WO2025193569A1 - Macrocyclic peptides useful as immunomodulators - Google Patents

Abstract

In accordance with the present disclosure, macrocyclic compounds have been discovered which inhibit the LAG-3/MHC Class II protein/protein interaction, and may be useful for the amelioration of various diseases, including cancer and infectious diseases.

Description

MACROCYCLIC PEPTIDES USEFUL AS IMMUNOMODULATORS

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No. 63/563,666 filed March 11, 2024 which is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

The present disclosure provides novel macrocyclic peptides which inhibit the LAG-3/MHC Class II protein/protein interaction and are thus useful for the amelioration of various diseases, including cancer and infectious diseases.

Lymphocyte activation gene-3 (LAG-3; LAG3; CD223) is a ty pe I transmembrane protein that is expressed on the cell surface of activated CD4+ T cells, CD8+ T cells. T regulatory cells, B cells, and subsets of natural killer (NK) and dendritic cells (Triebel F, et al., J. Exp. Med. 1990; 171 : 1393-1405; Huard, Eur. J. Immunol. 1994; 24:3216-21; Grosso, J. Clin. Invest. 2007; 117:3383-92; Huang, Immunity. 2004; 21:503-13; Kieslow, Eur. J. Immunol. 2005; 35:2081- 88; Workman CJ, et al., J. Immunol. 2009; 182(4): 1885- 91 ; Castelli, Oncoimmunology 2014; 3: 11). LAG-3 is closely related to CD4, which is a co-receptor for T helper cell activation. Both molecules have four extracellular Ig-Iike domains and require binding to their ligand, major histocompatibility⁷ complex (MHC) class II, for their functional activity⁷. In contrast to CD4, LAG-3 is only expressed on the cell surface of activated T cells and its cleavage from the cell surface terminates LAG-3 signaling. LAG-3 can also be found as a soluble protein but it does not bind to MHC class II and its function is unknown.

LAG-3 is composed of the intracellular signalling domain, a transmembrane domain and 4 extracellular domains, designated DI to D4 (Huard 1997 Proc. Natl. Acad. Sci. 94:5744-9). Domain 1-2 associates with MHC class II ligand and it has been shown that the tip of domain 1 (extra loop) forms the binding site (Huard 1997 Proc. Natl. Acad. Sci. 94:5744-9).

LAG-3 can also associate with alternative ligands, Galectin-3 and LSECtin, which induce its inhibitory signalling (Kouo 2015 Cancer Immunol Res. 3(4):412-23; Xu 2014 Cancer Res 74(13):3418-28). Association with Galectin-3 on cells or within the extracellular matrix could downregulate T cells that would not normally engage with MHC class II, such as CD8+ T cells. Therefore blockade of this ligand could serve as a mechanism for enhancing broad T cell function.

A role of LAG-3 on T cells is to regulate T cell activation (Huard 1994 Eur. J. Immunol. 24:3216- 21). LAG-3 engages with MHC class II and this leads to down regulation of CD4+ T cells (Huard 1996 Eur. J. Immunol. 26:1180-6). Upon T cell activation, LAG-3 surface expression increases. The engagement of LAG-3 dimer with ligand induces signalling through an intracellular KIEELE domain (Workman 2002 J. Immunol 169:5392-5) leading to downregulation of the T cell activity. Therefore, LAG-3 serves to modulate responses to antigens, preventing over-stimulation and maintaining immune homeostasis.

It has been reported that LAG-3 plays an important role in promoting regulatory T cell (Treg) activity and in negatively regulating T cell activation and proliferation (Workman CJ, et al., J. Tmmunok 2005; 174:688-695). Both natural and induced Treg express increased LAG-3, which is required for their maximal suppressive function (Camisaschi C. et al., J. Tmmunok 2010; 184:6545-6551 and Huang CT, et al, Immunity. 2004: 21 :503-513). Furthermore, ectopic expression of LAG-3 on CD4+ effector T cells reduced their proliferative capacity and conferred on them regulatory potential against third part)⁷ T cells (Huang CT, et al, Immunity⁷. 2004; 21 :503-513). Recent studies have also shown that high LAG-3 expression on exhausted lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells contributes to their unresponsive state and limits CD8+ T cell antitumor responses (Blackbum SD, et ak, Nat. Tmmunok 2009; 10:29-37 and Grosso JF, et ak, J. Clin. Invest. 2007; 117:3383-3392). In fact, LAG-3 maintained tolerance to self and tumor antigens via direct effects on CD8+T cells in 2 murine models (Grosso JF. et ak, J. Clin. Invest. 2007; 117:3383-3392).

Epstein-Barr virus infection is yet another factor to consider in the potential induction of T cell exhaustion in hematological malignancies. It is known that EBVassociated CLL, Richter’s syndrome, and lymphoma cases are usually more aggressive than their EBV(-) counterpart (Tsimberidou AM, et al., Leuk Lymphoma 2006:47:827; Ansell SM, et al., Am J Hematol 1999;60:99.; Dolcetti R. et al., Infectious Agents and Cancer 2010;5:22; Kanakry JA, et al., Blood 2013;121:3547). Interestingly, the expression of checkpoint inhibitors like PD-L1 and LAG-3 has also been documented in EBV-associated malignancies (Green MR, et al., Clin Cancer Res 2012; 18: 1611; Monti S, et al., Blood 2005; 105: 1851). High expression of LAG-3 has in fact been documented in chronic viral infections and its blockade with anti-LAG-3 antibodies has been able to reduce viral titers and the expression of checkpoint inhibitors in murine models (Blackbum SD, et al., Nat. Immunol. 2009;10:29-37). Furthermore, LAG-3 expression, alone or in combination with other markers, has been evaluated as a prognostic or predictive marker in CLL and Hodgkin lymphoma (Zhang J, et al., BMC Bioinformatics 2010;! l(Suppl 9):S5; Kotaskova J, et al., J Mol Diagn 2010;12(3):328 — 334). LAG-3 expression on tumor-infiltrating lymphocytes (TILs) and peripheral blood also mediates T cell exhaustion in hematological malignancies (Dickinson JD, et al., Leuk Lymphoma 2006;47(2):231-44). Moreover, LAG-3 blockade with specific antibodies has shown antitumor activity in leukemia (Berrien-Elliott. M, et al.. Cancer Research 2013; 73(2):605-616) and solid tumor models (Woo, S-R, et al., Cancer Research 2011; 72(4):917-927; Coding, S. R., et al., Journal of Immunology', Baltimore, Md. 1950; 190(9):4899-909). Therefore, LAG-3 is a potential therapeutic target in hematological malignancies.

Recent preclinical studies have documented a role for LAG-3 in CD8 T cell exhaustion, and blockade of the LAG-3/ MHC Class II interaction using LAG-3 blocking antibodies or LAG-3-Ig fusion proteins is being evaluated in a number of clinical trials in cancer patients.

Additional background information can be found in WO2015/042246 Al, WP2015/116539 Al, and W02014/008218 AL

LAG-3 blockade with macrocyclic peptide inhibitors, alone and in combination with standard of care (e.g., nivolumab. imatinib, lenalidomide) or with other checkpoint inhibitors deserves further exploration.

The molecules described herein demonstrate the ability to block the interaction of LAG-3 with MHC Class II, in both biochemical and cell-based experimental systems. These results are consistent with a potential for therapeutic administration to enhance immunity in cancer or chronic infection, including therapeutic vaccine.

The macrocyclic peptides described herein are capable of inhibiting the interaction of Lag-3 with MHC class II. These compounds have demonstrated highly efficacious binding to LAG-3, blockade of the interaction of LAG-3 with MHC Class II, and are capable of promoting enhanced T cell functional activity, thus making them candidates for parenteral, oral, pulmonary, nasal, buccal and sustained release formulations.

Additionally or alternatively, the macrocyclic peptides can possess one or more of the following functional properties described above, such as high affinity binding to human LAG-3, relatively good binding affinity to cyno LAG-3, and lack of binding to mouse LAG-3, the ability to inhibit binding of LAG-3 to MHC Class II molecules and/or the ability to stimulate antigen-specific T cell responses.

