CA2170158A1 - Inhibitors of tnf-alpha secretion - Google Patents

Abstract

Compounds and methods are disclosed that are useful in inhibiting the TNF-.alpha. converting enzyme (TACE) responsible for cleavage of TNF-.alpha. precursor to provide biological active TNF-.alpha.. The compounds employed in the invention are peptidyl derivatives having active groups capable of inhibiting TACE such as hydroxamates, thiols phosphoryls and carboxyls.

Description

Wo 95/06031 2 ~ 7 ~` ~ 5 8 PCT/US94/09343 TIT~ E
"Inhibitors of TNF-alpha Secretion"

~ROSS-RF.F~.RF.NC~. TO RF.~,ATFD APPT ICATTONS

This application is a continuation-in-part of application serial no. 08/110,601, filed August 23, 1993, pending.
F~FT,D OF THF. INVF.NT~ON
The invention pertains to compounds which are inhikitnrs of metalloproteases and, in particular, to compounds which inhibit the TNF-a converting enzyme.
RACKGROUND OF THF. INVF.NT~ON

Tumor necrosis factor-a (TNF-a, also known as cachectin) is a m~mm~ n protein capable of inducing a variety of effects on numerous cell types. TNF-a was initially 20 characterized by its ability to cause lysis of tumor cells and is produced by activated cells such as mononucLear phagocytes, T-cells, B-cells, mast cells and NK cells. In mononuclear phagocytes, TNF-a is initially synthesized as a membrane-bound protein of a~ro~ lalely 26 kD. A 17 kD fragment of the 26 kD membrane-bound TNF-a is "secreted" and combines with two other secreted TNF-a molecules to form a circulating 51 kD homotrimer.
25 TNF- a is a principal mediator of the host response to gram-negative bacteria.
Lipopolysaccharide (LPS, also called endotoxin), derived from the cell wall of gram-negative bacteria, is a potent stimulator of TNF-a synthesis. Because the deleterious effects which can result from an over-production or an unregulated-production of TNF aree~lGmely serious, considerable efforts have been made to control or regulate the serum level 30 of TNF. An important part in the effort to effectively control serum TNF levels is the und~ ndillg of the mechanism of TNF biosynthesis.

The mechanism by which TNF-a is secreted has only been recently elucidated.
Kriegler et al. Cell, 53, 45-53, (1988) conjectured that TNF-a "secretion" is due to the 35 cleaving of the 26 kD membrane-bound molecule by a proteolytic enzyme or protease.
Scuderi et. al., J. Immunology, 143, 168-173 (1989), suggested that the release of TNF-a from human leukocyte cells is dependent on one or more serine proteases, e.g., a leukocyte el~st~e or trypsin. A serine protease inhibitor, p-toluenesulfonyl-L-arginine methyl ester, was found to suppress human leukocyte TNF release in a concentration-dependent manner.
40 Scuderi et. al. suggested that the arginine methyl ester competes for the arginine-binding site 2 ~ PCT/US94/09343 in the enzyme's reactive center and thereby blocks hydrolysis. The lysine and phenylalanine analogs of the inhibitor reportedly failed to mimic the arginine methyl ester.

We have discovered that the protease which causes the cleavage of the TNF-a S molecule into the 17 kD protein is, in fact, a metalloprotease which is believed to reside in the plasma membrane of cells producing TNF-a. The physicochemical characteristics of the enzyme have not been published.

Most, but not all, proteases recognize a specific amino acid sequence. Some 10 proteases primarily recognize residues located N-terminal of the cleaved bond, some recognize residues located C-terminal of the cleaved bond, and some proteases recognize residues on both sides of the cleaved bond. Metalloprotease enzymes utilize a bound metal ion, generally Zn2+, to catalyze the hydrolysis of the peptide bond. Metalloproteases are implicated in joint destruction (the matrix metalloproteases), blood pressure regulation 15 (angiotensin converting enzyme), and regulation of peptide-hormone levels (neutral endopeptidase-24.1 1).
Numerous inhibitors have been developed against the previously described metalloproteases. A general family of inhibitors against matrix-metalloproteases, and in 20 particular collagenase, is reported in WO 92/09563. This document shows compounds having the general structure of a reverse hydroxamate - or a hydroxyurea - linked via an amide to an amino acid derivative, such as tryptophan or 2-naphthyl alanine. Inhibitors of collagenase are also reported in WO 88/06890; these compounds contain sulfhydryl moieties as well as phenylalanine and tryptophan analogs. Collagenase inhibitors are reported in WO
25 92/09556 and U.S. Patent No. 5,114,953 and possess hydroxamate moities and fused or conjugated bicycloaryl substituents. The myriad potential gelatinase inhibitors covered by the generic formula in EPA 489,577 are amino acid derivatives optionally possessing a hydroxamate group. Hydroxamate derivatives useful as angiotensin converting enzyme (ACE) inhibitors are reported in EPO 498,665.
Inhibition of the TNF-a converting enzyme (hereinafter referred to as "TACE"), anovel metalloprotease, inhibits release of TNF-a into the serum and other extracellular spaces. TACE inhibitors would therefore have clinical utility in treating conditions characterized by over-production or unregulated production of TNF-a. A particularly useful 35 TACE inhibitor for certain pathological conditions would selectively inhibit TACE while not affecting TNF-13 (also known as Iymphotoxin) serum levels. The over-production or unregulated production of TNF-a has been implicated in certain conditions and ~ e~es, for example:

wo 95/06031 2 17 015 g PCT/US94/09343 I. Systemic Tnfl~mm~tory Response Syndrome, which includes:
Sepsis syndrome gram positive sepsis gram negative sepsis culture negative sepsis fungal sepsis n~uLI~penic fever urosepsis meningococcemia Trauma/h~,l.c",hage Burns Ionizing radiation exposure Acute ~lc,-,alilis Adult respiratory distress syndrome.
II. Reperfusion Injury, which includes:
Post pump syndrome Ischemia-reperfusion injury III. Cardiovascular Disease, which includes:
Cardiac stun syndrome Myocardial infarction Congestive heart failure IV. Infectious Disease, which includes:
HIV infection/ HIV neuropathy Meningitis Hepatitis Septic arthritis Peritonitis Pneumonia Epiglottitis E. coli 0157:H7 Hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura Malaria Dengue hemorrhagic fever T f~ hm~ni~cic Leprosy WO 95/06031~ 2 ~ 7 ~15 8 PCT/US94/09343 Toxic shock syndrome Streptococcal myositis Gas gangrene Mycobacterium tuberculosis Mycobacterium avium intr~cell~ re Pneumocystis carinii pnt~llmoni~
Pelvic infl~ y disease Orchitis/epidydimitis Legionella Lyme disease Influenza A
Epstein-Barr Virus Viral-associated hemaphagocytic syndrome Viral encephalitis/aseptic meningitis V. Obstetrics/Gynecology, including:
Gl I l~lt Ul G labor Miscarriage Infertility VI. Tnfl~mm~toryDisease/Au~illlllllll~ity, which includes:
Rheumatoid arthritis/seronegative al ~l~opathies Osteoarthritis Tnfl~mm~tory bowel disease Systemic lupus erythematosis Iridocyclitis/uveitis/optic neuritis Idiopathic pulmonary fibrosis Systemic vasculitis/Wegener's granulomatosis Sarcoidosis Orchitis/vasectomy reversal procedures VII. Allergic/Atopic Diseases, which includes:
Asthma Allergic rhinitis F~.7~.m~
Allergic contact dermatitis Allergic conjunctivitis Hypersensitivity pneumonitis WO 95/06031 2 ~ 7 ~ i 5 8 PCT/US94/09343 VIII. ~lign~ncy, which includes:
A~
A~L
CML
~.
Hodgkin's disease, non-Hodgkin's lymphoma MM[ ' Kaposi's s~;o,l,a Colorectal ;~.;i,l~ ,lla Nasopharyngeal carcinoma Malignant histiocytosis Paraneoplastic syndrome/hypercalcemia of malignancy IX. Transplants, including:
Organ transplant rejection Graft-versus-host disease X. Cachexia XI. Congenital, which includes:
Cystic fibrosis Familial h~llla~c,phagocytic lymphohistiocytosis Sickle cell anemia XII. Dermatologic, which includes:
Psoriasis Alopecia XIII. Neurologic, which includes:
Multiple sclerosis Migraine he~ he " XIV. Renal,which includes:
Nephrotic syndrome Hemodialysis Uremia WO95/06031 2 ~7 01~ 8 PCT/USg4/09343 XV. Toxicity, which includes:
OKT3 therapy Anti-CD3 therapy Cytokine therapy Chemotherapy Radiation therapy Chronic salicylate intoxication XVI. Metabolic/ldiopathic, which includes:
Wilson's disease Hemachromatosis Alpha-1-antitrypsin deficiency Diabetes H~chimoto's thyroiditis Osteoporosis Hypoth~l~mic-~iLui~ y-adrenal axis evaluation Primary biliary cirrhosis Inhibitors of TACE would prevent the cleavage of cell-bound TNF-a thereby 20 reducing the level of TNF-a in serum and tissues. Such inhibitors would be of significant clirlical utility and could be potential therapeutics for treating the above TNF-a-related disorders.

SUMMARY OF THF, INVF.NTION
The invention relates to compounds of formula I:

O O

X-[CH]m-CH-C-N-CH-C-[A]n-N-B-NH2 (I) I I H I H

wherein:
X is hydroxamic acid, thiol, phosphoryl or carboxyl;
misO, 1 or2;
Rl, R2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl), oR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted Cl to C8 alkyl, Cl to Wo 95/06031 Z ~ ~ O l S 8 PCT/US94/09343 C8 aL~-ylenearyl, aryl, a p,oLe~Led or unprotected side chain of a naturally occurring a-amino acid; or the group -R6R7, wherein R6 is substituted or unsubstituted Cl to C8 alkyl and R7 is oR4, SR4, N(R4)(R5) or halogen, wherein R4 and R5 are, each independent of the other, hydrogen or substituted or unsubstituted C1 to C8 alkyl;
nisO, 1 or2;
provided that when n is 1, A is a ~ro~ecled or an unprotected a-amino acid radical;
when n is 2, A is the same or different protected or unprotected a-amino acid radical;
and B is unsubstituted or substituted C2 to C8 alkylene;
and the phaTm~elltir~lly acceptable salts thereof.

The compounds of formula I are useful as metalloprotease inhibitors, and particularly 15 useful as inhibitors of the TNF-a converting enzyme (TACE).

The invention also relates to a method of treating a mammal having a disease characterized by an overproduction or an unregulated production of TNF-a. The method comprises the steps of ~lmini~tering to the m~mm~l a composition comprising an effective amount of a biologically active compound of formula II:
O O
Il 11 X-[CH]m-CH-C-N-CH-C-[A]n-N-y (II) l l H l H

wherein:
X is hydroxamic acid, thiol, phosphoryl or carboxyl;
misO, 1 or2;
R1, R2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl), oR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted Cl to C8 alkyl, C1 toC8 aLkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring a-amino acid; or the group -R6R7, wherein R6 is Cl to C8 aLkyl and R7 is oR4, SR4, N(R4)(R5) or halogen, wherein R4 and R5 are each, independent of the other,hydrogen or substituted or unsubstituted Cl to C8 alkyl;
n is 0, 1 or 2;
Y is hydrogen, unsubstituted or substituted Cl to C8 alkyl, alkylene(cycloalkyl), the group -R8-COOR9 or the group -RlON(Rl l)(R12); wherein R8 is Cl to C8 WO 95/06031 2 17 0 1~ 8 PCTIUS94/09343 aL~ylene; R9 is hydrogen or Cl to C8 aL~cyl; R10 is unsubstituted or substituted Cl to C8 alkylene; and Rl 1 and R12 are each, independent of the other, hydrogen or Cl to C8 aL~cyl;
provided that when n is 1, A is a protected or an unprotected a-amino acid radical;
and when n is 2, A is the same or different protected or unprotected a-arnino acid radical;
and the pharm~ellti~lly acceptable salts thereof;
wherein the colll~oul,d is capable of reducing serum TNF-a levels by at least 80%
when ~tlmini~tered at 25mg/kg in a murine model of LPS-induced sepsis syndrome;
and a ~hal ,. ,~eutir~lly acceptable calrier.

The discovery of useful inhibitors of the TACE metalloprotease has led to the discovery of further embodiments of the invention, including pharmaceutical compositions for treating the above-listed disorders comprising a compound according to formula II and 15 protein having TNF-binding activity.

T)FT~IJ,FD DFSCRIPTTON OF THF INVFNTI20 The invention is directed to a compound of formula I:
O O
Il 11 X-rCH]m-CH-C-N-CH-C-[A]n-N-B-NH2 (I) I I H I H

wherein:
X is hydroxamic acid, thiol, phosphoryl or carboxyl;
mis 0, 1 or 2;
Rl, R2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl), oR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted Cl to C8 aL~yl, Cl toC8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring oc-amino acid; or the group -R6R7, wherein R6 is substituted or unsubstituted Cl to C8 aL~yl and R7 is oR4, SR4, N(R4)(R5) or halogen, wherein R4 and R5 are each, independent of the other, hydrogen or substituted or unsubstituted C1 to C8 alkyl;
n is 0, 1 or 2;
provided that when n is 1, A is a pl.~lecled or an unprotected oc-amino acid radical;

WO 95/06031 . r - PCT/US94/09343 ~170158 provided that when n is 1, A is a ,~lvle~;led or an unprotected oc-amino acid radical;
when n is 2, A is the same or different protected or unprotected a-amino acid radical;
and B is unsubstituted or substituted C2 to C8 alkylene;
and the pharmaceutically acceptable salts thereof.

J The compounds of formula I are useful as inhibitors of TNF-oc secretion, and pa~ticularly useful as inhibitors of the TNF-o~ converting enzyme (TAOE).

The invention also relates to a method for treating a m~mm~l having a condition or a disease characterized by overproduction or unregulated production of TNF-oc, comprising ~-lmini~tering to the ",~.1"~1 a composition comprising an effective amount of a biologically active compound of formula II:

O O
Il 11 X-[CH]m-CH-C-N-CH-C-[A]n-N-Y (II) H I H

wherein:
X is hydroxarnic acid, thiol, phosphoryl or carboxyl;
misO, 1 or2;
Rl, R~2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl), oR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted Cl to C8 alkyl, Cl toC8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring a-amino acid; or the group -R6R7, wherein R6 is C1 to C8 alkyl and R7 is oR4, SR4, N(R4)(R5) or halogen, wherein R4 and R5 are each, independent of the other,hydrogen or substituted or unsubstituted C1 to C8 alkyl;
n is 0, 1 or2;
Y is hydrogen, unsubstituted or substituted C1 to C8 alkyl, alkylene(cycloalkyl), the group -R8-COOR9 or the group -R1ON(R1 1)(R12); wherein R8 is Cl to C8 alkylene; R9 is hydrogen or Cl to C8 alkyl; R10 is unsubstituted or substituted Cl to C8 alkylene; and Rl 1 and R12 are each, independent of the other, hydrogen or Cl to C8 alkyl;
provided that when n is 1, A is a protected or an unprotected oc-arnino acid radical;
and W 095/06031 ~ ~ 7 ~ ~ ~ 8 PCTrUS94/09343 when n is 2, A is the same or different protected or unprotected a-amino acid radical;
and the pharmaceutically acceptable salts thereof;
wherein the co~ ulld is capable of reducing serum TNF levels by at least 80%
when administered at 25mg/kg in a murine model of LPS-induced sepsis syndrome;
and a ph~"laceutically acceptable carrier.
The invention includes pharmaceutical compositions containing a compound according to formula I as the active component. In addition, pharmaceutical compositions comprising a compound according to formula II and a protein which binds TNF are 10 described. An example of a protein which binds TNF is an anti-TNF antibody or a soluble TNF receptor which is described in EPA 0418014, ~ignecl to the assignee of the instant application. The disclosure of EPA 0418014iS incorporated herein by reference.

