EA045724B1 - CYCLIC PEPTIDE TYROSINE–TYROSINE COMPOUNDS AS MODULATORS OF NEUROPEPTIDE Y RECEPTORS - Google Patents

Description

Scope of the invention

The present invention generally relates to novel cyclic peptide tyrosine tyrosine (PYY) compounds that are neuropeptide Y2 receptor modulators. The present invention also relates to pharmaceutical compositions and methods of using them. The new compounds are useful for preventing, treating or ameliorating diseases and disorders such as obesity, type 2 diabetes, metabolic syndrome, insulin resistance and dyslipidemia, among others.

Cross references to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62/413,613, filed Oct. 27, 2016, and U.S. Provisional Patent Application No. 62/413,586, filed Oct. 27, 2016. Each disclosure is incorporated herein by reference.

Link to sequence listing submitted electronically

This document contains a sequence listing presented electronically via EFS-Web as a sequence listing in ASCII format, with the file name PRD3411 Sequence Listing and the creation date of October 23, 2017, having a size of 96 kb. The sequence listing provided by EFS-Web is part of the specification and is incorporated herein by reference in its entirety. In the event of any inconsistency regarding structures for SEQ ID NO: 1-111 between the information described herein and the sequence listing provided electronically via EFS Web under file name PRD3411 Sequence Listing, the information provided in this document.

Prerequisites for creating the invention

Neuropeptide Y (NPY) receptors are activated by a closely related group of peptide agonists called the NPY family, which have different affinities for each receptor subtype. NPY, peptide tyrosine-tyrosine (PYY), and pancreatic polypeptide (PP), all of which are 36 amino acids in length, are agonists of the NPY receptor family. NPY is a neurotransmitter that is synthesized, co-stored and released together with norepinephrine and epinephrine. NPY is one of the most abundant and widespread peptides in the central nervous system (CNS) of humans and rodents and is expressed in areas of the brain associated with nutrition and stress. In the peripheral nervous system, neurons containing NPY are predominantly sympathetic. PYY is predominantly synthesized and released by intestinal endocrine cells. Cleavage of NPY and PYY by the endothelial serine protease, dipeptidyl peptidase IV (DPP-IV), generates NPY3.36 and PYY3.36, which are selective ligands for the Y2 and Y5 subtypes of the NPY receptor family. PP is primarily found in pancreatic islet cells other than those containing insulin, glucagon, or somatostatin.

To date, five different NPY receptors have been identified, four of which are known to be relevant to human physiology. The Y1, Y2 and Y5 receptors preferentially bind NPY and PYY, whereas the Y4 receptor preferentially binds PP. Y2 and Y5 receptors are also highly activated by NPY3.36 ^and PYY3-36. In general, the NPY family of ligands has variable selectivity for each NPY receptor isoform, with PYY _3-36 previously reported to have moderate to strong selectivity for the Y2 isoform. Each of these receptors is coupled to inhibition of adenylate cyclase by toxin-sensitive Gai pertussis.

PYY is secreted from endocrine L cells in response to food and, in particular, after fat ingestion. PYY1.36 is dominant in the fasted state, with PYY3.36 being the major form found postprandially in humans, with plasma concentrations negatively correlated with caloric intake. PYY3-36 has been shown to reduce food intake in humans, monkeys, rats, rabbits and mice (Batterham RL et al. Nature, 2002 Aug 8; 418(6898):650-4; Batterham RL et al. N. Engl. J Med. 2003 Sep 4; 349(10):941-8; Challis BG et al., Biochem Biophys. Res. Commun. 2003 Nov 28; 311(4):915-9). The anorexigenic effects of PYY3.36 are thought to be mediated by Y2 based on preferential binding to this receptor and loss of feeding efficiency in Y2-deficient mice (Batterham RL, et al. Nature, 2002 Aug 8;418(6898):650-4) . Intra-arc injection of PYY _{3_36} reduces food intake in rats and mice (Batterham et al. Nature, 2002 Aug 8;418(6898):650-4), suggesting that hypothalamic Y2 receptor interactions may mediate _these effects. Acute feeding responses have also been shown to result in dose-dependent effects on body weight in ob/ob mice, DIO mice, and Zucker fa/fa mice (Pittner RA et al. Int. J. Obes relat Metab. Disord. 2004 Aug;28(8):963-71). In addition, PYY ₃ - ₃₆ has also been shown to improve insulin-mediated glucose disposal and insulin sensitivity in DIO rodents (Vrang N. et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. Aug; 291(2 ):R367-75). Bariatric surgery results in increased immunoreactivity to circulating PYY (le Roux CW et al., Ann Surg, 2006 Jan; 243(1):108-14), which appears to play a role in weight loss after surgery.

Given its role in appetite control and food intake, as well as its antisecretory effects

- 1 045724 and effects promoting gastrointestinal absorption in mammals, PYY3.36 may be effective in the treatment of obesity and related conditions, as well as a number of gastrointestinal disorders. However, the therapeutic utility of PYY3-36 itself as a therapeutic agent is limited by its rapid metabolism and resulting short half-life in the blood (Torang et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 310:R866-R874 (2016) ).

Thus, it is desirable to provide a PYY analogue or derivative with improved metabolic stability and pharmacokinetic profile compared to PYY _3-36 . Such derivatives with a long half-life in vivo will provide modulation of the Y2 receptor with a longer duration of action, making them acceptable as therapeutic agents for subjects in need of such modulation.

The above description is presented solely to better understand the nature of the problems facing the art and should not be construed in any way as an admission of prior art; nor should the citation of any reference herein be construed as an admission that such reference constitutes prior art to the present invention.

Summary of the invention

One general aspect of the present invention relates to a compound of formula I

Z ₄ PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNRYYZ22Z23LRZ _2e YLNZ ₃₀

I^/ 1 ^HN ,° and

.....ς \x(CH ₂ ) _m -----BRIDGE------(CH ₂ ) _n [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH ₂

Λ°

Formula I where p is 0 or 1;

m is 0, 1, 2, 3, 4 or 5;

n is 1, 2, 3 or 4;

q is 0 or 1;

provided that q is equal to 1 only if Z ₃₀ is absent;

BRIDGE represents -Ph-CH ₂ -S-, -triazolyl-, -NHC(O)CH ₂ S-, -SCH ₂ C(O)NH-,

- (OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

Z ₄ represents K, A, E, S or R;

Z ₇ represents A or K, wherein the side chain amino group of said K is optionally acylated

where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH ₂ Cl;

Z9 is G or K, wherein the side chain amino group of said K is optionally acylated

where t is 0, 1 or 2;

- 2 045724 u equals 0 or 1;

v is equal to 14, 16 or 18;

where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH2Cl;

Zu represents D or K, wherein the side chain amino group of said K is optionally acylated

where w is 0, 1, 2 or 4;

x is 0 or 1;

y is 14, 16 or 18;

O _n COaH O tCH ₂ ) ₁₄ CH ₃

where i is an integer from 0 to 24, and X=Br, I or Cl,

- 3 045724

-C(O)CH2Br, -C(O)CH2I or -C(O)CHCl;

Z ₂₂ represents A or K, wherein the side chain amino group of said K is optionally acylated

where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH2C1;

Z ₂₃ represents S or K, wherein the side chain amino group of said K is optionally acylated

- 4 045724

where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH2Cl;

Z ₂₆ represents A or H;

Z ₃₀ is L, W, absent or K, provided that Z ₃₀ is absent only if q is 1 and the side chain amino group of said K is optionally acylated

where r is 0, 1 or 2;

s=0 or 1;

q is 14, 16 or 18; or

- 5 045724

Z ₃₅ is

CF ₃

Z ₃₄ is

(CH2CH2O) ₁₂ CH ₃ .

(CH ₂ ) ₁₅ OH or a pharmaceutically acceptable salt thereof.

The present invention also provides methods for preventing, treating, delaying the onset of or alleviating a syndrome, disorder or disease or one or more symptoms of said syndrome, disorder or disease, wherein said syndrome, disorder or disease is selected from the group consisting of obesity, diabetes 2 type, metabolic syndrome (eg, syndrome X), insulin resistance, impaired glucose tolerance (eg, glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors relating to uncontrolled cholesterol and/or lipid levels, osteoporosis, inflammation and eczema, comprising administering to a subject in need thereof an effective amount of a compound of formula I, a derivative or pharmaceutically acceptable salt thereof, or a form thereof, composition or medicinal product, or any

- 6 045724 fight the combination described above.

The present invention also provides for the prevention, treatment, delay of the onset or amelioration of any of the diseases, disorders, syndromes or symptoms described herein by combination therapy which comprises administering to a subject in need thereof an effective amount of a compound of Formula I, a derivative or pharmaceutically acceptable compound thereof. a salt or form, composition or drug thereof in combination with any one or more of the following additional compounds: a dipeptidyl peptidase-4 (DPP-4) inhibitor (eg, sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); GLP-1 receptor agonist (eg, short-acting GLP-1 agonists such as exenatide and lixisenatide; intermediate-acting GLP-1 receptor agonists such as liraglutide; long-acting GLP-1 receptor agonists such as sustained-release exenatide, albiglutide , dilaglutide); sodium gluconate cotransporter-2 (SGLT-2) inhibitors (eg, canaglifosin, dapaglifosin, empaglifosin, etc.); bile acid excretion enhancers (eg, colesevelam, etc.) and dopamine receptor agonists (eg, immediate release bromocriptine). In some embodiments, the dose of the additional compound is reduced when administered in combination with a compound of Formula I, a derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, when used in combination with a compound of Formula I, the additional compounds may be used at lower dosages than when each is used alone.

The present invention provides for the prevention, treatment, delay of the onset or amelioration of any of the diseases, disorders, syndromes or symptoms described herein by combination therapy which comprises administering to a subject in need thereof an effective amount of a compound of Formula I, a derivative thereof or a pharmaceutically acceptable compound thereof. a salt or form, composition or drug thereof, in combination with one or more of the following additional compounds: biguanides (eg, metformin, etc.); insulin; oxyntomodulin; sulfonylureas (e.g. chlorpropamide, glimepiride, glipizide, glyburide, glibenclamide, glybornuride, glisoxepide, glycopyramide, tolazamide, tolbutamide, acetohexamide, carbutamide, etc.) and thiazolidinediones (e.g. pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone , englitazone, netoglitazone, rivoglitazone, troglitazone, etc.). In some embodiments, when used in combination with a compound of Formula I, a derivative thereof, or a pharmaceutically acceptable salt thereof, the additional compounds may be used at lower dosages than when each is used alone.

In other embodiments, the present invention provides for the prevention, treatment, delay of the onset or amelioration of any of the diseases, disorders, syndromes or symptoms described herein through combination therapy which comprises administering to a subject in need thereof an effective amount of a compound of Formula I, a derivative thereof or a pharmaceutically acceptable salt thereof or a form, composition or drug thereof, in combination with surgical treatment such as bariatric surgery (eg, gastric bypass surgery such as Roux-en-Y gastric bypass); sleeve gastrectomy; adjustable gastric banding; biliopancreatic bypass with exclusion of the duodenum; intragastric balloon; gastric plication and combinations thereof).

Additional aspects, features and advantages of the present invention will become more apparent upon reading the following detailed description of the present invention and the claims.

Detailed Description of the Invention

The Background section and the text of this document contain citations or descriptions of various publications, articles and patents; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, products, and the like that have been included in the present description is provided as context for the present invention. Such discussion is not an admission that any or all such references are part of the prior state of knowledge with respect to any inventions described or claimed.

All technical and scientific terms used herein, unless otherwise specified, have the same meaning as commonly understood by one skilled in the art to which this invention relates. Otherwise, certain terms used herein have the meanings set forth in the specification.

It should be noted that in this document and in the attached claims, the singular form includes objects in the plural unless the context clearly indicates otherwise.

Unless otherwise indicated, any numerical values, such as concentration or concentration range, described herein are to be understood as modified in all cases by the term about. Therefore, the numerical value typically includes ±10% of the specified

- 7 045724 values. For example, a concentration of 1 mg/ml includes 0.9 to 1.1 mg/ml. Likewise, the concentration range from 1 to 10% (w/v) includes from 0.9 to 11% (w/v). As used herein, the use of a numeric range explicitly includes all possible subranges, all individual numeric values within that range, including integers within such ranges, and fractional values, unless the context clearly requires otherwise.

Unless otherwise indicated, the term at least before a series of elements should be understood to refer to each element in the series. Those skilled in the art will be able to identify, or will be able to determine through routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the present invention.

As used herein, the terms contains, contains, includes, includes, has, having, contains or contains, or any other variation thereof, are intended to include the specified integer or group of integers, but not to exclude any other integer or group of integers, and are not exclusive or limiting. For example, a composition, mixture, process, method, article or device that contains a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article or device . Also, unless explicitly stated otherwise, the term or refers to the inclusive, not the exclusive or. For example, the condition A or B is satisfied by any of the following conditions: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true ( or present).

It should also be understood that the terms about, approximately, mainly, mainly and similar terms used herein when referring to the size or characteristic of a component of the preferred invention indicate that the described size/characteristic is not a strict boundary or parameter and does not exclude minor deviations therefrom, which are functionally the same or similar, as will be understood by one of ordinary skill in the art. At a minimum, such numerical references will include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), do not change the least significant digit.

The terms identical or percentage identity, in the context of two or more nucleic acid or polypeptide sequences (e.g., the cyclic polypeptide sequences PYY3-36), refer to two or more sequences or subsequences that are the same or have a specified proportion of amino acid residues or nucleotides that are identical, when compared and aligned for maximum consistency, as measured using one of the following sequence comparison algorithms or by visual inspection using methods known in the art in light of the present description.

For sequence comparisons, typically one sequence serves as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, the coordinates of the subsequence are determined, if necessary, and the parameters of the sequence algorithm program are determined. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the specified program parameters.

Optimal alignment of sequences for comparison can be carried out, for example, using the local homology algorithm according to Smith Waterman &, Adv. Appl. Math. 2:482 (1981), using the homology region alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), using the similarity search method of Pearson Lipman &, Proc. Nat'l. Acad. Sci. USA, 85:2444 (1988), using computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by visual inspection (see mainly Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture of Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (Supplement, 1995) (Ausubel)).

Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990), J. Mol. Biol. 215; 403-410 and Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402 respectively. Software for performing BLAST analyzes is publicly available from the National Center for Biotechnology Information.

A further indication that two nucleic acid sequences or two polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with the polypeptide encoded by the second nucleic acid.

- 8 045724 acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.

As used herein, the term subject means any animal, preferably a mammal, most preferably a human. As used herein, the term mammal includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

The term administration, as used in the methods of the present invention, means a method of therapeutically or prophylactically preventing, treating, or ameliorating a syndrome, disorder, or disease as described herein using a compound of the present invention or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety; or its form, composition or medicinal product. Such methods include administering an effective amount of the specified compound, form of the compound, composition or drug at different times during the course of treatment or simultaneously with other compounds in a combination form. The methods of the invention should be understood to include all known therapeutic regimens.

The term effective amount means that amount of an active compound or pharmaceutical agent that produces a biological or medical response in a tissue system, animal, or human being sought by a researcher, veterinarian, physician, or other practitioner, which includes the prevention, treatment, or amelioration of a syndrome, disorder, or diseases being treated or symptoms of the syndrome, disorder or disease being treated.

As used herein, the term composition is intended to cover a product containing specified ingredients in specified amounts, as well as any product that can be obtained, directly or indirectly, from combinations of specified ingredients in specified amounts.

Connections

In one general aspect, the present invention includes a compound of formula I

where p is 0 or 1;

m is 0, 1, 2, 3, 4 or 5;

n is 1, 2, 3 or 4;

q is 0 or 1;

provided that q is equal to 1 only if Z ₃₀ is absent;

BRIDGE represents -Ph-CH ₂ -S-, -triazolyl-, -NHC(O)CH ₂ S-, -SCH ₂ C(O)NH-,

-(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

Z ₄ represents K, A, E, S or R;

Z ₇ represents A or K, wherein the side chain amino group of said K is optionally acylated

- 9 045724 where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH2Cl;

Z9 is G or K, wherein the side chain amino group of said K is optionally acylated, where t is 0, 1 or 2; u is 0 or 1; and v is 14, 16 or 18;

N dsn ₂ ) _1В ср2Н

6. A SLS ₂ ) ₁₀ CO ₂ H where i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl ;

Zu represents D or K, wherein the side chain amino group of said K is optionally acylated

where w is 0, 1, 2 or 4;

x is 0 or 1;

y is 14, 16 or 18;

- 10 045724 where i is an integer from 0 to 24, and X=Br, I or Cl,

LSN2) _1v SO _i N

-C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

Z ₂₂ represents A or K, wherein the side chain amino group of said K is optionally acylated

L LSNL _1v CO ₂ H where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH2Cl ;

Z ₂₃ represents S or K, wherein the side chain amino group of said K is optionally acylated

- 11 045724

where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH2Cl;

Z ₂₆ represents A or H;

Z ₃₀ is L, W, absent or K, provided that Z ₃₀ is absent only if q is 1 and the side chain amino group of said K is optionally acylated

where r is 0, 1 or 2;

s=0 or 1;

q is 14, 16 or 18; or

- 12 045724

Z ₃₄ is

Z ₃₅ represents ci

Cl

CFj

_0O2H

_CO2H .

CF ₃

CO _? H

_CO2H

CF-

(CH ₂ ) ₁₀ CF ₃ . CO ₂ H ₀ O^(CH ₂ ) ₁₅ OCH ₂ CH _E

CO ₂ H (ch ₂ ) ₁₃ cf ₃

CO ₂ H ^ΝΗ Co ₂ n °у( ^ΝΗ

CO ₂ H o

£^(CH ₂ CH ₂ O) _1b CH ₃ .

ABOUT . X^(CH ₂ CH ₂ O) ₁₂ CH ₃ . g g ₀ O_(CH ₂ ) ₁₅ OH

_CO2H

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention includes a compound of Formula I or a derivative thereof, wherein:

p is 0 or 1;

m is 0, 1, 2, 3 or 5;

n is 1, 2 or 4;

q is 0 or 1;

provided that q is equal to 1 only if Z ₃₀ is absent;

The BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

Z ₄ represents K, A, E, S or R;

Z ₇ represents A or K, wherein the amine side chain of said K is replaced by

O _n Ν (CH^CC^H ^N hoV ? N . _n A^4^N_(CH ₂ ) ₁₄ CH ₃ “ YES or

ABOUT

AND.

'VG;

- 13 045724

Z ₉ represents G or K, wherein the amine side chain of said K is replaced by and."

where t is 0;

uequals 1;

v is equal to 14;

Zn represents D or K, wherein the amine side chain of said K is optionally replaced by where w is 0 or 4;

x is equal to 1;

y is 14;

COjH o ^Λ N^(CH ₂ ) ₁₄ CH ₃

Vg

I o

'Vg,

O N' 'Mp;

, -C(O)CH ₂ VG,

O and CO ₂ H O

OR

Z22 is A or K, wherein the amine side chain of said K is replaced by o

y(CH ₂ ) ₁₄ CH ₃ o o

N BUT

(CH ₂ ) ₁₀ CO ₂ H o

y.

'VG ;

Z ₂₃ represents S or K, wherein the amine side chain of said K is replaced by

- 14 045724

Z ₂₆ represents A or H;

Z ₃₀ is L, W, absent, or K, provided that Z ₃₀ is absent only if q is 1 and the amine side chain of said K is replaced by

where r is 0 or 2;

s equals 1;

q is 14, 16 or 18; or

Z ₃₄ is

- 15 045724

Z ₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention contains a compound of Formula I or a derivative thereof, wherein:

p is 0 or 1;

m is 0, 1, 2, 3 or 5;

n is 1, 2 or 4;

q is 0 or 1;

provided that q is equal to 1 only if Z ₃₀ is absent;

The BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

Z ₄ represents K, A, E, S or R;

Z ₇ represents A or K, wherein the amine side chain of said K is replaced by

Z9 is G or K, wherein the amine side chain of said K is replaced by

where t is 0;

u equals 1;

v is equal to 14;

Z11 is D or K, wherein the amine side chain of said K is optionally substituted with wherein w is 0 or 4;

x is equal to 1;

y is 14;

Z ₂₂ represents A or K, wherein the amine side chain of said K is replaced by

- 16 045724

Z ₂₃ represents S or K, wherein the amine side chain of said K is replaced by

Z26 is A or H;

Z ₃₀ is L, W, absent, or K, provided that Z ₃₀ is absent only if q is 1 and the amine side chain of said K is replaced by

where r is 0 or 2;

s equals 1;

q is 14, 16 or 18; or

- 17 045724

Z ₃₄ is

Z ₃₅ is

or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a compound of formula I or a derivative thereof selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 110, or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a compound of formula I or a derivative thereof selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 72, or a pharmaceutically acceptable salt thereof.

Another general aspect of the present invention relates to a conjugate containing a compound of Formula I, a derivative thereof, or a pharmaceutically acceptable salt thereof, and a half-life prolonging moiety conjugated thereto. As used herein, the term conjugated refers to a compound of the present invention covalently linked or covalently linked to a half-life prolonging moiety, directly or via a linker. As used herein, in connection with a compound of Formula I, a derivative thereof, or a pharmaceutically acceptable salt thereof, the phrase conjugate comprising the compound and a half-life prolonging moiety conjugated thereto is used interchangeably with the phrase compound conjugated to a half-life prolonging moiety.

As used herein, the term linker refers to a chemical module containing a covalent or atomic chain that covalently connects a compound of the present invention to a half-life extending moiety. The linker may, for example, include, without limitation, a peptide linker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a hybrid linker consisting of PEG and an intercalated heterocycle, and a hydrocarbon chain. The linker may, for example, be first covalently linked to a compound of the present invention and then covalently linked to a moiety to prolong the half-life.

As used herein, the term half-life prolonging fragment is used interchangeably with the term half-life prolonging fragment. Examples of fragments for extending half-life include, but are not limited to, monoclonal antibodies or fragments thereof, albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments thereof, and analogs. Additional half-life extension moieties that may be incorporated into the conjugates of the present invention include, for example, polyethylene glycol (PEG) molecules such as PEG5000 or PEG20,000, α-tocopherolyl, fatty acids and fatty acid esters of various chain lengths, for example, laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedicarboxylic acid, tetradecanedicarboxylic acid, octadecanedicarboxylic acid, docosanedicarboxylic acid, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) to obtain desired properties.

The compounds of the present invention may be covalently linked to one or more moieties to prolong the half-life using methods known to those skilled in the art as used herein. For example, as shown in the examples below, a half-life extending moiety, such as a PEG moiety or a lipophilic moiety, can be attached to a peptide molecule of the present invention, for example, by introducing a cysteine or lysine residue into the molecule and attaching the half-life extending moiety to the cysteine or lysine using well known methods. Examples with

- 18,045,724 units of the present invention, conjugated to a monoclonal antibody as a fragment to extend half-life, are also described in US provisional patent application No. 62/413,586, filed October 27, 2016, and in US patent application entitled Cyclic peptide tyrosine α-tyrosine compounds subjected to binding to antibodies as modulators of neuropeptide receptors, filed on the same day as the application for this invention, with registration number PRD3436; the contents of both applications are incorporated herein by reference in their entirety.

In accordance with embodiments of the present invention, an electrophile is introduced into the side chain of a cyclic PYY of the present invention, such as bromoacetamide or maleimide, that reacts site-specifically with the sulfhydryl group of a Cys residue incorporated into a half-life extending fragment, such as a monoclonal antibody or fragment thereof , thus creating a covalent bond between the cyclic PYY and the moiety to prolong the half-life. The compound of the present invention may be covalently linked to one or more moieties to prolong the half-life, directly or via a linker. Linkers suitable for the present invention include, but are not limited to, a peptide linker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, or a hybrid linker consisting of PEG and an embedded heterocycle. In some embodiments, the half-life extension moiety with the linker may be conjugated to a compound of the present invention at one or more amino acid positions of cyclic PYY, such as amino acid residue 4, 7, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 30 or 31 PYY, using methods known in the art. In some embodiments, the half-life extension moiety without a linker may be conjugated to a compound of the present invention at one or more amino acid positions of cyclic PYY, such as amino acid residue 4, 7, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 30 or 31 PYY, using methods known in the art. The numbering of amino acid residues corresponds to the numbering of hPYY _3-36 . Any of the compounds of the present invention, including, without limitation, SEQ ID NO: 1 to SEQ ID NO: 110, can be conjugated to a half-life extension moiety, directly or indirectly via a linker. According to embodiments of the present invention, a compound selected from the group consisting of SEQ ID NOs: 74, 95, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 and 110, or the pharmaceutically acceptable salt may be covalently linked to a half-life extending moiety, such as a monoclonal antibody or fragment thereof, via a linker.

The peptide molecule of the present invention, or a conjugate containing a peptide molecule covalently linked to one or more half-life prolonging moieties, can be assayed for functionality using known assay methods in light of the present disclosure. For example, the biological or pharmacokinetic activity of a peptide molecule of the present invention, alone or in a conjugate, in accordance with the present invention, can be analyzed using known in vitro or in vivo assays and compared.

In one embodiment, the present invention includes a compound of formula II

where p is 0 or 1;

m is 0, 1, 2, 3, 4 or 5;

n is 1, 2, 3 or 4;

The BRIDGE is -Ph-CH ₂ -S-, -triazolyl- or NHC(O)CH ₂ S-;

Z ₄ represents K, A, E or S;

Z ₉ represents G or K, wherein the amine side chain of said K is replaced by

1.".

where t is 0, 1 or 2;

u is 0 or 1;

v is equal to 14, 16 or 18;

- 19 045724

Z _n represents D or K, wherein the amine side chain of said K is replaced by

CH^CHZ where w is 0, 1 or 2;

x is 0 or 1;

y is 14, 16 or 18;

(СНа) _1в СО _g Н

Z ₂₆ represents A or H;

Z ₃₀ represents L or K, wherein the amine side chain of said K is replaced by

where r is 0, 1 or 2;

- 20 045724 s=0 or 1;

q is 14, 16 or 18; or

Z ₃₄ is

Z ₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention includes a compound of Formula II wherein: p is 0 or 1;

m is 0, 2, 3 or 5;

n is 1, 2 or 4;

The BRIDGE is -Ph-CH ₂ -S-, -triazolyl- or -NHC(O)CH ₂ S-;

Z ₄ represents K, A, E or S;

Z9 is G or K, wherein the amine side chain of said K is replaced by

Z ₁₁ represents D or K, and the amine side chain of said K is replaced by ί H ¹¹ “(CHzJuCHg

But

Z ₂₆ represents A or H;

Z ₃₀ represents L or K, wherein the amine side chain of said K is replaced by

- 21 045724

where r is 0 or 2;

s=0 or 1;

q is 14, 16 or 18;

Z ₃₄ represents _u o I o

VyS* ^yS* _or

Z ₃₅ represents and? I? । n ^NH ^NH ^NH ^NH η^Αιη, h ₂ n^nh^ h ₂ nAjh _mj1m η ₂ νΑ or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention includes a compound of Formula II wherein: p is 0 or 1;

m is 0, 2, 3 or 5;

n is 1, 2 or 4;

The BRIDGE is -Ph-CH ₂ -S-, -triazolyl- or -NHC(O)CH ₂ S-;

Z ₄ represents K, A, E or S;

Z ₉ represents G or K, wherein the amine side chain of said K is replaced by

Zu represents D or K, wherein the amine side chain of said K is replaced by

- 22 045724

Z26 is A or H;

Z ₃₀ represents L or K, wherein the amine side chain of said K is replaced by

where r is 0 or 2;

s=0 or 1;

q is 14, 16 or 18;

Z ₃₄ is

Z ₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention includes a compound of Formula II wherein: p is 0 or 1;

m is 0, 2, 3 or 5;

n is 1, 2 or 4;

The BRIDGE is -Ph-CH ₂ -S-, -triazolyl- or -NHC(O)CH ₂ S-;

Z ₄ represents K, A, E or S;

Z9 is G or K, wherein the amine side chain of said K is replaced by

- 23 045724

Zu represents D or K, wherein the amine side chain of said K is replaced by

Z ₂₆ represents A or H;

Z ₃₀ represents L or K, wherein the amine side chain of said K is replaced by

where q is 14, 16 or 18; Z ₃₄ is

Z ₃₅ is or its pharmaceutically acceptable salt.

In another embodiment, the present invention includes a compound of Formula II wherein: p is 0 or 1;

m is 0, 2, 3 or 5;

n is 1, 2 or 4;

The BRIDGE is -Ph-CH ₂ -S-, -triazolyl- or -NHC(O)CH ₂ S-;

Z ₄ represents K, A, E or S;

Z9 is G or K, wherein the amine side chain of said K is replaced by

Zu represents D or K, wherein the amine side chain of said K is replaced by

- 24 045724

Z ₂₆ represents A or H;

Z ₃₀ represents L or K, wherein the amine side chain of said K is replaced by

where q is 14, 16 or 18;

Z ₃₄ is

Z ₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention includes a compound of Formula II wherein: p is 0 or 1;

m is 0, 2, 3 or 5;

n is 1, 2 or 4;

The BRIDGE is -Ph-CH ₂ -S-, -triazolyl- or -NHC(O)CH ₂ S-;

Z ₄ represents K, A, E or S;

Z9 represents G; Z11 represents D;

Z ₂₆ represents A or H;

Z ₃₀ represents L or K, and the amine side chain of said K is replaced by where q is 14, 16 or 18; Z34 is

Z ₃₅ represents n but^o

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention includes a compound of formula II selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 46.

Another embodiment of the present invention includes N termini with cyclic side chain PYY analogues exhibiting at least 70, 75, 80, 85, 90, 95 or 99% sequence identity to hPYY( _3-36 ). As an example of a method for determining sequence identity between two analogues, two peptides

- 25 045724

(SEQ ID NO: 111) are aligned with each other.

The sequence identity of an analogue with respect to hPYY( _3-36 ) is expressed by the total number of aligned residues minus the number of distinct residues (ie, the number of identical residues aligned) divided by the total number of residues in hPYY _3-36 . In this example, the distinct residues are D11, which was replaced by substituted K11, then V31, which was replaced by hC31, and finally R35 was decarbonylated. Accordingly, in this example the sequence identity is (34-3)/34x100.

Cyclic PYY peptides.

PYY _3-36 is an endogenous hormone secreted by L cells in the distal intestine that acts as a Y2 receptor agonist to inhibit food intake. Given its role in the control of appetite and food intake, as well as its antisecretory and absorption promoting effects in the gastrointestinal tract in mammals, PYY _3-36 may be effective in the treatment of obesity and related conditions, as well as a number of gastrointestinal disorders. However, the therapeutic utility of PYY _3-36 itself as a therapeutic agent is limited by its rapid metabolism and short half-life. Thus, the present invention essentially relates to modified PYY _3-36 conjugates that prolong the half-life of the PYY _3-36 peptide and reduce the metabolism of the peptide in vivo.

