl and cycloheptyl.

The term “disubstituted amino” is defined as a nitrogen radical containing two substituents independently selected from alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl. Preferred disubstituted amino radicals are “lower disubstituted amino” radicals, wherein the substituent is lower alkyl. Also preferred are disubstituted amino radicals are amino radicals in which one substituent is a lower alkyl group and the other substituent is an α-carboxy amino acid side chain.

"Fmoc" group is a 9-fluorenylmethoxycarbonyl group.

The term "guanidino" is defined as a nitrogen radical of the formula:

.

.

^Wherein R ^x5 , R ^x7 and R ^x8 are each independently selected from hydrido, alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl groups; R ^x6 is selected from alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl groups.

The term "halo" means a bromo, chloro, fluoro or iodo radical.

A "heteroaryl" or "heteroaryl ring" contains one to four heteroatoms or hetero groups selected from O, N, S or SO in a single or fused heterocyclic ring system containing 5 to 15 membered rings. It is defined as an aromatic radical. In a preferred embodiment, the heteroaryl ring system contains 6 to 10 membered rings. One or more hydrogen atoms are also acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxamido, carboxyamino, sia Furnace, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio , Thioacylamino, thioureido or ureido. Examples of heteroaryl groups include, but are not limited to, pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl and pyrrolyl groups.

The term “heterocyclyl”, “heterocyclic” or “heterocyclyl ring” refers to O, N, NH, N (lower alkyl), in a single or fused heterocyclic ring system containing 3 to 12 membered rings, A saturated or partially unsaturated ring containing 1 to 4 heteroatoms or hetero groups selected from S, SO or SO ₂ is meant. In a preferred embodiment, the heterocyclyl is a ring system containing 3 to 7 membered rings. One or more hydrogen atoms are also acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxamido, carboxyamino, sia Furnace, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio , Thioacylamino, thioureido or ureido. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, piperidinyl and pyrrolidinyl.

The term "hydrido" is defined as a single hydrogen atom (H).

The term "iminoamino" means a nitrogen radical of the formula:

.

.

^Wherein R ^x9 and R ^x11 are each independently selected from hydrido, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl groups; R ^x10 is selected from alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl groups.

The term "N-methyl amino acid" means a compound of the formula:

.

.

In the above formula, R ^aa is an amino acid side chain. Examples of amino acid side chains of N-methyl amino acids include

Can be mentioned.

The term "monosubstituted amino" refers to a nitrogen radical containing a hydrido group and a substituent selected from alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl. Preferred monocyclic amino radicals are "lower monocyclic amino" radicals, wherein the substituent is a lower alkyl group. More preferred monocyclic amino radicals are amino radicals containing an α-carboxy amino acid side chain.

The term "phosphonamino" is defined as a nitrogen radical of the formula:

.

.

^Wherein R ^x12 is selected from hydrido, alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl; Wherein R ^x13 and R ^x14 are each independently selected from alkyl, alkoxy, aryl, aryloxy, cycloalkyl, heteroaryl and heterocyclyl.

The term "protecting group" is used to mean any chemical compound that can be used to prevent a group of a molecule from undergoing a chemical reaction while a chemical change occurs at any other position in the molecule. Groups that may require protection include hydroxyl, amino, carboxylic acid and carboxyamino groups. There is a number of savers are known to those skilled in the art, for example, is found in the literature [ "Protective Groups in Organic Synthesis" by Theodora W. Greene and Peter GM Wuts, John Wiley and ^{Sons, New York, 3 rd Edition} 1999, hereinafter referred to as Greene] Can be.

The term “amino protecting group” means any chemical compound that can be used to prevent the amino groups of a molecule from undergoing a chemical reaction while chemical changes occur at any other position in the molecule. Numerous amino protecting groups are known to those skilled in the art, examples can be found in Greene. Examples of “amino protecting groups” include phthalimido, trichloroacetyl, STA-base, benzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, adamantyloxycarbon Carbonyl, chlorobenzyloxycarbonyl, nitrobenzyloxycarbonyl and the like. Preferred amino protecting groups are amino protecting groups which form carbamate when bound to an amino group, or "carbamate amino protecting group" defined as azido groups. Preferred amino carbamate protecting groups are allyloxycarbonyl (alloc), carbenzyloxy (CBZ), 9-fluorenylmethoxycarbonyl (Fmoc) and tert -butoxycarbonyl protecting groups.

The term “hydroxyl protecting group” is used to mean any chemical compound that can be used to prevent the hydroxyl group of a molecule from undergoing a chemical reaction while a chemical change occurs at any other position in the molecule. Numerous hydroxyl protecting groups are known to those skilled in the art and examples can be found in Greene (see below). Examples of hydroxyl protecting groups include esters such as formate, acetate, substituted acetate, crotonate, benzoate, substituted benzoate, methyl carbonate, ethyl carbonate, alkyl and aryl carbonates, borate and sulfonate It may be, but is not limited to such. Examples of hydroxyl protecting groups are also ethers such as methyl, benzyloxylmethyl, siloxymethyl, tetrahydropyranyl, substituted tetrahydropyranyl, ethyl, substituted ethyl, allyl, tert -butyl, propargyl, phenyl, substituted phenyl , Benzyl, substituted benzyl, alkyl silyl and silyl ether, and the like, but are not limited thereto. Preferred hydroxyl protecting groups are "acid labile ethers" which are defined as ether protecting groups which can be removed by treatment with an acid. Preferred hydroxyl ether protecting groups are trityl (Trt), tert -butyl (tBu), benzyl (Bzl) and tert -butyldimethylsilyl (TBDMS) protecting groups.

The term “carboxylic acid protecting group” means any chemical compound that can be used to prevent the carboxylic acid of a molecule from undergoing a chemical reaction while chemical changes occur at any other position in the molecule. Numerous carboxylic acid protecting groups are known to those skilled in the art and examples can be found in Greene (see below). Examples of carboxylic acid protecting groups include amides, hydrazides and esters such as methyl esters, substituted methyls, phenacyl, tetrahydropyranyl, tetrahydrofuranyl, cyanomethyl, triisopropylsilylmethyl, decyl, ethyl 2-substituted Ethyl, phenyl, 2,6 dialkyl phenyl, benzyl, substituted benzyl, silyl and stanyl, and the like, but are not limited thereto. Preferred carboxylic ester protecting groups are allyl (All), tert -butyl (tBu), benzyl (Bzl), 4- {N- [1- (4,4-dimethyl-2,6-dioxocyclohexylideneene ) -3-methylbutyl] -amino} benzyl (ODmab), 1-adamantyl (1Ada) and 2-phenylisopropyl (2-PhiPr) protecting groups.

The term "sulfinyl" means a tetravalent sulfur radical substituted by an oxo substituent and a second substituent selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl groups.

The term sulfonamino is defined as the amino radical of the formula:

.

.

^Wherein R ^x15 is selected from hydrido, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl groups; R ^x16 is selected from alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl groups.

The term "sulfonyl" refers to a hexavalent sulfur radical substituted by two oxo substituents and a third substituent selected from alkyl, cycloalkyl, heterocyclyl aryl or heteroaryl.

The term "thio" may be defined as a radical, such as methylthio and phenylthio, bound to a divalent sulfur atom and containing substituents independently selected from hydrido, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. have.

The term "thioacylamino" means an amino radical of the formula:

.

.

^Wherein R ^x17 is selected from hydrido, alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl groups; R ^x18 is selected from alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl groups.

The term "thioureido" is defined as the sulfur radical of the formula:

.

.

^Wherein R ^x19 and R ^x20 are each independently selected from hydrido, alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl groups; R ^x21 is selected from alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl groups.

Trityl group is a triphenylmethyl group.

The term "ureido" is defined as a nitrogen radical of the formula:

.

.

^Wherein R ^x21 and R ^x22 are each independently selected from hydrido, alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl groups; R ^x23 is selected from alkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl groups.

The terms " lptA ", " lptB "" lptC " and " lptD " refer to nucleic acid molecules that encode subunits of A54145 NRPS. In a preferred embodiment, the nucleic acid molecule is derived from Streptomyces MRS (Streptomyces), more preferably the nucleic acid molecule is derived from Streptomyces MRS plastic DI (S. fradiae). The lptA nucleic acid encodes for amino acids 1-5. lptB nucleic acid encodes for amino acids 6 and 7. lptC nucleic acid encodes for amino acids 8-11. The lptD nucleic acid encodes for amino acids 12 and 13 (FIG. 1). The term "lptA", "lptB", "lptC" and "lptD" also refers to other Streptomyces MRS allelic variants (allelic variant) of the species or the gene, which may be obtained from other Streptomyces MRS plastic DI system.

The terms " dptA ", " dptBC " and " dptD " refer to nucleic acid molecules encoding subunits of daptomycin NRPS. In a preferred embodiment, the nucleic acid molecule is derived from Streptomyces , and more preferably the nucleic acid molecule is derived from Streptomyces roseosporus . dptA nucleic acid encodes amino acids 1-5. dptBC nucleic acid encodes amino acids 6-11. dptD nucleic acid encodes amino acids 12-13 (FIG. 1). The term "dptA", "dptBC" and "dptD" also, which can be obtained from other Streptomyces species or any other Streptococcus MRS MRS Rosetta agarose porous grid, means for allelic variants of the gene.

Salts of the compounds of the present invention include acid addition salts and base addition salts. In a preferred embodiment, the salt is a pharmaceutically acceptable salt of a compound of formula (I) or any compound of formula (F1-F22). The term "pharmaceutically acceptable salts" is generally used to include salts which form alkali metal salts and form addition salts of free acids or free bases. If pharmaceutically acceptable, the nature of the salt is not critical. Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention may be prepared from inorganic or organic acids. Examples of the inorganic acid include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonate, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic acids of organic acids, examples of which are formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, Maleic acid, embonic acid (palmic acid), methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, pantothenic acid, benzenesulfonic acid, toluenesulfonic acid, sulfanilic acid, mesylic acid, cyclohexylaminosulfonic acid, stearic acid, Algenic acid, β-hydroxybutyric acid, malonic acid, galactic acid and galacturonic acid, but are not limited thereto. Suitable pharmaceutically acceptable base addition salts of the compounds of the invention include metal salts formed from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or N, N'-dibenzylethylenediamine, chloroprocaine, choline, Organic salts formed from diethanolamine, ethylenediamine, N-methylglucamine, lysine and procaine, but are not limited thereto. All of these salts can be prepared by conventional methods, for example from the corresponding compounds of the invention, by treating the compounds of the invention with a suitable acid or base.

The compounds of the present invention may contain one or more asymmetric carbon atoms and therefore may exist in the form of optical isomers, as well as in racemic or non-racemic mixtures thereof. The compounds of the present invention can be used in the present invention as single isomers or as mixtures of stereochemically isomeric forms. Diastereomers, ie nonsuperimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization and sublimation. The optical isomers can be obtained by partitioning the racemic mixture according to conventional methods, for example by forming diastereomeric salts by optically active acid or base treatment. Examples of suitable acids include tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, dytoluoyltartaric acid and camphorsulfonic acid. The mixture of diastereomers may be crystallized and then separated by freeing the optically active base from the optically active salt. Alternative methods of optical isomer separation include the use of chiral chromatographic columns that are optimally selected to maximize separation of the enantiomers. Another method involves the synthesis of diastereomeric molecules of covalent atoms by the reaction of the compounds of the invention with optically pure acids or optically pure isocyanates in the active form. The synthesized diastereomers can be separated in a conventional manner such as chromatography, distillation, crystallization or sublimation and then hydrolyzed to yield enantiomeric pure compounds. Optically active compounds of the invention can likewise be obtained by using optically active starting materials. The isomer may be in the form of a free acid, free base, ester or salt.

The invention also includes an isolated compound, preferably a compound of formula (I) or any of formulas F1-F22. Isolated compound means a compound, preferably a compound of formula I or any of formulas F1-F22, which is at least about 1%, preferably at least about 10%, more preferably said compound present in the mixture At least about 20%, even more preferably at least about 50%, even more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 99%. In one embodiment of the invention, said compound, preferably a compound of formula (I) or any of formulas F1-F22, is present in the composition in about 80% to about 90% or more. In another embodiment, said compound, preferably a compound of formula (I) or any of formulas F1-F22, is present in at least 90% of the composition. In another embodiment, said compound, preferably a compound of formula (I) or any of formulas F1-F22, is present in greater than 90% of the composition.

The percentage of the compound, preferably the compound of formula I or any of formulas F1-F22, is determined by nuclear magnetic resonance (NMR), gas chromatography / mass spectrometer (GC / MS), liquid chromatography / mass spectrometer (LC / MS Or microbiological analysis. Preferred methods of determining compound purity are by analytical high pressure liquid chromatography (HPLC) or LC / MS.

In one embodiment of the invention, the compound, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound is detectable (ie, statistically) when tested by conventional biological assays, such as the assays described herein. Effective bactericidal activity).

Depsipeptide Compound

In one aspect, the present invention provides a compound of formula (I) and salts thereof.

이다. 본 발명의 일 구체예에서, 아미노산 측쇄는

이다. 본 발명의 또 다른 구체예에서, 아미노산 측쇄는 D-아미노산으로부터 유도된다. 본 발명의 또 다른 구체예에서, 아미노산 측쇄는The group R ² of formula I is an amino acid side chain,

to be. In one embodiment of the invention, the amino acid side chain is

to be. In another embodiment of the invention, the amino acid side chains are derived from D-amino acids. In another embodiment of the invention, the amino acid side chain is

이며,

Is,

^Wherein R ^aa1 and R ^aa2 are each independently amino, single substituted amino, double substituted amino, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, Noamino, or phosphonamino.

Substituent R ^{2 *} is H. Alternatively, R ² and R ^{2 *} together with the atoms to which they are attached form a five or six membered heterocyclic ring. In one embodiment of formula I, R ² and R ^{2 *} together with the atoms to which they are attached form a pyrrolidine ring.

또는 비단백질원성 아미노산 측쇄이다. 본 발명의 일 구체예에서, 화학식 I의 기 R³은

이다. 본 발명의 또 다른 구체예에서, 비단백질원성 아미노산은 The group R ³ in formula (I)

Or a nonproteinaceous amino acid side chain. In one embodiment of the invention, the group R ³ in formula I is

to be. In another embodiment of the invention, the nonproteinaceous amino acid is

이다.

to be.

Substituent R ⁵ of formula I is H or methyl and substituent R ^{5 *} of formula I is an amino acid side chain derived from H or N-methylamino acid. In one embodiment of the invention, R ^{5 *} is methyl,

이다.

to be.

Alternatively, R ⁵ and R ^{5 *} together with the atoms to which they are attached form a five or six membered heterocyclic ring. In one embodiment of formula I, R ⁵ and R ^{5 *} together with the atoms to which they are attached form a piperidine or pyrrolidine ring.

이다. The group R ⁶ in formula I is methyl or

to be.

이다. 본 발명의 일 구체예에서, 화학식 I의 치환기 R⁸은 수소, 메틸,

이다. 본 발명의 또 다른 구체예에서, 아미노산 측쇄는 D-아미노산으로부터 유도된다. 본 발명의 또 다른 구체예에서, 치환기 R⁸은 글리신, D-알라닌, D-아스파라긴, D-세린 또는 D-리신으로부터 유도된 아미노산 측쇄이다. 본 발명의 또 다른 구체예에서, 아미노산 측쇄는 Substituent R ⁸ in Formula I is amino acid side chain, hydrogen, methyl

to be. In one embodiment of the invention, substituent R ⁸ in formula I is hydrogen, methyl,

to be. In another embodiment of the invention, the amino acid side chains are derived from D-amino acids. In another embodiment of the invention, substituent R ⁸ is an amino acid side chain derived from glycine, D-alanine, D-asparagine, D-serine or D-lysine. In another embodiment of the invention, the amino acid side chain is

이며,

Is,

^Wherein R ^aa1 and R ^aa2 are each independently amino, single substituted amino, double substituted amino, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, Noamino, or phosphonamino.

Substituent R ^{8 *} of Formula I is H. Alternatively, R ⁸ and R ^{8 *} together with the atoms to which they are attached form a five or six membered heterocyclic ring. In one embodiment of formula I, R ⁸ and R ^{8 *} together with the atoms to which they are attached form a pyrrolidine ring.

, 또는 하나 이상의 카르복실산으로 치환된 아미노산 측쇄이다. 본 발명의 일 구체예에서, 화학식 I의 기 R⁹는

이다. 본 발명의 또 다른 구체예에서, 아미노산 측쇄는

이다. The group R ⁹ in formula (I)

Or an amino acid side chain substituted with one or more carboxylic acids. In one embodiment of the invention, the group R ⁹ in formula (I) is

to be. In another embodiment of the invention, the amino acid side chain is

to be.

이다. 본 발명의 일 구체예에서, 화학식 I의 치환기 R¹¹은 메틸,

이다. 본 발명의 일 구체예에서, 아미노산 측쇄는 D-아미노산으로부터 유도된다. 본 발명의 또 다른 구체예에서, 화학식 I의 R¹¹은 D-알라닌, D-세린 또는 D-아스파라긴으로부터 유도된 아미노산 측쇄이다. 본 발명의 또 다른 구체예에서, 아미노산 측쇄는 Substituent R ¹¹ in Formula I is amino acid side chain, methyl,

to be. In one embodiment of the invention, substituent R ¹¹ in formula I is methyl,

to be. In one embodiment of the invention, the amino acid side chains are derived from D-amino acids. In another embodiment of the invention, R ¹¹ in formula (I) is an amino acid side chain derived from D-alanine, D-serine or D-asparagine. In another embodiment of the invention, the amino acid side chain is

이며,

Is,

^Wherein R ^aa1 and R ^aa2 are each independently amino, single substituted amino, double substituted amino, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, Noamino, or phosphonamino.

Substituent R ^{11 *} is H. Alternatively, R ¹¹ and R ^{11 *} together with the atoms to which they are attached form a five or six membered heterocyclic ring. In one embodiment of formula I, R ¹¹ and R ^{11 *} together with the atoms to which they are attached form a pyrrolidine ring.

Substituent R ¹² in formula (I) is H or CH ₃ .

이다. Substituents R ¹³ in formula (I) are CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

to be.

이다. In one embodiment of the invention, R ¹³ is CH (CH ₂ CH ₃ ) CH ₃ or

to be.

R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thio Acylamino, thioureido, iminoamino, or phosphonamino. In one embodiment of the invention, R ¹ is amino, NH-amino protecting group or acylamino. In another embodiment of the invention, R ¹ is amino. In another embodiment of the invention, R ¹ is an NH-amino protecting group. In another embodiment of the invention, R ¹ is acylamino. In another embodiment of the invention, R ¹ is alkanoylamino. In another embodiment of the invention, R ¹ is C ₁₀ -C ₁₃ alkanoylamino. In another embodiment of the invention, R ¹ is

이다.

to be.

In another embodiment of the invention, R ^{6 *} and R ^{8 **} are each independently amino or NH-amino protecting groups. In another embodiment of the invention, R ^{6 *} and R ^{8 **} are each independently amino. In another embodiment of the invention, R ^{6 *} and R ^{8 **} are each independently NH-amino protecting group.

Exemplary compounds of formula I are shown in Table I below.

TABLE I

Compound of formula (I)

이고; R¹³은 CH(CH₂CH₃)CH₃이다. 이 구체예는 하기 화학식 II의 화합물을 제공한다:In one embodiment of the invention, R ^{2 *} , R ^{5 *} , R ^{8 *} , R ^{11 *} and R ¹² are each H and R ⁹ is

ego; R ¹³ is CH (CH ₂ CH ₃ ) CH ₃ . This embodiment provides a compound of Formula II:

Wherein R ^{9 *} is H or OMe, R ¹ , R ² , R ³ , R ⁵ , R ⁶ , R ⁸ and R ¹¹ are as defined above.

In Table II below, exemplary compounds of Formula II are shown.

TABLE II

Compound of formula II

In another embodiment of the invention,

이고;R ² is

ego;

이며;R ³ is

Is;

이고;R ⁹ is

ego;

R ^{2 *} , R ⁵ , R ^{5 *} , R ^{8 *} and R ^{11 *} are each H;

이다.R ⁶ is

to be.

This embodiment provides a compound of Formula III:

Wherein R ¹ , R ^{6 *} , R ⁸ , R ¹¹ , R ¹² and R ¹³ are as defined above.

In Table III below exemplary compounds of Formula III are shown.

TABLE III

Compound of formula III

In another embodiment of the invention, R ^{2 *} , R ^{8 *} and R ^{11 *} are each H. This embodiment provides a compound of Formula IV:

Exemplary compounds of Formula IV are shown in Table IV below.

TABLE IV

Compound of formula IV

In another embodiment, the present invention provides a compound of Formula F1 and salts thereof:

Where

이고;a) R ⁸ is hydrogen,

ego;

이며;b) R ¹¹ is methyl,

Is;

c) R ¹² is H or CH ₃ ;

이며;d) R ¹³ is CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

Is;

e) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

이다. In another embodiment of the invention, substituent R ¹³ in formula F1 is CH (CH ₂ CH ₃ ) CH ₃ ,

to be.

In another embodiment of the invention, the compound of formula F1

Is selected from.

인 경우 C₁₀-알카노일이 아니다. In one embodiment of the invention, substituent R ¹ in formula (F1), substituent R ^{8 **} is hydrogen or

Is not C ₁₀ -alkanoyl.

Exemplary compounds of Formula F1 include, but are not limited to, compounds C22, C189, C201, C210, C37, and C39 (see above).

In another embodiment, the present invention provides a compound of Formula F2 and salts thereof:

Where

이고;a) R ⁸ is hydrogen, methyl,

ego;

b) R ¹² is H or CH ₃ ;

이고;c) R ¹³ is CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

ego;

d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another embodiment of the invention, the compound of formula F2

Is selected from.

Exemplary compounds of Formula F2 include, but are not limited to, compounds C46, C49, and C61 (see above).

In another embodiment, the present invention provides a compound of Formula F3 and salts thereof:

Where

이고;a) R ⁸ is hydrogen,

ego;

이며;b) R ¹¹ is methyl,

Is;

c) R ¹² is H or CH ₃ ;

d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another aspect, the present invention provides a compound of formula F4 and salts thereof:

Where

이고;a) R ⁸ is hydrogen, methyl,

ego;

이며;b) R ¹¹ is methyl, or

Is;

c) R ¹² is H or CH ₃ ;

d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another embodiment, the present invention provides a compound of Formula F5 and salts thereof:

Where

이고;a) R ⁸ is hydrogen, methyl,

ego;

이며;b) R ¹¹ is methyl,

Is;

c) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting groups, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another embodiment, the present invention provides a compound of Formula F6 and salts thereof:

Where

이고;a) R ⁸ is

ego;

이며;b) R ⁹ is

Is;

이고;c) R ¹¹ is methyl,

ego;

d) R ¹² is H or CH ₃ ;

e) R ¹ is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, imino Amino, or phosphonamino.

In another embodiment of the invention, the compound of formula F6

Is selected from.

Exemplary compounds of Formula F6 include, but are not limited to, compounds C292, C289, C307, and C304 (see above).

In another embodiment, the present invention provides a compound of Formula F7 and salts thereof:

Where

이고;a) R ⁸ is methyl,

ego;

이며;b) R ⁹ is

Is;

c) R ¹² is H or CH ₃ ;

d) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In another embodiment of the invention, the compound of formula F7

Is selected from.

Exemplary compounds of Formula F7 include, but are not limited to, compounds C337 and C328 (see above).

In another embodiment, the present invention provides a compound of Formula F8 and salts thereof:

Where

a) R ^{3 **} is hydroxyl or hydrogen;

이고;b) R ⁸ is methyl,

ego;

이며;c) R ¹¹ is amino acid side chain, methyl,

Is;

d) R ¹² is H or CH ₃ ;

e) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In one embodiment of the invention, the group R ^{3 **} in formula F8 is hydroxyl. This gives a compound of formula F8A:

Wherein R ¹ , R ⁸ , R ^{8 **} , R ¹¹ and R ¹² are as defined for Formula F8.

In one embodiment of the invention, the compound of formula F8A is

Is selected from.

Exemplary compounds of formula F8A include, but are not limited to, compounds C87 and C111 (see above).

In another embodiment of the invention, the group R ^{3 **} in formula F8 is hydrogen. This gives a compound of formula F8B:

Wherein R ¹ , R ⁸ , R ^{8 **} , R ¹¹ and R ¹² are as defined for Formula F8.

In one embodiment of the invention, the compound of formula F8B is

Is selected from.

Exemplary compounds of formula F8B include, but are not limited to, compounds C102 and C99 (see above).

In another embodiment, the present invention provides a compound of Formula F9 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In one embodiment of the invention, substituent R ¹² in formula F9 is methyl.

In another embodiment of the invention, the compound of formula F9 is

Is selected from.

Exemplary compounds of formula F9 include, but are not limited to, compounds C105 and C108 (see above).

In another embodiment, the present invention provides a compound of Formula F10 and salts thereof:

Where

a) R ^{13 *} is H or CH ₃ ;

b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another embodiment of the invention, the compound of formula F10

Is selected from.

Exemplary compounds of Formula F10 include, but are not limited to, compounds C259 and C262 (see above).

In another embodiment, the present invention provides a compound of Formula F11 and salts thereof:

Where

a) R ^{13 *} is H or CH ₃ ;

b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another embodiment of the invention, the compound of formula F11

Is selected from.

Exemplary compounds of formula F11 include, but are not limited to, compounds C4 and C8 (see above).

In another embodiment, the present invention provides a compound of Formula F12 and salts thereof:

Where

이고;a) R ¹³ is CH (CH ₂ CH ₃ ) CH ₃ or

ego;

b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another embodiment of the invention, the compound of formula F12

Is selected from.

Exemplary compounds of Formula F12 include, but are not limited to, compounds C233 and C221 (see above).

In another aspect, the present invention provides a compound of Formula F13 and salts thereof:

Wherein R, R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another embodiment of the invention, the compound of formula F13 is

Is selected from.

Exemplary compounds of Formula F13 include, but are not limited to, compounds C236, C237 and C238 (see above).

In another embodiment, the present invention provides a compound of Formula F14 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino.

In another embodiment of the invention, the compound of formula F14 is

Is selected from.

Exemplary compounds of Formula F14 include, but are not limited to, compounds C283 and C277 (see above).

In another embodiment, the present invention provides a compound of Formula F15 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In one embodiment of the invention, substituent R ¹² in formula F15 is methyl.

In another embodiment of the invention, the compound of formula F15

Is selected from.

Exemplary compounds of Formula F15 include, but are not limited to, compounds C325 and C153 (see above).

In another embodiment, the present invention provides a compound of Formula F16 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino.

In one embodiment of the invention, substituent R ¹² in formula F16 is methyl.

In another embodiment of the invention, the compound of formula F16 is

Is selected from.

Exemplary compounds of Formula F16 include, but are not limited to, compounds C90 and C114 (see above).

In another embodiment, the present invention provides a compound of Formula F17 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, imino Amino, or phosphonamino.

In another embodiment of the invention, the compound of formula F17 is

Is selected from.

Exemplary compounds of Formula F17 include, but are not limited to, compounds C316 and C319 (see above).

In another aspect, the invention provides compounds of Formula F18 and salts thereof:

Where

R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thi Oureido is also iminoamino or phosphonamino.

In another embodiment of the invention, the compound of formula F18

to be.

Exemplary compounds of formula F18 include, but are not limited to, compound C180 (see above).

In another embodiment, the present invention provides a compound of Formula F19 and salts thereof:

Where

이고;a) R ² is

ego;

이며;b) R ⁶ is methyl or

Is;

이고;c) R ⁸ is methyl or

ego;

b) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting groups, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino.

In another embodiment of the invention, the compound of formula F19 is

Is selected from.

Exemplary compounds of Formula F19 include, but are not limited to, compounds C86, C359, and C356 (see above).

In another embodiment, the present invention provides a compound of Formula F20 and salts thereof:

Where

a) R ¹² is H or CH ₃ ;

b) R ¹ and R ^{8 **} are amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thi Oureido is also iminoamino or phosphonamino.

In another embodiment of the invention, the compound of formula F20

Is selected from.

Exemplary compounds of Formula F20 include, but are not limited to, compounds C343 and C340 (see above).

In another embodiment, the present invention provides a compound of Formula F21 and salts thereof:

Where

이고; a) R ¹ is

ego;

b) R ¹² is H or CH ₃ ;

c) R ^{8 **} is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, Iminoamino, or phosphonamino.

In another embodiment of the invention, the compound of formula F21 is

Is selected from.

Exemplary compounds of Formula F21 include, but are not limited to, compounds C265 and C271 (see above).

In another embodiment, the present invention provides compounds of Formula F22 and salts thereof:

                                                       F (22)

Where

R ^{6 *} is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, iminoamino Or phosphonamino.

In another embodiment of the invention, the compound of formula F22

to be.

Exemplary compounds of formula F22 include, but are not limited to, compound C3 (see above).

In one embodiment of the invention, the substituent R ¹ in any compound of Formula F1-F20 is an amino, acylamino, NH-amino protecting group or carbamoyl. In another embodiment of the invention, substituent R ¹ in any compound of Formula F1-F20 is C ₁₀ -C ₁₃ alkanoylamino. In another embodiment of the invention, substituent R ¹ in any compound of Formula F1-F20 is

이다.

to be.

이다. In another embodiment of the invention, substituent R ¹ in any compound of Formula F1-F20 is

to be.

In one embodiment of the invention, the substituents R ^{6 *} in any of the compounds of the formulas F1-F5, F10-F14, F19 and F22 are amino, NH-amino protecting groups or carbamoyl. In another embodiment of the invention, the substituents R ^{6 *} in any of the compounds of Formulas F1-F5, F10-F14, F19 and F22 are amino.

In one embodiment of the invention, the substituents R ^{8 **} in any of the compounds of the formulas F2-F5, F7-F9, F13, F15, F16, F18 and F20-F21 are amino, NH-amino protecting groups or carbamoyl . In another embodiment of the invention, the substituents R ^{8 **} in any compound of formula F2-F5, F7-F9, F13, F15, F16, F18 and F20-F21 are amino.

Those skilled in the art will understand that the compounds of the present invention, in particular the compounds of formula (I) and formula (F1-F22), are useful as intermediates for the preparation of other compounds of formula (I) and (F1-F22). Particularly useful intermediate compounds include compounds of formula I, F2-F5, F13 and F19, wherein at least one of R ¹ , R ^{6 *} or R ^{8 **} is amino, NH-amino protecting group or carbamoyl; A compound of formula F1 or F10-F14, wherein at least one of R ¹ or R ^{6 *} is amino, NH-amino protecting group or carbamoyl; Compounds of formula F7-9, F15-16, F18 and F20, wherein at least one of R ¹ or R ^{8 **} is amino, NH-amino protecting group or carbamoyl; A compound of Formula F22, wherein R ^{6 *} is amino, NH-amino protecting group or carbamoyl; A compound of Formula F21, wherein R ^{8 **} is amino, NH-amino protecting group or carbamoyl; And R ¹ is amino, NH-amino protecting group or carbamoyl.

Pharmaceutical Compositions and Methods of Use thereof

The present invention provides, in one embodiment, a pharmaceutical composition or formulation comprising a compound of Formula I or a compound of any of Formulas F1-F22, or salts thereof.

Compounds of the invention, preferably compounds of formula (I) or compounds of any one of formulas (F1-F22), or pharmaceutically acceptable salts thereof, are orally or intravenously for the treatment or prophylactic treatment of diseases, in particular bacterial infections. It may be formulated for intramuscular, subcutaneous or parenteral administration. For oral or parenteral administration, the compounds of the present invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like. Compositions comprising a compound of the present invention contain from about 0.1% to about 99% by weight of active compound, more typically from about 10% to about 30% of active compound.

The pharmaceutical formulations disclosed herein are prepared according to standard procedures and administered in dosages selected to reduce, prevent or eliminate infections (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA and "Goodman and Gilman's The Pharmaceutical Basis of Therapeutics ", Pergamon Press, New York, NY, the contents of which are incorporated herein by reference, which provides a general description of how to administer various fungicides for the treatment of the human body. Compositions of the invention, preferably compositions of Formula (I) or compositions of any of Formulas F1-F22, may be delivered using a controlled (e.g., capsule) or sustained release delivery system (e.g., a biodegradable matrix). Can be. Representative delayed release delivery systems for drug delivery suitable for administration of a composition of the invention, preferably a composition of Formula (I) or any of Formulas F1-F22, are described in US Pat. No. 4,452,775 (Kent), 5,239,660 to Leonard and 3,854,480 to Zaffaroni.

Pharmaceutically acceptable compositions of the present invention, in the case of one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants and / or excipients, collectively referred to herein as " carrier " Together with other active ingredients, at least one compound of the invention, preferably a compound of formula (I) or a compound of any one of formulas (F1-F22). This composition may comprise conventional carriers and excipients such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The composition may comprise croscarmellose sodium, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid.

Purifying binders that may be contained are acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (povidone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearate or other metal-based stearates, stearic acid, silicone fluids, talc, waxes, oils and colloidal silica.

Flavoring agents such as peppermint, methyl salicylate (oil of wintergreen), cherry flavor and the like can also be used. It may also be desirable to add colorants to the formulation to make the appearance more aesthetic or to aid in the identification of the product.

For oral use, solid preparations such as tablets and capsules are particularly useful. Sustained release or enteric coating formulations may also be considered. Suspensions, syrups and chewing tablets are particularly suitable for use in pediatric and geriatric medicine. For oral administration, the pharmaceutical compositions are, for example, in the form of tablets, capsules, suspensions or solutions. The pharmaceutical composition is preferably prepared in the form of a dosage unit containing a therapeutically effective amount of the active ingredient. Such dosage units include tablets and capsules. For therapeutic purposes, tablets and capsules may be used in addition to the active ingredient in conventional carriers such as binders such as acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; Fillers such as calcium phosphate, glycine, lactose, corn starch, sorbitol, or sucrose; Lubricants such as magnesium stearate, polyethylene glycol, silica, or talc; Disintegrants, for example potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations are generally in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs and may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous preparations, preservatives, colorants and flavoring agents. Examples of additives for liquid formulations include acacia, almond oil, ethyl alcohol, fractional distillation coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol Or sorbic acid.

For intravenous (IV) use, the compounds of the present invention may be dissolved or suspended in any of the intravenous fluids commonly used and administered by infusion. Intravenous fluids include, but are not limited to, saline or Ringer's solution. Intravenous administration can be performed using, but not limited to, syringes, minipumps or intravenous lines.

Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injectable solutions or suspensions. Such solutions or suspensions may be prepared from sterile powders or granules containing one or more of the carriers mentioned for use in the formulation for oral administration. This compound can be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride, and / or various buffers.

For intramuscular preparations, sterile preparations of the invention, or suitable soluble salt forms of the compounds, for example hydrochloride, are administered by dissolving in pharmaceutical diluents such as water for injection (WFI), physiological saline or 5% glucose. can do. Suitable insoluble forms of this compound can be prepared and administered as suspensions in aqueous bases or in pharmaceutically acceptable oil bases, for example esters of long chain fatty acids such as ethyl oleate.

Doses of intravenous, intramuscular or parenteral preparations of the compounds of the invention may be administered as bolus or slow infusion. Bolus is a dose administered in less than 30 minutes. In a preferred embodiment, the bolus is administered in less than 15 minutes or less than 10 minutes. In a more preferred embodiment, the bolus is administered in less than 5 minutes. In even more preferred embodiments, the bolus is administered within 1 minute. Infusion is a dose administered at a rate of 30 minutes or more. In a preferred embodiment, the infusion is administered at a rate of at least 1 hour. In another embodiment, the infusion is administered at a substantially constant rate.

For topical use, the compounds of the invention, preferably compounds of formula (I) or compounds of any of formulas (F1-F22) may be prepared to be applied to the skin, or to the mucous membranes of the nose and throat, in a suitable form, It may take the form of a cream, ointment, liquid propellant or inhalant, lozenge, or throat paint. Such topical preparations may further contain chemical compounds such as dimethylsulfoxide (DMSO) to promote surface penetration of the active ingredient.

For use on the eyes or ears, the compounds of the invention, preferably compounds of formula (I) or compounds of any one of formulas (F1-F22) are liquids formulated in hydrophobic or hydrophilic bases such as ointments, creams, lotions, paints or powders or It may be provided in semi-liquid form.

For rectal administration, the compounds of the present invention, preferably one of Formula I or Formula F1-F22, may be administered in the form of suppositories mixed with conventional carriers such as cocoa butter, wax or other glycerides. .

Alternatively, the compound of the invention, in one embodiment, the compound of formula (I) or any one of formulas F1-F22 may be in powder form for reconstitution in a suitable pharmaceutically acceptable carrier at the time of administration. In another embodiment, the unit dosage form of the compound may be a solution of the compound or preferably its salt in a suitable diluent included in a sterile sealed ampoule or sterile syringe. The concentration of the compound in the unit dose may vary, for example, from about 1% to about 50%, depending on the compound used and its solubility and dose chosen by the physician. If the composition contains dosage units, each dosage unit preferably contains 1 to 500 mg of active substance. For adult treatment, it is desirable to use a dosage of 5 mg to 10 g per day, depending on the route of administration and frequency of administration.

In another aspect, the invention provides a method of inhibiting the growth of microorganisms, preferably bacteria, comprising contacting said organism with a compound of the invention under conditions that permit contact of said compound with said organism and said microorganism. It provides a method to include. Such conditions are known to those skilled in the art and are exemplified in the Examples. The method comprises contacting the microbial cells in vivo or in vitro with a therapeutically effective amount of a compound (s) of the invention, preferably a compound (s) of formula (I) or a compound of any one of formulas F1-F22. Include.