The following definitions are used herein. "Alkyl" means a straight or branched,15 univalent, saturated or unsaturated hydrocarbon group of 1 to 8 carbon atoms. Alkyl groups include the straight-chain groups methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, vinyl, allyl, butenyl, pentenyl, hexenyl, heptenyl and octenyl as well as the branched isomers thereof.

"Substituted alkyl" means an alkyl group substituted with one or more of hydroxy, amino, halogen, or thiol.

"Alkylene" means a bivalent alkyl group as defined above.

"Substituted alkylene" means an alkylene group substituted with one or more of hydroxy, amino, halogen or thiol groups.

"Aryl" means an aromatic or heteroaromatic group, including for example, phenyl,naphthyl, pyridyl, quinolyl, thienyl, furyl and the like, optionally substituted with one or more of Cl to C8 alkyl, hydroxy, amino, halogen, thiol or alkyl groups.

"Alkylene(cycloalkyl)" refers to groups of the structure -R13-R14 wherein R13 is an alkylene as defined above, and R14 is a univalent cyclic alkane radical, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
"Alkylenearyl" means the group -R15-R16, wherein R15 is a substituted or -n.cllbstitllte~l alkylene group as defined above, and R16 is a substituted or unsubstituted aryl group as defined above.

~ 21~01~

"a-Amino acid" refers to any of the 22 common amino acids, e.g., alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, S ~I~eulline, tryptophan, tyrosine and valine.

"~olecled amino acid" and "~lulecled side chain of an a-amino acid" means the side chains of the amino acid are permanently or L~ ,ol~ily coupled to a chemical group which protects or prevents the side chain from undesired branching, structural modification or 10 rearrangement which can occur during subsequent synthetic steps. Use of such protecting groups for these purposes is well known in the art, as are the protecting groups themselves.
Examples of common protecting groups are N-tert-butyloxycarbonyl (Boc) and N-9-fluorenylmethyloxycarbonyl (Fmoc).

"Biologically active" as used in defining certain compounds of formula II, designates a compound capable of (a) inhibiting secretion of TNF-a; (b) preventing cleavage of membrane-bound TNF-a by TACE; or (c) reducing serum TNF levels by at least 80% when ~rlministered at 25 mg/kg in a standard murine model of LPS-indl~e-l sepsis syndrome.

In the compounds of formulas I and II, pl~rt;llc;d radicals for X are hydroxamic acid, thiol and phosphoryl. More preferred X radicals are hydroxamic acid and thiol, while the most preferred radical is hydroxamic acid. The plere~ d value for m is 1.

Preferred R1 or R2 radicals are hydrogen, C1 to C8 alkyl and Cl to C8 alkylenearyl. Where Rl or R2 is alkyl, preferred is Cl to C6 alkyl and most ~l~rell~,d is Cl to C4 alkyl. Where R1 or R2 is alkylenearyl, preferred alkylene groups are C1 to C6 alkylene, and more ~iefelled is Cl to C4 alkylene; and preferred aryl groups are phenyl and substituted phenyl. The most preferred alkylenearyl group for R1 or R2 is C1 to C4 alkylenephenyl. The most preferred group for R1 is hydrogen and the most preferred group for R2 is isobutyl.

Preferred R3 radicals are substituted and unsubstituted C1 to C8 alkyl and C1 to C8 alkylenearyl. Where R3 is alkyl, pl~fellt;d is Cl to C6 alkyl and more preferred is Cl to C4 alkyl, with t-butyl being most preferred. Where R3 is C1 to C8 alkylenearyl, preferred alkylene groups are Cl to C6 alkylene, and more pl~;fell ;;d is Cl to C4 alkylene; and preferred aryl groups are phenyl, naphthyl, and thienyl, each optionally substituted with hydlu~y, amino, halogen, thiol or alkyl groups. F'lcfelled groups for R3 are therefore Cl to C4 alkylenephenyl, Cl to C4 alkylenenaphthyl, and C1 to C4 alkylenethienyl. More _ WO 95/06031 ~17 01~ 8 PCT/US94/09343 ~

plcrell~d is C1 to C4 alkylenenaphthyl, with methylenenaphthyl being most ~lefel,cd.
Where R3 is a protected or unprotected side chain of a naturally occurring a-amino acid, R3 preferably is an arginine, lysine, tryptophan or tyrosine side chain. However, the most rellGd radicals for R3 are t-buyl, methylene(cyclohexyl) and methylene-(2'naphthyl).
S r The radical A is preferably an unprotected naturally-occurring amino acid residue.
More preferred naturally-occurring residues are the alanyl radical or an wlplolec~ed seryl radical. The most plc~ll~d radical for A is an alanyl residue. Further preferred compounds are those where n is 0 or 1, while most preferably n is 1.
~er~ d radicals for B are C2 to C6 alkylene. More ~lc;~lled radicals are C2 to C4 aLkylene, with dimethylene being most ~lt;rt;lled.

For compounds according to formula II, Y is preferably hydrogen, unsubstituted or substituted Cl to C8 alkyl or the group -R10N(Rl l)(R12). Most preferred is the group -R10N(R11)(R12) with Rl0 preferably being unsubstituted or substituted C1 to C6 alkylene, Rl 1 and R12 preferably are each independently hydrogen or Cl to C6 alkyl. More pl~rtill ;;d R10 radicals are unsubstituted or substituted Cl to C4 alkylene, with dimethylene 'oeing most preferred. More preferred radicals for R10 and Rl 1 are hydrogen or Cl to C4 alkyl, with hydrogen being most plef~llGd.

Compounds according to the invention can be prepared utilizing the procedures outlined below, the appended reaction Schemes and the procedures detailed in the Examples below.
General Synthesis With reference to Scheme 1, the inhibitor compounds may be prepared by converting the carboxylic acid or ester compound (Io), wherein R is H or C1 to C8 alkyl, and P is CBZ, BOC, FMOC or other suitable protective group (Greene T., Wuts P., "Protective Groups in Organic Synthesis", 2nd Ed.; Wiley: New York, 1991; Chapter 7), to thecorresponding hydroxamic acid or hydroxamic ester compound (Ip). In compound (Ip), R' is H, TMS, t-Bu, Bzl or other group made by treating these compounds, or an activated form of the carboxylic acid, (Bodanszky, M., Bodanszky, A., "The Practice of Peptide Synthesis"; Springer-Verlag: Berlin, 1984; Chapter II) with a hydroxylamine reagent under conditions which effect the conversion. This is followed by the subsequent removal of the protective group P and R' to generate compound (Iq). The abbreviations used above correspond to the following: Bzl=benzyl; BOC=t-butoxycarbonyl; tBu=t-butyl;
CB~ben~yloxyc~'~nyl; FMOC=9-fluorenylmethoxycarbonyl; TMS=trimethylsilyl.

~ WO 95/06031 - . PCT/US94/09343 2~0158 A hydl~xylamine reagent described above can be hydroxylamine or ~ltern;ltively, it can be an O-protected hydroxylamine such as commercially available O-trimethylsilyl hydroxylamine, O-tert-butylhydroxylamine, or O-benzylhydroxylamine.
The ~.re~)aldlion of precursor compound (Io) may be carried out by condensing the dicarboxylate compound (Ie), with the amine (In), wherein R" is an activating group (Bod~n~7~y, M.; et al., suprà.) such as an active ester, anhydride or other group that causes con-1~n~tion with the amine terminus of compound (In) to occur with formation of a peptide 10 bond.

The preparation of compound (Ie) may be typically carried out as follows: the sodium salt of the 2-oxocarboxylate compound (Ia), is esterified with benzyl bromide to produce the benzyl ester (Ib). Several examples of compound (Ia) are commercially 15 available as various salts or carboxylic acids. Others can be made synthetically (see, for example, Nimitz, J. et al., J. Org. Chem. 46:211, 1981; and Weinstock, L.et al., Synth.
Commun. 11:943, 1981). The benzyl ester compound (Ib) is treated with a Wittig reagent, typically methyl or tert-butyl triphenylphosphoranylidene acetate, to form the alkene (Ic), as a mi~Lule of E- and Z- isomers. Reduction of the alkene compound (Ic) is carried out with 20 hydrogen, in the presence of an appropriate catalyst (typically palladium on activated charcoal), to both hydrogenate the double bond and to remove the benzyl ester, giving the mono-ester compound (Id) as a enantiomeric mixture. Compound (Ie) is obtained bytreating the mono-ester compound (Id) using any of a variety of conventional carboxylate activation procedures.
The preparation of the amine compound (In) is achieved by condensing the com~ou,ld (Il~ with the amine compound (Ik), wherein P' is an amine protective group other than P, and R" is an activating group such as an active ester, anhydride or other group that causes condensation with the amine terminus of (Ik) to occur with formation of a peptide 30 bond, to give compound (Im). Removal of P' is accomplished under a~p~ iate conditions (Bodanszky, M.; Bodanszky, A., "The Practice of Peptide Synthesis"; Springer-Verlag:
Berlin, 1984; Chapter III) to produce compound (In), either as corresponding amine or the amine salt.

3~ Compound (Il) is prepared from the commercially available N-protected carboxylic acid, or which can be synthesized by standard methods.

WO 9S/06031 ~ ~ 7 (1 15 8 PCTIUS94/09343 Preparation of (Ik) is carried out by condensing the compound (Ii) with mono-protected diamine (Ih) wherein P is an amine protective group such as CBZ, BOC, FMOC or other suitable protective group; and P' is an amine protective group other than P, and R" is an activating group such as an active ester, anhydride or other group that causes 5 con~ens~tion with the unprotected amine terminus of compound (Ih) to occur with formation of a amide bond to give compound (Ij). Removal of P' under a~ .-iate conditions is accomplished to produce compound (Ik), either as the corresponding amine or the arnine salt.

Precursor compound (Ih) is ~ paled in two steps from the amine-nitrile (If).
Several examples of compound (If) are available commercially and others can be easily synthesized by classical methods. The amine-nitrile (If) is protected with an a~p-u~-iate ploleclive group reagent to produce the protected amine-nitrile (Ig). In compound Ig, P is typically CBZ, BOC or FMOC groups, but can be any other suitable group. The protected 15 amine-nitrile (Ig) undergoes reduction with a reagent such as borane-methyl sulfide complex or sodium borohydride/cobalt (II) chloride, to give the mono-protected diamine (Ih) which can be isolated as its amine salt.

Compound (Ii) is p-~l)al~d from the carboxyl form of the co--Gs~-onding P'-pl~ule~;led 20 dipeptide or P'-protected amino acid by conventional methods, or can be purchased cc" . " "~ ;ially.

The compounds of formula II may be aclmini~tered orally, parenterally, via inh~l~tion, transdermally, intra-nasally, intra-ocularlly, mucosally, rectally and topically.
25 Such a~lminictration may be in dosage unit formulations containing conventional adjuvants and carrier materials. The term "pal~nte-~l" as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection or infusion techniques.

The amount of active ingredient that may be combined with the carrier materials to 30 produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Such carrier materials are well known, and are described, for example, in European Patent Application No. 0 519 748, incorporated herein by reference.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, 35 the age, body weight, general health, sex, diet, time of administration, route of arlmini~tration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

WO95/060~ Q 1~ g PCT/US94/09343 The following examples are illustrative of the invention. Thin layer chromotagraphy was ~elrcl"led using silica gel 60 F2s4 plates. Reaction schemes for Examples 1 through 9 are appende~d and follow Example 14. As used heren, "Compound A" refers to the compound N- { D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl } L-3-(2'naphthyl)-5 alanyl-L-alanine amide described by Spatola et. al., Peptides: Chemistry and Biology, Proceedings of the 12th American Peptide Symposium, eds. Smith, J.A., Rivier, J.E., ESCOM, Leiden, Netherlands. Compound A was ~ ,ar~d using the following procedure, and a reaction scheme therefor is appended as reaction scheme A.
10 ~ ion of Compound A
Referring to reaction scheme A and scheme 2, a mixture of 2.0g (6.3 mmol) of N-BOC-L-3-(2'-naphthyl)alanine and 0.80g (6.9 mmol) of N-hydroxysuccinimi(le7 and 1.8g (9.5 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in anhydrous N,N-dimethylfo,,.~ -ifle (10 ml) was stirred for 90 minutes at room lelllp~ldture. To this was added 1.2g (9.5 mmol) of L-alanine amide hydrochloride, followed by 1.4 ml (9.5 mmol) of triethylamine dissolved in S ml of anhydrous N,N-dimethylful".~...i(le. After stirring at room temperature for 14 hours, the solvent was removed in vacuo. The residue was dissolved in ethyl acetate (200 ml) and washed with lM HCl (3x50 ml), water (2x50 ml), saturated sodium bicarbonate (2x50 ml) and finally brine (50 ml). After drying over anhydrous magnesium sulfate, the solution was filtered and concentrated in vacuo to give 2.1g (86%) yield) of N-BOC-L-3-(2'-naphthyl)alanyl-L-alanine amide (Al) as a white solid. TLC: Rf 0.16 (chloroform-isopropanol 19:1); NMR (d6-DMSO) o 1.15 (m,3H), 1.24(s,9H), 3.05(m,2H), 4.23(m,2H), 7.02(s,1H), 7.07(s,2H), 7.35(s,1H), 7.47(m,2H), 7.71(s,1H), 7.82(m,3H), 7.98(d,1H).
A suspension of 1.8g (4.7 mmol) of (Al) in dichloromethane (15 ml) was cooled with an ice bath. Trifluoroacetic acid (15 ml) was added and the homogeneous solution was stirred at ca.5 C for 5 minutes, then allowed to warm to room temperature. After 1 hour the dichloromethane and the trifluoroacetic acid were removed in vacuo. The residue was dissolved in anhydrous N,N-dimethylform~micle (18 ml) containing 5.6 ml (33 mmol) of triethylamine. To this was added 1.2g (4.2 mmol) of (ld) in one portion. After stirring for 14 hours, the N,N-dimethylformamide was removed in vacuo to give a residue. The residue was dissolved in ethyl acetate (250 ml) and washed with lM HCl (2x75 ml), water (75 ml), saturated sodium bicarbonate solution (2x75 ml) and finally brine (75 ml). After drying over anhydrous magnesium sulfate, the solution was filtered and concentrated to produce l.5g (89% yield) of N-{D,L-2-(methoxycarbonyl)methyl-4-methylpentanoyl}-L-3-(2'-naphthyl)alanyl-L-alanine amide (A23 as a white solid. TLC: Rf 0.57 (chloroform-iS~~ ol 9:1);
MS: mle 455 (M+) ~170~ 58 Under an atmosphere of argon, a mixture of 0.62g (11 mmol) of KOH in 2.8ml of hot methanol was combined with a mixture of 0.61g (8.8 mmol) of hydroxylamine hydrochloride in 2.8 ml of hot methanol. After cooling in an ice bath, the reaction was filtered into a flask containing 1.0g (2.2 mmol) of (A2) and 1 ml of anhydrous N,N-dimethylf~ .icle After stirring for 18 hours, the solvent was removed in vac~o . The solid was dissolved in hot ethyl acetate (250 ml) and washed with 16 ml of 10% potassium bisulfate solution. The organic phase was heated to its boiling point before drying over anhydrous sodium sulfate. Filtration and subsequent concentration of the filtrate in vacuo 10 produced a solid, which was ~ uldled with ether (50 ml) and collected by filtration to give 0.77g (77% yield) of N-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-~3-(2'-naphthyl)alanyl-L-alanine amide (A) as a white solid The diastereomers of (A) were separated and purified by reverse phase HPLC using a C1g column, eluting with water col~ i,-g 0 1% trifluoracetic acid with a gradient of acetonitrile (0-60% in 30 minutes) and 15 also containing 0 1% ~ifluoroacetic acid, ("Method A"), to give a purified early eluting dia~Lcreolller and a purified late eluting diastereomer, which had retention times of 21 and 23 esrespectively. TLC: Rf0.13(chloroform-methanol9:1) lH NMR(d6-DMSO) o 0.63~d,3H), 0.72(d,3H), 0.90(m,1H), 1.21(d,3H), 1.26(m,2H), 1.86(m,2H), 2.63(m,1H), 2.99(m,1H), 3.24(m,1H), 4.18(q,1H), 4.55 (m,lH), 20 7.05(s,1H), 7.28(s,1H), 7.48(m,3H), 7.72(s,1H), 7.83(m,3H), 7.91(d,1H), 8.27(d,1H);
13C NMR (D2O/CD3CN) o 17.7, 21.8, 23.1, 26.0, 36.3, 37.4, 41.5, 42.2, 50.1, 55.5, 126.7, 127.1, 128.2, 128.5, 128.8, 129.0, 133.2, 134.2, 135.6, 170.4, 173.0, 177.4, 177.5.
MS: mle 456 (M+) FXA~PJ,F 1 Synthesis of N- ~ D.L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl ~ -L-3-(2'-naphthyl)alanyl-L-alanine.2-aminoethyl amide (Compound 1) HO-H N ~N V--N ~ N _ N H2 HOA~
0~< 0 ~