In certain embodiments of the present invention, the modified PYY _3-36 peptides are cyclic PYY peptides. The terms cyclic PYY peptide, cyclic PYY _3-36 analogue, and cyclic PYY _3-36 peptide analogue may be used interchangeably.

As used herein, the term NTSC-PYY is intended to describe the cyclic analogues of PYY between the N-terminus and the side chain.

The peptide sequences described herein are written according to the usual procedure, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomeric forms of amino acids are known, they are the L-form of the amino acid presented unless explicitly stated otherwise. For convenience in describing the molecules of the present invention, traditional and non-traditional abbreviations for various amino acids (including both one- and three-letter encodings) and functional fragments are used. These abbreviations are well known to those skilled in the art, but for the sake of clarity they are listed as follows: A = Ala = alanine; R = Arg = arginine; N = Asn = asparagine; D = Asp = aspartic acid; βΆ = eAla = beta-alanine; C = Cys = cysteine; hC = hCys = homocysteine; E = Glu = glutamic acid; Q = Gln = glutamine; C = Gly = glycine; H = His = histidine; I = Ile = isoleucine; L = Leu = leucine; K = Lys = lysine; Nle = norleucine; F = Phe = phenylalanine; P = Pro = proline; S = Ser = serine; T = Thr = threonine; W = Trp = tryptophan; Y = Tyr = tyrosine and V = Val = valine.

For convenience, the numbering order of amino acid residues used to name the NTSC-PYY peptides of the present invention corresponds to the numbering order of hPYY _3-36 . Specific amino acid substitutions that have been introduced into NTSC-PYY peptides, relative to native residues at corresponding positions in hPYY _3-36 , are indicated by the corresponding amino acid code followed by the position of the substitution. Thus, S4 in the NTSC-PYY peptide refers to a peptide in which the corresponding native lys4 hPYY residue _3-36 has been replaced by serine. Similarly, hC31 in the NTSC-PYY peptide refers to a peptide in which the corresponding native val31 hPYY residue _3-36 has been replaced by homocysteine. Additional amino acid substitutions occurring in the NTSC-PYY peptides are described in accordance with this order and will be recognized by those skilled in the art as such.

Also for convenience, the naming order used for the NTSC-PYY peptides of the present invention includes the amino acid residues included in the ring along with the linking group(s) therebetween, in a left-to-right direction, starting with the N-terminal residue included in the ring. In all cases, the N-terminal amino acid residue of the cycle is connected through its α-amine functional group to a linking group, which, in turn, is connected to the bo residue

- 26 045724 of the amino acid chain at position 31 of the NTSC-PYY peptide. Thus, cyclo-(I3-m-COPhCH ₂ hC31) is used to describe the NTSC-PYY peptide cycle in which the α-amine functional group of Ile3 is acylated to a metatoluic acid residue whose methyl group is further linked via a thioester bond to the side chain of the hCys31 residue . Similarly, the term cyclo-(K4-CO(CH ₂ ) ₂ NHCOCH ₂ -hC31) is used to describe the cycle of the NTSC-PYY peptide in which the native Ile3 residue has been removed and whose (now N-terminal) α-amine functional group lys4 is acylated A 3-acetamidopropanoyl group in which the acetamidomethylene carbon atom is attached to the side chain of the hCys31 residue via a thioether bond.

Lysine residues can be introduced at various positions in the hPYY _{3_36} sequence to provide _a convenient functional subchain for additional derivatization. Lysine residues can be modified for either direct or indirect binding to the monoclonal antibody. In indirect binding to a monoclonal antibody, the lysine residue can be modified to contain a linker that allows the cyclic PYY peptide to be coupled to the monoclonal antibody. One skilled in the art will appreciate that the corresponding orthologs discussed herein can also be effectively used as such.

The term K(y-Glu) present in the peptide sequence is a lysinyl residue whose side chain ε-amino group is acylated by the γ-carboxyl group of glutamic acid.

The term K^-Glu-Pal (palmitoyl) means a lysinyl residue whose side chain ε-amino group is acylated by the γ-carboxyl group of N-hexadecan-1-oylglutamic acid.

The term K^-Glu-Stear (stearoyl) means a lysinyl residue whose side chain ε-amino group is acylated by the γ-carboxyl group of N-octadecane-1-oylglutamic acid.

The term K(γ-Glu-Arach (arachidoyl)) means a lysinyl residue whose side chain ε-amino group is acylated by the γ-carboxyl group of N-dodecane-1-oylglutamic acid.

The term K(OEG) (8-amino-3,6-dioxaoctanoyl) means a lysinyl residue whose side chain ε-amino group is acylated with 8-amino-3,6-dioxaoctanoic acid.

The term (OEG) ₂ means two OEG units sequentially linked to each other via an amide bond (ie 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid).

The term K(OEG) ₂ means a lysinyl residue whose side chain ε-amino group is acylated with 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid.

The term K((OEG) ₂ -y-G1u means a lysinyl residue whose side chain ε-amino group is acylated (22S)-22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18- diazatricosanedicarboxylic acid via its 1-carboxylic acid functionality.

The term K((OEG) ₂ -γ-Glu-Stear) means a lysinyl residue whose side chain ε-amino group is acylated (22S)-10,19-dioxo-22-stearamido-3,6,12,15-tetraoxa-9 ,18-diazatricosanedicarboxylic acid via its 1-carboxylic acid functionality.

The term K((OEG) ₂ -y-G1u-COC ₁₆ CO ₂ H) means a lysinyl residue whose side chain ε-amino group is acylated (21S)-9,18,23-trioxo-2,5,11,14-tetraoxa -8,17,22-triazanonantriacontane 1,21,39-tricarboxylic acid via its 1-carboxylic acid functional group.

Similarly, the term K((OEG) ₂ -y-G1u-COC ₁₈ CO ₂ H) means a lysinyl residue whose side chain ε-amino group is acylated (21S)-9,18,23-trioxo-2,5,11, 14-tetraoxα-8,17,22triazagentetracontane-1,21,41-tricarboxylic acid via its 1-carboxylic acid functional group.

The term K((OEG) ₂ -COC ₁₆ CO ₂ H) means a lysinyl residue whose side chain ε-amino group is acylated by 10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazahexatriacontanedicarboxylic acid via its 1 -carboxylic acid functional group.

The term K(PEG24-AcBr) means a lysinyl residue whose side chain ε-amino group is acylated with N-bromoacetyl-75-amino4,7,10,13,16,19,22,25,28,31,34,37,40, 43,46,49,52,55,58,61,64,67,70,73-tetracosaoxapentaheptacontanoic acid via its 1-carboxylic acid functional group.

The term K(PEG12-AcBr) means a lysinyl residue whose side chain ε-amino group is acylated with N-bromoacetyl-39-amino-4,7,10,13,16,19,22,25,28,31,34,37- dodecaoxanonatriacontanoic acid through its 1-carboxylic acid functional group.

The term K(PEG6-AcBr) means a lysinyl residue whose side chain ε-amino group is acylated with N-bromoacetyl-3-[(17-amino-3,6,9,12,15-pentaoxaheptadec-1-yl)oxy]-propane acid through its 1-carboxylic acid functional group.

The term K(PEG8-triazolyl-CH ₂ CH ₂ CO-PEG ₄ -AcBr) means a lysinyl residue whose side chain ε-amino group is acylated 27-[4-[2-[3-[2-[2-[3-( M-bromoacetylamino)propoxy]ethoxy]ethoxy]propylaminocarbonyl]ethyl]tetrazol-1-yl]-4,7,10,13,16,19,22,25-octahepta

- 27 045724 cosanoic acid through its 1-carboxylic acid functional group.

The term K(mPEG16) means a lysinyl residue whose side chain ε-amino group is acylated with 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49-hexaoxapentacontanoic acid via its 1-carboxylic acid functional group.

The term K(mPEG12) means a lysinyl residue whose side chain ε-amino group is acylated with 4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxaoctatriacontanoic acid via its 1-carboxylic acid moiety.

The term VitE refers to the α-tocopherol unit in the molecule.

The term AcVitE refers to an α-tocopherol unit whose phenolic group has a methylenylcarboxyl functional group linked to an ester.

The term K-Y-Glu-AcVitE means a lysinyl residue whose side chain ε-amino group is acylated (2-(((2R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12 -trimethyltridecyl)chroman-6-yl)oxy)αcetyl)L-glutamic acid through its γ-carboxylic acid functional group.

Many of the compounds of the present invention include a reduced amide bond between the C-terminal sequence residue, Y36, and the adjacent residue, R35. This reduced amide bond is represented by the term psi-(R35,Y36).

Various amino acid residues containing certain sequences that form the subject of the present invention contain methylated α-amino groups. Thus, the terms N-Me-Q34 or N-Me-R35 mean a-N-methylated glutamine at position 34 of the sequence, and a-N-methylated arginine at position 35 of the sequence, respectively.

The term N-Me-Q34, psi-(R35,Y36) in the sequence description refers to a sequence containing both an a-methylglutamine residue at position 34 and a reduced amide bond between residues R35 and Y36.

Likewise, the term N-Me-R35, psi-(R35,Y36) in the sequence description refers to a sequence containing both an a-methylarginine residue at position 35 and a reduced amide bond between this residue and Y36.

As used herein, the term PEGylation refers to covalent conjugates of one or more polyethylene glycol (PEG) molecules and one or more NTSC-PYY peptides. These conjugates may include, without limitation, from 1 to 24 PEG molecules on a single NTSC-PYY peptide. These conjugates may further include suitable linkers between the PEG molecules and the NTSC-PYY molecule, including, without limitation, γ-glutamate, -NHC(O), C(O) and C( _{1_4} )alkyl.

As used herein, the phrase lipidation refers to covalent conjugates of an NTSC-PYY peptide and one or more lipophilic groups. Preferred lipophilic groups include long chain hydrocarbon groups. Other lipophilic groups include steroids, terpenes, fat-soluble vitamins, phytosterols, terpenoids, phospholipids, glycerols, and natural or synthetic fatty acids. Examples of lipophilic groups include, but are not limited to, a-tocopherolyl, stearic acid, palmitic acid and arachidic acid. These conjugates may further include suitable linkers between the lipophilic molecules and the NTSC-PYY molecule, including, without limitation, γ-glutamate, -NHC(O), C(O), and C^-^alkyl.

The term PYY ₃ - ₃₆ refers to the following compound (SEQ ID NO: 111):

He

Pharmaceutical compositions.

In another general aspect, the present invention relates to a pharmaceutical composition containing the conjugates and compounds of the present invention, and a pharmaceutically acceptable carrier. The term pharmaceutical composition as used herein means a product containing a conjugate of the present invention together with a pharmaceutically acceptable carrier. The conjugates and compounds of the present invention and compositions containing them can also be used in the manufacture of a medicament for the therapeutic purposes mentioned herein.

As used herein, the term carrier refers to any excipient, diluent, excipient, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vial, microsphere, liposomal encapsulation or other material well known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.

- 28 045724

As used herein, the term pharmaceutically acceptable carrier refers to a non-toxic material that does not interfere with the effectiveness of the composition of the present invention or the biological activity of the composition of the present invention. In accordance with specific embodiments, in light of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody pharmaceutical composition can be used in the present invention.

Pharmaceutically acceptable acid/anionic salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate, tosylate and triethiodide. Organic or inorganic acids also include, without limitation, hydroiodic, perchloric, sulfuric, phosphoric, propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, saccharic or trifluoroacetic acid.

Pharmaceutically acceptable basic/cationic salts include, but are not limited to, aluminum salts, 2-amino-2-hydroxymethylpropane-1,3-diol (also known as tris-(hydroxymethyl)aminomethane, tromethane or TRIS), ammonium salts, benzathine, tert- butylamine, calcium salts, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium salts, L-lysine, magnesium salts, meglumine, N-methylD-glucamine, piperidine, potassium salts, procaine, quinine, sodium salts, triethinolamine or zinc salts.

Some embodiments of the present invention provide pharmaceutical compositions containing the compounds of the present invention in an amount of from about 0.001 to about 100 mg/ml, from about 0.01 to about 50 mg/ml, or from about 0.1 to about 25 mg/ml . The pharmaceutical compositions may have a pH of from about 3.0 to about 10, such as from about 3 to about 7 or from about 5 to about 9. The composition may further contain at least one ingredient selected from the group consisting of a buffer system, a preservative (s), tonicity regulator(s), chelating agent(s), stabilizer(s) and surfactant(s).

A composition containing pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, in Remington: The Science and Practice of Pharmacy (eg, edition 21 (2005) and any subsequent editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity regulators, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers may be used in the formulation of the pharmaceutical compositions of the present invention.

In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid composition is an aqueous composition, i.e. composition containing water. The liquid composition may comprise a solution, suspension, emulsion, microemulsion, gel, or the like. The aqueous composition usually contains at least 50% wt./wt. water, or at least 60, 70, 75, 80, 85, 90 or at least 95% wt./wt. water.

In one embodiment, the pharmaceutical composition can be formulated as an injectable preparation, which can be administered, for example, through an injection device (eg, a syringe or infusion pump). The injection may, for example, be delivered subcutaneously, intramuscularly, intraperitoneally or intravenously.

In yet another embodiment, the pharmaceutical composition is a solid formulation, such as a lyophilized or spray-dried composition, that can be administered as is or to which solvents and/or diluents are added by the physician or patient prior to use. Solid dosage forms may include tablets, such as compressed tablets and/or film-coated tablets, and capsules (eg, hard or soft gelatin capsules). The pharmaceutical composition may also be in the form of, for example, sachets, dragees, powders, granules, lozenges or powders for dissolution.

Dosage forms may be immediate release, in which case they may contain a water-soluble or dispersible carrier, or they may be delayed release, sustained release, or modified release, in which case they may contain water-insoluble polymers that control the rate of dissolution of the dosage form in gastrointestinal tract.

In other embodiments, the pharmaceutical composition may be delivered intranasally, intraperitoneally, or sublingually.

The pH of the aqueous composition may range from 3 to 10. In one embodiment of the present invention, the pH of the composition is from about 7.0 to about 9.5. In yet another embodiment of the present invention, the pH of the composition is from about 3.0 to about 7.0.

- 29 045724

In another embodiment of the present invention, the pharmaceutical composition contains a buffer. Non-limiting examples of buffers include arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate , tartaric acid, tricine and tris-(hydroxymethyl)aminomethane and mixtures thereof. The buffer may be present alone or as part of an aggregate at a concentration of from about 0.01 to about 50 mg/ml, such as from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific buffers are alternative embodiments of the present invention.

In another embodiment of the present invention, the pharmaceutical composition contains a preservative. Non-limiting examples of buffers include benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidomourea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomersal and mixtures thereof. The preservative may be present individually or as an aggregate at a concentration of from about 0.01 to about 50 mg/ml, such as from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific preservatives are alternative embodiments of the present invention.

In another embodiment of the present invention, the pharmaceutical composition contains an isotonic agent. Non-limiting examples of this embodiment include salt (such as sodium chloride), amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan and threonine), alditol (such as glycerol, 1,2-propanediol, propylene glycol, 1,3-propanediol, 1,3-butanediol), polyethylene glycol (for example PEG400) and mixtures thereof. Another example of an isotonic agent includes sugar. Non-limiting examples of sugars may be mono-, di- or polysaccharides or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, in which _{the term sugar alcohol is defined as a C(4.8 )} hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol and arabitol. Pharmaceutical compositions containing each isotonic agent specified in this paragraph are alternative embodiments of the present invention. The isotonic agent may be present individually or as an aggregate at a concentration of from about 0.01 to about 50 mg/ml, such as from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific isotonic agents are alternative embodiments of the present invention.

In yet another embodiment of the present invention, the pharmaceutical composition contains a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA) salts, and mixtures thereof. The chelating agent may be present individually or as an aggregate at a concentration of from about 0.01 to about 50 mg/ml, such as from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific chelating agents are alternative embodiments of the present invention.

In yet another embodiment of the present invention, the pharmaceutical composition contains a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and/or one or more protease inhibitors.

In yet another embodiment of the present invention, the pharmaceutical composition contains a stabilizer, wherein said stabilizer is carboxy-/hydroxypropylcellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur compounds such as monothioglycerol) or thioglycolic acid. The stabilizer may be present individually or as part of an aggregate at a concentration of from about 0.01 to about 50 mg/ml, for example from about 0 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific stabilizers are alternative embodiments of the present invention.

In further embodiments of the present invention, the pharmaceutical composition contains one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term surfactant refers to any molecules or ions that are formed from a water-soluble (hydrophilic) part and a fat-soluble part.

- 30 045724 (lipophilic) part. The surfactant may, for example, be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants and/or zwitterionic surfactants. The surfactant may be present individually or as part of an aggregate at a concentration of from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific surfactants are alternative embodiments of the present invention.

In a further embodiment of the present invention, the pharmaceutical composition contains one or more protease inhibitors, such as, for example, EDTA and/or benzamidine hydrochloride (HCl). The protease inhibitor may be present individually or as an aggregate at a concentration of from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific protease inhibitors are alternative embodiments of the present invention.

The pharmaceutical composition of the present invention may contain an amount of amino acid base sufficient to reduce the formation of polypeptide aggregates during storage of the composition. The term amino acid base refers to one or more amino acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine) or analogs thereof. Any amino acid may be present either in free base form or in salt form. Any stereoisomer (ie L, D or mixture thereof) of the amino acid base may be present. The amino acid base may be present alone or in combination with other amino acid bases at a concentration of from about 0.01 to about 50 mg/ml, such as from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific amino acid bases are alternative embodiments of the present invention.

It will also be apparent to one skilled in the art that the therapeutically effective dose of the compounds of the present invention or pharmaceutical composition thereof will vary depending on the desired effect. Therefore, one skilled in the art can readily determine the optimal dosages to administer, and these will vary depending on the particular compound used, the route of administration, the concentration of the drug, and the degree of progression of the disease state. In addition, factors associated with the particular subject being treated, including the subject's age, body weight, diet, and time of administration, necessitate dose adjustment to the appropriate therapeutic level.

For all indications, the compounds of the present invention are preferably administered peripherally at a dose of about 1 μg to about 5 mg per day in single or divided doses (for example, a single dose can be divided into 2, 3, 4, 5, 6, 7, 8, 9 or 10 subdoses) or from about 0.01 to about 500 μg/kg, more preferably from about 0.05 to about 250 μg/kg, most preferably below about 50 μg/kg. Dosages within these ranges will vary in the potency of each agonist and can be readily determined by one skilled in the art. Therefore, the doses given above are examples for the average case. There may, of course, be individual circumstances requiring the use of higher or lower dosage ranges, which are within the scope of this invention.

In some embodiments, the compounds of the present invention are administered at a dose of about 1 μg to about 5 mg, or at a dose of about 0.01 μg/kg to about 500 μg/kg, more preferably at a dose of about 0.05 μg/kg up to about 250 μg/kg, most preferably at a dose below about 50 μg/kg with a dose of a second therapeutic agent (eg, liraglutide) at a dose of about 1 μg to about 5 mg or at a dose of about 0.01 to about 500 μg/ kg, more preferably at a dose of from about 0.05 to about 250 μg/kg, most preferably at a dose of about 50 μg/kg.

Pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts or quaternary ammonium salts formed from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, benzoate, benzenesulfonate, citrate, camphorate, dodecyl sulfate, hydrochloride, hydrobromide, lactate, maleate, methanesulfonate, nitrate, oxalate, pivalate, propionate, succinate, sulfate and tartrate. Basic salts include ammonium salts, alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, organic base salts such as dicyclohexamine salts, and amino acid salts such as arginine. In addition, basic nitrogen-containing groups can be converted to the quaternary state using, for example, alkyl halides.

The pharmaceutical compositions of the invention can be administered by any means suitable for the intended purpose. Examples include parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, or eye drops. Administration can be done orally. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, e.g.

- 31 045724 salts, acidic solutions, alkaline solutions, solutions in water with the addition of dextrose, isotonic carbohydrate solutions and inclusion complexes with cyclodextrin.

The present invention also covers a method for preparing a pharmaceutical composition, comprising mixing a pharmaceutically acceptable carrier with any of the compounds of the present invention. Additionally, the present invention includes pharmaceutical compositions prepared by mixing one or more pharmaceutically acceptable carriers with any of the compounds of the present invention.

Additionally, the compounds of the present invention may exist in one or more polymorphic or amorphous crystalline forms and are intended to be included within the scope of the invention. In addition, the compounds can form solvates, for example, with water (ie, hydrates) or common organic solvents. As used herein, the term solvate means the physical association of the compounds of the present invention with one or more solvent molecules. Such physical bonding involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain cases, it is possible to release a solvate, for example, if one or more solvent molecules are included in the crystal lattice of the crystalline solid. The term solvate is intended to cover both solution phase solvates and releaseable solvates. Non-limiting examples of acceptable solvates include ethanolates, methanolates, and the like.

Polymorphs and solvates of the conjugates of the present invention are intended to be included within the scope of the present invention. Thus, in the methods of treatment of the present invention, the term administration includes means of treating, ameliorating or preventing a syndrome, disorder or disease described herein using the conjugates of the present invention or a polymorph or solvate thereof, which is obviously included in The scope of the present invention, although not specifically described.

In another embodiment, the present invention relates to the compounds of the present invention for use as a medicine.

Included within the scope of the present invention are prodrugs of the compounds of the present invention. In general, such prodrugs are functional derivatives of compounds that are readily converted in vivo to the desired compound. Thus, in the treatment methods of the present invention, the term administration covers the treatment of various described disorders using a specific described compound or using a compound that has not been specifically described but which converts to the established compound in vivo after administration to a patient. Conventional procedures for selecting and preparing suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. Bundgaard, Elsevier, 1985.

Moreover, it is intended that, within the scope of the present invention, any element, particularly those referred to in connection with the compounds of the present invention, will include all isotopes and mixtures of isotopes of said element that occur naturally or are produced synthetically, naturally occurring or in isotopically enriched form . For example, reference to hydrogen also covers 1H, ^2H (D) and ^3H (T). Likewise, references to carbon and oxygen cover ¹² C, ¹³ C, and ¹⁴ C, and ¹⁶ O, and ¹⁸ O, respectively. Isotopes can be radioactive or non-radioactive. The radiolabeled compounds of the present invention may contain a radioactive isotope selected from the group consisting of ³ H, ^PS , ¹⁸ F, ¹²² I, ¹²³ I, ¹²⁵ I, ¹³¹ I, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br and ⁸² Br . Preferably, the radioactive isotope is selected from the group consisting of ³ H, ^PS and ¹⁸ F.

Some compounds of the present invention may exist as atropisomers. Atropoisomers are stereoisomers produced by constrained rotation around single bonds, with the barrier to steric deformation upon rotation being high enough to permit isolation of conformers. It should be understood that all such conformers and mixtures thereof are included within the scope of the present invention.

When the compounds of the present invention have at least one stereocenter, they may suitably exist as enantiomers or diastereomers. It should be understood that all such isomers and mixtures thereof are included within the scope of the present invention.

If mixtures of stereoisomers are formed during the methods for preparing the compounds according to the invention, these stereoisomers can be isolated by conventional techniques such as preparative chromatography. The compounds can be obtained in racemic form or the individual enantiomers can be obtained by enantiospecific synthesis or by resolution. Compounds can, for example, be separated into their constituent enantiomers by standard techniques, such as forming diastereomeric pairs by forming a salt with an optically active acid such as (-)-di-p-toluoyl-D-tartaric acid and/or (+)-di -p-toluoyl-L-tartaric acid, followed by fractional crystallization and regeneration of the free base. Compounds can also be separated by formation of diastereomeric esters or amides followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, compounds can be separated using a chiral column via a high efficiency

- 32 045724 liquid chromatography (HPLC) or SFC. In some cases, there may be rotamers of compounds observed on 1H NMR, leading to the appearance of complex multiplets and clustering of peaks in the 1H NMR spectrum.

During any of the processes for preparing the compounds of the present invention, there may be a need and/or desire to protect sensitive or reactive groups on any of the molecules in question. For these purposes, standard protecting groups can be used, such as those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, each of which is incorporated herein by reference in its entirety for all purposes. The protecting groups can subsequently be removed at any convenient stage using methods known in the art.

Methods of application

The present invention relates to a method of preventing, treating, or ameliorating a Y2 receptor syndrome, disorder, or disease in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a prolonging moiety. half-life, or the pharmaceutical composition of the present invention.

The present invention also provides a method of preventing, treating, delaying the onset of, or alleviating a disorder, disease, or condition, or any one or more symptoms of said disorder, disease, or condition, in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a compound derivative thereof. or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention.

In certain embodiments, the disease disorder or condition is selected from the group consisting of obesity, type I or II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia , hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol levels and/or lipids, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and/or eczema.

In certain embodiments, a therapeutically effective amount refers to an amount of drug that is sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or alleviating the severity of the disease, disorder, or condition being treated, or associated symptom; (ii) reducing the duration of the disease, disorder or condition being treated, or a symptom associated therewith; (iii) preventing the development of a disease, disorder or condition being treated, or a related symptom; (iv) regression of the cause of the disease, disorder or condition being treated, or an associated symptom; (v) preventing the development or occurrence of a disease, disorder or condition being treated, or a related symptom; (vi) preventing recurrence of the disease, disorder or condition being treated, or a related symptom; (vii) reducing the need for hospitalization of a subject having a disease, disorder or condition being treated, or a related symptom; (viii) reducing the length of hospitalization of a subject having a disease, disorder or condition being treated, or a related symptom; (ix) increasing the survival rate of a subject with the disease, disorder or condition being treated, or a symptom associated therewith; (xi) inhibiting or suppressing the disease, disorder or condition being treated, or a symptom associated therewith in the subject; and/or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another therapy.

The therapeutically effective amount or dosage may vary depending on various factors, such as the disease, disorder or condition being treated, the means of administration, the target site, the physiological condition of the subject (including, for example, age, body weight, health), whether the subject human or animal, other drugs administered and whether the treatment is prophylactic or therapeutic. Treatment dosages were titrated optimally to optimize safety and efficacy.

As used herein, the terms treat, treater, and treatment refer to the alleviation or reversion of at least one measurable physical parameter associated with a disease, disorder, or condition that is not necessarily evident in the subject, but may be visible in the subject. The terms treat, treater and treatment can also refer to causing regression, preventing progression, or at least slowing down the progression of a disease, disorder or condition. In a specific embodiment, treat, le

- 33 045724 treatment and treatment refers to alleviating, preventing the development or occurrence of, or reducing the duration of one or more symptoms associated with a disease, disorder or condition. In a specific embodiment, treat, treat, and treat refer to preventing recurrence of a disease, disorder, or condition. In a specific embodiment, treat, treat and treat refer to improving the survival of a subject having a disease, disorder or condition. In a specific embodiment, the terms treat, treater, and treatment refer to eliminating a disease, disorder, or condition in a subject.

In one embodiment, the invention provides a method of preventing, treating, delaying the onset of, or ameliorating obesity or any one or more symptoms of obesity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof. , optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention. In some embodiments, the subject's body weight is reduced, for example, between about 0.01 to about 0.1%, between about 0.1 to about 0.5%, between about 0.5 to about 1%, between about 1 to about 5%, between about 2 to about 3%, between about 5 to about 10%, between about 10 to about 15%, between about 15 to about 20%, between about 20 to about 25 %, between about 25 to about 30%, between about 30 to about 35%, between about 35 to about 40%, between about 40 to about 45% or between about 45 to about 50%, relative to body weight of the subject prior to administration of any of the conjugates, compounds, pharmaceutical compositions, forms or drugs of the present invention described herein, or in comparison with control subjects not receiving the conjugates, compositions, forms, drugs or combinations of the present invention described in this document.

In certain embodiments, the weight loss is maintained for, for example, about 1 week, for about 2 weeks, for about 3 weeks, for about 1 month, for about 2 months, for about 3 months, for for about 4 months, for about 5 months, for about 6 months, for about 7 months, for about 8 months, for about 9 months, for about 10 months, for about 11 months, for about 1 years, for about 1.5 years, for about 2 years, for about 2.5 years, for about 3 years, for about 3.5 years, for about 4 years, for about 4.5 years, for about 5 years, for about 6 years, for about 7 years, for about 8 years, for about 9 years, for about 10 years, for about 15 years or for about 20 years.

The present invention provides a method of preventing, treating, delaying the onset of, or ameliorating a syndrome, disorder, or disease, or any one or more symptoms of said syndrome, disorder, or disease, in a subject in need thereof, wherein said syndrome, disorder, or disease is selected from the group consisting of: obesity, type I or type II diabetes, metabolic syndrome (ie, syndrome X), insulin resistance, impaired glucose tolerance (eg, glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and/or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema, comprising administering to a subject in need thereof an effective amount of a compound, a derivative thereof or a pharmaceutically acceptable salt, optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention.

As used herein, metabolic syndrome refers to a subject having one or more of the following: high blood sugar (eg, high fasting blood sugar), high blood pressure, abnormal cholesterol levels (eg, low HDL levels ), abnormal triglyceride levels (i.e., high triglyceride levels), large waist (i.e., waist circumference), increased abdominal fat deposition, insulin resistance, glucose intolerance, increased levels of C-reactive protein (i.e., proinflammatory state) and elevated levels of plasma plasminogen activator inhibitor-1 and fibrinogen (i.e., prothrombotic state).

The present invention provides a method of reducing food intake in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or pharmaceutical composition of the present invention. In some embodiments, the subject's food intake is reduced, for example, between about 0.01 to about 0.1%, between about 0.1 to about 0.5%, between about 0.5 to about 1%, between about 1 to about 5%, between about 2 to about 3%, between about 5 to about 10%, between about 10 to about 15%, between about 15 to about 20%, between

- 34 045724 from about 20 to about 25%, between about 25 to about 30%, between about 30 to about 35%, between about 35 to about 40%, between about 40 to about 45% or between about 45 to about 50% relative to the subject's food intake prior to administration of any of the conjugates, compounds, compositions, forms, drugs or combinations of the present invention described herein, or compared to control subjects not receiving the conjugates, compounds, compositions, forms, drugs or combinations of the present invention described herein.

In some embodiments, the reduction in food intake is maintained, for example, for about 1 week, for about 2 weeks, for about 3 weeks, for about 1 month, for about 2 months, for about 3 months, for about 4 months, for about 5 months, for about 6 months, for about 7 months, for about 8 months, for about 9 months, for about 10 months, for about 11 months, for about 1 year , for about 1.5 years, for about 2 years, for about 2.5 years, for about 3 years, for about 3.5 years, for about 4 years, for about 4.5 years , for about 5 years, for about 6 years, for about 7 years, for about 8 years, for about 9 years, for about 10 years, for about 15 years or for about 20 years.