According to this aspect of the invention, the novel compositions disclosed herein are contained in a pharmaceutically acceptable carrier and delivered to a receiving subject (preferably human) in accordance with known drug delivery methods. In general, the method of the present invention for delivering a composition of the present invention in vivo employs a protocol for the delivery of a formulation recognized in the art, wherein the drug in the art recognized protocol is a compound of the present invention, preferably a formula Substitution with the compound of I or any one of formulas F1-F22 is the only substantial procedural modification. Likewise, methods of using the compositions of the invention to treat cells in culture, for example for the purpose of removing or reducing bacterial contamination of the cell culture, are in the art for treating cell cultures with antibacterial agent (s). The only practical procedural is to replace the agents used in the protocols recognized in the art with the compounds of the present invention, preferably the compounds of formula I or the compounds of formulas F1-F22, using the protocols recognized by It is a variation.

In one embodiment, the invention provides a method of treating an infection of a subject, in particular an infection caused by a gram positive bacterium, with a therapeutically effective amount of a compound of the invention. Representative methods for administering antibacterial agents are described in US Pat. No. 5,041,567 and PCT patent application EP94 / 02552 (published number WO 95/05384), which is incorporated by reference in its entirety herein. As used herein, the phrase “therapeutically effective amount” means an amount of a compound of the present invention that prevents the onset of a bacterial infection or alleviates the symptoms or stops its progression. The term "treatment" is defined as administering to a subject a therapeutically effective amount of a compound of the present invention to prevent the occurrence of an infection and to control or eliminate the infection. The phrase “subject” as described herein is defined as a mammal, plant or cell culture. In a preferred embodiment, the subject is a human or other animal patient in need of antibacterial treatment.

The method comprises administering to the subject an effective amount of a compound of the present invention. An effective amount is generally from about 0.1 mg / kg to about 100 mg / kg of a compound of the present invention or a pharmaceutically acceptable salt thereof. Preferred doses are from about 1 mg / kg to about 50 mg / kg of a compound of the present invention or a pharmaceutically acceptable salt thereof. More preferred doses are about 1 mg / kg to about 25 mg / kg of a compound of the present invention or a pharmaceutically acceptable salt thereof. The effective amount for cell culture is generally 0.1-1000 μg / mL, more preferably 0.1-200 μg / mL.

Compositions containing a compound of the present invention can be administered as a single daily or multiple daily dosage. Treatment regimens may require long-term administration, for example for several days or for two to four weeks. Dosages per dose or total dosage will vary depending on factors such as the nature of the infection and the severity of the infection, the patient's age and systemic health, the patient's resistance to the compound and the microorganism (s) associated with the infection. A method for administering daptomycin, another member of the depsipeptide compound class, to a patient is disclosed in US Pat. Nos. 6,468,967 and 6,852,689, the contents of which are incorporated herein by reference.

The compounds of the invention can also be administered in the diet or feed of a patient or animal. If administered as part of the total dietary intake, the amount of the compound used may be less than 1% by weight of the diet, preferably 0.5% by weight or less. An animal diet may be a normal diet to which the compound may be added or as a premix.

The invention also provides a compound of the invention, preferably a compound of formula (I) or a compound of formula (F1-F22), or a subject thereof in need thereof in an amount effective in reducing, ameliorating or eliminating bacterial infection. A method of administering a pharmaceutical composition is provided. The compound may be administered orally, parenterally, inhaled, topically, rectally, nasal, buccal, vaginal administration or by implanted reservoir, external pump or catheter. This compound may be prepared for ophthalmic or aerosol use. The compounds of the present invention can be administered as aerosols. Preferred aerosol delivery media are anhydrous or dry powder inhalants. The compound of formula I or a compound of any one of formulas F1-F22, or a pharmaceutical composition thereof, may also be injected or administered directly into the boil, chamber or joint. Parenteral administration includes subcutaneous, intravenous, intramuscular, intraarticular, synovial, cisternal, intradural, intrahepatic, intralesional and intracranial injections or infusions. In a preferred embodiment, the compounds of the invention are administered intravenously, subcutaneously or orally. In a preferred embodiment for administering a compound of Formula (I) or any of Formulas F1-F22 to a cell culture, the compound may be administered in a nutrient medium.

The compounds of the present invention can be used to treat subjects with bacterial infections caused or exacerbated by any type of bacteria, in particular gram positive bacteria. In one embodiment, a compound of the invention or a pharmaceutical composition thereof is administered to a patient according to the methods of the invention. In a preferred embodiment, the bacterial infection is one that can be caused or exacerbated by gram positive bacteria. Such as Gram-positive bacteria appeared methicillin susceptible and methicillin-resistant Staphylococcus [Staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis (S. epidermidis), Staphylococcus Mori's Tea Syracuse (S. haemolyticus ), including S. hominis , S. saprophyticus and coagulase negative Staphylococcus], glycopeptide mediated susceptible Staphylococcus aureus ( GISA), vancomycin-resistant Staphylococcus aureus (VRSA), penicillin-susceptible and penicillin-resistant Streptococcus - Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus payioh Jens (S. pyogenes), Streptococcus Agar rakti (S. agalactiae), Streptococcus Oh Away (S. avium), Streptococcus bovis (S. bovis), Streptococcus lactis (S. lactis), Streptococcus S. sangius and Streptococcus group C, including Streptococcus group G and Viridans streptococci ], Enterococcus [vancomycin susceptible and vancomycin resistant strains such as Enterococcus fascus faecalis) and Enterococcus passive help (including E. faecium)], Clostridium difficile (Clostridium difficile), Clostridium Claus tree form di (C. clostridiiform e), Clostridium Ino kyum (C. innocuum ), Clostridium perfringens (C. perfringens), L'isle Clostridium (C. ramosum), by a brush Russ influenza (Haemophilus influenzae ), Listeria monocytogenes ( Listeria monocytogenes ), Corynebacterium jeikeium ), Bifidobacterium spp., Eubacterium aerofaciens), Euro tumefaciens renteom (E. lentum), Lactobacillus Ash's also a filler (Lactobacillus acidophilus), Casey Lactobacillus (L. casei), Lactobacillus Planta Rum (L. plantarum), Lactococcus species (Lactococcus spp ), Leuconostoc spp., Pediococcus , Peptostreptococcus Inner Aerobis anaerobius), pepto Streptococcus Asaka a utility kusu (P. asaccarolyticus), pepto Streptococcus Magnus (P. magnus), pepto Streptococcus My cross (P. micros), pepto Streptococcus boti frame (P. prevotii), pepto Streptomyces P. productus , Propionibacterium acnes ), Actinomyce s spp., Moraxella spp. (including M. catarrhalis ) and Escherichia spp. ( E. coli E. coli ), but is not limited to such.

In a preferred embodiment, the antibacterial activity of a compound of formula (I) or of any of formulas F1-F22 against a traditionally "resistant" strain is comparable to the resistance to a traditionally "sensitive" strain in in vitro experiments. be worth. In another preferred embodiment, the minimum inhibitory concentration (MIC) value of the compound according to the invention for susceptible strains is generally less than or equal to the value of vancomycin or daptomycin. Thus, in a preferred embodiment, a compound of the invention or a pharmaceutical composition thereof is administered according to the method of the invention to a patient exhibiting a bacterial infection resistant to other compounds, including vancomycin or daptomycin. In addition, unlike glycopeptide antibiotics, depsipeptides such as those disclosed herein exhibit rapid and concentration dependent bactericidal activity against gram positive organisms. Thus, in a preferred embodiment, the compound according to the invention or a pharmaceutical composition thereof is administered according to the method of the invention to a patient in need of rapid action antibiotic treatment.

The methods of the invention can be used for any bacterial infection of any organ or tissue of the body. In a preferred embodiment, the bacterial infection is caused by gram positive bacteria. Such organs or tissues include, but are not limited to, skeletal muscle, skin, blood flow, kidneys, heart, lungs, and bones. The methods of the present invention can be used in the treatment of skin and soft tissue infections, bacteremia and urinary tract infections, but are not limited thereto. The methods of the invention can also be used for the treatment of mixed infections comprising different types of gram positive bacteria bacteria or both gram positive bacteria and gram negative bacteria. Infections of this type include abdominal infections and obstetric / gynecological infections. The methods of the invention can also be used for the treatment of infections including, but not limited to, endocarditis, nephritis, septic arthritis, intraperitoneal sepsis, bone and joint infections and osteomyelitis. In a preferred embodiment, the compounds according to the invention or pharmaceutical compositions thereof can be used to treat any of the aforementioned diseases.

The method of the present invention may also be practiced while simultaneously administering one or more other antimicrobial agents such as antibacterial agents (antibiotics) or antifungal agents. In one embodiment, the method may be carried out by administering one or more compounds according to the invention. In another embodiment, the process comprises the compounds according to the invention, for example in US Pat. Nos. 6,911,525 and 6,794,490 and in International Patent Applications WO 01/44272; WO 01/44274; It may be carried out by administration with a lipopeptide compound such as WO 01/44271 and WO 03/014147 or a lipopeptide compound such as daptomycin.

Antibacterial agents and classes thereof which may be coadministered with the compounds according to the invention include penicillin and related drugs, carbapenem, cephalosporin and related drugs, aminoglycosides, bacitracin, gramicidine, mupirosine, Chloramphenicol, thiamphenicol, pushdate sodium, lincomycin, clindamycin, macrolide, novobiocin, polymyxin, rifamycin, spectinomycin, tetracycline, vancomycin, teicoplanin, streptogramamine; Pyrimethamine with antifolates, such as sulfonamides, trimethoprim and combinations thereof; Synthetic antibacterial agents including nitrofuran, methenamin mandelate and methenamin hypurate, nitroimidazole, quinolone, fluoroquinolone, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid (PAS), cycloserine, capreo Mycin, ethionamide, prothionamide, thiacetazone, biomycin, evernimycin, glycopeptide, glycylsilcline, ketolide, oxazolidinone; Imiphenene, amikacin, netylmycin, fosfomycin, gentamicin, ceftriaxone, ZIRACIN®, LY 333328, CL 331002, HMR 3647, ZYVOX®, SYNERCID®, aztreo South Metronidazole, Epiproprim, OCA-983, GV-143253, San Petrinen Sodium, CS-834, Viafenem, A-99058.1, A-165600, A-179796, KA 159, Dynemycin A, DX8739, DU 6681; Cefluprenam, ER 35786, cephalis, sanpetrinem selecetyl, HGP-31, cepypyrom, HMR-3647, RU-59863, musacidin, KP 736, ripalazil; AM 1732, MEN 10700, Renafenem, BO 2502A, NE-1530, PR 39, K130, OPC 20000, OPC 2045, Beneprim, PD 138312, PD 140248, CP 111905, Sulfenem, Lipefenam acornsil, RO-65 -5788, cyclothialidine, Sch-40832, SEP-132613, mycacosidin A, SB-275833, SR-15402, SUN A0026, TOC 39, carumonem, zozofran, cepetetamet cladding and T 3811 It may include, but is not limited to.

Antifungal agents which may be co-administered with the compounds according to the invention include caspofungen, voriconazole, cetaconazole, IB-367, FK-463, LY-303366, Sch-56592, citafloxacin, DB-289 poly Enes such as amphotericin, nystatin, primaricin; Azoles such as fluconazole, itraconazole and ketoconazole; Allylamines such as naphthypine and terbinafine; And metabolic agents such as flucytosine. Other antifungal agents include, but are not limited to, those described in Fostel, et al., 2000, Drug Discovery Today 5: 25-32, incorporated herein by reference. Fostel et al. Disclose antifungal compounds including corynecandine, Mer-WF3010, fusacandines, atrikitin / LL 15G256, sodarins, cispentasin, azoxyvacillin, oreovacidine and caprefungin.

The compounds according to the invention can be administered according to the methods of the invention until the bacterial infection is eradicated or reduced. In one embodiment, the compound of formula (I) or any one of formulas F1-F22 is administered for a period of 2 days to 6 months. In a preferred embodiment, in one embodiment, the compound of formula (I) or any one of formulas F1-F22 is administered for 7 to 56 days. In a more preferred embodiment, the compound of formula (I) or any one of formulas F1-F22 is administered for 7 to 28 days. In even more preferred embodiments, the compound of formula (I) or any one of formulas F1-F22 is administered for 7-14 days. The compound of formula (I) or any of formulas F1-F22 may be administered for a longer time or for a shorter time, if necessary.

The invention provides, in one embodiment, an antibacterial composition or formulation comprising a compound of Formula (I) or a compound of any of Formulas (F1-F22), or salts thereof. In one embodiment, the antibacterial composition may be contained in an aqueous solution. In yet another embodiment, the aqueous solution can be a buffered aqueous solution. In yet another embodiment, the buffer may have an acidic, neutral or basic pH.

new Depsipeptide  Produce

1. Synthetic Method

In one embodiment of the invention, the compound of formula (I) or compound of formula (F1-F22) may be prepared using solid support chemistry. According to three preferred methods, namely methods A-C, resin-bonded linear precursors nn3, nn3a or nn3b are prepared.

As outlined in Scheme I, Method A uses nn1, a polypeptide fragment derived from 7 amino acids of resin binding type, and nn2, a polypeptide fragment derived from 6 amino acids. This method is called "7 + 6 fragment synthesis".

Scheme I

Method A "7 + 6" fragment synthesis

Alternatively, as shown in Scheme II, Method B utilizes nn1a, a polypeptide fragment derived from six amino acids of the resin bound type, and nn2a, a polypeptide fragment derived from seven amino acids. This method is called "6 + 7 fragment synthesis".

Scheme II

Method B. Synthesis of "6 + 7" Fragments

Another method, Method C, uses a polypeptide derived from six amino acids, a resin bound amino acid, and a polypeptide derived from a second six amino acids. This method is called "1 + 6 + 6 fragment synthesis".

Scheme III

Method C. Synthesis of "1 + 6 + 6" Fragments

Depsi Peptide  Solid support synthesis of compounds, method A: synthesis of 7 + 6 fragments

Depsipeptide compounds of formula I can be synthesized on a solid support as outlined in Scheme IV, Scheme V and Scheme VI below.

Scheme IV

In a first step, such as commercially available Na-Fmoc-L-glutamic acid a-allyl ester or N-Fmoc-L-3-methyl glutamic acid a-allyl ester (see Examples 1-68 and 1-69, see below) The protected glutamic acid-derivative was coupled to the resin to form compound nn5, wherein R ¹² is as defined above. Non-limiting examples include Wang, HMPA, Safety Catch, Rink Acid, 2-Chlorotrityl-chloride resin, Trityl-chloride resin, 4-methyltrityl-chloride resin, Resin or solid support such as 4-methoxytrityl-chloride resin or PAM resin can be used for this reaction. The protecting groups P ₁ and P ₂ are chosen such that they can be removed independently of each other without causing cleavage of the peptide from the resin. Examples of protecting groups can be found in "Protecting Groups in Organic Synthesis" by Theodora W. Greene (see above), below "Greene". Non-limiting examples of protecting group combinations such as P ₁ is allyl ester and P ₂ is Fmoc are also suitable for the reaction.

이고; R^{6 *A}는 보호된 아미노, 단치환 아미노, 이치환 아미노, 아실아미노, 우레이도, 구아니디노, 카르바모일, 설폰아미노, 티오아실아미노, 티오우레이도, 이미노아미노, 또는 포스폰아미노이며, 단, R^{6 *A}는 펩티드로부터 수지를 제거하는 데 필요한 조건과 상용성이며; R^8A는 아미노산 측쇄, 보호된 아미노산 측쇄, 메틸, CH₂-OP₆, CH₂-CONHP_{5 *} 또는

이고; 여기서 P_{5 *} 및 P₆은 각각 독립적으로 적절한 보호기이며; 여기서 R^{8 **A}는 보호된 아미노, 단치환 아미노, 이치환 아미노, 아실아미노, 우레이도, 구아니디노, 카르바모일, 설폰아미노, 티오아실아미노, 티오우레이도, 이미노아미노, 또는 포스폰아미노이며, 단, R^{8 **A}는 펩티드로부터 수지를 제거하는 데 필요한 조건과 상용성이며; 여기서 R^9A는

, 또는 화학식

의 카르복실산기 하나 이상으로 치환된 아미노산 측쇄이고; P₇은 수지로부터의 펩티드의 절단을 초래하지 않고 P₁과 독립적으로 제거될 수 있는 보호기이며; P₈ 및 P₉는 각각 독립적으로, P₁ 및 P₇이 P₈ 및 P₉ 각각과 독립적으로 제거될 수 있고 P₈ 및 P₉ 각각이 수지로부터 절단시 절단되는 적절한 보호기이며; P₁, R^{8 *}, R^9A, R^{11 *}, R^11A 및 R¹²는 상기에 정의된 바와 같다.After deprotecting the amine of compound nn5, coupling free amino with an amino acid or a protected amino acid forms compound nn6, wherein P ₃ can be removed independently of P ₁ without causing cleavage of the peptide from the resin. A protecting group; R ^11A is an amino acid side chain, protected amino acid side chain, methyl, CH ₂ -OP ₄ , or CH ₂ -CONHP ₅ ; P ₄ and P ₅ are each independently a suitable protecting group, and P ₁ and R ^{11 *} are each as defined above. This peptide coupling process, ie deprotection of the alpha-amino group, followed by coupling to the protected amino acid is repeated until the desired number of amino acids are coupled to the resin. In Scheme IV, a total of seven amino acids are coupled to form compound nn1, wherein R ^6A is methyl or

ego; R ^{6 * A} is protected amino, mono-substituted amino, di-substituted amino, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, iminoamino, or phosphonamino Provided that R ^{6 * A} is compatible with the conditions necessary to remove the resin from the peptide; R ^8A is an amino acid side chain, a protected amino acid side chain, methyl, CH ₂ -OP ₆ , CH ₂ -CONHP _{5 *} or

ego; Wherein P _{5 *} and P ₆ are each independently a suitable protecting group; Wherein R ^{8 ** A} is protected amino, mono-substituted amino, di-substituted amino, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, iminoamino, or phosph Phonamino, provided that R ^{8 ** A} is compatible with the conditions necessary to remove the resin from the peptide; Where R ^9A is

Or formula

An amino acid side chain substituted with one or more carboxylic acid groups of; P ₇ is a protecting group that can be removed independently of P ₁ without causing cleavage of the peptide from the resin; P ₈ and P ₉ are, each independently, P ₁ and P ₇ P ₈ and P ₉ are to be removed independently of each, and P ₈ and P ₉ each a suitable protecting group to be cut when cutting from the resin and; P ₁ , R ^{8 *} , R ^9A , R ^{11 *} , R ^11A and R ¹² are as defined above.

As outlined in Scheme V, the second peptide is coupled to the resin in a similar manner.

Scheme V

In step 1, N-protected glycine, such as commercially available Fmoc-N-glycine, is coupled to the resin to yield compound nn7, wherein R ^5A and R ^{5 * A} are independently hydrido and P ₁₀ is from the resin Is a protecting group selected such that it can be removed without causing cleavage of the peptide. The choice of resin used in step 1 depends on the nature of the amino acids coupled in steps 2-6. If the amino acid side chain contains a protecting group, the resin should be selected such that the protecting group remains intact when the resin is removed from the peptide in step 7. Resins that can be cleaved while preserving the protecting group of the peptide include safety catch, link acid, 2-chlorotrityl-chloride resin, trityl-chloride resin, 4-methyltrityl-chloride resin, 4-methoxytrityl -Chloride resins or PAM resins.

Deprotection of protected amino from compound nn7 followed by coupling of free amino with n14 below gives compound nn8.

     (n14)

Wherein P ₁₁ is a protecting group selected such that it can be removed without causing cleavage of the peptide from the resin. This peptide coupling process, ie deprotection of the alpha-amino group followed by coupling to the protected amino acid, is repeated until the desired number of amino acids are coupled to the resin. In Scheme V, five amino acids are coupled to form compound n11, wherein R ^1A is a protected amino, monosubstituted amino, disubstituted amino, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thio Acylamino, thioureido, iminoamino, or phosphoneamino, provided that R ^1A is compatible with the conditions necessary to remove the resin from the peptide; R ^2A is an amino acid side chain, protected amino acid side chain, CH ₂ -CH ₂ -CO ₂ P ₁₄ , or CH ₂ -CONHP ₁₅ ; R ^3A is CH ₂ —CO ₂ P ₁₆ , CH (OP ₁₇ ) CONH ₂ , CH ₂ CONH ₂ , a nonproteinaceous amino acid side chain, or a protected nonproteinaceous amino acid side chain; P ₁₂ And P ₁₃ are each a protecting group selected such that they can be removed without causing cleavage of the peptide from the resin; P ₁₄ , P ₁₅ , P ₁₆ and P ₁₇ are each independently a suitable protecting group; R ^{2 *} , R ^5A and R ^{5 * A} are as defined above.

와 커플링되어 화합물 n12를 형성하며, 여기서 P₁₈은 적절한 보호기이고, R^13A는 CH(CH₃)₂, CH(CH₂CH₃)CH₃,

이다.Compound n11 is a compound

Is coupled to form compound n12, wherein P ₁₈ is a suitable protecting group, and R ^13A is CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

to be.

Peptide n12 is then removed from the resin to yield compound nn2, where P ₁₉ is a suitable protecting group.

Coupling of peptide fragments nn1 and nn2 is outlined in Scheme VI below.

Scheme VI

Peptide fragments nn1 and nn2 are coupled to form a resin-bound peptide nn3, wherein R ^1A , R ^{2 *} , R ^2A , R ^3A , R ^5A , R ^{5 * A} , R ^6A , R ^{8 *} , R ^8A , R ^9A , R ^11A , R ^{11 *} , R ¹² , R ^13A , P ₁ , P ₈ and P ₁₈ are as defined above. Deprotection of the P ₁ and P ₁₈ protecting groups followed by cyclization yields the resin-bound depsipeptide nn 4, wherein R ^1A , R ^{2 *} , R ^2A , R ^3A , R ^5A , R ^{5 * A} , R ^6A , R ^{8 *} , R ^8A , R ^9A , R ^11A , R ^{11 *} , R ¹² , R ^13A , and P ₈ are as defined above. The cleavage of the depsipeptide from the resin and the deprotection of any residual protecting groups yield the compound of formula (I).

Depsi Peptide  Solid support synthesis of compounds, method B:

6 + 7 fragment synthesis

Depsipeptide compounds of formula I can be synthesized on a solid support as described in Schemes VII, VIII, and IX.

Scheme VII

Compound nn6 is prepared as described in Method A. The peptide coupling process (see above), ie deprotection of the alpha-amino groups, followed by coupling to the protected amino acids is repeated until the desired number of amino acids are coupled to the resin. In Scheme VII, a total of six amino acids are coupled to form compound nn1a, wherein R ^{8 *} , R ^8A , R ^9A , R ^{11 *} , R ^11A , R ¹² , P ₁ and P ₈ are as defined above. And P ₂₀ is a protecting group that can be removed independently of P ₁ without causing cleavage of the peptide from the resin, such as P ₁ is allyl and P ₂₀ is Fmoc.

As outlined in Scheme VIII, the second peptide is coupled to the resin in a similar manner.

Scheme VIII

In step 1, the N-protected amino acid is coupled to the resin to form compound n16, wherein P ₂₁ is a protecting group selected to be removed without causing cleavage of the peptide from the resin and R ^6A is as defined above. same. The choice of resin used in step 1 depends on the nature of the amino acids coupled in steps 2-6. If the amino acid side chain contains a protecting group, the resin should be selected so that the protecting group remains intact when the resin is removed from the peptide in step 8. Resins that can be cleaved while preserving the protecting group of the peptide include safety catch, link acid, 2-chlorotrityl-chloride resin, trityl-chloride resin, 4-methyltrityl-chloride resin, 4-methoxytrityl -Chloride resins or PAM resins.

Deprotection of the protected amino of compound n16 followed by coupling of the free amino to the second protected amino acid yields compound n17, wherein P ₂₂ can be removed without causing cleavage of the peptide from the resin. Is a protector selected to be; R ⁵ , R ^{5 *} , and R ⁶ are as defined above.

Deprotection of the protected amino of compound n17 followed by coupling of free amino groups with n14 (see above) yields compound n18, wherein R ⁵ , R ^{5 *} , R ^6A and P ₉ are defined above As shown. The peptide coupling process, ie deprotection of the alpha-amino group, followed by coupling to the protected amino acid is repeated until the desired number of amino acids are coupled to the resin. In Scheme VIII, six amino acids were coupled to form compound n21, wherein P ₂₃ and P ₂₄ are each protecting groups that can be removed without causing cleavage of the peptide from the resin; R ^1A , R ^2A , R ^{2 *} , R ^3A , R ⁵ , R ^{5 *} , and R ^6A are as defined above.

Compound n21 couples with n15 (see above) to form compound n22, wherein R ^1A , R ^2A , R ^{2 *} , R ^3A , R ⁵ , R ^{5 *} , R ^6A , R ^13A and P ₁₈ are As defined.

Peptide n22 is then removed from the resin to yield compound nn2a, wherein R ^1A , R ^2A , R ^{2 *} , R ^3A , R ⁵ , R ^{5 *} , R ^6A , R ^13A and P ₁₈ are as defined above. same.

Coupling of peptide fragments nn1a and nn2a is outlined in Scheme IX below.

Scheme IX

Coupling peptide fragments nn1a and nn2a yields resin-bound peptide nn3a, wherein R ^1A , R ^2A , R ^{2 *} , R ^3A , R ⁵ , R ^{5 *} , R ^6A , R ^{8 *} , R ^8A , R ^9A , R ^{11 *} , R ^11A , R ¹² , R ^13A , P ₁ , P ₈ , P ₉ and P ₁₈ are as defined above.

Deprotection of the P ₁ and P ₁₈ protecting groups followed by cyclization yields the resin-bound depsipeptide nn4a, wherein R ^1A , R ^2A , R ^{2 *} , R ^3A , R ⁵ , R ^{5 *} , R ^6A , R ^{8 *} , R ^8A , R ^9A , R ^{11 *} , R ^11A , R ¹² , R ^13A , and P ₈ are as defined above.

The cleavage of the depsipeptide from the resin and the deprotection of any residual protecting groups yield the compound of formula (I).

Depsi Peptide  Solid support synthesis of compounds, method C

1 + 6 + 6 fragment synthesis

In another embodiment of the present invention, the depsipeptide compound of Formula I can be synthesized as described in Scheme X-XII.

Scheme X

In step 1, protected β-methyl glutamic acid derivatives such as commercially available Na-Fmoc-L-glutamic acid a-allyl ester or N-Fmoc-L-3-methyl glutamic acid a-allyl ester (Examples 1-68 and 1- 69, see below), is coupled to the resin to form compound n23, wherein R ^12A is methyl. Non-limiting examples include Wang, HMPA, safety catch, link acid, 2-chlorotrityl-chloride resin, trityl-chloride resin, 4-methyltrityl-chloride resin, 4-methoxytrityl-chloride resin Or a resin or solid support such as a PAM resin can be used for this reaction. The protecting groups P ₂₅ and P ₂₆ are selected such that they can be removed independently of each other without causing cleavage of the peptide from the resin. As a non-limiting example, a combination of protecting groups such as P ₂₅ is allyl ester and P ₂₆ is Fmoc is suitable for the reaction.

As outlined in Scheme XI below, the second peptide is coupled to the resin in a similar manner.

Scheme XI

In step 1, the protected amino acid is coupled to the resin to yield compound n24, wherein P ₂₇ is a protecting group that can be removed without causing cleavage of the peptide from the resin; R ^{11 *} and R ^11A are as defined above. The choice of resin used in the first step depends on the nature of the amino acids coupled in the previous step. If the amino acid side chains contain protecting groups, the resin should be selected such that these protecting groups remain intact when the peptide is removed from the resin. Resins that can be cleaved while preserving the protecting group of the peptide include safety catch, link acid, 2-chlorotrityl-chloride resin, trityl-chloride resin, 4-methyltrityl-chloride resin, 4-methoxytrityl -Chloride resins or PAM resins.

The peptide coupling process, ie deprotection of the alpha-amino group, followed by coupling to the protected amino acid is repeated until the desired number of amino acids are coupled to the resin. In Scheme XI, a total of six amino acids were coupled to form compound n25, wherein P ₂₈ is a protecting group that can be removed without causing cleavage of the peptide from the resin; R ^6A , R ^{8 *} , R ^8A , R ^9A , R ^{11 *} , R ^11A and P ₈ are as defined above.

Cleavage of the peptide from the resin yields compound n26. Coupling of the three peptide fragments is outlined in Scheme XII below.

Scheme XII

Deprotection of the resin-bonded 3-methylglutamate n23, where R ^12A is as described above, affords the free amine, which is then coupled to fragment n26 to give the resin-bonded fragment nn1b, wherein R ^11A , R ^{11 *} , R ^9A , R ^8A , R ^{8 *} , R ^6A , P ₈ , P ₂₅ , and P ₂₈ are as described above. It is then coupled to the fragment nn2 described above to form nn3b, wherein R ^1A , R ^2A , R ^{2 *} , R ^3A , R ^{5 * A} , R ^{5 * A} , R ^6A , R ^{8 *} , R ^8A , R ^9A , R ^{11 *} , R ^11A , R ^12A , R ^13A , P ₁ , P ₈ , and P ₁₈ are as described above. Deprotection and cyclization as described in Method A to form a resin bound depsipeptide nn4b, wherein R ^1A , R ^2A , R ^{2 *} , R ^3A , R ^{5 * A} , R ^{5 * A} , R ^6A , R ^{8 *} , R ^8A , R ^9A , R ^{11 *} , R ^11A , R ^12A , R ^13A , and P ₈ are as described above. Depsipeptides are cleaved from the resin and then deprotected of any residual protecting groups to afford compounds of formula (I).

Following the synthetic scheme (Scheme IV-XII), it should be understood that both amino acid amino groups and amino acid side chain functionalities must be orthogonal protected before attaching them to the growing peptide chain. Suitable protecting groups can be any protecting group useful for peptide synthesis. Such pairs of protecting groups are well known. See, eg, "Synthesis Notes" in the Novabiochem Catalog and Peptide Synthesis Handbook, 1999, pages S1-S93, and references cited therein.

Those skilled in the art will also understand that the selection of protecting groups for amino acid side chain functional groups allows the protecting groups to be cleaved or not to be cleaved at the same time that the peptide is finally cleaved from the resin, thereby forming a natural amino acid functional group or a protected derivative thereof. Additional deprotection may be necessary if the protecting groups are not simultaneously cleaved when the depsipeptide is cleaved from the resin.

It will be apparent to those skilled in the art that the linear precursors nn3, nn3a or nn3b, and thus the intermediates nn4, nn4a and nn4b and the final product I can be obtained by combining any two fragment pairs as well as by the method AC as described above. something to do. Such fragment pairs can be designed by fragmenting a compound of formula (I) between any two amino acids in a sequence, for example 1 + 12, 2 + 11, 3 + 10 and the like.

Alternatively, the compounds may be prepared by linear assembly prior to ester formation by the methods described in US Pat. Nos. 6,911,525 and 6,794,490 and in international patent applications WO 01/44272, WO 01/44274, WO 01/44271 and WO 03/014147. Can be formed. Alternatively, this compound may be formed by the assembly of multiple fragments.

The method described above uses resin chemistry, but this method will also be suitable for solution phase peptide chemistry.

Alternatively, the compounds of the present invention may be formed by the method described in international patent application WO 2005/012541.

2. Biosynthetic Method

Birribosome  Peptide Synthetase Pathway

Bacteria and fungi, including actinomycetes, synthesize various low molecular weight peptides and polyketide compounds (approximately 2-48 residues in length). Biosynthesis of these compounds is promoted by non-ribosomal peptide synthetase (NRPS) and by polyketide synthase (PKS). NRPS processes not involved in ribosomal mediated RNA translation according to the genetic code can produce peptides that exhibit vast structural diversity compared to peptides translated from RNA templates by ribosomes. These include the integration of hydroxy acids with D- and L-amino acids; Mutations in the peptide backbone that form a linear, cyclic, or branched ring structure; Additional structural modifications including oxidation, acylation, glycosylation, N-methylation, and heterocyclic ring formation. Useful pharmacology (eg antibiotic, antiviral, antifungal, antiparasitic, iron siderophore, cytostatic, immunosuppressive, anticholesterolic and anticancer), agrochemical or physical Many nonribosomal synthetic peptides have been found that have chemical (eg biosurfactant) properties.

Nonribosome synthetic peptides are assembled by large (eg, about 200-2000 kDa) multifunctional NRPS enzyme complexes comprising one or more subunits. Examples include daptomycin, A54145, vancomycin, echinocandine and cyclosporin. Similarly, polyketides are assembled by large multifunctional PKS enzyme complexes comprising one or more subunits. Examples include erythromycin, tyrosine, monensin and avermectin. In some cases, complex molecules can be synthesized by a mixed PKS / NRPS system. Examples include rapamycin, bleomycin and epothilones.

An NRPS generally consists of one or more open reading frames that make up the NRPS composite. NRPS complexes contain a series of protein biosynthetic units that are configured to bind to and activate specific building block substrates and promote peptide chain formation and extension (Konz and Marahiel, 1999, Chem. Biol. 6). : 39-48 and references cited therein; von Doehren et al., 1999, Chem. Biol. 6: 273-279 and references cited therein; and Cane and Walsh, 1999, Chem. Biol. 6: 319-325 and references cited therein, each of which is incorporated herein by reference in its entirety). Each NRPS or NRPS subunit includes one or more modules. A "module" is defined as a catalytic unit that integrates one building block (eg, one amino acid) into a growing peptide chain. The order and specificity of the biosynthetic module forming the NRPS protein template determines the sequence and structure of the final peptide product.

Each module of NRPS acts as a semi-autonomous active site containing folded individual protein domains that serve to promote the specific response required for peptide chain extension. The minimum module (in a single module complex) may comprise at least two core domains; That is, 1) an adenylation domain, which serves to activate amino acids (or sometimes hydroxy acids), and 2) thiolated or acyl carrier domains, which serve to transfer the activated intermediates to enzyme-linked pantein cofactors. Most modules also include condensation domains that serve to promote peptide bond formation between 3) activated intermediates. Complementing these three core domains may include, for example, N-methylation (M or methylation domain) and L to D conversion (E or epimerization domain) of the bound amino acid intermediate, and heterocyclic ring formation (Cy or Cyclization domains). These domains are generally characterized by having specific amino acid motifs or features. The combination of such additional domains that locally act on tethered intermediates in nearby modules contributes to the vast structural and functional diversity of the mature peptide product assembled by NRPS and the mixed NRPS / PKS enzyme complex.

The adenylation domain of each minimal module promotes specific recognition and activation of cognate amino acids. In this early stage of nonribosome peptide biosynthesis, the cognate amino acids of each NRPS module are bound to the adenylation domain and activated as unstable acyl adenylate (with ATP hydrolysis) (Ref. Stachelhaus et al., 1999, Chem., Biol. 6: 493-505 and Challis et al., 2000, Chem. Biol. 7: 211-224, each of which is incorporated herein by reference in its entirety). In most NRPS modules, the acyl adenylate intermediate is then transferred to the T (thiolated) domain (also called peptidyl carrier protein or PCP domain) of the module, where it is converted to a thioester intermediate and transthiolation reactions. Through a covalently bound enzyme cofactor (4'-phosphopanthethenyl (4'-PP) intermediate). Modules that serve to integrate D-form conformations or N-methylated amino acids may have extra modification domains, which are located between the A and T domains in some studied NRPS.

Enzyme-bound intermediates of each module are then assembled into peptide products by staged condensation reactions, wherein the intermediates remain covalently bound to the NRPS, while one residue of the thioester activated carboxyl group of one module, For example, transfer to a contiguous amino group of the next amino acid in the next module. Each condensation reaction is generally facilitated by a condensation domain located between two minimal modules. The number of condensation domains in the NRPS generally corresponds to the number of peptide bonds present in the final (linear) peptide. Extra C domains have been found in some NRPS (e.g., at the amino terminus of cyclosporin synthase and at the carboxyl terminus of rapamycin; see, eg, Konz and Marahiel, supra), which is a peptide chain termination ring. It has been suggested to be involved in the reaction. However, many other NRPS complexes release full length chains in reactions promoted by C-terminal thioesterase (Te) domains (relative molecular weight about 28-35K).

The thioesterase domain of most NRPS complexes utilizes a catalytic triad (similar to that of the well known chymotrypsin mechanism), which preserves three-dimensional conformations conserved for histidine and acidic residues. Serine (sometimes cysteine or aspartate) residues. For example, V. De Crecy Lagard in "Comprehensive Natural Products Chemistry", Volume 4, ed. J.W. Kelly, Elsevier, New York, 1999, pp. 221-238, which is incorporated herein by reference in its entirety. Thioester cleavage is a two-step process. In the first (acylation) step, the full length peptide chain is transferred from a thiol linked enzyme intermediate in the thiolation domain (see above) to a conserved serine residue in the Te domain to form an acyl-O-Te ester intermediate. In the second (deacylation) step, depending on whether the ester intermediate is attacked by water (hydrolysis) or by activated intramolecular nucleophiles (ringing), the Te domain serine ester intermediate is hydrolyzed (by Linear full length product is liberated), or cyclization occurs.

By sequence comparison of C-terminal thioesterase domains from various members of the NRPS superfamily, about 25 from the conserved motifs comprising serine catalytic residues (GXSXG motifs), and often subsequent conserved serine residues Aspartic acid residues downstream of the amino acids were found. The second type of thioesterase, free thioesterase enzyme, is known to participate in the biosynthesis of some peptides and polyketide secondary metabolites. See, eg, Schneider and Marahiel, 1998, Arch. Microbiol. 169: 404-410 and Butler et al., 1999, Chem. Biol. 6: 87-292, which is incorporated herein by reference in its entirety. These thioesterases are often required for efficient natural product synthesis (see US Patent Application 2002-0192773). Butler et al. Hypothesized that the free thioesterase found in the polyketided tyrosine gene clusters required for efficient tyrosine production may be involved in editing and correction functions.