WO 95/06031 2 ~ 7 0 i 5 ~ PCT/US94/09343 With reference to reaction Scheme 2, a slurry of 25g (0.164 mol) of the sodium salt of 4-methyl-2-oxopentanoic acid, sodium salt in anhydrous N,N-dillleLhylfc" ."~.-,;de (50 ml) cont~ining 19.6 ml (0.164 mol) of benzyl bromide was ~git~te,(l at room temperature for 4 days. The solvent was removed in vacuo. The residue was dissolved in 250 ml of hexane S and washed with water (3x50 ml) and brine (50 ml) . After drying over anhydrous m~nçsillm sulfate, the solution was filtered and concentrated in vacuo to give 33.2g (92%
yield) of benzyl 4-methyl-2-oxopentanoate (la) as a viscous, colorless oil. TLC: Rf 0.70 (ethyl acetate-hexane 1:4); 1H NMR(CDCl3) o 0.94(d, 6H), 2.18(m,1H), 2.71(d, 2H), 5.26(s, 2H), 7.37(m, 5H); 13C NMR (CDCl3) o 22.5, 24.2, 48.1, 67.9, 128.7, 128.8, 128.9, 134.7, 161.3, 194Ø

A solution of 26.4g (0.120 mol) of benzyl ester (la) and 40.1g (0.120 mol) of methyl (triphenylphosphoranylidene)acetate in dichl~ l~",lethane (410 rnl) was sti~red at room t~ p~ UlG for 18 hours. Removal of the dichloromethane in vacuo produced a solid which lS was triturated with several volumes of hexane (4xlO0 ml). The hexane volumes were collected by filtration, combined and concentrated in vacuo to produce an oil which was fli~tille~l at reduced pressure (bp.138-157 C/0.8mm Hg) to obtain 27.8g (84% yield) of purified benzyl E,Z-2-isobutyl-3-(methoxycarbonyl)propenoate (lb) as a yellow oil. TLC:
Rf 0.53 and 0~67; E and Z isomers (ethyl acetate-hexane 1:4); NMR(CDC13) o O.91(m, 6H, CH(CH3)2), 1.85(m,1H, CH(CH3)2), 2.23(Z) and 2.79(E) (d, 2H, C=CCH2), 3.62(Z) and 3.74(O (s, 3H, C02CH3), 5.23(E) and 5.27(Z) (s, 2H, CO2CH2C6Hs), 5.82(Z) and6.82(E) (s, 1~, CH=C), 7.35(m, SH, C6Hs).

A suspension of 4.0g of 10% palladium on activated carbon in a solution of 27.2g(0.098 mol) of (lb) dissolved in 75 ml of methanol was agit~te~l under 4 atmospheres of hydrogen for 24 hours. Removal of the catalyst by filtration and conce~ tion of the filtrate invacuo gave an oil which was distilled at reduced pressure (bp.115-121C/O.Smm Hg) to obtain 12.7g (68%) of D,L-2-isobutyl-3-(methoxycarbonyi)propionic acid (lc) as acolorless oil. lH NMR(CDC13) â 0.94 (m, 6H), 1.36(m,1H), 1.63(m, 2H), 2.58(m, 2H), 2.95(m, lH), 3.70(s, 3H), 10.8(bs,1H); 13C NMR (CDCl3) o 22.1, 22.3, 25.6, 35.8,39.2, 40.8, 51.7, 172.2, 181.3.

A solution of 12.3g (0.065 mol) of (lc) and 7.5g (0.065 mol) of N-hydroxysuccinimitle dissolved in anhydrous tetrahydrofuran (100 ml) was cooled to ca. 5 C
with an ice bath. A solution of 13.5g (0.065 mol) of 1,3-dicyclohexylcarbodiimide dissolved in anhydrous tetrahydlofuldn (50 ml) was added. The n~ ul~; was stirred at ca. 5 C for 1 hour, then allowed to stand overnight under refrigeration. After removal of the dicyclohexylurea by-product by filtration, the filtrate was concentrated invacuo to produce a WO 95/06031 ~ 1 7 0 ~ PCT/US94/09343 solid, which was recryst:~lli7çd from ethyl acetate-hexane to give 14.5g (78% yield) of D,L-2-isobutyl-3-(methoxycarbonyl)propionic acid, N-hydroxysuccinimidyl ester (ld) as a white solid. TLC: Rf 0.46 (chloroforrn-isopropanol 19:1); lH NMR(CDC13) ~ 0.97(m, 6H), 1.61(m,2H), 1.80(m, lH), 2.72(m, 2H), 2.84(s, 4H), 3.74(s, 3H); 13C NMR
(CDC13) ~ 21.9, 22.5, 25.5, 36.2, 37.2, 41.0, 52.0, 168.8, 170.6, 171Ø

To a solution of 24.9g (0.10 mol) of benzyl succinimidylcarbonate and 10.2g (0.llmol) of aminoacetonitrile hydrochloride dissolved in anhydrous N,N-dimethylrc,~ ,.-ide (100 ml) was added 15.4 ml (0.1 lmol) of triethylamine over a period of 10 30 l~ u~es at room temperature. The Il~ ul~ was stirred at room temperature for 12 hours.
Removal of the N,N-dimethylformamide in vacuo produced a residue which was dissolved in 350 ml of ethyl acetate. The solution was washed with water (350 ml), 2M HCl (3x50 ml) and brine (50 ml). After drying over anhydrous magnesium sulfate, the solution was ~lltered and concentrated in vacuo to give 17.3g (91% yield) of N-CBZ-aminoacetonitrile 15 (le) as an amber solid. TLC: Rf 0.65 (ethyl acetate-hexane 1:1); lH NMR(CDC13) 4.05(d, 2H), 5.13(s, 2H), 5.46(bt, lH), 7.35(bs, 5H); 13C NMR (CDCl3) ~ 29.5, 67.9, 116.2, 128.3, 128.5, 128.7, 135.5, 155.7.

Under an atmosphere of dry argon, 24.3g (0.128 mol) of N-CBZ-aminoacetonitrile 20 (le) was dissolved in anhydrous tetrahydrofuran (32 ml). The solution was stirred and 64 ml of borane-methylsulfide complex (2M in tetrahydrofuran) was added via syringe. The Lulc was heated to reflux and stirred overnight. The llli~UlG was cooled with an ice bath as 5 ml of water was added slowly, with vigorous stirring. The stirring was continued for ca. 5 minutes, then 75 ml of 6M HCl was slowly added. The mixture was stirred for 1 25 hour, then the excess tetrahydroruldn and dimethyl sulfide was removed ~n vacuo. The aqueous residue was extracted with ether (2x50 ml). The ether extracts were then discarded.
The pH of the aqueous residue was raised to 11 by adding concentrated NH40H. Theresulting aqueous solution was extracted with ethyl acetate (3x100 ml) and the ethyl acetate extracts were combined and washed with brine (50 ml). After drying over anhydrous 30 magnesium sulfate, the solution was filtered and concentrated in vacuo. The resulting oil was dissolved in 30 ml of anhydrous methanol, treated with cold methanolic HCl and concentrated invaCuo to produce a solid. The solid was triturated with ether and collected by filtration to give 15.1g (51% yield) of N-CBZ-ethylenefli~mine hydrochloride tlf) as a white powder. lH NMR(D20) â 3.15(m, 2H), 3.46(m, 2H), 5.14(s, 2H), 7.46(bs, 5H);35 13C NMR (D2O) ~ 41.1, 42.6, 70.4, 131.0, 131.3, 131.7, 132.0, 139.4, 161.7.

A solution of 10.0g (0.043 mol) of (lf) and 10.3g (0.036 mol) of N-BOC-L-~l~nine7 N-hydroxysuccinimide ester in anhydrous N,N-dimethylformamide (50 ml) was WO 9S/06031 ~ ~ 7 ~ ~ 5 8 PCTIUS94/09343 cooled with an ice bath. To this was added 7.6 ml (0.054 mol) of triethylamine in anhydrous N,N-dimethylformamide (20 ml) over a period of 30 minutes. The reaction was stirred at ca. 5 C for 1 hour, then at room temperature for 1 hour. The N,N-dimethylrc,~ ,-lide was removed in vacuo and the resulting residue was dissolved in 300 ml of ethyl acetate. The solution was washed with lM HCl (3x100 ml), water (100 ml), saturated sodium bicarbonate solution (3x100 ml) and finally, with brine (100 ml). After drying over anhydrous magnesium sulfate, the solution was ffltered and concentrated in vacuo to give 12.4g (94% yield) of N-BOC-L-alanine, 2-(benzyloxycarbonylamino)ethyl amide (lg) as a white solid. TLC: Rf 0.67 (chloroform-iso~r~allol 9:1); lH NMR(CDC13) ~ 1.27(d, 3H), 1.40(s, 9H), 3.32(m, 4H), 4.15(m, lH), 5.06(s, 2H), 5.51(d, lH), 5.90(m,1H), 7.19(m, lH), 7.31(bs, 5H); 13C NMR (CDC13) ~ 18.5, 28.2, 39.6, 40.5,50.1, 66.5, 79.8, 127.9, 128.3, 136.3, 155.4, 156.9, 173.7.

A solution of 12.0g (0.033 mol) of (lg) in 25 ml of dichloromethane was cooled with an ice bath and 25 ml of trifluoroacetic acid was added. The solution was stirred at ca 5 C for 20 minutes, then allowed to stir to room telll~cl~ture. After 90 minutes, the dichlc,~ Ll.~l-e and trifluoroacetic acid were removed in vacuo . The resl-lting residue was dissolved in 200 ml of ethyl acetate and washed with 2M sodium hydroxide (200 ml) and brine (100 ml). After drying over anhydrous magnesium sulfate, the solution was filtered and concentrated in vacuo to produce 7.86g (90% yield) of L-alanine, 2-(benzyloxycarbonylamino)ethyl amide (lh) as a white solid. 1H NMR(CDCl3) o 1.28(d, 3H), 2.09(m, 2H), 3.33(m, 4H), 3.47(q, lH), 5.07(s, 2H), 5.59(bt, lH), 7.33(bs, 5H), 7.69(bt, lH);13C NMR (CDC13) ~ 21.3, 39.5, 40.9, 50.4, 66.6, 128.0, 128.1, 128.4, 136.4, 156.9, 176.7.
Under an atmosphere of dry argon, a solution of 8.9g (0.028 mol) of N-BOC-L-3-(2'-naphthyl)alanine and 3.2 ml (0.028 mol) of 4-methylmorpholine in anhydrous N,N-dimethylform~mide (20 ml) was cooled to -15 C and treated with 3.67 ml (0.028 mol) of isobutyl chlcloru~ a~e. The Illi~Lul~; was stirred at -15 C for 30 minutes, then a solution of 7.5g (0.028 mol) of (lh) and 3.2 ml (0.028 mol) of 4-methylmorpholine in anhydrous N,N-dimethylfollllalllide (20 ml) was added slowly, over 10 minutes. The reaction was stirred at -15 C for 2 hours, then at room temperature for 18 hours. The N,N-dimethylform~mi-1e was removed in vaCuo and the resulting solid was dissolved in 1 liter of hot ethyl acetate. The hot solution was washed with lM HCl (3x150 ml), water (150 ml), saLul~ted sodium bicarbonate (3x150 ml) and finally with brine (150 ml). After drying over anhydrous magnesium sulfate, the hot solution was concentrated in vacuo. The resulting yellow solid was triturated with 400 ml of cold 1:3 ethyl acetate-hexane and collected by filtration to give 14.5g (91% yield) of N-BOC-L-3-(2'-naphthyl)alanyl-L-alanine, 2-WO 95/06031 2 ~ 7 ~ 1 ~ 8 PCT/US94/09343 l (benzyloxycarbonylamino)ethyl amide (li) as a white solid. TLC: Rf 0.59 (chlol~ fol.n-isopropanol 9:1); lH NMR(CDCl3) ~ 1.26(d, 3H), 1.35(s, 9H), 3.16(m, 6H), 4.42(m,lH), 4.50(m, lH), 5.07(s, 2H), 5.25(d, lH), 5.69(m, lH), 6.82(m, lH), 6.90(d, lH), 7.29(s, lH), 7.31(bs, SH), 7.45(m, 2H), 7.61(s, lH), 7.76(m,3H);13C NMR (CDCl3) S 18.0, 28.2, 38.2, 39.7, 40.6, 49.0, 55.9, 66.6, 80.6, 125.8, 126.2, 127.2, 127.5, 127.6, 127.9, 128.0, 128.4, 132.4, 133.3, 133.8, 134.2, 155.4, 156.7, 171.4, 172.4.

A suspension of 2.5g (0.0044 mol) of (li) in dichlol~"llethane (10 ml) was cooled with an ice bath and 10 ml of trifluoroacetic acid was added. The homogeneous solution was stirred at ca. 5 C for 20 minutes, then allowed to warm to room temperature. After 90 minlltes the dichlolu..~ nt- and trifluoroacetic acid were removed in vacuo . The resulting residue was dissolved in 100 ml of ethyl acetate and washed with 2M NaOH (3x50 ml), water (50 ml) and brine (50 ml). The non-homogeneous solution was transferred to a flask containing 100 ml of absolute ethanol, and heated until it became homogeneous. The hot solution was dried over a small amount of anhydrous sodium sulfate, filtered, and conce~ ted in vacuo to obtain a solid. The solid was triturated with cold 1:3 ethyl acetate-hexane and collected by filtration to give 1.46g (71% yield) of L-3-(2'-naphthyl)alanyl-L-~l~nine, 2-(benzyloxy-carbonyl-amino)-ethyl amide (lj) as a white solid. lH NMR(CDC13) ~ 1.33(d, 3H), 1.60(bs, 2H), 2.83(m, lH), 3.34(m, 5H), 3.82(m, lH), 4.44(m, lH),5.07(s, 2H), 5.33(t, lH), 6.92(t, lH), 7.31(bs, 5H), 7.36(s, lH), 7.48(m, 2H), 7.65(s, lH), 7.72(d, lH), 7.81(m, 3H);13C NMR (CDC13) ~ 17.6, 40.6, 40.7, 40.9, 48.6, 56.1, 66.9, 125.4, 125.8, 127.2, 127.4, 127.5, 127.8, 127.9, 128.4, 132.4, 133.4, 135.1, 136.5, 156.1, 172.7, 174.7.