The present invention provides a method of lowering glycemic hemoglobin (A1C) in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention. inventions. In some embodiments, the subject's A1C is reduced, for example, by between about 0.001 to about 0.01%, between about 0.01 to about 0.1%, between about 0.1 to about 0.2%, between about 0.2 to about 0.3%, between about 0.3 to about 0.4%, between about 0.4 to about 0.5%, between about 0.5 to about 1%, between about about 1 to about 1.5%, between about 1.5 to about 2%, between about 2 to about 2.5%, between about 2.5 to about 3%, between about 3 to about 4% , between about 4 to about 5%, between about 5 to about 6%, between about 6 to about 7%, between about 7 to about 8%, between about 8 to about 9%, or between about 9 up to about 10% based on the subject's A1C prior to administration of any of the conjugates, compounds, compositions, forms, drugs or combinations of the present invention described herein, or compared to control subjects not receiving the conjugates, compounds, compositions, forms, drugs or combinations of the present invention described herein.

In other embodiments, methods are provided for reducing fasting blood glucose levels in a subject in need thereof, the methods comprising administering to the subject in need thereof an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a moiety to prolong the half-life, or pharmaceutical composition of the present invention. Fasting blood glucose levels may be reduced to less than about 140 to about 150 mg/dL, less than about 140 to about 130 mg/dL, less than about 130 to about 120 mg/dL, less than about 120 to about 110 mg/dL, less than about 110 to about 100 mg/dL, less than about 100 to about 90 mg/dL, or less than about 90 to about 80 mg/dL, relative to levels fasting blood glucose in a subject prior to administration of any of the conjugates, compounds, compositions, forms, drugs or combinations of the present invention described herein, or in comparison with control subjects not receiving the conjugates, compounds, compositions, forms, drugs agents or combinations of the present invention described herein.

The present invention provides a method of modulating Y2 receptor activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention. As used herein, the term modulation refers to an increase or decrease in receptor activity.

In some embodiments, an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention is administered to a subject in need thereof once daily, twice daily, three times daily, four times daily. once a day, five times a day, six times a day, seven times a day or eight times a day. In other embodiments, an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention is administered to a subject in need thereof every other day, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, twice a month, three times a month or four times a month.

- 35 045724

Another embodiment of the present invention includes a method of preventing, treating, delaying the onset of or alleviating a disease, disorder or syndrome, or one or more symptoms of any of these diseases, disorders or syndromes in a subject in need thereof, the method comprising administering to the subject in need thereof an effective an amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention in combination therapy. In some embodiments, the combination therapy is a second therapeutic agent. In some embodiments, the combination therapy is a surgical therapy.

As used herein, the term combination, in the context of administering two or more drugs to a subject, refers to the use of more than one therapy.

As used herein, combination therapy refers to the administration to a subject in need thereof of one or more additional therapeutic agents or one or more surgical therapies, together with an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a moiety to prolong the half-life , or the pharmaceutical composition of the present invention. In some embodiments, one or more additional therapeutic agents or surgical therapeutic agents may be administered on the same day as an effective amount of a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or pharmaceutical composition of the present of the invention, and in other embodiments, one or more additional therapeutic agents or surgical therapeutic agents can be administered in the same week or in the same month as an effective amount of a compound, derivative, or pharmaceutically acceptable salt thereof, optionally conjugated to a period extending moiety half-life, or the pharmaceutical composition of the present invention.

In some embodiments, in which the disease or disorder is selected from the group consisting of obesity, type II diabetes, metabolic syndrome, insulin resistance, and dyslipidemia, the second therapeutic agent may be an antidiabetic agent. In some embodiments, the antidiabetic agent may be a glucagon-like peptide-1 (GLP-1) receptor modulator.

The present invention also provides for the prevention, treatment, delay of the onset or amelioration of any of the diseases, disorders, syndromes or symptoms described herein in a subject in need thereof using combination therapy, which includes administering to the subject in need thereof an effective amount of a compound, derivative or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or a pharmaceutical composition of the present invention in combination with one or more of the following therapeutic agents: a dipeptidyl peptidase-4 (DPP-4) inhibitor (e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); GLP-1 receptor agonist (eg, short-acting GLP-1 agonists such as exenatide and lixisenatide; intermediate-acting GLP-1 receptor agonists such as liraglutide; long-acting GLP-1 receptor agonists such as sustained-release exenatide, albiglutide , dilaglutide); sodium gluconate cotransporter-2 (SGLT-2) inhibitors (eg, canaglifosin, dapaglifosin, empaglifosin, etc.); enhancers of bile acid excretion (for example, colesevelam, etc.); dopamine receptor agonists (eg, immediate release brompiraptin); biguanides (for example, metformin, etc.); insulin; oxyntomodulin; sulfonylureas (for example, chlorpropamide, glimepiride, glipizide, glyburide, glibenclamide, glybornuride, glisoxepide, glycopyramide, tolazamide, tolbutamide, acetohexamide, carbutamide, etc.); and thiazolidinediones (eg; pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone, etc.). In some embodiments, the dosage of the additional therapeutic agent(s) is reduced when administered in combination with a compound, a derivative thereof, or a pharmaceutically acceptable salt thereof, optionally conjugated to a half-life prolonging moiety, or pharmaceutical composition of the invention. In some embodiments, when used in combination with a conjugate or compound of the present invention, the additional therapeutic agent(s) may be administered at lower doses than when each is used alone.

In certain embodiments, wherein the disease or disorder is selected from the group consisting of obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia , hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and/or lipid levels, osteoporosis,

- 36 045724 inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema, the second therapeutic agent may be liraglutide.

The present invention provides for the prevention, treatment, delay of the onset or amelioration of any of the diseases, disorders, syndromes or symptoms described herein in a subject in need thereof using combination therapy which comprises administering to the subject in need thereof an effective amount of a compound, a derivative thereof or a pharmaceutically acceptable salt thereof, optionally conjugated to a moiety to prolong the half-life, or a pharmaceutical composition of the present invention, in combination with surgical therapy. In some embodiments, the surgical therapy may be bariatric surgery (e.g., gastric bypass surgery such as Roux-en-Y gastric bypass; sleeve gastrectomy; adjustable gastric banding; biliopancreatic duodenal bypass; intragastric balloon; gastric plication; and combinations thereof) .

In embodiments in which one or more additional therapeutic agents or surgical therapeutic agents are administered on the same day as an effective amount of a conjugate or compound of the present invention, the conjugate or compound of the present invention may be administered before, after, or simultaneously with the additional therapeutic agent or surgical therapeutic agent. Use of the term in combination does not limit the order in which the drugs are administered to a subject. For example, the first therapeutic agent (e.g., a composition described herein) can be administered before (e.g., 5, 15, 30, 45 minutes, 1, 2, 4, 6, 12, 16, 24, 48, 72, 96 hours , 1, 2, 3, 4, 5, 6, 8 or 12 weeks), together with or after (for example, after 5, 15, 30, 45 minutes, 1, 2, 4, 6, 12, 16, 24, 48, 72, 96 hours, 1, 2, 3, 4, 5, 6, 8 or 12 weeks) of administration of a second therapeutic agent to a subject in need thereof.

Abbreviations

Here and throughout the rest of the application text, the following abbreviations may be used:

Abu: 4-aminobutyric acid;

Ac ₂ O: acetic anhydride;

aq.: aquatic;

alloc: allyloxycarbonyl;

arach: arachidyl;

Boc: tert-butoxycarbonyl;

BSA: bovine serum albumin;

CDI: 1,1'-carbonyldiimidazole;

DCM: dichloromethane;

Dde: 1 -(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl;

DIBAL-H: diisobutylaluminum hydride;

DIC: diisopropylcarbodiimide;

DIEA: diisopropylethylamine;

DMA: K,K-dimethylacetamide;

DMF: K,K-dimethylformamide;

DMSO: methyl sulfoxide;

DODT: 2,2'-(ethylenedioxy)diethanethiol;

EDC: K-(3-dimethylaminopropyl)-N-ethylcarbodiimide;

EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

Et: ethyl;

EtOAc: ethyl acetate;

EtOH: ethyl alcohol;

FBS: fetal bovine serum;

Fmoc: 9-fluorenylmethoxycarbonyl;

g: gram(s);

h: hour(s);

HATU: 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate;

HBSS: Hank's Balanced Salt Solution;

HBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate);

HCTU: 2-(6-chloro-1H-benztriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate;

HCl: hydrochloric acid;

HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid;

HOBT: 1-hydroxybenztriazole;

HPLC: high performance liquid chromatography;

ivDde: 1-(4,4-dimethyl-2,6-dioxycyclohex-1-ylidene)-3-methylbutyl;

LAH: lithium aluminum hydride;

LCMS: high pressure liquid chromatography with mass spectrometry;

- 37 045724

Me: methyl;

MeCN: acetonitrile;

MeOH: methyl alcohol;

mg: milligram;

min: minute(s);

Mmt: 4-methoxytrityl;

ml/min: 1 ml per minute;

Mtt: 4-methyltrityl;

NHS: N-hydroxysuccinimide;

NMP: 1-methyl-2-pyrrolidone;

OEG: 8-amino-3,6-dioxaoctanoyl;

Oxime: ethylcyano(hydroxyimino)acetate;

Pal: palmitoyl;

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;

Pd(PPh ₃ ) ₄ : tetrakis-(triphenylphosphine)palladium(0);

PhSiH ₃ : phenylsilane;

psi: reduced amide bond (between adjacent amino acids);

PyBroP: bromo-tris-pyrrolidinophosphonium hexafluorophosphate;

Pyoxim: 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidinophosphonium hexafluorophosphate; r.t.: room temperature;

RT: retention time;

saturated: saturated;

SPPS: solid phase peptide synthesis;

Stear: stearoyl;

t-Bu: tert-butyl;

TBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate;

TFA: trifluoroacetic acid;

THF: tetrahydrofuran;

THP: tetrahydropyranyl;

TIPS: triisopropylsilane;

Tris: tris-(hydroxymethyl)aminomethane;

Trt: triphenylmethyl.

Synthesis.

The compounds of formula I in the present invention can be synthesized in accordance with general synthetic methods known to those skilled in the art. The following description of the synthesis is provided by way of example and is in no way intended to limit the present invention.

Analogs or derivatives of NTSC cyclic PYY (NTSC-PYY) can be synthesized using various known standard techniques for forming sequential peptide bonds between amino acids and are preferably carried out by solid-phase peptide synthesis (SPPS), as generally described in Merrifield (J. Am. Chem. Soc., 1963, 85, 2149-2154), using an automated peptide synthesizer, traditional laboratory synthesis, or a combination of both approaches. Traditional peptide synthesis techniques involve the condensation of the free amino group of one amino acid residue, the other reactive functional groups of which are suitably protected, and the free carboxyl group of another amino acid, the reactive functional groups of which are also suitably protected. Examples of condensation agents commonly used to form a peptide bond include diisopropylcarbodiimide (DIC) with or without 1-hydroxybenztriazole (HOBT) or ethyl cyano(hydroxyamino) acetate (oxime pure), 2-(1H-benzotriazol-1-yl)-1, 1,3,3-tetramethylaminium hexafluorophosphate (HBTU), 2-(1H-7-azabenztriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HATU), 2-(6-chloro-1H-benztriazole -1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyOxim), 2-(1H-benzotriazol-1-yl) )-1,1,3,3tetramethylaminium tetrafluoroborate (TBTU), bromo-tris-pyrrolidinephosphonium hexafluorophosphate (PyBroP), etc.

The methodology for automated peptide synthesis can be carried out at room temperature or at elevated temperatures, preferably by using microwave heating, as described in Yu (J. Org. Chem., 1992, 57, 4781-4784) and later improved by Palasek (J. Pept. Sci., (2007), 13, 143-148).

The compounds of the present invention (C-terminal amides) can be conveniently prepared using N-a-Fmoc-protected amino acid methodology, in which the carboxy terminus of a suitably N-a-Fmoc-protected amino acid is coupled to a conventional solid phase resin

- 38 045724 using a suitable binding agent. Suitable conventional commercially available solid phase resins include Rink amide MBHA resin, Rink amide AM resin, Tentagel S RAM resin, Fmoc-PAL-PEG PS resin, SpheriTide Rink amide resin, ChemMatrix Rink amide resin, Sieber TG amide resin, Sieber TG resin and so on. The resin-bound Fmoc amino acid can then be deprotected by exposure to 20% piperidine in DMF or NMP, which serves to selectively remove the Fmoc protecting group. Additional Fmoc-protected amino acids are then sequentially coupled and sequentially deprotected, thereby generating the desired protected peptide bound to the resin. In certain cases, it may be necessary to use an orthogonally reactive protecting group for another amine in the peptide sequence that will withstand Fmoc deprotection conditions. Protecting groups such as 4-methyltrityl (Mtt) or 4-methoxytrityl (Mmt), both of which are removed by treatment with 1% TFA/DCM, or preferably alloxycarbonyl (alloc; which is removed by treatment with Pd(PPh ₃ ) ₄ /PhSiH ₃ ), 1-(4,4-dimethyl-2,6-dioxycyclohex-1-ylidene)ethyl (Dde; which is removed by treatment with 2-3% hydrazine/DMF), and 1-(4,4-dimethyl-2,6 -dioxycyclohex1-ylidene)-3-methylbutyl (ivDde; which is removed by treatment with 2-3% hydrazine/DMF) can be used effectively in such cases.

In traditional peptide synthesis methodologies, reactive alpha amino acid side chains are largely protected throughout the synthesis process with suitable protecting groups to render them inert to coupling and deprotection protocols. Although many protecting groups for amino acid side chains are known in the art, the most preferred protecting groups herein are: tert-butyl (t-Bu) for serine, threonine, glutamic acid, aspartic acid and tyrosine; trityl (Trt) for asparagine, glutamine, cysteine, homocysteine and histidine; tert-butoxycarbonyl (Boc) for tryptophan and the ε-amino group of lysine; and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine. These protecting groups are removed by strong acid treatment, such as high concentrations of trifluoroacetic acid (TFA).

Once SPPS is complete, the resin-bound side chain protected peptide is deprotected and simultaneously cleaved from the resin using a digestion mixture that consists predominantly of (TFA) along with various combinations of carbocation scavengers such as triisopropylsilane (TIPS), water, phenol and anisole. The crude solid peptide is then isolated by precipitation of the peptide filtrate/mixture with cold ether. In the particular case of protected peptides bound to Sieber resin, cleavage of the protected peptide from the resin can be effectively achieved after repeated treatment with 1-2% TFA in DCM without compromising the side chain protection. After isolation, further manipulation of the protected peptide can be carried out through reactions in solution. Finally, the protected peptide can be deprotected by a separate treatment with the cleavage mixture and precipitated as described above. The crude peptide thus obtained is then dissolved to a low concentration (approximately <4 mg/ml) in a predominantly aqueous solvent system containing an organic co-solvent such as acetonitrile or ethanol. After raising the pH of the solution to >5, the peptide is then subjected to an intramolecular cyclization reaction to form the corresponding crude NTSC PYY analogue of the present invention. The NTSC PYY analogues thus formed can be purified using purification techniques well known in the art. The preferred method for purifying the peptide used herein is reverse phase high pressure liquid chromatography (HPLC). The purified peptides are then analyzed using liquid chromatography mass spectrometry (LCMS).

General schemes.

The general synthetic procedure for the synthesis of C-terminal NTSC-PYY amides, in which the BRIDGE is -Ph-CH ₂ -S-, is shown in Scheme 1.

- 39 045724

Scheme 1

Synthesis of toluoyl-thioether-linked NTSC-PYY peptides: C-terminal amides

Rink's Fmoc resin (PAL-PEG PS resin; -0.16-0.2 mmol/g)

Automated SPPS stage A stage B eq. Ptos-AA(protected)-OH, DIC/Oxime or HBTU/DIEA

[l] _p Z _4P EZ ₇ PZ ₉ EZ ₁₁ AS _P EELNRY _Y Z ₂₂ Z ₂ 3LRZ _26Y LNZ3 ₀ Z3 ₁ [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-Q nh ₂

I 20 eq. CHCH ₂ RbSOgN, 10 eq. DIC, microwave

I irradiation, 75 °C, 15 min

[l] _p Z _4p E _Z7P Z ₉ EZ ₁₁ AS _P EELNR _Y YZ ₂₂ Z ₂₃ LRZ _26Y LNZ ₃₀ Z ₃₁ [V ₃₁ ] _q TR _Z34 Z ₃₅ Y-NH-Q

HN

stage C

TFU/N ₂ O/phenol/TIPS (88:5:5:2)

[l] _p Z ₄ PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNR _YY Z ₂₂ Z ₂ 3LRZ ₂₆ yLNZ3oZ3 ₁ [V3 ₁ ] _q TRZ3 ₄ Z _35Y -NH ₂

stage D

MeCN/H ₂ O or EtOH/H,O, ph ~7 - 9 [< 4 mg/ml]

H

[I1PZ4PEZ7PZ9EZHASPEELNRYYZ22Z23LRZ26YLNZ3Q-N

I ___

O --------------------------s ^x (V ₃₁ ] _q TRZ ₃₄ Z _35Y -NH ₂ * Amino acids are protected, Z ₃ j = Cys , homoСу s

X represents Cl or Br

A) Synthesis of resin-bound C-terminal amide peptide.

The protected peptidyl resin can be synthesized using the Fmoc strategy as described above on a CEM Liberty Blue microwave peptide synthesizer using low loading Rink amide resins, preferably Fmoc-PAL-PEG PS resin (approximately 0.16-0.2 m- eq/g available from Applied Biosystems) on a 0.1 mmol scale as depicted in Scheme 1. Standard Fmoc-protected amino acids (available from Novabiochem (EMD Millipore), Bachem, Peptides International or Chem-Impex) can be coupled at 5 -fold excess of the resin load using DIC/oxime as coupling agents and a reaction temperature of approximately 90°C for 4 minutes. Double coupling of Fmoc-Arg(Pbf)-OH can be performed at 90°C for 4 min each, and Fmoc-His(Trt)-OH can be coupled using a two-step protocol: 4 min at RT, then 8 min at 50°C. Individual Fmoc deprotections can be performed using 20% piperidine in DMF (deprotection solution) at 90°C for 1.5 min.

B) Method for binding halomethylbenzoic acids.

The deprotected Fmoc peptide resin (0.1 mmol) can be treated with a solution of the desired isomer (meta or para) of either chloro- or bromomethylbenzoic acid (20 eq) and DIC (10 eq) in DMF (4 ml) in the microwave reactor at 75°C for 15 minutes. The completeness of the reaction can be determined using the Kaiser ninhydrin test (Kaiser, et al., Anal. Biochem., 1970, 34, 595-598). In cases where binding is determined to be incomplete, binding can be repeated with fresh reagents.

C) Method for removing peptides from the resin.

Once SPPS is complete, the resin can be extensively washed with DMF and then DCM, and dried. The resin can then be treated with a cleavage mixture (on a 10 ml/0.1 mmol scale) consisting of TFA/water/TIPS (95:2.5:2.5) (Digestion Mix A) or more preferably with TFA/TIPS water/phenol/TIPS (88:5:5:2) (cleavage mixture B) and microwave at 38°C for 40 min, then filter. The resin can be washed with TFA and the combined filtrates concentrated under a stream of nitrogen to a volume of approximately 2.5 ml, and then

- 40 045724 The tide can be precipitated by adding cold diethyl ether (40 ml). The peptide/ester suspension can be centrifuged and the ester layer decanted. The peptide pellet can be resuspended in ether, centrifuged and decanted, and this process can be repeated a third time. The crude peptide thus obtained can then be dried under a gentle stream of nitrogen.

D) Method of peptide cyclization (thioester formation).

Crude cysteine- or homocysteine-containing peptide can be dissolved in anoxic MeCN/water (50-60% MeCN) or EtOH/water (60% EtOH) at a concentration of <4 mg/ml. The pH of the peptide solution can then be raised to approximately 7-9 by adding either solid NaHCO ₃ or saturated aq. NaHCO ₃ or 1 M aq. Tris buffer (pH 7.5) and the resulting solution can be stirred at room temperature. within 3-16 hours. Typically, cyclization is completed within 3-4 hours, as determined by LCMS analysis.

E) Peptide purification procedure.

The cyclization reaction mixture can be acidified to pH 1.5-3 by adding TFA and the solution concentrated to remove most of the organic solvent (MeCN or EtOH) to the point where little cloudiness occurs. If necessary, a minimal amount of co-solvent can be added back to achieve homogeneity of the mixture, and the resulting solution can then be directly purified using preparative multiple injection HPLC. Purification can be performed on either an Agilent PrepStar LC System or a Gilson 2020 Personal Purification System LC using a C18 or C8 reverse phase column selected from the following: Varian Pursuit XRs C18 (21x250 mm, 100 A, 5 µm) ; Varian Pursuit XRs Diphenyl (30x100 mm, 100 A, 5 µm); Zorbax 300 SB-C8 (21x250 mm, 300 A, 5 µm); Waters Atlantis T3 C18 (19x250 mm, 100 A, 5 microns); Agilent Polaris 5 C18-A (30x250 mm, 180 A, 5 µm). The mobile phase may consist of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 10-20% B to final concentrations of 40-90% B with values execution time in the range of 36-80 minutes. UV detection can be monitored at 220 and 254 nm. Fractions containing product can be analyzed by analytical HPLC on an Agilent 1100 HPLC system using the appropriate column type listed above (4.6 x 250 mm, 5 µm). The pure fractions can be combined, concentrated to remove most of the organic phase, and then lyophilized. The TFA/HCl salt exchange can then be carried out by lyophilization three times from 2 mM HCl according to the procedure described in Andrushchenko, et al., (J. Pept. Sci., 2006, 13, 37-43).

A general synthetic method for the synthesis of C-terminal amide NTSC-PYY peptides, where the BRIDGE is -NHC(O)CH2S, is shown in Scheme 2.

- 41 045724

Scheme 2

Synthesis of acetamidoyl-thioester-linked NTSC-PYY peptides: C-terminal amides Ptoc-Rink resin (PAL-PEG PS resin; -0.16-0.2 mmol/g)

Automated SPPS stage A eq. Ptos-AA(protected)-OH, DIC/Oxime or HBTU/DIEA

[l] _p Z4PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNRY¥Z ₂₂ Z ₂₃ LRZ ₂₆ YLNZ ₃ oZ ₃₁ [V3 ₁ ] _q TRZ ₃ 4Z ₃₅ Y-NH-Q

HN _x .(CH ₂ ) _i y”NH Stage ₂ B > (6 equiv.) (BgCH ₃ CO) ₂ O, microwave irradiation, 50 °C, 5 min

[l] _p Z ₄ PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ ₃₀ Z ₃₁ [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-Q

I n

HN.ACH ₂ )-N

Br

O stage C

TFU/N ₂ O/phenol/TIPS (88:5:5:2)

[l]pZ ₄ PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNRYYZ ₂₂ Z23LRZ ₂₆ YLNZ3 ₀ Z ₃₁ [V3 ₁ ]qTRZ ₃ 4Z3 ₅ Y-NH ₂ stage I)

[l] _p Z4PEZ ₇ PZ ₉ EZ _u ASPEELNRYYZ ₂₂ Z ₂ 3LRZ ₂₆ YLNZ ₃₀ -N

MeCN/HjO or EtOH/H ₂ O, ph -7 - 9 [< 4 mg/ml]

N?

[V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH ₂ ,(CH ₂ ) _P

S * Amino acids protected, Ζ _3ί = Cys, homoSuv

Steps A, C, D and purification E are essentially identical to those described in Scheme 1. However, an alternative BRIDGE can be introduced in step B as described below.

The deprotected Fmoc peptide resin (0.1 mmol) can be treated with a solution of bromoacetic anhydride (6-20 eq) in DMF (5 ml) in a microwave reactor at 50°C for 5 min; after this time, the reaction is usually determined to be complete according to the Kaiser ninhydrin test. In cases where binding is determined to be incomplete, binding can be repeated with fresh reagents.

General synthetic method for the synthesis of C-terminal amide NTSC-PYY peptides, where Z ₃₅ represents

shown in Diagram 3.

- 42 045724

Scheme 3

Loading the structural element psi-(R35,Y36) onto Sieber resin.

Fmoc-Sieber resin (Sieber resin NovaSyn TG; -0.19 mmol/g)

Fmoc (manual) stage A 2.5 3KB.Fmoc-Arg(Pbf)T[CH ₂ N(Boc)]Tyr(t-Bu)-OH,

NATi/DIEA or DIC/Oxima/DIEA, b.t., overnight

Fmoc-Z ₃₅ Y-NH-Q (automated) ^paws 5 eq. Fmoc-AA, [shushdtsed, microwave irradiation, 50 °C, 15 min

Fmoc-NH-[l]pZ ₄ PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNRY¥Z2 ₂ Z ₂ 3LRZ ₂₆ ¥LNZ ₃₀ Z ₃₁ [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-Q * Amino acids are protected; Z ₃₁ = Cys, homoSys, Glue, azido-Lys

A) HATU-mediated binding.

In a fused microwave reaction vessel (available from CEM Corporation), NovaSyn TG Sieber resin (available from Novabiochem) (0.1 mmol) can be treated with deprotection solution (5 ml) and heated at 50°C for 2.5 minutes. The reaction mixture is drained, washed with DMF and treated again with deprotection solution at 50°C for 5 minutes. After draining and rinsing the resin with DMF, perform a third deprotection treatment at 50°C for 5 minutes. The resin is drained and washed intensively with DMF and then with DCM. The resin is then treated with a solution of Fmoc-Arg(Pbf)-psi-(N-Boc)Tyr(tBu)-OH from Scheme 14 (3-5 eq.), HATU (2.75-4.5 eq.) and DIEA ( 6-10 eq.) in DMF (4 ml) and stir at room temperature. for 24 hours. The mixture is drained and the resin is washed intensively with DMF. The resin is then capped by treatment with 20% Ac ₂ O in DMF (5 ml) under microwave conditions at 50°C for 5 minutes. The reaction mixture is drained and the resin is washed extensively with DMF and then DCM.

A) (alternate) DIC/oxime mediated binding.

In a fused microwave reaction vessel, Sieber's NovaSyn TG resin (0.1 mmol) can be deprotected as described in step A above, then treated with a solution of moc-Arg(Pbf)-psi-(NBoc)Tyr(tBu)-OH (2 .75 eq.), DIC (2.75 eq.), Oxime (2.75 eq.) and DIEE (0.275 eq.) in MeCN (4 ml) and stir at room temperature. for 24 hours. The reaction mixture is drained and the resin is washed extensively with DMF and then DCM and can be used directly without capping.

B) Improvement of reduced amide (psi-R35,Y36) peptide on preloaded Sieber resin.

Additional amino acid extensions on preloaded Sieber resin (psi-R35,Y36) can be performed on a CEM Liberty Blue microwave peptide synthesizer. Standard Fmoc-protected amino acids were coupled at a 5-fold excess of the original resin load using HBTU/DIEA as coupling agents and a reaction temperature of approximately 50°C for 15 minutes. Double coupling of Fmoc-Arg(Pbf)-OH can be carried out using a two-step protocol for each coupling: 25 min at RT, then 15 min at 50°C and double coupling of Fmoc-His(Trt)-OH can be carried out using two-step protocol for each binding: 4 min at RT, then 8 min at 50°C. Fmoc deprotection can be accomplished in two stages, using fresh deprotection solution for each stage: 1) 50°C for 2.5 min and 2) 50°C for 5 min. In some cases, double couplings can be effectively applied throughout the process to improve the quality of the isolated crude peptide.

Installation of the thioether BRIDGE moiety can also occur by coupling with bromomethylbenzoic acid, shown in Scheme 1, Step B, with a reaction temperature of 50°C. Alternatively, the thioether BRIDGE moiety can be introduced using the bromoacetylation coupling shown in Scheme 2, Step B.

Once SPPS is complete, the resin is extensively washed with DMF and then DCM and dried. The protected peptide can then be cleaved from the Sieber resin using a solution of 1-2% TFA in DCM (10 ml/0.1 mmol scale), stir for approximately 10 min and filter. This treatment can be repeated an additional 9 times, using fresh mixture each time. The combined filtrates are then concentrated to yield the crude protected peptide as a syrup/yellow solid that can be used directly after treatment.

- 43 045724 for removing general protection. The above-obtained protected peptide is treated with cleavage mixture B (10 ml) at room temperature. for 2.5 hours and then concentrated under a stream of nitrogen to a volume of approximately 2.5 ml. The crude peptide can be precipitated by adding cold diethyl ether (40 ml). The peptide/ester suspension can be centrifuged and the ester layer decanted. The peptide pellet can be resuspended in ether, centrifuged and decanted, and this process can be repeated a third time. The crude peptide thus obtained can be dried under a gentle stream of nitrogen. Cyclization and purification of the reduced amide peptides (psi-R35,Y36) can be accomplished according to the procedures described in Scheme 1, Step D and Step E.

The synthesis of lipated c-terminal amide NTSC-PYY peptides can be carried out according to Scheme 4.

Scheme 4

Method for introducing lipidated lysine residues into peptide sequences synthesized on Rink amide resin PAL-PEG PS

Rink's Fmoc resin (PAL-PEG PS resin; ~0.16-0.2 mmol/g)

Fmoc-O

Automated SPPS eq. Ptos-AA(protected)-OH, DIC/oxime, 90 °C, 4 min.

ivDde-K(Fmoc)- Z ₃₁ [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-Q*

1) 5 eq. FMOC-OEG-CO ₂ H, DIC/Oxime, microwave irradiation, 90 °C, 4 min and/or ^gap A 5 eq. FMOC-Glu-CO ₂ tBu, DIC/Oxime, microwave irradiation, 90 °C, 4 min

2) 5 eq. Lipophilic acid, DIC/Oxime, or , HBTU/DIEA microwave irradiation, 75 °C, 15 min ivDde-Z3 ₀ Z3 ₁ [V ₃₁ ] _q TRZ34Z3 ₅ Y-NH-Q* step B step C (manual)

[2-3% H ₂ NNH ₂ in DMF; 90 °C, 3.5 min] x 3

H2N-Z30Z31 [V ₃₁ ] _q TRZ ₃₄ Z3sY-NH- Q* (automated) eq. Fmoc-AA, DIC/Oxime, microwave irradiation, 90 °C, 4 min.

Fmoc-NH-[l] _p Z ₄ PEZ ₇ PZ _g EZ ₁₁ ASPEELNRYYZ ₂₂ Z ₂ 3LRZ ₂₆ YLNZ ₃₀ Z ₃₁ [V ₃₁ ] _q TRZ ₃ 4Z ₃₅ Y-NH-Q * Amino acids are protected; Z _3i = Cys, roMoCys, Glue, azido-Lys

A) Method for introducing derivatized lysine residues into peptide sequences built on standard Rink amide resin.