In its first embodiment the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

A is selected from a bond, wherein:

* denotes the point of attachment to the carbonyl group and denotes the point of attachment to the nitrogen atom; n is 0 or 1;

R¹³ and R¹⁴ are independently selected from hydrogen and methyl; R¹⁵ is selected from H, COOH. CONR¹⁶R¹⁷, wherein R¹⁶ and R¹⁷ can be independently selected from, hydrogen, 1-3 alkyl, (l-3)alkyl-5-6-carbocycle or heterocycle wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy, -CHR¹⁸C(O)NR¹⁶R¹⁷, -CHR¹⁸C(O)OH, -CHR¹⁸C(O)NHCHR¹⁹C(O)NR¹⁶R¹⁷, -CHR¹⁸C(O)NHCHR¹⁹C(O)OH, -CHR¹⁸C(O)NHCHR¹⁹C(O)NHCHR²⁰C(O)NR¹⁶R¹⁷, -CHR¹⁸C(O)NHCHR¹⁹C(O)NHCHR²⁰C(O)OH; -CHR¹⁸C(O)NHCHR¹⁹C(O)NHCHR²⁰C(O)NHCHR²¹C(O)NR¹⁶R¹⁷, -CHR¹⁹C(O)NHCHR' ⁹C(O)NHCHR²⁰C(O)NHCHR²¹ C(O)OH; wherein R¹⁸ is selected from hydrogen and sidechain of a natural or unnatural amino acid and wherein R¹⁹ is selected from hydrogen and sidechain of a natural or unnatural amino acid and wherein R²⁰ is selected from hydrogen and sidechain of a natural or unnatural amino acid and wherein R²¹ is selected from hydrogen and sidechain of a natural or unnatural amino acid; and 0-2 PEG9 spacers in between any one of the amino acids

X is bond, S, O, CHR²², NR²³ wherein R²² is hydrogen or 1-3 alkyl;

R^b, R^d, R^e, R^g, R^h, R¹, R>, R^k, R¹, and R^m, are each independently selected from hydrogen or methyl;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² are independently selected from a natural amino acid side chain and an unnatural amino acid side chain or form a ring with the corresponding vicinal R group as described below;

R^a is hydrogen, or methyl, or R^a and R¹ together with the atoms to which they are attached can form a ring selected from azetidine, pyrrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy;

R^c is hydrogen, or methyl, or R^c and R³ together with the atoms to which they are attached can form a ring selected from azetidine, py rrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy;

R^e is hydrogen, or methyl, or R^e and R⁵ together with the atoms to which they are attached can form a ring selected from azetidine, pyrrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole: wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy;

R^f is hydrogen, or methyl, or R^f and R⁶ together with the atoms to which they are attached can form a ring selected from azetidine, pyrrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy;

In another embodiment the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein A is n = 0, 1,

X is bond, S, O, CHR²², NR²² wherein R²² is hydrogen or methyl;

In another embodiment the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein A is n = 0. 1;

X = S, CH2, NH, O;

R⁴ is the side chain of L-Asp:

R^b, R^d, R^g, R^h, R¹, R'. R^k, R¹, and R^m, are each independently selected from hydrogen;

R^a is hydrogen, or R^a and R¹ together with the atoms to which they are attached can form a ring selected from pyrrolidine; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy;

R^c is hydrogen, or R^c and R³ together with the atoms to which they are attached can form a ring selected from pyrrolidine; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy; R^e is hydrogen, or R^e and R⁵ together with the atoms to which they are attached can form a ring selected from pyrrolidine; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy:

R^f is hydrogen, or R^f and R⁶ together with the atoms to which they are attached can form a ring selected from pyrrolidine; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy;

In another embodiment the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein A is

X = S, CH₂, and O;

R^a, R^b, R^c,R^d, R^e, R^f, R^g, R^h, R‘, R^j, R^k, R¹. and R^m, are each independently selected from hydrogen;

R¹⁰ is selected from the side chain of L-Asp, L-Phe(4-COOH), L-Trp(l- CH2COOH), L-Tyr(CH₂COOH);

In another embodiment the present disclosure provides a method of enhancing, stimulating, and/or increasing the immune response in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of at least one macrocyclic peptide described herein. In another embodiment the method further comprises administering an additional agent prior to, after, or simultaneously with the macrocy clic peptide or peptides described herein. In another embodiment the additional agent is an antimicrobial agent, an antiviral agent, a cytotoxic agent, and/or an immune response modifier.

In another embodiment the present disclosure provides a method of inhibiting growth, proliferation, or metastasis of cancer cells in a subject in need thereof, said method comprising administering to the subj ect a therapeutically effective amount of one or more macrocyclic peptides described herein. In another embodiment the cancer is selected from melanoma, renal cell carcinoma, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, squamous cell carcinoma of the head and neck, carcinomas of the esophagus, gastrointestinal tract and breast, and a hematological malignancy. In another embodiment the present disclosure provides a method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one macrocyclic peptide described herein. In another embodiment the infectious disease is caused by a virus. In another embodiment, the virus is selected from HIV, Hepatitis A, Hepatitis B, Hepatitis C, herpes virus, and influenza.

In another embodiment the present disclosure provides a method of treating septic shock in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more macrocyclic peptides described herein.

In another embodiment the present disclosure provides a method blocking the interaction of LAG-3 with MHC Class II molecule in a subject, said method comprising administering to the subject a therapeutically effective amount of at least one macrocyclic peptide described herein.

In compounds of formula (I), where R¹ is not part of a ring, preferred R¹ groups are side chains of the following amino acids: phenylalanine, tyrosine, tryptophan, leucine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, 3,4- difluorophenylalanine, 3,5-difluorophenylalanine, 3,4-dichlorophenyl, 3,4,5- difluorophenylalanine, pentafluorophenylalanine, 3-trifluoromethylphenylalanine, 4- methylphenylalanine, 4-chlorophenylalanine, 3-methoxyphenylalananie, 4- methoxy phenyl alanine, 1 -naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), 3- cyanophenylalanine, 4-cyanophenylalanine, 4-difluoromethylphenylalanine, biphenylalanine, 3-(2-thienyl)-alanine, 3-(3-thienyl)-alanine, 3 -benzothienylalanine (Bzt), 4-bromophenylalanine, 3-pyridinylalanine, 4-trifluoromethylphenylalanine, 4- benzoylphenylalanine (Bpa). 3-chlorophenylalanine, 4-aminophenylalanine, 4- aminomethylphenylalanine, 4-carbamoylphenylalanine, 4-carboxyphenylalaine, 3-(4- thiazolylj-alanine, 4-acetamide-phenylalanine, homo-cyclohexylalanine, 2-fuanylalanine, homo-tyrosine, 4-phenoxyphenyl alanine, 4-cyclohexyloxyphenylalanine, 4- propargyloxyphenylalamne, tyrosine(O-acetic acid), 4-benoxyphenylalanine, tryptophane(l -acetic acid), and 4-tert-buty [phenylalanine;

In compounds of formula (I), preferred R² groups are side chains of the following amino acids: alanine, 2-aminobutyric acid (Abu), cyclopropylglycine, threonine, and valine; In compounds of formula (I), preferred R³ groups are side chains of the following amino acids: alanine. 3-(2-thienyl)-alanine, 3-(3-thienyl)-alanine. 3-benzothienylalanine (Bzt). 4-benzoylphenylalanine (Bpa), allothreonine, aspartic acid, citrulline (Cit), asparagine, glutamic acid, homo-glutamic acid, 2,3-diaminopropionic acid (Dap), phenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, 3- methylphenylalanine, 4-methylphenylalanine, 3-methoxyphenylalanine, 4- aminophenylalanine. 7-quinolinylalanine, homophenylalanine, homoglutamide, homoserine, leucine, 4-carboxyphenylalamine, tyrosine, glutamine, serine, 2-amino-4- aminobutyric acid (Dab), threonine, 4-carboxymethoxyphenyl-alanine, tryptophan, tryptophan (1 -acetic acid), and tryptophane (5-benzoxy), tryptophan (1 -methyl), tyrosine, and tyrosine(O-acetic acid).