The modular construction of the NRPS polyenzyme complex is reflected at the level of genomic DNA encoding these modules. The construction and DNA sequence of genes encoding several different NRPS have been studied (Marahiel, 1997, Chem. Biol. 4: 561-567, which is hereby incorporated by reference in its entirety). By comparing NRPS sequences from many different organisms, conserved sequences that characterize specific NRPS functional domains have been identified, and these conserved sequence motifs can be used to design probes useful for identifying and isolating new NRPS genes and modules. come.

The modular structure of the PKS and NRPS enzyme complexes can be used to design new enzymes with new specificities by changing the number and location of modules at the DNA level by genetic engineering and in vivo recombination. The functional hybrid NRPS can be configured based on total module fusion, for example. See, eg, Gokhale et al., 1999, Science 284: 482-485 and Mootz et al., 2000, Proc. Natl. Acad. Sci. USA 97: 5848-5853, which are incorporated herein by reference in their entirety. Recombinant techniques may also be used to successfully exchange domains derived from heterologous PKS or NRPS complexes. See, eg, Schneider et al., 1998, Mol. Gen. Genet. 257: 308-318; McDaniel et al., 1999, Proc. Natl. Acad. Sci. USA. 96: 1846-1851; U.S. Patents 5,652,116 and 5,795,738; And International Patent Publication No. WO 00/56896, which are hereby incorporated by reference in their entirety.

It is also known to design new substrate specificities within the module by changing the residues that form the substrate binding pocket of the adenylation domain. See, eg, Cane and Walsh, 1999, Chem. Biol. 6: 319-325; Stachelhaus et al., 1999, Chem. Biol. 6: 493-505; And International Patent Application No. WO 00/52152, each of which is incorporated herein by reference in its entirety. By comparing the sequence of the Bacillus subtilis peptide synthetase GrsA adenylation domain (PheA, whose structure is known) with the sequences of 160 other adenylation domains from prokaryotic and eukaryotic NRPS, for example, see Stachelhaus et al. Challis et al. Chem. Biol. 7: 211-224 (2000) defined an adenylated (A) domain signature sequence (similar to codons in the gene code) for various amino acid substrates. From the collection of these symbolic sequences, putative NRPS selectivity giving codes (having degeneracy like gene codes) were constructed.

The ability to design NRPS with new module template structure and new substrate specificity by adding, deleting or replacing modules (or by adding, deleting or replacing domains in one or more modules) may alter new peptides with altered and potentially beneficial properties. To be created. For example, a combination comprising 50 or more new polyketides by systematically modifying the PKS synthesizing the erythromycin precursor (DEBS) by replacing the counterpart sequence from rapamycin PKS (coding another substrate specificity). The library was prepared. See International Patent Application No. WO 00/63361 and McDaniel et al., 1999, supra, which are hereby incorporated by reference in their entirety.

Daptomycin is an example of a nonribosome synthetic peptide prepared by NRPS (FIG. 1). Modifications of genes encoding proteins involved in the daptomycin biosynthetic pathway, including daptomycin NRPS, result in improved antimicrobial (eg greater efficacy) as well as modified Streptomyces roseosporus (NRRL 11379 ). Can exert; Natural or novel antibiotics can be produced in increased amounts; Or in the production of other host strains capable of producing other peptide products having useful biological properties. Compositions and methods related to Streptomyces roseosporus daptomycin biosynthetic gene clusters, including isolated nucleic acids and isolated proteins, are described in International Patent Application No. WO 03/014297, which is incorporated herein by reference. .

A54145 is another example of a nonribosomal synthetic peptide prepared by NRPS. A54145 is Streptomyces fradiae ) (NRRL 18158 ) is a cyclic lipopeptide antibiotic prepared by fermentation. A54145 comprises a fatty acid chain linked through three amino acid chains to the N-terminal tryptophan of a cyclic peptide consisting of 10 amino acids (FIG. 2). This compound is similar to the A21978C / Daptomycin factor for various strains of S. aureus , S. epidermidis , Streptococcus pyogenes and Enterococcus. In vitro anti-bacterial activity is shown. Compositions and methods related to Streptomyces Pradiai A54145 biosynthetic gene clusters, including isolated nucleic acids and isolated proteins, are described in International Patent Application No. WO 03/060127, which is incorporated herein by reference.

Genes encoding proteins involved in the A54145 biosynthetic pathway, including A54145 NRPS, may exhibit improved antibioticity (eg greater efficacy) as well as modified Streptomyces pradia; Natural or novel antibiotics can be produced in increased amounts; Or in the production of other host strains capable of producing other peptide products having useful biological properties.

NRPS To change gene clusters to produce new compounds by

NRPS  Alteration of Polypeptide Modules and Domains

In one aspect, the present invention provides a method of altering the number or position of modules in NRPS to obtain a compound of formula (I) or a compound of any of formulas F1-F22. In one embodiment, one or more domains can be deleted from the NRPS. In this case, the product produced by NRPS will have a chemical change compared to the peptide produced without deletion, for example if the epimerized and / or methylated domains are deleted.

In yet another embodiment, one or more domains can be added to the NRPS. In this case, the peptide synthesized by NRPS will have additional chemical changes. For example, if epimerized or methylated domains are added, the resulting peptides will each contain extra D-amino acids or methylated amino acids. In another embodiment, one or more modules can be mutated, such as the adenylation domain can be mutated such that it may have amino acid specificities different from the naturally occurring adenylation domain. If one knows the amino acid code, one skilled in the art can alter the final amino acid composition and / or sequence of the resulting peptide synthesized by the altered NRPS by performing mutagenesis by a variety of known techniques to replace the code of one module with another. In yet another embodiment, one or more subunits can be added or deleted in the NRPS.

In another embodiment, one or more domains, modules, or subunits can be substituted with other domains, modules, or subunits to produce novel peptides by complementary phenomenon (see International Patent Application No. WO 01/30985, Publications particularly provide a method of substitution of modules). In this case, the peptide produced by the altered NRPS will have one or more other amino acids as compared to, for example, a naturally occurring peptide. In addition, different combinations of insertions, deletions, substitutions and mutations of domains, modules or subunits may be used to generate the peptide of interest. For example, a naturally occurring module may be substituted with a modified module, domain or subunit, or a naturally occurring module, domain or subunit from an NRPS from one organism may be replaced by a module, domain or subunit of NRPS from another organism. It may be replaced by a unit. Modification of modules, domains and subunits can include site directed mutagenesis, domain exchange (for module or subunit modifications), deletion, insertion or substitution of domains within a module or subunit, or deletion, insertion or substitution of a module within a subunit. It can be performed by. In addition, any method known in the art can be used to disrupt domains, modules or subunits, resulting in loss of function. Such disruption includes techniques such as single crossover destruction or substitution via homologous recombination, eg, by other genes (eg, genes that allow for selection or screening).

The product produced by the modified NRPS complex will have several amino acids incorporated, several chemical changes of the amino acids (eg methylation and epimerization). A domain, module or subunit can be derived from any desired number of NRPS, including two, three or four NRPS. The present invention also encompasses modified NRPS complexes with or without the integral thioesterase domain.

The source of modules, domains and / or subunits may be derived from daptomycin biosynthetic gene cluster NRPS, A54145 biosynthetic gene cluster NRPS, or may be from any NRPS or other peptide source encoding other lipopeptides. These peptide sources include glycopeptide gene clusters, mixed pathway gene clusters, and iron trapping gene clusters. Artificial NRPS and methods for its preparation are described in International Patent Application No. WO 01/30985, which is incorporated herein by reference. In addition, sources of modules, domains and / or subunits can be obtained from any suitable source, including streptomyces and non-streptomyces sources. Non-by Streptomyces sources include actinomycetes e.g., ahmiko ratop sheath (Amycolatopsis)], prokaryotic non- Streptomyces e.g., Bacillus (Bacillus) and cyanobacteria], and non-bacterial sources (e.g. Fungi).

The NRPS or a portion thereof may be heterologous to the host cell of interest or endogenous to the host cell. In one embodiment, the NRPS or portion thereof (eg, domain, module or subunit thereof) is introduced into a host cell on any vector (eg, plasmid, cosmid, bacteriophage or BAC) known to those skilled in the art. The host cell into which the NRPS or part thereof is introduced may contain an endogenous NRPS or part thereof (eg, a domain, a module, or a subunit thereof). Alternatively, the heterologous NRPS or part thereof may be introduced into a host cell containing the heterologous NRPS or part thereof. The first NRPS, or other NRPS, or domain, module, or subunit of the NRPS may have a naturally occurring sequence or a modified sequence. In another embodiment, the NRPS or part thereof is endogenous to the host cell, for example the host cell is Streptomyces pradiai for A54145 and Streptomyces roseosporus for daptomycin. Naturally occurring or modified NRPS, or domains, modules or subunits thereof, can be introduced into host cells comprising endogenous NRPS or portions thereof. Heterologous domains, modules, subunits or NRPS may comprise constitutive or regulatory promoters, which are known to those skilled in the art. The promoter may be homologous or heterologous to the nucleic acid molecule introduced into the cell. In certain embodiments, the promoter may be from a daptomycin biosynthetic gene cluster, as described above.

Nucleic acid molecules comprising NRPS or portions thereof (eg, domains, modules, subunits) may be maintained within the episomes or integrated into the genome. Nucleic acid molecules can be introduced into the genome, for example, at phage integration sites. In addition, nucleic acid molecules may be introduced into the genome at sites or in other locations within the genome of endogenous or heterologous NRPS or portions thereof. Nucleic acid molecules may be introduced in a way that destroys all or part of the function of a domain, module or subunit of an NRPS already present in the genome, or may be introduced in a way that does not destroy the function of NRPS or a portion thereof.

Peptides produced by these NRPS may be useful as new compounds, or may be useful for producing new compounds. In a preferred embodiment, the novel compounds are useful as antibiotic compounds or can be used to produce antibiotic compounds. In another preferred embodiment, the novel compounds are antibiotic, antifungal, antiviral, antiparasitic, antimitotic, cytostatic, antitumor, immunomodulatory, anticholesterolic, iron trapping, agrochemical ( For example, it may be useful or used to produce other peptides with useful activity, including but not limited to pesticidal) or physicochemical (eg, surfactant) properties.

In addition, the additional diversity of nonribosomal synthetic peptides and polyketides can be achieved by one or more NRPS and PKS genes (genes encoding natural, hybrid or otherwise altered modules or domains) that are heterologous host cells, i.e. It can obtain by expressing in host cell other than a host cell derived from.

3. Subsequent Modification of Peptides

Compounds of the present invention may be prepared by first assembling the core of the molecule by any of the methods described above, followed by US Pat. No. 6,911,525; And 6,794,490 and International Patent Application No. WO 01/44272; WO 01/44274; And all or part of the residual primary amino groups, as described in WO O1 / 44271.

Treating the primary amino group (s) with reagents such as isocyanates, isothiocyanates, activated esters, acid chlorides, sulfonylchlorides or activated sulfonamides, heterocycles with easy-to-substituted groups, imidates, lactones or the like When reductively treated with, at least one of substituents R ¹ , R ^aa1 , R ^aa2 , R ^{6 *} and R ^{8 **} is independently mono-substituted amino, disubstituted amino, acylamino, ureido, guanidino, carba Compounds are produced that are moyl, sulfonamino, thioacylamino, thioureido, iminoamino or phosphonamino.

To carry out such modifications it may be necessary to protect certain functional groups in the molecule. Protection of such functional groups can be carried out within the skill level of those skilled in the art in accordance with the teachings of the present invention (see, e.g., Greene, supra).

Recombination NRPS Cells capable of expressing and methods for producing the cells

The present invention includes cells capable of expressing recombinant NRPS gene clusters capable of expressing recombinant NRPS and producing the various compounds of the present invention and methods of making such cells. In certain specific embodiments, the cells are Streptomyces lividans ), Streptomyces coelicolor , or Gram positive cells, including Streptomyces roseosporus . In another specific embodiment of the invention, the recombinant NRPS is assembled from a module derived from daptomycin or A54145 NRPS gene cluster. Such genes may be "exchanged" using recombinant techniques known in the art or exemplified herein. In another embodiment, certain genes in recombinant NRPS are inactivated or "knocked out" to prevent the production of expression products or to prevent their activity in cells [JILL, SHOULD WE MENTION 3MG HERE AND lptl]. ?]

In a preferred embodiment, bacterial host cells are used to express the nucleic acid molecules of the invention. Useful expression vectors for bacterial hosts include pBluescript, pGEX-2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and derivatives thereof. Bacterial plasmids, such as plasmids derived from E. coli or Streptomyces, for example, phage DNA such as numerous derivatives of phage lambda (e.g., NM989, λGT10 and λGT11) and a wider range of host plasmids such as RP4, and other phages ( For example, M13 and filamentary single stranded phage DNA) can be mentioned. Preferred vectors are bacterial artificial chromosomes (BAC). More preferred vector is pStreptoBAC described in Example 2 of International Patent Application No. 03/014297.

In other embodiments, eukaryotic host cells, such as yeast, insect or mammalian cells, can be used. Yeast vectors include yeast integration plasmids (e.g., YIp5) and yeast replication plasmids (YRp and YEp series plasmids), yeast isoplasmic plasmids (YCp family plasmids), pGPD-2, 2μ plasmids and derivatives thereof; Sugino, Gene, 74, pp. 527-34 (1988), and improved shuttle vectors such as those disclosed in YIplac, YEplac and YCplac. Expression in mammalian cells can be expressed in various plasmids including pSV2, pBC12BI, and p91023, as well as lytic viral vectors (eg vaccinia virus, adenovirus and baculovirus), episomal viral vectors (eg, Bovine papillomavirus) and retroviral vectors (eg murine retroviruses). Useful vectors for insect cells include baculovirus vectors and pVL 941.

Another aspect of the invention provides a compound described herein and a method of making the compound from recombinant cells. These compounds can be prepared by culturing cells using techniques and conditions known in the art or described herein. Culture conditions of the cells may include a process of fermenting the cells with a lipopeptide tail precursor that promotes the production of certain compounds of the invention. The precursor can be taken up by the cell during the fermentation process and can increase the productivity of certain compounds in the cell. The precursors supplied to the cells during the fermentation process are sometimes called fermentation feeds and the resulting compounds are called feed products. Compounds of the invention prepared by culturing or fermenting cells of the invention may be further isolated and / or purified from fermentation products.

new Depsipeptide  Produce

1. Synthetic Method

The following examples are set forth to more fully understand the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Example  1-1: Synthesis of Peptide Resin Compound 1:

Suzy- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NH ₂ (One)

Reaction 1: resin Gly - Thr - NHFmoc (2) manufacturing

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-threonine (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide (N-methylpyrrolidine 2 mL of 0.5 molar solution in) and 1-hydroxy-benzotriazole (2 mL of 0.5 molar solution in N-methylpyrrolidine) were added to commercially available glycine 2-chlorotrityl resin (334 mg). . The mixture was shaken for 1 hour, filtered through a glass sinter funnel, and several beads were subjected to standard Kaiser test [E. Kaiser, et al., 1970, Anal. Biochem. 34: 595; And "Advanced Chemtech Handbook of Combinatorial, Organic and Peptide Chemistry" 2003-2004, p. 208] for the presence of free amines. The Kaiser test showed blue, indicating that the reaction was incomplete and thus the coupling conditions were repeated. After filtration through a glass sinter funnel, the product containing resin was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing with 2 gave compound 2.

Reaction 2: resin Gly - Thr - NH ₂ (3) Preparation

Compound 2 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing with 3 gave compound 3.

Reaction 3: resin Gly - Thr -Asp ( OtBu )- NHFmoc (4) manufacturing

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-tert-butyl ester (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide (2 mL of 0.5 molar solution in N-methylpyrrolidine) and 1-hydroxy-benzotriazole (2 mL of 0.5 molar solution in N-methylpyrrolidin) were added to compound 3. The mixture was shaken for 1 hour, filtered through a glass sinter funnel and the coupling repeated. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing with 4 gave compound 4.

Reaction 4: resin Gly - Thr - ASp ( OtBu )- NH ₂ (5) Preparation

Compound 4 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel, washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again with N-methylpyrrolidine (3 x 6 mL). Compound 5 was obtained.

Reaction 5: resin Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- NHFmoc (6) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-asparagine δ-N-trityl (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide ( A solution of 2 mL of 0.5 molar solution in N-methylpyrrolidine) and 1-hydroxy-benzotriazole (2 mL of 0.5 molar solution in N-methylpyrrolidine) was added to Resin 5. The mixture was shaken for 1 hour, filtered through a glass sinter funnel and the coupling repeated. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with compound 6 to obtain.

Reaction 6: Resin Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- NH ₂ Manufacture of 7

Compound 6 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with to give compound 7.

Reaction 7: Resin Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NHFmoc (8) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-tryptophan (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide (N-methylpyrrolidine 2 mL 0.5 molar solution in) and 1-hydroxy-benzotriazole (2 mL 0.5 molar solution in N-methylpyrrolidine) were added to Resin 7. The mixture was shaken for 1 hour, filtered through a glass sinter funnel and the coupling repeated. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing with 2 gave compound 8.

Reaction 8: Resin Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NH ₂ Manufacture of (1)

Compound 8 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solids were washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). ) To obtain Resin Peptide Compound 1.

Example  1-2: Synthesis of Peptide Resin Compound 9:

Suzy- Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp- Orn ( NHBoc )- NH ₂ (9)

Reaction 1: resin Glu (αO allyl)- NHFmoc Manufacture of 10

1,3-diisopropylcarbodiimide (0.940 mL) in a suspension of commercially available 4-hydroxymethylphenoxy resin (Wang resin, 5 g, 0.4 mmol / g) in dichloromethane (60 mL), 4-dimethylaminopyridine (24 mg in N-methylpyrrolidine (1 mL)) and commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-glutamic acid α-allyl ester (N-methylpyrrolidine 2.46 g) in (9 mL) was added. The reaction mixture was stirred for 16 h, filtered through a glass sinter funnel, the solid was washed with N-methylpyrrolidine and dichloromethane and dried under reduced pressure to afford compound 10.

Reaction 2: resin Glu (αO allyl)- NH ₂ Manufacture of 11

Compound 10 (526 mg) was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with to give compound 11.

Reaction 3: resin Glu (αO allyl)- DSer ( OtBu )- NHFmoc Manufacture of 12

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-serine-tert-butyl ether (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide (N 2 mL of 0.5 molar solution in methylpyrrolidine) and 1-hydroxy-benzotriazole (2 mL of 0.5 molar solution in N-methylpyrrolidine) were added to Resin 11. The reaction mixture was shaken for 1 hour, then filtered through a glass sinter funnel and the coupling was repeated. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with to give compound 12.

Reaction 4: resin Glu (αO allyl)- DSer ( OtBu )- NH ₂ Manufacture of 13

Compound 12 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with to give compound 13.

Reaction 5: resin Glu (αO allyl)- DSer ( OtBu )- Gly - NHFmoc Manufacture of 14

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-glycine (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide (N-methylpyrrolidine 2 mL 0.5 molar solution in) and 1-hydroxy-benzotriazole (2 mL 0.5 molar solution in N-methylpyrrolidine) were added to Resin 13. The reaction mixture was shaken for 1 hour, then filtered through a glass sinter funnel and the coupling was repeated. The reaction mixture was filtered through a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with compound 14 to obtain.

Reaction 6: Resin Glu (αO allyl)- DSer ( OtBu )- Gly - NH ₂ Manufacture of 15

Compound 14 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing to yield Compound 15.

Reaction 7: Resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- NHFmoc Manufacture of 16

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-tert-butyl ester (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide (2 mL of 0.5 molar solution in N-methylpyrrolidine) and 1-hydroxy-benzotriazole (2 mL of 0.5 molar solution in N-methylpyrrolidine) were added to Resin 15. The reaction mixture was shaken for 1 hour, then filtered through a glass sinter funnel and the coupling was repeated. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing to yield Compound 16.

Reaction 8: Resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- NH ₂ Manufacture of 17

Compound 16 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with to give compound 17.

Reaction 9: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla - NHFmoc Manufacture of 18

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-alanine (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide (N-methylpyrrolidine A solution of 2 mL 0.5 molar solution in water) and 1-hydroxy-benzotriazole (2 mL 0.5 molar solution in N-methylpyrrolidine) was added to Resin 17. The reaction mixture was shaken for 1 hour, then filtered through a glass sinter funnel and the coupling was repeated. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing to give compound 18.

Reaction 10: Resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla - NH ₂ Manufacture of 19

Compound 18 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with compound 19 to obtain.

Reaction 11: Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -NHFmoc (20) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-tert-butyl ester (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-diisopropylcarbodiimide (2 mL of 0.5 molar solution in N-methylpyrrolidine) and 1-hydroxy-benzotriazole (2 mL of 0.5 molar solution in N-methylpyrrolidine) were added to Resin 19. The reaction mixture was shaken for 1 hour, then filtered through a glass sinter funnel and the coupling was repeated. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing to yield Compound 20.

Reaction 12: Resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -NH ₂ Manufacture of 21

Compound 20 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washing to afford Compound 21.

Reaction 13: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu )- Orn -Preparation of NHFmoc (22)

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -Nδ- (tert-butoxycarbonyl) -L-ornithine (2 mL of 0.5 molar solution in N-methylpyrrolidine), 1,3-di A solution of isopropylcarbodiimide (2 mL of 0.5 molar solution in N-methylpyrrolidine) and 1-hydroxy-benzotriazole (2 mL of 0.5 molar solution in N-methylpyrrolidine) was added to Resin 21. . The reaction mixture was shaken for 1 hour, then filtered through a glass sinter funnel and the coupling was repeated. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with to give compound 22.

Reaction 14: Resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Orn (NHBoc) -NH ₂ (9) Preparation

Compound 22 was shaken for 30 minutes in 20% piperidine (6 mL) in N-methylpyrrolidine. The resin was filtered through a glass sinter funnel and resuspended in 20% piperidine (6 mL) in N-methylpyrrolidine and shaken for 30 minutes. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again N-methylpyrrolidine (3 x 6 mL). Washed with to give compound 9.

Example  1-3: Synthesis of Peptide Resin Compound 23:

Reaction 1: Preparation of Compound 24

Pentafluorophenol (3.68 g) was dissolved in dichloromethane (40 mL) and cooled to 0 ° C. in an ice / NaCl bath. Decanoylchloride (4.15 mL) was added dropwise to maintain the temperature below 2 ° C. Once the addition was complete, the reaction was stirred for 2.5 hours at 0 ° C. The cooling bath was then removed and the reaction was allowed to warm to room temperature and stirred for 17 hours. The volatiles were removed under reduced pressure to afford crude product pentafluorophenyl ester 24, which could be used in subsequent processes without further purification.

Reaction 2: Preparation of Compound 23

Resin peptide compound 1 (2 g) was added to a solution of pentafluorophenyl ester (24) of decanoic acid (24) in dichloromethane. The mixture was shaken for 17 hours, filtered through a glass sinter funnel and the Kaiser test (see above) was used to determine if the reaction was incomplete. Decanoic acid (517 mg), 1-hydroxy-benzotriazole (446 mg) and 1,3-diisopropylcarbodiimide (438 μl) were dissolved in N-methylpyrrolidine (8 mL) and 1 hour Was stirred. The resin was then added to the decanoic acid mixture and stirred for 8 hours, filtered through a glass sinter funnel, washed with N-methylpyrrolidine (3 x 6 mL), methanol (3 x 6 mL) and again Wash with N-methylpyrrolidine (3 × 6 mL). A Kaiser test was used to confirm the reaction was completed and resin bound lipopeptide 23 was obtained.

Example  1-4: Compound C352 Synthesis of:

Reaction 1: Preparation of Compound (25)

Commercial Kynurenine (3 g) was suspended in acetonitrile (100 mL) and water (30 mL). Diisopropylethylamine (DIPEA, 5.01 mL) was added dropwise to the solution and stirring continued until the solution was homogeneous. The solution was then cooled to 0 ° C. in an ice / sodium chloride bath and allyloxycarbonyl oxysuccinimide (AllocOSu, 4.3 g) in acetonitrile (30 mL) was added. The reaction mixture was stirred for 3 hours, then concentrated to remove acetonitrile, basified with 5% K ₂ CO ₃ solution (220 mL), ethyl acetate (5 x 90 mL) and dichloromethane (1 x 90 mL). )). The aqueous portion was then acidified to pH 1 and extracted with ethyl acetate (4 x 90 mL). The combined acidic organic washes were dried over anhydrous MgSO ₄ and evaporated to afford crude product (4.85 g). Purification by column chromatography on silica gel using 19: 1 dichloromethane and methanol as eluent and the desired intermediate L-2-N- (allyloxycarbonyl) -4- (2-aminophenyl) after solvent evaporation 4-Oxobutanoic acid was obtained as a yellow solid (2.92 g). This solid (2.9 g) was dissolved in 4 N HCl (100 mL) and cooled to 0 ° C. in ice / sodium chloride bath. A solution of NaNO ₂ (0.76 g) in water (10 mL) was added dropwise to maintain the temperature below 3 ° C. and the resulting solution was stirred at 0 ° C. for 2.5 h. A solution of NaN ₃ (1.95 g) in water (10 mL) was added dropwise to maintain the temperature below 3 ° C., and the resulting solution was allowed to warm to room temperature and stirred for 19 hours. The reaction mixture was poured into water (250 mL) and extracted with dichloromethane (4 x 100 mL). The combined organic washes were dried over anhydrous MgSO ₄ and evaporated to afford the desired product compound 25 (2.86 g).

Reaction 2: Preparation of Compound (26)

L-2-N- (allyloxycarbonyl) -4- (2-azidophenyl) -4-oxobutanoic acid 25 (636 mg), 4-dimethylaminopyridine (25 mg) and N-methyl-2- Chloropyridinium iodide (511 mg) was sufficiently flushed with argon and then suspended in dichloromethane (10 mL). Triethylamine (560 μl) was added and the reaction mixture was stirred to give a homogeneous solution. Resin lipopeptide 23 (667 mg) was added to the solution and the flask was washed with argon again and shaken for 17 hours. 20 mg of the resin sample was taken to test whether the reaction was complete (20 mg of resin in dichloromethane (0.6 mL) was treated with 2,2,2-trifluoroethanol (0.2 mL) and acetic acid (0.2 mL) and for 3 hours. The reaction mixture was filtered through a glass sinter funnel and the solvent was evaporated to give a residue, liquid chromatography / mass spectral analysis of the residue showed the reaction was incomplete). The coupling was judged to be incomplete, and the resin was dried for 5 days under reduced pressure, and the coupling was repeated for an additional 17 hours. The reaction mixture was filtered through a glass sinter funnel and the solid was washed sufficiently with dichloromethane. The solid was then suspended in dichloromethane (6 mL), 2,2,2-trifluoroethanol (2 mL), acetic acid (2 mL) and shaken for 5 hours. The reaction mixture was filtered through a glass sinter funnel and the filtrate was evaporated to afford crude target peptide 26 (44 mg). The crude product was eluted with 25% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid over 25 minutes with reversed phase HPLC (C18 10 μM Jupiter). Column 250 × 21.2 mm). The product containing fractions were lyophilized to give pure product 26 (10.6 mg).

Reaction 3: Preparation of Compound (27)

Hydroxy-benzotriazole (5 mg), 1,3-diisopropylcarbodiimide (6 μl) and peptide resin compound 9 (12.3 mg) were added to compound 26 (10.6) in N-methylpyrrolidine (0.7 mL). mg), followed by shaking for 22 hours. The resin was filtered through a glass sinter funnel and the Kaiser test (see above) was used to determine if the coupling was complete and a resin bound lipopeptide 27 was obtained.

Reaction 4: Preparation of Compound (28)

The dried resin 27 was placed under argon atmosphere and tetrakis- (triphenylphosphine) palladium (0) (19 mg) in dichloromethane (1.47 mL), acetic acid (74 μl) and N-methylmorpholine (37 μl) Treated with a solution of. The mixture was shaken at room temperature for 4 hours, filtered through a glass sinter funnel, and the solid was washed twice with N-methylmorpholine, twice with methanol and again with N-methylmorpholine. 1-hydroxy-benzotriazole (0.5 mL of 0.5 molar solution in N-methylmorpholine) and 1,3-diisopropylcarbodiimide (0.5 mL of 0.5 molar solution in N-methylmorpholine) were added to the resin It was. The reaction was shaken for 17 hours, filtered through a glass sinter funnel and washed thoroughly with N-methylmorpholine to yield resin bound cyclized depsipeptide 28.

Reaction 5: compound ( C352 Manufacturing

The dried resin 28 was suspended in dichloromethane (4 mL), trifluoroacetic acid (6 mL), ethanedithiol (250 μl) and triisopropylsilane (250 μl), and the reaction mixture was stirred at room temperature for 3 hours. It was. The resin was filtered through a glass sinter funnel and the combined filtrates were evaporated under reduced pressure. The crude product was then partitioned between diethyl ether (6 mL) and water (3 mL). The aqueous layer was freeze dried to afford the crude product. The crude product was eluted with 25% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid over 25 minutes with reversed phase HPLC (C18 10 μM Jupiter). Column 250 × 21.2 mm). The product containing fractions were combined and lyophilized to give pure product C352 (1.0 mg).

Example  1-5: Compound C369 Synthesis of:

Reaction 1: Preparation of Compound (30)

Compound 30 is obtained from compound 23 using Method D or Method E (see below).

Method D

Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (618 mg), bromo-tris-pyrrolidino commercially available in dichloromethane (5 mL) in resin bound lipopeptide 23 (1 g). A solution of phosphonium hexafluorophosphate (PyBrOP, 815 mg) and di-isopropylethylamine (914 μl) was added. Dimethylaminopyridine (5 mg) was added and the mixture was shaken for 2 hours. The mixture was filtered through a glass sinter funnel for 2 hours and washed with dichloromethane (3 × 10 mL) and the coupling procedure was repeated. The resulting resin was filtered through a glass sinter funnel, washed with dichloromethane (3 × 10 mL) and methanol (3 × 10 mL) and dried over potassium hydroxide pellets under reduced pressure. The dried resin was suspended in dichloromethane (3 mL), 2,2,2-trifluoroethanol (1 mL) and acetic acid (1 mL) and shaken for 3 hours. This resin was filtered through a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 30 (400 mg) as a white solid.

Method E

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (95 mg), 4-dimethylaminopyridine (6 mg) and N-methyl-2-chloropyridinium iodide (69 mg) Was washed with argon sufficiently and then suspended in dichloromethane (2.7 mL). Triethylamine (76 μl) was added and the reaction mixture was stirred to give a homogeneous solution. Resin lipopeptide 23 (200 mg) was added to the solution, the flask was washed again with argon and the reaction mixture was shaken for 14 hours. The resin obtained was then filtered through a glass sinter funnel and washed thoroughly with dichloromethane. The solid was suspended in dichloromethane (6 mL), 2,2,2-trifluoroethanol (2 mL) and acetic acid (2 mL) and shaken for 3 hours. The resin was filtered through a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 30 (54 mg) as a white solid.

Reaction 2: Preparation of Compound (31)

1-hydroxy-benzotriazole (26 mg), 1,3-diisopropylcarbodiimide (30 [mu] l) and peptide resin compound 9 (64 mg) were depsipeptide 30 in N-methylmorpholine (3.8 mL). (54 mg) was added to the solution and the resulting mixture was shaken for 22 hours. The resin was filtered through a glass sintered funnel and the Kaiser test (see above) was used to determine if the coupling was complete and a resin bound depsipeptide 31 was obtained.

Reaction 3: Preparation of Compound (32)

The dried resin 31 was placed under argon atmosphere to give tetrakis- (triphenylphosphine) palladium (0) (48 mg (7.63 mL) in dichloromethane), acetic acid (0.38 mL) and N-methylmorpholine (0.19 mL ) Solution. The mixture was shaken at room temperature for 4 hours, filtered through a glass sinter funnel, and the solid was washed twice with N-methylmorpholine, twice with methanol and again with N-methylmorpholine. The solid resin was suspended in 20% piperidine (7 mL) in N-methylmorpholine for 105 minutes, filtered through a glass sinter funnel, and the solid was sufficiently washed with N-methylmorpholine. 1-hydroxy-benzotriazole (0.3 mL of 0.5 molar solution in N-methylmorpholine) and 1,3-diisopropylcarbodiimide (0.3 mL of 0.5 molar solution in N-methylmorpholine) were added to the resin It was. The reaction mixture was shaken for 17 hours, filtered through a glass sinter funnel, and the precipitate was washed sufficiently with N-methylmorpholine to give resin bound cyclized depsipeptide 32.

Reaction 4: Compound C369 Manufacture

The dried resin 32 was suspended in dichloromethane (4 mL), trifluoroacetic acid (6 mL), ethanedithiol (250 μl) and triisopropylsilane (250 μl) and stirred at room temperature for 3 hours. The reaction mixture was filtered through a glass sinter funnel and washed with dichloromethane (2 x 2 mL) and the combined filtrates were evaporated under reduced pressure. The crude product was then partitioned between diethyl ether (6 mL) and water (3 mL). The aqueous layer was separated and freeze dried to afford crude product 33 (21.5 mg). The crude product was then eluted with 25% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid over 25 minutes with reverse phase HPLC (C18 10 μM). Purified on a Jupiter column 250 × 21.2 mm). The product containing fractions were combined and lyophilized to give pure product C369 (1.8 mg).

Example  1-6: Synthesis of Peptide Resin Compound 34:

Suzy- Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp- ( OtBu ) NH ₂ (34)

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -NHFmoc (35) Preparation

As a solution in N-methylpyrrolidine (20 mL), commercially available Nα- (9-fluorenylmethoxycarbonyl) -Nε- (t-butyloxycarbonyl D-lysine) (1.48 g), 1,3 -Diisopropylcarbodiimide (0.49 mL), 1-hydroxy-benzotriazole (425 mg) and 4-dimethylaminopyridine (37 mg) were added to Resin 17 (see above). The reaction mixture was shaken for 3 hours, filtered through a glass sinter funnel and the coupling repeated for 15 hours. After the reaction mixture was filtered through a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 x 15 mL), methanol (3 x 15 mL), and N-methylpyrrolidine (3 x 15 mL). Washed again to give compound 35.

Reaction 2: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc )- NH ₂ Preparation of 36

Compound 35 was shaken in 20% piperidine (25 mL) in N-methylpyrrolidine for 1 hour. The reaction mixture was filtered through a glass sinter funnel and the solid was washed with N-methylpyrrolidine (3 x 15 mL), methanol (3 x 15 mL) and with N-methylpyrrolidine (3 x 15 mL). Wash again to give compound 36.

Reaction 3: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -NHFmoc (37)

As a solution in N-methylpyrrolidine (20 mL), commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-tert-butyl ester (2.16 g), 1,3-diiso Propylcarbodiimide (822 μl) and 1-hydroxy-benzotriazole (710 mg) were added to Resin 36. The reaction mixture was shaken for 4 hours. After the reaction mixture was filtered through a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 x 15 mL), methanol (3 x 15 mL), and N-methylpyrrolidine (3 x 15 mL). ) Again to give compound 37.

Reaction 4: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -NH ₂ Preparation of 34

Compound 37 was shaken in 20% piperidine (25 mL) in N-methylpyrrolidine for 1 hour. After the reaction mixture was filtered through a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 x 15 mL), methanol (3 x 15 mL), and N-methylpyrrolidine (3 x 15 mL). ) Again to afford compound 34.

Example  1-7: Synthesis of Peptide Resin Compound 38:

Resin-Glu (αOallyl) -DSer (OtBu) -Gly-Asp (OtBu) -DAla-Asp (OtBu) -Ala-NH _{2 (} 38)

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Ala-NHFmoc (39) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-alanine (1.62 g), 1,3-diisopropylcarbodiimide (825 μl) and 1-hydroxy-benzotriazole (715 mg) Was added to Resin 21 (see above) as a solution in N-methylpyrrolidine (20 mL). The reaction mixture was stirred for 17 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). It washed with and obtained compound 39.

Reaction 2: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Ala-NH ₂ Preparation of 38

Compound 39 (227 mg) was stirred in 20% piperidine in N-methylpyrrolidine (1 mL) for 0.5 h. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 5 mL), methanol (3 × 5 mL) and then again N-methylpyrrolidine (3 × 5 mL). Compound 38 was obtained by washing with.

Example  1-8: Synthesis of Peptide Resin Compound 40:

Suzy- Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu )- NH ₂ 40

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- NHFmoc Manufacture of 41

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-asparagine (NMTrt) OH (3.1 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluro A solution of nium tetrafluoroborate (TBTU, 1.67 g), hydroxy-benzotriazole (0.56 g) and diisopropylethylamine (DIPEA, 2.7 mL) in N-methylpyrrolidone (NMP, 40 mL) To the solution was added to Resin-Glu-NH ₂ (11, supra, 4 g). The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 40 mL) and methanol (3 × 40 mL), and then again N-methylpyrrolidine (3 × 40 mL). ML) to give compound 41.

Reaction 2: resin Glu (αO allyl)- DAsn ( NHTrt )- NH ₂ Preparation of 42

Compound 41 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (30 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (30 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 30 mL), methanol (3 × 30 mL) and then again N-methylpyrrolidine (3 × 30 mL). It was washed with to give compound 42.

Reaction 3: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly - NHFmoc Manufacture of 43

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -glycine (1.55 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate ( TBTU, 1.67 g), hydroxy-benzotriazole (HOBt, 0.56 g) and diisopropylethylamine (DIPEA, 2.7 mL) as a solution in N-methylpyrrolidone (NMP, 40 mL) as compound 42 (4 to g). The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 40 mL) and methanol (3 × 40 mL), and then again N-methylpyrrolidine (3 × 40 mL). ML) to give compound 43.