To a solution of 1.4g (0.003 mol) of (lj) and 0.42 ml (0.003 mol) of triethylamine dissolved in anhydrous N,N-dimethylform~mide (2 ml) was added 0.87g (0.003 mol) of (ld). The mixture was stirred at room temperature for 18 hours. The N,N-dimethylform~mide was removed in vacuo. The resulting residue was dissolved in 200 ml of hot ethyl acetate and washed with lM HCI (3x50 ml), water (50 ml), saturated sodium bicarbonate solution (3x50 ml) and finally brine (50 ml). After drying over anhydrous m~.lesil.." sulfate, the hot ethyl acetate solution was filtered and concentrated in vacuo to give 1.7g (89% yield) of D,L-2-(methoxycarbonyl)methyl-4-methylpentanoyl-L-3-(2'-naphthyl)-alanyl-L-alanine, 2-(benzyloxycarbonylamino)ethyl amide (lk) as an off-white solid. TLC: Rf 0.32 (chlororol,ll-isopropanol 19 Under an atmosphere of argon, a I~ ul~; of 2.66g (0.047 mol) of KOH in 12 ml ofhot methanol was combined with a mixture of 2.63g (0.037 mol) of hydroxylamine hydrochloride in 12 ml of hot methanol. After cooling in an ice bath, the reaction was WO 95/06031 ,~ ~ 7 ~ 1 58 Pcrtuss4/o9343 filtered into a flask containing 6.0g (0.0095 mol) of (lk) and 12 ml of anhydrous N,N-dimethylro-..l~.-.i~le. After stirring under argon for 18 hours, the solvent was removed in vacuo. The resulting solid was triturated with 100 ml of ethyl acetate and collected by filtration to give 5.2g (86% yield) of D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl-L-3-(2'-naphthyl)alanyl-L-alan-ine, 2-(benzyloxycarbonylamino)ethyl amide (lm) as an off white solid. TLC: Rf 0.23 and 0.36 (chlolofol,.,-isopropanol 9:1);
13C NMR(d6-DMSO) o 18.0, 21.7, 23.2, 25.1, 35.7, 36.6, 37.3, 38.7, 40.7, 40.8, 48.5, 54.0, 65.3, 125.3, 125.9, 127.3, 127.4, 127.7, 127.9, 128.3, 131.8, 132.9, 135.7, 136.0, 137.1, 156.1, 167.1, 170.7, 172.7, 174.7.
MS: )nle 634 (M+).

A suspension of l.Og of 10% palladium on activated carbon in a solution of 2.0g (0.0031 mol) of (lm) dissolved in glacial acetic acid (75 ml) was agitated under 4 atmospheres of hydrogen for 24 hours. Removal of the catalyst by filtration, andconcentration of the filtrate in vacuo produced a residue which was triturated with 50 ml of ether and dried in vacuo to give 2.0g of crude D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl-L-3-(2'-naphthyl)alanyl-L-~l~ninç, 2-(amino)ethyl amide (1).

The diastereomers of (1) were separated by reverse phase HPLC using a Clg column and eluting with water containing 0.1% trifluoroacetic acid with a gradient of acetonitrile (0-60% in 30 minutes) also containing 0.1% trifluoroacetic acid (hereinafter "Method A"). The purified diastereomers (ln) and (lo) had retention times of 20 and 22 minutes, respectively. Diastereomer (ln) showed the following NMR data. 13C
NMR(D2O) o 24.6, 28.9, 29.1, 30.3, 33.2, 43.4, 44.8, 47.0, 48.6, 49.1, 57.6, 62.8, 134.2, 134.6, 135.3, 135.6, 135.8, 135.9, 136.4, 140.2, 141.2, 142.1, 178.3, 180.8, 183.1, 185.4.
MS: mle 500 (M+).

The following is an alternative method, which is a plerell~id method, for preparing compound ~(c) such that a greater ratio of the desired stereoisomer (R) is produced as coll~paled to the undesired stereoisomer (S). The reaction steps and reference numerals for the respective compounds are shown in Reaction Scheme 10.

- By following the procedure of Newman, M. S.; Kutner, A. J. Am. Chem. Soc.
1951, 73, 4199, a solution of sodium methoxide was prepared by dissolving 1.29g (0.056 mol) of sodium in l5ml of anhydrous methanol, which was added to a slurry of 25g (0.242 mol) of L-valinol in 500 ml of diethyl carbonate. The reaction mixture was then heated for 2 hours, with 200ml of distillate collected in the temperature range of 75-123 C. The distillate WO 95/06031 2 ~ 5 ~ PCT/US94/09343 was discarded and the reaction n~ ulc was allowed to cool to room l~l"~e-~ture and stand overnight. The excess diethyl carbonate was removed from the reaction ",i~ in vacuo by rotary evaporation to give a residue. The residue was dissolved in 500ml of ethyl acetate and washed with water (3 x 200ml) and brine (200ml). After drying over anhydrous magnesium S sulfate, the solution was filtered and concentrated in vacuo to give a white solid. The solid was recryst~lli7e-1 from ethyl acetate-hexane to produce 23.2g (74% yield) of (S)-4-isopropyl-2-oxazolidinone 12(a) as white needles. TLC of 12(a): Rf 0.50 (ethyl acetate-hexane 3:1); lH NMR (CDC13) d 0.90(d, J = 6.7Hz, 3H), 0.97(d, J = 6.7Hz, 3H), 1.72(m, lH), 3.63(m, lH), 4.10(dd, J = 8.7, 6.4Hz, lH), 4.45(m, lH), 7.32(bs, lH);
10 13C NMR (CDC13) d 17.5, 17.8, 32.6, 58.3, 68.5, 160.7.

Following the procedure of Vogel, A. In Vogel's Practical Organic Chemistry, 4thEd.; Wiley & Sons: New York, 1978; p 498 and 1208, 4-methylpentanoyl chloride 12(b) was ~lc~aled by adding dropwise with stirring, 38ml (0.52 mol) of thionyl chloride to 50g 15 (0.43 mol) of 4-methylvaleric acid over 30 minutes. The mixture was heated during the addition, leading to vigorous HCl gas evolution. When the thionyl chloride addition was completed, the reaction IlliX.~UlG was refluxed for 1 hour. The reaction ll~ G was ~ tilled, with collection of the ~ till~te between 135 and 148 C. The material was re-distilled and 47.3g (81% yield) of 4-methylvaleroyl chloride 12(b) was collected between 143 and 148 20 C as a colorless liquid. lH NMR (CDC13) d 0.92(d, J = 6.2 Hz, 6H), 1.62(m, 3H), 2.90(t, J = 7.4 Hz, 2H); 13C NMR (CDC13) d 22.0, 27.2, 33.6, 45.3, 173.9.

Following the procdure of Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem.
Soc. 1981,103, 2127, a solution of 32.3g (0.25 mol) of 12(a) in 500ml of anhydrous 25 tetrahydrofuran was cooled to -78 C and lOOml of 2.5M (0.25 mol) n-butyllithium in hexanes was added. When the addition was complete, the mixture was stirred at -78 C for 10 minutes, then warmed to 0 C and stirred for 20 minutes. The reaction mixture was cooled to -78 C and 34.6ml (0.25 mol) of 12(b) was added over 10 minutes. Stirring was continued at -78 C for one hour, then the reaction mixture was allowed to stir at room 30 t~ ture overnight. The tetrahydrofuran was removed in vacuo by rotary evaporation to produce an orange residue.

The residue was dissolved in 750ml of ethyl acetate and washed with water (2 x 250ml) and brine (3 x lOOml). After drying over anhydrous magnesium sulfate, the solution 35 was filtered and concenlldled in ~acuo to give 60g of orange oil.

o 95/06031 ~ ~ 7 01 ~ B . PCT/US94/09343 The oil was purified in two batches by flash cl~ atography on silica gel 60 (500 g).
The product was eluted with 1 :4 ethyl acetate:hexane to produce 48.6g (86%) of 12(c) as a pale yellow oil. TLC: Rf 0.42 (1 :4 ethyl acetate-hexane) lH NMR (CDCL3) d 0.88(d, J = 6.9Hz, 3H), 0.92(m, 9H), 1.57(m, 3H), 2.37(m, lH), 2.93(m, 2H), 4.25(m, 2H), 4.44(m, lH); 13C NMR (CDCl3) d 14.5, 17.9, 22.2, 27.6, 28.3, 33.2, 33.5, 58.3, 63.2, 153.9, 173.5.

Following the procedure of Evans, D.A.; Ennis, M.D.; Mathre, D.J. J. Am. C~em.
Soc. 1982, 104, 1737, a mixture of 16.3ml (0.116 mol) of diisopropylamine and 200ml of anhydrous tetrahydrofuran was cooled to -S C under an atmosphere of dry argon, and 46.5ml (0.116 mol) of n-butyllithium (2.5 M in hexanes) was added. The mixture was stirred at -S C for 25 minutes, then cooled to -78 C. A solution of 24.0g (0.106 mol) of 12(c) in 67ml of anhydrous tetrahydrofuran was added, and the reaction mixture was stirred at -78 C for 30 minutes. The reaction was allowed to warm to -5 C and 27.4 ml (0.317 mol) of allyl bromide was added. The Illi~LLllG was stirred at -S C for4 hours then 10 ml of water was added, followed by removal of the tetrahydlc~rulall by rotary evaporation to give an oil. The oil was dissolved in ethyl acetate (SOOml) and washed with water (125 ml) and brine ~3 x 125 ml). After drying over anhydrous magnesium sulfate, the solution was filtered and concentrated in vacuo by rotary evaporation to produce an oil. The oil was puri~led by filtering it through lOOg of silica gel 60 with 1.25 liters of 1:4 ethyl acetate-hexane. Five fractions of 250 ml each were collected. Each fraction was checked by TLC.
The fractions containing purified product were combined and the solvent was removed by rotary evaporation to give 26.8g (95% yield) of 12~d3 as a colorless oil. TLC: Rf 0.52 (1:4 ethyl acetate-hexane). IH NMR (CDCl3) d 0.89(m, 12H), 1.28(m,1H), 1.53(m, lH), 1.65(m, lH), 2.33(m, 3H), 4.06(m, lH), 4.23(m, 2H), 4.46(m, lH), 5.04(m, 2H), 5.80(m, lH); 13C NMR (CDC13) d 14.5, 18.0, 22.5, 22.8, 26.0, 28.3, 37.5, 40.2, 40.3, 58.5, 62.9, 117.0, 135.1, 153.6, 176.1.

Generally following the methods of Evans, D.A.; Ennis, M.D.; Mathre, D.J. J. Am.Chem. Soc. 1982,104, 1737, a solution of 20.2g (0.187 mol) of anhydrous benzyl alcohol dissolived in 63ml of anhydrous tetrahydrofuran was cooled to -5 C under a dry argon atmosphere and 56.1ml (0.140 mol) of n-butyllithium (2.5 M in hexanes) was added over 10 minlltes~ The reaction mixture was stirred at -5 C for 20 minutes, then a solution of 25.0g (0.0934 mol) of 12(d) dissolved in 380 ml of anhydrous tetrahydror.lldn (pre-cooled to -5C) was added. The reaction was stirred at -5 C for 2 hours, then water (SOml) was added. The reaction was allowed to warm to room temperature. The tetrahydrofuran was removed by rotary evaporation to produce a residue. The residue was dissolved in ethyl acetate (250nnl) and washed with water (125ml) and brine (125ml). After drying over WO 95/06031 ~ ~ 7 ~1 5 8 PCT/US94/09343 anhydrous magnesium sulfate, the solution was filtered and concentrated by rotary evaporation to produce an oil. The oil was purified by flash chromatography on silica gel (240g). The product was eluted with 97:3 hexane-ethyl acetate to give 38.9g (85%) of 12(e) as a pale yellow oil. The chiral auxiliary 12(a) was eluted with ethyl acetate for re-use (40% recovery). TLC of 12(e): Rf 0.80 (1:4 ethyl acetate-hexane). lH NMR
(CDC13) d 0.86(d, J = 6.8 Hz, 3H), 0.88(d, J = 6.8 Hz, 3H), 1.27(m, lH), 1.57(m, 2H), 2.23(m, lH), 2.33(m, lH), 2.58(m, lH), S.Ol(m, 2H), 5.10(s, 2H), 5.71(m, lH), 7.33(m, SH); 13C NMR (CDCl3) d 21.9, 22.9, 26.0, 37.0, 41.0, 43.4, 65.9, 116.7, 128.0, 128.1, 128.4, 135.3, 136.0, 175.5.
By generally following the procedures of Carlsen, P.H.J.; Katsuki, T.; Martin, V.S.; Sharpless, K.B. J. Org. Chem. 1981, 46, 3936, a suspension of 38.0g (0.154 mol) of 12(e~ and 145g (0.679 mol) of sodium periodate in 330 ml of acetonitrile, 330 ml of carbon tetrachloride and 497 ml water was stirred at O C, while 0.83g (2.4 mol%) of 15 r~lthenillm trichloride hydrate was added. The mixture was stirred at O C for lS minutes, then allowed to stir to room temperature for 4 hours. The reaction was filtered to remove the solid, using SOOml of dichloromethane and 250ml of water to rinse the solid collected. The filtrate was Lldn~ll~d to a separatory funnel and the layers were separated. After drying over anhydrous magnesium sulfate, the lower(dichloromethane) layer was filtered and 20 cf~.~ce~ Led in vacuo by rotary evaporation to produce a dark oil. The oil was purified with two successive flash chromatography columns [each column: 500 grams of silica gel 60, eluted with l900ml of 1:4 ethyl acetate:hexane, and 1000 ml of ethyl acetate] to produce 26.6 (65% yield) of 12(f~ as a viscous oil.
TLC of 12(f): Rf 0.10 (1:4 ethyl acetate-hexane). lH NMR (CDC13) d 0.88(d, J
25 = 6.2 Hz, 3H), 0.92(d, J = 6.4 Hz, 3H), 1.33(m, lH), 1.60(m, 2H), 2.49(dd, J = 17.0, 4.8 Hz, lH), 2.77(dd, J = 17.0, 9.S Hz, lH), 2.94(m, lH), 5.15(s, 2H), 7.35(m, SH), ll.l(bs, lH); 13C NMR (CDC13) d 22.2, 22.4, 25.7, 36.1, 39.2, 41.0, 66.4, 128.0,128.1, 128.4, 135.8, 174.9, 178.2.

Ethereal diazo~ llane (Aldrich Chemical Co. Technical Information Bulletin No.
AL-180) was slowly added to a solution of 22g (0.083 mol) of 12(f) in 50 ml of diethyl ether until the reaction mixture remained yellow with swirling. The reaction mixture was back titrated to colorlessness with 1 :9 acetic acid-diethyl ether. After drying over anhydrous magnesium sulfate the colorless solution was filtered and concentrated in vacuo by rotary 35 evaporation to produce a viscous oil. The oil was dissolved inlOOml of methanol and transferred to a Parr bottle containing l.Og of 10% p~ r~ m on charcoal catalyst and shaken under 4 atm. of hydrogen for 6 hours at room temperature. The IlliXlul~ was filtered through celite and the filtrate was concentrated in vacuo by rotary evaporation to produce an oil. The ~ W095/06031 2~ 7~15~ ~ PCr/US94109343 oil was vacuu7m distilled to give 13.9 g (89% yield) of 12(f) as a colorless oil; b.p. 110-123 C / 0.2mmHg.
TLCofl2(f~: Rf 0.15 (3:7 ethylacetate-hexane) TLC of methyl ester intenneAi~te: Rf 0.73 (3:7 ethylacetate-hexane) TLC of l(c):Rf 0.23 (3:7 ethyl acetate-hexane). IH NMR of l(c) (CDC13) dO.91(d, J = 6.3 Hz, 3H), 0.95(67.4 Hz, 3H), 1.33(m, lH), 1.64(m, 2H), 2.45(dd, J = 16.7, 11.43(bs, lH);
3C NMR of ~I(c) (CDC13) d 22.2, 22.4, 25.7, 35.8, 39.3, 40.9, 51.8, 172.3, 181.6.