In compounds of formula (I), preferred R⁴ is the side chain of aspartic acid.

In compounds of formula (I), where R⁵ is not part of a ring, preferred R⁵ groups are side chains of the following amino acids: alanine, citrulline, 1 -aminocyclopentane- 1 - carboxylic acid (Cle), 3-hydroxy 1 -aminocyclopentane- 1 -carboxylic acid (Cle(3-OH)). aspartic acid, D-alanine, 2-amino-4-aminobutyric acid (Dab), Dab(COCH2NH2), Dab(COCH₂NHBoc), Dab(COCH₂CH₂NH₂), Dab(COCH₂CH₂NHBoc), (S)-2-(X2- azaneyl)-4-guanidinobutanoic acid (Dab(-CH(=NH)NH₂)), Dab(CO-ethyl), Dab(CH₂-4- (CH₂NH₂)phenyl), Dab(CH₂-4-(CH₂NHBoc)phenyl), Dab(CONH-isopropyl), 2,3- diaminopropionic acid (Dap), Dap(CH(=NH)NH₂), D-glutamic acid, D-histidine, D- homoarginine, D-lysine, D-lysine(COCH3), D-asparagine, D-glutamine, D-arginine, D- serine, D-valine, glutaminic acid, phenylalanine, glycine, histidine, homoarginine, lysine, leucine, asparagine, ornithine (Om), phenylglycine, propargylalanine, glutamine, arginine, valine, try ptophan (1 -acetic acid), tyrosine (O-acetic acid), tyrosine (O- propargyl), and P-alanine;

In compounds of formula (I), where R⁶ is not part of a ring, preferred R⁶ groups are side chains of the following amino acids: 1 -naphthylalanine (1-Nal), 2- naphthylalanine (2-Nal), alanine, biphenylalanine (Bip), 3 ’-carboxylic acid- biphenylalanine, 4’-carboxylic acid-biphenylalanine, 3-benzothienylalanine (Bzt), 4- benzoylphenylalanine (Bpa), phenylalanine, 3,4,5-trifluorophenylalanine, 3,4- dichlorophenylalanine, 3,5-difluorophenylalanine. 3-chlorophenylalanine, 4- chlorophenylalanine. 4-cyanophenylalanine. 4-aminomethylphenylalanine. 4- carbamoylphenylalanine, 4-carboxyphenylalaine, 4-aminophenylalanine, alphamethylphenylalanine, beta-hydroxy-phenylalanine, isoleucine, histidine, leucine, phenyglycine (Phg), valine, tryptophan, tryptophan (1-acetic acid), tryptophan (5- benzoxy), tyrosine, tyrosine (O-acetic acid), tyrosine (O-propargyl), tyrosine (O-phenyl), and 2S)-2-amino-3-({4-[(2S)-2-amino-2-carboxyethyl]phenyl}formamido)propanoic acid;

In compounds of formula (I), preferred R⁷ geoups are side chains of the following amino acids: alanine, aspartic acid, citrulline, 3-diaminopropionic acid (Dap), 2-amino-4- aminobutyric acid (Dab), Dap(COEt), Dap(CH(=NH)NH2), glutamic acid, glycine, histidine, 4-carboxyphenylalanine. homoarginine, lysine, asparagine, ornithine (Om), arginine, serine, threonine, tryptophan, try ptophan (1-acetic acid), tyrosine, tyrosine (Ci- acetic acid), and tyrosine (O-propargyl);

In compounds of formula (I), preferred R⁸ groups are side chains of th following amino acids: 4-cyclohexyloxyphenylalanine, 1 -naphthylalanine (1-Nal), 2- naphthylalanine (2-Nal), (2S)-2-amino-3-[6-(2-methylphenyl)pyridin-3-yl]propanoic acid, biphenylalanine, 2'-methoxycarbonyl-biphenylalanine, 2'-ethyl-biphenylalanine, 2'- ethyl, 4'-hydroxy-biphenylalanine, 2'-ethyl, 4'-methyoxy-biphenylalanine, 2'-fluoro- biphenylalanine, 2'-methyl-biphenylalanine, 2'-n-butoxyl-biphenylalanine, 2'-methyoxy, 4'-fluoro-biphenylalanine, 3',5'-difluoro-biphenylalanine, 3'-cyanobiphenylalanine, 3'- carboxybiphenylalanine. 3'-methyl biphenylalanine. 4'-chlorobiphenylalanine, N- cyclopropyl-4'-carboxamide-biphenylalanine, 4'-dimethylcarbamoyl-biphenylalanine, 4'- carboxybiphenylalanine, 4'-acetamido-biphenylalanine, 4'-phenoxy -biphenylalanine, (3S)-3-amino-4-[5-(2-methylphenyl)pyrimidin-2-yl]-2-oxobutanoic acid, 4- benzoylphenylalanine (Bpa). (2S)-2-amino-3-[4-(l-benzofuran-2-yl)phenyl]propanoic acid, (2S)-2-amino-3-[4-(3-cyanothiophen-2-yl)phenyl]propanoic acid, (2S)-2-amino-3- {4-[(E)-2-phenylethenyl]phenyl}propanoic acid (Phe(4-CH=CH-phenyl)), 4- iodophenylalanine, 4-isopropylphenylalanine, 4-tert-butylphenylalanine, (2S)-2-amino-3- [4-(6-methoxypyridin-3-yl)phenyl]propanoic acid, (2S)-2-amino-3-[4-(pyrrolidine-l- carbonyl)phenyl] propanoic acid. homosenne(O-benzyl). tryptophan, tryptophan(5- benzoxy), tyrosine(O-propargyl), and tyrosine(O-phenyl);

In compounds of formula (I), preferred R⁹ groups are side chains of the following amino acids: 1 -naphthylalanine (1-Nal), 6-quinolinylalanine, 8-isoquinolinylalanine, 4- isoquinolinylalanine, alanine (3-methoxy-isoquinolin-7-yl), 6-isoquinolinylalanine, 2- naphthylalanine (2-Nal), biphenylalanine, 4-benzoylphenylalanine (Bpa). 3- benzothienylalanine (Bzt), phenylalanine, 2-chlorophenylalanine, 3,4- dichlorophenylalanine, 3,4-difluorophenylalanine, 3,5-difluorophenylalanine, 3- trifluoromethylphenylalanine, 3 -chlorophenylalanine, 3-methoxyphenylalanine, 4- chlorophenylalanine. 4-fluorophenylalanine, 4-aminophenylalanine, 4- carbamoylphenylalanine, 4-tert-butylphenylalanine. homohomophenylalanine, homoserine (O-2-methyl-4-chlorophenyl), homotyrosine, tryptophan, and tryptophan(l- acetic acid);

In compounds of formula (I) preferred R¹⁰ geoups are side chains of aspartic acid, 4-carboxyphenylalanine. tyrosinefO-acetic acid), and tryptophan(l -acetic acid);

In compounds of formula (I) preferred R¹¹ groups are side chains of the following amino acids: 1 -naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), 2-amino-3-(lH-indol- 4-yl)propanoic acid, phenylalanine, 3,4-dimethoxyphenylalanine, 3- methoxyphenylalanine, 4-aminophenylalanine, 1 -methyltryptophane, asparagine, tryptophan, tyrosine. 5 -hydroxy tryptophane, and tyrosine(O-acetic acid);

In compounds of formula (I) preferred R¹² groups are side chains of the following amino acids: alanine, alpha, alpha-dimethylglycine (Aib), aspartic acid, D-alanine, D- aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D-homo-arginine, D-lysine, D-leucine, D-asparagine. D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptophan, D-tyrosine, glutamic acid, glycine, serine, tryptophan(l -scetivc acid), tyrosine(O-acetic acid), and tyrosine(O-propargyl);

As shown below in Tables 1-5. the following compounds of the invention show activity at less than or equal to 1.0 nM in the assay included later in the application.