Reaction 4: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly - NH ₂ Preparation of 44

Compound 43 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (30 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (30 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 30 mL), methanol (3 × 30 mL) and then again N-methylpyrrolidine (3 × 30 mL). Compound 44 was obtained by washing with.

Reaction 5: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- NHFmoc Manufacture of 45

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-t-butyl ester (2.14 g), 2- (1H-benzotriazol-yl) -1,1,3,3- Tetramethyluronium tetrafluoroborate (TBTU, 1.67 g), HOBt (0.56 g) and diisopropylethylamine (DIPEA, 2.7 mL) were used as a solution in N-methylpyrrolidone (NMP, 40 mL) 44 (4 g) was added. The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration through a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 40 mL) and methanol (3 × 40 mL), and then again N-methylpyrrolidine (3 × 40 mL). ML) to give compound 45.

Reaction 6: Resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu ) NH ₂ Manufacture of 46

Compound 45 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (30 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (30 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 30 mL), methanol (3 × 30 mL) and then again N-methylpyrrolidine (3 × 30 mL). The mixture was washed with to obtain Compound 46.

Reaction 7: Resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla - NHFmoc Manufacture of 47

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-alanine (0.81 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoro Borate (TBTU, 0.84 g), HOBt (0.28 g) and diisopropylethylamine (DIPEA, 1.4 mL) were added to compound 46 (2 g) as a solution in N-methylpyrrolidone (NMP, 20 mL). . The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 20 mL), methanol (3 × 20 mL), and then again N-methylpyrrolidine (3 × 20 mL) to give compound 47.

Reaction 8: Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla - NH ₂ Manufacture of 48

Compound 47 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (15 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (15 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 10 mL), methanol (3 × 10 mL), and then again N-methylpyrrolidine (3 × 10 mL). Compound 48 was obtained by washing with.

Reaction 9: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -NHFmoc (49) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-t-butyl ester (1.07 g), 2- (1H-benzotriazol-yl) -1,1,3,3- Tetramethyluronium tetrafluoroborate (TBTU, 0.84 g), HOBt (0.28 g) and diisopropylethylamine (DIPEA, 1.4 mL) were obtained as a solution in N-methylpyrrolidone (NMP, 20 mL). (2 g) was added. The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 20 mL) and methanol (3 × 20 mL), and then again N-methylpyrrolidine (3 × 20 mL). ML) to give compound 49.

Reaction 10: Glu (αO allyl)- DAsn ( NHTrt )- Gry -Asp ( OtBu )- DAla -Asp ( OtBu )- NH ₂ Manufacture of 40

Compound 49 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (15 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (15 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 10 mL), methanol (3 × 10 mL), and then again N-methylpyrrolidine (3 × 10 mL). Compound 40 was obtained by washing with.

Example  1-9: Synthesis of Peptide Resin Compound 50:

Suzy- Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu )- Orn ( NHBoc )- NH ₂ 50

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Orn (NHBoc) -NHFmoc (51) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-ornithine (Boc) -OH (1.17 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetra Methyluronium tetrafluoroborate (TBTU, 0.83 g), HOBt (0.31 g) and diisopropylethylamine (DIPEA, 1.4 mL) were prepared as a solution in N-methylpyrrolidone (NMP, 20 mL) as compound 40 ( 2.8 g). The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 20 mL) and methanol (3 × 20 mL), and then again N-methylpyrrolidine (3 × 20 mL). ML) was washed to give compound 51.

Reaction 2: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Orn (NHBoc) -NH ₂ Manufacture of 50

Compound 51 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (15 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (15 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 10 mL), methanol (3 × 10 mL), and then again N-methylpyrrolidine (3 × 10 mL). Compound 50 was obtained by washing with.

Example  1-10: Synthesis of Peptide Resin Compound 52:

Suzy- Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Ala- NH ₂ (52)

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Ala-NHFmoc (53) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-alanine (63 mg), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoro Borate (TBTU, 64 mg), HOBt (27 mg) and diisopropylethylamine (DIPEA, 70 μl) were added to compound 40 (340 mg) as a solution in N-methylpyrrolidone (NMP, 1 mL). . The mixture was stirred for 30 minutes, filtered through a glass sinter funnel, and several beads were tested using the standard Kaiser test (see above) for the presence of free amines. The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 2 mL) and methanol (3 × 2 mL), and then again N-methylpyrrolidine (3 × 2 mL). ML) to give compound 53.

Reaction 2: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Ala-NH ₂ Manufacture of 52

Compound 53 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (1.5 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (1.5 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 1 mL), methanol (3 × 1 mL), and then again N-methylpyrrolidine (3 × 1 mL). Compound 52 was obtained by washing.

Example  1-11: Synthesis of Peptide Resin Compound 54:

Suzy- Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp ( OtBu ) -Orn ( NHBoc )- NH ₂ (54)

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -NHFmoc (55)

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-ornithine (Boc) -OH (0.44 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetra Methyluronium tetrafluoroborate (TBTU, 0.31 g), HOBt (0.13 g) and diisopropylethylamine (DIPEA, 0.3 mL) were dissolved in compound 34 (NMP, 20 mL) as a solution in N-methylpyrrolidone (NMP, 20 mL). See above, 0.8 g). The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 20 mL) and methanol (3 × 20 mL), and then again N-methylpyrrolidine (3 × 20 mL). ML) to give compound 55.

Reaction 2: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Orn (NHBoc) -NH ₂ Manufacture of 54

Compound 55 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (8 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (8 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 8 mL), methanol (3 × 8 mL), and then again N-methylpyrrolidine (3 × 8 mL). Compound 54 was obtained by washing with water.

Example  1-12: Synthesis of Peptide Resin Compound 56:

Suzy- Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp ( OtBu )- NH ₂ (56)

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -NHFmoc (57) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-Nα- (9-fluorenylmethoxycarbonyl) -Nε- (t-butyloxycarbonyl L-lysine (1.28 g), 2- ( 1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 0.84 g), HOBt (0.28 g) and diisopropylethylamine (DIPEA, 1.4 mL) Was added to compound 46 (2 g) as a solution in N-methylpyrrolidone (NMP, 20 mL) The mixture was stirred for 30 min, filtered through a glass sinter funnel and several beads were subjected to standard Kaiser test (homology). Was tested for the presence of free amines The Kaiser test indicated yellow, indicating that the coupling was complete After filtration with a glass sinter funnel, the product containing the resin was subjected to N-methylpyrrolidine ( 3 × 20 mL) and methanol (3 × 20 mL), followed by washing with N-methylpyrrolidine (3 × 20 mL) to give Compound 57.

Reaction 2: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc )- NH ₂ Manufacture of 58

Compound 57 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (15 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (15 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 10 mL), methanol (3 × 10 mL), and then again N-methylpyrrolidine (3 × 10 mL). Compound 58 was obtained by washing with.

Reaction 3: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -NHFmoc (59) Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-t-butyl ester (1.07 g), 2- (1H-benzotriazol-yl) -1,1,3,3- Tetramethyluronium tetrafluoro borate (TBTU, 0.84 g), HOBt (0.28 g) and diisopropylethylamine (DIPEA, 1.4 mL) were obtained as a solution in N-methylpyrrolidone (NMP, 20 mL) as compound 58 (2 g) was added. The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 20 mL) and methanol (3 × 20 mL), and then again N-methylpyrrolidine (3 × 20 mL). ML) to give compound 59.

Reaction 4: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) NH ₂ Manufacture of 56

Compound 59 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (15 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (15 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 10 mL), methanol (3 × 10 mL), and then again N-methylpyrrolidine (3 × 10 mL). Compound 56 was obtained by washing with.

Example  1-13: Synthesis of Peptide Resin Compound 60:

Suzy- Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp ( OtBu ) -Orn ( NHBoc ) -NH (60)

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -NHFmoc (61)

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-ornithine (Boc) -OH (0.54 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetra Methyluronium tetrafluoroborate (TBTU, 0.38 g), HOBt (0.12 g) and diisopropylethylamine (DIPEA, 0.63 mL) were used as a solution in N-methylpyrrolidone (NMP, 12 mL) as compound 56 ( 1.2 g). The mixture was stirred for 30 minutes, filtered through a glass sinter funnel, and several beads were tested using the standard Kaiser test (see above) for the presence of free amines. The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 20 mL) and methanol (3 × 20 mL), and then again N-methylpyrrolidine (3 × 20 mL). ML) to give compound 61.

Reaction 2: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Orn (NHBoc) -NH ₂ Manufacture of 60

Compound 61 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (12 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (12 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 10 mL), methanol (3 × 10 mL), and then again N-methylpyrrolidine (3 × 10 mL). The mixture was washed with to obtain compound 60.

Example  1-14: Synthesis of Peptide Resin Compound 62:

Suzy- Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp ( OtBu ) -Ala-  NH ₂ (62)

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Ala-NHFmoc (63)

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-alanine (0.78 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoro Borate (TBTU, 0.80 g), HOBt (0.27 g) and diisopropylethylamine (DIPEA, 0.81 mL) were added to compound 56 (2 g) as a solution in N-methylpyrrolidone (NMP, 20 mL). . The mixture was stirred for 30 minutes, filtered through a glass sinter funnel, and several beads were tested using the standard Kaiser test (see above) for the presence of free amines. The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 20 mL) and methanol (3 × 20 mL), and then again N-methylpyrrolidine (3 × 20 mL). ML) to give compound 63.

Reaction 2: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Ala-NH ₂ Manufacture of 62

Compound 63 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (20 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (20 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 20 mL), methanol (3 × 20 mL) and then again N-methylpyrrolidine (3 × 20 mL). Compound 62 was obtained by washing with.

Example  1-15: Synthesis of Peptide Resin Compound 64:

Resin-Ala- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NH ₂ (64)

Reaction 1: Resin-Ala- Sar - NMeFmoc Manufacture of 65

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -sarcosine (1.56 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1.61 g) and a solution of diisopropylethylamine (DIPEA, 871 μl) as a solution in N-methylpyrrolidone (NMP, 25 mL) commercially available alanine 2-chlorotrityl resin (66, 2.5 g )). The mixture was stirred for 30 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 15 mL) and methanol (3 × 15 mL), and then again N-methylpyrrolidine (3 × 15 mL). ML) to give compound 65.

Reaction 2: Resin-Ala- Sar - NMeH Manufacture of 67

Compound 65 was stirred in 20% piperidine in N-methylpyrrolidine (20 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Compound 67 was obtained by washing with.

Reaction 3: Resin-Ala- Sar - Thr - NHFmoc Manufacture of 68

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-threonine (853 mg), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrOP, 1.165 g) and DIPEA (1.31 mL) To solution 67 (334 mg) was added as a solution in dichloromethane (25 mL). The mixture was stirred for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Compound 68 was obtained by washing with.

Reaction 4: Resin-Ala- Sar - Thr - NH ₂ Preparation of 69

Compound 38 (see above) was stirred in 20% piperidine in N-methylpyrrolidine (25 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The mixture was washed with to obtain Compound 69.

Reaction 5: Resin-Ala- Sar - Thr -Asp ( OtBu )- NHFmoc Manufacture of 70

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-t-butyl ester (2.06 g), TBTU (1.61 g) and DIPEA (871 μl) as a solution in NMP (25 mL) Was added to compound 69. The mixture was stirred for 3 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The mixture was washed with to obtain compound 70.

Reaction 6: Resin-Ala- Sar - Thr -Asp ( OtBu )- NH ₂ Preparation of 71

Compound 70 was stirred in 20% piperidine in N-methylpyrrolidine (25 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL), and then again with N-methylpyrrolidine (3 × 15 mL). Compound 71 was obtained.

Reaction 7: Resin-Ala- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- NHFmoc Manufacture of 72

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-asparagine δ-N-trityl (1.49 g), TBTU (1.61 g) and DIPEA (871 μl) as a solution in NMP (25 mL) Was added to 71. The reaction mixture was stirred for 17 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The mixture was washed with to obtain Compound 72.

Reaction 8: Resin-Ala- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- NH ₂ Manufacture of 73

Compound 72 was stirred in 20% piperidine in N-methylpyrrolidine (25 mL) for 2 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). It was washed with to give compound 73.

Reaction 9: Resin-Ala- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NHFmoc Manufacture of 74

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-tryptophan (1.07 g), TBTU (802 mg) and DIPEA (435 μL) were added to Resin 73 as a solution in NMP (10 mL). The reaction mixture was stirred for 43 hours. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL), and then again N-methyl pyrrolidine (3 × 15 mL). Compound 74 was obtained by washing with.

Reaction 10: Resin-Ala- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NH ₂ Manufacture of 64

Compound 74 was stirred in 20% piperidine in N-methylpyrrolidine (25 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). It wash | cleaned with and obtained resin peptide compound 64.

Example  1-16: Synthesis of Peptide Resin Compound 75:

Resin-Ala- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - Undecano  Amide (75)

Commercially available undecanoic acid (930 mg), 1,3-diisopropylcarbodiimide (0.78 mL) and 1-hydroxy-benzotriazole (676 mg) in N-methylpyrrolidine (20 mL) As a compound 64. The mixture was stirred for 23 hours, filtered through a glass sinter funnel and the reaction was determined to be incomplete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL), and then again with N-methylpyrrolidine (3 × 15 mL). . The reaction was confirmed to be complete using the Kaiser test to give resin bound Compound 75.

Example  1-17: Synthesis of Peptide Resin Compound 76:

Suzy- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp -8- Methyldecano  Amide (76)

Commercially available 8-methyldecanoic acid (1.55 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 2.67 g), diisopropyl Ethylamine (DIPEA, 2.9 mL) and 1-hydroxy-benzotriazole (1.12 g) were added to compound 1 (7.6 g) as a solution in N-methylpyrrolidine (80 mL). The mixture was stirred for 18 hours, filtered through a glass sinter funnel, and the reaction was judged to be complete using the Kaiser test (see above), giving a resin bound Compound 76.

Example  1-18: Synthesis of Peptide Resin Compound 77:

Suzy- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - Tridecano  Amide (77)

Commercially available tridecanoic acid (2.39 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 3.47 g), diisopropylethylamine (DIPEA, 3.75 mL) and 1-hydroxy-benzotriazole (1.46 g) were added to compound 1 (10 g) as a solution in N-methylpyrrolidine (80 mL). The mixture was stirred for 17 hours, filtered through a glass sinter funnel, and the reaction was judged to be complete using the Kaiser test (see above), giving a resin bound Compound 77.

Example  1-19: Synthesis of Peptide Resin Compound 78

Suzy- Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  Amide (78)

Reaction 1: resin Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- NHFmoc Manufacture of 79

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-glutamic acid γ-t-butyl ester (1.14 g), TBTU (0.87 g), HOBt (0.37 g) and DIPEA (940 μL) were added to NMP (20). To compound 5 as a solution in mL). The reaction mixture was stirred for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Washed with. The reaction was judged to be complete using the Kaiser test (see above) and gave resin bound Compound 79.

Reaction 2: resin Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- NH ₂ Manufacture of 80

Compound 79 was stirred for 15 min in 20% piperidine in N-methylpyrrolidine (20 mL). The resin was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (20 mL) and stirred for 15 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The resulting mixture was washed with to obtain a resin-bonded compound 80.

Reaction 3: resin Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - NHFmoc Manufacture of 81

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-tryptophan (1.15 g), TBTU (0.87 g), HOBt (0.37 g) and DIPEA (940 μL) were obtained as a solution in NMP (20 mL). Added to 80. The reaction mixture was stirred for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Washed with. The reaction was judged to be complete using the Kaiser test (see above) and gave resin bound Compound 81.

Reaction 4: resin Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - NH ₂ Manufacture of 82

The resin bound compound 81 was stirred for 15 min in 20% piperidine in N-methylpyrrolidine (20 mL). The resin was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (20 mL) and stirred for 15 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The resulting mixture was washed with resin to obtain compound 82

Reaction 5: resin Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  Preparation of Amides (78)

Commercially available 8-methyldecanoic acid (0.71 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1.21 g), diisopropyl Ethylamine (DIPEA, 2.0 mL) and 1-hydroxy-benzotriazole (0.508 g) were added to compound 82 (4.0 g) as a solution in N-methylpyrrolidine (80 mL). The mixture was stirred for 18 hours, filtered through a glass sinter funnel and the reaction was judged to be complete using the Kaiser test (see above). The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 6 mL), methanol (3 × 6 mL), and then again N-methylpyrrolidine (3 × 6 mL). The resulting mixture was washed with resin 78 to obtain a resin bonded compound.

Example  1-20: Synthesis of Peptide Resin Compound 83

Resin-Ala- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp -8- Methyldecano  Amide (83)

Commercially available 8-methyldecanoic acid (0.71 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 0.60 g), diisopropyl Ethylamine (DIPEA, 0.64 mL) and 1-hydroxy-benzotriazole (0.25 g) were added to compound 34 (1.8 g) as a solution in N-methylpyrrolidine (20 mL). The mixture was stirred for 18 hours, filtered with a glass sinter funnel and the reaction was judged to be complete using the Kaiser test (see above). The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 6 mL), methanol (3 × 6 mL), and then again N-methylpyrrolidine (3 × 6 mL). It wash | cleaned with and obtained the resin-bonded compound 83.

Example  1-21: Synthesis of Peptide Resin Compound 84

Resin-Ala- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  Amide (84)

Reaction 1: Resin-Ala- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- NHFmoc Manufacture of 85

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-glutamic acid γ-t-butyl ester (0.98 g), TBTU (0.74 g), HOBt (0.31 g) and DIPEA (810 μL) were added to NMP (20). To solution 71 (1.8 g) as a solution in mL). The reaction mixture was stirred for 17 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The compound was washed with to obtain compound 85.

Reaction 2: Resin-Ala- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- NH ₂ Manufacture of 86

Compound 85 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (20 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (20 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 20 mL), methanol (3 × 20 mL) and then again N-methylpyrrolidine (3 × 20 mL). The mixture was washed with to obtain compound 86.

Reaction 3: Resin-Ala- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - NHFmoc Manufacture of 87

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-tryptophan (0.98 g), TBTU (0.74 g), HOBt (0.31 g) and DIPEA (810 μL) as a solution in NMP (25 mL) To 86 (2.2 g). The reaction mixture was stirred for 17 hours. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 25 mL), methanol (3 × 25 mL) and then again N-methylpyrrolidine (3 × 25 mL). Compound 87 was obtained by washing with.

Reaction 4: Resin-Ala- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp -NH 88 Preparation

Compound 87 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (25 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (25 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 30 mL), methanol (3 × 30 mL) and then again N-methylpyrrolidine (3 × 30 mL). Compound 88 was obtained by washing with.

Reaction 5: Resin-Ala- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  amides( 84)  Produce

Commercially available 8-methyldecanoic acid (0.34 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 0.60 g), diisopropyl Ethylamine (DIPEA, 0.64 mL) and 1-hydroxy-benzotriazole (0.25 g) were added to compound 88 (2.0 g). The mixture was stirred for 18 hours, filtered through a glass sinter funnel and the reaction was judged to be complete using the Kaiser test (see above). The solid was washed with N-methylpyrrolidine (3 × 15 mL), washed with methanol (3 × 16 mL) and then again with N-methylpyrrolidine (3 × 1 6 mL) to give a resin-bound compound 84 Got.

Example  1-22: Synthesis of Peptide Resin Compound 89:

Resin-Ala- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - Undecano  Amide (89)

Reaction 1: Resin-Ala- Gly - NHFmoc Manufacture of 90

Commercially available solutions of Nα- (9-fluorenylmethoxycarbonyl) -glycine (1.49 g), TBTU (1.61 g) and DIPEA (871 μl) as a solution in NMP (25 mL), commercially available alanine-2- To chlorotrityl-resin (66, 2.5 g) was added. The mixture was stirred for 3 hours, filtered through a glass sinter funnel, and several beads were tested for the presence of free amine using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 15 mL) and methanol (3 × 15 mL), and then again N-methylpyrrolidine (3 × 15 mL). ML) to give compound 90.

Reaction 2: Resin-Ala- Gly - NH ₂ Manufacture of 91

Compound 90 was stirred in 20% piperidine in N-methylpyrrolidine (20 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Compound 91 was obtained by washing with.

Reaction 3: Resin-Ala- Gly - Thr - NHFmoc Preparation of 92

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-threonine (853 mg), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrOP, 1.165 g) and DIPEA (1.31 mL) To compound 91 (334 mg) was added as a solution in dichloromethane (25 mL). The mixture was stirred for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The mixture was washed with to obtain compound 92.

Reaction 4: Resin-Ala- Gly - Thr - NH ₂ Manufacture of 93

Compound 92 was stirred in 20% piperidine in N-methylpyrrolidine (20 mL) for 1.5 h. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The resulting mixture was washed with, to obtain a compound 93 having a resin bond.

Reaction 5: Resin-Ala- Gly - Thr -Asp ( OtBu )- NHFmoc Manufacture of 94

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-t-butyl ester (2.06 g), TBTU (1.61 g) and DIPEA (871 μl) as a solution in NMP (25 mL) Was added to compound 93. The mixture was stirred for 3 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Compound 94 was obtained by washing with.

Reaction 6: Resin-Ala- Gly - Thr -Asp ( OtBu )- NH ₂ Manufacture of 95

Compound 94 was stirred in 20% piperidine in N-methylpyrrolidine (20 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The compound 94 was obtained by washing with water.

Reaction 7: Resin-Ala- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- NHFmoc Manufacture of 96

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-asparagine δ-N-trityl (1.49 g), TBTU (0.80 g) and DIPEA (435 μL) were obtained as a solution in DMF (10 mL). Added to 95. The mixture was stirred for 17 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The mixture was washed with to obtain compound 96.

Reaction 8: Resin-Ala- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- NH ₂ Manufacture of 97

Compound 96 was stirred in 20% piperidine in N-methylpyrrolidine (20 mL) for 2 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). It was washed with to give compound 97.

Reaction 9: Resin-Ala- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NHFmoc Preparation of 98

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-tryptophan (1.07 g), TBTU (0.80 g) and DIPEA (435 μL) were added to compound 97 as a solution in NMP (25 mL). The mixture was stirred for 43 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). It was washed with to give compound 98.

Reaction 10: Resin-Ala- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NH ₂ Manufacture of 99

Compound 98 was stirred in 20% piperidine in N-methylpyrrolidine (20 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Compound 99 was obtained by washing with.

Reaction 11: Resin-Ala- Gly - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - Undecano  Preparation of Amides (89)

Commercially available undecanoic acid (930 mg), 1,3-diisopropylcarbodiimide (0.78 mL) and 1-hydroxy-benzotriazole (676 mg) in N-methylpyrrolidine (20 mL) As a compound 99. The mixture was stirred for 23 hours, filtered through a glass sinter funnel and the reaction was determined to be incomplete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL), and then again washed with N-methylpyrrolidine (3 × 15 mL). The reaction was confirmed to be complete using the Kaiser test and compound 89 was obtained.

Example  1-23: Synthesis of Peptide Resin Compound 100

Resin-Ala- Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - Undecano  Amide (100)

Reaction 1: Resin-Ala- Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- NHFmoc Preparation of 101

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-glutamic acid γ-t-butyl ester (1.06 g), TBTU (0.80 g) and DIPEA (435 μL) as a solution in DMF (10 mL) Added to 95. The mixture was stirred for 17 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). The mixture was washed with to give compound compound 101.

Reaction 2: Resin-Ala- Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- NH ₂ Manufacture of 102

Compound 101 was stirred in 20% piperidine in N-methylpyrrolidine (20 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Compound 102 was obtained by washing with.

Reaction 3: Resin-Ala- Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - NHFmoc Manufacture of 103

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-tryptophan (1.07 g), TBTU (0.80 g) and DIPEA (435 μL) were added to compound 102 as a solution in NMP (25 mL). The mixture was stirred for 43 hours. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Compound 103 was obtained by washing with.

Reaction 4: Resin-Ala- Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - NH ₂ Manufacture of 104

Compound 103 was stirred in 20% piperidine in N-methylpyrrolidine (20 mL) for 1 hour. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL) and then again N-methylpyrrolidine (3 × 15 mL). Compound 104 was obtained by washing with.

Reaction 5: Resin-Ala- Gly - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - Undecano  Preparation of Amide 100

Commercially available undecanoic acid (930 mg), 1,3-diisopropylcarbodiimide (0.78 mL) and 1-hydroxy-benzotriazole (676 mg) in N-methylpyrrolidine (20 mL) As compound 104. The mixture was stirred for 23 hours, filtered through a glass sinter funnel and the reaction was determined to be incomplete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with N-methylpyrrolidine (3 × 15 mL), methanol (3 × 15 mL), and then again with N-methylpyrrolidine (3 × 15 mL). . The reaction was confirmed to be complete using the Kaiser test and compound 100 was obtained.

Example  1-24: Synthesis of Peptide Resin 105:

Suzy- Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp -8- Methyldecano  Amide (105)

Reaction 1: resin Orn ( NHBoc )- NHFmoc Manufacture of 106

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -N-δ-t-butoxycarbonyl-L-ornithine (8.73 g) was converted to dichloromethane (100 mL) and diisopropylethylamine (DIPEA, As a solution in 13.4 mL), it was added to preswelled commercial 2-chlorotrityl resin 107 (10.0 g). The mixture was stirred for 1 hour, filtered through a glass sinter funnel and the reaction was judged to be complete using the Kaiser test (see above). The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 100 mL), methanol (3 × 100 mL), and then again N-methylpyrrolidine (3 × 100 mL). It was washed with to give compound 106.

Reaction 2: resin Orn ( NHBoc )- NH ₂ Manufacture of 108

Compound 106 was stirred in 20% piperidine in N-methylpyrrolidine (100 mL) for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (100 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 130 mL), methanol (3 × 130 mL), and then again N-methylpyrrolidine (3 × 130 mL). Compound 108 was obtained by washing with.

Reaction 3: resin Orn ( NHBoc )- Sar - NMeFmoc 109 Preparation

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -sarcosine (2.6 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 2.7 g), HOBt (1.13 g) and a solution of diisopropylethylamine (DIPEA, 2.9 mL) were added to compound 108 (10 g) as a solution in N-methylpyrrolidone (10 mL). The mixture was stirred for 60 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 115 mL) and methanol (3 × 115 mL), and then again N-methylpyrrolidine (3 × 115 mL). ML) to give compound 109.

Reaction 4: resin Orn ( NHBoc )- Sar - NMeH Preparation of 110

Compound 109 was stirred for 30 minutes in 20% piperidine in N-methylpyrrolidine (100 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (100 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 130 mL), methanol (3 × 130 mL), and then again N-methylpyrrolidine (3 × 130 mL). Compound 110 was obtained by washing with water.

Reaction 5: resin Orn ( NHBoc )- Sar - Thr - NHFmoc Manufacture of 111

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-threonine (2.9 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoro Borate (TBTU, 2.7 g), HOBt (1.13 g) and diisopropylethylamine (DIPEA, 2.9 mL) were added to compound 110 (11 g) as a solution in N-methylpyrrolidone (100 mL). The mixture was stirred for 60 minutes, filtered through a glass sinter funnel, and several beads were tested using the standard Kaiser test (see above) for the presence of free amines. The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 115 mL) and methanol (3 × 115 mL), and then again N-methylpyrrolidine (3 × 115 mL). ML) to give compound 111.

Reaction 6: Resin Orn ( NHBoc )- Sar - Thr - NH ₂ Manufacture of 112

Compound 111 was stirred in 20% piperidine in N-methylpyrrolidine (110 mL) for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (110 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 110 mL), methanol (3 × 110 mL), and then again N-methylpyrrolidine (3 × 110 mL). Compound 112 was obtained by washing with.

Reaction 7: Resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- NHFmoc Manufacture of 113

Commercially available solution of Nα- (9-fluorenylmethoxycarbonyl) -L-aspartic acid β-t-butyl ester, TBTU (2.7 g), HOBt (1.13 g) in N-methylpyrrolidone (100 mL) As a compound 112 (11 g), diisopropylethylamine (DIPEA, 2.9 mL) was added. The mixture was stirred for 60 minutes and filtered through a glass sinter funnel. Several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration, the product containing the resin was washed with N-methylpyrrolidine (3 × 115 mL) and methanol (3 × 115 mL), and then again with N-methylpyrrolidine (3 × 115 mL). Compound 113 was obtained.

Reaction 8: Resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- NH ₂ Manufacture of 114

Compound 113 was stirred in 20% piperidine in N-methylpyrrolidine (115 mL) for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (115 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 120 mL), methanol (3 × 120 mL) and then again N-methylpyrrolidine (3 × 120 mL). The compound was washed with, obtaining Compound 114.

Reaction 9: resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- NHFmoc Manufacture of 115

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-asparagine (5.0 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluor Roborate (TBTU, 2.7 g), HOBt (1.13 g) and diisopropylethylamine (DIPEA, 2.9 mL) were added to compound 114 (12 g) as a solution in NMP (120 mL). The mixture was stirred for 60 minutes, filtered with a glass sinter funnel, and several beads were tested for the presence of free amines using standard Kaiser test (homologous). The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 125 mL) and methanol (3 × 125 mL), and then again N-methylpyrrolidine (3 × 125 mL). ML) to give compound 115.

Reaction 10: Resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- NH ₂ 116 Preparation

Compound 115 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (130 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (130 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 130 mL), methanol (3 × 130 mL), and then again N-methylpyrrolidine (3 × 130 mL). It washed with and obtained compound 116.

Reaction 11: resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NHFmoc Synthesis of 117

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-tryptophan (3.57 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoro Borate (TBTU, 2.7 g), HOBt (1.13 g) and diisopropylethylamine (DIPEA, 2.9 mL) were added to compound 116 (13 g) as a solution in N-methylpyrrolidone (130 mL). The mixture was stirred for 60 minutes, filtered through a glass sinter funnel, and several beads were tested using the standard Kaiser test (see above) for the presence of free amines. The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 135 mL) and methanol (3 × 135 mL), and then again N-methylpyrrolidine (3 × 135 mL). ML) to give compound 117.

Reaction 12: Resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp - NH ₂ Preparation of 118

Compound 117 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (130 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (130 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 130 mL), methanol (3 × 130 mL), and then again N-methylpyrrolidine (3 × 130 mL). It was washed with to give compound 118.

Reaction 13: resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DAsn ( NHTrt )- Trp -8- Methyldecano  Preparation of Amide 105

Commercially available 8-methyldecanoic acid (1.56 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 2.7 g), diisopropyl Ethylamine (DIPEA, 2.9 mL) and 1-hydroxy-benzotriazole (1.25 g) were added to compound 118 (13.8 g) as a solution in N-methylpyrrolidine (120 mL). The mixture was stirred for 18 hours, filtered through a glass sinter funnel and the reaction was judged to be complete using the Kaiser test (see above). The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 120 mL), methanol (3 × 120 mL), and then again N-methylpyrrolidine (3 × 120 mL). Compound 105 was obtained by washing with.

Example  1-25: Synthesis of Peptide Resin Compound 119:

Suzy- Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  Amide (119)

Reaction 1: resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- NHFmoc Manufacture of 120

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -D-glutamic acid γ-t-butyl ester (2.29 g), TBTU (1.73 g), HOBt (0.73 g) and DIPEA (1.9 mL) were converted into NMP (25). To solution 114 (3.3 g) as a solution in mL). The mixture was stirred for 3 hours. The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 25 mL), methanol (3 × 25 mL) and then again N-methylpyrrolidine (3 × 25 mL). The mixture was washed with to obtain compound 120.

Reaction 2: resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- NH ₂ Manufacture of 121

Compound 120 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (25 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (25 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 30 mL), methanol (3 × 30 mL) and then again N-methylpyrrolidine (3 × 30 mL). Compound 121 was obtained by washing with.

Reaction 3: resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - NHFmoc Manufacture of 122

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-tryptophan (2.30 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoro Borate (TBTU, 1.7 g), HOBt (0.73 g) and diisopropylethylamine (DIPEA, 1.9 mL) were added to compound 121 (3.5 g) as a solution in N-methylpyrrolidone (25 mL). The mixture was stirred for 60 minutes, filtered through a glass sinter funnel, and several beads were tested using the standard Kaiser test (see above) for the presence of free amines. The Kaiser test appeared yellow, indicating that the coupling is complete. After filtration with a glass sinter funnel, the product containing the resin was washed with N-methylpyrrolidine (3 × 25 mL) and methanol (3 × 25 mL), and then again N-methylpyrrolidine (3 × 25 mL). ML) to give compound 122.

Reaction 4: resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp - NH ₂ Preparation of 123

Compound 122 was stirred for 30 min in 20% piperidine in N-methylpyrrolidine (25 mL). The reaction mixture was filtered with a glass sinter funnel, resuspended in 20% piperidine in N-methylpyrrolidine (25 mL) and stirred for 30 minutes. The reaction mixture was filtered with a glass sinter funnel, the solids were washed with N-methylpyrrolidine (3 × 30 mL), methanol (3 × 30 mL) and then again N-methylpyrrolidine (3 × 30 mL). The mixture was washed with to obtain Compound 123.

Reaction 5: resin Orn ( NHBoc )- Sar - Thr -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  Preparation of Amide (119)

Commercially available 8-methyldecanoic acid (0.50 g), 2- (1H-benzotriazol-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 0.86 g), diisopropyl Ethylamine (DIPEA, 0.94 mL) and 1-hydroxy-benzotriazole (0.35 g) were added to compound 123 (3.8 g) as a solution in N-methylpyrrolidine (30 mL). The mixture was stirred for 18 hours, filtered through a glass sinter funnel and the reaction was determined to be complete using the Kaiser test (see above). The reaction mixture was filtered with a glass sinter funnel, the solid was washed with N-methylpyrrolidine (3 × 36 mL), methanol (3 × 36 mL), and then again N-methylpyrrolidine (3 × 36 mL). The mixture was washed with to give compound 119.

Example  1-26: Peptide Resin Compound 76 Esterification  And cleavage

Gly - Thr ( OIleNHFmoc ) -Asp ( OtBu )- DAsn ( NHTrt )- Trp -8- Methyldecano  Amide (126)

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (1.7 g), 4-dimethylaminopyridine (117 mg) and N-methyl-2-chloropyridinium iodide (1.23 g) to argon After washing well, it was suspended in dichloromethane (20 mL). Triethylamine (76 μl) was added and the reaction mixture was stirred to give a homogeneous solution. Compound 76 (2.0 g) was added to the solution. The flask was washed again with argon and stirred for 14 hours. The resulting resin was filtered through a glass sinter funnel and washed well with dichloromethane. The solid was suspended in dichloromethane (21 mL), 2,2,2-trifluoroethanol (7 mL) and acetic acid (7 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 126 (285 mg) as a white solid.

Example  1-27: Peptide Resin Compound 77 Esterification  And cleavage

Gly - Thr ( OIleNHFmoc ) -Asp ( OtBu )- DAsn ( NHTrt )- Trp - Tridecano  Preparation of Amide (127)

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (1.48 g), 4-dimethylaminopyridine (102 mg) and N-methyl-2-chloropyridinium iodide (1.07 g) as argon After washing, it was suspended in dichloromethane (20 mL). Triethylamine (1.17 mL) was added and the reaction mixture was stirred to give a homogeneous solution. Compound 77 (1.75 g) was added to the solution. The flask was washed with argon and stirred for 14 hours. The resulting resin was filtered with a glass sinter funnel and washed with dichloromethane. The solid was suspended in dichloromethane (15 mL), 2,2,2-trifluoroethanol (5 mL) and acetic acid (5 mL) and stirred for 4 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 127 (490 mg) as a white solid.

Example  1-28: Peptide Resin Compound 78 Esterification  And cleavage

Gly - Thr ( OIleNHFmoc ) -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  Preparation of Amide 128

To compound 78 (5.9 g) is commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (4.9 g), bromo-tris-pyrrolidinophosphonium hexafluoro in dichloromethane (60 mL). A solution of phosphate (PyBrOP, 6.5 g) and diisopropylethylamine (7.3 mL) was added. Dimethylaminopyridine (25 mg) was added and the mixture was stirred for 2 hours. After 2 hours, the mixture was filtered with a glass sinter funnel, washed with dichloromethane (3 × 60 mL) and the coupling procedure was repeated. The resulting resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 60 mL) and methanol (3 × 60 mL) and dried under reduced pressure on potassium hydroxide pellets. The dried resin was suspended in dichloromethane (27 mL), 2,2,2-trifluoroethanol (9 mL) and acetic acid (9 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford peptide 128 (2.1 g) as the desired white solid.

Example  1-29: Peptide Resin Compound 83 Esterification  And cleavage

Ala- Sar - Thr ( OIIeNHFmoc ) -Asp ( OtBu )- DAsn ( ONHTrt )- Trp -8- Methyldecano  amides( 129)  Produce

To compound 83 (3.3 g) commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (3.2 g), bromo-tris-pyrrolidinophosphonium hexafluoro in dichloromethane (60 mL) A solution of phosphate (PyBrOP, 4.2 g) and diisopropylethylamine (4.7 mL) was added. Dimethylaminopyridine (23 mg) was added and the mixture was stirred for 2 hours. After 2 hours, the mixture was filtered with a glass sinter funnel, washed with dichloromethane (3 × 30 mL) and the coupling procedure was repeated. The resulting resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 30 mL) and methanol (3 × 30 mL) and dried under reduced pressure on potassium hydroxide pellets. The dried resin was suspended in dichloromethane (24 mL), 2,2,2-trifluoroethanol (6 mL) and acetic acid (6 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 129 (2.9 g) as a white solid.

Example  1-30: Peptide Resin Compound 75 Esterification  And cleavage

Ala- Sar - Thr ( OIleNHAlloc ) -Asp ( OtBu )- DAsn ( NHTrt )- Trp - Undecano  Preparation of Amide 130

Nα- (allyloxycarbonyl) -L-isoleucine 124 (1.34 g, see below), 4-dimethylaminopyridine (15 mg) and N-methyl-2-chloropyridinium iodide (1.59 g) were washed with argon and It was suspended in dichloromethane (30 mL). Triethylamine (1.74 mL) was added and the reaction mixture was stirred to give a homogeneous solution. Compound 75 (1.25 g) was added to the solution. The flask was washed with argon and stirred for 14 hours. The resulting resin was filtered with a glass sinter funnel and washed with dichloromethane. The solid was suspended in dichloromethane (12 mL), 2,2,2-trifluoroethanol (4 mL) and acetic acid (4 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 130 (154 mg) as a white solid.