F.XAMPl,F 2 Synthesis of N-~D.L-2-(hydroxyaminocarbonvl)methvl-3-methylbutanoyl~-L-3-(2'-naphthyl)-L-alanine amide (Compounds 2 and 3) Y H
HO-HN ~ .~ NH2 0~<

Referring to Scheme 3, Compound (2d) was synthesized from the sodium salt of the3-methyl-2-oxobutanoic acid by the sequence of reactions used to prepare compound (ld) from 4-methyl-2-oxol~e,~ oic acid, sodium salt.

Compound (2a): 73% yield; bp. 100-121 C/0.3mmHg;
lH NMR(CDC13) o 1.13(d,6H), 3.24(m,1H), 5.27(s,2H), 7.37(m,5H); 13C
NMR(CDC13) o 17.0, 37.0, 67.6, 128.4, 128.5, 128.6, 134.5, 161.5, 197.7.

Compound (2b): 58% yield; bp. 125-147 C/0.6mmHg;
TLC: Rf 0.54(ethyl acetate-hexane 1:4), lH NMR(CDC13) o l.ll(d,6H), 2.66(m,1H), 3.62(s, 3H), 5.27(s,2H), 5.79(s,1H), 7.35(m,5H); 13C NMR (CDC13) ~ 20.4, 32.7, 25 51.5, 67.0, 117.0, 128.2, 128.3, 128.5, 135.3, 156.2, 165.4, 168.4.

Compound (2c): 76% yield; bp. 115-119 C/0.7mmHg; TLC: Rf 0.09 (ethyl acetate-hexane 1:4); lH NMR(CDC13) ~ 0.96(d,3H), O.99(d,3H), 2.09(m,1H), 2.43(m,1H), 2.76(m,3H), 3.69(s,3H); 13C NMR(CDC13) ~ 19.1, 19.8, 29.7, 32.1, 47.0, 51.7, 172.8,. 30 180.4.

WO 9S/06031 PCT/US94/09343 ~

Compound (2d3: 55% yield; TLC: Rf0.60(chloroform-isopropanol 19:1); 1H
NMR(CDC13) ~ 1.06(d,3H), 1.08(d,3H), 2.12(m,1H), 2.58(m,1H), 2.84(m,5H), 3.07(m,lH), 3.72(s,3H); 13C NMR(CDC13) o 19.4, 19.6, 25.6, 30.3, 33.1, 45.2, 52.1, 168.9, 169.6, 171.5.
The diastereomers (2) and ~3) can be made from L-3-(2'-naphthyl)zll~nine amide hydrochloride (8b) and compound (2d), using the sequence of reactions used to prepare Compound (1) from Compounds (lj) and (ld). Compounds (2) and (3) were separated by reverse phase HPLC as described above.
Compound (2): HPLC retention time (Method A) 21 ,~inl-~es lH NMR(CD3CN/D2O) ~ 0.19(d,3H), 0.50(d,3H), 1.38(m,1H), 2.24(m,3H), 2.95(m,1H), 3.50(m,1H), 4.68(m,1H), 7.48(m,3H), 7.76(s,1H), 7.83(m,3H); 13C NMR
(CD3CN/D2O) o 20.2, 20.3, 31.1, 33.4, 38.0, 50.2, 55.5, 126.7, 127.2, 128.4, 128.6, 129.1, 129.2, 133.8, 134.4, 136.6, 171.5, 176.3, 176.4.
MS: mle 371 (M+).

Compound (3): HPLC retention time (Method A) 23.1 minutes.
MS: mle 371 (MH+).
FXAMPI,F 3 Synthesis of N-r3-(hyd,v~cyaminocarbonyl)propanoyll-L-3-(2'-naphthyl~alanyl-L-alanine amide (Compound 4) HO-H N -~L~~r N _~--N I N H2 ~<

~_d Referring to Scheme 4, to a solution of 1.74g (10 mmol) of tert-butyl hydrogen succinate (Buchi, G.; Roberts, C. J. Org Chem., 33:460, 1968) and 1.15g (10 mmol) of N-hydroxy-succinimide in anhydrous tetrahydrofuran (20 ml) was added 2.06g (10 mmol) 30 of 1,3-dicyclohexylcarbodiimide After stirring at room temperature overnight, the reaction was filtered to remove the dicyclohexylurea by-product. The filtrate was concentrated in vacuo to give a residue. Chromatography on silica gel using ethyl acetate-hexane (1:1), 1~ Wo 95/06031 ~ ~ 7 0 1 ~ 8 PCT/US94/09343 provided 2.3g (84% yield) of tert-butyl succinimidyl succinate (4a) as a white solid. TLC:
Rf 0.50 (ethyl acetate-hexane 1: 1); NMR(d6-DMSO) ~ 1.39(s,9H), 2.56(m,2H), 2.80(bs,4H), 2.86 (m,2H).

A solution of 0.70g (1.8 mmol) of (A13 dissolved in 5.0 ml of trifluoroacetic acid was stirred at room temperature for 90 minutes. The trifluoroacetic acid was removed in vacuo to give a residue which was triturated with ether (20 ml) and dried in vacuo to give 0.72g of a pink solid. A portion (0.35g) of the solid was dissolved in 2.0 ml of anhydrous N,N-dilllt;Ll,ylr~ --H.-.i~le. To this was added 0.24g (0.87 mmol) of (4a) and 0.18 ml (1.3 mmol) of triethylamine. After stirring at room temperature for 2 hours, the solvent was removed in vacuo to produce a residue. Chromatography on silica gel using chloroform-isopr~anol 9:1 provided 0.32g (84% yield) of N-[3-(tert-butoxycarbonyl)propanoyl]-L-3-(2'-naphthyl)alanyl-L-alanine amide (4b) as white solid. TLC: Rf 0.33 (chloroform-isopropanol 9:1); 1H NMR(d6-DMSO) ~ 1.23(d,3H), 1.30(s,9H), 2.27(m,4H), 2.93(m,1H), 3.20(m,1H), 4.22(m,1H), 4.61(m,1H), 7.03(s,1H), 7.22(s,1H), 7.46(m,3H), 7.75(s,1H), 7.83(m,3H), 8.07(d,1H), 8.19(d,1H); 13C NMR(d6-DMSO) o 18.3, 27.8, 30.1, 30.3, 37.6, 48.1, 54.1, 79.6, 125.4, 126.0, 127.4, 127.5, 127.9, 131.9, 133.0, 135.8, 170.8, 171.1, 171.6, 174.1.

A solution of 0.29g (0.64 mmol) of (4b) dissolved in lOml of trifluoroacetic acid was stirred at room temperature for 30 minutes. The trifluoroacetic acid was removed in vacuo to give a residue which was triturated with ether (20ml) and dried in vacuo to give 0.24g (95% yield) of N-[3-carboxypropanoyl]-L-3-(2'-naphthyl)alanyl-L-alanine amide (4c) as a white solid. TLC: Rf 0.04 (chloroform-isopropanol 9:1); 1H NMR(d6-DMSO) 1.23(d,3H), 2.29(m,4H), 2.92(m,1H), 3.21(m,1H), 4.21(m,1H), 4.58(m,1H), 7.04(s,1H), 7.23(s,1H), 7.46(m,3H), 7.75(s,1H), 7.83(m,3H), 8.06(d,1H), 8.21(d,1H);
3C NMR (d6-DMSO) o 18.3, 29.1, 30.0, 37.6, 48.2, 54.1, 125.4, 126.0, 127.4, 127.5, 128.0, 131.9, 133.0, 135.8, 170.8, 171.3, 173.9, 174.1.

Under an atmosphere of dry argon, a solution of 0.22g (0.56 mmol) of (4c) and 0.062 ml (0.56 mmol) of 4-methylmorpholine anhydrous N,N-dimethylrc"",al,lide (2 ml) was cooled to -15 C and treated with 0.073 ml (0.56 mmol) of isobutyl chloroformate. The ulc was stirred at -15 C for 15 minutes, then a solution of O.lOg (0.81 mmol) of (O-benzyl)hydroxylamine in anhydrous N,N-dimethylformamide (0.5 ml) was added. The llli~Lul~ was stirred at -15 C for 1 hour, then at room temperature for 1 hour. The solvent was removed in vacuo. The resulting solid was triturated with ethyl acetate and collected by filtration to obtain 0.20g (73% yield) of N-[3-(benzyloxyaminocarbonyl)propanoyl]-L-3-(2'-naphthyl)alanyl-L-alanine amide (4d) as a white solid. TLC: Rf 0.46 (chloroform-WO 95/06031 ~ 17 ~ ~ ~ 8 PCT/US94/09343 iso~r~anol 8:2); lH NMR (d6-DMSO) ~1.26(d,3H), 2.25(m,4H), 2.95(m,1H), 3.22 (m,lH), 4.23(m,1H), 4.57(m,1H), 4.74(s,2H), 7.03(s,1H), 7.16(s,1H), 7.36(bs,5H),7.46(m,3H), 7.77(s,1H), 7.83(m,3H), 8.12(d,1H), 8.32(d,1H), 11.03(s,1H); 13C
NMR(d6-DMSO) ~18.3, 27.9, 30.4, 37.6, 48.4, 54.5, 77.0, 125.6, 126.1, 127.6, 128.1, 128.4, 128.5, 129.0, 132.0, 133.2, 136.0, 136.2, 169.0, 171.0, 171.7, 174.3.

A suspension of 0.20g of 5% palladium on activated carbon in a solution of O.lOg(0.20 mmol) of (4d) in 4 ml of glacial acetic acid was agitated under 4 atmospheres of hydrogen for 18 hours. Removal of the catalyst by filtration, and col~ce.,lldlion of the filtrate in vacuo produced a residue which was Llilulated with 10 ml of ether and dried in vacuo to give a solid. Ch,u",alography on Baker octadecyl reverse phase gel, eluting with water-acetonitrile-acetic acid(57:40:3), provided 0.065g (79% yield) of N-[3-(hydlo~ya~l~inocarbonyl)-propan-oyl]-L-3-(2'-naphthyl)alanyl-L-alanine amide (4), as a white solid. TLC: Rf 0.05 (chloroform-isopropanol 8:2); lH NMR(d6-DMSO) ~
1.24(d,3H), 2.08(m,2H), 2.28(m,2H), 2.92(m,1H), 3.22(m,1H), 4.20(q,1H), 4.54(m,1H), 7.02(s,1H), 7.20(s,1H), 7.46(m,3H), 7.76(s,1H), 7.84(m,3H), 8.12(d,1H), 8.27(m,1H), 10.39(s,1H); 13c NMR(d6-DMSO) ~18.0, 27.6, 30.4, 37.3, 47.9, 54.0, 125.3, 125.8, 127.2, 127.3, 127.7, 131.7, 132.8, 135.7, 168.3, 170.5, 171.3, 174Ø

F~AMPT F 4 Synthesis of N -~DL-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl~-L-ar~inyl-T -alanine, 2-aminoethyl amide (Co~ ,ound 5) HO-HN~--~N--LN~N--~NH2 ~NH
HN~ NH2 With reference to Scheme 5, Compound (5a) was synthesized from Compound (lh) and Na-Boc-Ng-(di-cBz)-L-arginine in 79% yield, by following the method used to prepare Compound (li). TLC: Rf 0.59 (chloroform-isopropanol 9:1); lH NMR (CDC13)~ 1.18(d,3H), 1.40(s,9H), 1.62(m,4H), 3.27(m,4H), 3.89(m,2H), 4.09(m,1H), 4.21(m,1H), 5.06(s,2H), 5.13(m,2H), 5.22(s,2H), 5.58(m,1H), 5.67(m,1H), 6.70(d,1H), 6.80(m,1H), 7.33(bm,15H), 9.30(m,1H), 9.42(m,1H); 13C NMR (CDC13) 17.3, 25.0, 27.9, 28.3, 39.8, 40.7, 44.0, 49.3, 54.7, 66.6, 67.1, 69.0, 80.4, 127.9, WO 95/06031 ~ 1 7 0 :~ 5 ~ PCT/US94/09343 128.0, 128.3, 128.4, 128.5, 128.8, 128.9, 134.5, 136.6, 155.7, 156.9, 160.7, 163.5, 172.2, 172.4.

Compound (5b) was ~ Gd from Compound (5a) in 87% yield, by the method 5 used to ~JlG~)al~;; Compound (lj). TLC: Rf 0.11 (chlol~rc~ isopropanol 9:1); lH NMR
(CDC13) ~ 8(d,3H), 1.43(m,1H), 1.70(m,4H), 3.30(m,6H), 3.91(m,2H) 4.34(m,1H), 5.03(s,2H), 5.1 l(s,2H), 5.22(s,2H), 5.50(m,1H), 7.01(m,1H), 7.33(bm,15H), 7.76(d,1H), 9.25(m,1H), 9.41(m,1H); 13C N~R (CDC13) ~ 17.7, 24.5, 31.1, 40.3, 40.6, 44.1, 48.6, 54.1, 66.7, 66.9, 68.9, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 10 128.8, 134.6, 136.3, 136.8, 155.7, 157.1, 160.4, 163.7, 172.8, 175.4.

Compound (5c) was ~ alGd from Compounds (5b) and (Id) in 88% yield, as a ule of diastereomers, with the method used to prepare Compound (lk).
1HNMR (d6-DMSO; mixture of diastereomers) o 0.79(bm,6H), 1.06(m,1H), 1.13 &
1.20(d, 3H), 1.52(bm,6H), 2.40(m,1H), 2.71(m,1H), 3.03(bm,5H), 3.47 & 3.54(s,3H), 3.88(m, 2H), 4.18(m,2H), 5.00(s,2H), 5.04(s,2H), 5.24(s,2H), 7.35(bm,18H), 7.59 &
7.71(d,1H), 7.66 & 7.94(t,1H), 8.13 & 8.45(d,1H); 13C NMR(d6-DMSO); mixture of diastereomers) ~ 17.8 & 18.3, 21.8 & 22.2, 22.9 & 23.0, 25.0 & 25.2, 25.4, 28.4 &
28.7, 36.4 & 36.5, 39.6, 40.0, 41.2 & 41.3, 44.3 & 44.4, 48.1 & 48.2, 51.1 & 51.4, 52.4 & 53.1, 65.3, 66.1, 68.2, 127.5, 127.6, 128.3, 128.6, 135.2, 135.3, 137.0, 155.0, 156.1, 156.2, 159.5, 162.8, 162.9, 170.9, 171.0, 171.9, 172.0, 172.8, 174.0, 174.8.

Hydroxamate (5d) was prepared from Compound (~c) in 78% yield as a Illi~Ul~ of diastereomers.
Hydroxamate ~5d) was de~rotec~ed by hydrogenolysis to give Compound (5) in 59% yield as a IllixLulG of diastereomers. HPLC retention times (method A) 10.1 and 10.3 minutes; lH NMR(D2O; mixture of diastereomers) o 0.89(m,6H), 1.25(m,1H), 1.39(m,3H), 1.69(bm,6H), 2.38(m,2H), 2.85(m,1H), 3.15(dd,2H), 3.22(dd,2H), 3.53(m,2H), 4.32(m, 2H); 13C NMR (D2O; mixture of diastereomers) o 24.3 & 24.5, 28.9 & 29.1, 30.4 & 30.5, 32.4 & 32.6, 33.4 & 33.5, 35.7 & 35.8, 43.4 & 43.6, 44.9, 47.0 & 47.1, 48.4 & 48.5, 49.0 & 49.1, 49.2, 57.8 & 58.0, 61.1 & 61.4, 164.8, 178.4 &
178.5, 181.4 & 181.8, 183.5 & 183.8, 185.6 & 186.4.
MS: mle 459 (M+).