Compounds <= 1.0 nM

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Definitions

The definitions provided herein apply, without limitation, to the terms as used throughout this specification, unless otherwise limited in specific instances. Those of ordinary' skill in the art of amino acid and peptide chemistry are aware that an amino acid includes a compound represented by the general structure:

L- or S- -amino acid D- or R-a-amino acid

(if R=H) (if R=H) where R and R' are as discussed herein.

Unless otherwise indicated, the term "amino acid" as employed herein, alone or as part of another group, includes, without limitation, an amino group and a carboxyl group linked to the same carbon, referred to as "a" carbon, where R and/or R' can be a natural or an un-natural side chain, including hydrogen. The absolute "S" configuration at the "a" carbon is commonly referred to as the "L" or "natural" configuration. In the case where both the "R" and the "R'"(prime) substituents equal hydrogen, the amino acid is glycine and is not chiral.

The term “naturally occurring amino acid side chain,” as used herein, refers to side chain of any of the naturally occurring amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, -histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) usually in the S -configuration (i.e., the L-amino acid).

The term “non-naturally occurring amino acid side chain,” as used herein, refers to a side chain of any naturally occurring amino acid usually in the R-configuration (i.e., the D-amino acid) or to a group other than a naturally occurring amino acid side chain in R- or S-configuration (i.e., the D- or L-amino acid, respectively) selected from:

The "inhibitory⁷ concentration" of LAG-3 inhibitor is intended to mean the concentration at which a compound screened in an assay of the disclosure inhibits a measurable percentage of the interaction of LAG-3 with MHC Class II molecules. Examples of "inhibitory concentration" values range from IC50 to IC90, and are preferably, IC50, ICeo, IC70, ICso, or IC90, which represent 50%, 60%, 70%, 80% or 90% reduction in LAG-3/MHC Class II molecules binding activity', respectively. More preferably, the "inhibitory concentration" is measured as the IC50 value. It is understood that another designation for IC50 is the half-maximal inhibitory concentration. Binding of the macrocyclic peptides to LAG-3 can be measured, for example, by methods such as homogeneous time-resolved fluorescence (HTRF), Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC), nuclear magnetic resonance spectroscopy (NMR), and the like. Further, binding of the macrocyclic peptides to LAG- 3 expressed on the surface of cells can be measured as described herein in cellular binding assays.

Administration of a therapeutic agent described herein includes, without limitation, administration of a therapeutically effective amount of therapeutic agent. The term "therapeutically effective amount" as used herein refers, without limitation, to an amount of a therapeutic agent to treat or prevent a condition treatable by administration of a composition of the LAG-3/MHC Class II molecules binding inhibitors described herein. That amount is the amount sufficient to exhibit a detectable therapeutic or preventative or ameliorative effect. The effect may include, for example and without limitation, treatment or prevention of the conditions listed herein. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance.

In another aspect, the disclosure pertains to methods of inhibiting growth of tumor cells in a subject using the macrocyclic peptides of the present disclosure. As demonstrated herein, the macrocyclic peptides of the present disclosure are capable of binding to LAG-3, disrupting the interaction between LAG-3 and MHC class II molecules. As a result, the macrocyclic peptides of the present disclosure are potentially useful for modify ing an immune response, treating diseases such as cancer or infectious disease, stimulating a protective autoimmune response or to stimulate antigen-specific immune responses (e.g, by coadministration of LAG-3 blocking peptides with an antigen of interest).

In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The terms "Programmed Death Ligand 1", "Programmed Cell Death Ligand 1", "Protein PD-L1", "PD-L1", "PDL1", "PDCDL1", "hPD-Ll", "hPD-LI", "CD274" and "B7-H1" are used interchangeably, and include variants, isoforms, species homologs of human PD-L1, and analogs having at least one common epitope with PD-L1. The complete PD-L1 sequence can be found under GENBANK® Accession No. NP_054862.

The terms "Programmed Death 1", "Programmed Cell Death 1", "Protein PD-1", "PD-1", "PD1", "PDCD1", "hPD-1" and "hPD-I" are used interchangeably, and include variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The complete PD-1 sequence can be found under GENBANK® Accession No. U64863.

The terms "cytotoxic T lymphocyte-associated antigen-4", "CTLA-4", "CTLA4", "CTLA-4 antigen" and "CD152" (see, e.g., Murata, Am. J. Pathol., 155:453-460 (1999)) are used interchangeably, and include variants, isoforms, species homologs of human CTLA-4. and analogs having at least one common epitope with CTLA-4 (see, e.g.. Balzano, Int. J. Cancer Suppl., 7:28-32 (1992)). The complete CTLA-4 nucleic acid sequence can be found under GENBANK® Accession No. L15006.

The term "immune response" refers to the action of, for example, lymphocy tes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including macrocyclic peptides, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

A "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. As used herein, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal across the plasma membrane of a cell. An example of a "cell surface receptor" of the present disclosure is the PD-1 receptor.

The term "macrocyclic peptide derivatives" refers to any modified form of the macrocyclic peptides disclosed herein, e.g., mutations, isoforms, peptides with altered linker backbones, conjugates with an antibody and/or another agent, etc..

As used herein, a (preferred?) macrocyclic peptide of the present disclosure that "specifically binds to human LAG-3" is intended to refer to a macrocyclic peptide that binds to human LAG-3 with an IC50 of less than about 1000 nM, less than about 300 nM, less than about less than about 100 nM, less than about 80 nM, less than about 60 nM, less than about 40 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less. In this context, the term "about" shall be construed to mean anywhere between ± 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nM more or less than the cited amount.

The term "treatment" or "therapy" refers to administering an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (e.g., a disease), the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a disease, or otherwise arrest or inhibit further development of the disease, condition, or disorder in a statistically significant manner.

As used herein, "about" or "comprising essentially of mean within an acceptable error range for the particular value as determined by one of ordinary' skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, "about" or "comprising essentially of can mean within one or more than one standard deviation per the practice in the art. Alternatively, "about" or "comprising essentially of can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of should be assumed to be within an acceptable error range for that particular value.

As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

Variant Macrocyclic Peptides

In yet another embodiment, a macrocyclic peptide of the disclosure comprises amino acid sequences that are homologous to the amino acid sequences of the macrocyclic peptides described herein, and wherein the macrocyclic peptides retain the desired functional and/or biological properties of the macrocyclic peptide of the disclosure.

For example, the disclosure provides a macrocyclic peptide, or antigen-binding portion thereof, comprising: an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the compounds described herein; and the macrocyclic peptide exhibits one or more of the following properties:

(a) the macrocyclic peptide binds to human LAG-3 with an IC50 of 200 nM or less;

(b) the macrocyclic peptide does not substantially bind to human CD4;

(c) the macrocyclic peptide binds to human LAG-3 and one or more of the following: cynomolgus monkey LAG-3, and/or mouse LAG-3;

(d) the macrocyclic peptide inhibits the binding of LAG-3 to MHC Class II molecules;

(e) the macrocyclic peptide inhibits tumor cell growth in a cellular assay and/or in vivo assay;

In other embodiments, the macrocyclic peptide amino acid sequences may be about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%. about 93%, about 94%, about 95%, about 96%, about 97%. about 98% or about 99% homologous to the sequences set forth above. In this context, the term "about" shall be construed to mean anywhere between 1, 2, 3, 4, or 5 percent more or less than the cited amount. A macrocyclic peptide of the present disclosure having sequences with high identity (/.e., 80% or greater) to the sequences set forth above, can be obtained by mutating the sequences during chemical synthesis, for example, followed by testing of the altered macrocyclic peptide for retained function (z.e., the functions set forth in (a) through (i) above) using the functional assays described herein. The biological and/or functional activity of the variant macrocyclic peptide amino acid sequences may be at least about lx, 2x, 3x, 4x, 5x, 6x,7x, 8x, 9x, or lOx more than the reference macrocyclic peptide on which the variant is based. In this context, the term "about" shall be construed to mean anywhere between 0. lx, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, or 0.9x more or less than the cited amount. As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (z.e., % homology = # of identical positions / total # of positions, times. 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the algorithm of Meyers E. et al., (Comput. Appl. Biosci.. AA - 1 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman et al. (J. Mol. Biol., 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG® software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Macrocyclic Peptides with Conservative Modifications

In yet another embodiment, a macrocyclic peptide of the disclosure comprises amino acid sequences that are homologous to the amino acid sequences of the macrocyclic peptides described herein, and wherein the macrocyclic peptides retain the desired functional and/or biological properties of the macrocyclic peptide of the disclosure.