Example  1-31: Peptide Resin Compound 84 Esterification  And cleavage

Ala- Sar - Thr ( OIleNHFmoc ) -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  amides( 131)  Produce

To compound 84 (4.8 g) commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (3.2 g), bromo-tris-pyrrolidinophosphonium hexafluoro in dichloromethane (60 mL) A solution of phosphate (PyBrOP, 4.2 g) and diisopropylethylamine (4.7 mL) was added. Dimethylaminopyridine (23 mg) was added and the mixture was stirred for 2 hours. After 2 hours, the mixture was filtered with a glass sinter funnel, washed with dichloromethane (3 × 30 mL) and the coupling procedure was repeated. The resulting resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 30 mL) and methanol (3 × 30 mL) and dried under reduced pressure on potassium hydroxide pellets. The dried resin was suspended in dichloromethane (24 mL), 2,2,2-trifluoroethanol (6 mL) and acetic acid (6 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 131 (2.92 g) as a white solid.

Example  1-32: Peptide Resin Compound 89 Esterification  And cleavage

Ala- Gly - Thr ( OIleNHAlloc ) -Asp ( OtBu )- DAsn ( NHTrt )- Trp - Undecano  Preparation of Amide 132

Nα- (allyloxycarbonyl) -L-isoleucine 124 (1.34 g, see below), 4-dimethylaminopyridine (15 mg) and N-methyl-2-chloropyridinium iodide (1.59 g) were washed with argon and It was suspended in dichloromethane (30 mL). Triethylamine (1.74 mL) was added and the reaction mixture was stirred to give a homogeneous solution. Compound 89 (1.25 g) was added to the solution. The flask was washed with argon and stirred for 14 hours. The resulting resin was filtered with a glass sinter funnel and washed with dichloromethane. The solid was suspended in dichloromethane (12 mL), 2,2,2-trifluoroethanol (4 mL) and acetic acid (4 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 132 (147 mg) as a white solid.

Example  1-33: of Peptide Resin Compound 100 Esterification  And cleavage

Ala- Gly - Thr ( OIleNHAlloc ) -Asp ( OtBu )- DGlu ( OtBu )- Trp - Undecano  Preparation of Amide 133

Nα- (allyloxycarbonyl) -L-isoleucine 124 (1.34 g, see below), 4-dimethylaminopyridine (15 mg) and N-methyl-2-chloropyridinium iodide (1.59 g) were washed with argon and It was suspended in dichloromethane (30 mL). Triethylamine (1.74 mL) was added and the reaction mixture was stirred to give a homogeneous solution. Compound 100 (1.25 g) was added to the solution. The flask was washed with argon and stirred for 14 hours. The resulting resin was filtered with a glass sinter funnel and washed with dichloromethane. The solid was suspended in dichloromethane (12 mL), 2,2,2-trifluoroethanol (4 mL) and acetic acid (4 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 133 (95 mg) as a white solid.

Example  1-34: Peptide Resin Compound 105 Esterification  And cleavage

Orn ( NHBoc )- Sar - Thr ( OIleNHFmoc ) -Asp ( OtBu )- DAsn ( NHTrt )- Trp -8- Methyldecano  Preparation of Amide 134

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (2.0 g), 4-dimethylaminopyridine (140 mg) and N-methyl-2-chloropyridinium iodide (1.46 g) in argon It was washed and suspended in dichloromethane (20 mL). Triethylamine (1.6 mL) was added and the reaction mixture was stirred to give a homogeneous solution. Compound 105 (2.0 g) was added to the solution. The flask was washed with argon and stirred for 14 hours. The resulting resin was filtered with a glass sinter funnel and washed with dichloromethane. The solid was suspended in dichloromethane (21 mL), 2,2,2-trifluoroethanol (7 mL) and acetic acid (7 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford the desired peptide 134 (890 mg) as a white solid.

Example  1-35: Peptide Resin Compound 119 Esterification  And cleavage

Orn ( NHBoc )- Sar - Thr ( OIleNHFmoc ) -Asp ( OtBu )- DGlu ( OtBu )- Trp -8- Methyldecano  amides( 135)  Produce

Commercially available Nα- (9-fluorenylmethoxycarbonyl) -L-isoleucine (2.0 g), 4-dimethylaminopyridine (140 mg) and N-methyl-2-chloropyridinium iodide (1.46 g) in argon It was washed and suspended in dichloromethane (20 mL). Triethylamine (1.6 mL) was added and the reaction mixture was stirred to give a homogeneous solution. Compound 119 (1.75 g) was added to the solution. The flask was washed with argon and stirred for 14 hours. The resulting resin was filtered with a glass sinter funnel and washed with dichloromethane. The solid was suspended in dichloromethane (21 mL), 2,2,2-trifluoroethanol (7 mL) and acetic acid (7 mL) and stirred for 3 hours. The resin was filtered with a glass sinter funnel and the filtrate was evaporated to afford peptide 135 (761 mg) as the desired white solid.

Example  1-36: Compound C16 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu )-Orn (NHBoc) -Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide ( 137)  Produce

Hydroxy-benzotriazole (17 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 55 mg) and diisopropylethylamine (22 [Mu] l) was added to a solution of compound 126 (174 mg) in dimethylformamide (3 mL), compound 9 (300 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL), and dried under reduced pressure to give a resin-bonded compound 137.

Reaction 2: Preparation of Compound 138

Compound 137 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 ML) and dichloromethane (10 mL) and dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 138.

Reaction 3: Compound C16 Manufacture

The dried compound 138 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give pure product C16 (3.7 mg).

Example  1-37: Compound C76 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu )-Orn (NHBoc) -Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide ( 140)  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 128 (174 mg) in dimethylformamide (3 mL). Compound 9 (300 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sintered funnel, washed with dichloromethane (3 × 3 mL), and dried under reduced pressure to give a resin-bonded compound 140.

Reaction 2: Preparation of Compound 141

Compound 140 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 141.

Reaction 3: Compound C76 Manufacture

The dried compound 141 is suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture is stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give pure product C76 (9.0 mg).

Example  1-38: Compound C75 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu )-Orn (NHBoc) -Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide ( 143)  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 134 (243 mg) in dimethylformamide (3 mL). Compound 21 (322 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 143.

Reaction 2: Preparation of Compound 144

A compound of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) was placed under argon atmosphere. Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sintered funnel and washed with N-methylmorpholine to give compound 144.

Reaction 3: Compound C75 Manufacture

The dried compound 144 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C75 (8.1 mg).

Example  1-39: Compound C74 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp- ( OtBu Preparation of) -Ala-Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide (146)

Hydroxy-benzotriazole (27 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 88.5 mg) and diisopropylethylamine (100 [Mu] l) was added to a solution of compound 126 (278 mg) in dimethylformamide (3 mL). Compound 38 (227 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was judged to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to give compound 146.

Reaction 2: Preparation of Compound 147

Compound 146 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarbohydrate in dimethylformamide (10 mL), 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 147.

Reaction 3: Compound C74 Manufacture

The dried compound 147 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give Compound C74 (3.9 mg).

Example  1-40: Compound C86 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu Preparation of) -Ala-Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide (149)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 129 (228 mg) in dimethylformamide (3 mL). Compound 21 (280 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 149.

Reaction 2: Preparation of Compound 150

Compound 149 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture is stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid is 0.5% sodium thiocarboateate (10 ml) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide ( 10 mL) and dichloromethane (10 mL) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 7 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 150.

Reaction 3: Compound C86 Manufacture

The dried compound 150 is suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture is stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C86 (2.8 mg).

Example  1-41: Compound C79 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Orn (NHBoc) -Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide ( 152)  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (52 [Mu] l) was added to a solution of compound 135 (217 mg) in dimethylformamide (3 mL). Compound 21 (278 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL), and dried under reduced pressure to give a resin-bonded compound 152.

Reaction 2: Preparation of Compound 153

Compound 151 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture is stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid is 0.5% sodium thiocarboateate (10 ml) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide ( 10 mL) and dichloromethane (10 mL) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 7 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 153.

Reaction 3: Compound C79 Manufacture

The dried compound 153 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C79 (1.5 mg).

Example  1-42: Compound C81 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu Preparation of) -Ala-Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtB) -Trp-8-methyldecanoamide (155)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 128 (183 mg) in dimethylformamide (3 mL). Compound 38 (227 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sintered funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 155.

Reaction 2: Preparation of Compound 156

Compound 155 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture is stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid is 0.5% sodium thiocarboate in dimethylformamide (1 mL), 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 156.

Reaction 3: Compound C81 Manufacture

The dried compound 156 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C81 (2.3 mg).

Example  1-43: Compound C80 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu Preparation of) -Ala-Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide (158)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 131 (196 mg) in dimethylformamide (3 mL). Compound 21 (278 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sintered funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 158.

Reaction 2: Preparation of Compound 159

Resin 158 was placed under argon and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL). Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarbohydrate in dimethylformamide (10 mL), 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 159.

Reaction 3: Compound C80 Manufacture

The dried compound 159 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give Compound C80 (6.2 mg).

Example  1-44: Compound C72 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn - Gly -Asp ( OtBu )- DAla -Asp ( OtBu )- Orn ( NHBoc Preparation of) -Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide (161)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 126 (274 mg) in dimethylformamide (3 mL). Compound 50 (303 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 161.

Reaction 2: Preparation of Compound 162

Compound 161 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarbohydrate in dimethylformamide (10 mL), 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) was added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 162.

Reaction 3: Compound C72 Manufacture

The dried compound 162 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give Compound C72 (2.9 mg).

Example  1-45: C352 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Orn (NHBoc) -Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide ( 164)  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 127 (183 mg) in dimethylformamide (2 mL). Compound 50 (265 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was judged to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to give compound 164.

Reaction 2: Preparation of Compound 165

Compound 164 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 165.

Reaction 3: Compound C352 Manufacture

The dried compound 165 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C352 (4.7 mg).

Example  1-46: Compound C85 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Orn (Boc) -Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide ( 167)  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (30 [Mu] l) was added to a solution of compound 134 (248 mg) in dimethylformamide (3 mL). Compound 40 (238 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sintered funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 167.

Reaction 2: Preparation of Compound 168

Compound 167 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 168.

Reaction 3: Compound C85 Manufacture

The dried compound 168 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 25O × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C85 (3.7 mg).

Example  1-47: Compound C353 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu Preparation of) -Ala-Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide (170)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (52 [Mu] l) was added to a solution of compound 126 (209 mg) in dimethylformamide (2 mL). Compound 52 (340 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropyl ethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to afford compound 170.

Reaction 2: Preparation of Compound 171

Resin 170 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours and filtered through a glass sinter funnel. The solid was washed with 0.5% sodium thiocarboate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine (10 mL) and dichloromethane (10 mL) in dimethylformamide, then dried under reduced pressure. I was. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 171.

Reaction 3: Compound C353 Manufacture

The dried compound 171 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C353 (6.8 mg).

Example  1-48: Compound C82 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu Preparation of) -Ala-Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide (173)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 129 (221 mg) in dimethylformamide (3 mL). Compound 40 (238 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL), and then dried under reduced pressure to give compound 173.

Reaction 2: Preparation of Compound 174

Compound 173 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 174.

Reaction 3: Compound C82 Manufacture

The dried compound 174 is suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture is stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 25O × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give Compound C82 (3.8 mg).

Example  1-49: Compound C83 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu ) -Orn (NHBoc) -Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide ( 176)  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 135 (221 mg) in dimethylformamide (3 mL). Compound 40 (238 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 176.

Reaction 2: Preparation of Compound 177

Compound 176 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 177.

Reaction 3: Compound C83 Manufacture

The dried compound 177 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C83 (4.3 mg).

Example  1-50: Compound C84 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu Preparation of) -Ala-Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide (179)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 128 (183 mg) in dimethylformamide (3 mL). Compound 52 (212 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sintered funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 179.

Reaction 2: Preparation of Compound 180

Compound 179 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sintered funnel and washed with N-methylmorpholine to give compound 180.

Reaction 3: Compound C84 Manufacture

The dried compound 180 is suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture is stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C84 (6.6 mg).

Example  1-51: Compound C354 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DAla -Asp ( OtBu Preparation of) -Ala-Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide (182)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 131 (196 mg) in dimethylformamide (2 mL). Compound 40 (238 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to afford compound 182.

Reaction 2: Preparation of Compound 183

A compound of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) was placed under argon atmosphere. Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 183.

Reaction 3: Compound C354 Manufacture

The dried compound 183 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C354 (4.7 mg).

Example  1-52: Compound C73 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of Asp (OtBu) -Orn (NHBoc) -Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide (185)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 126 (298 mg) in dimethylformamide (3 mL). Compound 54 (312 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 185.

Reaction 2: Preparation of Compound 186

Compound 185 was placed under argon and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL). Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 186.

Reaction 3: Compound C73 Manufacture

The dried compound 186 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C73 (24.6 mg).

Example  1-53: compound C355 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) u-Trp-8-methyldecanoamide (188)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 128 (183 mg) in dimethylformamide (2 mL). Compound 54 (322.6 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was judged to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to give compound 188.

Reaction 2: Preparation of Compound 189

Compound 188 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 189.

Reaction 3: Compound C355 Manufacture

The dried compound 189 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C355 (8.2 mg).

Example  1-54: Compound C356 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide (191)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 134 (243 mg) in dimethylformamide (2 mL). Compound 34 (263 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to give compound 191.

Reaction 2: Preparation of Compound 192

Compound 191 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and then dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 192.

Reaction 3: Compound C356 Manufacture

The dried compound 192 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C356 (8.7 mg).

Example  1-55: compound C357 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Ala-Gly-Thr (OIleNHAlloc) -Asp (OtBu) -DAsn (NHTrt) -Trp-Undecanoamide ( 194)  Produce

Hydroxy-benzotriazole (14 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 44 mg) and diisopropylethylamine (20 [Mu] l) was added to a solution of compound 132 (147 mg) in dimethylformamide (3 mL). Compound 34 (200 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropyl ethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to give compound 194.

Reaction 2: Preparation of Compound 195

A compound of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 ml), acetic acid (0.5 ml) and N-methylmorpholine (0.25 ml) was placed under argon atmosphere. Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 195.

Reaction 3: Compound C357 Manufacture

The dried compound 195 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was lyophilized to give crude product (43 mg). Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C357 (4.5 mg).

Example  1-56: Compound C358 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp-Ala-Sar-Thr (OIleNHAlloc) -Asp (OtBu) -DAsn (NHTrt) -Trp-Undecanoamide (197)

Hydroxy-benzotriazole (14 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 44 mg) and diisopropylethylamine (20 [Mu] l) was added to a solution of compound 130 (154 mg) in dimethylformamide (3 mL). Compound 34 (200 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropyl ethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was judged to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to give compound 197.

Reaction 2: Preparation of Compound 198

Compound 197 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 198.

Reaction 3: Compound C358 Manufacture

The dried compound 198 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C358 (2.7 mg).

Example  1-57: Compound C359 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide (200)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 135 (217 mg) in dimethylformamide (2 mL). Compound 34 (263 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropyl Ethylamine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to give compound 200.

Reaction 2: Preparation of Formula 201

Compound 200 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed well with N-methylmorpholine to give compound 201.

Reaction 3: Compound C359 Manufacture

The dried compound 201 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C359 (4.7 mg).

Example  1-58: Compound C360 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Ala-Gly-Thr (OIleNHAlloc) -Asp (OtBu) -DGlu (OtBu) -Trp-Undecanoamide ( 203 of  Produce

Hydroxy-benzotriazole (14 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 44 mg) and diisopropylethylamine (20 [Mu] l) was added to a solution of compound 133 (95 mg) in dimethylformamide (3 mL). Compound 34 (200 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to afford compound 203.

Reaction 2: Preparation of Compound 204

Compound 203 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL), followed by addition of 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbo diimide (157 μl). The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 204.

Reaction 3: Compound C360 Manufacture

The dried compound 204 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C360 (3.4 mg).

Example  1-59: Compound C361 Manufacture

Reaction 1: resin Glu (αO allyl)- DSer ( OtBu )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Ala-Sar-Thr (OIleNHAlloc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide ( 206 of  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 131 (196 mg) in dimethylformamide (2 mL). Compound 34 (270 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to afford compound 206.

Reaction 2: Preparation of Compound 207

Compound 206 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 207.

Reaction 3: Compound C361 Manufacture

The dried compound 207 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C361 (6.3 mg).

Example  1-60: Compound C77 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide (209)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 126 (243 mg) in dimethylformamide (3 mL). Compound 60 (217 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sintered funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 209.

Reaction 2: Preparation of Compound 210

Compound 209 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture is stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid is 0.5% sodium thiocarboateate (10 ml) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide ( 10 mL) and dichloromethane (10 mL) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 210.

Reaction 3: Compound C77 Manufacture

The dried compound 210 is suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture is stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C77 (3.8 mg).

Example  1-61: Compound C362 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide (212)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 128 (183 mg) in dimethylformamide (2 mL). Compound 60 (217 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to give compound 212.

Reaction 2: Preparation of Compound 213

A compound of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) was placed under argon atmosphere. Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 213.

Reaction 3: Compound C362 Manufacture

The dried compound 213 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C362 (3.1 mg).

Example  1-62: Compound C363 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide 215

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of 134 (342 mg) in compound dimethylformamide (2 mL). Compound 56 (416 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropyl Ethylamine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to afford compound 215.

Reaction 2: Preparation of Compound 216

A compound of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 ml), acetic acid (0.5 ml) and N-methylmorpholine (0.25 ml) was placed under argon atmosphere. Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 216.

Reaction 3: Compound C363 Manufacture

The dried compound 216 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C363 (6.1 mg).

Example  1-63: compound C364 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Ala-Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-tridecanoamide ( 218  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 127 (146 mg) in dimethylformamide (3 mL). Compound 62 (171 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 218.

Reaction 2: Preparation of Compound 219

Compound 218 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 219.

Reaction 3: Compound C364 Manufacture

The dried compound 219 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C364 (4.8 mg).

Example  1-64: Compound C365 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Ala-Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DAsn (NHTrt) -Trp-8-methyldecanoamide ( 221  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine ( 26 μl) was added to a solution of compound 129 (195 mg) in dimethylformamide (2 mL). Compound 56 (400 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was judged to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to afford compound 221.

Reaction 2: Preparation of Compound 222

Compound 221 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 222.

Reaction 3: Compound C365 Manufacture

The dried compound 222 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C365 (10.2 mg).

Example  1-65: Compound C366 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc Preparation of) -Asp (OtBu) -Orn (NHBoc) -Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide (224)

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 135 (217 mg) in dimethylformamide (2 mL). Compound 56 (217 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropylethyl Amine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was judged to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to afford compound 224.

Reaction 2: Preparation of Compound 225

Compound 224 was placed under argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and then dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 225.

Reaction 3: Compound C366 Manufacture

Compound 225 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl) and the reaction mixture was stirred at room temperature for 4.5 hours. The resin was filtered through a glass sinter funnel washed with dichloromethane, and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C366 (1.1 mg).

Example  1-66: Compound C367 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Ala-Gly-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide ( 227)  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 128 (183 mg) in dimethylformamide (3 mL). Compound 62 (294 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was judged to be complete using the Kaiser test (see above). The resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 3 mL) and dried under reduced pressure to give compound 227.

Reaction 2: Preparation of Formula 228

A compound of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) was placed under argon atmosphere. Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours and then filtered through a glass sinter funnel. The solid was washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and dried under reduced pressure. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 228.

Reaction 3: Compound C367 Manufacture

The dried compound 228 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give the product C366 (6.9 mg).

Example  1-67: Compound C368 Manufacture

Reaction 1: resin Glu (αO allyl)- DAsn ( NHTrt )- Gly -Asp ( OtBu )- DLys ( NHBoc ) -Asp (OtBu) -Ala-Sar-Thr (OIleNHFmoc) -Asp (OtBu) -DGlu (OtBu) -Trp-8-methyldecanoamide ( 230)  Produce

Hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 66 mg) and diisopropylethylamine (26 [Mu] l) was added to a solution of compound 131 (296 mg) in dimethylformamide (2 mL). Compound 56 (416 mg) was added and the mixture was stirred for 17 hours. A portion of the resin was removed and the coupling was determined to be incomplete using the Kaiser test (see above). Additional portion of hydroxy-benzotriazole (20 mg), benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphonate (BOP, 60 mg) and diisopropyl Ethylamine (30 μl) was added and the mixture was stirred for 26 hours. Coupling was determined to be complete using the Kaiser test, the resin was filtered with a glass sinter funnel, washed with dichloromethane (3 × 5 mL) and dried under reduced pressure to afford compound 230.

Reaction 2: Preparation of Compound 231

Compound 230 was placed under an argon atmosphere and a solution of tetrakis- (triphenylphosphine) palladium (0) (340 mg) in dichloromethane (9.25 mL), acetic acid (0.5 mL) and N-methylmorpholine (0.25 mL) Treated with. The mixture was stirred at room temperature for 4.5 hours, filtered with a glass sintering funnel, and the solid was 0.5% sodium thiocarboateate (10 mL) in dimethylformamide, 0.5% diisopropylethylamine in dimethylformamide (10 Ml) and dichloromethane (10 ml) and then dried under reduced pressure. The resin was washed with DMF: piperidine 4: 1 (10 mL) for 4 hours, then filtered with a glass sinter funnel, the solids were washed with dimethylformamide (10 mL) and dichloromethane (10 mL) and decompressed To dryness. The resin was suspended in N-methylmorpholine (3 mL) and then 1-hydroxy-benzotriazole (135 mg) and 1,3-diisopropylcarbodiimide (157 μl) were added. The reaction was stirred for 17 hours, filtered with a glass sinter funnel and washed with N-methylmorpholine to give compound 231.

Reaction 3: Compound C368 Manufacture

The dried compound 231 was suspended in dichloromethane (2.5 mL), trifluoroacetic acid (2.5 mL), ethanedithiol (125 μl) and triisopropylsilane (125 μl), and the reaction mixture was stirred at room temperature for 4.5 hours. It was. The resin was filtered with a glass sinter funnel washed with dichloromethane and the combined filtrates were evaporated under reduced pressure. The crude product was partitioned between diethyl ether (10 mL) and water (2.5 mL). The aqueous layer was freeze dried to afford the crude product. Reverse phase HPLC (C18 10 μM Jupiter column 250 × 21.2 mm) eluting the crude product with a gradient of 20% acetonitrile 0.5% formic acid: 80% water 0.5% formic acid to 80% acetonitrile 0.5% formic acid: 20% water 0.5% formic acid ) For 25 minutes. Fractions containing the product were combined and lyophilized to give compound C368 (11.8 mg).

Example  1-68: 2S, 3R-N- Fmoc -L-3- methyl Stereoselective Synthesis of -glutamic Acid Alpha Allyl Ester 232

Reaction 1

Tetrabutyl ammonium iodide (39.4 g) was added to a solution of commercial Garner Aldehyde 233 (98 g) in 3 M potassium carbonate (K ₂ CO ₃ , 100 mL) under a nitrogen atmosphere to obtain a heterogeneous solution. After 15 minutes, t-butyl-diethyl phosphonoacetate (130 g) was added and the reaction mixture was stirred vigorously for 18 hours. Water (500 mL) was added and the resulting mixture was extracted with methyl t-butyl ether (MTBE, 3 × 250 mL). The combined organic fractions were combined, washed with saturated sodium chloride (1 × 250 mL), dried over magnesium sulfate (MgSO ₄ ), filtered and concentrated to afford the crude product as a yellow oil. Purification by column chromatography on silica gel eluting with ethyl acetate: hexanes 1: 9 gave the desired product 234 (95.3 g).

Reaction 2

A solution of copper iodide (CuI, 137 g) in anhydrous tetrahydrofuran (THF, 2250 mL) was cooled to -10 DEG C under a nitrogen atmosphere and stirred for 30 minutes. To this solution was added a 1.6 M solution of methyl lithium (MeLi) in diethyl ether (900 mL) to keep the temperature below -10 ° C. The resulting mixture was stirred at −10 ° C. for 30 minutes, then cooled to −78 ° C. and stirred for 45 minutes. Trimethyl chloride silyl TMSCl (91 mL) was added to maintain the temperature below −78 ° C., and then the reaction mixture was stirred for 15 minutes. A solution of base ester 234 (85.45 g) in THF (250 mL) was added dropwise over 1 hour. The reaction mixture is stirred at -78 ° C for 1 hour and warmed to -40 ° C before slowly adding a quench solution of 90% saturated ammonium chloride (NH ₄ Cl): 10% ammonium hydroxide (NH ₄ OH, 1500 mL) I was. The reaction mixture was stirred for 30 minutes, warmed to -30 ° C, and then worked up into three separate 1,500 mL fractions. Each fraction was partitioned and the aqueous layer was extracted with MTBE (500 mL). The combined organic phases were filtered through celite, washed with 90% saturated NH ₄ Cl: 10% NH ₄ OH solution (4 × 400 mL), dried over sodium sulfate (Na ₂ SO ₄ ), filtered and concentrated to product Got. The volatiles were removed from the product under high vacuum to yield product 235 (85.45 g). Compound 235 was used without further purification.

Reaction 3

A solution of oxazolidine 235 (70 g) in methanol (1800 mL) was cooled to 0 ° C. and stirred for 1 hour. Boron trifluoride acetic acid complex (BF ₃ .2HOAc, 450 mL) was added dropwise for 2 hours to maintain the internal temperature below 3 ° C. Thereafter, 20% sodium bicarbonate (Na ₂ CO ₃ , 3,000 ml) was added over 2 hours to quench the reaction mixture and the resulting solution was worked up into five separate 1,000 ml fractions. Extract each 1,000 ml fraction with dichloromethane (3 × 300 mL), combine organic extracts, wash with NaHCO ₃ (1 × 300 mL), saturated sodium chloride (1 × 30 mL), dry over MgSO ₄ , Filtration and concentration gave the crude product. The combined products were purified by column chromatography on silica gel eluting with a gradient elution from 20% ethyl acetate: 80% hexanes to 50% ethyl acetate: 50% hexanes. Fractions containing product were combined and evaporated to afford the desired product 236 (36.3 g).

Reaction 4

A solution of alcohol 236 (24 g) in acetonitrile (23 mL) and water (29.7 mL) was cooled to 0 ° C, and periodic acid (52.2 g) was added in portions to maintain a temperature of 0 ° C. The reaction mixture was stirred at 0 ° C. for 45 min and chromium trioxide (CrO ₃ , 460 mg) was added. The reaction mixture was stirred for 15 minutes before quenching by the slow addition of 0.4 M dibasic sodium phosphate solution (Na ₂ HPO ₄ , 560 mL, pH 9.0). The resulting mixture was extracted with MBTE (4 × 300 mL) and the combined organic extracts were washed with saturated sodium chloride (1 × 250 mL), NaHCO ₃ (1 × 250 mL) and saturated sodium chloride (1 × 250 mL). The organic fractions were then dried over MgSO ₄ , filtered and concentrated to afford crude product. Purification by preparative thin layer chromatography on silica gel eluting with 20% ethyl acetate: 80% hexane and extraction from silica gel with dichloromethane gave the desired product 237 (15.32 g).

Reaction 5

Potassium bicarbonate (KHCO ₃ , 9.66 g) was added to a solution of acid 237 (15.32 g) in N, N-dimethylformamide (DMF, 200 mL), and the resulting suspension was stirred for 15 minutes. A solution of allyl bromide (21 mL) in DMF (200 mL) was added dropwise over 30 minutes, and the reaction mixture was stirred for 19 hours. Water (500 mL) was added and the resulting mixture was extracted with ethyl acetate (5 × 200 mL) and the combined organic extracts washed with water (2 × 200 mL) and saturated sodium chloride (1 × 200 mL). The organic fractions were dried over Na ₂ S0 ₄ , filtered and concentrated to afford the crude product as a yellow oil. Purification by column chromatography on silica gel eluting with ethyl acetate: hexane 1: 4 gave the desired product 238 (9.2 g).

Reaction 6

Trifluoroacetic acid (TFA, 25 mL) and triisopropyl silane (TIPS, 1 mL) were added to a solution of ester 238 (9.2 g) in dichloromethane and the reaction mixture was stirred for 1 hour. Then the mixture was concentrated in vacuo and the resulting residue was dissolved in hexane (100 mL) and redevaporated three times. The residue is then dissolved in saturated NaHCO ₃ (53 mL) and 1,4-dioxane (50 mL) and 9-fluorenylmethoxycarbonyl-N-hydride in 1,4-dioxane (50 mL) A solution of oxysuccinimide (EmocOSu, 9.52 g) was added dropwise for 30 minutes. At this time, the reaction mixture became cloudy and an additional portion of 1,4-dioxane (20 mL) was added to obtain a heterogeneous solution, which was stirred for an additional 17 hours. The reaction mixture was filtered and the residue was washed with 1,4-dioxane (50 mL). The combined organic fractions were evaporated, redissolved in ethyl acetate (250 mL) and acidified before washing with potassium sulfate (KHSO ₄ , 3 × 50 mL) and saturated sodium chloride (1 × 50 mL). The organic fractions were then dried over Na ₂ SO ₄ , filtered and concentrated to afford crude product. The product was purified by column chromatography on silica gel using a gradient elution of 20% ethyl acetate: 80% hexanes to 40% ethyl acetate: 60% hexanes. Fractions containing product were combined and evaporated to afford the desired product 232 (6.32 g).

Example  1-69: 2S, 3S-N- Fmoc -L-S- methyl Stereoselective Synthesis of -glutamic Acid Alpha Allyl Ester 239

Reaction 1

Glycine benzyl ester tosylate salt (6.75 g) was partitioned between dichloromethane (100 mL) and aqueous 10% w / v K ₂ CO ₃ (100 mL). The aqueous fractions were extracted with dichloromethane (2 x 50 mL) and the combined organic fractions were dried over MgSO ₄ , filtered and evaporated to a glassy solid (3.29 g). This solid was dissolved in anhydrous dichloromethane (80 mL) and a solution of benzophenone imine (3.62 g) in dichloromethane (20 mL) was added. The resulting mixture was stirred at room temperature for 17 hours. The mixture was concentrated in vacuo to an oil, redissolved in ether (80 mL) and washed with water (2 x 40 mL). The organic layer was dried over MgSO ₄ , filtered and evaporated to afford the crude product as a clear oil. Purification by recrystallization from warm ether / hexanes gave pure compound 241 (3.82 g).

Reaction 2

Benzyl-N- (diphenylmethylene) glycinate 241 (5.7 g) and O-allyl-N- (9-anthracenylmethyl) cinconidinium in dichloromethane (80 mL) cooled to -78 ° C under a nitrogen atmosphere. To a suspension of bromide (1.05 g) was added cesium hydroxide (14.53 g). The mixture was stirred for 20 minutes and t-butyl crotonate (9.13 mL) was added dropwise to maintain the temperature at -78 ° C. After stirring at −78 ° C. for 2 hours, the mixture was warmed to −50 ° C. for 30 minutes, and then the mixture was allowed to warm to room temperature for 2 hours. The mixture was then poured into diethyl ether (600 mL) and water (200 mL) and partitioned and the organic layer washed with water (2 x 170 mL) and saturated sodium chloride (1 x 150 mL). The ether fractions were dried over MgSO ₄ , filtered and evaporated to afford product 242 (4.46 g) which was used later without further purification.

Reaction 3

To a solution of protected 3-methyl glutamate 64 (4.46 g) in tetrahydrofuran (250 mL) was added a solution of 10% w / v citric acid (120 mL) and the mixture was stirred for 17 h. The solution was concentrated in vacuo to remove tetrahydrofuran and diethyl ether (100 mL) and 1 N HCl (250 mL) was added. After partitioning, the aqueous layer was washed with diethyl ether (2 × 100 mL), solid K ₂ CO ₃ was added to basify the pH to 14, and extracted with ethyl acetate (4 × 100 mL). Acetic acid (3 mL) and 10% palladium on carbon (500 mg) were added to the combined ethyl acetate fractions and the resulting suspension was stirred for 16 h under a hydrogen atmosphere. Methanol (300 mL) was added and the reaction mixture was filtered through celite. The filtrate was evaporated to oil, it was dissolved, first evaporated in ethyl acetate (300 mL) and then evaporated in diethyl ether (300 mL) to give a white gel. This residue is dissolved in tetrahydrofuran (200 mL) and 10% w / v K ₂ CO ₃ (100 mL) and 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide (5.83 g) Added. The reaction mixture was stirred for 22 hours and concentrated in vacuo to remove tetrahydrofuran. To the concentrated solution was added diethyl ether (170 mL) and water (300 mL). After partitioning, the aqueous layer was washed with diethyl ether (3 x 130 mL), acidified to pH 2 with concentrated HCl and extracted with ethyl acetate (3 x 200 mL). The ethyl acetate fractions were dried over MgSO ₄ , filtered and evaporated to afford product 243 (3.31 g) which was used later without further purification.

Reaction 4

N, N'-diisopropylcarbodiimide polystyrene resin in a solution of 2S, 3S-N-Fmoc-L-3-methyl-glutamic acid γ-t-butyl ester 243 (3.3 g) in dichloromethane (150 mL) 10.8 g) and 4-dimethylaminopyridine (92 mg) were added and the reaction mixture was stirred for 5 minutes. Allyl alcohol (0.612 mL) was added and the reaction mixture was stirred for an additional 90 minutes. Filtration and evaporation of the solvent gave the desired diester 244 (2.02 g).

Reaction 5

To a solution of diester 244 in dichloromethane (42 mL) cooled to 0 ° C. was added triisopropylsilane (0.82 mL) and trifluoroacetic acid (4 mL). The reaction mixture was stirred at 0 ° C. for 10 minutes, warmed to room temperature and stirred for 90 minutes. Hexane (600 mL) was added, the mixture was evaporated and the residue was dissolved in diethyl ether (150 mL) and 5% w / v K ₂ CO ₃ (200 mL). The aqueous layer was washed with diethyl ether (2 x 80 mL), acidified to pH 2 with concentrated HCl and extracted with ethyl acetate (3 x 100 mL). The ethyl acetate fractions were dried over MgS0 ₄ , filtered and evaporated to give product 239 (1.48 g).

Example  1-70: 2S-N- Fmoc -L-3-O- (t- Butyldimethylsilyl ) -Manufacture of Asparagine

Reaction 1

To a suspension of commercial aspartic acid ester 246 (2.75 g) in 70% perchloric acid (HClO ₄ , 3 mL) was added t-butyl acetate ester (100 mL). After 24 hours, the solution was poured into saturated K ₂ CO ₃ (200 mL). The resulting dibasic mixture is extracted with diethyl ether (3 × 100 mL) and the combined organic extracts are washed with saturated K ₂ CO ₃ (3 × 50 mL), dried over Na ₂ SO ₄ , filtered and depressurized Concentrated under to give a clear colorless oil. The oil was then dissolved in cold (0 mL) diethyl ether (50 mL) and 1 N HCl in diethyl ether (15 mL) was added. After 20 minutes, the solution was concentrated under reduced pressure to give product 247 (2.0 g).

Reaction 2

To a suspension of diester 247 (4.78 g) in methyl t-butyl ether (100 mL) was added saturated aqueous K ₂ CO ₃ (100 mL). The resulting aqueous phase was washed with methyl t-butyl ether (3 x 100 mL). The organic extracts were combined and washed with saturated K ₂ CO ₃ (2 × 100 mL), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting colorless oil was mixed with trityl chloride (5.57 g), CH ₃ CN (100 mL) and triethylamine (5.60 mL). After 16 h, the mixture was filtered and diluted with methyl t-butyl ether (200 mL). The resulting organic extract was washed with 1N citric acid (3 × 100 mL), saturated NaHCO ₃ (3 × 100 mL), saturated NaCl (3 × 100 mL), dried over Na ₂ SO ₄ , filtered, Concentrated under reduced pressure. Chromatography with flash silica gel (20 × 4 cm) base washed with 1:11 ethyl acetate-hexane with 1% triethylamine present gave product 248 (7.12 g).

Reaction 3

To a cooled (-78 ° C.) solution of compound 248 (2.22 g) in tetrahydrofuran (20 mL) was added 16.5 mL of 0.91 M potassium hexamethyldisilazane solution in tetrahydrofuran. After 1 hour at −78 ° C., oxodiperoxy (pyridine) (1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidinone) molybdenum (IV) (MoOPD, 3.68 g , Strem Chemicals, Inc .; see Anderson, J.C. and Smith, SC, 1990, Synlett 107-109 for the synthesis of the formulations. The mixture was stirred at -78 ° C for 1 hour, warmed to -55 ° C and stirred for an additional 1 hour. The mixture was quenched with saturated aqueous Na ₂ SO ₃ (20 mL) and warmed to room temperature. The resulting biphasic mixture was washed with methyl t-butyl ether (3 × 100 mL), the organic layers combined, 1 N citric acid (3 × 50 mL), saturated aqueous NaHCO ₃ (3 × 50 mL) and saturated aqueous NaCl ( 3 x 50 mL), dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. Chromatography on flash silica gel (20 × 3 cm) with 1: 5 ethyl acetate-hexane gave product 249 (1.64 g).

Reaction 4

Lithium hydroxide (507 mg) was added to a solution of compound 249 (650 mg) in 1: 1 dioxane-water (50 mL). After 1 h, the solution was washed with methyl t-butyl ether (3 x 50 mL) and the resulting aqueous phase was acidified to pH about 4 with 1 N citric acid. The resulting solution was extracted with methyl t-butyl ether (3 × 100 mL). The organic phase was washed with 1N citric acid (3 × 100 mL) and saturated NaCl (3 × 100 mL), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound 250 (630 mg).