WO 9S/06031 ~ i 8 PCTIUS94/09343 F,XAMPT.F, 5 Synthesis of N - ~D.L-2-(hydroxyaminocall~nyl)methyl-4-methylpentanoyl ~L-Iysinyl-L-alanine amide (Compound 6) O ~ H O CH3 HO-HN ~L~N----~N _~NH2 Referring to Scheme 6, a solution of 5.0g (0.010 mol) of Na-BOC-N-CBZ-L-lysine p-nitrophenyl ester and 1.5g (0.012 mol) of L-alanine amide hydrochloride and 1.67 ml (0.012 mol) of triethylamine in anhydrous N,N-dhnt;Lhylro~ ,,ide. (50 ml) was stirred at 10 room temperature for 16 hours before the solvent was removed in vacuo. The resulting residue was dissolved in ethyl acetate (200 ml) and washed with 3M NaOH (3x100 ml), water (3x100 ml), lM HCl (2x100 ml) and finally with brine (100 ml). After drying over anhydrous sodium sulfate, the solution was filtered and concentrated in vacuo to give 4.3g (96% yield) of Noc-BOC-N-CBZ-L-lysyl-L-alanine amide (6a) as a white solid.
15 TLC: Rf 0.32 (chlolorc,~,n-isopropanol 9:1); lH NMR (d6-DMSO) o 1.20(d,3H), 1.35(bm, 6H), 1.37(s,9H), 2.97(m,2H), 3.86(m,1H), 4.21(m,1H), 5.00(s,2H), 6.95(d,1H), 7.06(s, lH), 7.24(t,1H), 7.34(m,6H), 7.78(d,1H); 13C NMR (d6-DMSO) 18.6, 22.8, 28.2, 29.2, 31.4, 40.1, 47.8, 54.5, 65.2, 78.2, 127.8, 128.4, 137.3, 155.5, 156.1, 171.7, 174.2.
Compound (6b) was prepared from Compounds (6a) and (ld) in 69% yield using the method previously described to prepare Compound (A2). TLC: Rf 0.21 and 0.29 (chloroform-isopropanol 9:1); lH NMR (d6-DMSO; mixture of diastereomers) ~
0.81(m,3H), 0.88(m,3H), 1.17 & 1.23(d,3H), 1.40(bm,8H), 2.46(m,3H), 2.78(m,1H), 25 2.98(m,2H), 3.54 & 3.56(s, 3H), 4.08(m,1H), 4.16(m,1H), 5.00(s,2H), 7.04(m,1H), 7.23(t,1H), 7.34(m,6H), 7.58 & 7.68(d,1H), 8.10 & 8.42(d,1H).

Compound (6c) was prepared from Compound (6b3 in 48% yield, using the method previously described to prepare (A3). TLC: Rf 0.16 (chloroform-isopropanol 8:2).
30 MS: 7nle 522 (M+).

WO 95/06031 ~ 1 7 ~ Pcrluss4lo9343 The diastereomers (6A) and (6B) were l~el,ared from Compound (6c) by the method used to prepare Co~ ound (1) from Compound (lm). HPLC purification (method A) produced an early-eluting isomer (6A) and a late-eluting isomer (6~).

- 5 Compound (6A): HPLC retention time (method A): 9.2 minutes;
H NMR ~d6-DMSO) ~ 0.81(d,3H), 0.88(d,3H), 1.06(m,1H), 1.28(d,3H), 1.40(bm,7H), 1.75(m,1H), 2.03(m,1H), 2.22(m,1H), 2.73(m,3H), 4.01(m,1H), 4.13(m,1H), 7.04(s,1H), 7.11(s,1H), 7.78(bs,3H), 8.06(d,1H), 8.48(d,1H), 10.61(s,1H); 13C NMR(d6-DMSO) o 17.6, 21.8, 22.4, 23.5, 25.5, 26.4, 30.1, 35.7, 39.2, 40.0, 41.3, 48.4, 53.1, 168.1, 171.4, 174.8, 175.5;
MS: mle 387 (M+).

Cc,lll~x)ulld (6B): HPLC retention time (method A): 9.9 minutes;
1H NMR(d~-DMSO) o 0.81 (d,3H), 0.87(d,3H), 1.08(m, lH), 1.18(d,3H), 1.46(bm,7H),1.68(m,1H), 2.05(m,1H), 2.17(m,1H), 2.76(m,3H), 4.16(m,2H), 7.04(s,1H), 7.35(s,1H), 7.67(d,1H), 7.73(bs,3H), 8.08(d,1H), 10.58(s,1H); 13C NMR(d6-DMSO) 18.5, 22.1, 22.2, 23.2, 25.1, 26.3, 30.5, 35.5, 39.2, 40.1, 41.3, 47.8, 52.0, 167.9, 171.1, 174.0, 174.3;
MS: mle 387 (MH+).
FXAMPI,F 6 Synthesis of N- f D.L-2-(hydroxyaminocarbonyl)methyl-4-methylpen~ oyl ~L-tyrosyl-L-alanine amide (Compound 7) HO-HN)~_~N~ N~NH2 OH

With reference to Scheme 7, Compound (7a) was prepared from N-BOC-(O-benzyl)-L-tyrosine p-nitrophenyl ester and L-alanine amide hydrochloride in 99% yield, with - the method used to pl~pa-e Compound (6a). TLC: Rf 0.51 (chl~r~ fo"l~-is~,~allol 9:1);
1H NMR (d6-DMSO) o 1.22(d,3H), 1.30(s,9H), 2.67(m,1H), 2.91(m,1H), 4.09(m,1H), 4.22(m,1H), 5.05(s,2H), 6.90(m,3H), 7.06(s,1H), 7.18(m,2H), 7.28(s,1H), WO 95/06031 ~ ~ 7 ~1~ g PCT/IJS94/09343 2.38(bm,5H), 7.88(d,1H); 13C NMR (d6-DMSO) ~ 18.5, 28.1, 36.4, 47.8, 56.0, 69.1,78.1, 114.3, 127.5, 127.7, 128.3, 130.1, 130.2, 137.2, 155.2, 156.8, 171.2, 174Ø
Compound ~7b) was plepal~,d from Compound ~7a3 as a nli~lule of dia~
in 64% yield with the method used to synthesize Compound (6b). TLC: Rf 0.53 and 0.57 (chlorof{,.lll-is~",lol)anol 9:1); lH NMR (d6-DMSO; nlixLu~e of dia~l~re~ 0.60 &0.68(d,3H), 0.76 & 0.82(d,3H), 1.04(m,1H), 1.19 & 1.26(d,3H), 1.40(m,2H), 2.31(bm, 2H), 2.68(m,2H), 3.05(m,1H), 3.48 & 3.55(s,3H), 4.20(m,1H),4.44(m,1H), 5.03 &
5.04(s,2H), 6.87(m,2H), 7.06(bs,1H), 7.15(m,3H), 7.38(bm,5H), 7.69 & 7.78(d,1H),8.15 & 8.39 (d,lH); 13C NMR (d6-DMSO; IlliX.IUlG of diasl~l~olll~ 18.0 & 18.4, 21.9 & 22.1, 22.9 & 23.1, 24.6 & 25.1, 35.8 & 36.0, 36.4 & 36.6, 39.4 & 39.7, 41.1 &
41.2, 47.9 & 48.0, 51.2 & 51.4, 53.9 & 54.6, 69.1 & 69.2, 114.2 & 114.3, 127.5, 127.7, 128.4, 130.1, 130.2, 137.2, 156.8 & 156.9, 170.6 & 170.8, 171.9 & 172.7, 173.8 &173.9, 174.0 & 174 4 Compound (7c3 was prepared from Compound (7b) in 48% yield with the method used to prepare Colllpoul~d (6c3. A single diastereomer of Compound (7c3 was isolated by HPLC (method A). 1H NMR (CD30D). ~ 0.46(m,6H), 0.61(m,1H), 0.76(m,1H), 1.13(m,1H), 1.28(d,3H), 1.89(m,1H), 2.17(m,1H), 2.45(m,2H), 3.10(m,1H), 4.18(m,1H), 4.39(m,1H), 4.83(s,2H), 6.70(m,2H), 6.97(m,2H), 7.17(m,5H);
3C NMR(CD3OD) ~ 17.8, 22.2, 23.9, 26.3, 36.8, 37.2, 42.2, 43.0, 50.8, 56.7, 71.0, 115.9, 128.5, 128.9, 129.5, 131.1, 138.8, 159.1, 170.9, 173.8, 178.2, 178.6.
The diastereomer ~7c) was deprotected under 4 atmospheres of hydrogen in the presence of 10% p~ m on carbon in methanol to produce C~,lllpound (73 in 92% yield.
FXAMP~,FSynthesis of N-rD~L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl~-L-3-(2'-naphthyl)alanine amide (Compounds 8 and 9) HO-HN ~N_~NH2 F<

WO 9S/06031 . PCT/US94/09343 2~iO~'S'8' With reference to Scheme 3, a solution of 3.2g (0.010 mol) of N-BOC-L-3-(2'-naphthyl)alanine and 1.3g (0.011 mol) of N-hydroxysuccinimide dissolved in 10 ml of anhydrous tetrahy.lloruld,l was cooled to ca. 5 C. A solution of 2.3g (0.011 mol) of 1,3-dicyclohexylcarbotliimide dissolved in 5 ml of anhydrous tetrahy(llofu~dn was added, and the mi~lule was stirred at ca. 5 C for 30 minutes, then at room lelll~ldture for 30 minutes.
The dicyclohexylurea by-product was removed by filtration, and the filtrate was transferred to a flask containing 1.5 ml (0.022 mol) of concentrated NH40H. After the ~ ul~; had stirred at room temperaturè for 1 hour, the solvent was removed in vacuo to give a residue.
The residue was dissolved in ethyl acetate (350 ml) and washed with water (100 ml), lM
10 HCL (100 ml), water (100 ml), saturated sodium bicarbonate solution (100 ml) and finally with brine (100 ml). After drying over anhydrous magnesium sulfate, the solution was filtered and concentrated in vaCuo to produce a solid. The solid was recry~t~lli7ed from ethyl acetate to give 2.2g (70% yield) of N-BOC-L-3-(2'-naphthyl)alanine amide (8a) as a white solid. TLC: Rf 0.50 (chloroforrn-isopropanol 9: 1);
15 lH NMR(d6-DMSO) ~ 1.27(s,9H), 2.92(m,1H), 3.12(m,1H), 4.22(m,1H), 6.91(d,1H),7.07(s,1H), 7.44(s,1H), 7.50(m,3H), 7.75(s,1H), 7.85(m,3H); 13C NMR (d6-DMSO) 28.3, 37.9, 55.7, 78.1, 125.5, 126.1, 127.5, 127.6, 128.0, 132.0, 133.1, 136.2, 155.4, 173.7.

A stream of hydrogen chloride gas was bubbled into a solution of 1.95g (0.0062 mol) of N-BOC-L-3-(2'-naphthyl)alanine dissolved in 60 ml of anhydrous 1,4-dioxane, for 15 minutes. Ether (400 ml) was added, causing a solid to precipitate. The solid was collected by filtration and dried in vacuo to give 1.36g (88% yield) of L-3-(2'-naphthyl)alanine amide hydrochloride (8b). 1H NMR(d6-DMSO) ~ 3.27(m,2H), 25 4.10(m,1H), 7.48(m,3H), 7.55(s,1H), 7.79(s,1H), 7.86(m,3H), 8.14(s,1H), 8.40(bm,3H); 13C NMR(d6-DMSO) ~ 37.0, 53.6, 125.9, 126.3, 127.7, 127.9, 128.1, 128.4, 132.4, 133.0, 133.1, 169.8.

The diastereomers (8) and (9) can be made from L-3-(2'-naphthyl)alanine amide 30 hydrochloride (8b) and (ld), using the sequence of reactions used to prepare Compound (1) from Compounds (lj) and (ld).

Compound (8): HPLC retention time (method A) 22.6 minutes. 1H NMR
(CD3CN/D2O) ~ 0.71(m,6H), 1.09(m,2H), 1.28(m,1H), 2.12(m,2H), 2.59(m,1H), 35 2.84(m,1H), 3.11(m,1H), 4.45(m,1H), 6.94(m,7H).
MS: mle 385 (M+).
Compound (9): HPLC retention time (method A) 24.3 minutes, MS: mle 385 (M+)-W O 95/06031 ~17 0 t ~ 8 ; PCTrUS94/09343 ~". 1 ' ''' ' ~XAlV~PT,F, X
Synthesis of N- rD~L-2-fhydroxyaminocarbonyl)methyl-4-methylpentanovl ~ -L-3-(2'-naphthyl~-alanyl-L-serine amide (C~ )ound 10) HO-HN~N _N~NH2 0~9< 0 With reference to Scheme 8, N-BOC-L-3-(2'-naphthyl)alanyl-L-(O-benzyl)serine amide (lOa) was prepared from N-BOC-L-3-(2'-naphthyl)alanine and L-(O-benzyl)serine 10 amide in 80% yield with the method used to prepare (7a). TLC: Rf 0.51 (chloroform-isopropanol 9:1); lH NMR (d6-DMSO) o 1.24(s,9H), 2.93(m,1H), 3.19(m,1H), 3.65(m,2H), 4.34(m,1H), 4.48(m,1H), 4.51(s,2H), 7.16(d,1H), 7.27(s,1H), 7.34(m,5H), 7.46(m,4H), 7.78(s,1H), 7.82(m,3H), 8.04(d,1H); 13C NMR (d6-DMSO) ~ 28.0, 37.4, 52.5, 55.9, 70.0, 72.1, 78.2, 125.4, 125.9, 127.3, 127.4, 127.5, 127.8, 15 128.2, 131.8, 132.9, 135.9, 138.2, 155.4, 171.3, 171.5.

L-3-(2'-naphthyl)alanyl-L-(O-benzyl)serine amide (lOb) was prepared from Com-pound (lOa) in 95% yield with the method used to prepare Compound (lj). TLC: Rf 0.08 (chloroform-isopropanol 9:1); IH NMR d6-DMSO) o2.81(m,1H), 3.15(m,1H), 20 3.42(m,3H), 3.63(m,2H), 4.37(s,2H), 4.43(m,1H), 7.32(m,6H), 7.46(m,4H), 7.72(s,1H), 7.82(m,3H), 8.14(d,1H); 13C NMR (d6-DMSO) ~ 40.6, 52.0, 55.8, 70.0, 72.0, 125.3, 125.9, 127.4, 127.5, 127.7, 128.0, 128.2, 131.8, 133.0, 136.2, 138.1, 171.5, 174Ø

Compound (lOc) was prepared from Compounds (lOb) and (ld) as a mixture of diastereomers in 97% yield following the method used to prepare Compound (lk). TLC:
Rf 0.69 and 0.73 (chloroform-isopropanol 9:1); 1H NMR (d6-DMSO; mixture of diastereomers) oO.25 & 0.40(d,3H), 0.68 & 0.79(d,3H), l.OO(m,lH), 1.32(m,2H), 2.31(bm,3H), 2.~S4(m,1H), 2.98(m,1H), 3.37 & 3.50(s,3H), 3.68(m,2H), 4.48(m,1H),30 4.49 & 4.53(s,2H), 4.72(m,1H), 7.35(bm,6H), 7.44(m,4H), 7.78(m,4H), 7.93 &
7.99(d,1H), 8.30 & 8.49(d,1H); 13C NMR (d6-DMSO; mixture of diastereomers) ~ 21.4 &

~ WO 95/06031 2 ~ 7 0 1 ~ 8 Pcr/uss4l09343 22.1, 22.8, 24.5 & 25.1, 36.3 & 36.6, 37.1, 39.6, 41.0 & 41.1, 51.1 & 51.4, 52.6 &
52.7, 53.7 & 54.2, 69.8 & 69.9, 72.1, 125.3, 125.8, 127.4, 127.5, 127.6, 127.8, 128.2, 131.8 & 131.9, 132.9 & 133.0, 135.7 & 135.8, 138.1, 170.0, 171.2, 171.3, 171.8, 172.5, 174.0, 174.2.