For example, the disclosure provides a macrocyclic peptide, or antigen-binding portion thereof, comprising: an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the macrocyclic peptides described herein, wherein one or more amino acids have been substituted with a conservative amino acid; and the macrocyclic peptide exhibits one or more of the following properties:

(a) the macrocyclic peptide binds to human LAG-3 with an IC50 of 200 nM or less; (b) the macrocyclic peptide does not substantially bind to human CD4;

(c) the macrocyclic peptide binds to human LAG-3 and one or more of the following: cynomolgus monkey LAG-3, and/or mouse LAG-3;

(d) the macrocyclic peptide inhibits the binding of LAG-3 to MHC Class II molecules;

(e) the macrocyclic peptide inhibits tumor cell growth in a cellular assay and/or in vivo assay;

As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the macrocyclic peptide containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as substitution of peptide amidites during chemical synthesis, site- directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, try ptophan, histidine). Thus, one or more amino acid residues within the antigen binding regions of macrocyclic peptides of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (a) thru (i) above) using the functional assays described herein. Conservative amino acid substitutions may also be selected from one or more non-naturally occurring amino acids disclosed herein.

Peptide Synthesis

The description of the present disclosure herein should be construed in congruity with the laws and principals of chemical bonding. It should be understood that the compounds encompassed by the present disclosure are those that are suitably stable for use as pharmaceutical agent. One of skill in the art will know what compounds would and would not be stable based on the general principles of chemical bonding and stability⁷.

The macrocyclic peptides of the present disclosure can be produced by methods known in the art, such as they can be synthesized chemically, recombinantly in a cell free system, recombinantly within a cell or can be isolated from a biological source. Chemical synthesis of a macrocyclic peptide of the present disclosure can be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semisynthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. A preferred method to synthesize the macrocyclic peptides and analogs thereof described herein is chemical synthesis using various solid-phase techniques such as those described in Chan, W.C. et al, eds., Fmoc Solid Phase Synthesis, Oxford University⁷ Press, Oxford (2000); Barany, G. et al, The Peptides: Analysis, Synthesis, Biology, Vol. 2 : "Special Methods in Peptide Synthesis, Part A", pp. 3-284, Gross, E. et al, eds., Academic Press, New York (1980); in Atherton, E.. Sheppard, R. C. Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, England (1989); and in Stewart, J. M. Young, J. D. Solid-Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co., Rockford, IL (1984). The preferred strategy is based on the (9-fluorenylmethyloxy carbonyl) group (Fmoc) for temporary protection of the a-amino group, in combination with the tert-butyl group (tBu) for temporary protection of the amino acid side chains (see for example Atherton, E. et al, "The Fluorenylmethoxycarbonyl Amino Protecting Group", in The Peptides: Analysis, Synthesis, Biology. Vol. 9 : "Special Methods in Peptide Synthesis. Part C", pp. 1-38, Undenfriend, S. et al, eds., Academic Press, San Diego (1987).

The peptides can be synthesized in a stepwise manner on an insoluble polymer support (also referred to as "resin") starting from the C-terminus of the peptide. A synthesis is begun by appending the C-terminal amino acid of the peptide to the resin through formation of an amide or ester linkage. This allows the eventual release of the resulting peptide as a C-terminal amide or carboxylic acid, respectively.

The C-terminal amino acid and all other amino acids used in the synthesis are required to have their a-amino groups and side chain functionalities (if present) differentially protected such that the a-amino protecting group may be selectively removed during the synthesis. The coupling of an amino acid is performed by activation of its carboxyl group as an active ester and reaction thereof with the unblocked a-amino group of the N-terminal amino acid appended to the resin. The sequence of a-amino group deprotection and coupling is repeated until the entire peptide sequence is assembled. The peptide is then released from the resin with concomitant deprotection of the side chain functionalities, usually in the presence of appropriate scavengers to limit side reactions. The resulting peptide is finally purified by reverse phase HPLC.

The synthesis of the peptidyl-resins required as precursors to the final peptides utilizes commercially available cross-linked polystyrene polymer resins (Novabiochem, San Diego. CA; Applied Biosystems, Foster City. CA). Preferred solid supports are: 4- (2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methyl benzhydrylamine resin (Rink amide MBHA resin); 9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin); 4-(9-Fmoc)aminomethyl-3,5- dimethoxyphenoxy)valerylaminomethyl-Merrifield resin (PAL resin), for C-terminal carboxamides. Coupling of first and subsequent amino acids can be accomplished using HOBt, 6-Cl-HOBt or HO At active esters produced from DIC/HOBt, HBTU/HOBt, BOP, PyBOP, or from DIC/6-Cl-HOBt, HCTU, DIC/HOAt or HATU, respectively. Preferred solid supports are: 2-chlorotrityl chloride resin and 9-Fmoc-amino-xanthen-3-yloxy- Merrifield resin (Sieber amide resin) for protected peptide fragments. Loading of the first amino acid onto the 2-chlorotrityl chloride resin is best achieved by reacting the Fmoc- protected amino acid with the resin in dichloromethane and DIEA. If necessary', a small amount of DMF may be added to solubilize the amino acid.

The syntheses of the peptide analogs described herein can be carried out by using a single or multi-channel peptide synthesizer, such as an CEM Liberty Microwave synthesizer, or a Protein Technologies, Inc. Prelude (6 channels) or Symphony (12 channels) or Symphony X (24 channels) synthesizer.

Useful Fmoc amino acids derivatives are shown below.

Examples of Orthogonally Protected Amino Acids used in Solid Phase Synthesis

The peptidyl-resin precursors for their respective peptides may be cleaved and deprotected using any standard procedure (see, for example. King, D.S. et al, Int. J. Peptide Protein Res., 36:255-266 (1990)). A desired method is the use of TFA in the presence of water, TIS as scavenger, and DTT or TCEP as the disulfide reducing agent. Typically, the peptidyl-resin is stirred in TFA/water/TIS/DTT (94:3:3: 1). v:v:v:w; 1 mL/100 mg of peptidyl resin) for 1.5-3 hrs at room temperature. The spent resin is then filtered off and the TFA solution was cooled and Et2O solution was added. The precipitates were collected by centrifuging and decanting the ether layer (3 x). The resulting crude peptide is either redissolved directly into DMF or DMSO or CH3CN/H2O for purification by preparative HPLC or used directly in the next step.

Peptides with the desired purity can be obtained by purification using preparative HPLC, for example, on a Waters Model 4000 or a Shimadzu Model LC-8A liquid chromatography. The solution of crude peptide is injected into a YMC S5 ODS (20 x 100 mm) column and eluted with a linear gradient of MeCN in water, both buffered with 0.1% TFA. using a flow rate of 14-20 mL/min with effluent monitoring by UV absorbance at 220 nm. The structures of the purified peptides can be confirmed by electro-spray MS analysis.

Analytical Data:

Mass Spectrometry: ^L‘ESI-MS(+)” signifies electrospray ionization mass spectrometry performed in positive ion mode; “ESI-MS(-)” signifies electrospray ionization mass spectrometry performed in negative ion mode; “ESI-HRMS(+)” signifies high-resolution electrospray ionization mass spectrometry' performed in positive ion mode; “ESI-HRMS(-)'’ signifies high-resolution electrospray ionization mass spectrometry performed in negative ion mode. The detected masses are reported following the “nCz” unit designation. Compounds with exact masses greater than 1000 were often detected as double-charged or triple-charged ions.