Reaction 5

Solution of compound 250 (650 mg), benzotriazol-1-yloxy-tris (dimethylamino) -phosphonium hexafluorophosphate (997 mg) and NH ₄ Cl (153 mg) in dimethylformamide (30 mL). To this was added diisopropylethylamine (0.78 mL). After 1 h, ethyl acetate (150 mL) was added and the resulting solution was washed with 10% K ₂ CO ₃ (3 × 100 mL), water (3 × 100 mL), 1N citric acid (3 × 100 mL) and saturated Washed with NaCl, dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give compound 251 (640 mg).

Reaction 6

To a solution of compound 251 (1.91 g) in dichloromethane (5 mL) was added water (1 mL) followed by trifluoroacetic acid (10 mL). After 4 hours, the solution was concentrated under reduced pressure and the resulting slurry was concentrated twice from toluene. The resulting solid was triturated with diethyl ether and the resulting solid was filtered off and washed with diethyl ether. The resulting solid was dried under reduced pressure to give compound 252 (952 mg).

Reaction 7

To the solution was added a solution of 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide (3.37 g) in 1,4-dioxane (50 mL). After 16 h, the resulting solution was diluted with 5% aqueous K ₂ CO ₃ solution (25 mL) and extracted with diethyl ether (3 × 50 mL). The resulting aqueous extract was acidified to pH about 2 with 1 N HCl solution and diethyl ether (50 mL) was added. The resulting solid was partitioned between acidic solution and diethyl ether. The solid was collected and washed with 1N HCl and diethyl ether to give compound 253 (1.50 g).

Reaction 8

To a solution of compound 253 (370 mg) in dimethylformamide (10 mL) was added t-butyldimethylsilyl chloride (300 mg) followed by imidazole (200 mg). After 8 h the solution was diluted with ethyl acetate and washed with 1N HCl (3 × 100 mL) and saturated sodium chloride, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Compound 245 (300 mg) was obtained by chromatography on flash silica gel (25 × 2 cm) using 19: 1: 0.1 CH ₂ Cl ₂ : MeOH: AcOH as eluent.

Example  1-71: 2S-N- Fmoc -L- (3- Methoxy ) -β- tert -Butyl Aspartic acid  Preparation of Ester 254

Scheme 1

To a suspension of diester 255 (4.78 g) in commercially available methyl tert-butyl ester (100 mL) was added saturated aqueous K ₂ CO ₃ (100 mL). The final aqueous layer was washed with methyl tert-butyl ether (3 × 100 mL). The organic extracts were combined and washed with saturated K ₂ CO ₃ (2 × 100 mL), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The final colorless oil was dissolved in a solution of triethylamine (5.60 mL), and trityl chloride (5.57 g) in acetonitrile (100 mL). After 16 h, the mixture was filtered and diluted with methyl tert-bunyl ether (200 mL). The final organic extract was washed with 1 N citric acid (3 × 100 mL), saturated NaHCO ₃ (3 × 100 mL), and saturated NaCl (3 × 100 mL), Na ₂ SO ₄ Dried over, filtered and concentrated under reduced pressure. Chromatography was performed on flash silica gel (20 × 4 cm) washed with base using ethyl acetate: hexane 1:11 in the presence of 1% triethylamine to give 256 (7.12 g).

Scheme 2

16.5 mL of 0.91 M potassium hexamethyldisilazane solution in tetrahydrofuran was added to 256 (2.22 g) cold solution (-78 ° C.) in tetrahydrofuran (20 mL). After 1 hour at −78 ° C., oxodiperoxy (pyridine) (1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidinone) molybdenum (IV) (STREM Chemicals, Inc.) 3.68 g, the synthesis of this reagent (see Anderson, JC. And Smith, SC., 1990, Synlett 107-109), added in portions. The mixture was stirred at −78 ° C. for 1 h, warmed to −55 ° C. and stirred for an additional hour. The mixture was quenched with saturated aqueous Na ₂ SO ₃ (20 mL) and allowed to come to room temperature. The final biphasic mixture was washed with methyl tert-butyl ether (3 × 100 mL). The organic layer was combined and washed with 1N citric acid (3 × 50 mL), saturated aqueous NaHCO ₃ (3 × 50 mL), saturated aqueous NaCl (3 × 50 mL), Na ₂ SO ₄ Dried over filtration, and concentrated under reduced pressure. Chromatography was carried out on flash silica gel (20 × 3 cm) using ethyl acetate-hexane 1: 5 to give 257 (1.64 g).

Scheme 3

To a solution of 257 (4.61 g) in dichloromethane (100 mL) was added trifluoroacetic acid (2 mL). After 1 h, the final solution was concentrated under reduced pressure, aqueous 5% K ₂ CO ₃ (50 mL) was added and benzyloxycarbonyl-N-hydroxysuccinimide (2.49 g) in dioxane (50 mL). The solution was added. After 16 hours, the final solution was extracted with ethyl acetate (3 × 100 mL). The organic extract was washed with 1N citric acid (3 × 50 mL), saturated NaHCO ₃ (3 × 50 mL) and NaCl (3 × 50 mL), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Chromatography was performed on flash silica gel (25 × 3 cm) using ethyl acetate-hexane 1: 3 to give 258 (2.01 g).

Scheme 4

Iodine methane (0.62 mL) was added to a cold suspension (0 ° C.) of 258 (353 mg) and silver oxide (462 mg) in tetrahydrofuran (25 mL). The mixture was allowed to warm to room temperature over 4 hours. After 48 hours, the suspension was filtered through Celite and concentrated under reduced pressure. Chromatography was performed on flash silica gel (25 × 2 cm) using ethyl acetate-hexane 1: 3 to give 259 (300 mg).

Scheme 5

To a cooled (0 ° C.) solution containing 259 (300 mg, 0.81 mmol) in 25 mL of dioxane was added 240 mg (10 mmol) of LiOH in 25 mL water. After 1 h, the mixture was acidified to 1-4 with 1 N citric acid and extracted with ether (3 x 50 mL). The organic extract was washed with 1N citric acid (3 × 50 mL) and saturated NaCl (3 × 30 mL), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to afford 260 (280 mg) as a clear, colorless oil. Got it.

Scheme 6

Under the hydrogen atmosphere described above for converting 242 to 243, a 260 ethyl acetate solution was treated with 10% palladium on carbon and the amine protection reaction was carried out as described above for converting 252 to 253. 260 was converted to 254.

Example  1-72: Nα- ( Allylcarbonyl ) -L- Isoleucine  124

Scheme 1

Commercial isoleucine (22 g) was added to a solution of allyloxycarbonyl oxysuccinimide (AllocOSu, 51 g) in tetrahydrofuran (150 mL). To the suspension was added 10% K ₂ CO ₃ aqueous solution (100 mL) and the mixture was stirred for 17 h and then concentrated under reduced pressure to approximately 120 mL. The solution was added to 10% K ₂ CO ₃ aqueous solution (100 mL) and water (200 mL) and washed with diethyl ether (4 × 150 mL). The aqueous portion was then acidified to pH 1 and extracted with dichloromethane (4 x 200 mL). The combined acidic dichloromethane washes were dried over anhydrous MgSO ₄ and evaporated to the crude product (38.1 g). Purification via column chromatography on silica (eluted using a dichloromethane / methanol concentration gradient with a dichloromethane: methanol ratio of 9: 1 in 100% dichloromethane) and evaporated the solvent to give 124 (36 g) of a yellow oil. )

Example  2-1: for the creation of new biosynthetic pathways s . Roseosporus - base Construction of In-Trans Expression System

For expression of the hybrid nonribosomal polypeptide synthetase (NRPS) pathway, S. all lacking the NPRS gene . Dapto azithromycin of Rosedale agarose porous-high producing strain variants (NPRS 11379) was constructed. S at ΦC31 attB site . Rosedale agarose portion to a neutral region of the porous genome was specifically bonded to a hybrid route on the BAC- based vector that is integrated into the inside of the strain.

Es. In order to fruition the NRPS genes are present in all Rosedale porous agarose, it was constructed the deletion cassette containing DNA in contact with the side downstream of the upstream and dptH of dptEF (Fig. 2).

Regions flanking upstream of dptEF (5 ') and downstream of dptH (3') were cloned into a selection cassette containing tsr ( thiostrepton resistance) and cat (chloramphenicol resistance). The 5 'fragment is 1478 bp long and the 3' fragment is 1862 bp long. These two fragments were cloned into copies of pUC19 (New England Biolabs) already containing tsr and cat resistant cassettes to generate the deletion cassette. The cassette was then delivered with a temperature sensitive replication origin and dominant allele of rpsL (streptomycin sensitization) and called pRHB538 (Hosted, TJ and Baltz, RH, 1997, J Bacteriol . 179 (1): 180-6). Transfer to plasmid. Es the plasmid held by the rpsL recessive allele that gives the streptomycin resistance. It was introduced into Roseosporus strains. The recombinant strains were then incubated overnight in liquid broth and the cells were applied to plates containing streptomycin and thiostrepton and incubated at 39 ° C. Under these conditions, only those strains whose dptA- H locus was exchanged for deletion cassettes (containing tsr and cat ) via homologous recombination survived this screening method; Other genotypes have been eliminated.

PCR and Southern blot confirmed the genotype of the dptA- H deletion mutants. PCR fragments were designed to be amplified by primers located outside the 5 'and 3' flanking regions and inside the tsr and cat genes. In this way, PCR products can only be formed when cells exchange dptA- H loci with deletion cassettes. Southern blots showed that dptA-H was deleted, further confirming that abnormal integration or recombination did not occur around the locus. Genetic identification of dptA- H deletions was then performed to confirm that they are phenotypically true null mutants.

Example  2-2: Streptomyces Roseosporus  Fermentation culture

Streptomyces Spores of Roseosporus UA 431 were collected on 10 days of medium A (2% crude oat (Quaker), 0.7% tryptone (Difco), 0.2% soy peptone (Sigma), 0.5% sodium chloride (BDH), 0.1% trace). A slant culture of salt solution, 1.8% agar no. 2 (Lab M), 0.01% apramycin (Sigma) was recovered by suspending in 5 ml of 10% aqueous glycerol (BDH). 1 ml of the suspension, present in a 1.5 ml frozen vial, recovered after storage at −135 ° C., contains the starting material. 0.3 ml of the starting material was aseptically placed on the slope of the medium A and incubated at 28 ° C. for 9 days to generate a preculture.

The preculture was aseptically treated with 4 ml of 0.1% Tween 80 (Sigma) solution to gently immerse the slope to produce a suspension of trophic mycelium and spores. Aliquots of 2 ml of the suspension were added to 40 ml of nutrient solution S (1% D-glucose (BDH), 1.5% glycerol (BDH), 1.5% soy peptone (Sigma), 0.3% sodium chloride (BDH), 0.5 Sterilized by% Malt Extract (Oxoid), 0.5% Yeast Extract (Lab M), 0.1% Junlon PW100 (Honeywell and Stein Ltd.), 0.1% Tween 80 (Sigma), 4.6% MOPS (Sigma), pH 7.0 and autoclaved ) Into a 250 ml baffled flask and shake incubated at 30 rpm for 30 hours at 240 rpm.

50 ml medium P (1% glucose (BDH), 2% soluble starch (Sigma), 0.5% yeast extract (Difco), 0.5% casein (Sigma), 4.6% MOPS (Sigma), pH 7 and autoclaved 5% of the species culture was aseptically transferred to a 250 ml baffle flask containing and shaken at 240 ° C. for up to 7 days to produce a production culture.

Example  2-3: Streptomyces Roseosporus  A21978C obtained by fermentation culture Lipopeptide  analysis

Prior to analysis, 2 ml of the total embryos of the production culture described in Example 2-2 were aseptically taken and centrifuged for 10 minutes to prepare samples for analysis. Streptomyces In order to monitor the production of natural lipoic peptide (A21978C) produced by Rosedale porous agarose and analyzed up to 50 microliter volumes of the supernatant. This assay was performed at ambient temperature using a Waters Alliance 2690 HPLC system equipped with a 4.6 × 50 mm Symmetry C8 3.5 μm column and a Phenomenex Security Guard C8 cartridge and a 996 PDA detector. The concentration gradient was initially maintained with 90% water and 10% acetonitrile for 2.5 minutes, followed by a linear concentration gradient up to 100% acetonitrile over 6 minutes. The flow rate was 1.5 ml per minute and the concentration gradient was buffered with 0.01% trifluoroacetic acid. Up to 2 days of fermentation culture, the UV / visible spectrum is the same as daptomycin under the conditions mentioned above, and the retention times are HPLC at retention times of 5.62 minutes, 5.77 minutes and 5.90 minutes (? Max 223,8, 261.5 and 364.5 nm). It was confirmed that three kinds of natural lipopeptides, A21978C ₁ , A21978C _2, and A21978C _3, which produced peaks, were produced. The lipopeptides were clearly present during the fermentation culturing process at each sample extraction time point for the next 7 day period. The total yield of lipeptides A21978C ₁ , A21978C ₂ , and A21978C ₃ ranged from 10-20 mg per liter of fermentation material.

Liquid phase chromatography-mass spectrometry (LC-MS) was performed on a Finnigan SSQ710c LC-MS system using cationic mode electrospray ionization with a scan range of 200-2000 Daltons and a 2 second scan. Chromatographic separation was performed using a Water Symmetry C8 column (2.1 × 50 mm, 3.5 μm particle size), eluted using a linear water-acetonitrile concentration gradient containing 0.01% formic acid, specifically, After an initial 0.5 minute delay, 10% to 100% acetonitrile was increased over a 6 minute period, and then eluted by holding in 100% acetonitrile for an additional 3.5 minutes before rebalancing. The flow rate was 0.35 ml / min and the method was carried out at ambient temperature.

As indicated by molecular ions ([M + H] ⁺ ) measured at m / z of 1634.7, 1648,7 and 166.2, it was confirmed that there were three natural lipopeptides in the control ( S. roseosporus wild type) . The molecular ion measured is Streptomyces It is consistent with the weight reported for each of the major peptide factor A21978C lipoic A21978C1, A21978C2 and A21978C3 produced in Rosedale porous agarose (lit. [Debono, etc., 1987, J. Antibiotics 40: 761-777 reference). The UA 431 mutant did not produce all A21978C lipopeptides, confirming that the mutant was a true deletion mutant.

Example  2-4: pDA300 Building and s . Roseosporus dptA Complementing the -H Deletion

Unlike yeast and some natural competent bacteria, E. coli cannot be easily transformed with linear DNA fragments. This is due to the degradation of foreign DNA by intracellular exonucleases such as RecBCD (Lorenz, MG, and Wackernagel, W., 1994, Microbiol . Rev. 58: 563). Traditionally, homologous recombination utilizes mutant strains that lack RecBCD (Jasin, M., and Schimmel, P., 1984, J. Bacteriol 159: 783) or helper plasmids that cannot replicate in hosts under limited conditions. With the help of the vector, this could be accomplished by transporting DNA. Recombination events are rare and require several kilobases with homology.

Recently, several laboratories have developed strains with the advantages of bacteriophage λ-induced "hyper recombination" status (Datsenko, KA, and Wanner, BL, 2000, Proc. Nat . Acad. Sci. USA 97: 6640; US Pat. Nos. 6,355,412 and 6,509,156B; Yu, D., et al. , 2000, Proc . Nat. Acad . Sci . USA 97: 5978). DNA intermolecular recombination of sequences with as little as 40 to 50 bp also occurs when using linear DNA. λ Red genes ( exo, bet and ga m ) increase the rate of recombination. λ-exonuclease and β-protein are responsible for recombination through repair of double helix breaks, while the gam gene product binds to and inhibits the function of the host's RecBCD complex (Murphy, K., 1998, J. Bacteriol . 180: 2063). In the present invention, this technique is referred to as the "Red" system or Red-mediated recombination system.

PDA300 (a truncated form of B12: 03A05 containing only the dapA- H gene) was constructed using the "Red" system. The plasmid is B12: were constructed from the 03A05, which in BAC plasmid containing all dpt biosynthetic gene cluster, S. Will disconnect from the chromosomal libraries of Rosedale porous agarose (Miao, etc., 2005, Microbiology 151: 1507-1523) , all gene present downstream of the gene, and all dptA -H present upstream of dptA-H are Red- mediated recombination It was deleted using homologous recombination through the system. This was done by introducing B12: 03A05 into E. coli carrying the Red gene on plasmids (pKD78, Datsenko, KA., And Wanner, BL., 2000, Proc . Nat. Acad . Sci . USA 97: 6640). The strain was then transformed with a PCR product for the tet resistance gene flanked by oligonucleotides homologous to the upstream or downstream region of the dpt gene group. After constructing pDA300, it was introduced into UA431 via conjugation to generate strain UA493. Plasmids from E. coli, S. pDA300 is. Comprises: (76-83 (1998)}, see, it includes them by reference to the present invention Baltz, 1998, Trends Microbiol 6 in.) Of porous agarose Rosedale plasmid RK2 origin for bonding to oriT. Coli the plasmid pDA 300 (E. coli) S17.1, or self-S through the junction from a strain containing a clone plasmid RK2 (same as above). Introduced to Roseosporus . Es. Rosedale agarose porous UA493 the embodiment 2-2, and performed using each method described in Example 2-3 was cultured fermentation and analysis.

As indicated by the molecular ions ([M + H] ⁺ ) measured at m / z of 1634.7, 1648,7 and 166.2, it was identified as three natural lipopeptides in the control ( S. roseosporus wild type). Streptomyces Is consistent with the weight reported for each of the major peptide factor A21978C lipoic A21978C1, A21978C2 and A21978C3 produced in Rosedale porous agarose (literature: see [Debono, etc., 1987, J. Antibiotics 40 761-777] ). This demonstrates that pDA300 can successfully compensate for dptA- H deletion and restores lipopeptides production in UA493.

Example  2-5: promoting the introduction of different amino acid (s) and non- Ribosome  Peptide synthase ( NRPS Subunit of  exchange

The gene encoding the third subunit of daptomycin NRPS (see FIG. 1) is for the introduction of amino acids 12 (3-methyl-glutamic acid (3-MeGlu)) and 13 (L-kynurenine (L-Kyn)). It includes two modules that encode specificity. Such genes include cyclic lipopeptide CDA (Kempter et al., 1997, Angew. Chem. Int. Ed. Engl. 36: 498-501; Chong et al., 1998, Microbiology 144: 193-199; each of these in its entirety The third subunit for biosynthesis of the reference) and also the last two amino acids, in this case amino acids 10 (3-MeGlu) and 11 (L-Tryptophan (L-Trp); FIG. 1). Derivatives of daptomycin containing L-Trp instead of L-Kyn at position 13 were generated by deleting the gene dptD and replacing it with a gene encoding PS3 instead of CDA (Hojati et al . , 2002, Chem Biol. 9 (11): 1175-87). Vector pMF23 expresses the PS3 gene from a strong promoter (e.g., the ermEp * promoter; Baltz, 1998, Trends Microbiol . 6: 76-83, incorporated herein by reference in its entirety), and interspecies conjugation) (Baltz, 1998, Trends Microbiol 6: Dar through 76-83). By introducing a porous agarose Rosedale, S. Rosedale agarose porous genome within the neutral region sites on-when the specific insert, cdaPS3 is to compensate for dptD mutant L-Kyn this replacement strain A. Toma, who is the L-Trp is generated and the compound C1, compound C2, and the compound C3 You get Recombinant strains were fermented and cultured as described in Examples 2-2 and Example 2-3 and the products of the recombinant strains were analyzed by LC-MS. Similar experiments were performed, where the dptD deletion was compensated for by the gene encoding the third subunit for the biosynthesis of cyclic lipopeptides A54145 (pMF30 was expressed from ermEp * (Motamedi et al., 1995, Gene 160: 25-31). A derivative of pHM11a containing lptD , which also encodes the last two amino acids, in this case amino acids 12 (3-MeGlu) and 13 (L-isoleucine (L-Ile) or L-valine (L-Val)). daptomycin containing L-Ile or L-Val instead of L-Kyn at position 13, destroying dptD and replacing it with a gene encoding lptD for A54145 (compounds C4, C5, C6, C7, C8, C9) 2 derivatives of were produced.

Similar manipulations were made for the trans-compensation of other subunits, ie, destroying or deleting subunits of the daptomycin biosynthetic gene group or A54145 biosynthetic gene group, and then at the trans position with one or more natural or modified subunits from NRPS. Compensation (the latter may include trans-compensation with daptomycin or variants of the A54145 biosynthetic genogroup subunit). Trans-compensation between NRPS subunits yields new nonribosomal peptides that can be analyzed as described in the above examples.

To perform trans-compensation experiments using the daptomycin or A54145 biosynthetic gene group and part of the calcium dependent antibiotic (CDA) biosynthetic gene group, a daptomycin biosynthetic gene set, or daptomycin biosynthetic genes and auxiliary genes such as BAC clones The gene set contained in B12: 03A05 is introduced into other natural or genetically modified Actinomyces species or strains through transformation or conjugation. Such recipients may be known producers or uncharacterized strains that produce secondary metabolites, or may be produced using recombinant techniques to retain biosynthetic pathways other than those for biosynthesis of daptomycin. Transformants or conjugates are fermented and cultured in various media and their entire liquid medium or extract is used to screen for novel daptomycin-like compounds or biological activity against daptomycin-resistant test organisms.

Often this compensation can be facilitated by inactivating a portion of the subunit gene of daptomycin or the A54145 biosynthetic pathway (as described above for the deletion of dptD and compensation by cdaPS3 or lptD ). Sequences encoding the subunits of NRPS can be deleted or replaced with marker genes to form modified NRPS biosynthetic pathways; This original strain produced (dapto azithromycin is S. Rosedale porous agarose, A54145 is S. Dia plastic or S. Le pyuyi Neuss, CDA is S. Ko elementary color) or may be carried out in the plasmid held by these biosynthetic pathways.

To produce novel lipopeptides, the homologous recombination across the flanking DNA sequence was used to exchange the giant of the coding region of dptD in pDA300 with a heterologous marker gene. In order to perform homologous recombination, two oligonucleotides were designed to amplify the regions immediately upstream ("5 'fragment") and downstream ("3'fragment") of dptD . The 5 'and 3' fragments using the primer set of the S to having a restriction site of specific introduction 5'-end extension portion (underlined). It was amplified from chromosomal DNA of Roseosporus .

5 'fragment (1122 bp)

5 'GCG AAG CTT CTG GTG GCG CAT CAC CTG G 3 '(SEQ ID NO: 1)

5 'GC T CTA G AT GGA AGT ATG TCC TCC ATC GC 3 '(SEQ ID NO: 2)

3 'fragment (1535 bp)

5 'C GG ATC C CG CCG GCA CCT GAC CC 3' (SEQ ID NO: 3)

5 'CC G AAT TC C GCC TCC GAG TAC ATC GAG G 3 '(SEQ ID NO: 4)

Amplified fragments were cloned to follow corresponding unique sites in the multiple cloning site of pNEB193 (New England Biolabs). The final construct, pSD002, was directed by restriction enzyme digestion, and sequencing confirmed that there were no errors in PCR generated portions. SpeI fragments containing the marker gene, ermE (erythromycin resistance gene; Hopwood reference, homology) were inserted into the XbaI site of pSD002 and verified by restriction enzyme digestion. Thus, the final plasmid, pSD005 contains a cassette consisting of a DNA strand ermE the side in contact with the DNA sequence and the homology of the upstream and downstream portions of the dptD. When inserted into the daptomycin biosynthetic gene family pathway by homologous recombination, the cassette essentially replaces all parts of dptD with ermE except for the first bp 31 bp and the last posterior 12 bp. This region containing the replaced cassette was then transferred to the vector (cloning-site morphotype of pRHB538; Hosted and Baltz, 1997, J. Bacteriol. 179) having a temperature sensitive replication origin and rpsL (a gene conferring sensitivity to streptomycin) . : S by subcloning the 180 to 186). The final plasmid, pSD300, is generated in a series of plasmids for introduction into Roseosporus.

The plasmid, pSD300, via S- conjugation . It was introduced into Roseosporus (Balz 1998, Trends Microbiol ., 6: 76-83). Next, 1 ml of water containing 1.2 mg of erythromycin was introduced into each plate, and the final concentration was 50 µg / ml when the liquid was absorbed into the medium. Seven days later, the erythromycin-tolerant colony generated on the transfection plate was inoculated in 25 ml of TSB (Hopwood, homology) to which erythromycin was added and incubated at 30 ° C. for 48 hours. The mycelium was recovered, the mycelium mass was immersed and transferred to a fresh aliquot of 25 ml TSB containing erythromycin. The final solution was then incubated at 40 ° C. and selected via a temperature sensitive replica of pSD030. After 48 hours, the mycelium was recovered by centrifugation, immersed and resuspended in a final volume of 2 ml TSB. The suspension (100 μl) was applied to SPMR plates (Babcock et al., 1988, J. Bacteriol . 170: 2802-2808) containing 50 μg / ml erythromycin and 30 μg / ml streptomycin. Surviving colonies were screened, S, for example the strain. In order to confirm whether dptD was successfully replaced by ermE in Roseosporus UA378 , it was confirmed that the PCR had the correct genotype. The mutant was then compensated for in-trans status via the initial dptD , which dptD was expressed under the control of the constitutive promoter ermEp * and expressed plasmid pHM11a (Motamedi et al., 1995, Gene 160 (1): 25-31 ) Is expressed from.

Starting materials for UA378 are described in Medium A (see "Practical Streptomyces Genetics" by Kiers, et al., John Innes Foundation, Norwich, 2000, where "Kieser"; 2% irradiated oats in 5 ml of 10% aqueous glycerol (BDH), 0.7% Tryptone (Difco), 0.2% Soy Peptone (Sigma), 0.5% Sodium Chloride (BDH), 0.1% Trace of Salt Solution, 1.8% Agar No. 2 (Lab M), 0.01% Apramycin (Sigma) 10 day old gradient cultures) were suspended and regenerated. 1.5 ml frozen vials containing 1 ml of starting material, stored at −135 ° C., were recovered and thawed rapidly. 0.3 ml of the starting material was aseptically placed on the slope of medium A and incubated at 28 ° C. for 9 days to produce preculture. The material for inoculation of the species culture was made by aseptically treating the preculture with 4 ml of 0.1% Tween 80 (Sigma) solution and gently immersing the slope to produce nutrient mycelium and spore suspension.

Species cultures were produced by aseptically inoculating 1 ml of the inoculum into a 2 L baffle Elenmeyer flask containing 250 ml of nutrient solution S (see Keiser, homologous) and shaking culture at 30 at 240 rpm for 30 days.

Production cultures were produced by aseptic inoculation of the seed culture into a 20 L fermentor containing 14 liters of nutrient solution P (Kieser, homologous). The production fermenter was stirred at 350 rpm, air was injected at 0.5 vvm, and the temperature was controlled to 30 ° C. After 20 hours of incubation, 50% (v / v) glucose solution was fed to the culture at 5 g / h throughout the fermentation culture.

After 40 hours of incubation, a 50:50 (w / w) mixture of decanoic acid: methyl oleate (Sigma and Acros Organics, respectively) was fed to the fermentor at 0.5 g / hr for the rest of the fermentation culture. Cultures were recovered after 112 hours, and biomass was removed from the culture supernatant by a batch treatment process via bowl centrifugation.

Biomass was removed from the 20 L fermentation culture and the clear liquid was applied to an open glass column (60 × 300 mm) filled with Mitsubishi HP ₂ 0 resin and adjusted with methanol and water. Before eluting, the column was washed with 2 L of water and then with 2 L of methanol / water (1: 4). The column was then eluted with 1 L methanol / water (4: 1) and again with 1 L methanol and two separate fractions were recovered.

Liquid Chromatography-Mass Spectrometry (LC-MS) Electrospray Ionization (IESI) analysis revealed that both fractions contained A21978C / CDA hybrid molecules and were subsequently treated with the less complex methanol / water (4; 1) fraction. . This was vacuum evaporated to an aqueous residue followed by up to 500 mL of water. Next, 500 mL × 3 times back extraction with ethyl acetate was carried out in the 2 L separatory funnel, and the aqueous and organic fractions were obtained. According to LC-MS (ESI) the hybrid molecule was not present in the organic phase and thus discarded. The aqueous fractions were lyophilized overnight under reduced pressure.

Hybrids were purified by preparative high performance liquid chromatography (HPLC) using a Waters Prep LC system and a Waters 40 × 200 mm Nova-Pak C18 60 × 6 μm radial-compression dual cartridge with 40 × 100 mm guard. The freeze-dried material was dissolved in water and purified using a concentration gradient. The method was maintained for 2 minutes in 90% water and 10% acetonitrile followed by a linear concentration gradient over 13 minutes until 25% water and 75% acetonitrile. The flow rate was 55 ml / min and the total concentration gradient was buffered with 0.04% trifluoroacetic acid. Fractions were recovered and analyzed by LC-MS on a Finnigan SSQ710c LC-MS system using cationic mode electrospray ionization (ESI), with a scan range of 200-2000 Daltons and a 2 second scan. Chromatographic separation for the LC-MS analysis was performed on a Waters Symmetry C8 column (4.6 × 50 mm, 3.5 μm particle size), using a linear water-acetonitrile concentration gradient containing 0.01% formic acid, 0.5 min Acetonitrile was increased from 10% to 100% over a period of 6 minutes after the initial delay, followed by eluting with 100% acetonitrile for an additional 3.5 minutes before rebalancing. The flow rate was 1.5 ml / min and the method was carried out at ambient temperature.

The assay identified Compound C1 and Compound C2. Both fractions required subsequent purification before NMR testing. Compound C1 was further purified using an isocratic method using 60% water and 40% acetonitrile buffered with 0.04% trifluoroacetic acid. Approximately 1.8 mg of material was isolated. Final purification of Compound C2 was performed using an isocratic method of 58% water and 42% acetonitrile buffered with 0.04% trifluoroacetic acid. Approximately 1.5 mg of material was isolated. UV maxima and ESI0MS molecular ion information (double charged ions observed in anion mode) for Compounds C1 and C2 are shown in the table below.

Compound c1 Compound c2 ESI-MS (m / z) 814 (M-2H) ^2- 821 (M-2H) ^2- UV-vis? Max / nm 221, 280 221, 280

Example  2-6: dptBC Modules built at positions 8 and 11

Plasmids bearing dptBC pKN24 were constructed through cleavage of B12: 03A05 with the daptomycin biosynthesis ( dpt ) gene family. Target dpt Red-mediated recombination systems were used to introduce linear PCR products of antibiotic resistance genes flanked by 45 bp sequences having homology to the upstream or downstream regions of the gene (as described in Examples 2-4). The upstream (5 ') region of dptBC (pKN24-26) or dptD (pKN27) was deleted by a spectinomycin resistance gene ( spec ) and a spec- ermEp * cassette containing the potent , always expressed ermEp * . This fragment was amplified using primers Sp6Del-1-2 and dptBC-ermEp. The downstream (3 ′) region of dptBC (pKN24) was deleted with the beta-lactamase gene ( amp , derived from pRB322). The fragment was amplified using primers GTC del2 and dptD-3 ':: amp. The selection cassette for deletion of the CAT module under investigation was amplified using PCR primers having 50 bp homology to the linker region of the module (see linker position in FIG. 5; see WO 01/30985). These PCR fragments were digested with pKN24 (a truncated form of pDA300 containing only dptBC , expressed from the constitutive promoter ermEp * ; Bibb, MJ. Et al., 1985, Gene, 38 (1-3): 215-26) and the induction Red system. Introduced into the containing electro-competent cells and the resistance cart was site-specifically integrated into the target site of pDA300 via homologous recombination (FIG. 3).

5 'fruition, 3' fruition

Sp6Del-l-2

5'-GCCAGCATGGAGCCGAACTGCCGGAACACCGCGTCCCGGTCCACC TGTGTAGG

CTGGAGCTGCTTC -3 '(SEQ ID NO: 5)

GTC del2

5'-GCCGACTGGGAGTGGGTCAAGTGGCTGCCGCACGTGCTGGATCCG CATATGAATA

TCCTCCTTA -3 '(SEQ ID NO: 6)

dptBC-ermEp

5'-CCGAGACAGGCAGGATCTCCTCGACTACCTTCGACCGGCGGTT CATATG TCC

GCCTCCTTTGGTCAC -3 '. (SEQ ID NO: 7)

DptD-3 ':: am

5'-CATACTTCCTCTCACTCCGCTGCAGGAGGGACTGCTGTTCCACAG TGTGTAGGCTG

GAGCTGCTTC -3 '(SEQ ID NO: 8)

These cells were then selected for the presence of tet resistance markers, and the final colonies were genetically analyzed to verify that proper deletion or destruction was established. Part of the primer design design included restriction enzyme sites located within the linker region of the subject (FIG. 3). Once the deletion BAC was verified, the selection cassette was incised using a unique restriction enzyme site introduced into the linker region (FIG. 3 Avr II and Pme I ).

Replacement modules (serine; alanine) were subcloned again in pBR322 (Yanisch-Perron et al., 1985, Gene 33 (1): 103-19; flanking the appropriate site) using Red-mediated recombination. This technique is called a gap-filling scheme, in which a primer contains 50 bp that overlaps the inner region of the linker of the preferred module (Lee, EC et al., 2001, Genomics 73:56). The primers were used to amplify a portion of pBR322 including the origin of replication and amp ^r to produce a linear fragment flanking the homology region within the preferred module (serine; alanine). Was introduced into competent E. coli - DH10B electro containing: (6640.. Datsenko, KA , and Wanner, BL, 2000, Proc .Nat Acad Sci .USA 97..) Of these (see above) and the PCR fragment pKN24 pKD78 . Recombination through the two homology regions causes the module to move from the original vector to pBR322, converting it into a circular form that can be replicated and screened (Figure 3). It is preferred that the original vector to which the module was cloned has an F-plasmid replication origin (as opposed to a replication origin with a high copy number).

The cloned module was excised from pBR322 and linked to the deletion form of pKN24 using a compatibility restriction enzyme site introduced around the deletion. This produced 2 plasmids: 1) pDR2155 in which D-serine-11 of daptomycin was replaced with D-alanine through module exchange and 2) pDR2160 in which daptomycin replaced D-alanine-8 by D-serine. to be. Both pDR2155 and pDR2160 were confirmed by PCR and sequencing.

Since S to the plasmid. An appropriate expression host in Rosedale porous agarose was built. The dptB- D mutant KN100 containing the chromosomal deletion from which dptBC , d was removed was constructed using the technique described in Example 2-1. Both pKN24 and pRB04 (plasmids constructed in vector pHM11a expressing the dptD subunit under the control of the ermE * constitutive promoter) were introduced by cross-conjugation with the KN100 strain to generate KN101. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strain was found to produce natural lipopeptides A21978C ₁ , A21978C ₂ , and A21978C ₃ . Es. Rosedale agarose porous when verifying the strain KN100, it had been generated the secondary derivatives, KN156 (KN100 holds the pRB04). This strain was then used as a host for all module exchanges performed in dpBC . PB103 was constructed by introducing pDR2155 into KN156. Fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3 showed that this strain produced compounds C46, C47 and C48 (FIG. 4).

PDR2160 was introduced into KN156 to construct strain PB118. Fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3 showed that the strain produced compounds C22, C23 and C24 (FIG. 4).

The recombinant strains described above were fermented and analyzed under the conditions described in Example 2-2 and analyzed using the method described in Example 2-3 (results are summarized in Table VI).

Derivatives having Asn at position 8 were prepared via module exchange using fusion sites TC (B) and TE (CAT). Using a red-mediated recombination system, modules 8 or 11 of pKN24 were genetically engineered with the gentamicin resistance gene ( ahp2 ) flanking the Avr II and Pme I restriction enzyme sites (Chow JW, Kak V, You I, Kao SJ, Petrin J, Clewell DB, Lerner SA, Miller GH, Shaw KJ. 2001, Antimicrob . Agents Chemother . 45, 2691-2694). Since the DNA sequences of modules 8 and 11 are very homologous, the same primer pair was used to delete both modules in linker B and CAT.

DNA fragments encoding Asn modules (B-CAT), ie the 11th module from A54145 NRPS, were cloned by gap-repair. A linear fragment flanked with 45 bp introduced homology with Nhe I and Hpa I restriction enzyme sites and preferred module fragments by PCR amplification of a portion of pBR322 containing an amp resistance gene and a replication origin using gap-repair primers Was generated. SF1: 10D08 (A54145 is a BAC clone of the biosynthetic gene> 100 kb DNA to encode a part of the group, the clones: constructed using the protocol described in [Mial etc., 2005, Microbiology 151 1507-1523] S. PRA From the genome BAC-based library of Dia . The clone BAC-P13 was isolated from a library using the protocol of [Mial et al., 2005, Microbiology 151: 1507-1523] and tetR-pKD119 encoding the Red Recombination System. The electro-competent Escherichia coli carrying the was transformed with the linear PCR fragment. If Red-induced recombination occurs in both homology regions, the Asn module migrates from BAC-P13 to linear pBR322, resulting in a circular, replicable plasmid. Modular fragments with correct sequence (validated by sequencing) were cleaved by Nhe I and Hpa I restriction enzyme digestion and linked with appropriately deleted pKN24 forms to generate hybrid plasmids.