The resin-coupled C-terminal amide peptide, advanced to a point prior to the desired derivatization point, and prepared as described in Scheme 1, Step A, can be sequentially coupled to Dde-Lys(Fmoc)-OH or ivDde-Lys(Fmoc)- OH and then Fmoc-Glu-OtBu (all supplied by Novabiochem) under microwave irradiation (either manually or on a Liberty Blue peptide synthesizer) using DIC/oxime coupling methods as described in Scheme 1, Step A. After removal Fmoc protection, the resin can be treated with a solution of lipophilic acid [palmitic acid or α-tocopheryloxyacetic acid (AcVitE)] (5-10 eq.), DIC (5-10 eq.) and either HOBT or Oxime (5-10 eq.) in DMF under microwave irradiation at 90°C for 10 min. The reaction mixture is then drained and the resin is washed with DMF.

B) Procedure for deprotecting Dole- or ivDde-protected lysinyl peptide.

The derivatized lysinyl peptide resin can be treated with a solution of 3% hydrazine in DMF (6 ml/0.1 mmol resin) under microwave irradiation at 90°C for 3.5 min. The reaction mixture is drained, and this procedure can be repeated two more times. The reaction mixture is drained and the resin is washed extensively with DMF and then with DCM.

C) Method for direct inclusion of the Fmoc-Lys(Pal-Glu-OtBu)-OH residue.

In cases where a palmitoylated-γ-Glu-lysinyl residue must be included in the sequence, Fmoc-Lys(Pal-Glu-OtBu)-OH (available from Peptides International or ActivePeptide) can be used directly in the procedure described in Scheme 1, step A.

The compounds of the present invention, with lipidated lysine residues, can be prepared

- 44 045724 following the methods presented in Diagram 1, stages B, C, D and E.

The synthesis of lipidated, reduced amide (psi-R35,Y36), C-terminal amide, NTSC-PYY peptides can be carried out according to Scheme 5.

Scheme 5

Method for introducing derivatized lysine residues into peptide sequences synthesized on preloaded (psi-R35,Y36) Sieber resin.

Fmoc-Z _3S Y-NH- Sieber resin (automated) eq. Fmoc-AA, HBTU/DIEA, microwave irradiation, 50 °C, 15 min.

Alloc-K(Fmoc)-Z3 ₁ [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-Q stage A

1) 5 eq. FMOC-OEG-CO _2H

HATU/DIEA, microwave irradiation, 50 °C, 15 min, and/or eq. FMOC-Glu-CO ₂ tBu, HATU/DIEA, microwave irradiation, 50 °C, 15 min.

2) 5 eq. Lipophilic acid, HAUT/DIEA, microwave irradiation, 50 °C, 15 min.

Alloc-Z ₃₀ Z ₃₁ [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-Q* (manual)

[Pd(PPhJ ₄ / Ph ₃ SiH, DCM; k.g., 30 min] x 2

H ₂ NZ ₃₀ Z ₃₁ [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH- Q* (automated) eq. Fmoc-AA, HBTU/DIEA, microwave irradiation, 50 °C, 15 min.

Fmoc-NH-[l]pZ4PEZ ₇ RZ ₉ EZ ₁₁ ASREELNRYYZ ₂ 2Z ₂₃ LRZ ₂₆ YLNZ3 ₀ Z ₃₁ [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH *Amino acids are protected; Z ₃ , = Cys, homoSuya, azido-Lys

A) Method for introducing derivatized lysine residues into peptide sequences built on preloaded (psi-R35,Y36) Sieber resin.

The partially enhanced reduced amide (psi-R35,Y36) peptide prepared as described in Scheme 3, Step A, can be sequentially coupled to Alloc-Lys(Fmoc)-OH (available from Chem-Impex or AAPPTes, LLC) and then with Fmoc-Glu-OtBu and then with palmitic acid (each at 5 eq., using either HATU/DIEA or HBTU/DIEA coupling methods at 50°C for 15 min. When the lipophilic acid to be coupled is stearic acid, arachidic acid, octadecanedicarboxylic acid, mono-tert-butyl ester (available from AstaTech, Inc.) or 20-(tert-butoxy)-20-oxoicosanic acid (available from Key Organics, Inc.), as solvent NMP can be used due to better solubility of the reagents, and the coupling reaction can be mediated using HATU/DIEA at 50°C for 30 min. Alternatively, for arachidic acid, coupling can be accomplished using the DIC/oxime mediated technique described in Scheme 1, stage A, but with reagent concentrations of 5 eq. and THF as the reaction solvent.

B) Method for removing Alloc protection.

The above resin (0.1 mmol) was washed with oxygen-free DCM and then treated with PhSiH ₃ (12.5 eq.) in oxygen-free DCM (5 ml). After 2 minutes, a solution of Pd(PPh ₃ ) ₄ (0.25 eq) in oxygen-free DCM (5 ml) can be added and the reaction mixture is stirred for 0.5 hours under nitrogen. The reaction mixture is decanted and the resin is washed 1x with oxygen-free DCM. The resin is again treated with PhSiH ₃ (12.5 eq.) and Pd(PPh ₃ ) ₄ (0.25 eq.) as described above and reacts for an additional 0.5 h. The reaction mixture is drained and The resin is successively washed intensively with DCM, DMF and DCM.

Further development and completion of the peptide can be performed as described above herein, starting with Scheme 3, Step B.

The synthesis of C-terminal amide analogs of NTSC-PYY in which the BRIDGE is triazolyl is shown in Scheme 6.

- 45 045724

Scheme 6

Synthesis of triazole-linked NTSC-PYY analogues: C-terminal amide peptides

Rink's Fmoc resin (PAL-PEG PS resin; -0.16-0.2 mmol/g)

Fmoc-θ (Automated SPPS) 1) 5 eq. Rptos-AA(protected)-OH, stage A DIC/Oxima, 90 °C, 4 min. 2) 5 eq. 4-Pentinoic acid, DIC/Oxime, 90 °C, 4 min, double coupling

[llpZ4PEZ ₇ PZ9EZ ₁₁ ASPEELNRY¥Z2 ₂ Z2 ₃ LRZ ₂₆ YLNK(Dde)Z ₃₁ TRZ ₃₄ Z ₃₅ Y-NH-Q

O (manual)

1) [2-3% H ₂ NNH ₂ in DMF; k.t., 5 min] x 7 stage B

2) 5 eq. FMOC-Glu-CO ₂ tBu, DIC/Oxime, microwave irradiation, 90 °C, 4 min

3) 5 eq. Lipophilic acid, DIC/Oxime or HBTU/DIEA, microwave irradiation, 75 °C, 15 _0]pZ4 p _EZ7 p _Z9EZiiAS p _EELNR YY _Z22Z23LRZ26 Y _LN ^^

TFA/water/TIPS/DODT (92.5:2.5:2.5:2.5), microwave irradiation, 38 °C, 40 min.

stage C stage D

ABOUT

Η 9

ASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ ₃₀ -N

TRZ ₃₄ Z ₃₅ Y-NH _z .(CH ₂ ) ₄

1) CuSO ₄ , TVTA, water/EtOH/MeCN

2) sodium ascorbate in water

Η Η

[l]pZ ₄ PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNRYYZ ₂₂ Z23LRZ ₂₆ YLNZ ₃₀ -N^A

TRZ34Z35Y-NH2.(CH) ₄

HN n=n * Amino acids are protected; Z _3l = k-azido-Nle;

A) Synthesis of azido- and alkynyl-containing C-terminal amide peptides bound to resin.

The resin-bound ε-azidonorleucine-containing protected peptide, N-terminally capped with 4-pentinoic acid, can be prepared according to Scheme 1, Step A. A double coupling protocol can be used to incorporate 4-pentinoic acid.

B) Procedure for introducing derivatized lysine residues into azido- and alkynyl-containing C-terminal amide peptides bound to the resin.

Fmoc-Lys(Dde)-OH can be introduced into SPPS at the position of the sequence to be derivatized, following the procedure described in Scheme 1, Step A. After completion of the linear sequence (after introduction of 4-pentonic acid), the resin can be treated with 3% hydrazine in DMF (on a scale of 8 ml/0.1 mmol) for 5 min at room temperature, after which the mixture is drained. This procedure can be repeated approximately 6 times, after which the resin is extensively washed with DMF and then DCM. Fmoc-Glu-OtBu is then sequentially coupled to the Dde-deprotected resin following the procedure described in Example 6A.

C) Method for removing azido- and alkynyl-containing peptide from the resin.

Once SPPS is complete, the resin can be extensively washed with DMF and then DCM, and dried. The resin is then treated with a digestion mixture (10 ml/0.1 mmol scale) consisting of TFA/water/DODT/TIPS (92.5:2.5:2.5:2.5) (Digestion Mixture C) and heated in a microwave reactor at 38°C for 40 minutes, then filtered. The resin is washed with TFA and the combined filtrates are concentrated under a stream of nitrogen to a volume of approximately 2.5 ml and the peptide can be precipitated by adding cold diethyl ether (40 ml). The peptide/ester suspension can be centrifuged and the ester layer decanted. The peptide pellet can be resuspended in ether, centrifuged and decanted, and this process can be repeated a third time. The crude peptide thus obtained can be dried under a gentle stream of nitrogen.

- 46 045724

D) Method of peptide cyclization (triazole formation).

The following solutions can be prepared using oxygen-free water:

1) CuSO ₄ (7 mg in 2 ml of water),

2) 30 mg TBTA in 5.4 ml EtOH and 0.6 ml MeCN,

3) premix solution 1 (943 μl) and solution 2 (4.8 ml),

4) sodium ascorbate (30 mg in 3 ml of water).

To a solution of the crude peptide from step C (0.021 mmol) in either oxygen-free water or HEPES buffer solution (pH 7.4) (20 ml), solution 3 can be added, then solution 4 (2.4 ml) and the resulting milky solution heat to 35-40°C until cyclization is complete, as determined by LCMS (approximately 1-5 hours). The reaction solution can be diluted to 40 ml with either water (0.1% TFA) or 60% MeCN/water (0.1% TFA), filtered and purified directly by preparative HPLC using multiple injections as described in Scheme 1, step E.

The synthesis of C-terminal amide analogs of NTSC-PYY in which the BRIDGE is a lactam is shown in Scheme 7.

Scheme 7

Synthesis of lactam-bridged cyclic peptides

Sieber or Rink Fmoc resin (PAL-PEG PS resin; -0.16-0.2 mmol/g)

Fmoc(Automated SPPS) (6 eq.) Ptos-AA(protected)-OH

NATU/NMM 25 °C, 10 min stage C stage B

[l] _p Z ₄ PEZ ₇ PZ ₉ EZii ASPEELNRYYZ ₂₂ Z ₂₃ LRZ;

HN

ABOUT

NYE, O

[l] _p Z ₄ PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ ₃₀ E(OAII)[V3 ₁ ] _q TRZ3 ₄ Z3 ₅ Y-NHQ*

NH (manual)

1) [0.2% Pd(PPh ₃ ) ₄ , phenylsilane (15 equiv), DCM, 25 °C, 10 min] x 2

2) [N-hydroxysuccinimide, NATI/DIEA 25 °C, 20 min] x 2

O ¹

ABOUT

ABOUT

1) TFA/TIPS/water (95%/2.5% 2.5%) 2 h, 25 °C

2) DMSO, TEA (10 eq.), 20 min., 25 °C

3) 2% hydrazine, DMF, 10 min., 25 °C, x 2

[V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y ₃₆ -NH ₂

[l] _p Z ₄ PEZ ₇ PZ ₉ EZ^ AS PE ELN RYYZ22Z2 ₃ LRZ _2e YLNZ ₃₀

*Amino acids are protected; Lys is protected as Dde

A) Synthesis of the C-terminal amide peptide bound to the resin.

The protected peptidyl resin can be synthesized using the Fmoc strategy on Protein Technologies' Symphony X peptide synthesizer. Couplings can be carried out on either Rink amide resins or Sieber resins using HATU and NMM in DMF for 10 min at RT. Fmoc amino acids can be used in 6-fold excess and double-linked. For peptides containing the psi-(R35,Y36) modification, Fmoc-Arg(Pbf)Ψ[CH ₂ N(Boc)]Tyr(t-Bu)-OH was coupled in triple excess using HATU and NMM in DMF for 1 hour at room temperature The α-amino group of the terminal residue of the linear sequence can be protected with Boc and the γ-terminal group of the glutamic acid residue that forms the lactam bridge can be protected with allyl. The lysine(s) in the peptide can be orthogonally protected using Dole.

B) Synthesis of γ-glutamate-N-hydroxysuccinimide ester.

Once the linear sequence is complete, the allylic protecting group of the glutamate side chain can be removed using Pd(PPh ₃ ) ₄ and PhSiH ₃ in DCM. N-Hydroxysuccinimide (NHS) can then be synthesized by double coupling of NHS using HATU and DIEA for

- 47 045724 min at room temperature

C) Lactam cyclization procedure.

The peptide can be cleaved from the resin and deprotected by treatment with TFA/TIPS/water (95:2.5:2.5) for 2 hours at RT, then precipitated in ether and collected by centrifugation, and dry. The crude product can be dissolved in DMSO; Add TEA (10 eq.) and continue cyclization for 1 hour at room temperature. The crude product can be diluted with water and purified using preparative RP-HPLC. Lysines orthogonally protected with dde can be deprotected by dissolving the peptide in 2% hydrazine/DMF (at a concentration of 5 mM) and stirring for 1 h at room temperature. The reaction mixture can be diluted with water and adjusted to pH 2 by adding 10% TFA/water and the solution can be directly purified by preparative RP-HPLC as described in Scheme 1, Step E. Synthesis of lipidated C-terminal amide analogs of NTSC-PYY in which BRIDGE is a lactam, shown in solution phase in Scheme 8.

The solution-phase synthesis of lipidated C-terminal amide analogues of NTSC-PYY, in which the BRIDGE is a lactam, is shown in Scheme 8.

Scheme 8

Solution phase lipidation of lactam-bridged C-terminal amide cyclic peptides shown for Zu lysine

Only one of Z _{4 >} Z ₇ , Z ₉ , Zu , Z ₂₂ , Z ₂₃ , or Z ₃₀ can be lysine; Shown for Zn=Lys

The peptide obtained above in Scheme 7, in which only one of Z ₄ , Z ₇ , Z ₉ , Zu, Z ₂₂ , Z ₂₃ or Z ₃₀ can be lysine, is dissolved in DMF (at a concentration of 5 mM), TEA is added ( 5 equiv.), and then N-hydroxyester of the protected lipid (2 equiv.). The reaction can be carried out overnight at room temperature and then purified by preparative HPLC. The t-butyl ethers can be deprotected in TFA/TIPS/water (95:2.5:2.5) for 30 min at RT, and the reaction mixture is concentrated and purified by preparative RP-HPLC as described in Scheme 1, stage E.

General methods for the synthesis of bromoacetylated cyclic thioether peptides.

Each of Schemes 9-11 demonstrates different routes for preparing the bromoacetylated cyclic thioether peptidos of the present invention. In Scheme 9, the thioester bridge is formed by the reaction of a thiol-containing amino acid side chain in the peptide sequence with a bromoacetyl group at the amino terminus of the peptide. Scheme 10 shows a similar approach to Scheme 9, but with a discrete PEG spacer introduced between the lysine side chain and the bromoacetyl group. Scheme 11 illustrates a strategy for the synthesis of another class of bromoacetylated cyclic thioester peptides in which a bridge is formed by the reaction of a thiol nucleophile at the amino terminus of the peptide with a bromoacetyl group covalently attached to a lysine side chain within the peptide sequence.

A. Synthesis of C-terminal resin-bound amide peptide.

The protected peptide on the resin can be synthesized on Sieber amide resins or Ring amide resins using the FMOC strategy. Standard FMOC-protected amino acids (available from Novabiochem (EMD Millipore), Bachem, Peptides International or Chem-Impex) can be coupled in 3-6 times excess of the resin load using

- 48 045724

DIC/oxime, HBTU/DIPEA or HATU/NMM as coupling agents at RT. or elevated temperature. To increase purity, double coupling can be performed, and especially for the amino acid coupled to aN-alkylated amino acids. For peptides containing the psi-(R35,Y36) modification, Fmoc-Arg(Pbf)Ψ[CH ₂ N(Boc)]Tyr(t-Bu)-OH can be coupled in 3-fold excess using HATU and NMM in DMF for 1 hour at room temperature.

B. Procedure for bromoacetylation of resin-bound peptide.

The lysine to be bromoacetylated can be orthogonally protected with an alloc, ivDDE or DDE protecting group. Once the linear sequence is complete on the resin, the orthogonally protected lysine can be deprotected (for alloc, Pd(Ph ₃ ) ₄ and phenylsilane in DCM; for DDE or ivDDE, 2% hydrazine in DMF) and the amino group can be subjected to bromoacetylation under various conditions such as as 1) by reacting with a large excess of bromoacetic anhydride in DMF in a microwave reactor at 50°C for 5 min, at which point the reaction can be determined to be substantially complete according to the Kaiser ninhydrin test; 2) by reaction with a large excess of bromoacetic anhydride in DMF or DCM in the presence of a base such as TEA or DIPEA at room temperature; or 3) by coupling with bromoacetic acid using DIC or a DIC/oxime mixture.

C. Method of cleaving the peptide from the resin.

Once SPPS is complete, the resin can be extensively washed with DMF and then DCM, and dried. In the case of a peptide bound to a Sieber amide resin, the dried resin can be treated with a solution of 1-2% TFA in DCM (10 ml) for 5-10 minutes, then filtered. This treatment can be repeated several more times using fresh mixture for each treatment. The filtrates are then combined and concentrated to obtain the crude protected peptide as a yellow foam. This foam can then be treated with a breakdown mixture of TFA/phenol/H ₂ O/TIPS (88/5/5/2), or TFA/water/TIPS (95:2.5:2.5), or TFA/phenol/ H ₂ O/TIPS/DTT (84/10/2.5/2.5/1) and heat in a microwave reactor at 38°C for 30-45 min or at room temperature for 2-3.5 h. Crude peptide can be precipitated in cold diethyl ether. The peptide/ester suspension can be centrifuged and the ester layer decanted. The peptide pellet can be resuspended in ether, centrifuged and decanted, and this process can be repeated a third time. The crude peptide thus obtained can be dried under a gentle stream of nitrogen.

Alternatively, the peptide coupled to the Sieber amide resin or Rink amide resin can be treated with the digestion mixture as described above, without pretreatment with 1-2% TFA in DCM to produce a completely unprotected peptide.

D. Method of peptide cyclization (thioester formation).

The crude free thiol and bromoacetamide-containing peptide can be dissolved in anoxic MeCN/water or EtOH/water at a concentration of 4 to 10 mg/ml with the optional addition of EDTA. The pH of the peptide solution can then be raised to approximately 7-9 by adding a base such as NaHCO ₃ , NaOH, DIPEA or TEA and the resulting solution stirred at room temperature for 0.25-2.5 hours.

Alternatively, the crude peptide can be purified by HPLC, the peptide fractions pooled and made alkaline to approximately pH 7-9 and stirred at room temperature, optionally in the presence of EDTA, for 0.25-2.5 hours. After acidification, the reaction solution can be concentrated at room temperature to remove organic solvent and then purified by HPLC.

E. Peptide purification procedure.

The cyclization reaction mixture can be acidified with TFA and the solution concentrated to remove most of the organic solvent (MeCN or EtOH), and the resulting solution can then be directly purified by preparative HPLC on a reverse phase column. The mobile phase consists of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from initial concentrations of 0-20% B to final concentrations of 40-90% B over the duration of the operation 20-60 min. UV detection can be monitored at 220 and 254 nm. Fractions containing product can be analyzed by HPLC on an Agilent 1100 HPLC system using a Waters T3 Atlantis C18 column (4.6 x 250 mm, 5 µm). The pure fractions can be combined, concentrated to remove most of the organic phase, and then lyophilized.

- 49 045724

Scheme 9

Synthesis of bromoacetylated cyclic thioester C-terminal amide peptides in which the thioester bridge is formed by the reaction of a thiol-containing amino acid side chain in the peptide sequence with a bromoacetyl group at the amino terminus of the peptide. In this scheme, the dde protecting group is used to protect the lysine side chain and is removed by treatment with 2% hydrazine in DMF.

Sieber or Rink Fmoc resin (PAL-PEG PS resin; -0.16-0.2 mmol/g) (Automated SPPS) (6 eq.) Ptos-AA(protected)-OH,

HATU/NMM 25 °C, 10 min

[l] _p Z ₄ PEZ ₇ PZ ₉ EZ ₁₁ ASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ ₃₀ -N^V(V ₃₁ ] _q TRZ _M Z ₃₅ Y-NH-Q*

I n about

NH cr η νη ₂

1) 2% hydrazine, DMF, 10 min., 25 °C, to 2

2) bromoacetic anhydride; DIEA, DMF, 15 min., 25 °C o

U _Rr (CH ₂ ) ₄ --N'^^ ^Br Η (Trt)S

[I] z ₄ pez ₇ pz ₉ en

I N

N.H.

ASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ ₃₀ -N' ^oh (CH ₂ ) _P

About cr η

ΗΝ. Br

ABOUT

I 1) TFA/TIPS/water (95% 2.5%/2.5%) 2 h, 25 °C

I 2) pH 7-7.5, 30% ACN/water, 20 min., 25 °C

[Ι] _ρ Ζ ₄ ΡΕΖ ₇ ΡΖ ₉ Ε-Ν

I ⁿ F n

ASPEELNRYYZ ₂₂ Z ₂ 3LRZ ₂₆ YLNZ ₃₀ TI^JL[V3 ₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH ₂ o a

JCH ₂ ), S *Amino acids are protected; Zn=Lys(dde)

- 50 045724

Scheme 10

Synthesis of bromoacetylated cyclic thioester peptides, in which a thioether bridge is formed by reacting a thiol-containing amino acid side chain in the peptide sequence with a bromoacetyl group at the amino terminus of the peptide, and a discrete PEG separator is introduced between the bromoacetyl group and the lysine side chain. In this scheme, the alloc protecting group is used to protect the lysine side chain and is removed by treatment with Pd(PPh ₃ ) ₄ and phenylsilane (Trt)S.

(CH ₂ ) _P

Fmoc-(p-Ala)-[l] _p Z ₄ PEZ ₇ PZ ₉ EKASPEELNRYYZ22Z23LRZ2 ₆ YLNZ3o-N>T—(V ₃₁ ] _q TRZ3 ₄ Z3 ₅ Y-NH-Q* I ₍ ^A o (9 ^H 2> 4 I 1. Pd(PPh ₃ ) ₄ , PhSiH ₃

2. Fmoc-PEGi2-OH, HATU/DIPEA

NH(Alloc) (Trt)s.

( _CH2 ) _n

Fmoc-O}-Ala)-[l]pZ4REZ ₇ RZ ₉ EpSPEELNRYYZ22Z ₂ 3LRZ2 ₆ YLNZ ₃₀ -b^jp [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-Q (CH ₂ ) ₄

HN. JPEG12)—Fmoc

Vg O

1. 20% piperidine

2. Bromoacetic anhydride

HN—(P-AlaHI] _p Z ₄ PEZ ₇ PZ ₉ EKASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ ₃₀ -N ^X J]-[V3 ₁ ] _q TRZ34Z3 ₅ Y-NH-Q IH _o (CH ₂ ) ₄ hn^Upegi2 )—nh

About Vg

Removing general protection

1. 1-2% TFAvDCM, b.t., 1 h

2. TFA-SO-TIPS-phenol (88-5-2-5%)

Br ,0 ^HS lCH ₂ ) _n

HN—(3-Ala)-[l]pZ ₄ PEZ ₇ PZ ₉ EKASPEELNRYYZ2 ₂ Z ₂ 3LRZ ₂₆ YLNZ ₃₀ -^ %HV ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH ₂ IH o (CH ₂ ) ₄

HN^JpEG12PnH

About Vg

NaHCO ₃ , pH -7.5 (CH ₂ ),

HN— (p-Ala)-[l] _p Z ₄ PEZ ₇ PZ ₉ EKASPEELNRYYZ ₂ 2Z ₂ 3LRZ ₂₆ YLNZ

I o nh ₂ * Amino acids are protected (CH ₂ ) ₄

HN.

About Vg

- 51 045724

Scheme 11

Synthesis of bromoacetylated cyclic thioether peptides in which a thioether bridge is formed by reacting a thiol group at the amino terminus of the peptide with a bromoacetylated lysine side chain in the peptide sequence, and a discrete PEG spacer is introduced between the bromoacetylate group and the other lysine side chain

Dde-HN.

CKx—S-Trt

Η N— (l] _p Z ₄ PEZ ₇ PZ ₉ EKAS PE ELN RYYZ ₂₂ Z23LRZ ₂₆ YLNZ3 ₀ IH (CH ₂ ) ₄ *

[V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-O

ABOUT

0,. S-Trt (CH ₂ ) ₄

HN

moc

ABOUT

1. 20% piperidine

2. NH ₂ NH ₂ (2%) in DMF

3. Bromoacetic anhydride

W

HN—[l]pZ ₄ PEZ ₇ PZ ₉ E|<ASPEELNRYYZ22Z23LRZ ₂₆ YLNZ ₃₀ -N ° _H (CH ₂ ) ₄

(CH ₂ ) ₄

Vg

[V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-Q o

HN

Removing general protection

1. 1 - 2% TFA in DCM, b.t., 1 h

2. TRA-N ₂ O-TIPS-phenol (88-5-2-5%) o

ABOUT

NaHCOj, pH -7.5

[V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH ₂ ♦ Amino acids protected

General methods for the synthesis of bromoacetylated cyclic lactam peptides

Cyclic lactam peptides can be synthesized according to the procedures presented in Scheme 12. The cyclic lactam peptide is first synthesized according to Scheme 7, where only one of Z ₄ , Z ₇ , Z ₉ , Zu, Z ₂₂ , Z ₂₃ or Z ₃₀ is lysine . Lysine is then subjected to bromoacetylation using N-hydroxysuccinimide bromoacetic acid ester (3-7 equiv) in 10% ACN/water at pH 10, bt, 20 min. The resulting bromoacetylated peptide can be purified by RP-HPLC as described for cyclic thioester peptides.

- 52 045724

Scheme 12

Synthesis of bromoacetylated cyclic lactam C-terminal amide peptides

H II

[IIAPEZ^EZ^ASPEELNRYYZ^^^

Bromoacetic acid N-hydroxysuccinimide ester, 10% ACN/water, pH 10.20 min, 25 °C

ABOUT

[I^PEZ^E-N^^SPEELNRYYZ^L^

Illustrated for Zu = Lys, only one of Z ₄ , Z ₇ , Z ₉ , Z _lb Z ₂₂ , Z ₂₃ , Z ₃₀ can be Lys

General procedures for the synthesis of bromoacetylated cyclic triazole-linked peptides.

Bromoacetylated cyclic triazole-linked peptides can be synthesized according to the procedure shown in Scheme 13. The linear protected peptide on a resin can be synthesized in a manner similar to that described for the cyclic thioester peptides in Scheme 10, except that L-azidolysine can be introduced to form triazole, and the N-terminal residue may be 4-pentinoic acid. Fmoc can be removed with 20% piperidine in DMF and then reacted with bromoacetic anhydride. The linear sequence can be deprotected overall (TFA/TIPS water: 95%/2.5%/2.5%), and the crude peptide can be precipitated in cold ether, collected by centrifugation, and purified by preparative RP-HPLC. The purified linear peptide can be cyclized in the presence of CUSO4/TBTA and NaASrb in a buffer solution (HEPES, MOPS, etc.) to yield cyclic triazole-linked peptides, which can be purified by preparative RP-HPLC as described in Scheme 1. stage E.

An alternative class of cyclic triazole-linked peptides can be synthesized by those skilled in the art starting with a linear sequence in which the N-terminal residue is an azidocarboxylic acid (e.g., 5-azidopentanoic acid) and the residue at position 30 or 31 is an alkynyl amino acid ( for example, 2-amino-7-octynic acid), in a manner similar to that described above.

- 53 045724

Scheme 13

Synthesis of bromoacetylated cyclic triazole-linked C-terminal amide peptides

O %—[i]pZ ₄ PEZ ₇ PZ ₉ EKASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ3 ₀ (Lys(N ₃ ))[V ₃₁ ] _q TRZ34Z35Y-NH-Q (CH ₂ ) ₄

HN^JPEG12j—Fmoc

ABOUT

1.20% piperidine

2. Bromoacetic anhydride

N—[l]pZ4PEZ ₇ PZ ₉ EKASPEELNRY¥Z ₂₂ Z ₂ 3LRZ ₂₆ YLNZ ₃₀ (Lys(N ₃ ))[V3 ₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH-O ⁿ I „ (CH ₂ ) ₄

HN^ApEG12Pn-4

Removing general protection

1.1-2% TFA in DCM, b.t., 2 h

2. TFU-N ₂ O-TIPS (95-2.5-2.5%)

N—[l]pZ ₄ PEZ ₇ PZ ₉ EKASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ3 ₀ (Lys(N ₃ ))[V ₃₁ ] _q TRZ3 ₄ Z3 ₅ Y-NH ₂

H (CH ₂ ) ₄

HN^JPEG12>-N· O

Vg

CuSO ₄ /TBTA, NaAsrb, HEPES

N=N

HN %CH ₂ ) ₂ (CH ₂ ) ₃

[l]pZ ₄ PEZ ₇ PZ ₉ EKASPEELNRYYZ ₂₂ Z ₂₃ LRZ ₂₆ YLNZ3o N [V ₃₁ l _q TRZ ₃₄ Z ₃₅ Y-NH ₂

I _ ⁿ O (CH ₂ ) ₄

HN. J PEG1 *Amino acids protected

Peptide analysis and characterization.

Purified peptides were analyzed by LCMS on a Hewlett Packard 1100 MSD series system equipped with an HP 1100 series HPLC using a Waters Atlantis T3 C18 column (4.6x250 mm, 300 A, 5 μm). Depending on the polar/non-polar nature of the peptide, one of three gradients was used (buffers A and B, as described above) with a flow rate of 1 ml/min and a column temperature of 35°C: Method 1) 15-60% B for 22 min; Method 2) 30-60% B for 22 minutes; Method 3) 40-90% B for 22 minutes. Mass analysis for each peptide was performed using electrospray analysis (ES-API, scanning positive ions). In all cases, multiply charged particles were observed with the characteristic, most clearly observed ions 1/3[M+3]+ and 1/4[M+4]+. All products produced the expected multiply charged ions within acceptable limits. The results of mass spectral analysis of the peptides and the observed LC retention times (RT) are shown in Table. 1.