The crude material was purified via preparative LC/MS. Fractions containing the desired product were combined and dried via centrifugal evaporation.

Analytical LC/MS Condition A: Column: Waters Acquity UPLC BEH Cl 8. 2.1 x 50 mm,

1.7-pm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate;

Temperature: 50 °C; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analytical LC/MS Condition B: Column: Waters Acquity UPLC BEH Cl 8, 2.1 x 50 mm,

1.7-pm particles; Mobile Phase A: 5:95 acetonitrile:water with 0. 1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50 °C; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analytical LC/MS Condition C: Column: Waters Acquity UPLC BEH C18, 2. 1 x 50 mm,

1.7-pm particles; Mobile Phase A: 5:95 acetonitrile: water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate;

Temperature: 70 °C; Gradient: 0-100% B over 3 minutes, then a 2.0-minute hold at 100% B; Flow: 0.75 mL/min; Detection: UV at 220 nm.

Analytical LC/MS Condition D: Column: Waters Acquity' UPLC BEH Cl 8, 2.1 x 50 mm,

1.7-pm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 70 °C; Gradient: 0-100% B over 3 minutes, then a 2.0-minute hold at 100% B; Flow: 0.75 mL/min; Detection: UV at 220 nm.

Analytical LC/MS Condition E: Column: Kinetex XB C18, 3.0 x 75 mm, 2.6-pm particles; Mobile Phase A: 10 mM ammonium formate in water: acetonitrile (98:2); Mobile Phase B: 10 mM ammonium formate in Water: acetonitrile (02:98); Gradient: 20- 100% B over 4 minutes, then a 0.6-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 254 nm.

Analytical LC/MS Condition F: Column: Ascentis Express C18, 2.1 x 50 mm, 2.7-pm particles; Mobile Phase AMO mM ammonium acetate in water: acetonitrile (95:5); Mobile Phase B: 10 mM ammonium acetate in Water: acetonitrile (05:95), Temperature: 50 °C; Gradient: 0-100% B over 3 minutes; Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analytical LC/MS Condition G: Column: X Bridge C18, 4.6 x 50 mm, 5-[im particles; Mobile Phase A: 0.1% TFA in water; Mobile Phase B: acetonitrile, Temperature: 35 °C; Gradient: 5-95% B over 4 minutes; Flow: 4.0 mL/min; Detection: UV at 220 nm.

[0001] The following abbreviations are employed in the Examples and elsewhere herein:

Ph = phenyl Bn = benzyl i-Bu = iso-butyl i-Pr = iso-propyl Me = methyl Et = ethyl Pr = n-propyl Bu = n-butyl t-Bu = /c/7-but l Trt = trityl TMS = trimethylsilyl TIS =triisopropylsilane Et20 = diethyl ether

HOAc or AcOH = acetic acid MeCN or AcCN = acetonitrile

DMF = N,N-dimethylformamide

EtOAc = ethyl acetate

THF = tetrahydrofuran

TFA = trifluoroacetic acid

TFE = a,a,a-trifluoroethanol

Et2NH = diethylamine

NMM = 4-methylmorpholine

NMP = N-methylpyrrolidone

DCM = dichloromethane

TEA = triethylamine min. = minute(s) h or hr = hour(s)

L = liter mL or ml = milliliter pL = microliter g = gram(s) mg = milligram(s) mol = mole(s) mmol = millimole(s) meq = milliequivalent rt or RT = room temperature sat or sat'd = saturated aq. = aqueous mp = melting point

BOP reagent = benzotriazol-l-yloxy-tris-dimethylamino-phosphonium hexafluorophosphate (Castro's reagent)

PyBOP reagent = benzotriazol-l-yloxy-tripyrrolidino phosphonium hexafluorophosphate HBTU = 2-(lH-Benzotriazol-l-yl)-EE3,3-tetramethyluronim hexafluorophosphate HATU = O-(7-Azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronim hexafluorophosphate

HCTU = 2-(6-Chl oro-1 -H-benzotriazol- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate T3P = 2,4,6-tripropyl-l,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide

DMAP = 4-(dimethylamino)pyridine

DIEA = diisopropylethylamine

Fmoc or FMOC = fluorenylmethyloxycarbonyl

Boc or BOC = /e/V-butyloxycarbonyl

HOBT or HOBT*H2O = 1 -hydroxy benzotriazole hydrate

Cl-HOBt = 6-Chloro-benzotriazole

HO AT = l-hydroxy-7-azabenzotriazole

HPLC = high performance liquid chromatography

LC/MS = high performance liquid chromatography /mass spectrometry MS or Mass Spec = mass spectrometry

NMR = nuclear magnetic resonance

Sc or SC or SQ = sub-cutaneous IP or ip = intra-peritoneal

General Procedures:

All manipulations were performed under automation on a Prelude Prelude, or a Symphony, or Symphony X peptide synthesizer (Protein Technologies). All procedures were performed according to the published methods (e.g.. WO 2023/225661).

Prelude: Resin-swelling procedure. Single-coupling procedure, Single-coupling extended time procedure. Chloroacetic Anhydride coupling, Single-Coupling Manned Addition Procedure A, Single-Coupling Manual Addition Procedure B, Manual removal of Fmoc group procedure:

Symphony : Resin-swelling procedure. Single-coupling procedure, Single-coupling extended time procedure, Double-coupling extended time procedure, Chloroacetic Anhydride coupling:

Symphony X: Resin-swelling procedure. Single-coupling procedure, Single-coupling 3 deprotections procedure. Single-coupling extended time procedure. Single-coupling 3 deprotections extended time procedure. Pre-activated single-coupling procedure, SingleCoupling Manual Addition Procedure A, Single-Coupling Manual Addition Procedure B:

Chloroacetic Anhydride coupling, Final rinse and dry procedure. The following procedures were performed according to the published methods (e.g., WO 2023/225661).

Global Deprotection Method. Cyclization Method, N -Methylation on-Resin Method A. N- Methylation On-resin Method B (Turner, R.A. et al, Org. Lett., 15(19):5012-5015 (2013)), N-Alkylation On-resin Procedure Method A, N-Alkylation On-resin Procedure

Method B, N-Nosylate Formation Procedure, N-Nosylate Removal Procedure, General Procedure for Preloading amines on the PL-FMP resin. General Procedure for Preloading Fmoc-Amino Acids on Cl-trityl resin. Click Reaction On-Resin Method A. Click Reaction On-Resin Method B, Suzuki Reaction On-resin Procedure. Fatty acid chain coupling procedure A, Fatty acid chain coupling procedure B, General Purification Procedures.

Unnatural Fmoc-Amino Acid Synthesis and fatty acid tails were prepared according to the published methods (e.g., WO 2023/225661).

To a 45-mL polypropylene solid-phase reaction vessel was added Sieber resin (141 mg, 0.1 mmol), and the reaction vessel was placed on the Prelude peptide synthesizer. The following procedures were then performed sequentially: "Prelude Method A: Resin-swelling procedure” was followed;

“Prelude Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH; “Prelude Method A: Single -coupling procedure” was followed with Fmoc-Cys(Trt)-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH;

“Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Asp( tBu)-OH; “Prelude Method A: Single -coupling procedure” was followed with Fmoc-Trp(Boc)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Bip-OH;

“Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH;

“Prelude Method A: Single -coupling procedure” was followed with Fmoc-Asp(tBu)-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Val-OH;

“Prelude Method A: Single-coupling procedure” was followed with Fmoc-Phe-OH; “Prelude Method A: Manual removal ofFmoc group procedure ”; “Chloroacetic acid coupling procedure A ” was followed;

“Global Deprotection Method A" was followed;

“Cyclization Method A ” was followed.

The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19 x 150 mm, 5-pm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10- mM ammonium acetate; Gradient: 10-50% B over 20 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 14.6 mg, and its estimated purity by LCMS analysis was 91%.