Module 8 deletion primer

pKN24-Mod8 :: Gen. B-CAT

8_B

TTGTTCGAGGCGCCGACGGTGAGCCGTTTGGAGCGGTTGCTGCGGGAGCG CCTAGG ACGTTGACACCATCGAATGG. (SEQ ID NO: 9)

8_CAT-Pme

ACAATCTCAGCACCCCCCACCACACCAACCGCCCCAGCGTCCGAACCAC GTTTAAAC CCTCATTCATCGGGCGAAAG (SEQ ID NO: 10)

Deletion Primer of Module 11

pKN24-Modll :: Gen. B-CAT

8_B

TTGTTCGAGGCGCCGACGGTGAGCCGTTTGGAGCGGTTGCTGCGGGAGCG CCTAGG ACGTTGACACCATCGAATGG (SEQ ID NO: 11)

8_CAT-Pme

ACAATCTCAGCACCCCCCACCACACCAACCGCCCCAGCGTCCGAACCAC GTTTAAAC CCTCATTCATCGGGCGAAAG (SEQ ID NO: 12)

gap-repair primer for lptAsn11

Lpt - Nll -B- P13

TCGGGGCGCGGGTCGGCGGGGCGCAGCCGGGGTCCGGCCTCGCCC GCTAGC TTCTTAGACGTCAGGTGGCAC (SEQ ID NO: 13)

Lpt - Nll  -CAT- Pl  4

CGCGACATCTTCGAACAGCGCACGCCCGCCGCCCTCGCCGGCCGC GTTAAC CGATACGCGAGCGAACGTGA (SEQ ID NO: 14).

Plasmids were screened by PCR and restriction enzyme digestion, and the plasmids with the correct genotype were subsequently named pKN45 (inserted D-Asn module at position 8) and pKN47 (inserted D-Asn module at position 11). These two plasmids were then conjugated to expression host KN156 ( Δ dptBCD + pRB04). Conjugates were selected on ASI plates containing apramycin (50 μg / ml) and the recombinant strains selected on the plates were then fermented in culture and assayed using the protocol described in Examples 2-2 and 2-3. It was. Analysis of the fermentation broth of KN392 by LC-MS revealed novel lipopeptides C189, C190 and C191 with molecular weights consistent with the insertion of Asn at position 8 of A21978C _1,2,3 (see Table VI). . Novel lipopeptides C233, C234 and C235 with molecular weights consistent with the insertion of Asn at position 11 of A21978C _1,2,3 were detected by LC-MS analysis of fermentation broth of KN404 (see Table VI).

Result of module change in positions 8 and 11 Dpt amino acid # Alternate Amino Acids (Source Pathway) 5 'linker 3 'linker result D-Ala-8 D-Ser ( dpt ) TC ^# TE ^# Lipopeptides with molecular masses of 1650 (Compound C22), 1664 (Compound C23), and 1678 (Compound C24) are detected D-Asn-8 D-Ser ( dpt ) TC ^# TE ^# Lipopeptides with molecular masses of 1677.72 (compound C189), 1691.75 (compound C190), and 1705.78 (compound C191) are detected D-Ser-11 Ala ( dpt ) TC ^# TE ^# Lipopeptides with molecular masses of 1618 (Compound C46), 1632 (Compound C47), and 1646 (Compound C48) are detected D-Asn-11 Ala ( dpt ) TC ^# TE ^# Lipopeptides with molecular masses of 1661.72 (compound C243), 1675.75 (compound C244) and 1689.78 (compound C245) are detected (See Fig. 5 for the location of ^# TC and TE)

The presence of the predicted mass ions was confirmed and PB103, PB118, KN392 and KN404 were fermented in bulk and C22, C46, C189, C233 were purified using the method as described in Example 2.5.

Example  2-7: dptD The module at position 13

A module exchange was constructed at position 13 of the dpt gene family to replace kynurenine. The construct subunit expression plasmid pRB04 (plasmid constructed in vector pHM11a expressing the dptD subunit under the ermE * constitutive promoter control described in Examples 2-5). Unique AvrII sites were introduced inside the TC linker. A second unique PmeI was introduced just downstream of the coding region of dptD . This removes the terminal module for Kyn from dptD along with the thioesterase . The replacement of two modules containing the domain arrangement CATTe was prepared in an AvrII fragment and a PmeI site facing side. Isoleucine and tryptophan modules are responsible for the introduction of terminal amino acids in the A54145 (Ile) and CDA (Trp) pathways. After the replacement module cloned into the deletion pRB04 dptD deletion es the hybrid construct. Roseosporus Fermentation culture and analysis were carried out using the method described in Example 2-1. The results are summarized in Table VII.

Result of module change in position 13 Replacement Amino Acids (Source Pathway) Dpt amino acid # 5 'linker 3 'linker result Trp (CDA) Kyn-13 TC ^# 3 'of dptD Lipopeptides with molecular masses of 1630 (Compound C1), 1644 (Compound C2), and 1658 (Compound C3) are detected Ile (A54145) Kyn-13 TC ^# 3 'of dptD Lipopeptides with molecular masses of 1577 (Compound C4), 1571 (Compound C5), and 1585 (Compound C6) are detected ( ^# Linker is defined in Figure 5)

Example  2-8: Daptomycin NRPS  In the genome dptI  By deleting the gene at position 12 Glutamate  Possessed Lipopeptide  production

dptI , lptI And sequence comparisons between the glmT genes predict that dptI plays an important role in the methylation of glutamate at position 12 (the glmT gene product is believed to methylate glutamate at analogous positions of related lipopeptides CDA; lptI gene product). Is believed to methylate glutamate during the synthesis of A54145). To test this theory, S containing pDA300 . Rosedale Austrian porous US431 my dptI It made a deletion in the gene. A deletion plasmid containing 2 × 1 kb fragment flanked upstream and downstream of dptI was constructed. These fragments were linked in-frame with the deletion of dptI . Cloning the cassette into the pRHB538 and S (see Examples 2-1). Rosedale OSU was introduced into the porous UA431 / pDA300. Under appropriate selection conditions (see Example 2-1), the deletion cassette was exchanged for the dptI gene on the chromosome to construct an in-frame deletion of dptI . Genotype of this mutant was confirmed by PCR and Southern blot. The mutants were fermented in culture and analyzed using the method as described in Example 2-1. According to the analysis results, the strains were lipopeptides containing glutamate instead of 3-methyl-glutamate at position 12; Only lipopeptides with masses of 1620, 1634, and 1648 corresponding to the masses predicted for Compound C10, Compound C11, and Compound C12, respectively, could be produced. These results concluded that dptI plays an important role in the methylation of glutamate during the synthesis of daptomycin.

Example  2-9: Recombination Streptomyces From Roseosporus  new Lipopepti Building a combinatorial library of de

Successful implementation module change who have reached in Example 2-5 pDR2155 and pDR2160 the lptD (S cloning the Ile / Val in the expression plasmid encoded a 3MeGlu. PRA Dia-terminal subunit of the A54145 biosynthetic pathway) and cdaPS3 (the expression plasmid S for encoding the cloned 3MeGlu and Trp in the calcium-dependent antibiotic nose elementary color, in combination with the subunit exchange for dptD which may include a terminal subunit of CDA biosynthetic pathway) can be further strengthened. This combination results in expression in a host that contains a dptI (methyl-transferase presumed to be involved in the methylation of glutamate present at position 12 of daptomycin ) resulting in the inclusion of glutamate instead of 3-methyl-glutamate at position 12. It was further strengthened as much as possible. One or more of the above methods, i.e. 1) a module exchange method effective for changing positions 8 and 11, 2) a dptI deletion effective for altering position 12, and 3. a subunit compensation method effective for altering position 13 It was used to build a combinatorial library containing 48 new lipopeptides.

Example 2-5 Construction of a technical as well as KN100 from the secondary S. Rosedale OSU has built a porous mutants, KN125 (carried out using the method described in Example 2-1) was named LA, which has a chromosomal deletion of the dptBC, D, G, H, I, J removed. After confirming that KN125 is a deletion mutant, pKN24 and pRB04 were introduced into KN125 and used to construct KN159. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strain was found to produce compounds C10, C11 and C12 lacking all the methyl groups of glutamate 12 shown in A21978C.

Strain KN107 was constructed by introducing pKN24 and pMF23 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C1, C2 and C3.

Strain KN110 was constructed by introducing pKN24 and pMF30 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C4, C5, C6, C7, C8 and C9.

Strain KN160 was constructed by introducing pKN24 and pMF23 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C13, C14 and C15.

Strain KN161 was constructed by introducing pKN24 and pMF30 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were produced by compounds C16, C17, C18, C19, C20 and C21.

The combination mentioned above was then enhanced by introducing a modified dptBC construct into pDR2155 and pDR2160. Strain PB105 was constructed by introducing pDR2155 and pMF23 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C49, C50 and C51.

Strain PB108 was constructed by introducing pDR2155 and pMF30 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C52, C53, C54, C55, C56, and C57.

Strain PB110 was constructed by introducing pDR2155 and pRB04 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C58, C59, and C60.

Strain PB113 was constructed by introducing pDR2155 and pMF23 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C61, C62, and C63.

Strain PB116 was constructed by introducing pDR2155 and pMF30 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C64, C65, C66, C67, C68, and C69.

Strain PB120 was constructed by introducing pDR2160 and pMF23 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C25, C26, and C27.

Strain PB123 was constructed by introducing pDR2160 and pMF30 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C28, C29, C30, C31, C32, and C33.

Strain PB128 was constructed by introducing pDR2160 and pRB04 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C34, C35, and C36.

Strain PB130 was constructed by introducing pDR2160 and pMF23 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C37, C38, and C39.

Strain PB131 was constructed by introducing pDR2160 and pMF30 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C40, C41, C42, C43, C44, and C45.

Strain KN393 was constructed by introducing pKN45 and pMF23 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strain was found to produce compounds C198, C199, and C200.

Strain KN394 was constructed by introducing pKN45 and pMF30 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C201, C202, C203, C210, C211, and C212.

Strain KN395 was constructed by introducing pKN45 and pRB04 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C192, C193, and C194.

Strain KN396 was constructed by introducing pKN45 and pMF23 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C195, C196, and C197.

Strain KN397 was constructed by introducing pKN45 and pMF30 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C204, C205, C206, C207, C208, and C209.

Strain KN405 was constructed by introducing pKN47 and pMF23 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strain was found to produce compounds C224, C225, and C226.

Strain KN406 was constructed by introducing pKN47 and pMF30 into KN100. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C221, C222, C223, C213, C214, and C215.

Strain KN407 was constructed by introducing pKN47 and pRB04 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C230, C231, and C232.

Strain KN408 was constructed by introducing pKN47 and pMF23 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strain was found to produce compounds C227, C228, and C229.

Strain KN409 was constructed by introducing pKN47 and pMF30 into KN125. Upon fermentation culture and analysis under the conditions described in Examples 2-2 and 2-3, the strains were found to produce compounds C72, C219, C220, C216, C217, and C218.

Example  2-10: Daptomycin  Module change built in positions 2 to 4

Multi-module exchanges were performed with dptA , dptBC , or a combination of dptA and BC , which required a new expression plasmid of pKN24 containing only d ptBC to complete this exchange. New vector, pKN18 is dptA and dptBC A cleavage product of B12: 03A05 (a BAC clone containing the entire daptomycin biosynthetic pathway, see Example 2-1) capable of expressing both. Plasmid pKN18 was constructed by cleaving B12: 03A05 using Red-mediated recombination (see Example 2-5) through two consecutive deletions of B12: 03A05. These two deletions resulted in the deletion of all genes upstream of dptR and all genes downstream of dptBC (pKN18 has the locus dptR - drrAB - dptEFABC ). First, the upstream 5 'region of the locus (insertion is equivalent to 0.552 kb to 45,576 kb on B12: 03A05, primers are shown in Table VI) was deleted using the spectinomycin resistance gene. the downstream region (3 ') of dptBC (insertion portion B12: Reference is comparable with 91,093 ~ 127,392 kb kb on the 03A05, primers are provided in Table V) was the deletion using the amp gene.

To combine the modular exchange of dptA , BC with subunit replacement for dptD (see Examples 2-6) and peptide custom methyl transferase dptI (see Examples 2-8), the dpt deletion host UA431 with the modified pKN18 plasmid ( In Example 2-1), it was required to construct a novel expression plasmid capable of expressing the dptIJ gene. To express glutamate methyltransferase in UA431 (Δ dptE-J ), pKN54, a plasmid that possesses the strong promoter permEp * and has the function of integrating into chromosomes from phi-BT1 phage, was constructed based on kanR pRT802 (Gregory , MA; Till, rl; Smith, MC; 2003. J Bacteriol . 185: 5320-5323). 1.8kb Bal II / Sma I fragment of pHM11a with ermEp * and transcription terminator (integrated vector responsible for heterologous gene expression in Streptomyces spp., Motamedi, H; Safiee, A; Cai, SJ; 1995, Gene , 160: 25-31) of pRT802 (Gregory, MA; Till, R .; Smith, MC; 2003, J. Bacteriol . 185: 5320-5323), encoding the phi-BT1 integration system. Cloned to the BamH I / EcoR V site. The plasmid was replicated in selection media containing kanamycin (50 μg / ml).

B12: Using as a template dptI 03A05 DNA fragments encoding both and dptJ were PCR amplified. Two primers (with genetically engineered restriction sites, underlined) dptJ-C-HindIII: 5'-GGCGG AAGCTT ACGGCACGGCAAGGCCGTTTC-3 '(SEQ ID NO: 15) and dptl-N-Ndel: 5'-GGCGG CATATG ACCGTGCACGACTACCAC- S '(SEQ ID NO: 16) was used for PCR amplification. The PCR fragment was cloned into pKN54 at the Nde I and Hind III sites to generate pKN55.

Finally, a series of expression hosts were generated for use in the multi-module exchanges described in this example. KN576 was constructed by introducing pRB04 (expressing dptD at the minicircle integration site, see Example 2-6) into UA431 (Δ dptE- J ). KN580 was constructed by introducing pRB04 (see Example 2-6) and pKN55 (expressed dptIJ from the phi-BT integration site) into UA431 (Δ dptE- J ). KN577 was constructed by introducing pMF30 (expressing lptD from the minicircle integration site, see Example 2-6) into UA431 ( ΔdptE- J ). KN587 was constructed by introducing pMF30 (see Example 2-6) and pKN55 into UA431 ( ΔdptE- J ).

Sp6 del3

5'GCATCCGATGCAAGTGTGTCGCTGTCGACGGTGACCCTATAGTCG TGTAGGCTGGAGCTGCTTC (SEQ ID NO: 17)

Sp6 del4

5'-CCGAGGAAAAGAGGGAACGGGACAGGTCAGTGACCGGCGACCGTG CATATGAATATCCTCCTTA -3 '(SEQ ID NO: 18)

DptD-3 ':: amp

5'-CATACTTCCTCTCACTCCGCTGCAGGAGGGACTGCTGTTCCACAG TGTGTAGGCTGGAGCTGCTTC -3 '(SEQ ID NO: 19)

GTC del2

5'-GCCGACTGGGAGTGGGTCAAGTGGCTGCCGCACGTGCTGGATCCG CATATGAATATCCTCCTTA -3 '(SEQ ID NO: 20)

Multiple module exchanges at pKN18 were performed using a Red-mediated recombination system to simultaneously change several amino acid residues on the daptomycin core. First, the genR (Wohlleben, W. et al. , 1989, Mol . Gen. Gene. 217: 202-208) gene was introduced into pKN18 to encode modules 2-3-4 (2-4) between linker region B and CAT. DNA fragments were replaced (exchange 2-4). The genR gene was then removed by AvrI I / Pme I digestion.

From the A54145 pathway, DNA fragments encoding modules 2-4 were cloned into pBR322 via the gap-repair method as described in the single module exchange of Example 2-5. The fragment was sectioned by Nhe I and Hpa I digestion and linked with deletion pKN18 to generate pKN51 (having D-Glu at position 2 of daptomycin and Asn at position 3). This plasmid, pKN51, was recombinantly introduced into expression host: KN576 to produce KN630, introduced into expression host KN580 to produce KN631, introduced into KN577 to produce KN632, and introduced into KN587 to produce KN633. The recombinant strains were fermented in culture and analyzed using the method described in Examples 2-2 and 2-3. Only the fermentation broth of KN633 was the only strain containing mass ions consistent with the production of C259, C260, C261, C262, C263, and C264. LC / MS analysis of the fermentation broths of the other strains KN630, KN631, and KN632 confirmed that no new lipopeptides were present.

primer for deletion of dpt2-4

dpt-Asn2-Del-B:

GTTCGCCTTCCCCACCGTCGCCGGCCTTCTCCCGCTCCTGGACGACAA CCTAGG TGTGTAGGCTGGAGCTGCTTCG (SEQ ID NO: 21)

dpt-Thr4-Del-CAT:

TCAGGGCGCCGGTCGATCCTGGTCACAGGTGGCAGGGCGGTGCCGG GTTTAAAC CATATGAATATCCTCCTTA (SEQ ID NO: 22)

Primer for lpt2-4 gap-repair cloning

LptGlu2-Pickup-B:

5 'TCC GGG CGG GGC CGG ACG GGA CGG ACG TGG TCG TCC GGC ACG GCC GCTAGC TTCTTAGACGTCAGGTGGCAC 3' (SEQ ID NO: 23)

lpt-Thr4-pickup-CAT:

5 'TTC GAG GCG CCCACG CCC GCC GCG CTGTCC CGG CGC CTC GACACC GTTAAC CGATACGCGAGCGAACGTGA 3' (SEQ ID NO: 24)

Example  2-11: Daptomycin  Build module exchange in positions 8-11

A daptomycin derivative containing 2 modifications at positions 8 and 11 was generated using a Red-mediated recombination system as described in Examples 2-5. Briefly, DNA fragments encoding 4 modules (D-Ala8-Asp9-Gly10-D-Ser11) were deleted at pKN24 via the gentamycin resistance gene flanked by Avr II and Pme I restriction enzyme sites. The genR gene was then removed by Avr II / Pme I digestion.

Corresponding DNA fragments encoding module 8-9-10-11 (D-Lys-Asp-Gly-D-Asn) from A54145 BAC-P13 subcloned on pBR322 by the gap-repair method (Example 2-5) Was used to link the deletion pKN24 to generate pKN50. KN156 the pKN50 (Examples 2-9, see, S. Rosedale agarose porous Δ dptBC, D + pRB04 [ermE * embodiment constructed in the vector pHM11a expressing dptD subunit under the control of a constitutive promoter plasmid of, for example, 2-5 KN410 was generated by introducing the same. KN410 was fermented in culture and analyzed using the protocol described in Examples 2-2 and 2-3. LC / MS analysis revealed that there were mass ions consistent with the insertion of lysine at position 8 of A21978C _1,2,3 and asparagine at position 11. The compounds were named C236, C237, C238.

Deletion Primers of Modules 8-11

8_B

TTGTTCGAGGCGCCGACGGTGAGCCGTTTGGAGCGGTTGCTGCGGGAGCG CCTAGG ACGTTGACACCATCGAATGG (SEQ ID NO: 25)

11_SUE ( delete termination at 3 'end of dptBC )

CAGCTCGCTGATGATATGCTGACGCTCAATGCCGTTTGGCCTCCGACTAA GTTTAAA CCCTCATTCATCGGGCGAAAG (SEQ ID NO: 26)

Gap repair primer in module 8-11

lptK8-B-P13

GTCCTCCGACCGCGACATCCGTCGCAACGCGGGGCGGGTGTCAGGGCGG GCTAGC TTCTTAGACGTCAGGTGGCAC (SEQ ID NO: 27)

lpt-Nll-SUE-P14 (cloned fragment extending to 3 'end of lptBC)

CACCGAACTCGACCAGCTCGAAGCAGAGTGGAAGGCCGGCTGATG GTTAAC CGATACGCGAGCGAACGTGA (SEQ ID NO: 28)

Example  2-12: New Lipopeptides  For production s . Pradia - Based Inn -Construction of trans expression system

For expression of the hybrid nonribosomal polypeptide synthetase (NRPS) pathway, S. lacks all NRPS and possible amino acid modification genes . Dia Pra of A54145 factors were building a high-producing strain variants. φC31 attB S in the site . Plastic parts in neutral regions of the genome dia - a path in which a genetically modified strain on BAC--based vector which is integrated into specific bonding was the strain.

Es. In order to deletion of all NRPS genes are present in the plastic dia deletion cassette was constructed which contained the DNA from the side facing upstream and downstream of the lptI lptEF.

Regions flanking the upstream 5 'of lptEF and downstream 93' downstream of lptI were cloned near the selection cassette containing tsr and cat . The 5 'fragment was 3665 bp long and the 3' fragment was 2004 bp long. The two fragments, together with the tsr and cat resistant cassettes, are temperature sensitive replication origins called pRHB538 (Hosted, TJ. And Baltz, RH., 1997, J. Bacteriol. 179 (1): 180-6) and rpsL (streptomycin sensitive). Cloned into plasmid copy containing the dominant allele. Es the plasmid held by the rpsL recessive allele that gives the streptomycin resistance. Introduced in Pradia strains. The recombinant strains were then incubated overnight at 39 ° C. in liquid culture, and then the cells were applied to plates containing streptomycin and thiostrepton and incubated at 39 ° C. Under these conditions, only those strains whose lptE- I locus was exchanged for deletion cassettes (containing tsr and cat ) through homologous recombination would survive the screening method; Other genotypes are eliminated. S after the above strain. PRA was named DI DA1187.

Example  2-13: s . Pradia  Fermentation Culture of Strains

S. who kept in -80 ℃. Plastic DI medium for mycelium glycerol stock of DA1187 R [10.3% sucrose (Sigma), 0.025% potassium persulfate (Sigma), 1.01% magnesium chloride hexahydrate (Sigma), 1% glucose (Sigma), 0.01% casamino acids (Difco ), 0.5% Yeast Extract (Difco), 0.57% TES Buffer (Sigma), 2.2% Agar (MBI), 0.005% Potassium Phosphate (Sigma), 0.29% Calcium Chloride Dihydrate (Sigma) and 0.07% Sodium Hydroxide (Sigma) ] (See Kiers) and grown at 30 ° C. for 3-5 days.

Nutritional mycelium and spores obtained by gently immersing the material on the surface of the agar medium in C medium [3% trypticase soy solution (Difco), 0.3% yeast extract (Difco), 0.2 in a 50 ml culture tube containing appropriate antibiotics % Magnesium sulfate (Sigma), 0.5% glucose and 0.4% maltose (Sigma); Hosted, TJ., And Baltx, RH., 1996, Microbiology 142: 2803-2813 were added to generate suspensions to make starting cultures. The starting culture was shaken at 30 ° C. for 24 to 26 hours at 240 rpm.

A 1 ml aliquot of the culture was added to nutrient solution S (1% D-glucose (BDH), 1.5% glycerol (BDH), 1.5% soy peptone (Sigma), 0.3% sodium chloride (BDH), 0.5% malt extract (Oxoid) ), 0.5% yeast extract (Lab M), 0.1% Junlon PW100 (Honeywell and Stein Ltd), 25 ml of 0.1% Tween 80 (Sigma), 4.6% MOPS (Sigma), adjusted to pH 7.0 and autoclaved) Transfer to a 125 ml baffle flask and shake culture at 30 ° C. for 24 to 36 hours at 200 rpm.

The production culture was 50 ml of medium D (3% glucose (Sigma), 2.5% soybean meal (Arkady), 0.5% molasses (DSM Bakeries), 0.06% ammonium ferrous sulfate (Sigma), 0.79% L-isoleucine (Sigma) And aseptically sterilize 5% of the above cultures in a 250 ml baffle flask containing 6% calcium carbonate (Sigma), adjusted to pH 7, autoclaved; Boeck et al ., 1990, J. Antibiotics 43: 607-615). And generated by shaking culture at 30 ° C. at 200 ° C. for up to 7 days. Addition of L-isoleucine to medium D resulted in an increase in the proportion of isoleucine (Ile13) with factor 13 and a decrease in the proportion of valine (Val13) with position 13 (Boeck et al., 1990, J. Antibiotics). 43: 607-615). Occasionally, fermentation cultures were performed in medium D free of isoleucine, where the fermentation broth contained a mixture having both Ile13 and Val13 factors.

Example  2-14: Streptomyces Pradia's  Analysis of A54145 Lipopeptides Produced by Fermentation Culture

Prior to analysis, 2 mL of the whole culture of the production culture described in Example 2-2 was aseptically extracted and centrifuged for 10 minutes to prepare a sample for analysis. Streptomyces volumes up to 50 microliters in the supernatant It was analyzed to monitor the production of natural lipopeptides (A21978C) produced by Roseosporus. This assay was performed at ambient temperature using a Waters Alliance 2690 HPLC system equipped with a 4.6 × 50 mm Symmetry C8 3.5 μm column and a Phenomenex Security Guard C8 cartridge and a 996 PDA detector. The concentration gradient was initially maintained with 90% water and 10% acetonitrile for 2.5 minutes, followed by a linear concentration gradient up to 100% acetonitrile over 6 minutes. The flow rate was 1.5 ml per minute and the concentration gradient was buffered with 0.01% trifluoroacetic acid. By 2 days of fermentation culture, the UV / visible spectrum is the same as daptomycin and shows HPLC peaks at retention times of 5.62, 5.77 and 5.90 (? Max 223,8, 261.5 and 364.5 nm) under the conditions mentioned above It was confirmed that three kinds of natural lipopeptides, A21978C ₁ , A21978C ₂ and A21978C _3, were produced. The lipopeptides then remained evident during the fermentation culture process at each sample extraction time point for a 7 day period. The total yield of the lipeptides A21978C ₁ , A21978C ₂ , and A21978C ₃ ranged from 10-20 mg per liter of the presentation material.

Liquid phase chromatography-mass spectrometry (LC-MS) was performed on a Finnigan SSQ710c LC-MS system using cationic mode electron-part ionization with a scan range of 200-2000 Daltons and a 2 second scan. Chromatographic separations were performed using a Water Symmetry C8 column (2.1 × 50 mm, 3.5 μm particle size), eluting using a linear water-acetonitrile concentration gradient containing 0.01% formic acid, specifically delaying the initial 0.5 min. Then eluted by increasing 100% acetonitrile at 10% over a 6 minute period and then holding 100% acetonitrile for an additional 3.5 minutes before rebalancing. The flow rate was 0.35 ml / min and the method was carried out at ambient temperature.

As 1630.7, 1644.7, 1644.7, 1658.7, 1658.7, 1658.7, 1672.7 of the bar that appears in the molecular ion ([M + H] ⁺⁾ at m / z, the control (S grown in a culture medium D without isoleucine. PRA dia wild-type) in Identification of three natural lipopeptides was performed, consistent with the reported mass for each of the major A54145 lipopeptiide factors F, A, A1, B1, B, D, E produced in Streptomyces pradia. (See Boeck et al., 1990, J. Antibiotics 43: 587-593). The DA1187 mutant did not produce any A54145 lipopeptides in medium D with or without isoleucine, confirming that the mutant is a true deletion mutant.

Example  2-15: pDA2002 Building and s . Pradia lptE -I reward of fruition

The "Red" system was used to construct the Streptomyces integrated BAC vector pDA2002 (containing the lpt biosynthetic gene family). The plasmid is SF1: was constructed from 10D08 (S PRA coli BAC plasmid that including one, cheukjeop DNA isolated from chromosome library containing both lpt biosynthetic gene cluster of dia.). Said Streptomyces integration cassette; flanking DNA region and S. containing the phiC31 integrase and attP sites, oriT from plasmid RK2 , and apramycin ( apr ) resistance markers and having identity to the backbone of the BAC vector . It was genetically engineered through DNA cloning to have orf21 of Pradia insert. The Streptomyces integration cassette was inserted into the BAC vector and the region of the BAC insert was deleted using homologous recombination via a Red-mediated recombination system. This is accomplished by introducing SF1: 10D08 into a strain of E. coli carrying the Red gene on plasmid (pKD119; see Datsenko, KA., And Wanner, BL., 2000, Proc . Nat. Acad . Sci . USA 97: 6640). Can be. The strain was then transformed with gel tablet fragments containing the Streptomyces integration cassette flanked by regions suitably described as having homology. Cells were then selected with or without resistance to both apr and cat , and the final colonies were genetically analyzed to verify insertion of the integration cassette and deletion of the upstream sequence of orf21 . Constructs in which the plasmid is pDA2002 S via the joint. By introducing a plastic DA1187 DI was created a strain DA1116. PDA2002 plasmid is S in E. coli. For conjugation to Pradia , it contains oriT from plasmid RK2 (see Baltz, 1998, Trends in Microbiol. 6: 76-83 (1998), incorporated herein by reference in its entirety). A plasmid pDA2002 through the junction E. coli S17.1, or self-S isolates from the clone containing the plasmid RK2 (same as above). It was introduced into a plastic dia. Es. Pradiai DA1116 was fermented in culture and analyzed using the methods described in Examples 2-13 and 2-14, respectively.

Streptomyces when grown in media D without isoleucine PRA key to Dia produce A54145 lipoic peptide factors of F, A, A1, B1, B, D, E a mass in the same manner, 1630.7, 1644.7, 1644.7, 1658.7, 1658.7, 1658.7, 1672.7 reported for each m / Molecular ions ([M + H] ⁺ ) measured at z appear, confirming the presence of native A54145 lipopeptides (see Boeck et al., 1990, J. Antibiotics 43: 587-593). When grown in medium D containing isoleucine, the native A54145 lipopeptide predominantly contained Ile13, with the same mass reported for each of the major A54145 lipopeptide factors A, A1, B1, B, D, E, 1644.7 , was confirmed to appear are 1644.7, 1658.7, 1658.7, 1658.7, 1672.7 of the m / molecular ions (([M + H] ^+, as measured in a z). this hayeoseo pDA2002 successfully compensate for the lipoic lptE -I deletion peptides DA1116 It shows that production can be restored.

Example  2-16: Removal of Downstream Genetic Groups through Insertion of Termination Cassettes for Identification of Genes Presumed to Be Amino Acid Modified Genes

The termination cassette was genetically engineered so that the t ₀ termination from lambda phage was located in front of the amp resistance gene. The termination cassette (including the t ₀ termination and amp ) was amplified using PCR primers having 40-50 bp homology to the potent amino acid modification gene and the ends of the BAC vector. When these PCR fragments were introduced into electro-competent cells containing pDA2002 and an induction Red-system, the terminations were site specificly integrated into pDA2002 via homologous recombination. These cells were then selected for the presence of apr and amp resistance markers and the final colonies were genetically analyzed to verify insertion of the termination cassette and sequence deletion at the end of the BAC vector.

The terminator cassette is amplified by PCR primers inserting end portion downstream of orf46 it was generate pDA2080. The Estonian pDA2080. So as to attain joining to the plastic DA1187 DI was created a strain DA1339. When fermented incubated and analyzed under the conditions described in Examples 2-13 and 2-14, the strain was streptomyces Matching (see [587-593 Boeck, etc., 1990, J.Antibiotics 43]), the production of plastic dia, main A54145 lipoic peptide factor A, A1, B1, B, D, E, respectively the mass reported for the It has been shown to produce lipopeptides with molecular ions (([M + H] ⁺ ) measured at m / z of 1644.7, 1644.7, 1658.7, 1658.7, 1658.7, and 1672.7, which pDA2080 successfully compensates for the lptE- I deletion. This confirms that even if there is a termination cassette inserted into the BAC, it is possible to restore the lipopeptide production in DA1339.

The termination cassette was amplified with a PCR primer inserting the termination into the IptI gene (presumed to be the methyltransferase of glutamate 12, see 2-8) to generate pDA2054. The Estonian pDA2054. So as to attain joining to the plastic DA1187 DI was created a strain DA1312. When fermented incubation and analysis under the conditions described in Examples 2-13 and 2-14, DA1312 yielded lipopeptides with molecular ions ([M + H] ⁺ ) measured at m / z of 1644.7, 1644.7 and 1658.7. It was produced. It has Streptomyces with glutamic acid (Glu12) instead of 3-methyl-glutamic acid (mGlu12) at position 12. It is consistent with the mass reported for each of the major A45145 lipopeptide factors A, A1, and D, produced in Pradia (see Boeck et al., 1990, J. Antibiotics 43: 587-593). These results concluded that lptI plays an important role in the methylation of glutamic acid during the synthesis of A54145.

The termination cassette was amplified using PCR primers inserting the termination into the lptL gene (presumed as oxygenase of asparagine 3) to generate pDA2076. The Estonian pDA2076. By plastic DI DA1187 and DA1336 junction was created a strain. When fermented incubation and analysis under the conditions described in Examples 2-13 and 2-14, DA1336 yielded lipopeptides with molecular ions ([M + H] ⁺ ) measured at m / z of 1628.7, 1628.7 and 1642.7. It was produced. Which coincides with the mass of each of A, A1, and D, analogs of the Glu12 factor, bearing asparagine (Asn3) instead of 3-hydroxy-asparagine (hAsn3) at position 3. Under such culture conditions it was also not confirmed whether the strain was capable of producing factors with Val13 C144, C145, and C146, but DA1336 was shown to produce Ile13 compounds C93, C94, and C95. These results conclude that lptL plays an important role in the addition of hydroxyl groups to the asparagine at position 3 during the synthesis of A54145.

LptK terminating cassette PDA2074 was generated by amplification with a PCR primer inserting the termination into the gene (presumed to be O-methyltransferase involved in methoxylation of aspartic acid at position 9). The Estonian pDA2074. So as to attain joining to the plastic DA1187 DI was created a strain DA1333. When fermented incubation and analysis under the conditions described in Examples 2-13 and 2-14, DA1333 has lipopeptides with molecular ions ([M + H] ⁺ ) measured at m / z of 1614.7, 1614.7, and 1628.7 Appeared to produce. This coincides with the mass of analogs A, A1, and D of the factor Glu 12, each having 3-hydroxy-aspartic acid (hAsp9) instead of 3-methoxy-aspartic acid (moAsp) at position 9 and Asn3 instead of hAsn3. , will be. Under these fermentation culture conditions it was not confirmed whether the strain also produced factors with Val13 C132, C133, and C134, but DA1333 was shown to produce Ile3 compounds C102, C103, and C104. These results conclude that lptK plays an important role in the methoxylation of hydroxyl-aspartic acid at position 9 during the synthesis of A54145.

The termination cassette was amplified with a PCR primer inserting the termination into the lptJ gene (estimated by the syrP control) to generate pDA2060. The Estonian pDA2060. So as to attain joining to the plastic DA1187 DI was created a strain DA1327. When fermented incubation and analysis under the conditions described in Examples 2-13 and 2-14, DA1327 has lipopeptides with molecular ions ([M + H] ⁺ ) measured at m / z of 1598.7, 1598.7, and 1612.7 Appeared to produce. This is consistent with the masses of analogs A, A1, and D of the Glu 12 factor, respectively, with aspartic acid (Asp9) instead of moAsp9 at position 9 and Asn3 instead of hAsn3. Under these fermentation culture conditions it was not confirmed whether this strain is also likely to produce factors with Val13 C135, C136, and C137, but DA1327 has been shown to produce Ile3 compounds C105, C106, and C107. These results suggest that lptJ plays an important role in the hydroxylation of aspartic acid at position 9 during the synthesis of A54145, although lptJ is not a regulator of A54145 biosynthesis.

Example  2-17: Φ for compensation experiment BT1 -base Plasmid  build

S at ΦC31 attB site . Plastic dia region a neutral site of the genome within lptE -I mutants using apr resistance BAC- based that are specifically incorporated into the vector, were expressed a modified A54145 biosynthesis. For subsequent compensation of these strains it is necessary to use compatible integrating plasmids with other selection markers. The ΦBT1--based vectors (such as Gregory, J. Bacteriol 2003: 5320-5323) is S in ΦBT attB sites it has a neomycin (neo) or hygromycin (hyg) resistance marker. Site-specific integration into the neutral region of the Pradia genome. This can be achieved in apr resistant strains containing already integrated ΦC31-based BAC vectors.

ΦBT1 integrase-based streptomyces integration cassette was obtained from MS82 (Gregory et al., J. Bacteriol 2003: 5320-5323) and contains ΦBT1 integrase and attP sites, plasmid RK2 derived oriT , and hyg resistance markers, and BAC Flanking DNA regions and S that have identity to the backbone of the vector . It was genetically engineered by DNA cloning to have a Pradia insert. The φBT1 integration cassette also contains an ermE * constitutive promoter that drives expression of downstream genes. Homologous recombination between the Streptomyces integration cassette flanking the appropriate homology region and the BAC vector can be achieved by transducing the E. coli strain carrying SF1: 10D08 and the Red gene containing plasmid pKD119 into gel purified fragments. These cells were selected for the presence of hyg and cat resistance and the final colonies were genetically analyzed to verify the deletion of the integration cassette and upstream sequences.

The Streptomyces integrated BAC vector pJR2012 was constructed using the "Red" system. The ΦBT1 integrase-based Streptomyces integration cassette is a framework of the BAC vector and S. aureus . Bordering a DNA region having a plastic insertion portion dia lptK and identity side. Homologous recombination between the BAC vector and the Streptomyces integration cassette was achieved by transforming cells containing SF1: 10D08 BAC and the induction Red-system (see Example 2-5). The insert of the φBT1 integration cassette is located directly in front of lptK at the ermE * constitutive promoter to ensure its expression, including the downstream genes remaining on the BAC vector.

The "Red" system was used to construct Streptomyces integrated BAC vector pJR2015. The ΦBT1 itasease-based Streptomyces integration cassette is a framework of the BAC vector and S. aureus . The DNA region having identity with lptL of the Pradia insertion portion is flanked . Homologous recombination between the BAC vector and Streptomyces integration cassette was achieved by transforming cells containing SF1: 10D08 BAC and the induction Red system. The insert of the integration cassette ΦBT1 hayeoseo position and directly to ermE * promoter in front lptL configuration ensures the expression thereof, including the downstream gene remaining in the BAC vector.

inserting a cassette containing an ermE * constitutive promoter driving the expression of the fd and t ₀ terminator portion-side facing streptomycin (spec) resistance marker was converted by the neo resistance ΦBT1 pRT802 plasmid as a expression plasmid gave the pDA2113. The PCR amplified expression cassette was inserted into pRT802 plasmid enzymatically digested with EcoR V and Not I. The spec marker of pCA2113 and the t ₀ termination were replaced by PCR- enhanced biosynthetic gene and cat termination cassette (t ₀ termination genetically engineered before cat resistance gene) to either lptI (mGlu12 methyltransferase) or lptL (hAsn3 oxygenase ) Generated a compensating plasmid expressing together.