Compound (lOd~ was prepared from Compound (lOc) in 74% yield with the method used to ,~lG~al`e Co-llpoul~d (lm). TLC: Rf 0.12 (chlclo~l"l-isopl~opallol 9:1).

Compound (10) was l~,Gpa,~;d from Compound (lOd) in 84% yield with the metho~l used to pl~l.ale Compound (ln). HPLC retention times: 25.2 and 27.1 minutes (method A).
MS: mle 472 (M+).

Synthesis of N- rD,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanovl ~ -L-3-(2'-naphthyl)-alanyl-L-alanine methylamide (Compound 11) HO-HN~N~LN~NH-CH3 1~<
~_q Referring to Scheme 9, Compound (lla) was prepared from N-BOC-L-3-(2'-naphthyl)alanine and L-alanine methylamide hydrochloride, in 89% yield using the method previously described to prepare Compound (li).
TLC: Rf 0.58 (chloroform-isopropanol 9:1); 1H NMR (d6-DMSO) ~ 1.21(d,3H), 1.25(s,9H), 2.54(d,3H), 2.91(m,1H), 3.18(m,1H), 4.28(m,2H), 7.04(d,1H), 7.46(m,3H~, 7.75(s,1H), 7.83(m,4H), 8.07(d,1H); 13C NMR (d6-DMSO) o 18.5, 25.5, 28.0, 37.5, 48.1, 55.7, 78.1, 125.4, 125.9, 127.3, 127.4, 127.5, 127.9, 131.8, 132.9, 135.9, 155.3, 171.1, 172.3.

Cornpound (11~) was prepared from Compounds (lla) and (ld), in 86% yield using the method previously described to prepare Compound (A2).
TLC: Rf 0.57 and 0.62 (chloroform-isol~lol~anol 9:1);

WO 95106031 ~ 1 7 ~1 5 8 PCT/US94/09343 1H NMR (d6-DMSO; mixture of diastèreomers) ~ 0.23 & 0.40(d,3H), 0.70 & 0.79(d,3H), 1.01(m,2H), 1.18 & 1.26(d,3H), 1.32(m,2H), 2.22(m,2H), 2.53(d,3H), 2.92(m,1H), 3.22(m,1H), 3.38 & 3.39(s,3H), 4.22(m,1H), 4.63(m,1H), 7.44(m,4H), 7.73(s,1H), 7.81(m,4H), 8.22 & 8.46(d,1H).
Compound (11~ was prepared from Compound (llb) in 23% yield using the method previously described to prepare Compound (A3). TLC: Rf 0.18 (chlorofo iso~r~,~anol 9: 1).

~XAMP~,F 10 Synthesis of N- ~D.L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl ~ -L-3-amino-2-dimethylbutanoyl-L-alanine~ 2-aminoethyl amide (Compound 13) l ~H CH3 H
HO-HN- ~N~N~N- - ~JI ij~
o - H o 7~
Following Reaction Scheme 10, N-Boc-L-tert-leucine 13(b) was prepared by ¢eating L-tert-leucine (Aldrich Chemical) with di-tert-butyl dicarbonate and diisopropylethyl an~ine in dimethylfluoride (DMF). Then (13b) was treated with NHS and dicyclohexylcarbodiimide (DCC) in anhydrous tertrahyd-oru,~n to produce N-Boc-L-tert-20 leucine N-hydroxysuccinimidyl ester, which then is coupled with (lh) from Reaction Scheme 2 and Example 1 to produce (13c). Compound (13) was prepared from (13c) by following procedures similar to those described in Example 1 and shown in Reaction Scheme 2 for the synthesis of compound (1). 1H NMR (d6-DMSO) o 0.76(d, J = 5.6 Hz, 3H), 0.82(d, J = 6.1 Hz, 3H), 0.90(s,9H), 1.06(m, lH), 1.17(d, J = 6.6 Hz, 3H), 1.39(m, 2H), 2.08(m, 2H), 2.69(m, 2H), 2.86(m, lH), 3.18(m, 2H), 4.19(m, 2H), 8.30(m, lH), 8.03(d, J = 7.0 Hz, lH), 7.86(d, J = 8.9 Hz, lH), 13C NMR (d6-DMSO)18.4, 22.6, 23.5, 25.7, 27.1, 34.5, 36.2, 39.2, 40.0, 41.1, 48.8, 60.3, 167.8, 170.1, 172.6, 174.5.

F.XAI~P~,li`. 11 Inhibition of TNF-a Release by T-cells The following example demonstrates the selective in vitro inhibition of T-cell TNF-a secretion, as co~ Jal~d to TNF-~ and IFN-~ secretion, by Compound 1.

WOg5/06031 21 ~ ~15 8 PCT/US94/09343 Human peripheral blood T-cells were purified from peripheral blood mononuclear cells by rosetting with 2-aminoethylisothiouronium bromide hydrobromide-treated sheep erythrocytes. After hypotonic lysis of sheep erythrocytes, monocytes were depleted by 5 plastic adherence for one hour at 37 C. The peripheral blood T-cells were stim~ t~(l with anti-CD3 antibody (OKT3) which was i""llobilized on the culture wells at 10 ~Lg/ml in PBS
plus 10 ng/ml of the phorbol ester, PMA. Culture medium comprised RPMI 1640 medium containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 ,ug/ml ~L,eptc""ycin. The stim~ tion was performed in the presence or absence of the inhibitor Compound 1 t200 10 ~lM), and TN~-a in the metlil-m was assayed by ELISA. Results are shown in Table I.
TABLE I
Effect of Compound l on Cytokine Production by Peripheral Blood T Cells TNF-a(pg/ml) 3 Hrs. 24 Hrs.48 Hrs.
with Compound 1 ~ 100 300 without Compound ll00 325 800 TNF-~ (pg/ml) with Co",~ou,ld 1 ~ 160 1050 without Compoulld 1 ~ 160 830 T~N- y (ELTSA OD) with Compound 1 0.2 0.9 1.08 without Compound l0.3 0.65 1.15 ~ lln~et~ct~ble After 3 hours, there was 100 pg/ml of TNF-a in the m~ lm of cells without Compound 1 and no detectable TNF-a in the medium of cells with 200 ,uM of Compound 1.
At 24 and 48 hours, Compound 1 inhibited TNF-a release by 72% and 63%, respectively, while there was no inhibitory effect on the release of TNF-13 or interferon-~. Compound 1 clearly demon~l,dles selective inhibition of TNF-a secretion and has no effect on either TNF-~ or interferon-~y secretion.
FX~MP~,F 12 Compound l Induced Increase in Cell Surface TNF-a on PMA+Ionomycin-Stimulated Human T-cells This example describes the effects of Compound 1 on cell surface TNF-a for humanT-cells which have been stim~ tecl by PMA and ionomycin.

WO 95/06031 ' . . ~ PCT/US94/09343 The alloreactive human T-cell clone, PL-l, does not express cell surface TNF-a in the absence of stimulation. However, after stim~ tion with PMA plus ionomycin, cell surface TNF-a, as well as the ligands for CD40 and 41BB, are rapidly induced on the cell S s--rfa~e Detection of cell surface TNF-a was performed by staining with an Fc fusion protein consisting of an Fc portion of a human IgG1 molecule (IgGFc) coupled with an extr~cell~ r domain of TNF receptor (p80). Detection of cell surface ligands for 41BB and CD40 was pe rc ",led by st~ining with analogous Fc fusion proteins consisting of IgGFc and extracellular domains of 41BB and CD40, respectively. A fusion molecule consisting of 10 IgGFc and the extr~cell~ r portion of the IL-4 receptor (IL-4R:Fc) was utilized as a negative control for staining, since PL-l cells do not express cell-surface IL-4 in response to PMA
stim--l~tion. TNFR:Fc and IL-4R:Fc fusion proteins are described in EP 0 464 533, incorporated herein by reference. The same general procedures used to construct the ~NFR:Fc and IL-4R:Fc fusion molecules were utilized in the construction of the 41BB:Fc 15 and CD40:Fc molecules. Fc fusion proteins bound to their respective cell-surface ligands were then detected with a biotinylated anti-human IgG1 followed by streptavidin-phycoerythrin. The intensity of staining was measured by a FACS (fluorescence activated cell sorting) scan flow cyLoll-etel. The results are shown in Table II.
TABLE II
Effects of Compound 1 on Expression of Cell Surface TNF-a, IL-4, 41BBL and CD40L on PMA and lonomvcin-Stimulated Human T-Cells (MF~. arbitrary units) TNF-a 41BBL C~40L IL-4 No stimulation 10 10 10 10 4h after stimulation + Compound 13040 344 107 10 - Compound 1 83 428 107 10 18h after stimulation + Compound 1 616 9 46 10 - Compound 1 7 5 19 10 The specificity of Compound 1 for increasing cell surface TNF-a is apparent. Cells 3~ stim--l~te~l with PMA and ionomycin for four hours in the presence of Compound 1, followed by staining with TNFR:Fc as described above, displayed a MFI of 3040 ascolllp~d to 83 in the absence of Compound 1. The effect of Compound 1 was specific for TNFR:Fc binding as no increase on 41BB:Fc or CD40:Fc binding was detected. A
substantial increase in cell-surface TNF-a resulted in a 100-fold increase in TNFR:Fc WO 95/06031 ~ :L 7 0 1 5 ~ PCTIIJS94/09343 binding in the presence of Compound 1 (MFI was 616) as compared to an MFI of 7 in absence of Compound 1, after 18 hours of stim~ tion. Under the same conditions, 41BB:Fc and CD40:Fc binding were enhanced only approximately 2-fold.
F.XAMPT.F 13 In vivo Inhibition of TACE
~g Compound A versus Compound 1 versus control Female Balb/c mice (18-20g) were injected i.v. with 400 ~lg of LPS.
Simnlt~neously, the mice were injected subcutaneously with 500 ,ug of Compound A or Compound 1 in 0.5 ml of saline containing 0.02% DMSO. Control mice received LPS
intravenously and saline/DMSO subcutaneously. Two hours following the LPS injection, serum was obtained and pooled from two mice in each treatment group. TNF-a levels were ~let~,rmined by ELISA and are shown in the following Table m.
TABLE III
Comparison of 500 ~g Each of Compound 1 versus Compound A on LPS-Induced Serum TNF Levels in Balb/c Mice (pglml) Compound 1 CompoundA Saline/DMSO
Serum TNF-a level lln~ tect~hle 65 157 Compound 1 inhibits the secretion of TNF-a at least by 80%, and essentially by 100%, as the TNF-a levels were undetectable. Comparatively, Compound A reduced serum 25 TNF-a levels by ~p-~ ~i,ately 60% as cc,~ ,d to the saline/DMSO control.

In a similar manner to the procedure described above, mice were injected i.v. with 400 ~g LPS. Simultaneously, the mice were injected subcutaneously with 500 ~Lg Compound 1 in 0.5 ml saline containing 0.02% DMSO. Two hours later, serum was 30 obtained and pooled. TNF-a levels were determined by ELISA. Results are shown in Table IV in pg/ml.

TABLE IV
Effect of 500 ~g Compound 1 on LPS-Induced Serum TNF Levels in Balb/c Mice (pg/ml) ExperimentNo. LPS + Cpmd 1 LPS only LPS + Saline WO 95/06031 ~ 'I 7 ~ 1 ~ 8 - PCT/US94/09343 ~

In e~y~,lhllent 1, Compound 1 reduced serum TNF-a levels by 82% as compared to TNF-a levels in mice that received LPS only. As compal~d to mice that received LPS +
saline, Compound 1 reduced serum TNF-a levels by 76%. In experiment 2, Compound 1 S reduced serum TNF-a levels by 89% as compared to TNF-a levels in mice that received LPS only. As compared to mice that received LPS + saline, Compound 1 reduced serum TNF-a levels by 85%. In experiment 3, Compound 1 reduced serum TNF-a levels by 85%
as cc~llp~,d to TNF-a levels in mice that received LPS only. As coll~ ed to mice that received LPS + saline, Compound 1 reduced serum TNF-a levels by 84%. Overall, 10Cc,l-lpou~ld 1 reduced serum TNF-a levels by 85.4 i 2.98% as compalGd to TNF-a levels in mice that received LPS only. From Tables III and IV, Compound 1 effectively reduces serum TNF-a levels by at least 80% when ~clmini~tered at 25 mg/kg in a murine model of LPS-induced sepsis syndrome.

15 j~ Compound A versus Compound 1 versus control Female Balb/c mice (18-20g) were injected i.v. with 450 ~Lg of LPS.
Simnlt~neously, the mice were injected subcutaneously with 250 ~g of Compound A or Compound 1 in 0.25 ml of saline containing 0.02% DMSO. Control mice received LPSintravenously and saline/DMSO subcutaneously. Two hours following the LPS injection, 20 serum was obtained from three mice in each tre:~tment group. TNF-a levels were deterrnined by ELISA. The results are expressed as the mean optical density (OD) obtained in the ELISA from each treatment group, and are shown in Table V. The background OD of the control sample was 0.162 + 0.003.

Comparison of 250 ~g Each of Compound l versus Compound A on LPS-Induced Serum TNF Levels in Balb/c Mice T PS+Saline LPS+Saline+DMSO Cmpd 1+DMSO Cmpd A+DMSO
300.271 + 0.022 0.268 + 0.040 0.147 + 0.004 0.299 + 0.023 Table V illustrates the effect of Compound 1 and Compound A on inhibiting serum TNF-a release in Lps-s~im~ te~l mice. Compound 1 reduced serum TNF-a levels to those 35 of the control, thereby indicating a complete inhibition of TNF-oc secretion at 250 ~lg/ml.
Compound A had no effect in reducing serum TNF-a levels as shown by the similarlity in OD readings between LPS+Saline, LPS+Saline+DMSO, and Compound A.

WO9S/06031 1 7~ S8~ ~ PCT/~S94/09343 FXAMP~,F 14 Serum stability of Compound A and Compound 1 Each of Compound 1 and Compound A was diluted to 50 ~LM in normal mouse S serum and incubated at 37 C. At various times, aliquots were withdrawn, diluted 100-fold into ice-cold PBS, and tested for inhibitory efficacy against purified TACE. After 40 minutes, Cvlllpou-ld A showed a decrease in inhibitory effect corresponding to a 3-4 fold loss in concentration of the col-,poulld, and Compound 1 showed no decrease in inhibitory effect.