Analysis condition A: Retention time = 1.18 min; ESI-MS(+) m/z [M+2H]²⁺: 936.1. Analysis condition B: Retention time = 1.38 min; ESI-MS(+) m/z [M+2H]²⁺: 936.1.

To a 25-mL polypropylene solid-phase reaction vessel was added Sieber resin (70 mg, 0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially: “Symphony Method A: Resin-swelling procedure ’’ was followed;

“ Symphony Method A: Single-coupling procedure ’’ was followed with Fmoc-Gly-OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Cys(T rt)- OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Gly- OH: “Symphony Method A: Single-coupling procedure ” was followed with Fmoc- Tyr(tBu)-OH; “Symphony Method A: Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH, “ Symphony Method A: Single-coupling procedure” was followed with Fmoc-Trp(Boc)-OH;

“ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Bip-OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)- OH; “Symphony Method A: Single -coupling procedure ” was followed with Fmoc- Tyr(tBu)-OH: “Symphony Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH: “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “ Symphony Method A: Single-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Val-OH; “ Symphony Method A: Single-coupling followed by removal ofFmoc group procedure ” was followed with Fmoc-Phe-OH; “Chloroacetic acid coupling procedure A ” was followed: “Global Deprotection Method A ” was followed; “Cyclization Method A ” was followed. The crude material was purified via preparative LC/MS. The yield of the product was 18.2 mg, and its estimated purity by LCMS analysis was 96%.

Analysis condition A: Retention time = 1.14 min; ESI-MS(+) m/z [M+2H]²' : 976.6. Analysis condition B: Retention time = 1.54 min; ESI-MS(+) m/z [M+2H]²⁺: 976.3.

To a 45-mL polypropylene solid-phase reaction vessel was added Sieber resin (352 mg, 0.25 mmol), and the reaction vessel was placed on the Prelude peptide synthesizer. The following procedures were then performed sequentially: “Prelude Method A: Resin-swelling procedure” was followed “Prelude Method A: Single-coupling procedure " was followed with Fmoc-Gly-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc- Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Bip-OH; “Prelude Method A: Single -coupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc- Bip-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Arg(Pbf)-OH; “Prelude Method A: Double-coupling procedure” was followedwith Fmoc-Asp(tBu)- OH;

“Prelude Method A: Single-coupling procedure” was followedwith Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Val-OH; “Prelude Method A: Manual removal of Fmoc group procedure ” was followed; The resin was removed from the Prelude and dried under house vacuum. It was divided into Bio-Rad columns with each column containing about 0.05 mmol of the resin. “Manual -coupling procedure A ” was followed with (2S)-3-(4-chlorophenyl)-2-({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)propanoic acid on a 0.05 mmol scale using 2.5 equiv of the amino acid. “Chloroacetic acid coupling procedure A ” was followed; “Global Deprotection Method A ” was followed; “Cyclization Method A ’’ was followed.

The crude material was purified via preparative LC/MS. The yield of the product was 28.7 mg, and its estimated purity by LCMS analysis was 94%.

Analysis condition A: Retention time = 1.45 min; ESI-MS(+) m/z [M+2H]²⁺: 1021.1. Analysis condition B: Retention time = 1.51 min; ESI-MS(+) m/z [M+2H]²⁺: 1021.0.

The following examples were prepared, using Sieber resin following the general synthetic sequence described for the preparation of Example 1001.

To a 45-mL polypropylene solid-phase reaction vessel was added Sieber resin (352 mg, 0.25 mmol), and the reaction vessel was placed on the Prelude peptide synthesizer. The following procedures were then performed sequentially: “Prelude Method A: Resin-swelling procedure ” was followed;

“Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH;

“Prelude Method A: Single -coupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH;

“Prelude Method A: Single-coupling procedure ” was followed with Fmoc- Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Bip-OH; “Prelude Method A: Single -coupling procedure ” was followed with Fmoc-Tyr( tBu)-OH;

“Prelude Method A: Manual removal of Fmoc group procedure” was followed; The resin was removed from the Prelude and dried under house vacuum. It was divided into Bio-Rad columns with each column containing about 0.05 mmol of the resin. “Manual -coupling procedure A ” was followed with (2S)-3-(4-chlorophenyl)-2-({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)propanoic acid on a 0.05 mmol scale using 2.5 equiv of the amino acid. To a 25-mL polypropylene solid-phase reaction vessel was added the above resin (ca. 0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially: “Symphony Method A: Single-coupling procedure” was followed with Fmoc-Arg(Pbf)-OH; “ Symphony Method A: Double-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Val-OH; “ Symphony Method A: Single-coupling followed by removal of Fmoc group procedure ” was followed with Fmoc-Phe-OH;

“Chloroacetic acid coupling procedure A ” was followed;

“Global Deprotection Method A ” was followed; “Cyclization Method A ” was followed.

The crude material was purified via preparative LC/MS. The yield of the product was 17.1 mg, and its estimated purity by LCMS analysis was 97%.

Analysis condition A: RT= 1.36 min; [M+2H]²⁺: 983.3. Analysis condition B: RT= 1.47 min; [M+2H]²⁺: 983.0.

To a 45-mL polypropylene solid-phase reaction vessel was added 2-Chlorotrityl resin preloaded with Fmoc-Cys(Trt)-OH (758 mg, 0.25 mmol), and the reaction vessel was placed on the Prelude peptide synthesizer. The following procedures were then performed sequentially:

“Prelude Method A: Resin-swelling procedure” was followed:

“Prelude Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH;

“Prelude Method A: Single-coupling procedure” was followed with Fmoc-Cys(Trt)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH;

“Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Prelude Method A: Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH; “Prelude Method A: Single -coupling procedure” was followed with Fmoc-Trp(Boc)-OH; “Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Bip-OH;

“Prelude Method A: Single-coupling procedure ” was followed with Fmoc-Dap(Boc)- OH; “Prelude Method A: Manual removal ofFmoc group procedure ” was followed;

The resin was removed from the Prelude and derided under house vacuum. It was divided into Bio-Rad columns with each column containing about 0.05 mmol of the resin.

“Manual-coupling procedure A ” was followed with (2S)-3-(4-chlorophenyl)-2-( {[(9H- fluoren-9-yl)methoxyJcarbonyl}amino)propanoic acid on a 0.05 mmol scale using 2.5 equiv of the amino acid.

To a 25-mL polypropylene solid-phase reaction vessel was added the above resin (ca. 0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer.

The following procedures were then performed sequentially: “ Symphony Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH; “ Symphony Method A: Single -coupling procedure” was followed with Fmoc-Asp(tBu)-OH;

“Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)- OH; “ Symphony Method A: Single-coupling followed by removal ofFmoc group procedure ” was followed with Fmoc-Val-OH;

The resin was removed from the Symphony synthesizer and dried under house vacuum. The resin was transferred into a Bio-Rad column. “Manual-coupling procedure A ” was followed with (2S)-3-{4-[(tert-butoxy)carbonyl]phenyl}-2-({[ (9H-fluoren-9- yl)methoxy]carbonyl}amino)propanoic acid on a 0.05 mmol scale using 2.5 equiv of the amino acid. “Chloroacetic acid coupling procedure A ” was followed;

“Global Deprotection Method A ” was followed; “Cyclization Method A ” was followed. The crude material was purified via preparative LC/MS. The yield of the product was

16.7 mg, and its estimated purity by LCMS analysis was 94%. Analysis condition A: RT= 1.28 min; [M+2H]²⁺: 900.6. Analysis condition B: RT= 1.52 min; [M+2H]²⁺: 900.6. , , , , , , , , , , , , , acid (40 mg, 0.021 mmol) was stirred in DMF (0.5 mL) at rt. Acetic anhydride (4.21 mg, 0.021 mmol) was added. The reaction was stirred overnight.

The crude material was purified via preparative LC/MS. The yield of the product was 3.0 mg, and its estimated purity by LCMS analysis was 92%. Analysis condition A: RT= 1.16 2H]²⁺: 992.4.