- 54 045724

Analytical data for NTSC-PYY connections

Table 1

Seq. I.D. No. Mol. Formula MM 1/3[M+3] ⁺ (Calculate otherwise) 1/3 ⁺ [M+3] ⁺ (Measured) 1/4[M+4] ⁺ (Calculate otherwise) 1/4[M+4] ⁺ (Measured) Met. HPLC. RT (min.) 1 C21sH345BrN5gOg7S 4898.41 1633.80 1633.8 1225.60 1225.4 1 12.48 2 C187H281N53O55S 4183.66 1395.55 1395.4 1046.92 1046.6 1 11.7 3 CiegHaeiNggOgg 4184.63 1395.88 1395.6 1047.16 1047.0 1 11.1 4 C210H325N53O57S 4564.27 1522.42 1522.2 1142.07 1141.9 2 17.0 5 C-225B345N55O59S 4796.59 1599.86 1599.6 1200.15 1199.9 3 15, 8 6 C224H348N58O59 4797.56 1600.19 1600.1 1200.39 1200.3 3 15.7 7 C212H327N55O59S 4622.31 1541.77 1541.8 1156.58 1156.7 2 15, 8 8 C208H319N55O59S 4566.20 1523.07 1522.8 1142.55 1142.4 2 14.7 9 C187H283N53O54S 4169.68 1390.89 1390.6 1043.42 1043.3 1 11.3 10 C ₂₂₃ H ₃₃₉ N ₅₅ O ₆₁ S 4798.52 1600.51 1600.4 1200.63 1200.6 3 13.4 eleven C ₂ 23H341N55O ₆₀ S 4784.54 1595.85 1595.7 1197.14 1197.1 3 12.8 12 C188H283N53O55S 4197.69 1400.23 1400.2 1050.42 1050.3 1 11, 9 13 C208H321N55O58S 4552.22 1518.41 1518.2 1139.06 1139.0 2 13, 8 14 C209H321N55O59S 4580.23 1527.74 1527.8 1146.06 1145.9 2 15.2 15 C199H309N55O59S 4448.02 1483.67 1483.7 1113.01 1112.9 2 14.1 16 C202H308N54O58S 4453.04 1485.35 1485.1 1114.26 1114.2 2 13, 9 17 C205H312N54O59S 4509.10 1504.03 1503.9 1128.28 1128.2 2 16, 6 18 C ₂₀₇ H ₃₁₄ N ₅₄ O ₆₁ S 4567.14 1523.38 1523.2 1142.79 1142.7 2 16, 4 19 C ₂₀₈ H ₃₁₉ N550 ₅₉ S 4566.20 1523.07 1523.1 1142.55 1142.5 2 15, 0 20 c ₂ o ₈ h ₃₁₉ n ₅ 50 ₅₉ s 4566.20 1523.07 1522.8 1142.55 1142.4 2 15.1 21 c ₂ 02H ₃₁ 0N5 ₂ o ₅₉ s 4443.04 1482.01 1481.9 1111.76 1111.6 2 17.2 22 C ₂ 0 ₄ H ₃ 1 ₂ I ₅₂ O ₆₁ 3 4501.08 1501.36 1501.2 1126.27 1126.2 2 16, 8 23 S222Нз45М ₅₇ О ₆₆ 3 4900.57 1634.52 1634.4 1226.14 1225.9 2 16.7 24 C ₂₂₄ H ₃₄₉ N ₅₇ OggS 4928.62 1643.87 1643.4 1233.16 1233.0 2 18.4 25 C210H325N55O58S 4580.27 1527.76 1527.7 1146.07 1146.1 2 17.0 26 C212H329N55O58S 4608.32 1537.11 1537.1 1153.08 1153.0 2 19.7 27 C ₂₁₇ H ₃₃₈ N ₅₆ O ₆₃ S 4771.45 1591.48 1591.3 1193.86 1193.8 2 12.0 28 C202H310N54O57S 4439.06 1480.69 1480.6 1110.77 1110.8 2 13.3 29 C199H311N55O58S 4434.04 1479.01 1478.9 1109.51 1109.3 2 13.7 thirty C199H311N55O58S 4434.04 1479.01 1479.0 1109.51 1109.3 2 14.2 31 C ₂₂₂ H ₃₄₇ N ₅₇ O ₆₄ S 4870.58 1624.53 1624.6 1218.65 1218.6 2 18.5

- 55 045724

32 C205H314N54O58S 4495.12 1499.37 1499.3 1124.78 1124.8 2 15, 9 33 C209H323N55O58S 4566.24 1523.08 1522.9 1142.56 1142.4 2 14.6 34 C201H315N55O58S 4462.09 1488.36 1488.0 1116.52 1116.4 2 13, 9 35 C209H323N55O58S 4566.24 1523.08 1522.8 1142.56 1142.5 2 14.4 36 C200H313N55O58S 4448.07 1483.69 1483.5 1113.02 1112.9 2 13, 8 37 C205H322N56O59S 4547.20 1516.73 1516.6 1137.80 1137.7 2 14.2 38 C206H324N56O59S 4561.22 1521.41 1521.7 1141.31 1141.3 2 13.7 39 C209H322N54O59S 4567.23 1523.41 1523.2 1142.81 1142.7 3 10.3 40 S21eNz4bMboOb4 4843.52 1615.50 1615.4 1211.9 1211.8 2 15.1 41 C207H324N58O58 4553.20 1518.70 1518.4 1139.3 1139.3 2 13, 6 42 C221H348N ₆₀ O ₆₍ 5 4901.56 1634.90 1634.8 1226.4 1226.4 2 11, 0 43 C-220H343N57O64S 4842.53 1615.18 1615.0 1211.63 1211.5 2 15.5 44 C224H351N ₅₇ O ₆ 4S 4898.64 1633.88 1633.9 1225.66 1225.6 2 12.7 45 C261H428N ₅₆ O81S 5678.63 1893.88 1893.4 1420.66 1420.6 2 19.4 46 C205H322N56O59S 4547.20 1516.73 1516.6 1137.80 1137.6 2 13.7 47 C225H351N ₅₇ O ₆₆ S 4942.65 1648.55 1648.2 1236.66 1236.6 2 11.7 48 C225H351N ₅₇ O ₆₆ S 4942.65 1648.55 1648.6 1236.66 1236.6 2 11, 8 49 C208H321N55O59S 4568.22 1523.74 1523.6 1143.06 1142.9 1 15.2 50 C225H351N5 ₇ O ₆₅ S 4926.65 1643.22 1643.2 1232.66 1232.5 2 11, 6 51 C205H314N54O59S 4511.12 1504.71 1504.6 1128.78 1128.6 2 15, 6 52 C237H380N56O69S 5150.00 1717.67 1717.5 1288.50 1288.4 2 18.6 53 C234H371N59O ₆₈ S 5130.92 1711.31 1710.9 1283.73 1283.6 2 16, 6 54 C222H349N ₅₇ O ₆₂ S 4840.60 1614.53 1614.3 1211.15 1211.1 2 16, 6 55 C223H349N ₅₇ O ₆₃ S 4868.61 1623.87 1623.6 1218.15 1218.0 2 14.9 56 C206H300CIN55O58S 4542.48 1515.16 1515.0 1136.62 1136.4 1 13, 6 57 C207H301CI2N55O59 s 4606.95 1536.65 1536.3 1152.74 1152.5 1 14.5 58 C209H314FN55O58S 4576.17 1526.39 1526.2 1145.04 1144.9 2 10, 9 59 S22зНз49М ₅₇ О ₆ 43 4884.61 1629.20 1629.0 1222.15 1221.9 2 15.5 60 S22зНз49М ₅₇ О ₆ 43 4884.61 1629.20 1629.1 1222.15 1222.1 2 15.4 61 C210H314F3N55O58S 4626.18 1543.06 1542.7 1157.55 1157.4 2 12.7 62 C204H310F3N55O58S 4550.08 1517.69 1517.6 1138.52 1138.3 2 9.1 63 C207H316F3N55O58S 4592.16 1531.72 1531.7 1149.04 1149.0 2 12.1 64 C210H325N55O59S 4596.27 1533.09 1532.7 1150.07 1150.0 2 11, 8 65 C208H312D9N55O58S 4561.15 1521.38 1521.3 1141.29 1141.2 2 14.1 66 SgiNzhzGbYzzOzzZ 4694.17 1565.72 1565.4 1174.54 1174.6 2 14.1 67 SgiNzhzEbYzzOzzZ 4694.17 1565.72 1565.6 1174.54 1174.4 2 14.2 68 C219H348N58O65S 4865.57 1622.86 1622.7 1217.39 1217.3 1 17.3 69 C181H280N54O54 4076.53 1359.84 1359.7 1020.13 1020 1 11.4 70 C180H278N54O54 4062.50 1355.17 1355 1016.63 1016.7 1 12.2

- 56 045724

71 C181H278N54O55 4090.51 1364.50 1364.4 1023.63 1023.5 1 12.8 72 C215H339N57O66 4778.38 1593.79 1593.4 1195.60 1195.6 1 15.8 73 C ₂ 03H3 ₂₁ BrN ₅₆ O ₆₁ S 4634.09 1545.70 1545.4 1159.52 1159.6 1 12.12 74 C188H292BrN5 ₅ O ₅ 4S 4298.69 1433.90 1433.9 1075.67 1075.6 1 11.96 75 C ₂₁₁ H338BrN ₅₅ O _6S S 4813.30 1605.43 1605.5 1204.32 1204.4 1 12.27 76 C ₂ 4 ₉ H4 ₂₂ Bl?N 57O84S 5660.31 1887.77 1887.4 1416.08 1416.0 1 13.15 77 C ₂₁ 4H34 3BrN ₅₆ O ₆ 7S 4884.38 1629.13 1629.1 1222.09 1222.1 1 12.24 78 S21bNz4 7VgM5 ₆ O ₆ 73 4912.44 1638.48 1638.4 1229.11 1229.1 1 12.49 79 C ₂ logH34 ₇ BrN ₅ gOg ₇ S 4912.44 1638.48 1638.1 1229.11 1228.9 1 12.58 80 C ₂₂₀ Nz4 4VgN ₅₉ O ₆₇ 3 4999.48 1667.49 1667.3 1250.87 1250.8 1 12.55 81 C ₂₂₀ H344BrN ₅₉ O ₆₇ S 4999.48 1667.49 1667.4 1250.87 1250.7 1 12.43 82 C ₂ iiH33 ₈ BrN ₅ 5O ₆₈ S 4845.30 1616.10 1616.0 1212.32 1212.4 1 12.44 83 C217H ₃ 4 ₇ BrN5 ₈ O ₆₆ 4904.39 1635.80 1635.9 1227.10 1227.0 1 12.55 84 S ₂ 25Нз54ВгЦ5 ₉ ObbZ 5053.61 1685.54 1685.4 1264.40 1264.3 1 12.80 85 С21бНз45ВгЦ ₅₆ О ₆₈ 3 4926.42 1643.14 1642.9 1232.61 1232.6 1 11.72 86 S21bNz4 5VgTs5bOb ₉ 3 4942.42 1648.47 1648.3 1236.61 1236.5 1 11.92 87 C ₂₁ gH3 ₄₅ BrN ₅ gOg ₉ S 4942.42 1648.47 1648.3 1236.61 1236.4 1 11.82 88 C ₂₁₂ H ₃ 3 ₉ BrN ₅₆ O ₆₇ S 4856.33 1619.78 1619.5 1215.08 1214.9 1 12.43 89 C ₂ i3H ₃ 3 ₉ BrN ₅₆ O ₆ gS 4884.32 1629.11 1629.0 1222.08 1222.0 1 BUT 90 C ₂ 14Нз41ВгЦ ₅₆ О ₆₈ 3 4898.32 1633.77 1633.7 1225.58 1225.5 1 BUT 91 C ₂ 44H34 ₂ BrN5gOggS 4898.32 1633.77 1633.3 1225.58 1225.5 1 BUT 92 C ₂₁ gH345BrN ₅ gOggS 4926.42 1643.14 1642.7 1232.61 1232.6 1 BUT 93 C ₂ 1зНз41ВгЦ5 ₆ О ₆₇ 3 4870.35 1624.3 1624.1 1218.5 1218.4 1 12.67 94 C ₂ 1зНз41ВгЦ ₅₆ О ₆₇ 3 4870.35 1624.3 1624.3 1218.5 1218.4 1 12.42 95 C ₁₈₂ H ₂₇₉ BrN54O55 4183.45 1395.5 1395.3 1046.9 1046.7 1 13.41 96 S ₂ zenz ₉ zVgTs _5b O ₇₉ 3 5427.04 1810.0 1809.7 1357.8 1357.8 1 12.44 97 S ₂ 1 ₄ NZ4зВгЦ ₅ bOb ₇ 3 4884.4 1629.1 1628.8 1222.1 1221.9 1 11.74 98 S18bN28bVgC ₅ 5O ₅₅ 3 4284.6 1429.2 1428.9 1072.1 1071.9 1 12.28 99 C187H ₂ 8 8BrN55O ₅₅ S 4298.6 1433.9 1433.6 1075.6 1075.6 1 12.03 100 S18bN ₂ 8 8VgTs55O5 ₄ 3 4270.6 1424.5 1424.3 1068.6 1068.6 1 12.59

Intermediate connections.

It is intended that the following examples and embodiments described herein are for illustrative purposes only and that various changes and modifications therein will be readily apparent to those skilled in the art and are intended to be included within the scope and scope of this application and the appended claims. inventions. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Intermediate connection 1.

Synthesis of α-tocopherylacetic acid (AcVitE) (8).

A) tert-Butyl-a-tocopheryloxyacetate (7):

A mixture of a-tocopherol (1.14 g, 2.65 mmol), tert-butyl bromoacetate (470 μl, 3.18 mmol) and K ₂ CO ₃ (1.1 g, 7.94 mmol) in acetone (10 ml) stirred at room temperature. within 2-3 days. The mixture was then filtered through a small K ₂ CO ₃ pad and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography, eluting with EtOAc/heptanes (0-5%) to give tert-butyl-a-tocopheryloxyacetate (7) as a colorless oil.

^1H NMR (CDCl3) δ 4.17 (s, 2H), 2.56 (t, J=6.57 Hz, 2H), 2.18 (s, 3H), 2.14 (s, 3H), 2.07 (s, 3H), 1.65-1.86 (m, 2H), 1.52 (s, 9H), 0.98-1.46 (t, 22H), 0.80-0, 90 (m, 14H).

B) α-Tocopheryloxyacetic acid (AcVitE) (8)

- 57 045724

To a solution of tert-butyl-a-tocopheryloxyacetate (7) (1.4 g, 2.57 mmol) in DCM (12 ml) was added TFA (6 ml) and the resulting solution was stirred at room temperature. After 2 hours, the solution was concentrated under reduced pressure and the resulting dark oil was purified by chromatography on silica gel, eluting with MeOH/DCM (0-2.5% containing 0.5% HOAc) to give α-tocopheroxyacetic acid (AcVitE) (8) as an amber-colored syrup that slowly solidified under vacuum.

1H NMR (CDClj) δ 4.34 (s, 2H), 2.57 (t, J=6.82 Hz, 2H), 2.16 (s, 3H), 2.12 (s, 3H), 2 .08 (s, 3H), 1.79 (qt, J=6.76, 13.01 Hz, 2H), 1.48-1.59 (m, 3H), 1.18-1.48 (m , 12H), 1.01-1.17 (m, 7H), 0.77-0.93 (m, 14H).

LCMS: mass calculated for C ₃₁ H ₅ 2O ₄ : 488.75; measured: 489.5 [M+H]+.

Intermediate 2.

1. Synthesis of (S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetracontane -1-carboxylic acid (16).

A) Benzyl-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecane-13-oate (9)

2,2-dimethyl-4-oxo-3,8,11-trioxa-5azatridecane-13-carboxylic acid (18 g, 68.366 mmol), benzyl bromide (23.386 g, 136.732 mmol), potassium carbonate were placed in a 100 ml round bottom flask (28.346 g, 205.099 mmol) and DMF (200 ml). The resulting solution was stirred at room temperature. overnight and extracted with EtOAc (500 ml). The mixture was washed with water (300 ml) and highly concentrated saline solution (2x150 ml). The organic phase was evaporated and purified by a silica gel column (EtOAc/propyl ether, 1:5) to give benzyl 2,2-dimethyl-4-oxo-3,8,11trioxa-5-azatridecane-13-oate as a colorless oils (9).

LCMS: mass calculated for C ₁₈ H ₂₇ NO ₆ : 353.2, measured: 354.05 [M+H]+.

B) Benzyl 2-(2-(2-aminoethoxy)ethoxy)acetate (10) o n9

J η m TFU Π PMU

VpSG^ ----- VpSG^

9YU

Benzyl 2,2-dimethyl-4-oxo-3,8,11-trioxa-5azatridecane-13-oate (12 g, 33.955 mmol), TFA (19.358 g, 169.774 mmol) and DCM were placed in a 500 ml round bottom flask. (150 ml). The resulting solution was stirred at room temperature. during the night. The mixture was evaporated and dried to give benzyl 2-(2(2-aminoethoxy)ethoxy)acetate as a light yellow oil (10).

LCMS: mass calculated for C ₁₃ H ₁₉ NO ₄ : 253.13, measured: 254.05 [M+H]+.

WITH) . Benzyl 2,2-dimethyl-4,13-dioxo-3,8,11,17,20-pentaoxa-5,14-diazadocosane-22-oate (11)

Place 2,2-dimethyl-4-oxo-3,8,11-trioxa-5azatridecane-13-carboxylic acid (8.835 g, 33.558 mmol), benzyl-2-(2-(2-aminoethoxy) in a 250 mL round bottom flask )ethoxy)acetate (8.5 g, 33.558 mmol), HATU (15.312 g, 40.270 mmol), DIEA (8.674 g, 67.116 mmol) and DMF (100 ml). The resulting solution was stirred at room temperature. overnight and extracted with EtOAc (500 ml). The organic phase was washed with water (200 ml) and highly concentrated saline solution (2x100 ml). The organic phase was evaporated and purified by a silica gel column (DCM/MeOH, 10:1) to give benzyl-2,2-dimethyl-4,13-dioxo-3,8,11,17,20-pentaoxa-5,14diazadocosane -22-oate in the form of a light yellow oil (11).

LCMS: mass calculated for C ₂₄ H ₃₈ N ₂ O ₉ : 498.26, measured: 499.50 [M+H]+.

D) Benzyl-17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecane-1-oate (12)

O U

ВпО^ ⁰ '^'^^

Η n DXM:

Benzyl 2,2-dimethyl-4,13-dioxo-3,8,11,17,20pentaoxa-5,14-diazadocosane-22-oate (15 g, 30.086 mmol), TFA ( 17.153 g, 150.431 mmol) and DXM (200 ml). The resulting solution was stirred at room temperature. during the night. The mixture was evaporated and dried to give benzyl 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecane-1-oate as a light yellow oil (12).

LCMS: mass calculated for C ₁₉ H ₃₀ N ₂ O ₇ : 398.21, measured: 399.2 [M+H]+.

E) (S)-1-Benzyl-23-tert-butyl-22-(((9H-fluoren-9-yl)methoxy)carbonylamino)-10,19-dioxo-

- 58 045724

3,6,12,15-tetraoxa-9,18-diazatricosan-1,23-dioate (13)

Benzyl-17-amino-10-oxo-3,6,12,15-tetraoxa-9azaheptadecane-1-oate (11 g, 27.607 mmol), (S)-4-(((9H) was placed in a 250 ml round bottom flask -fluoren-9-yl)methoxy)carbonylamino)-3-tert-butoxy-3-oxopentanoic acid (11.746 g, 27.607 mmol), HATU (12.596 g, 33.128 mmol), DIEA (7.136 g, 55.214 mmol) and DMF (100 ml ). The resulting solution was stirred at room temperature. overnight and extracted with EtOAc (500 ml). The organic layer was washed with water (2x200 ml) and highly concentrated saline (200 ml). The organic phase was evaporated and purified by silica gel chromatography (DCM/MeOH, 10:1) to give ^)-1-benzyl-23-tert-butyl-22(((9H-fluoren-9-yl)methoxy)carbonylamino) -10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23dioate in the form of a light yellow oil (13).

LCMS: mass calculated for C ₄₃ H ₅₅ N ₃ O ₁₂ : 805.38, measured: 806.80 [M+H]+.

F) (S)-1-Benzyl-23-tert-butyl-22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23dioate (14)

(S)-1-Benzyl-23-tert-butyl-22-(((9H-fluoren-9yl)methoxy)carbonylamino)-10,19-dioxo-3,6,12,15 was placed in a 250 ml round bottom flask. -tetraoxa-9,18-diazatricosane-1,23-dioate (10 g, 12.408 mmol) and DBU in DCM (3%, 100 ml). The resulting solution was stirred at room temperature. overnight, then washed with water (2x200 ml). The organic phase was concentrated. The residue was dissolved in water (200 ml) and extracted with ether (2x200 ml). The aqueous phase was extracted with DCM (200 ml). The organic phase was evaporated and dried to obtain (S)-1-benzyl-23-tert-butyl-22-amino-10,19-dioxo3,6,12,15-tetraoxa-9,18-diazatricosane-1,23-dioate in the form of a light yellow oil (14).

LCMS: mass calculated for C ₂₈ H ₄₅ N ₃ O ₁₀ : 583.31, measured: 584.65 [M+H]+.

G) (S)-1-Benzyl-21,39-di-tert-butyl-9,18,23-trioxo-2,5,11,14-tetraoxa-8,17,22-triazanonantriacontane-1,21, 39-tricarboxylate (15) ^ΒηΟ γ-σ ο

Η Ο^ ^Ν

o ⁰ HO ₂ C(CH ₂ ) _ie CO ₂ -tBu

HATU

ΝΗ ₂ -------DIEA, DMF

(S)-1-Benzyl-23-tert-butyl-22-amino-10,19dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23-dioate was placed in a 50 ml round bottom flask. (1.575 g, 2.699 mmol), 18-tert-butoxy-18oxooatecanoic acid (1 g, 2.699 mmol), HATU (1.231 g, 3.239 mmol), DIEA (697.654 mg, 5.398 mmol, 2 eq) and DMF (15 ml) . The resulting solution was stirred at room temperature. overnight and diluted with EtOAc (200 ml). The organic phase was washed with water (2x100 ml) and highly concentrated saline solution (100 ml). The organic phase was concentrated and purified by silica gel chromatography (DCM/MeOH, 10:1) to give (S)-1-benzyl-21,39-di-tert-butyl-9,18,23-trioxo2,5,11 ,14-tetraoxa-8,17,22-triasanonatriacontane-1,21,39-tricarboxylate as a light yellow oil (15).

LCMS: mass calculated for C ₅₀ H ₈₅ N ₃ O ₁₃ : 935.61, measured: 936.6 [M+H]+.

H) (S)-22-(tert-Butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa- 59 045724

9,18,23-triazatetratetracontane-1-oic acid (16)

(S)-1-Benzyl-21,39-di-tert-butyl-9,18,23trioxo-2,5,11,14-tetraoxa-8,17,22-triazanonanthriacontane-1 was placed in a 100 ml round bottom flask ,21,39-tricarboxylate (2.5 g, 3.041 mmol), Pd/C (10 wt.%, 500 mg) and MeOH (50 ml). The resulting solution was stirred at room temperature. overnight in an _H2 atmosphere (3.5 atm). The residue was filtered, concentrated and purified by reverse phase silica gel chromatography (NH ₄ HCO ₃ /H ₂ O, 0.05%) to give (S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24 ,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontanoic acid in the form of a light yellow semi-solid (16).

LCMS: mass calculated for C ₄₃ H ₇₉ N ₃ O ₁₃ : 845.56, measured: 846.55 [M+H]+.

Ή NMR (300 MHz, CD3OD) δ: 4.24-4.29 (m, 1H), 4.07 (s, 2H), 4.03 (s, 2H), 3.69-3.72 (m , 8H), 3.57-3.67 (m, 4H), 3.45-3.49 (m, 2H), 3.34-3.42 (m, 2H), 2.23-2.35 (m, 6H), 2.12-2.21 (m, 1H), 1.93-1.96 (m, 1H), 1.521.70 (m, 4H), 1.45-1.51 (m , 18H), 1.33 (s, 24H).

Examples

The compounds of the present invention can be prepared using methods known to those skilled in the art. It is intended that the following examples are only representative examples of the present invention and are not intended to limit the invention in any way.

Example 1. Synthesis of a cyclic analogue of PYY SEQ ID NO: 1.

Scheme 14

Synthesis of Fmoc-psi-[Arg(Pbf)-(N-Boc)Tyr(tBu)]-OH

Fmoc'

2) NaBH ₃ CN,

FmocHN

Pd(PPh ₃ ) ₄ , PhNHMe

Fmoc

H

1) NH ₂ -Tyr(tBu)-Oall HOAc, THF-MeOH

- 60 045724

1. Synthesis of Fmoc-psi-[Arg(Pbf)-(N-Boc)Tyr(tBu)]-OH.

A. Synthesis of H2N-Tyr(tBu)-OAll.

Allyl bromide (72 g, 595.16 mmol) was added to an ice bath cooled solution of Fmoc-Tpyr(tBu)-O (69 g, 150.15 mmol) and K2CO3 (62 g, 445.36 mmol) in DMF (500 mL). ) and the resulting mixture was stirred for 3 hours. Ice/water (1 L) was then added and the mixture was extracted with EtOAc. The combined organic extracts were dried (Na ₂ SO ₄ ) and concentrated under reduced pressure to give Fmoc-Tyr(tBu)-OAll as a yellow oil. To an ice-bath cooled solution of Fmoc-Tyr(tBu)-OAll (70 g, 140.1 mmol) in DMF (600 mL), piperidine (150 mL) was added dropwise over 20 min. After 3 hours, the reaction solution was poured into water/ice (1 L) and extracted with EtOAc (2x2 L). The combined organic extracts were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue thus obtained was purified by silica gel chromatography, eluting with EtOAc/petroleum ether (10:1) to obtain 34 g of H ₂ N-Tyr(tBu)-OAll as a yellow oil.

B. Synthesis of Fmoc-Arg(Pbf)-N(Me)OMe (2).

To an ice bath cooled mixture of Fmoc-Arg(Pbf)-OH (1) (64.8 g, 99.88 mmol), N,O-dimethylhydroxylamine hydrochloride (20 g, 206.2 mmol) and HATU (57 g, 149.91 mmol) in DCM (500 mL), DIEA (52 g, 402.2 mmol) was added dropwise over 10 min and the resulting mixture was stirred at room temperature overnight. The reaction mixture was then poured into water/ice (1 L) and extracted with (1 L). The organic extracts were dried (Na ₂ SO ₄ ) and concentrated under reduced pressure to obtain 70 g of crude Fmoc-Arg(Pbf)-N(Me)OMe (2) as a yellow solid, which was then used without further purification.

C. Synthesis of Fmoc-Arg(Pbf)-CHO (3).

To a cooled (-78°C) solution of LAH in THF (1 M, 107 ml, 0.107 mmol) in an inert nitrogen atmosphere, a cooled (-50°C) solution of Fmoc-Arg(Pbf)-N(Me) was added dropwise through a hollow needle )OMe (2) (50 g, 72.3 mmol) in THF (100 ml) for 1 hour. After stirring at -78°C for 5 hours, the mixture was poured into 1N. HCl solution (300 ml) and additionally added 1N. HCl, as needed, to adjust pH to 4, was then extracted with EtOAc (2x2 L). The combined organic extracts were dried (Na2SO4) and concentrated under reduced pressure to give 45 g of crude Fmoc-Arg(Pbf)-CHO (3) as a yellow solid, which was used without further purification.

D. Synthesis of Fmoc-psi-[Arg(Pbf)-Tyr(tBu)]-OAll (4).

To an ice bath cooled solution of Fmoc-Arg(Pbf)-CHO (3) from step C (45 g, 71.12 mmol) and H ₂ N-Tyr(tBu)-OAll from step A (32 g, 115.37 mmol) in THF (200 ml), MeOH (200 ml) and HOAc (15 ml) sodium cyanoborohydride (18.0 g, 286.4 mmol) was added in portions over 30 min and the resulting solution was stirred at room temperature overnight . The reaction was quenched by adding saturated aqueous NaHCO ₃ (500 ml) and the mixture was extracted with EtOAc (2x2 L). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by chromatography on silica gel, eluting with EtOAc/petroleum ether (10:1) to give 40 g of Fmoc-psi-[Arg(Pbf)-Tyr(tBu)]-OAll (4) as a yellow solid colors.

E. Synthesis of Fmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OAll (5).

To a solution of Fmoc-psi-[Arg(Pbf)-Tyr(tBu)-OAll] (4) (53 g, 59.28 mmol) in MeCN (240 ml) was added di-tert-butyl dicarbonate (20 g, 91.3 mmol) and the resulting solution was stirred at 50°C overnight. The mixture was then concentrated under reduced pressure and the residue was purified by silica gel chromatography, eluting with EtOAc/petroleum ether (1:1) to obtain 32 g of Fmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]- OAll (5) as a yellow solid.

F. Synthesis of Fmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OH (6).

To a cooled (-30°C) solution of Fmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OAll (5) (32 g, 32 mmol) in DCM (600 ml) under an inert nitrogen atmosphere Pd(PPh ₃ ) ₄ (3.0 g, 4.33 mmol) was added followed by dropwise addition of N-methylaniline (10 g, 93 mmol) over 30 min. The resulting mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with EtOAc/petroleum ether (1:1) to give 26.8 g of Fmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OH (6) in as a yellow solid.

1H NMR (300 MHz, CD3OD) δ 7.75-7.77 (2H, m), 7.59-7.60 (2H, m), 7.32-7.33 (4H, m), 7, 09-7.11 (2H, m), 6.87-7.00 (2H, m), 4.27-4.50 (3H, m), 3.30-3.50 (4H, m), 3.02-3.23 (3H, m), 2.75-2.98 (3H, m), 2.57 (3H, s), 2.48 (3H, s), 2.00 (3H, s), 1.31-1.41 (28H, m).

LCMS (ES, m/z): mass calculated for C ₅₂ H ₆₇ N ₅ O ₁₀ S: 953.46, measured: 954.55 [M+H]+.

2. Loading the dipeptide Fmoc-psi-(R35-N(Boc)-Y36) onto Sieber resin.

In a fused microwave reaction vessel (available from CEM Corporation), NovaSyn TG Sieber resin (available from Novabiochem) (0.2 mmol) was treated with 20% piperidine in DMF (10 mL) and heated at 50°C for 2.5 min in microwave reactor

- 61 045724

SEM. The reaction mixture was decanted and the resin was washed with DMF and treated again with 20% piperidine in DMF at 50°C for 5 min in a CEM microwave reactor. After draining and washing the resin with DMF, the deprotection treatment was repeated one more time. The resin was then treated with a solution of Fmoc-psi-[Arg(Pbf)-(N-Boc)Tyr(tBu)]-OH prepared above (3-5 eq), HATU (2.75-4.8 eq) and DIEA (6-10 eq.) in DMF (4 ml) and stirred at room temperature. for 6-24 hours. The mixture was drained and the resin was washed extensively with DMF and then capped by treatment with 20% Ac ₂ O in DMF (5 ml) under microwave conditions at 50°C for 5 minutes. The reaction mixture was decanted and the resin was washed extensively with DMF and DCM.