WO 95/06031 ! PCTIUS94/09343 217~158 o o R2--C--C--ONa (la) O O
R2_e--C--OCH2Ph (Ib) O O
RO--e--CH=C--C--OCH2Ph (Ic) O O
RO--C [CH~m CH - C--OH (Id) ( R1 = H, m = 1 ) O O
RO--C [CH]m CH-C--OR" (Ie) Rl R2 WO95106031 2 17 01~ 8PCT/US94/09343 SCHEME 1 -Continued N _ C~(CH2)n~N H2 (If) N_C-(CH2)n-NH-P(Ig) H2N--B--N--P(Ih) (Ii) P'--N--[A]n-C--OR"

P'--N--[A]n-C--N--B--N--P (Ij) H2N--[A]n--C--N--B--N--P (Ik) H H

WO 95/06031 ~ ~L 7 ~ l S 8 PCT/US94/09343 SCHEME 1-Continued H2N--[A]n-C--N--B--N--P (Ik) (Il) P'--N--CH-e--OR"
H

O O
P--H--Cl H-C--NH--[A]n--C--H--B--H--P (Im) O O
H2N--ICH-e--H~[A]n~e--HN--B--HN--P (In~

O O
Il 11 (Ie) RO--C [cH]m cH-C--OR'' O O O O
Il 11 11 11 RO--C [C IH]m~ ICH -C--HN ICH-C--H~[A]n~C--N--B--N--P ( lo ) WO 95/06031 t~ S 8 PCTIUS94/09343 SCHEME 1-Continued o o o o RO--C [C I ]m~ ICH-C--H ICH-C--H~[A]n~C--H--B--H--P ( Io ) O O O O
RO-HN--e [c IH]m~ jCH-C--H ICH-e--H~[A]n~C--N--B--H--P ( Ip ) Rl R2 R3 O O O O
Il ll 11 11 HO-HN--C [CH]m CH-C--N--ICH-C--N - [A]n - c--H--B--NH2 ( Iq ) -WO9~;106031 ;~ oi~i~ PCI/US94/09343 SCt~lEME A

BOC-HN ~L t . NHS, EDC, DMF , BOC-HN _~L N ~ NH2 2. ~aanine amide ~ HCl, EbN, DMF ~ ( A,) 1. TFA, CH2C12 2. ~ld), Et3N, DMF

CH3O ~--H ~-~ (A2) ~_Y

H2N-OH, CH30H, KOH

Ho-HN)~$H ~_ ~ ( A ) WO 95/06031 ;~ 17 015 8 PCT/US94/09343 O Bzl-Br, DMF I
- n-- Na+ ~ _ ~0_~
O (~) O

Ph3P=CH-CO2CH3 CH 0~~~

( 1b ) CH30~0_~ H2, Pd/C. CH30H CH o ~_~OH
H O O
( 1b) ( 1c) O ~ O ~
CH30~0H NHS, DCC, THF ~ CH30~0Succ.
O O
( 1 C) ( ~) WO 95/06031 PCT/US94/09343 ~I
~17~
SCHEME-2-Continued --0 OSucc+HCI H2N--CN DMF, Et3N CB
(1e) CBZ HN--CN 1- BH3 ~(CH3)2 . THFC NH2 HCI
( 1 e ) ( lf ) ~, (1e). DMF- Et3N ~ BOc HN~,N ) NH-CBZ

BOC-HN~N_--NH-CBZ 1. CF3CO2H. CH2C12~ H NQ~N----NH-CBZ
( 1C ) 2. NaOHaq ( 1h ) O 1. iBuOCOCI 4-methyl,,,u,~l, 1 ,e DMF o CH3 H
BOC-HN _lLoH 2. ( lh ) 4-methyl"~ h ' ,e, DMF BOC HN--IL H ~N _--NH-CBZ

( 1 i) O CH3 H o CH3 H
BOC-HN_ILN~N_--NH-CBZ H2N~LH~N----NH-cBz 1. CF3CO2H. CH2CI2 /~
( 1 i ) 2. NaOHaq. ~ ( 1j ) WO95/06031 ~ 7 ~15 ~ PCT/US94/09343 SCHEME-2-Continued ~ CH30~N--~Ln~N----NH-CnZ
CH30 OSucc. ~ H o ( 1d ) (~), DMF, Et3N ~ ( 1k ) /H2N-OH, CH30H, ~/ KOH

~H O CH3 H
HO-HN~o N H~N_~NH-CBZ

( 1m ) H2, Pd/C, HOAc O ~H O CH3 H
HO-HN~N .~LN~N~NH2- HOAc ~l .
(1) 217 ~158 - ~ a-- Na+ Bzl-Br, DMF ~O_~

(~) a Ph3p=cH-co2cH3 CH 0~~~
(~) (~) O ~ _~ H2, Pd/C, CH3HCH o ~OH
H O ( ~ ) O

NHS, DCC, THF CH o~OSucc.

WO 9S/06031 2 :~ 7 ~15 8 PCT/US94109343 SCHEME-3-Continued O ' O
BOC-HN_ILOH 1. NHS, DCC, THF BOC-HN~NH2 2. NH40H

HCI / dioxane HO-HN~N--LNH2 o 0,~ 1. (ld), Et3N, DMF HCI H2N - LNH2 (~) & (~ 2. H2N-OH, KOH, ~ (8b) 1. (2d), Et3N, DMF
2. H2N-OH, KOH, Y H
HO-HN ~L~ NH2 (2) & (3) ~

Wo 95/06031 2 1 7 ~1 5 8 PCT/US94/09343 >Lo ll n-oH NHS, DCC, THF >L ~_ ~,O-N~
(.~La) O

BOC-HN~LNQ~NH2 ~ >LO~ N~LN~NH2 H o 2. (4a), Et3N, DMF O ' H o ( ~ ( 4b ) TFA

H CH3 HO~N__ILN~NH2 HO-H N J~ r N ~L N ~ N H2 o ~ H o ~
~(4) ~ (4C) H2, Pd on C, HOAc 1. iBuOCOCI, DMF, 4-methylmorpholine 2. Bzl-O-NH2 ~3-- H ~ N ~L N Q~ NH2 ~ ( 4d ) ~2 WO95/06031 ~ 1 7 0 15 ~ PCT/US94109343 BOC-HN--_ILOH BOC-HN_ILNQ~N_ NH-CBZ
1. iBuOCOCI ~ 4-m~tl ~ ,,u,~h " ,e DMF ~
~NH 2. (1h) 4-,nell,yl",o,~.h 1 ,e DMF NH (~a) CBZ-N ~ NH-CBZ CBZ-N ~ NH-CBZ

1.CF3CO2H.CH2Cl2 2.NaOH~

H2N ~ H ~ NH-CBZ

( ) ~ NH
CBZ-N NH-CBZ

(ld), Et3N DMF

~ H CH3 H
CH30~N~LN~N----NH-cBz (5C) ~NH
CBZ-N~ NH-CBz ~3 WO 95/06031 ~ PCTIUS94/09343 SCHEME-5-Continued CH20~N _~L N ~ N ~ NH-CBZ

( 5~) NH
CBZ-N~ NH-CBz H2N-OH, CH30H, KOH

HO-HN Q~NV - N~N - - NH-CBZ

~1) NH
CBz-N~ NH-CBz H2, Pd/C, CH30H. HCIaq.

HO-H N ~ N _~L N ~ N ~ NH2 (5) NH
HN~ NH2 wo gS/06031 2 ~ 7 ~15 8 PCT/US94/09343 BOC-HN__lLo_~No2 BOC-HNV_H o L-alanine amide- HCI, ~

NH-CBZ Et3N, DMF NH-CBZ

1. TFA, CH2C12 2. (:~!) . Et3N, DMF

CH30~N _~L N ~f NH2 - H
(6b) ,~ NH-CBZ
HO-HN~H O ~_ ( ~iA ) & ( 6B ) H2N-OH, CH30H, ~ H2, Pd on C, CH30H

HO-HN~H
O ~ O

(~) ' N H -CBz WO9S/06031 ~ 70~5g PCT/US94/09343 ~b BOC-HN_Lo_~NO2 BOC-HN_~LN~NH2 L-alanineamide-HCI, ~ (~ ) O-Bzl Et3N, DMF O-Bzl 1. TFA. CH2C12 2. (~), Et3N, DMF

CH30 ~ H
O ~ O
(~) ~
J--` O O-Bzl HO-HN~N~_LN~NH2 O ~ O
k~ ( 7 ) H2N-OH, CH30H, KOH
OH ~\H2. Pd on C, CH30H

HO-HN--~ , H~
O ~ O
(7Ç) O-Bzl WO 95/06031 ~ 17 û 15 8 PCT/US94/09343 O O-Bzl BOc-HN_JLOH 1. iBU0C0CI.4~ ul,ull ' ,e, DMF BOC-HN_~LH O
2. L-(O-benzyl)-serine amide, ~
&~ 4~ tl~ ul~JI ' ,e, DMF ~ ( 1 Oa ) 1. CF3CO2H. CH2C12 2. NaoHaq.

CH30~ N_JLN~NH2 ~ (ld) - DMF, EbN H2N--~N~NH2 ( 10c) ~ ( 10b ) H2N-OH, CH30H, KOH

HO-HN~ - H~o HO-HN~N_~N~NH2 H2, Pd on C, ~
( 10d ) HOAC ~ ( 10 ) WO 95/06031 ~17 Q 1~ 8 PCTIUS94/09343 : S(~HEME 9 BOC-HN__ILOH
1. iBuOCOCI, 4-methylmorpholine, DMF ~
2. L-alanine, methylamide- HCI, ~ (~) 4-methylmorpholine, DMF ~

/ 1. TFA
2. ~), Et3N, DMF

CH3O~N_LNS~NH-CH3 0~0< 0 (11b) H2N-OH, CH30H, KOH

HO-HN ~ , N
o - H o l( ( 11 ) ~58 1~WO 95/06031 PCT/US94/09343 2i701~

HO NH EtO2CO, NaOCH3,~;74% OJ~NH

L - Valinol (1 2a) SOCI2, ~; 81%
COOH ~ ~--COCI
4-Methylvaleric acid (1 2b) O O

O~NH a) nBuLi,THF.0C b)12b ,-78;C O~N~<
H` ~ 86/~
(1 2a) (1 2c) O N~< a) LDA, OC O~_~N
\~ b) allyl bromide ~ 100% H
H ~~ (1 2c) /~ (1 2d) 0~ ~0~

(1 2d) /=\ ~
~~0- Li + NH
~HF, 0 C; 8~% \~ ~40% recovery WO 95/06031 ~ PCTIUS94/09343 ~It ~ 7~
SCHEME-1 O-Continued ~~' H \~
( 1 2e) RuCl3, NalO4, CH3CN, CC4, H20; 65%

~~

(1 2f) a) CH2N2, Et20 b) H2, Pd / C, CH30H; 89%

H
--OH

(1c)

Claims (36)

What is claimed is:

1. A compound of the formula:

(I) wherein:
X is hydroxamic acid, thiol, phosphoryl or carboxyl;
m is 0, 1 or 2;
R1, R2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted C1 to C8 alkyl, C1 to C8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring a-amino acid; or the group -R6R7, wherein R6 is substituted or unsubstituted C1 to C8 alkyl and R7 is OR4, SR4, N(R4)(R5) or halogen, wherein R4 and R5 are each, independent of the other, hydrogen or substituted or unsubstituted C1 to C8 alkyl;
n is 0, 1 or 2;
provided that when n is 1, A is a protected or an unprotected .alpha.-amino acidradical;
when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical; and B is unsubstituted or substituted C2 to C8 alkylene; and the pharmaceutically acceptable salts thereof.

2. A compound according to claim 1, wherein B is C2 to C6 alkylene.

3. A compound according to claim 2, wherein B is dimethylene.

4. A compound according to claim 1, wherein X is hydroxamic acid.

5. A compound according to claim 3, wherein X is hydroxamic acid.

6. A compound according to claim 5, wherein R1 is hydrogen.

7. A compound according to claim 1, wherein R2 is C1 to C6 alkyl or a C1 to C6 alkylenearyl.

8. A compound according to claim 1, wherein R3 is selected from the group consisting of C1 to C6 alkyl, C1 to C6 alkylenephenol, C1 to C6 alkylene(cycloalkyl) or C1 to C6 alkylenearyl.

9. A compound according to claim 8, wherein R3 is C1 to C6 alkyl.

10. A compound according to claim 9, wherein R3 is t-butyl.

11. A compound according to claim 13, wherein R3 is methylenephenol.

12. A compound according to claim 8, wherein R3 is C1 to C6 alkylenearyl.

13. A compound according to claim 12, wherein R3 is methylene-(2'-naphthyl).

14. A compound according to claim 1, wherein A is an alanyl or seryl radical, and n is 1.

15. A compound according to claim 14, wherein A is alanyl, and n is 1.

16. The compound according to claim 1, which is N-{D,L-2-(hydroxyamino-carbonyl)methyl-4-methylpentanoyl}-L-3-(2'-naphthyl)alanyl-L-alanine, 2-(amino)ethyl amide.

17. The compound according to claim 1, which is N-{D,L-2-(hydroxyamino-carbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-nine, 2-(amino)ethyl amide.

18. A method for treating a mammal having a disease characterized by an overproduction or an upregulated production of TNF-.alpha., comprising administering to the mammal a composition comprising an effective amount of a biologically active compound of the formula:

(II) wherein:
X is hydroxamic acid, thiol, phosphoryl or carboxyl, m is 0, 1 or 2;

R1, R2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted C1 to C8 alkyl, C1 to C8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring .alpha.-amino acid; or the group-R6R7, wherein R6 is C1 to C8 alkyl and R7 is OR4, SR4, N(R4)(R5) or halogen, wherein R4 and R5 are each, independent of the other, hydrogen or substituted or unsubstituted C1 to C8 alkyl;
n is 0, 1 or 2;
Y is hydrogen, unsubstituted or substituted C1 to C8 alkyl, alkylene(cycloalkyl), the group -R8-COOR9 or the group -R10N(R11)(R12);
wherein R8 is C1 to C8 alkylene; R9 is hydrogen or C1 to C8 alkyl; R10 is C1 to C8 alkylene; and R11 and R12 are each, independent of the other, hydrogen or unsubstituted or substituted C1 to C8 alkyl; provided that when n is 1, A is a protected or an unprotected .alpha.-amino acid radical; and when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical;
and when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical; and the pharmaceutically acceptable salts thereof;
wherein the compound is capable of reducing serum TNF-.alpha. levels by at least80% when administered at 25 mg/kg in a murine model of LPS-induced sepsis syndrome;
and a pharmaceutically acceptable carrier.

19. The method according to claim 18, wherein B is C2 to C6 alkylene.

20. The method according to claim 19, wherein B is dimethylene.

21. The method according to claim 18, wherein X is hydroxamic acid.

22. The method according to claim 18, wherein R1 is hydrogen or C1 to C6 alkyl.

23. The method according to claim 22, wherein R1 is hydrogen.

24. The method according to claim 18, wherein R2 is hydrogen or C1 to C6 alkyl.

25. The method according to claim 24, wherein R2 is isobutyl.

26. The method according to claim 18, wherein R3 is selected from the group consisting of C1 to C6 alkyl, C1 to C6 alkylenephenol, C1 to C6 alkylene(cycloalkyl) or C1 to C6 alkylenearyl.

27. The method according to claim 26, wherein R3 is C1 to C6 alkyl.

28. The method according to claim 27, wherein R3 is t-butyl.

29. The method according to claim 26, wherein R3 is C1 to C6 alkylenearyl.

30. The method according to claim 29, wherein R3 is methylene-(2'-naphthyl).

31. The method according to claim 18, wherein A is an alanyl or seryl radical, and n is 1.

32. The method according to claim 31, wherein A is alanyl, and n is O or 1.

33. The method according to claim 18, which is N-{D,L-2-(hydroxyamino-carbonyl)methyl-4-methylpentanoyl}-L-3-(2'-naphthyl)alanyl-L-alanine, 2-(amino)ethyl amide.

34. The method according to claim 18, which is N-{D,L-2-(hydroxyamino-carbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine, 2-(amino)ethyl amide.

35. A pharmaceutical composition for treating TNF-.alpha. related disorders, conditions or diseases comprising a compound according to claim 1 as the active component.

36. A pharmaceutical composition for treating TNF-.alpha. related disorders, conditions or diseases comprising a compound according to claim 1 and a protein having TNF-.alpha. binding activity.