PCR amplification lptI digested neo- resistant ΦBT1 pDA2113 with Nde I and Hind III to remove spec markers and t ₀ terminations, and digested with Nde I and Xba I Linked with gene and cat termination cassette digested with Xab I and Hind III. The newly generated pDA2129 has the lptI gene, which encodes Glu12 methyltransferase, under the control of the constitutive ermE * promoter.

The neo- resistant ΦBT1 pDA2113 was digested with Nde I and Hind III to remove spec markers and t ₀ terminations, and the PCR-amplified lptL gene digested with Nde I and Xba I and the cat termination cassette digested with Xab I and Hind III. Connected together. The newly generated pDA2015 has an lptL gene encoding Asn3 oxygenase, under the control of the constitutive ermE * promoter.

Example  2-18: New Lipopeptides  Φ to produce BT1 -base Plasmid  ΦC31 BAC Containing s . Pradia  Compensation of Mutant Strains

Under configuration control of the ermE * promoter St. lptK (hAsp9 methoxy silanol claim), lptL (Asn3 oxide dioxygenase claim), and S through the junction pJR2012 a plasmid constructed to contain lptI (Glu12-methyl transferase). By introducing a plastic DA1333 DI was created a strain DA1449. When fermentation culture and analysis under the conditions described in Examples 2-13 and 2-14, S. PRA was DI DA1449 has appeared that for generating the Lipoic peptide having a molecular ion ([M + H] ^+), measured at 1644.7, 1644.7, 1658.7, 1658.7, 1672.7 of the m / z, which my process's bit repto It is consistent with the mass reported for each of the major A54145 lipopeptiide factors A, A1, B1, B, D, E produced by Pradia (Boeck et al., 1999, J. Antibiotics 43: 587-593). This shows that pJR2012 can successfully compensate for the DA1333 strain and restore lipopeptides production.

Under configuration control of the ermE * promoter St. lptK (hAsp9 methoxy silanol claim), lptL (Asn3 oxide dioxygenase claim), and S through containing the plasmid pJR2012 bonded to the lptI (Glu12-methyl transferase). By introducing a plastic DA1327 DI was created a strain DA1553. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. Plastic DI DA1553 has been shown to generate reports a peptide having a molecular ion ([M + H] ⁺⁾ at 1628.7, 1628.7 and 1642.7 of the m / z. This is consistent with the mass of each of the mGlu12 factor analogs B1, B, and E, with Asp9 instead of moAsp9. Under these fermentation culture conditions it was not confirmed whether the strain also had the potential to produce Val13 C117, C118, and C119, but DA1553 was shown to produce Ile13 compounds C114, C115, and C116. It is shown that the putative LptK protein requires lptJ protein to hydroxylate Asp9 before methoxylation occurs.

Constitutive ermE * S pJR2015 a plasmid constructed to contain lptL (Asn3 oxide dioxygenase agent) under the control of a promoter, and lptI (Glu12-methyl transferase) through the junction. By introducing a plastic DA1336 DI was created a strain DA1621. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. PRA was DI DA1621 has appeared that for generating the Lipoic peptide having a molecular ion ([M + H] ^+), as measured in the 1644.7, 1644.7, 1658.7, 1658.7, 1658.7, 1672.7 m / z, which Streptomyces It is consistent with the mass reported for each of the major A54145 lipopeptiide factors A, A1, B1, B, D, E produced by Pradia (Boeck et al ., 1999, J. Antibiotics 43: 587-593). This shows that pJR2015 can successfully compensate for the DA1336 strain and restore lipopeptides production.

Constitutive ermE * S pJR2015 a plasmid containing (a Asn3 oxide dioxygenase) under the control of a promoter lptL, and lptI (Glu12-methyl transferase) through the junction. Dia is introduced into the plastic by the strain DA1627 DA1333 was generated. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. Plastic DI DA1627 has been shown to generate reports a peptide having a molecular ion ([M + H] ^+), measured at 1644.7, 1644.7 and of 1658.7 m / z. This is consistent with the mass of each of the analogs B1, B, and E of the mGlu12 factor, with Asp9 instead of moAsp9. Under these fermentation culture conditions it was not confirmed whether the strain also had the potential to produce Val13 C126, C127, and C128, but DA1627 was shown to produce Ile13 compounds C111, C112, and C113.

Constitutive ermE * S pDA2129 a plasmid constructed to contain lptI (Glu12-methyl-transferase) under control of the promoter through the junction. By introducing a plastic strain DA1491 DI DA613 were generated. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. PRA was DI DA1491 has appeared that for generating the Lipoic peptide having a molecular ion ([M + H] ^+), measured at 1644.7, 1644.7, 1658.7, 1658.7, 1658.7, 1672.7 of the m / z, which Streptomyces It is consistent with the mass reported for each of the major A54145 lipopeptiide factors A, A1, B1, B, D, E produced by Pradia (Boeck et al., 1999, J. Antibiotics 43: 587-593). This shows that pDA2129 can successfully compensate for the DA612 strain and restore Glu12 and mGlu12 lipopeptides production.

Constitutive ermE * S pDA2129 a plasmid containing lptI (Glu12-methyl-transferase) under control of the promoter through the junction. By introducing a plastic DA1327 DI was created a strain DA1489. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. Plastic DI DA1489 has been shown to generate reports a peptide having a molecular ion ([M + H] ^+), as measured in the 1612.7, 1612.7 and 1626.7 m / z. This is consistent with the mass of each of the analogs B1, B, and E of the mGlu12 factor, with Asp9 instead of moAsp9 and Asn3 instead of hAsn3. Under these fermentation culture conditions it was not confirmed whether the strain also had the potential to produce Val13 C138, C139, and C140, but DA1489 was shown to produce Ile13 compounds C108, C109, and C110.

Constitutive ermE * S pDA2129 a plasmid containing lptI (Glu12-methyl-transferase) under control of the promoter through the junction. By introducing a plastic DA1327 DI was created a strain DA1489. Plasmid pDA2076 was introduced into this strain to generate DA2000. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. Plastic DI DA2000 has been shown to generate reports a peptide having a molecular ion ([M + H] ^+), measured at 1642.7, 1642.7 and of 1656.7 m / z. This is consistent with the mass of the analog of each of the mGlu12 factors B1, B, and E, with Asn3 instead of hAsn3. Under these fermentation culture conditions it was not confirmed whether the strain also had the potential to produce Val13 C141, C142, and C143, but DA1489 was shown to produce Ile13 compounds C96, C97, and C98.

Constitutive ermE * S pDA2129 a plasmid containing lptI (Glu12-methyl-transferase) under control of the promoter through the junction. By introducing a plastic DA1333 DI was created a strain DA1459. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. Plastic DI DA1459 has been shown to generate reports a peptide having a molecular ion ([M + H] ^+), as measured in the 1628.7, 1628.7 and 1642.7 m / z. This is consistent with the mass of each of the analogs B1, B, and E of the mGlu12 factor, with Asp9 instead of moAsp9 and Asn3 instead of hAsn3. Under these fermentation culture conditions it was not confirmed whether the strain also had the potential to produce Val13 C129, C130, and C131, but DA1489 was shown to produce Ile13 compounds C99, C100, and C101.

Constitutive ermE * S a plasmid containing Construction pDA2117 under control lptL (Asn3 oxide dioxygenase claim) of the promoter through the junction. By introducing a plastic DA1336 DI was created a strain DA1470. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. Plastic DI is DA1470, having a 1644.7, 1644.7, and was shown to produce the Lipoic born peptide having a molecular ion ([M + H] ⁺⁾ 1658.7, measured at the m / z, which Glu12 instead mGlu12 Streptomyces This is consistent with the mass reported for each of the major A54145 lipopeptiide factors A, A1, and D produced by Pradiai (Boeck et al., 1999, J. Antibiotics 43: 587-593). This shows that pDA2117 can successfully compensate for the DA1336 strain and restore Glu12 lipopeptide production.

Constitutive ermE * S pDA2117 a plasmid containing a control under lptL (Asn3 oxide dioxygenase claim) of the promoter through the junction. By introducing a plastic DA1327 DI was created a strain DA1484. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. Plastic DI DA1484 has been shown to generate reports a peptide having a molecular ion ([M + H] ^+), as measured in the 1614.7, 1614.7 and 1628.7 m / z. This is consistent with the mass of each of analogs A, A1, and D of the mGlu12 factor, with Asp9 instead of moAsp9. Under these fermentation culture conditions it was not confirmed whether the strain also had the potential to produce Val13 C120, C121, and C122, but DA1484 was shown to produce Ile13 compounds C90, C91, and C92.

Composition ermE * S pDA2117 a plasmid containing a control under lptL (Asn3 oxide dioxygenase claim) of the promoter through the junction. By introducing a plastic DA1333 DI was created a strain DA1453. Examples 2-13 and when under the conditions described in 2-14 hayeoteul fermentation culture and analysis, S. Plastic DI DA1453 has been shown to generate reports a peptide having a molecular ion ([M + H] ^+), measured at 1630.7, 1630.7 and of 1644.7 m / z. This is consistent with the mass of each of analogs A, A1, and D of the Glu12 factor, with hAsp9 instead of moAsp9. Under these fermentation culture conditions it was not confirmed whether the strain also had the potential to produce Val13 C123, C124, and C125, but DA1484 was shown to produce Ile13 compounds C87, C88, and C89.

Example  2-20: Module changeover build in position 8 or 11 of A54145

Module replacement to change positions 8 or 11 of A54145 was performed in plasmid pDA2054 (see Example 2-16, where the plasmid can express ltpABCD without the Pme I site). The plasmid is Δ lptE S. -I. It was recovered from the A54145 biosynthesis Pradesh Dia. Using the red-mediated recombination system described in Examples 2-6, in the linker region B and CAT, the DNA fragment encoding module 8 or module 11 of pDA2054 was replaced with the genR gene (see primers used below). . The genR gene was inserted into pDA2054 and the replaced lysine CAT module at position 8 or asparagine module at position 11 was removed via Nhe I / Pme I digestion (module 8) or Avr II / Pme I (module 11). Modules used to replace lysine or asparagine can be cloned by the gap-repair method described in Examples 2-6 and removed in pBR322 using restriction enzymes NheI / HpaI. The modules include DNA fragments encoding heterologous modules cloned from daptomycin d apD -Ala8, dptD -Ser 11 or A54145 lptD -Asn11. All possible combinations of Ser, Ala and Asn at positions 8 or 11 were constructed and named as follows: hybrid pKN56 (pDA2054 with D-Ala8), pKN57 (pDA2054 with D-Ser8), pKN58 (D-Asn8) the pDA2054), pDA2054), and pKN60 (pDA2054) containing D-Ser11 containing pKN59 (D-Ala11-S to. Plastic dia mutant DA1187 (see Examples 2-12 Δ lptE -I,) and DA740 (see Examples 2-18 DA1187, having a plasmid expressing pDA2129 lptI) 2 in position 8, or 11 and 12 is introduced to the species Hybrid lipopeptides with alterations in were generated.

lpt-D-Lys8 deletion primer

Ipt-Del-Lys8-B-Nhe

5'-GTGTTC GAG GCC CGAACG GTC GCC GCG CTGGCG GCC CGG CTG CGGACC GCGCTAGCTGTGTAG GCT GGA GCT GCTTCG-3 '(SEQ ID NO: 29)

lpt-Lys8-CAT-II:

5'-CGGCGA GAGCGG GGT CCT CGT CGC CTG CCG CGT CGGTCC TGC GGG GTTTAAAC CATATGAATATCCTCCTTA-3 '(SEQ ID NO: 30)

lpt-D-Asn11

lpt-Asn11-B

5'-CGAGACACCGACCGTGGCCGGTCTCGCCGCCGCGCTCTCCGCGGC CCTAGG TGTGTAGGCTGGAGCTGCTTCG-3 '(SEQ ID NO: 31)

lpt-Asn11-CAT

5'-GTCCCGCGACCGCCGAGTACCTCGGTCGCCGGACCGCCGGGGCGCG GTTTAA AC CATATGAATATCCTCCTTA-3 '(SEQ ID NO: 32)

Primer for gap- repair cloning of dptAla8 and dpt-Ser11 modules

 Ala / Ser-B-P13

5'-CGTCCGCTCCCGTGCCACCAGAGGCACCCGCACCCCCAAAGCCGAC GCTAGC TTCTTAGACGTCAGGTGGCAC-3 '(SEQ ID NO: 33)

Ser-CAT-P14-II

5'-GTTCGGGATGTTTTCGAGGGCCGTACGGTACGTGCTCTGGCGGCTGTG GTTAAC CGATACGCGAGCGAACGTGA-3 '(SEQ ID NO: 34)

Strain KN707 was constructed by introducing pKN56 into DA1187. Fermentation culture and analysis under the conditions described in Examples 2-13 and 2-14 showed that the strain produced compounds C313, C314, C315, C316, C317 and C318.

Strain KN681 was constructed by introducing pKN56 into DA740. When fermented and cultured under the conditions described in Examples 2-13 and 2-14, the strains were found to produce compounds C319, C320, C321, C322, C323 and C324.

Strain KN715 was constructed by introducing pKN57 into DA1187. When fermented and cultured under the conditions described in Examples 2-13 and 2-14, the strains were found to produce compounds C292, C293, C294, C295, C296 and C297.

Strain KN689 was constructed by introducing pKN57 into DA740. When fermented and cultured under the conditions described in Examples 2-13 and 2-14, the strains were found to produce compounds C289, C290, C291, C298, C299 and C300.

Strain KN723 was constructed by introducing pKN58 into DA1187. Fermentation culture and analysis under the conditions described in Examples 2-13 and 2-14 showed that the strain produced compounds C307, C308, C309, C310, C311, and C312.

Strain KN697 was constructed by introducing pKN58 into DA740. Fermentation culture and analysis under the conditions described in Examples 2-13 and 2-14 showed that the strain produced compounds C301, C302, C303, C304, C305 and C306.

Strain KN728 was constructed by introducing pKN59 into DA1187. Fermentation culture and analysis under the conditions described in Examples 2-13 and 2-14 showed that the strain produced compounds C334, C335, C336, C337, C338 and C339.

Strain KN701 was constructed by introducing pKN59 into DA740. Fermentation culture and analysis under the conditions described in Examples 2-13 and 2-14 showed that the strain produced compounds C328, C329, C330, C331, C332 and C333.

Strain KN730 was constructed by introducing pKN60 into DA1187. Fermentation culture and analysis under the conditions described in Examples 2-13 and 2-14 showed that the strain produced compounds C147, C148, C149, C325, C326 and C327.

Strain KN705 was constructed by introducing pKN60 into DA740. When fermented and cultured under the conditions described in Examples 2-13 and 2-14, the strains were found to produce compounds C150, C151, C152, C153, C154 and C155.

Example  2-21: Module changeover build in positions 2 to 4 of A54145

The exchange of module 2-4 (D-Glu-2 / hAsn-3 / Thr-4) at lptA was constructed on pDA2054 (this plasmid expresses lptABCD from BAC vector, construction of which is described in Example 2-16). . pDA2054 restored the biosynthesis of the glutamate derivative of A54145 in mutant DA1187 (see Examples 2-15). pDA2054 carried out on, a DNA fragment encoding the modules 2-4 of lptA using a red- mediated recombination system described in Example 2-6 was replaced with genR gene between the regions B and CAT (module replacement 2-4). The genR gene is flanking the restriction enzyme site of Nhe I / Pme I and can be easily removed from the BAC vector through restriction enzyme digestion. The replacement fragment from dptA was cloned into pBR322 using the gap-repair system described in Examples 2-6 and flanking the restriction enzyme site of Nhe I / Hpa I. The fragment is from the dptA encodes the module 2-4 (D-Asn-2 / Asp-3 / Thr4) dapto of azithromycin, by linking the fragment to 2-4 deletion variant of pDA2054 gave the pKN61. Plasmid pKN61 was introduced into DA1187 and XH1000 (DA1189 with pKN55, see Example 2-10) to produce recombinant strains KN650 (DA1187 with pKN61) and KBN665 (XH1000 with pKN61).

When fermented in culture and analyzed under the conditions described in Examples 2-13 and 2-14, strain KN650 was found to produce compounds C271, C272, C273, C274, C275 and C276.

When fermented and cultured under the conditions described in Examples 2-13 and 2-14, strain KN665 was found to produce compounds C265, C266, C267, C268, C269 and C270.

Lpt2-4 Deletion Primer

lpt-Del-Glu2-B-Nhe:

CCG GTC CCC GAC CGT CGC CCG CCT CGC GGA GGAACT GGG CGA CGG GCTAGC TGTGTAGGCTGGAGCTGCTTCG (SEQ ID NO: 35)

lptGlu2-CAT:

5'-CCT GCG GCG CGG GAC GCT CCG CGT CCG CGT CCG GTC CGG CGG ACC GTTTAAAC CATATGAATATCCTCCTTA-3 '(SEQ ID NO: 36)

Primer for gap repair cloning

Dpt2-4

dpt-Asn2-Pick-B:

AGGCGCTCCGGGCGCGGAGGCAGCGGCGGGGTGGTGTGCTGCCGTCCG GCTAGC TTCTTAGACGTCAGGTGGCAC (SEQ ID NO: 37)

dpt-Thr4-Pick-CAT:

CTCTTCGCCGCGCCCACGCCTGCCGGGCTCGCGACCGTACTGGCGGCC GTTAAC CGATACGCGAGCGAACGTGA (SEQ ID NO: 38)

Example  2-22: Module change build in positions 2 to 8 of A54145

The exchange of modules 2-8 (D-Glu-2 / hAsn-3 / Thr-4 / Sar-5 / Ala-6 / Asp-7 / D-Lys-8) at lptA , B, C with pDA2054 (this plasmid Expresses lptABCD from the BAC vector, its construction was constructed on Example 2-16). pDA2054 restored the biosynthesis of the glutamate derivative of A54145 in mutant DA1187 (see Examples 2-15). On pDA2054, the DNA fragments encoding modules 2-8 of lptA , B, C were replaced with the genR gene between regions B and CATE using the red-mediated recombination system described in Examples 2-6 (module exchange 2 -8). The genR gene is flanking the restriction enzyme site of Nhe I / Pme I and can be easily removed from the BAC vector through restriction enzyme digestion. The replacement fragment from dptA , B, C was cloned into pBR322 using the gap-repair system described in Examples 2-6 and flanking the restriction enzyme site of Nhe I / Hpa I. Said fragment from dptA , B, C shows module 2-8 of daptomycin (D-Glu-2 / hAsn-3 / Thr-4 / Sar-5 / Ala-6 / Asp-7 / D-Lys-8) Encoding the fragment, the fragment was linked to the 2-8 deletion variant of pDA2054 to generate pKN62. Plasmid pKN62 was introduced into DA1187 and XH1000 (DA1189 with pKN55, see Example 2-10) to produce recombinant strains KN653 (DA1187 with pKN62) and KBN669 (XH1000 with pKN62).

When fermented and cultured under the conditions described in Examples 2-13 and 2-14, strain KN653 was found to produce compounds C283, C284, C285, C286, C287 and C288.

When fermented and cultured under the conditions described in Examples 2-13 and 2-14, strain KN669 was found to produce compounds C277, C278, C279, C280, C281 and C282.

Deletion Primer

Lpt2-8

Ipt-Del-Glu2-B-Nhe:

CCG GTC CCC GAC CGT CGC CCG CCT CGC GGA GGAACT GGG CGA CGG GCTAGC TGTGTAGGCTGGAGCTGCTTCG (SEQ ID NO: 39)

lpt-Lys8-CATE2-II:

5 'GGGG GGC GAC CGG CAG GAT GTC CTC CAAGGC GGT GCC GGT GCG GC GTTTAAAC CATATGAATATCCTCCTTA 3' (SEQ ID NO: 40)

Primer for gap repair cloning

Dpt2-8

dpt-Asn2-Pick-B:

AGGCGCTCCGGGCGCGGAGGCAGCGGCGGGGTGGTGTGCTGCCGTCCG GCTAGC TTCTTAGACGTCAGGTGGCAC (SEQ ID NO: 41)

Ala-CATE2-P14-II

CGACGTGACGCTGGTGGAAGTGAACCAGGTGGAGCTCGACCGTCTGCAG GTTAAC CGATACGCGAGCGAACGTGA (SEQ ID NO: 42)

Example  2-23: for generating glycine at position 5 of A54145 Sarcosine  Methylation Deletion of Module

The selection cassette for deletion of the methylation domain in the lptA5- Sar module was amplified using PCR primers having 50 bp homology to the linker region of the domain under test (linker position see FIG. 5). The PCR fragment was introduced into electro-competent cells containing pDA2054 (cleaved form of lptBAC containing the entire lpt pathway, see Examples 2-16) and induction Red-system (see Examples 2-6) and were resistant The cassette was site-specifically integrated into the target site of pDA2054 via homologous recombination (see FIG. 3).

The cells were selected for the presence of gent resistance markers and the final colonies were genetically analyzed to verify that proper deletion or destruction was established. Part of the primer design included the restriction enzymes ( Pme I and Swa I) in the linker region of the subject (FIG. 3).

The BAC containing the gent deletion of the methylation domain was then digested using the unique restriction enzyme sites Pme I and Swa I to incise and relink the selectable markers, and the final cluster was genetically analyzed to ensure proper deletion of the methylation domain. It was verified that it was built. The final clone was called pSD409.

by introducing pSD409 in E. coli via the species bonded to DA1187 (see Examples 2-12 S. plastic dia of lptE -I Results entity,) was generated for SD409 S. Pradia SD409 was fermented in culture and analyzed using the method described in Examples 2-13 and 2-14, respectively.

Molecular ions ([M + H] ⁺ ), measured at m / z of 1616.7 (C183), 1630.7 (C182), 1630.7 (C181), and 1644.7 (C180), confirmed the presence of lipopeptides, which were indicated by Streptomyces pra This is consistent with the mass reported for the major A54145 lipopeptides produced in Dia. Under these conditions it was not confirmed whether the strains had the potential to produce factors with Val13 C184 and C185.

Example  2-24. Build module exchange in position 2 of A54145

Module exchange was performed in plasmid plpt-J14-P by replacing the D-Glu-2 module with a module for D-Asn, D-Ser or D-Ala. The plasmid pDA2054 can restore the biosynthesis of the glutamate derivative of A54145 in mutant DA1187. The DNA fragment encoding module 2 of pDA2054 was first replaced with the genR gene of the linker region CAT (see Example 2-20). genR gene was removed by NheI / PmeI digestion and described in Example 2-5 using NheK / Hpa DNA fragments encoding primers lpt D-Asn11 (primers Lpt-N11-B-P13 and Lpt-N11-CAT-P14). Cloned by gap-repair method). This produced hybrid plasmid pXH2000 and introduced into DA1189 and XH1000 (DA1189 with pKN55) to produce recombinant strains XH1001 (DA1889 with pXH2000) and XH1002 (XH1000 with pXH2000). XH1001 and XH1002 were fermented incubated and analyzed using the protocol described in Examples 2-13 and 2-14. Es. Plastic dia XH1001 has been shown to produce the Lipoic peptide having a molecular ion ([M + H] ^+), measured at 1629.7, 1629.7 and of 1643.7 m / z. This is consistent with the mass of derivatives A, A1, and D of the Glu12 factor, each with Asn-2. XH1001 has been shown to produce Ile13 compounds C343, C344, and C345. It was not confirmed whether the strain had the potential to produce factors with Val13 C346, C347, and C348 under these conditions. Es. Plastic dia XH1002 has been shown to produce the Lipoic peptide having a molecular ion ([M + H] ^+), measured at 1643.7, 1643.7, and 1657.7 of m / z. This is consistent with the mass of derivatives B, B1, and E of the mGlu12 factor, respectively, with Asn-2. XH1002 has been shown to produce Ile13 compounds C340, C341, and C342. It was not confirmed whether the strain under this condition had the potential to produce factors with Val13 C349, C350, and C351.

Deletion Primer of D-Glu-2 Module

lptGlu2-B:

5'-CCG GTC CCC GAC CGT CGC CCG CCTCGC GGA GGAACT GGG CGA CGG CCTAGG TGTGTAGGCTGGAGCTGCTTCG-3 '(SEQ ID NO: 43)

lptGlu2-CAT:

5'-CCT GCG GCG CGG GAC GCT CCG CGT CCG CGT CCG GTC CGG CGG ACC GTTTAAAC CATATGAATATCCTCCTTA-3 '(SEQ ID NO: 44)

gap repair primer for lpt-Asn-11

Lpt-Nll-B-PI3

TCGGGGCGCGGGTCGGCGGGGCGCAGCCGGGGTCCGGCCTCGCCC GCTAGC TTCTTAGACGTCAGGTGGCAC (SEQ ID NO 45)

Lpt-Nll -CAT-Pl 4

CGCGACATCTTCGAACAGCGCACGCCCGCCGCCCTCGCCGGCCGC GTTAAC CGATACGCGAGCGAACGTGA (SEQ ID NO: 46)

Example  3-1: Biological Activity

The antimicrobial activity against the panel of eogfms organisms was tested on a compound according to formula (I) according to the standard procedure described in the National Committee for Clinical Laboratory Stardards (NCCCLS document M7-A6, Vol. 23, 2 2003). And some exceptions were performed with all the tests under constant stirring at 37 ° C. and 200 rpm. The compound was dissolved in 100% dimethyl sulfoxide or water or a 50:50 volume ratio mixture of dimethyl sulfoxide and water depending on the solubility of the compound and diluted to a final reaction concentration (0.1 μg / ml-100 μg / ml) in the microbial growth medium. In all cases, the final concentration of dimethyl sulfoxide to be cultured with the cells was less than 1%. In order to calculate the minimum inhibitory concentration (MIC), the final volume 100 ㎕ medium of the compound in a microtiter plate containing 5 × 10 ⁴ bacterial cells in a (50 mg / L Ca ^{2 +} supplemented with Mueller-Hinton broth) wells Two-fold dilutions were added. The optical density (OD) of bacterial cells, which measures the growth and proliferation of the bacterial cells, was measured using a commercially available plate reader. The MIC value is defined as the minimum compound concentration that inhibits the growth of the test organism. MIC (μg / ml) values of representative compounds of the present invention are shown in Tables V-1 to V-4. Tables V-1 to V-4 below show the biological activities of the compounds of formula (I).

TABLE V-1

TABLE V-2

Table V-3

TABLE V-4

"+++" in the above table means that the compound has an MIC of 1 μg / ml or less or an ED ₅₀ of 1 mg / kg or less.

"++" means that the compound has a MIC (μg / ml) or ED ₅₀ higher than 1 μg / ml or 1 mg / kg, respectively, but a MIC of 10 μg / ml or less or an ED50 of 10 mg / kg or less To have;

"+" Means that the compound has a MIC higher than 10 μg / ml (μg / ml) or an ED ₅₀ higher than 10 mg / kg.

The strains used in the table are as follows.

Strain # Bell ATCC # Strain description SAU.42 Staphylococcus aureus 29213 NCCLS reference strain for liquid medium microdilution MIC analysis purchased from ATCC SAU.399 Staphylococcus aureus 4330 Clinically Isolated Strains of NCCLS Methicillin and Oxacillin Resistance Purchased from ATCC SAU.278 Staphylococcus aureus n / a Dapto neomycin resistant mutants (D10) - S matrix. Liquid Serial Passage Mutant Induced at Aureus 42 EFM.14 Enterococcus Passium 6569 FDA Test Organism Experimented with Bactericidal Activity of AOAC Purchased from ATCC EFM.384 Enterococcus Passium n / a Dapto hygromycin resistant mutant (E14-A) - the matrix. Liquid Serial Passage Mutant Induced in Passion 14 EFS.201 Enterococcus faecalis 49452 Quality Control Strains of API Products Purchased from ATCC EFS.312 Enterococcus faecalis n / a Dapto neomycin resistant mutants (EFA) - the matrix. Liquid Serial Passage Mutant Induced at Pacalis 201 SPN.402 Streptococcus pneumoniae 6303 Purchased from ATCC, see Public Health Record 59: 449-468 (serum 3)

Example  3-2: In vivo  activation

Mouse protection testing is an industry standard method of measuring the efficiency of test compounds in vivo (see, eg, Clemet, JJ., Et al., 1994, Antimicrobial Agents and Chemotherapy 38 (5): 1071-1078). ). As illustrated below, this test is to demonstrate the in vivo efficiency of the compounds of the present invention against bacteria.

The in vivo antibacterial activity of methicillin the outgoing female CD-1 mice (Charles River Lab, MA) weighing of 19 ~ 23 g resistant S. It was confirmed by infection intraperitoneally with an Aureus (MRSA) inoculum. The inoculum was prepared via methicillin resistant S. aureus (ATCC 4300). MRSA inoculum was incubated at 37 ° C. for 18 h in Mueller-Hinton (MH) liquid medium. Optical density (OD ₆₀₀ ) was measured at 600 nm to identify 1:10 dilutions of overnight cultures. Bacteria (8 × 10 ⁸ cfu) were added to 25 ml of phosphate buffered saline (Sigma P-0261) containing 5% swine gastric mucin (Sigma M-2378). All animals were injected with 0.5 ml of the inoculum, which is equivalent to 2 × 10 ⁷ cfu per mouse and a dose capable of ˜100% killing animals without treatment.

The test compound was dissolved in 10.0 mL of brine solution to give 1 mg / mL solution (pH = 7.0). This solution was serially diluted with vehicle four times (1.5 mL to 6.0) to give a 0.25, 0.063 and 0.016 mg / mL solution. All solutions were filtered through a 0.2 μm Nalgene syringe filter. One hour after bacterial inoculation, group 1 animals were injected subcutaneously (sc) with buffer (no test compound) and groups 2-5 were injected with 10.0, 2.5, 0.63, and 0.16 mg / kg of test compound, respectively. Group 6 animals were injected subcutaneously with only 10 mg / kg (or the highest therapeutic dose of a given compound) to observe acute toxicity. This injection was repeated once after 4 hours of inoculation for each group. Each hourly infusion dose was 10 ml per kilogram of body weight. A 50% protective dose (PD ₅₀ ) was calculated based on the number of surviving mice 7 days after inoculation.

All publications and patent applications mentioned in the specification of the present invention are incorporated by reference as if each individual publication or patent application were specifically and individually incorporated by reference. Although the foregoing invention has been described in detail with reference to examples and examples for purposes of understanding, some modifications and changes may be made in light of the present invention without departing from the scope or spirit of the invention. It will be apparent to those skilled in the art.

Claims (59)

A composition comprising a compound of Formula F11 and salts thereof:

Where a) R ^{13 *} is H or CH ₃ ; b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino. A composition comprising a compound of Formula F1 and salts thereof:

Where a) R ⁸ is hydrogen,

ego; b) R ¹¹ is methyl,

Is; c) R ¹² is H or CH ₃ ; d) R ¹³ is CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

Is; e) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfone amino, thioacylamino, Thiourei is also iminoamino or phosphonamino. A composition comprising a compound of formula F2 and salts thereof:

Where a) R ⁸ is hydrogen, methyl,

ego; b) R ¹² is H or CH ₃ ; c) R ¹³ is CH (CH ₃ ) ₂ , CH (CH ₂ CH ₃ ) CH ₃ ,

ego; d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F3 and salts thereof:

Where a) R ⁸ is hydrogen,

ego; b) R ¹¹ is methyl,

Is; c) R ¹² is H or CH ₃ ; d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, monosubstituted amino, bisubstituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F4 and salts thereof:

Where a) R ⁸ is hydrogen, methyl,

ego; b) R ¹¹ is methyl, or

Is; c) R ¹² is H or CH ₃ ; d) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F5 and salts thereof:

Where a) R ⁸ is hydrogen, methyl,

ego; b) R ¹¹ is methyl,

Is; c) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting groups, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F6 and salts thereof:

Where a) R ⁸ is

ego; b) R ⁹ is

Is; c) R ¹¹ is methyl,

ego; d) R ¹² is H or CH ₃ ; e) R ¹ is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, imino Amino, or phosphonamino. A composition comprising a compound of Formula F7 and salts thereof:

Where a) R ⁸ is methyl,

ego; b) R ⁹ is

Is; c) R ¹² is H or CH ₃ ; d) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F8 and salts thereof:

Where a) R ^{3 **} is hydroxyl or hydrogen; b) R ⁸ is methyl,

ego; c) R ¹¹ is amino acid side chain, methyl,

Is; d) R ¹² is H or CH ₃ ; e) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F9 and salts thereof:

Where a) R ¹² is H or CH ₃ ; b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F10 and salts thereof:

Where a) R ^{13 *} is H or CH ₃ ; b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino. A composition comprising a compound of Formula F12 and salts thereof:

Where a) R ¹³ is CH (CH ₂ CH ₃ ) CH ₃ or

ego; b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino. A composition comprising a compound of Formula F13 and salts thereof:

Wherein R, R ^{6 *} and R ^{8 **} are each independently amino, monosubstituted amino, double substituted amino, NH-amino protecting groups, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F14 and salts thereof:

Where a) R ¹² is H or CH ₃ ; b) R ¹ and R ^{6 *} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, Thiourei is also iminoamino or phosphonamino. A composition comprising a compound of Formula F15 and salts thereof:

Where a) R ¹² is H or CH ₃ ; b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F16 and salts thereof:

Where a) R ¹² is H or CH ₃ ; b) R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino , Thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F17 and salts thereof:

Where a) R ¹² is H or CH ₃ ; b) R ¹ is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, imino Amino, or phosphonamino. A composition comprising a compound of Formula F18:

Where R ¹ and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thi Oureido is also iminoamino or phosphonamino. A composition comprising a compound of Formula F19 and salts thereof:

Where a) R ² is

ego; b) R ⁶ is methyl or

Is; c) R ⁸ is methyl or

ego; b) R ¹ , R ^{6 *} and R ^{8 **} are each independently amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino , Thioacylamino, thioureido, iminoamino, or phosphonamino. A composition comprising a compound of Formula F20 and salts thereof:

Where a) R ¹² is H or CH ₃ ; b) R ¹ and R ^{8 **} are amino, single substituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thi Oureido is also iminoamino or phosphonamino. A composition comprising a compound of Formula F21 and salts thereof:

Where a) R ¹ is

ego; b) R ¹² is H or CH ₃ ; c) R ^{8 **} is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfonamino, thioacylamino, thioureido, Iminoamino, or phosphonamino. A composition comprising a compound of Formula F22:

                                         F (22) Where R ^{6 *} is amino, monosubstituted amino, double substituted amino, NH-amino protecting group, acylamino, ureido, guanidino, carbamoyl, sulfoneamino, thioacylamino, thioureido, iminoamino Or phosphonamino. The compound of claim 10, wherein R ^{8 **} is an amino, NH-amino protecting group or carbamoyl. The compound of claim 23, wherein R ^{8 **} is amino. The compound of claim 10, wherein R ¹ is amino, alkanoylamino, NH-amino protecting group or carbamoyl. The compound of claim 25, wherein R ¹ is C ₁₀ -C ₁₃ alkanoylamino. The compound of claim 26, wherein R ¹ is

Phosphorus compounds. The compound of claim 10, wherein R ¹² is CH ₃ . The compound of claim 28, wherein R ¹ is alkanoylamino. 30. The compound of claim 29, wherein R ¹ is C ₁₁ -alkanoylamino. The compound of claim 30, wherein R ¹ is

Phosphorus compounds. 32. The compound of claim 31, wherein R ^{8 **} is amino. The method of claim 10,

Compound selected from.

Compound selected from. The compound of claim 1, wherein R ^{6 *} is an amino, NH-amino protecting group or carbamoyl. 36. The compound of claim 35, wherein R ^{6 *} is amino. The compound of claim 1, wherein R ¹ is an amino, acylamino, NH-amino protecting group. 38. The compound of claim 37, wherein R ¹ is C ₁₀ -C ₁₃ acylamino. The compound of claim 38, wherein R ¹ is

Phosphorus compounds. The compound of claim 1, wherein R ¹ is

Phosphorus compounds. The method of claim 1,

Compound selected from. The method of claim 1,

Compound selected from. Compounds of the formula

Compounds of the formula

A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier. An antibacterial composition comprising the compound of claim 1 in an aqueous buffer. As a method of treating bacterial infections in patients, A method comprising administering to a patient in need of such treatment a therapeutically effective amount of a composition according to claim 1 under constant temperature and conditions to mitigate said bacterial infection. Use of a composition according to claim 1 in the manufacture of a medicament for treating a bacterial infection in a patient. The composition of claim 1, wherein the compound is present in an amount of about 80% to about 90% of the composition. The composition of claim 1, wherein the compound is present in about 90% of the composition. The composition of claim 1, wherein the compound is present in greater than about 90% of the composition. A method of producing recombinant cells, a. Providing in the cell a recombinant NRPS gene cluster encoding NRPS that can be recombined intracellularly with one or more exogenous polynucleic acid encoding an NRPS module to produce a compound of Formula F11 in the cell. How to include. The method of claim 52, wherein the at least one exogenous polynucleic acid encoding the NRPS module is from a daptomycin NRPS gene cluster or A54145 gene cluster. A recombinant cell produced in the method of claim 52. As a method of preparing a compound of Formula F11, Culturing the cells of claim 54 under conditions suitable for producing a compound of formula F11 How to include. The method of claim 55, Purifying the compound of Formula F11 How to further include. The method of claim 55, wherein culturing the cells By the presence of a precursor of formula F11 line group determined R ¹ of the fermentation the cells increasing the production of a compound of Formula F11 with a group R ¹ as determined line Further comprising. The method of claim 57, a. Purifying the compound of Formula F11 How to further include. A compound produced by the method of claim 55.

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