3. Synthesis of Fmoc-βA-IKPEAPGEK(Alloc)ASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber resin.

Amino acid extension on preloaded (psi-R35,Y36)-Sieber resin (0.2 mmol) was carried out on a CEM Liberty Blue microwave peptide synthesizer. Standard Fmoc-protected α-amino acids were double-coupled at 3.8-fold excess of the original resin load at 50°C for 15 min using HBTU/DIEA as coupling agents. Fmoc-Arg(Pbf)-OH was double-coupled using a two-step protocol: 25 min at RT, then 15 min at 50°C and Fmoc-His (Trt)-OH was double-coupled using a two-step protocol: 4 min at RT, then 8 min at 50°C.

4. Synthesis of Fmoc-βA-IKPEAPGEK(NH ₂ )ASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber resin: Removal of the Alloc protecting group.

The resin obtained above was treated with a solution of phenylsilane (25 eq.) in oxygen-free DCM (10 ml). After stirring for ~2 min, a solution of Pd(PPh ₃ ) ₄ (0.5 eq) in DCM (10 ml) was added and the resin mixture was stirred for 30 under argon. The reaction mixture was decanted and the resin was washed with oxygen-free DCM. Deprotection was repeated using fresh reagents, after which the reaction mixture was decanted and the resin was washed extensively with DCM and DMF.

5. Synthesis of Fmoc-eA-IKPEAPGEK(NH-dPEG ₁₂ -NHFmoc)ASPEELNRYYASLRHYLNL(hC)TRQ(psiR35Y36)-Sieber resin: binding of N-Fmoc dPEG ₁₂ -carboxylic acids at 11K.

The above Alloc deprotected Sieber peptide resin was treated with a solution of N-Fmoc-dPEG12-carboxylic acid (5 eq), HBTU (4.8 eq) and DIEA (10 eq) in DMF (7 ml) in the microwave CEM reactor at 50°C for 15 min; after this time, the reaction showed a negative Kaiser test result. The reaction mixture was decanted and the resin was washed extensively with DMF and DCM.

6. Synthesis of BrCH ₂ COHN-eA-IKPEAPGEK(NH-dPEG ₁₂ -NHCOCH ₂ Br)ASPEELNRYYASLRHY-

LNL(hC)TRQ(psi-R35Y36)-Sieber resins: bis-bromoacetylation with BA and dPEG ₁₂ .

The above resin was subjected to Fmoc deprotection using fresh 20% piperidine in DMF at 50°C for 5 min in a CEM microwave reactor. Removal of protection was repeated twice. The thus obtained Fmoc deprotected peptide resin was treated with a solution of bromacetic anhydride (20 eq.) in DMF (5 ml) in a CEM microwave reactor at 50°C for 10 min; after this time, the reaction showed a negative Kaiser test result. The reaction mixture was decanted and the resin was washed extensively with DMF and DCM, and then dried.

7. Synthesis of BrCH ₂ COHN-eA-IKPEAPGEK(NH-dPEG ₁₂ -NHCOCH ₂ Br)ASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-CONH ₂ : cleavage from the resin and removal of general protection.

The dried resin was treated with a solution of 1.5% TFA in DCM (10 ml) and stirred for 5-10 minutes, then filtered. This treatment was repeated 9 more times, using fresh mixture for each treatment. The combined filtrates were then combined and concentrated to yield the crude protected peptide as a yellow foam. This foam was treated with 20 ml of digestion mixture (TFA/phenol/H ₂ O/TIPS = 88/5/5/2) at room temperature for 2.5 hours and then concentrated under a stream of nitrogen to a volume of ~2.5 ml, after which cold ether (40 ml) was added to precipitate the peptide. The mixture was centrifuged (5 min; 5000 rpm) and decanted. This process was repeated 2 more times to obtain the crude peptide as an almost white powder.

Alternatively, the resin was treated with a cleavage mixture without pretreatment with 1-2% TFA in DCM to produce a completely unprotected peptide.

8. Cyclic PYY Analog SEQ ID NO: 1: Cyclization Procedure A and Purification.

The crude peptide described above was dissolved in anoxic 50% MeCN/water (5-10 mg/ml), optionally adding EDTA (1 mM). The pH of the reaction solution was then raised to approximately 8 by adding 7.5 w/v% NaHCO ₃ solution. The resulting solution was stirred at room temperature for 0.5-2.5 hours and then acidified to pH <1 by adding TFA. The solution was then concentrated under reduced pressure at room temperature. to approximately half the original volume (~24 ml). The resulting solution was purified using reverse phase preparative HPLC. Purification was performed on a Gilson 2020 Personal Purification System HPLC using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm). The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from the starting con

- 62 045724 concentration of 20% B to a final concentration of 50% B within 36 minutes. UV detection was monitored at 220 and 254 nm. Fractions containing product were analyzed by analytical HPLC on an Agilent 1100 HPLC system using the same column type as above (4.6 x 250 mm, 5 µm). The pure fractions were combined and then lyophilized to give the product as a cotton-like solid.

LCMS: 1225.5 (M+4H)/4, 1633.4 (M+3H)/3 and 2450.0 (M+2H)/2 for product peak at 12.27 min (LC: Atlantis T3 C18 Column, 5 µm, 4.6x250 mm, 1.0 ml/min, gradient 15-60%).

Example 2. Synthesis of a cyclic PYY analogue SEQ ID NO: 2.

1. Synthesis of resin H ₂ N-IKPEAPGEDASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG.

The protective peptidyl resin was synthesized using the Fmoc strategy as described above on a CEM Liberty Blue microwave peptide synthesizer using low loading Rink amide resins, preferably Fmoc-PAL-PEG PS resin (approximately 0.16-0.2 meq/g, available from Applied Biosysterns) on a 0.1 mmol scale, as depicted in Scheme 1. Standard Fmoc protected amino acids were coupled at a 5-fold excess of the resin load, using DIC/oxime as coupling agents, and a reaction temperature of approximately 90°C. C for 4 min. Fmoc-Arg(Pbf)-OH was double coupled at 90°C for 4 min each, and Fmoc-His(Trt)-OH was coupled using a two-step protocol: 4 min at RT, then 8 min at 50 °C. Deprotection of individual Fmocs was carried out using 20% piperidine in DMF (deprotection solution) at 90°C for 1.5 min.

2. Synthesis of m-BrCH2PhCOHN-IKPEAPGEDASPEELNRYYASLRHYLNL(hC)TRQRY-PALPEG resin.

The deprotected Fmoc peptide resin (0.1 mmol) described above was treated with a solution of m-bromomethylbenzoic acid (20 eq) and DIC (10 eq) in DMF (4 ml) in a microwave reactor at 75°C within 15 minutes; after this time, the reaction as a whole was determined to be complete according to the Kaiser ninhydrin test (Kaiser, et al., Anal. Biochem., 1970, 34, 595-598). In cases where binding was determined to be incomplete, binding was repeated with fresh reagents. The reaction mixture was decanted and the resin was washed extensively with DMF and DCM.

3. Synthesis of m-BrCH2PhCOHN-IKPEAPGEDASPEELNRYYASLRHYLNL(hC)TRQRY-CONH2: deprotection and removal from the resin.

The resin mentioned above was then treated with a digestion mixture (10 mL/0.1 mmol scale) consisting of TFA/water/phenol/TIPS (88:5:5:2) and heated in a microwave reactor at 38°C for 40 min, then filtered. The resin was washed with TFA and the combined filtrates were concentrated under a stream of nitrogen to a volume of approximately 2.5 ml and the peptide was precipitated by adding cold diethyl ether (40 ml). The peptide/ester suspension was centrifuged and the ether layer was decanted. The peptide tablet was resuspended in ether, centrifuged and decanted, and this process was repeated a third time. The crude peptide thus obtained was dried under a gentle stream of nitrogen.

4. Cyclic PYY Analog SEQ ID NO: 2: Cyclization Procedure A and Purification.

The crude peptide mentioned above was dissolved in anoxic MeCN/water (60% MeCN) at a concentration of <4 mg/mL. The pH of the peptide solution was then increased to approximately 7-9 by adding aq. NH ₄ OAc (200 mM, pH 8.4) and the resulting solution was stirred at room temperature. until cyclization is complete, according to LCMS (usually 3-4 hours). The cyclization reaction mixture was acidified to pH 1.5-3 by adding TFA and the solution was concentrated to remove most of the organic co-solvent to the point where slight turbidity occurred. To homogenize the mixture, a minimal amount of MeCN was added back as needed and the resulting solution was then directly purified by preparative HPLC in multiple injections using a Varian Pursuit XRS C18 C18 column (21 x 250 mm, 100 A, 5 µm). The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 20% B to a final concentration of 40% B over 45 min. UV detection was monitored at 220 and 254 nm. Fractions containing the product were analyzed by analytical HPLC on an Agilent 1100 HPLC system using the appropriate column as shown in Table 1. 1. The pure fractions were pooled, concentrated to remove most of the organic phase and then lyophilized.

Example 3. Synthesis of a cyclic PYY analogue SEQ ID NO: 3.

1. Synthesis of resin (H ₂ N)-IKPEAPGEDASPEELNRYYASLRHYLNL-(azido-nor-Leu)-TRQRYPALPEG.

The resin-bound peptide was prepared on a 0.1 mmol scale according to the method described in Example 2, step 1, replacing Fmoc-hCys(trt)-OH with Fmoc-isido-nor-Leu-OH at position 31.

2. Synthesis of resin (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNL-(azido-nor-Leu)TRQRYPAL-PEG.

4-Pentinoic acid was bonded to the resin mentioned above under micro

- 63 045724 wave irradiation using the DIC/HOVT protocol (75°C, 10 min). The reaction mixture was decanted and the resin was washed extensively with DMF and DCM.

3. Synthesis (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNL-(azido-nor-Leu)-

TRQRYPAL-CONH2.

The resin mentioned above was treated with 10 ml of digestion mixture consisting of TFA/DODT/H ₂ O/TIS (92.5:2.5:2.5:2.5) under microwave irradiation (38°C , 40 min). The reaction mixture was decanted and the resin was washed with TFA (10 ml). The combined filtrate was then concentrated under a stream of nitrogen to a volume of ~2.5 mL. Cold ether (40 ml) was then added to precipitate the peptide and the mixture was centrifuged (5 min; 5000 rpm) and decanted. This process was repeated 2 more times to obtain the crude peptide as an almost white powder.

4. Analogue of cyclic PYY SEQ ID NO: 3.

Prepare 7 mg of CuSO ₄ in 2 ml of oxygen-free H2O. Prepare 30 mg of TBTA in 5.4 ml of EtOH and 0.6 ml of MeCN. Pre-mix 0.94 ml of CuSO ₄ solution and 4.8 ml of TBTA solution. Prepare 30 mg of Na ascorbate in 3 ml of oxygen-free H2O.

To a 20 mL solution of the crude azide-containing peptide from Step 3 (100 mg) was added the premixed CuSO4/TBTA solution, followed by the addition of 2.4 mL of Na ascorbate solution (the solution immediately took on a milky color). The mixture was heated to 40°C and stirred for 1.5 hours, after which LCMS analysis indicated completion of the reaction. The mixture was diluted to ~40 ml with water (0.1% TFA); the mixture was centrifuged and the supernatant was purified by reverse phase preparative HPLC. Purification was carried out using a Varian Pursuit XRs C18 column (21x250 mm, 100 A, 5 µm) at 35°C. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 10% B to an intermediate concentration of 18% B (21 ml/min), and then to a final concentration of 33% B (10.5 ml/min) for 35 min. UV detection was monitored at 220 and 254 nm. Fractions containing product were analyzed by analytical HPLC on an Agilent 1100 HPLC system using the same column type as above (4.6 x 250 mm, 5 µm). The pure fractions were combined and then lyophilized to give the product as a cotton-like solid.

Example 4. Synthesis of a cyclic PYY analogue SEQ ID NO: 4.

1. Synthesis of (Dde)K(NH ₂ )ASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG resin.

The resin-bound peptide was prepared using the method described in Example 2, Step 1.

2. Synthesis of resin (Dde)K(NH-Glu-(OtBu)NH2)ASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG.

Fmoc-Glu-OtBu (5 eq.) was coupled to the resin mentioned above under microwave irradiation using DIC/oxime coupling methods (90°C, 6 min; double coupling). The resin was drained and washed with DMF. Fmoc was then deprotected with 20% piperidine in DMF using a 3-step protocol (75°C for 0.5 min; 75°C for 3 min; 75°C for 3 min), washing with DMF at every stage.

3. Synthesis of resin (Dde)K(NH-Glu-(OtBu)NH-Pal)ASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG.

Palmitic acid (5 eq.) was coupled to the resin mentioned above under microwave irradiation using DIC/oxime coupling methods (90°C, 5 min). The resin was drained and washed extensively with DMF and DCM.

4. Synthesis of resin (H2N)K(NH-Glu-(OtBu)NH-Pal)ASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG.

After washing the resin mentioned above with DMF, it was treated with a solution of 2% hydrazine in DMF (6 ml/0.1 mmol resin) at room temperature. for 5 min, then drained and washed with DMF. The treatment was repeated 5 more times.

5. Synthesis of resin (H2N)IKPEAPGEK(NH-Glu-(OtBu)NH-Pal)ASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG.

The remaining amino acid couplings were carried out using the method described in Example 2, step 1.

6. Analogue of cyclic PYY SEQ ID NO: 4.

The remainder of the synthesis was carried out using the methods described in Example 2, steps 2-4. Product purification was performed using a Varian Pursuit XRS C18 column (21x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 23% B to an intermediate concentration of 33% B (21 ml/min) over 5 min and then to a final concentration of 48% B (10.5 ml/min) over 55 min.

Example 5. Synthesis of a cyclic PYY analogue SEQ ID NO: 5.

The title compound was prepared according to the procedure described in Example 4, replacing palmitic acid with α-tocopheryloxyacetic acid (AcVitE) (8) in step 3.

The product was purified using an Agilent 300SB C8 column (21x250 mm, 100 A, 5 µm) at room temperature. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 35% B to an intermediate concentration of 45% B (21 ml/min) in for 5 min, and then to a final concentration of 60% B (10.5 ml/min) for

- 64 045724 min.

Example 6. Synthesis of a cyclic PYY analogue SEQ ID NO: 6.

1. Synthesis of resin (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(Dde)-(azido-norLeu)-TRQRY-PAL-PEG.

The resin-bound peptide was prepared as described in Example 3, step 1, replacing Fmoc-Leu-OH with Fmoc-Lys(Dde)-OH at position 30 and introducing 4-pentinoic acid (coupled in two steps) at this point step at position 2 and then Fmoc-Ile-OH at position 3.

2. Synthesis of resin (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(NH ₂ )-(azido-norLeu)-TRQRY-PAL-PEG.

The above resin was treated with 3% hydrazine in DMF (8 ml/0.1 mmol scale) for 5 min at RT. and then the mixture was drained and washed with DMF. This procedure was repeated approximately 5x, after which the resin was washed extensively with DMF and then DCM.

3. Synthesis of resin (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(NH-γ-Glu-

AcVitE)-(azido-HopLeu)-TRQRY-PAL-PEG.

Fmoc-Glu-OtBu was coupled to the resin mentioned above using the coupling protocol described in Example 2, step 1 with a coupling time of 5 minutes. The resin was deprotected by treatment with 20% piperidine in DMF using a 3-step microwave protocol (75°C, 0.5 min; 75°C, 3 min; 75°C, 0.3 min), after which the resin was washed extensively with DMF and DCM. α-Tocopheryloxyacetic acid (AcVitE) (8) was then coupled to the resin using the same procedure used to couple Fmoc-Glu-OtBu.

4. Synthesis of (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(NH-γ-Glu-AcVitE)-(azido-nor-Leu)-TRQRY-CONH2.

Cleavage of the peptide from the resin mentioned above and its precipitation was carried out using the procedure described in Example 3, step 3.

5. Analogue of cyclic PYY SEQ ID NO: 6.

The title compound was prepared using the procedure described in Example 3, Step 4. Product purification was performed on an arian Pursuit XRs C8 column (21x250 mm, 100 A, 5 μm) at 35°C. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 35% B to an intermediate concentration of 48% B (21 ml/min) in for 5 min, and then to a final concentration of 63% B (10.5 ml/min) over 40 min.

Example 7. Synthesis of a cyclic PYY analogue SEQ ID NO: 7.

The title compound was prepared using the procedure described in Example 4, with the K(NH-y-Glu-Pal) residue introduced at position 9 instead of position 11. The product was purified using an Agilent 300SB C8 column (21x250 mm, 100 A, 5 µm) at r.t. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 23% B to an intermediate concentration of 43% B (21 ml/min) and then to a final concentration of 43% B (10.5 ml/min) for 40 minutes. The contaminated fractions containing the product were re-purified on a Waters T3 C18 column (250x19 mm, 100 A, 5 µm) at room temperature. using a gradient from an initial concentration of 25% B to an intermediate concentration of 35% B (21 ml/min) and then to a final concentration of 45% B (10.5 ml/min) over 80 minutes.

Example 8. Synthesis of a cyclic PYYy analogue SEQ ID NO: 8.

The title compound was prepared using the procedure described in Example 4, with the K(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11. Product purification was performed using an Agilent 300SB C8 column (21x250 mm, 100 A, 5 µm) at 35°C. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 21% B to an intermediate concentration of 31% B (21 ml/min) and then to a final concentration of 41% B (10.5 ml/min) over 40 min. The contaminated fractions containing the product were re-purified on a Waters T3 C18 column (250x19 mm, 100 A, 5 µm) at room temperature. using a gradient from an initial concentration of 21% B to an intermediate concentration of 31% B (21 ml/min) and then to a final concentration of 40% B (10.5 ml/min) over 80 minutes.

Example 9. Synthesis of a cyclic PYY analogue SEQ ID NO: 9.

1. Synthesis of H ₂ N-IKPEAPGEDASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber resin.

Amino acid extension on the preloaded (psi-R35, Y36) Sieber resin from Example 1, step 2 (0.1 mmol) was carried out as described in Example 1, step 3, with the modification of using a 5-fold excess of protected amino acids .

2. Synthesis of m-BrCH ₂ PhCOHN-IKPEAPGEDASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber resin.

m-Bromomethylbenzoic acid was coupled to the resin mentioned above in accordance with the procedure described in Example 1, with the modification that the coupling was carried out at 50°C instead of 75°C.

- 65 045724

3. Analogue of cyclic PYY SEQ ID NO: 9.

The title compound was prepared from the resin mentioned above according to the procedures described in Example 1, steps 7 and 8. Product purification was performed using a Varian Pursuit XRs C18 column (21x250 mm, 100 A, 5 µm) at 35 °C. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 10% B to an intermediate concentration of 18% B (21 ml/min) in for 10 min and then to a final concentration of 33% B (10.5 ml/min) over 35 min.

Example 10. Synthesis of a cyclic PYY analogue SEQ ID NO: 10.

The title compound was prepared according to the procedure described in Example 4, replacing Fmoc-Leu-OH with Dde-Lys(Fmoc)-OH at position 30 and palmitic acid with α-tocopheryloxyacetic acid (AcVitE) (8) with stage 3. The product was purified using an Agilent 300SB C8 column (21x250 mm, 100 A, 5 µm) at room temperature. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 30% B to an intermediate concentration of 40% B (21 ml/min) in for 10 min and then to a final concentration of 55% B (21 ml/min) over 35 min. Contaminated product fractions were repurified using a modified gradient from an initial concentration of 35% B to an intermediate concentration of 43% B (21 ml/min) over 5 min and then to a final concentration of 58% B (10.5 ml/min) within 40 min.

Example 11. Synthesis of a cyclic PYY analogue SEQ ID NO: 11.

1. Synthesis of (AΠoc)K(NH2)-(hC)-TRQ(psi-R35Y36)-Sieber resin.

The resin mentioned above was prepared according to the procedure described in Example 9, Step 1, using Alloc-Lys(Fmoc)-OH instead of Fmoc-Leu-OH at position 30.

2. Synthesis of (Alloc)K(NH-γ-Glu-AcVitE)-(hC)-TRQ(psi-R35Y36)-Sieber resin.

Fmoc-Glu-OtBu and α-tocopheryloxyacetic acid (AcVitE) (8) (5 equiv. each) were sequentially coupled to the resin mentioned above using HBTU/DIEA-mediated couplings under microwave irradiation at 50°C within 15-20 minutes.

3. Synthesis of H2N-K(NH-Y-Glu-AcVitE)-(hC)-TRQ(psi-R35Y36)-Sieber resin.

The alloc protecting group was removed following the procedure described in Example 1, step 4.

4. Analogue of cyclic PYY SEQ ID NO: 11.

The title compound was prepared from the resin mentioned above in accordance with the procedures described in Example 9, steps 1-3, with the modification that 1M TRIS/HCl buffer, pH 7.5, was used instead of NH4OAc buffer to carry out cyclization. Product purification was performed using a Varian Pursuit XRs C18 column (21x250 mm, 100 A, 5 µm) at 35°C. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 10% B to an intermediate concentration of 18% B (21 ml/min) in for 10 min and then to a final concentration of 33% B (10.5 ml/min) over 35 min.

Example 12: Synthesis of a Cyclic PYY Analogue SEQ ID NO: 12.

The title compound was prepared according to the procedure described in Example 2, replacing Fmoc-Arg(pbf)-OH with Fmoc-N-Me-Arg(pbf)-OH at position 35 in step 1 and with that modification, that 1 M NaHCO ₃ buffer was used instead of NH ₄ OAc buffer to carry out the cyclization. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 10–60% B (30 mL/min) over 36 min.

Example 13. Synthesis of a cyclic PYY analogue SEQ ID NO: 13.

The title compound was prepared according to the procedures described in Example 11, using palmitic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8) in step 2, and with the modification that 60% EtOH/H ₂ O was used in as a solvent instead of MeCN/H ₂ O and saturated aq. NaHCO ₃ was used instead of NH4OAc buffer to carry out the cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) ranging from an initial concentration of 15% B to an intermediate concentration of 20% B (100 ml/min) over 5 min. and then to a final concentration of 35% B (100 ml/min) for 40 minutes.

Example 14 Synthesis of a cyclic PYY analogue SEQ ID NO: 14.

The title compound was prepared according to the procedure described in Example 4, with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11, and with Fmoc-N-Me-Arg(pbf) -OH instead of Fmoc-Arg(pbf)-OH at position 35 in step 1, and with the modification that 1 M NaHCO ₃ buffer was used instead of NH ₄ OAc buffer in step 6 to effect cyclization. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) in the range of 20–70% H (30 ml/min) over 36 min. Contaminated fractions repeat

- 66 045724 but purified on a Varian Pursuit XRs biphenyl column (30x100 mm, 100 A, 5 µm) at RT. using a gradient of 30-50% B (30 ml/min) for 25 minutes.

Example 15. Synthesis of a cyclic PYY analogue SEQ ID NO: 15.

The title compound was prepared according to the procedure described in Example 4 with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11, replacing the Fmoc-Ile-OH linkage with Fmoc-eAla- OH at position 3, and with the modifications that 1 M NaHCO ₃ buffer was used instead of NH ₄ OAc buffer to carry out the cyclization, and coupling with bromoacetic anhydride was used instead of m-bromomethylbenzoic acid in step 6 (step 2 of Example 2) using the following Methods: Fmoc deprotected peptide resin (0.1 mmol) was treated with a solution of bromoacetic anhydride (10 eq.) in DMF (5 ml) in a microwave reactor at 50°C for 5-10 min; after this time, the reaction was usually determined to be complete according to the Kaiser ninhydrin test. In cases where binding was determined to be incomplete, binding was repeated with fresh reagents. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) in the range of 20–60% B (30 ml/min) over 36 min. Contaminated fractions were repurified on a Varian Pursuit XRs biphenyl column (30x100 mm, 100 A, 5 µm) at room temperature. using a gradient of 30-50% B (30 ml/min) over 25 minutes.

Example 16 Synthesis of a cyclic PYY analogue SEQ ID NO: 16.

The title compound was prepared according to the procedure described in Example 4 with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11, omitting the Fmoc-Ile-OH linkage at position 3, and using p-bromomethylbenzoic acid instead of m-bromomethylbenzoic acid in step 6 (step 2 of Example 2). In addition, instead of 1 M NaHCO ₃ buffer, 3 M NH4OAc buffer was used to carry out cyclization. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm) at RT.

The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) in the range of 20–70% H (30 ml/min) over 36 min. Contaminated fractions were repurified on a Varian Pursuit XRs biphenyl column (30x100 mm, 100 A, 5 µm) at room temperature. using a gradient of 30-50% B (30 ml/min) over 25 minutes.

Example 17 Synthesis of a cyclic PYY analogue SEQ ID NO: 17.

The title compound was prepared according to the procedure described in Example 4 with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11 and Fmoc-Ala-OH used instead of Fmoc-Lys( Boc)-OH at position 4 in step 1. TRIS/HCl buffer (1 M, pH 7.5) was used instead of NH ₄ OAc buffer in step 6 to perform cyclization. Product purification was performed using an Agilent Polaris C18-A column (30x250 mm, 100 A, 5 µm) at room temperature. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 20% B to an intermediate concentration of 35% B (40 ml/min) in for 5 min and then to a final concentration of 45% B (40 ml/min) over 40 min.

Example 18 Synthesis of a cyclic PYY analogue SEQ ID NO: 18.

The title compound was prepared according to the procedure described in Example 4, with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11, and Fmoc-Glu(OtBu)-OH used instead Fmoc-Lys(Boc)-OH at position 4 in step 1. TRIS/HCl buffer (1 M, pH 7.5) was used instead of NH4OAc buffer in step 6 to perform cyclization. The product was purified using an Agilent Polaris C18-A column (30x250 mm, 100 A, 5 µm) at room temperature. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 20% B to an intermediate concentration of 35% B (40 ml/min) in for 5 min and then to a final concentration of 45% B (40 ml/min) over 40 min.

Example 19. Synthesis of a cyclic PYY analogue SEQ ID NO: 19.

The title compound was prepared according to the procedure described in Example 4 with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11, Fmoc-N-Me-Arg(pbf)-OH instead of Fmoc-Arg(pbf)-OH at position 35 in step 1, Fmoc-Cys(trt)-OH instead of Fmoc-hCys(trt)-OH, and using p-bromomethylbenzoic acid instead of m-bromomethylbenzoic acid in step 6 ( stage 2 from Example 2).

The following modification was made in step 6 (steps 3 and 4 from Example 2). The crude peptide obtained before cyclization was purified using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 μm) at rt. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) in the range of 20–70% H (30 ml/min) for 36 min. Fractions containing the product were combined and treated with solid NaHCO ₃ to increase the pH to ~7-8; the resulting solution was stirred at room temperature. for 4 h, then acidified to pH 4 with TFA. The solution was concentrated to a volume of 5-10 ml and MeCN was added to solubilize a possible precipitate. Product purification was performed as described above with a 20-60% B gradient (30 ml/min) over 36 min.

- 67 045724

Example 20. Synthesis of a cyclic PYY analogue SEQ ID NO: 20.

The title compound was prepared according to the procedure described in Example 4 with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11, Fmoc-N-Me-Arg(pbf)-OH instead of Fmoc-Arg(pbf)-OH at position 35 in step 1 and Fmoc-Cys(trt)-OH instead of Fmoc-hCys(trt)-OH. The crude linear peptide was purified and cyclized according to the modification described in Example 19. Final product purification was performed using a 20-60% H gradient (30 ml/min) over 36 minutes.

Example 21. Synthesis of a cyclic PYY analogue SEQ ID NO: 21.

The title compound was prepared according to the procedure described in Example 4 with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11, Fmoc-Ala-OH used instead of Fmoc-His( trt)-OH at position 26, and Fmoc-Ala-OH used instead of Fmoc-Lys(Boc)OH at position 4 in step 1. TRIS/HCl buffer, (1 M, pH 7.5), was used instead of NH buffer ₄ OAc in step 6 to perform cyclization. Product purification was performed using an Agilent Polaris C18-A column (30x250 mm, 100 A, 5 µm) at room temperature. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 20% B to an intermediate concentration of 35% B (40 ml/min) in for 5 min and then to a final concentration of 45% B (40 ml/min) over 40 min.

Example 22. Synthesis of a cyclic PYY analogue SEQ ID NO: 22.

The title compound was prepared according to the procedure described in Example 4 with a Lys(NH-y-Glu-Pal) residue introduced at position 30 instead of position 11, Fmoc-Ala-OH used instead of Fmoc-His( trt)-OH at position 26, and Fmoc-Glu(OtBu)-OH used instead of Fmoc-Lys(Boc)-OH at position 4 in step 1. TRIS/HCl buffer, (1 M, pH 7.5), was used instead of NH ₄ OAc buffer in step 6 to carry out cyclization. The product was purified using an Agilent Polaris C18-A column (30x250 mm, 100 A, 5 µm) at room temperature. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 20% B to an intermediate concentration of 33% B (40 ml/min) in for 5 min and then to a final concentration of 43% B (40 ml/min) over 40 min.

Example 23. Synthesis of a cyclic PYY analogue SEQ ID NO: 23.

The title compound was prepared according to the procedures described in Example 11 using octadecanedicarboxylic acid, mono-tert-butyl ether (available from AstaTech, Inc.) instead of α-tocopheryloxyacetic acid (AcVitE) (8), protocol coupling using HATU/DIEA at 50°C for 30 min and NMP as solvent instead of DMF in step 2, and coupling two Fmoc-OEG-OH units sequentially before coupling Fmoc-Glu-OtBu in step 2. Crude linear The peptide was purified and cyclized according to the modification described in Example 19 using a 20-60% B gradient. Final product purification was performed using a 20-70% B gradient (30 ml/min) over 36 minutes.

Example 24 Synthesis of a cyclic PYY analogue SEQ ID NO: 24.

The title compound was prepared according to the procedures described in Example 23 using 20-(tert-butoxy)-20-oxoicosanoic acid (available from Key Organics, Inc.) in place of octodecanedicarboxylic acid, mono-tert-butyl ether. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-60% B gradient. Final product purification was performed using a 20-60% B gradient (30 ml/min) over 36 minutes.

Example 25 Synthesis of a cyclic PYY analogue SEQ ID NO: 25.

The title compound was prepared according to the procedures described in Example 11 using stearic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8), a coupling protocol using HATU/DIEA at 50°C for 30 min and NMP as solvent instead of DMF in step 2. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-80% B gradient. Final product purification was performed using a 20-80% B gradient (30 ml /min) for 36 min.

Example 26. Synthesis of a cyclic PYY analogue SEQ ID NO: 26.

The title compound was prepared according to the procedures described in Example 11 using arachidic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8), a coupling protocol using HATU/DIEA at 50°C for 30 min and NMP as solvent instead of DMF in step 2. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-90% bar gradient. Final product purification was performed using a 20-90% B gradient (30 ml/min) over 36 min.

Example 27 Synthesis of a cyclic PYY analogue SEQ ID NO: 27.

The title compound was prepared according to the procedures described in Example 23, but omitting the Fmoc-Glu-OtBu coupling after the sequential Fmoc-OEG-OH couplings.

- 68 045724

The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-60% B gradient. Final product purification was performed using a 20-60% B gradient (30 ml/min) over 36 minutes.

Example 28 Synthesis of a cyclic PYY analogue SEQ ID NO: 28.

The title compound was prepared according to the procedures described in Example 11, using palmitic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8), omitting the Fmoc-Ile-OH linkage at position 3, and using p-bromomethylbenzoic acid acid instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2). The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-60% B gradient. Final product purification was performed using a 20-60% B gradient (30 ml/min) over 36 minutes.

Example 29. Synthesis of a cyclic PYY analogue SEQ ID NO: 29.

The title compound was prepared according to the procedures described in Example 11 using palmitic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8), replacing Fmoc-Ile-OH with Fmoc-eAla-OH at position 3 and subjecting coupling of bromoacetic anhydride instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2), using the modification described in Example 15. The crude linear peptide was purified and cyclized according to the modification described in Example 19, using a 20- 60% B. Final product purification was performed using a 20-60% B gradient (30 ml/min) over 36 min.

Example 30. Synthesis of a cyclic PYY analogue SEQ ID NO: 30.

The title compound was prepared according to the procedures described in Example 11 using α-tocopheryloxyacetic acid palmitic acid (AcVitE) (8), replacing Fmoc-Ile-OH with Fmoc-Aeu-OH at position 3 and coupling bromoacetic anhydride instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2), using the modification described in Example 15. The crude linear peptide was purified and cyclized according to the modification described in Example 19, using a 20-60 gradient % B. Final product purification was performed using a 20-60% B gradient (30 ml/min) over 36 min.

Example 31 Synthesis of a cyclic PYY analogue SEQ ID NO: 31.

The title compound was prepared according to the procedures described in Example 23 using stearic acid instead of octodecanedicarboxylic acid mono-tert-butyl ester. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-60% B gradient. Final product purification was performed using a 20-60% B gradient (30 ml/min) over 36 minutes.

Example 32 Synthesis of a cyclic PYY analogue SEQ ID NO: 32.

The title compound was prepared according to the procedures described in Example 11, using palmitic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8) in step 2, Fmoc-Ala-OH used instead of Fmoc-His(trt) -OH at position 26 and Fmoc-Ala-OH used instead of Fmoc-Lys(Boc)-OH at position 4 in step 4 (Example 9, step 1). TRIS/HCl buffer (1 M, pH 7.5) was used instead of NH ₄ OAc buffer in step 6 to carry out cyclization. The product was purified using an Agilent Polaris C18-A column (30x250 mm, 100 A, 5 µm) at room temperature. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 20% B to an intermediate concentration of 35% B (40 ml/min) in for 5 min and then to a final concentration of 45% B (40 ml/min) over 40 min.

Example 33 Synthesis of a cyclic PYY analogue SEQ ID NO: 33.

The title compound was prepared according to the procedures described in Example 11 using palmitic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8) in step 2 and Fmoc-N(Me)-Gln(trt)-OH instead Fmoc-Gln(trt)-OH at position 34 in step 4 (Example 9, step 1). In this case, couplings were carried out at room temperature using NMP as solvent, and the HATU/DIEA protocol (1 h, one coupling); Fmoc-N(Me)-Gin(trt)-OH and Fmoc-Arg(pbf)-OH were double coupled. A two-step Fmoc deprotection protocol (20% piperidine in DMF; rt; 10 min, 15 min) was used during surgery. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-70% B gradient. Final product purification was performed using a 20-70% B gradient (30 ml/min) over 36 minutes.

Example 34 Synthesis of a cyclic PYY analogue SEQ ID NO: 34.

The title compound was prepared according to the procedures described in Example 11 using palmitic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8), replacing Fmoc-hCys(trt)-OH with Fmoc-Cys(trt)- OH at position 31, Fmoc-Ile-OH to 6-Fmoc-aminohexanoic acid at position 3, and coupling bromoacetic anhydride instead of m-bromomethylbenzoic acid in step 4 (example 9, step 2), using the modification opi

- 69 045724 sledge in Example 15. Aq. NaHCO ₃ (2N) was used instead of NH ₄ OAc buffer to carry out the cyclization. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 20–70% B (30 mL/min) over 36 min.

Example 35 Synthesis of a cyclic PYY analogue SEQ ID NO: 35.

The title compound was prepared according to the procedures described in Example 9 with the following modifications: Fmoc-psi-[N-Me-Arg(Pbf)-N(Boc)Tyr(tBu)]-OH, prepared from Fmoc -N-Me-Arg(pbf)-OH instead of Fmoc-Arg(Pbf)-OH, in accordance with the procedure described in Example 1, step 1, was used instead of Fmoc-psi-[Arg(Pbf)-N(Boc) Tyr(tBu)]-OH (6) to prepare the loaded Sieber resin used herein; Fmoc-Lys(Pal-Glu-OtBu)-OH (from Active Peptide) was used in place of Leu at position 30; m-chloromethylbenzoic acid was used instead of m-bromomethylbenzoic acid in step 2; Bindings were performed at room temperature. using NMP as solvent and the HATU/DIEA protocol (1 h, one coupling) was used; Fmoc-Arg(pbf)-OH was double coupled. A two-step Fmoc deprotection protocol (20% piperidine in DMF; rt; 10 min, 15 min) was used during surgery. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-70% B gradient. Final product purification was performed using a 20-70% B gradient (30 ml/min) over 36 minutes.

Example 36 Synthesis of a cyclic PYY analogue SEQ ID NO: 36.

The title compound was prepared according to the procedures described in Example 35, replacing Fmoc-Ile-OH with Fmoc-eAla-OH at position 3, and coupling bromoacetic anhydride in place of m-bromomethylbenzoic acid in step 4 (Example 9, step 2), using the modification described in Example 15. The modified treatment specified in Example 19 was omitted. Fmoc-eAla-OH was coupled under microwave irradiation at 50°C for 20 min. Saturated aq. NaHCO3 was used instead of NH4OAc buffer to perform cyclization. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 20–70% B (30 mL/min) over 36 min.

Example 37 Synthesis of a Cyclic PYY Analog SEQ ID NO: 37

The title compound was prepared according to the procedures described in Example 9, replacing Fmoc-hCys(trt)-OH with Fmoc-Cys(trt)-OH at position 31 and Fmoc-Leu-OH with Fmoc-Lys(Pal -Glu-OtBu)-OH (from Active Peptide) at position 30. Additionally, Fmoc-Abu-OH was added to the sequence at position 2 in step 1, and bromoacetic anhydride coupling was used in place of m-bromomethylbenzoic acid in step 2 , using the modification described in Example 15. Saturated aq. NaHCO ₃ was used instead of NH ₄ OAc buffer in step 3 to carry out the cyclization. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 20–70% B (30 mL/min) over 36 min.

Example 38 Synthesis of a cyclic PYY analogue SEQ ID NO: 38.

The title compound was prepared according to the procedures described in Example 35, with the following modifications: Fmoc-eAla-OH was added to the sequence at position 2 after step 1, using microwave irradiation at 50°C for 20 minutes, and coupling with bromoacetic anhydride was used instead of m-bromomethylbenzoic acid in step 2, using the modification described in Example 15. Saturated aq. NaHCO3 was used instead of NH4OAc buffer to perform cyclization. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) in the range of 20–80% H (30 ml/min B) over 36 min.

Example 39 Synthesis of a Cyclic PYY Analog SEQ ID NO: 39

The title compound was prepared according to the procedures described in Example 11 using arachidic acid instead of α-tocopheryloxyacetic acid (AcVitE) (8) in step 2. Fmoc-Ser(tBu)-OH was used instead of Fmoc-Lys( Boc)-OH at position 4 in step 4 (Example 9, step 1) and t-chloromethylbenzoic acid were used instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2). 60% EtOH/H ₂ O was used as a solvent instead of MeCN/H ₂ O and saturated aq. NaHCO ₃ was used instead of NH ₄ OAc buffer in step 4 to carry out the cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) ranging from an initial concentration of 20% B to an intermediate concentration of 25% B (100 ml/min) over 5 min and then to a final concentration of 40% B (100 ml/min) for 40 minutes.

Example 40. Synthesis of a cyclic PYY analogue SEQ ID NO: 40.

1. Synthesis (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(Dde)-(azido-nor-Leu)-

- 70 045724

TRQ(psi-R35Y36)-Sieber resins.

Amino acid extension on preloaded (psi-R35,Y36)-Sieber resin from Example 1, step 2, (0.1 mmol) was carried out at RT. using NMP as solvent, 5-fold excess of protected amino acids and HATU/DIEA protocol (1 h, one binding); Fmoc-Arg(pbf)-OH and Fmoc-His(trt)-OH were double coupled. A two-step Fmoc deprotection protocol (20% piperidine in DMF; rt; 10 min, 15 min) was used during surgery.

2. Synthesis of (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(NH ₂ )-(azido-nor-Leu)TRQ(psi-R35Y36)-Sieber resin.

The resin mentioned above was treated with 2% hydrazine in DMF (12 ml/0.2 mmol scale) for 2 min at RT. and then the mixture was drained. This procedure was repeated approximately 4x, after which the resin was washed extensively with DMF and then DCM.

3. Synthesis of (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK((OEG) ₂ -γ-Glu-Pal)-(azidonor-Leu)-TRQ(psi-R3 5Y 3 6)-Sieber resin.

The resin mentioned above was coupled with (S)-10,19-dioxo-22-palmitamido-3,6,12,15tetraoxa-9,18-diazatricosanedicarboxylic acid (5 eq.) [prepared according to the procedure described in synthesis of intermediate 3, by replacing 18-tert-butoxy-18-oxooctadecanoic acid with palmitic acid in step G], using the HBTU/DIEA protocol at RT. for 1.5 hours. The resin was drained and washed extensively with DMF and DCM.

4. Synthesis of (HCCH(CH ₂ ) ₂ CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK((OEG) ₂ -γ-Glu-Pal)-(azidonor-Leu)-TRQ(psi-R3 5Y3 6)-CONH ₂ .

The dried resin was treated with a solution of 2% TFA in DCM (20 ml) and stirred for 20 minutes, then filtered. This treatment was repeated 2 more times, using fresh mixture for each treatment. The combined filtrates were then combined and concentrated to yield the crude protected peptide as a yellow foam. This foam was treated with 20 ml of digestion mixture (TFA/H2O/TIPS=95/2.5/2.5) at RT. for 2.5 hours and then concentrated under a stream of nitrogen to a volume of about 2.5 ml. Cold ether (40 ml) was then added to precipitate the peptide and the mixture was centrifuged (5 min; 5000 rpm) and decanted. This process was repeated 2 more times to obtain the crude peptide as an almost white powder.

Alternatively, the resin was treated with a cleavage mixture without pretreatment with 1-2% TFA in DCM to produce a completely unprotected peptide.

The crude peptide was purified by reverse-phase preparative HPLC using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 μm). The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 20–70% B over 36 min. UV detection was monitored at 220 and 254 nm. Fractions containing product were analyzed by analytical HPLC on an Agilent 1100 HPLC system using the same column type as above (4.6 x 250 mm, 5 µm). The pure fractions were combined and then lyophilized to give the product as a cotton-like solid.

LCMS: 1211.8 (M+4H)/4, 1615.4 (M+3H)/3 and 2422.9 (M+2H)/2 for product peak at 16.87 min (LC: Atlantis T3 C18 Column, 5 µm, 4.6x250 mm, 1.0 ml/min, gradient 30-60%).

5. Analogue of cyclic PYY SEQ ID NO: 40.

Prepare 5.1 mg of CuSO ₄ in 1 ml of H ₂ O. Prepare 10.4 mg of TBTA in 3 ml of EtOH. Pre-mix 400 μl of CuSO ₄ solution and 3 ml of TBTA solution. Prepare 13 mg of Na ascorbate in 2 ml of H2O.

To a solution of the purified azide-containing peptide from step 4 (37 mg) in 4 ml of HEPES (0.1 M, pH 7.4), 1.7 ml of premixed CuSO4/TBTA solution was added, followed by 1 ml of Na ascorbate solution. Adjust the EtOH/H2O ratio until the reaction solution becomes clear. The mixture was stirred at room temperature. and monitored by HPLC. After 30 minutes the reaction was complete. The mixture was adjusted to pH 4 and purified using reverse phase preparative HPLC. Purification was carried out using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 µm). The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 20–60% B over 36 min. UV detection was monitored at 220 and 254 nm. Fractions containing product were analyzed by analytical HPLC on an Agilent 1100 HPLC system using the same column type as above (4.6 x 250 mm, 5 µm). The pure fractions were combined and then lyophilized to give the product as a cotton-like solid.

Example 41 Synthesis of a cyclic PYY analogue SEQ ID NO: 41.

The title compound was prepared according to the procedure described in Example 40, replacing (S)-10,19-dioxo-22-palmitamido-3,6,12,15-tetraoxa-9,18-diazatricosanedicarboxylic acid with L -glutamic acid, N-(1-oxohexadecyl)-, 1-(1,1-dimethylethyl) ester, in step 3.

Example 42 Synthesis of a cyclic PYY analogue SEQ ID NO: 42.

The title compound was prepared according to the procedure described in Example 40, replacing (S)-10,19-dioxo-22-palmitamido-3,6,12,15-tetraoxa-9,18-diazatricosanedicarboxylic acid

- 71 045724 to (S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23triazatetratetetracontane -1-carboxylic acid (16) (intermediate 2), in step 3.

Example 43 Synthesis of a cyclic PYY analogue SEQ ID NO: 43.

1. Synthesis of (Alloc)Lys((OEG) ₂ -γ-Glu-NH ₂ )-(hC)-TRQ(psi-R35Y36)-Sieber resin.

Amino acid extension on preloaded (psi-R35,Y36)-Sieber resin from Example 1, step 2, (0.1 mmol) was carried out at RT. using NMP as solvent, 5-fold excess of protected amino acids and HATU/DIEA protocol (1 h, one binding); Fmoc-Arg(pbf)-OH was subjected to double coupling. A two-step Fmoc deprotection protocol (20% piperidine in DMF; RT; 10 min, 15 min) was used during surgery.

2. Synthesis of (Alloc)Lys((OEG) ₂ -γ-Glu-Pal)-(hC)-TRQ(psi-R35Y36)-Sieber resin.

Palmitic acid was coupled to the resin from step 1 using microwave irradiation conditions using HATU/DIEA at 50°C for 20-30 min and NMP as solvent.

3. Synthesis of (H ₂ N)Lys((OEG) ₂ -γ-Glu-Pal)-(hc)-TRQ(psi-R35Y36)-Sieber resin.

The alloc protecting group of the resin mentioned above was removed following the procedure described in Example 1, step 4.

4. Analogue of cyclic PYY SEQ ID NO: 43.

The title compound was prepared according to the procedures described in Example 9, steps 1-3, using m-chloromethylbenzoic acid instead of m-bromomethylbenzoic acid in step 2. The crude linear peptide was purified and cyclized according to the modification described in Example 19, using a 20-60% B gradient. Final product purification was performed using a 20-60% B gradient (30 ml/min) over 36 minutes.

Example 44 Synthesis of a cyclic PYY analogue SEQ ID NO: 44.

The title compound was prepared according to the procedures described in Example 43, modified so that the paired Fmoc-OEG-OH units and the Fmoc-Glu-OtBu unit were introduced in step 2 instead of step 1. Octodecanedicarboxylic acid, mono-tert-butyl ester (AstaTech, Inc.) was used instead of palmitic acid in step 2 and the linker lipid sequence was installed at position 11 instead of position 30. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-70% B gradient Final product purification was performed using a 20-70% B gradient (30 ml/min) over 36 min.

Example 45 Synthesis of a Cyclic PYY Analog SEQ ID NO: 45

The title compound was prepared according to the procedures described in Example 43, modified so that Fmoc-dPEG24-carboxylic acid was used in place of the tandem Fmoc-OEG-OH units and it, along with palmitic acid, was introduced in step 2. The linker-lipid sequence was installed at position 11 instead of position 30. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-90% bar gradient. Final product purification was performed using a 20-90% B gradient (30 ml/min) over 36 min.

Example 46 Synthesis of a cyclic PYY analogue SEQ ID NO: 46.

The title compound was prepared according to the procedures described in Example 9 using Fmoc-Lys(Pal-Glu-OtBu)-OH (obtained from Active Peptide) in place of Leu at position 30. Additionally, Fmoc-eAla -OH was added to the sequence at position 2, after step 1, using microwave irradiation conditions at 50°C for 20 minutes, and coupling with bromoacetic anhydride was used instead of m-bromomethylbenzoic acid in step 2, using the modification described in Example 15 The solid crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-70% B gradient. Final product purification was performed using a 20-70% B gradient (30 ml/min) over 36 min .

Example 47 Synthesis of a Cyclic PYY Analogue SEQ ID NO: 47

The title compound was prepared according to the procedures described in Example 44, installing the linker-lipid sequence at position 7 instead of position 11. Product purification was performed using a Varian Pursuit XRs C18 column (30x250 mm, 100 A, 5 μm) at k.t. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-60% B gradient. Final product purification was performed using a 20-60% B gradient (30 ml/min) over 36 minutes.

Example 48 Synthesis of a cyclic PYY analogue SEQ ID NO: 48.

The title compound was prepared according to the procedures described in Example 43, using octadecanedicarboxylic acid mono-tert-butyl ether (AstaTech, Inc.) instead of palmitic acid in step 2 with a coupling time of 30 minutes, and establishing the linker lipid sequence to position 22 instead of position 30. Crude linear peptide

- 72 045724 was purified and cyclized according to the modification described in Example 19 using a 20-70% B gradient. Final product purification was carried out using a 20-70% B gradient (30 ml/min) over 36 minutes.

Example 49 Synthesis of a Cyclic PYY Analogue SEQ ID NO: 49

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 16-tetrahydropyran-2yloxypalmitic acid in step 2, and using m-chloromethylbenzoic acid instead of m-bromomethylbenzoic acid stage 4 (Example 9, stage 2). 60% EtOH/H ₂ O was used as a solvent instead of MeCN/H2O and saturated aq. NaHCO ₃ was used instead of NH ₄ OAc buffer in step 4 to carry out the cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) ranging from an initial concentration of 20% B to an intermediate concentration of 10% B (100 ml/min) over 5 min and then to a final concentration of 30% B (100 ml/min) for 40 minutes.

Example 50 Synthesis of a cyclic PYY analogue SEQ ID NO: 50.

The title compound was prepared following the procedures described in Example 48 and installing the linker-lipid sequence at position 23 instead of position 30.

Example 51 Synthesis of a cyclic PYY analogue SEQ ID NO: 51.

The title compound was prepared according to the procedures described in Example 9 using Fmoc-Lys(Pal-Glu-OtBu)-OH (obtained from Active Peptide) instead of Fmoc-Leu-OH at position 30 and Fmoc- Ser(tBu)-OH instead of Fmoc-Lys(Boc)-OH at position 4, in step 1. 60% EtOH/H2O was used as a solvent instead of MeCN/H2O and saturated aq. NaHCO ₃ was used instead of NH ₄ OAc buffer in step 4 to carry out the cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) ranging from an initial concentration of 10% B to an intermediate concentration of 20% B (100 ml/min) over 5 min and then to a final concentration of 30% B (100 ml/min) for 40 minutes. The contaminated fractions were re-chromatographed using a gradient consisting of an initial concentration of 10% B and an intermediate concentration of 20% B (100 ml/min) over 5 min and then to a final concentration of 30% B (100 ml/min) over 60 min.

Example 52 Synthesis of a cyclic PYY analogue SEQ ID NO: 52.

The title compound was prepared according to the procedures described in Example 43, using Fmoc-dPEG12-carboxylic acid in place of the tandem Fmoc-OEGOH units and introducing it together with Fmoc-Glu-OtBu and palmitic acid in step 2. Linker- the lipid sequence was installed at position 11 instead of position 30. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20-70% B gradient. Final product purification was performed using a 20-70% B gradient (30 ml/min) for 36 min.

Example 53 Synthesis of a cyclic PYY analogue SEQ ID NO: 53.

The title compound was prepared according to the procedures described in Example 52 using four tandem Fmoc-OEG-OH units in place of the Fmoc-dPEG12carboxylic acid.

Example 54 Synthesis of a cyclic PYY analogue SEQ ID NO: 54.

The title compound was prepared according to the procedures described in Example 53, introducing two tandem Fmoc-OEG-OH units instead of two.

Example 55 Synthesis of a cyclic PYY analogue SEQ ID NO: 55.

The title compound was prepared according to the procedures described in Example 43, introducing a linker-lipid sequence at position 23 instead of position 30. The crude linear peptide was purified and cyclized according to the modification described in Example 19 using a 20 gradient -70% B. Final product purification was performed using a 20-70% B gradient (30 ml/min) over 36 min.

Example 56 Synthesis of a cyclic PYY analogue SEQ ID NO: 56.

The title compound was prepared according to the methods described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with (4'-chlorobiphenyl-4-yl)acetic acid in step 2, and using m- chloromethylbenzoic acid instead of mbromomethylbenzoic acid in step 4 (Example 9, step 2). 60% EtOH/H ₂ O was used as a solvent instead of MeCN/H ₂ O and saturated aq. NaHCO ₃ was used instead of NH ₄ OAc buffer in step 4 to carry out the cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 10-28% B (100 ml/min) for 40 min.

- 73 045724

Example 57 Synthesis of a cyclic PYY analogue SEQ ID NO: 57.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 3-[(2,4-dichlorophenoxy)phen-4yl]propionic acid in step 2, and using m-chloromethylbenzoic acid instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2). 60% EtOH/H ₂ O was used as a solvent instead of MeCN/H ₂ O and saturated aq. NaHCO ₃ was used instead of NH ₄ OAc buffer in step 4 to carry out the cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 10-30% B (80 ml/min) for 40 min. Fractions containing the product were pooled, acidified with TFA, concentrated and re-chromatographed on an Agilent Polaris C18-A column (30x250 mm, 100 A, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from an initial concentration of 20% B to an intermediate concentration of 15% B (40 ml/min) to final concentration 45% B (40 ml/min) for 45 min.

Example 58 Synthesis of a cyclic PYY analogue SEQ ID NO: 58.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 11-(4-fluorophenyl)undecanoic acid in step 2, and using m-chloromethylbenzoic acid instead m-bromomethylbenzoic acid in step 4 (example 9, step 2). 60% EtOH/H2O was used as a solvent instead of MeCN/H2O and saturated aq. NaHCO ₃ was used instead of NH ₄ OAc buffer in step 4 to carry out the cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 15-35% B (100 ml/min) for 40 min.

Example 59 Synthesis of a Cyclic PYY Analog SEQ ID NO: 59

The title compound was prepared according to the procedures described in Example 43, omitting step 2 and introducing palmitic acid coupling in step 1. The linker-lipid sequence was installed at position 22 instead of position 11. The crude linear peptide was purified and cyclized according to with the modification described in Example 19 using a 20-70% B gradient. Final product purification was performed using a 2070% B gradient (30 ml/min) over 36 minutes.

Example 60. Synthesis of a cyclic PYY analogue SEQ ID NO: 60.

The title compound was prepared according to the procedures described in Example 53, introducing two tandem FMOC-OEG-OH units instead of four, and installing the linker lipid sequence at position 7 instead of position 11. The crude linear peptide was purified and cyclized according to modification described in Example 19 using a 20-80% B gradient. Final product purification was performed using a 20-80% B gradient (30 ml/min) over 36 minutes.

Example 61 Synthesis of a cyclic PYY analogue SEQ ID NO: 61.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 11-[(4-trifluoromethyl)phenyl]undecanoic acid in step 2, and using m- chloromethylbenzoic acid instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2). 60% EtOH/H2O was used as a solvent instead of MeCN/H2O and saturated aq. NaHCO3 was used instead of NH4OAc buffer in step 4 to perform cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 15-35% B (100 ml/min) for 40 min.

Example 62. Synthesis of a cyclic PYY analogue SEQ ID NO: 62.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 11,11,11-trifluoroundecanoic acid in step 2, and using m-chloromethylbenzoic acid instead of m -bromomethylbenzoic acid in step 4 (Example 9, step 2). 60% EtOH/H ₂ O was used as a solvent instead of MeCN/H ₂ O and saturated aq. NaHCO ₃ was used instead of NH4OAc buffer in step 4 to perform cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 10-28% B (100 ml/min) for 40 min.

Example 63. Synthesis of a cyclic PYY analogue SEQ ID NO: 63.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 15,15,15-trifluoropentanedecanoic acid in step 2, and using m-chloromethylbenzoic acid instead of m -bromomethylbenzoic acid in step 4 (Example 9, step 2). 60% EtOH/H2O was used as a solvent instead of MeCN/H2O and saturated aq. NaHCO3 was used instead of NH4OAc buffer at the stage

- 74 045724 for cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 15-30% B (100 ml/min) for 40 min.

Example 64 Synthesis of a cyclic PYY analogue SEQ ID NO: 64.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 16-ethoxypalmitic acid in Step 2, and using m-chloromethylbenzoic acid instead of m-bromomethylbenzoic acid stage 4 (Example 9, stage 2). 60% EtOH/H ₂ O was used as a solvent instead of MeCN/H ₂ O and saturated aq. NaHCO ₃ was used instead of NH4OAc buffer in step 4 to perform cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM _NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 15-30% B (100 ml/min) for 40 min.

Example 65 Synthesis of a cyclic PYY analogue SEQ ID NO: 65.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 13,13,14,14,15,15,16,16,16-B9palmitic acid ( Cambridge Isotopes) in step 2, and using m-chloromethylbenzoic acid instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2). 60% EtOH/H2O was used as a solvent instead of MeCN/ _H2O and saturated aq. NaHCO ₃ was used instead of NH4OAc buffer in step 4 to perform cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 15-20% B (100 ml/min) for 5 min and then up to 35% B (100 ml/min) for 40 min.

Example 66 Synthesis of a cyclic PYY analogue SEQ ID NO: 66.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 11-[(2,4-bis(trifluoromethyl)phenyl]undecanoic acid in step 2. and using m-chloromethylbenzoic acid instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2) 60% EtOH/H ₂ O was used as a solvent instead of MeCN/H ₂ O and saturated aq NaHCO ₃ was used instead of NH buffer ₄ OAc in step 4 to perform cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT. The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 15-35% B (100 ml/min) for 40 min.

Example 67 Synthesis of a cyclic PYY analogue SEQ ID NO: 67.

The title compound was prepared according to the procedures described in Example 11, replacing α-tocopheryloxyacetic acid (AcVitE) (8) with 11-[(3,5-bis(trifluoromethyl)phenyl]undecanoic acid in step 2. and using m-chloromethylbenzoic acid instead of m-bromomethylbenzoic acid in step 4 (Example 9, step 2) 60% EtOH/H2O was used as a solvent instead of MeCN/H2O and saturated aq NaHCO3 was used instead of NH4OAc buffer in step 4 to carry out the cyclization. Product purification was performed using a Waters XBridge C18 OBD column (50x250 mm, 5 µm) at RT The mobile phase consisted of a gradient elution of buffer A (10 mM NH4OH in water, pH ~9) and buffer B (MeCN) in the range of 15 -35% B (100 ml/min) for 40 min.

Example 68 Synthesis of a cyclic PYY analogue SEQ ID NO: 68.

1. Synthesis of (Fmoc)-βA-IKPEAPGEK(Alloc)ASPEELNRYYASLRHYLNCVTRQ(psi-R35Y36)-Sieber resin.

Amino acid extension on preloaded (psi-R35,Y36)-Sieber resin from Example 1, step 2, (0.1 mmol) was carried out at RT. using DMF as solvent, 6-fold excess of protected amino acids and HATU/DIEA protocol (10 min, double coupling). A two-step Fmoc deprotection protocol (20% piperidine in DMF; RT; 10 min, 15 min) was used during surgery.

2. Synthesis of (Fmoc)-eA-IKPEAPGEK((OEG) ₂ -y-Glu-NHCO(CH ₂ ) ₁₆ CO ₂ tBu)-ASPEELNRYYASLRHYLNCVTRQ(psi-R35Y36)-Sieber resin.

Deprotection of the above resin was carried out in accordance with the method described in Example 1, step 4, using a modified reaction time of 10 minutes for each treatment. The resin was then coupled to intermediate 2 (15) (5 eq) using the HATU/DIEA protocol in DMF (1 h, RT).

3. Synthesis of (BrAc)-eA-IKPEAPGEK((OEG) ₂ -y-Glu-NHCO(CH ₂ ) ₁₆ CO ₂ tBu)-ASPEELNRYYASLRHYLNCVTRQ(psi-R35Y36)-Sieber resin.

After deprotection of Fmoc (20% piperidine/DMF), the above resin was treated with bromoacetic anhydride (10 equiv; r.t., 30 min) to obtain the bromoacetylated resin.

4. Synthesis of (BrAc)-eA-IKPEAPGEK((OEG) ₂ -y-Glu-NHCO(CH ₂ ) ₁₆ CO ₂ tBu)-ASPEELNRYYASLRHYLNCVTRQ(psi-R3 5Y3 6)-CONH ₂ .

The above resin was treated with a cleavage mixture containing TFA/H2O/TIPS

- 75 045724 (95:2.5:2.5), for 1.5 hours at room temperature. The crude peptide was precipitated with ether according to the procedure described in Example 1, step 7.

5. Analogue of cyclic PYY SEQ ID NO: 68.

The crude peptide obtained above was dissolved at a concentration of 10 mg/ml in 10% MeCN/H ₂ O and TEA was added to increase the pH of the solution to 8-9. After stirring at room temperature. for ~20 min. TFA was added to lower the pH to 2, and the solution was directly purified using preparative HPLC on a Kinetics C18 Evo column (30x100 mm, 100 A, 5 μm). The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 20–60% B over 22 min. UV detection was monitored at 220 and 254 nm. The pure fractions were combined and then lyophilized to give the product as a cotton-like solid.

Example 69 Synthesis of a cyclic PYY analogue SEQ ID NO: 69.

1. Synthesis of (Boc)-G-ISPEAPGEK(dde)ASPEELNRYYASLRHYLNLE(OAllyl)TRQ(psi-R35Y36)-Sieber resin.

Amino acid extension on preloaded (psi-R35,Y36) Sieber resin from Example 1, step 2 (0.1 mmol) was carried out at RT, using DMF as solvent, 6-fold excess of protected amino acids and protocol HATU/NMM (10 min, double binding). A two-step Fmoc deprotection protocol (20% piperidine in DMF; RT; 10 min, 15 min) was used during surgery.

2. Synthesis of (Boc)-G-ISPEAPGEK(dde)ASPEELNRYYASLRHYLNLE(NHS)TRQ(psi-R35Y36)-Sieber resin.

Alloc deprotection of the above resin was carried out in accordance with the method described in Example 1, step 4, using a modified reaction time of 10 minutes for each treatment. The resin was then coupled with NHS (10 eq) using the HATU/DIEA protocol in DMF (1 h, RT, double coupling).

3. Synthesis of (NH2)-G-ISPEAPGEK(dde)ASPEELNRYYASLRHYLNLE(NHS)TRQ(psi-R35Y36).

The above resin was treated with a digestion mixture containing TFA/H2O/TIPS (95:2.5:2.5) for 1.5 hours at RT. The crude peptide was precipitated with ether according to the procedure described in Example 1, step 7.

4. Analogue of cyclic PYY SEQ ID NO: 69.

The crude peptide obtained above was dissolved at a concentration of 80 mg/ml in DMSO and TEA (25 eq.) was added to effect lactamization. After stirring at room temperature for ~30 min, the reaction mixture was diluted 10-fold with 10% MeCN/water, adjusted to pH 2, and the crude peptide was purified directly by preparative HPLC on a Kinetics C18 Evo column (30x100 mm, 100 A, 5 µm). The mobile phase consisted of a gradient of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 10–60% B over 22 min. UV detection was monitored at 220 and 254 nm. Pure fractions were pooled and then lyophilized to obtain the K(Dde) protected peptide. The Dde protecting group was removed using 2% hydrazine/DMF (10 mg peptide/ml) for 30 min at RT. The reaction mixture was diluted 10-fold with 10% MeCN/water and adjusted to pH 2 with TFA, and the crude peptide solution was purified as described above to give the product as a cotton-like solid.

Example 70. Synthesis of a cyclic PYY analogue SEQ ID NO: 70.

The title compound was prepared according to the procedures described in Example 69, replacing Fmoc-E(OAll)-OH with Fmoc-Leu-OH at position 30 and replacing Fmoc-Val-OH with Fmoc-E(OAll)- HE is in position 31.

Example 71 Synthesis of a cyclic PYY analogue SEQ ID NO: 71.

1. Synthesis of resin (Boc)-G-ISPEAPGEK(dde)ASPEELNRYYASLRHYLNE(OAllyl)VTRQ(N-Me-R)YNovaSyn TGR.

Amino acid extension on NovaSyn TGR resin (0.1 mmol) was carried out using the procedure described in Example 69, step 1.

2. Analogue of cyclic PYY SEQ ID NO: 71.

The title compound was prepared from the resin mentioned above according to the procedures described in Example 69, steps 2-4.

Example 72. Synthesis of a cyclic PYY analogue SEQ ID NO: 72.

1. Synthesis of (S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetracontane -1-ovo N-hydroxysuccinimide ester.

To a solution of (S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42pentaoxa-9,18,23-triazatetratetetracontane-1- oic acid (Intermediate 2 (16)) (54.0 mg, 0.063 mmol), N-hydroxysuccinimide (14.6 mg, 0.127 mmol) and HATU (24.1 mg, 0.063 mmol) in 1.0 ml DMF were added DIEA (0.022 ml, 0.127 mmol), the mixture was stirred for 30 min at room temperature. and used directly in the next step without further purification.

- 76 045724

2. Synthesis of cyclic PYY analogue SEQ ID NO: 72.

To a solution of [cyclo-(G2-E30), S4, K11, psi-(R35,Y36)]-PYY2-36 (prepared in Example 70) (4 mg, 0.96 μmol) in DMF (0.2 ml) 24 µl of a solution of N-hydroxyester (prepared in step 1) and TFA (0.66 µl; 5 eq.) was added and the mixture was stirred overnight at room temperature. The reaction mixture was diluted 10-fold with 10% MeCN/water, adjusted to pH 2 with TFA, and the crude peptide was purified directly using preparative HPLC on a Kinetics C18 Evo column (30x100 mm, 100 A, 5 μm). The mobile phase consisted of a gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) ranging from 10–60% B over 22 min. UV detection was monitored at 220 and 254 nm. The pure fractions were pooled and then lyophilized to obtain the tert-butyl ether-protected peptide. The tert-butyl ether protecting groups were removed using TFA/H ₂ O/TIPS (95:2.5:2.5) for 1.5 hours at room temperature. The mixture was concentrated and the peptide was purified as described above to give the product as a cotton-like solid. Example 73 Synthesis of a Cyclic PYY Analogue SEQ ID NO: 73 The title compound was prepared according to the procedure described in Example 1, replacing N-Fmoc-dPEG6-carboxylic acid with N-Fmoc-dPEG12-carboxylic acid in step 5.

Example 74 Synthesis of a cyclic PYY analogue SEQ ID NO: 74.

The title compound was prepared according to the procedure described in Example 1, but omitting step 5 of linking the PEG linker.

Example 75 Synthesis of a cyclic PYY analogue SEQ ID NO: 75.

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-Cys(trt)-OH with Fmoc-hCys(trt)-OH at position 31 and omitting the Fmoc-βΑΙα-ΟΗ coupling step in step 3 .

Example 76 Synthesis of a cyclic PYY analogue SEQ ID NO: 76.

The title compound was prepared according to the procedure described in Example 1, with Step 3 and Step 4 modified. In Step 3, Fmoc-Lys(Alloc)-OH and Fmoc-Lys(dde)-OH were used at position 30 and provisions 11 respectively. After Alloc deprotection at position 30 with Pd(PPh ₃ )4-phenylsilane, mPEG16-carboxylic acid was coupled to HATU-DIPEA. In step 4, dde at position 11 was removed using 2% hydrazine in DMF.

Example 77 Synthesis of a Cyclic PYY Analog SEQ ID NO: 77

The title compound was prepared according to the procedure described in Example 76, replacing mPEG12-carboxylic acid with mPEG16-carboxylic acid in step 3 and omitting the Fmoc-dPEG12-carboxylic acid coupling step in step 5.

Example 78 Synthesis of a cyclic PYY analogue SEQ ID NO: 78.

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-N-Me-Gln(trt)-OH with Fmoc-Gln(trt)-OH in step 3.

Example 79 Synthesis of a Cyclic PYY Analog SEQ ID NO: 79

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-N-Me-Arg(pbf)-OH with Fmoc-Arg(pbf)-OH in step 1B.

Example 80 Synthesis of a cyclic PYY analogue SEQ ID NO: 80.

The title compound was prepared according to the procedure described in Example 79, replacing Fmoc-Arg(pbf)-OH with Fmoc-Lys(Boc)-OH at position 4 and replacing Fmoc-Trp(Boc)-OH with Fmoc -Leu-OH at position 30 in step 3.

Example 81 Synthesis of a cyclic PYY analogue SEQ ID NO: 81.

The title compound was prepared according to the procedure described in Example 80, replacing Fmoc-Cys(trt)-OH with Fmoc-hCys(trt)-OH at position 31 and replacing Fmoc-γ-aminobutanoic acid with Fmoc-eAla -OH in step 3.

Example 82 Synthesis of a cyclic PYY analogue SEQ ID NO: 82.

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-PEG2-carboxylic acid with Fmoc-eAla-OH and Fmoc-Cys(trt)-OH with Fmoc-hCys(trt)-OH at position 31 and also omitting the Fmoc-Ile-OH binding in step 3.

Example 83 Synthesis of a cyclic PYY analogue SEQ ID NO: 83.

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-Lys(N3)-OH with Fmoc-hCys(trt)-OH at position 31, replacing pent-4-ynic acid with Fmoc-eAla -OH in step 3, and following cyclization procedure B as described below.

Cyclization Procedure B: To a solution of the fully unprotected peptide with PEG12-AcBR installed at position 11 (38 mg, 0.0067 mmol) in 2 ml HEPES (pH 7.4), add 1.7 ml premixed CuSO ₄ /TBTA solution (a solution was prepared by mixing a solution of 2.2 mg CuSO ₄ in water (0.4 ml) with a solution of 11 mg TBTA in EtOH), followed by the addition of 7 mg sodium ascorbate in water (1 ml). The clear reaction solution was left to stir at room temperature. and monitored by HPLC. After 30 min, the reaction was complete and the reaction mixture was adjusted to pH 4 using TFA and subjected to HPLC purification (Pursuit XRS 5 250x30 mm C18 column, flow rate

- 77 045724 ml/min, controlling wavelength 214 nm, with a gradient ranging from 20-60% MeCN-water/water, both with 0.1% TFA, over 36 min). The desired fraction was collected and lyophilized.

Example 84 Synthesis of a cyclic PYY analogue SEQ ID NO: 84.

The title compound was prepared according to the procedure described in Example 1, omitting the coupling step of Fmoc-eAla-OH, replacing the N3-PEG8-carboxylic acid with Fmoc-dPEG12carboxylic acid in step 5, replacing the coupling with 3-(bromomethyl)benzoic acid with DIC to acylate the bromoacetic anhydride in step 3 and follow the cyclization procedure C as described below.

Cyclization procedure C. Aq. was added to a solution of the completely unprotected peptide (20 mg, 0.0035 mmol) in 5 ml of degassed water. NaHCO ₃ solution to bring the reaction mixture to pH 6.4 or higher. After 20 min, LCMS indicated that the reaction was complete, and the reaction mixture was adjusted to pH 4 with TFA and subjected to HPLC purification (Pursuit XRS 5 250x30 mm C18 column, flow rate 30 ml/min, monitoring wavelength 214 nm, with a gradient ranging from 10-60% MeCN-water/water, both with 0.1% TFA, for 36 min). The desired fraction was collected and lyophilized.

After cyclization, the cyclized intermediate was subjected to linker extension by click chemistry following cyclization procedure B with N-(1-bromo-2-oxo-7,10,13-trioxa-3-azahexadecan16-yl)pent-4-ynamide, which was prepared by coupling N-Boc-PEG4-NH ₂ to pent-4-ynic acid using HATU-DIPEA, followed by Boc deprotection with TFA and acylation with bromoacetic anhydride in the presence of TFA.

Example 85 Synthesis of a Cyclic PYY Analogue SEQ ID NO: 85

The title compound was prepared according to the procedure described in Example 1 with the PEG12-AcBr linker installed at position 23 instead of position 11.

Example 86 Synthesis of a cyclic PYY analogue SEQ ID NO: 86.

The title compound was prepared according to the procedure described in Example 1 with the PEG12-AcBr linker installed at position 22 instead of position 11.

Example 87 Synthesis of a Cyclic PYY Analog SEQ ID NO: 87

The title compound was prepared according to the procedure described in Example 1 with the PEG12-AcBr linker installed at position 7 instead of position 11.

Example 88 Synthesis of a Cyclic PYY Analogue SEQ ID NO: 88

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-V-OH with Fmoc-hCys(trt)-OH at position 31, replacing Fmoc-Cys(trt)-OH with Fmoc-Leu- OH at position 30 and replacing Fmoc-Gly-OH with Fmoc-eAla-OH in step 3.

Example 89 Synthesis of a Cyclic PYY Analogue SEQ ID NO: 89

The title compound was prepared according to the procedure described in Example 88, omitting step 1 to obtain the reduced dipeptide and replacing the loading with Fmoc-Tyr(tBu)-OH followed by coupling with Fmoc-(N-Me)Arg-OH with loading Fmoc-psi-(R35-N(Boc)-Y36)-OH at stage 2.

Example 90 Synthesis of a Cyclic PYY Analogue SEQ ID NO: 90

The title compound was prepared according to the procedure described in Example 89, replacing Fmoc-eAla-OH with Fmoc-Gly-OH in step 3.

Example 91 Synthesis of a cyclic PYY analogue SEQ ID NO: 91.

The title compound was prepared according to the procedure described in Example 89, replacing Fmoc-hCys(trt)-OH with Fmoc-Cys(trt)-OH at position 30 in step 3.

Example 92 Synthesis of a cyclic PYY analogue SEQ ID NO: 92.

The title compound was prepared according to the procedure described in Example 90, replacing Fmoc-hCys(trt)-OH with Fmoc-Val-OH at position 31 and replacing Fmoc-Leu-OH with Fmoc-Cys(trt)- OH at position 30 in step 3.

Example 93 Synthesis of a cyclic PYY analogue SEQ ID NO: 93.

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-Val-OH with Fmoc-hCys(trt)-OH at position 31, replacing Fmoc-Cys(trt)-OH with Fmoc-Leu- OH at position 30 and replacing Fmoc-Gly-OH with Fmoc-eAla-OH at the N-terminus in step 3.

Example 94 Synthesis of a cyclic PYY analogue SEQ ID NO:94.

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-Val-OH with Fmoc-hCys(trt)-OH at position 31 and replacing Fmoc-Cys(trt)-OH with Fmoc-Leu- OH at position 30 in step 3.

Example 95 Synthesis of a Cyclic PYY Analogue SEQ ID NO: 95

The title compound was prepared (0.05 mmol scale) according to the procedure described in Example 1, replacing Fmoc-Val-OH with Fmoc-hCys(trt)-OH at position 31, replacing Fmoc-Glu(OAlloc )-OH to Fmoc-Leu-OH at position 30, replacing Fmoc-Lys(Dde)-OH with Fmoc-Lys(Alloc)-OH at position 11, replacing Fmoc-Ser(tBu)-OH with Fmoc-Lys(Boc )-OH at position 4 and replacing Boc-Gly-OH with Fmoc-eAla-OH at the N-terminus in step 3.

To the resin obtained above, oxygen-free DCM (10 ml), phenylsilane (10 eq.) and a solution of Pd(PPh ₃ ) ₄ (0.2 eq.) in DCM (1 ml) were added and the mixture was stirred for 10 min. The reaction mixture was poured and

- 78 045724 the resin was washed with oxygen-free DCM and deprotection was repeated once.

To the resin obtained above, DMF (10 ml), HATU (5 eq) and DIEA (10 eq) were added and the mixture was stirred for 5 min, then a solution of N-hydroxysuccinimide (10 eq) in DMF was added and stirred for another 20 min. The resin was filtered and the procedure was repeated once.

The resin obtained above was deprotected for 2 hours at room temperature. in TFA/TIPS/water (95/2.5/2.5) (10 ml). The digestion mixture was concentrated to approximately 1 ml and then added to 40 ml of ether. The resulting precipitate was collected by centrifugation and dried in an N ₂ atmosphere.

The material obtained above was dissolved in 9 ml of DMSO, to which 10 equiv of TEA was added and the reaction proceeded for 3 hours at room temperature. The resulting solution was diluted to 30 ml with water, pH adjusted to 2 and purified by RP-HPLC on a 30x250 mm C18 column, eluting with a linear gradient of 20-40% MeCN in water (0.1% TFA) over 30 min. Fractions containing the product were lyophilized.

The material obtained above was then treated with 1-2% hydrazine/DMF (1 ml) to remove Dole from lysine. The resulting mixture was diluted to 10 mL with water, adjusted to pH 2, and purified by RP-HPLC as described above.

The resulting product was then dissolved in 10% MeCN/water, adjusted to pH 10 and a solution of N-hydroxysuccinimide bromoacetic ester (3 equiv 0.1 M/DMF solution) was added and the reaction proceeded for 10 min at room temperature. The resulting mixture was diluted to 10 ml with water, adjusted to pH 2 and then purified by RP-HPLC as described above to give the title product.

Example 96 Synthesis of a cyclic PYY analogue SEQ ID NO: 96.

The title compound was prepared according to the procedure described in Example 1, replacing N-Fmoc-dPEG24-carboxylic acid with N-Fmoc-dPEG12-carboxylic acid in step 5.

Example 97 Synthesis of a Cyclic PYY Analog SEQ ID NO: 97

The title compound was prepared according to the procedure described in Example 1, replacing Fmoc-Gly-OH with Fmoc-eAla-OH in step 3.

Example 98 Synthesis of a Cyclic PYY Analog SEQ ID NO: 98

The title compound was prepared according to the procedure described in Example 89, but omitting the Fmoc-dPEG12-carboxylic acid coupling step in step 5.

Example 99 Synthesis of a Cyclic PYY Analog SEQ ID NO: 99

The title compound was prepared according to the procedure described in Example 90, but omitting the Fmoc-dPEG12-carboxylic acid coupling step in step 5.

Example 100 Synthesis of a cyclic PYY analogue SEQ ID NO: 100.

The title compound was prepared according to the procedure described in Example 94, but omitting the Fmoc-dPEG12-carboxylic acid coupling step in step 5.

All of the following sequences are considered examples of the present invention.

List of sequences

SEQ ID NO: 1

Name: [Cyclo-( ₽A2-COCH ₂ -hC31), K(PEG12-AcBr)11, psi(35R,36Y)]-PYY2-36

Structure:

SEQ ID NO: 2

Name: [cyclo-(13- m-COPhCH ₂ -hC31)]-PYY3-36

Structure:

- 79 045724

- 80 045724

- 81 045724

- 82 045724

- 83 045724

- 84 045724

- 85 045724

- 86 045724

- 87 045724

- 88 045724

- 89 045724

- 90 045724

- 91 045724

- 92 045724

- 93 045724

- 94 045724

- 95 045724

- 96 045724

- 97 045724

psi-(R35,Y36)]-PYY3-36

Structure:

- 98 045724

- 99 045724

- 100 045724

(2,4-Cl ₂ Ph) 30, psi-(R35,Y36) ]-PYY3-3 6

Structure:

- 101 045724

- 102 045724

- 103 045724

- 104 045724

- 105 045724

- 106 045724

- 107 045724

SEQ ID NO: 70

Name: [cyclo-(G2-E30), S4, Kll, psi-(R35,Y36)]-PYY2-36

Structure:

hn nh ₂

SEQ ID NO: 71

Name: [cyclo-(G2-E30), S4, Kll, (N-Me-R35)]-PYY3-36

Structure:

hn^nh ₂

SEQ ID NO: 72

Name: [cyclo-(G2- E30), S4, K ( (OEG) ₂ -y-Glu- COCi ₆ CO ₂ H) 11, psi-(R35,Y36)]-PYY2-36

Structure:

- 108 045724

- 109 045724

Structure

- 110 045724

- 111 045724

- 112 045724

- 113 045724

- 114 045724

- 115 045724

- 116 045724

- 117 045724

- 118 045724

- 119 045724

Example 111: Human NPY2R cAMP in vitro potency assay (hY2 assay).

The method used to test the in vitro efficacy of PYY analogues was a cell-based assay designed to measure forskolin-induced cAMP inhibition produced by adenylate cyclase through modulation of the human Gi-protein coupled receptor NPY2R. Forskolin-induced cAMP production in NPY2R-transfected human HEK cells was reduced by activating NPY2R with PYY analogs and controls in a dose-dependent manner and measured in a FRET-based competitive cAMP LANCE immunoassay (PerkinElmer).

Cells were thawed from cryopreservation and added to 15 ml of cell medium (high glucose DMEM (Cellgro), 10% FBS (Hyclone), 1% penicillin/streptomycin (Life Technologies), 1% L-glutamine (Thermo Scientific), 1% Na pyruvate (Thermo Scientific)). Cells were centrifuged at 450xg for 5 minutes, supernatants were aspirated and cells were resuspended in cell medium at a density of 0.2x10 ⁶ cells/ml. Cells were distributed (25 μl/well) into a white 384-well

- 120 045724 Biocoat collagen-coated plate (Becton Dickinson) to a final density of 5000 cells/well and incubated at 37°C, 5% CO2 for 16-24 hours. Supernatants in the assay plate were decanted. Dilutions of PYY analogues and controls were prepared in 1x HBSS (Cellgro), 5 mM HEPES (Cellgro), 0.1% BSA (PerkinElmer) and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma) and 6 μl/well of each samples were added to the appropriate wells. Lance cAMP antibody (PerkinElmer) was diluted 1:100 in 1x HBSS (Cellgro), 5 mM HEPES (Cellgro), 0.005 mM forskolin (Sigma), 0.1% BSA (PerkinElmer) and 0.5 mM 3-isobutyl-1- methylxanthine (Sigma) and 6 μl of antibody mixture were added to the well, which was then incubated at RT. within 25 min. Then, 12 μl/well of LANCE cAMP development reagent mixture containing biotin-cAMP (1:750) and Europium-W8044 (1:2250) (PerkinElmer) was added to each assay plate, which was then incubated at RT. for 2 h. The plates were read on a PerkinElmer Envision plate reader (excitation 320 nm, emission 615 and 665 nm) and relative fluorescence units (RFU) were calculated as (615 nm/665 nm) x 10,000. All samples were analyzed in triplicate. Data were analyzed using Crucible in-house data analysis software developed by Eudean Shaw. The unknown cAMP concentrations in each well were interpolated from standard samples of known cAMP concentrations included in each plate. Parameters such as EC ₅₀ , Log(EC ₅₀ ), Hill curve slope (nH), top and bottom were determined by plotting cAMP concentrations against the logarithm of compound concentrations, fitting with a 4-P model using a nonlinear weighted least squares application. in the R environment (open source http://cran.us.r-project.org/), implemented by the non-clinical statistics and computing department of Janssen R&D.

Efficiency values for the NTSC-PYY analogues of the present invention compared to PYY ₃ . ₃₆ , which was used as a control in the same analysis, are presented in table. 2.

table 2

Efficacy _of compounds NTSC-PYY and PYY _{3_36} (SEQ ID NO: 111) towards the hY2 receptor

SEQ ID NO: Y2R EC ₅₀ (nM) SEQ ID NO: 2-39 Y2R EC ₅₀ (hM) SEQ ID NO: 111 2 0.14 0.19 3 0, 11 0, 12 4 0.74 0.05

- 121 045724

- 122 045724

Example 112: In vivo efficacy studies.

A) Food intake in lean mice C57BL6N: Acute dosage.

Male C57BL/6 mice (10–12 weeks old) were obtained from Taconic Laboratory. Mice were housed singly per cage with AlphaDri bedding in a temperature-controlled room with a 12-h light/dark cycle. Mice were given ad libitum access to water and maintained on their regular diet (Lab Diet Cat: 5001); Animals were acclimated to BioDAQ cages (Research Diets, inc., New Brunswick, NJ) at least 72 h before the start of the experiment.

After acclimation in BioDAQ cages, mice were grouped into cohorts of eight animals based on their individual food intake over the last 24 hours. At 16:00-17:00, animals were weighed and treated with either vehicle (2.7 mM disodium phosphate, 61.33 mM propylene glycol , 19.5 mM phenol, pH 8.2), or the test compound at a dose of 1 cmol/kg (500 nmol/ml) via subcutaneous administration. After administration of the compound, changes in feed mass for each cage were continuously recorded using a BioDAQ automatic control system for 24 hours. Crumbs were removed daily from the bins and areas around the cages using a vacuum. If necessary, the food was replenished. The percentage value of the average cumulative food consumption relative to the vehicle within 24 hours after administration of the compound was calculated, the results are presented in table. 3. Statistical analysis was performed using one-way analysis of variance with Tukey's post-experiment test in Prism software. All data are presented as average values.

B) Weight loss in diet-induced obese (DIO) mice: Acute dosage.

Male DIO C57BL/6 mice (20 weeks old, 14 weeks on a high-fat diet) were obtained from Taconic Laboratory. Mice were housed singly per cage with AlphaDri bedding in a temperature-controlled room with a 12-h light/dark cycle. Mice were given ad libitum access to water and maintained on a high-fat diet (D12492, Research Diet). Animals were acclimatized to the housing at least one week before the start of the experiment.

The day before dosing, mice were grouped into cohorts of eight animals based on

- 123 045724 individual body weight. The next day, at 15:00-16:00, animals were weighed and treated with either vehicle (2.7 mM disodium phosphate, 61.33 mM propylene glycol, 19.5 mM phenol, pH 8.2) or test compound at a dose of 100 nmol/kg (50 nmol/ml) by subcutaneous administration. Body weight was measured 24 hours after dosing and the percentage of weight loss was calculated, the results are presented in table. 3. Statistical analysis was performed using one-way analysis of variance with Tukey's post-experiment test in Prism software. All data are presented as average values.

C) Weight loss in diet-induced obese mice: Chronic dosing.

Male DIO C57BL/6 mice (20 weeks old, 14 weeks on a high-fat diet) were obtained from Taconic Laboratory. Mice were housed singly per cage with AlphaDri bedding in a temperature-controlled room with a 12-h light/dark cycle. Mice were given ad libitum access to water and maintained on a high-fat diet (D12492, Research Diet). Animals were acclimatized to the housing at least one week before the start of the experiment.

The day before dosing, mice were grouped based on individual body weight. At 15:0016:00 for each of the next 7 days, animals were weighed and then treated with either vehicle (2.7 mM disodium phosphate, 61.33 mM propylene glycol, 19.5 mM phenol, pH 8.2) or test compound at a dose of 100 nmol/kg (50 nmol/ml) via subcutaneous administration. After 7 days, body weight was measured and the percentage of weight loss was calculated, the results are presented in table. 3. Statistical analysis was performed using one-way analysis of variance with Tukey's post-experiment test in Prism software. All data are presented as average values.

Table 3

In Vivo Efficacy Studies of NTSC-PYY Compounds

Follow-up I.D. No. Food consumption by lean mice % carrier (dose=1cM/kg Acute weight loss in DIO mice % Weight change* (24 h) (Dose=100 nM/kg) Chronic weight loss in DIO mice % Weight change* (7 days) (DoZ = 100 nM/kg) PYY3-36 84 BUT BUT 2 65*** But But 3 76* But But 8 48*** -4.95*** -10.23*** 9 83 BUT BUT 10 26*** -2.06** But eleven BUT -5.10*** -9.65*** 13 BUT -3.58*** -10.20*** 14 BUT -2.56* -5.66** 25 BUT -3.95*** BUT 26 BUT -4.69*** BUT 39 BUT -4.73*** BUT 43 BUT -1.96** BUT 46 BUT -2.86* BUT 51 BUT -4.16*** BUT 68 BUT -5.68*** BUT

NO = not defined;

#% change in weight relative to control animals administered vehicle;

*p<0.05;

**p<0.01;

***p<0.001.

While the foregoing description sets forth the principles of the present invention with examples given for purposes of illustration, it is to be understood that the practice of the invention is intended to embrace all normal variations, adaptations and/or modifications included within the scope of the following claims and their equivalents.

All documents cited herein are incorporated by reference.

Claims (12)

1. Compound of formula I o and aspeelnryyz ₂₂ z ₂₃ lrz ₂₆ ylnz ₃₀ HN O -BRIDGE------(CH ₂ ) _P [V ₃₁ ] _q TRZ ₃₄ Z ₃₅ Y-NH ₂ Formula I where p is 0 or 1; m is 0, 1, 2, 3, 4 or 5; n is 1, 2, 3 or 4; q is 0 or 1; provided that q is equal to 1 only if Z ₃₀ is absent; BRIDGE represents -Ph-CH ₂ -S-, -triazolyl-, -NHC(O)CH ₂ S-, -SCH ₂ C(O)NH-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-; Z4 is K, A, E, S or R; Z7 is A or K, wherein the side chain amino group of said K is optionally acylated where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH ₂ Cl; Z9 is G or K, wherein the side chain amino group of said K is optionally acylated where t is 0, 1 or 2; u is 0 or 1; v is equal to 14, 16 or 18; - 125 045724 where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH ₂ Cl; Z _n represents D or K, wherein the side chain amino group of said K is optionally acylated where w is 0, 1, 2 or 4; x is 0 or 1; y is 14, 16 or 18; ABOUT _n so _? But N. J-. D. 1 N- chsn _g ) ₁₄ dc ₃ 2 o ^N (CH ₂ ) ₁₆ CO ₂ H ABOUT X, where i is an integer from 0 to 24, and X=Br, I or Cl, - 126 045724 rovana -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH ₂ Cl; Z ₂₂ represents A or K, where the amino group of the side chain of said K is optionally acyl- where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH2Cl; Z ₂₃ represents S or K, where the side chain amino group of said K is optionally acylated - 127 045724 where i is an integer from 0 to 24, and X=Br, I or Cl, -C(O)CH ₂ Br, -C(O)CH ₂ I or -C(O)CH ₂ Cl; Z ₂₆ represents A or H; Z ₃₀ is L, W, absent or K, provided that Z ₃₀ is absent only if q is 1 and the side chain amino group of said K is optionally acylated L y ⁴ T Y '° T - HcbY ⁰ , on ⁰ O and ⁰ ° '' O ' ^N ' Y O ' - ⁰ ~ 'Ν' ^L (CH.) _1b CO;H o ⁿ where r is 0, 1 or 2; s is 0 or 1; q is 14, 16 or 18; or - 128 045724 Z ₃₅ is CF ₃ Z ₃₄ is (CH ₂ CH ₂ O) ₁₂ CH ₃ . (CH ₂ ) _1S OH or a pharmaceutically acceptable salt thereof. 2. The connection according to claim 1, where m is 0, 1, 2, 3 or 5; n is 1, 2 or 4; Z7 is A or K, wherein the amine side chain of said K is replaced by .o. n Ν^ΠΟΗ^ΟΟ^ BUT'n N. (SNDpSNz BUT' or - 129 045724 Z9 is G or K, wherein the amine side chain of said K is replaced by /o\a where t is 0; u equals 1; v is equal to 14; Z11 is D or K, wherein the side chain amino group of said K is optionally acylated f 1 \/ ABOUT 4\s i [j '' - ) /χ where w is 0 or 4; x is equal to 1; y is 14; N. LSN ₂ )1 ₄ СНз Z22 is A or K, wherein the amine side chain of said K is replaced by Z23 is S or K, wherein the amine side chain of said K is replaced by n (SND ₁₄ CH ₃ •o° BUT' about And N' N or Z ₃₀ is L, W, absent, or K, provided that Z ₃₀ is absent only if q is 1 and the amine side chain of said K is replaced by - 130 045724 _CO2H 1CH ₂ CH ₂ OBCHz. SDS ₂ CH ₂ O), ₆ CH ₃ . _or where r is 0 or 2; s equals 1; q is 14, 16 or 18; or or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 1, selected from the group consisting of the sequences of SEQ ID NO: 1 to SEQ ID NO: 110 or a pharmaceutically acceptable salt thereof. 4. A pharmaceutical composition containing a compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. 5. A method of treating or alleviating obesity, comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 3. 6. A method of treating or alleviating a syndrome, disorder or disease, wherein said syndrome, disorder or disease is selected from the group consisting of obesity, type 2 diabetes, metabolic syndrome, insulin resistance and dyslipidemia, comprising administering to a subject in need thereof an effective amount of a compound according to claims 1-3. - 131 045724 7. The method of claim 6, wherein said syndrome, disorder or disease is type 2 diabetes. 8. A method of reducing food intake, comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 3. 9. A method of modulating Y2 receptor activity, comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 3. 10. A method of treating a disease, disorder or syndrome selected from the group consisting of obesity, type 2 diabetes, metabolic syndrome, insulin resistance and dyslipidemia, comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 3, in combinations with at least one antidiabetic agent. 11. The method of claim 10, wherein said antidiabetic agent is a glucagon-like peptide 1 receptor modulator. 12. A method for preparing a pharmaceutical composition, comprising combining a compound according to any one of claims 1 to 3 with a pharmaceutically acceptable carrier.