To a 25-mL polypropylene solid-phase reaction vessel was added Sieber resin (70 mg, 0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially: “Symphony Method A: Resin-swelling procedure ” was followed;

“Symphony Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Cys(T rt)- OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Gly- OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc- Tyr(tBu)-OH; “Symphony Method A: Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH; “ Symphony Method A: Single-coupling procedure” was followed with Fmoc-Trp(Boc)-OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Bip-OH: “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-His(Trt)-OH; “ Symphony Method A: Single -coupling procedure” was followed with (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(naphthalen-2- yl)propanoic acid; “ Symphony Method A: Single-coupling procedure ” was followed with (2S.4R)-4-(tert-butoxy)-l-{[(9H-fluoren-9-yl)methoxy]carbonyl}pyrrohdme-2-carboxylic acid; “ Symphony Method A: Double-coupling procedure” was followed with Fmoc- Asp(tBu)-OH; “ Symphony Method A: Single-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “ Symphony Method A: Single-coupling procedure followed by removal ofFmoc group procedure ” was followed with Fmoc-Val-OH;

“Manual-coupling procedure A ” was followed with (2S)-3-{4-[(tert- butoxy)carbonyl ]phenyl}-2-( {[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanoic acid on a 0.05 mmol scale using 2.5 equiv of the amino acid.

“Chloroacetic acid coupling procedure A ” was followed: “Global Deprotection Method A ” was followed; “Cyclization Method A ” was followed.

The crude material was purified via preparative LC/MS. The yield of the product was 24.5 mg, and its estimated purity by LCMS analysis was 100%.

Analysis condition A: Retention time = 1.57 min; ESI-MS(+) m/z [M+2H]²⁺: 990.2. Analysis condition B: Retention time = 1.69 min; ESI-MS(+) m/z [M+2H]²⁺: 990.1.

2-Chorotrityl resin (Mesh 50-150, 1.54 meq/gram) (0.499 g, 1.523 mmol) was swollen in DCM (20 mL) for 5 minutes. A solution of 2-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)ethoxy)acetic acid (0.52 g, 1.523 mmol) in DCM (15 mL) was added to the resin followed by DIEA (0.82 mL, 4.57 mmol). The reaction was shaken at room temperature for 2 h. DIEA (0.5 mL) and Methanol (3 mL) were added, and the mixture was shaken for an additional 15 minutes. The resin was rinsed with DCM (4 x 25 mL), DMF (4 x 20 mL), DCM (4 x 25 mL). Ether (4 x 25 mL), and then dried under house-vacuum. A sample of resin (19.5 mg) was treated with 2 mL of 20% piperidine /DMF for 10 minutes with shaking. A 1 mL of the solution was transferred to a 25-mL volumetric flask and diluted with methanol to a total volume of 25 mL. A blank solution 1 mL of 20% piperidine /DMF was diluted up with methanol in a volumetric flask to 25 mL. The UV was set to 301 nm and zero with the blank solution followed by the reading of the solution, Absorbance = 2.2320 (2.2320/19.5 mg)*6.94 = 0.794.

To a 25-mL polypropylene solid-phase reaction vessel was added the above resin (63 mg, 0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially: “Symphony Method A: Resin-swelling procedure ” was followed;

“Symphony Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)- OH; “Symphony Method A: Single-coupling procedure " was followed with Fmoc- Asp(tBu)-OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH; ‘‘Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Bip-OH: “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr( tBu)-OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH: “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH;

“ Symphony Method A: Single-coupling procedure followed by removal ofFmoc group procedure ” was followed with Fmoc-Val-OH;

“Manual-coupling procedure A ” was followed with (2S)-3-{4-[(tert- butoxy)carbonyl ]phenyl}-2-( {[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanoic acid on a 0.05 mmol scale using 2.5 equiv of the amino acid.

20% piperidine in DMF (3 mL) was added and shaken for 10 min. The solvents were drained. The resin was washed with DMF (4 x) and then CH2CI2 (2 x). The resin under house vacuum (20 min). It was then cleaved using 5 mL of HFIP/CH2CI2 (1 :4) cocktail for 2 h. The solution was drained to a 20-mL vial with 0.5 mL of toluene. The mixture was concentrated to dry, and CH2CI2 was added and evaporated (2 x). The resulting colorless crystalline film, (11S,14S,17S,2OS,23S,26S,32S,35S,38S,41S)-2O-([1,T- bi phenyl] -4-ylmethy l)-41 -amino- 14.32-bis(2-(tert-butoxy )-2-oxoethy 1)- 11.23 ,26.35- tetrakis(4-(tert-butoxy)benzyl)-l 7-(( 1 -(tert-butoxycarbonyl)-lH-indol-3-yl)methyl)-42- (4-(tert-butoxycarbonyl)phenyl)-38-isopropyl-7,10,13,16,19,22,25,28,31,34,37,40- dodecaoxo-3-oxa-6,9,12,15,18,21,24,27,30,33,36,39-dodecaazadotetracontan-l-oic acid, was used directly in the next step. This film (60 mg. 0.026 mmol) was dissolved in DMSO (5 mL). HATU (19.76 mg, 0.052 mmol) was added followed by DIEA (0.011 mL, 0.065 mmol). The reaction was stirred at rt for 3 h. The solvents were removed via V-10 under very high boiling point setting. The resulting residue was treated with TFA/TIS/water/ (94:3:3) (2 mL) for 1 h at rt. The excess TFA was removed and the residue was dissolved in DMSO/DMF and submitted to purification.

The crude material was purified via preparative LC/MS. The yield of the product was 10.5 mg, and its estimated purity by LCMS analysis was 98%.

Analysis condition A: Retention time = 1.20 min; ESI-MS(+) m/z [M+2H]²⁺: 899.5. Analysis condition B: Retention time = 1.56 min; ESI-MS(+) m/z [M+2H]²⁺: 899.1.

To a 25-mL polypropylene solid-phase reaction vessel was added the PL-FMP resin preloaded with cyclopropyl amine (ca. 100 mg, 0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:

“ Symphony Method A: Resin-swelling procedure” was followed;

“Symphony Method A: Single-coupling procedure” was followed with Fmoc-Gly-OH; “Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)- OH; “ Symphony Method A: Single-coupling procedure " was followed with Fmoc- Asp(tBu)-OH; “Symphony Method A: Single-coupling procedure” was followed with Fmoc-Trp(Boc)-OH; “ Symphony Method A: Single-coupling procedure” was followed with Fmoc-Bip-OH; “ Symphony Method A: Single-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “ Symphony Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH; “Symphony Method A: Single -coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Method A: Single-coupling procedure ” was followedwith Fmoc-Tyr(tBu)-OH; “ Symphony Method A: Single-coupling procedure followed by removal of Fmoc group procedure ” was followed with Fmoc-Val- OH; “Manual-coupling procedure A ” was followed with (2S)-3-{4-[(tert- butoxy)carbonyl ]phenyl}-2-( {[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanoic acid on a 0.05 mmol scale using 2.5 equiv of the amino acid.

“Chloroacetic acid coupling procedure A ” was followed;

“Global Deprotection Method A ” was followed; “ Cyclization Method A” was followed. The crude material was purified via preparative LC/MS. The yield of the product was 4.6 mg, and its estimated purity by LCMS analysis was 92%.

Analysis condition A: Retention time = 1.20 min; ESI-MS(+) m/z [M+2H]²⁺: 977.4.

Analysis condition B: Retention time = 1.53 min; ESI-MS(+) m/z [M+H]⁺: 1953.7.

To a 45-mL polypropylene solid-phase reaction vessel was added Sieber resin (35 mg, 0.025 mmol), and the reaction vessel was placed on the Symphony X peptide synthesizer. The following procedures were then performed sequentially: “Symphony X Resin-swelling procedure” was followed;

“Symphony X Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OEI;

“Symphony X Single-coupling procedure " was followed with Fmoc-D-Ser(tBu)-OH;

“Symphony X Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Symphony X Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony X Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

“Symphony X Pre-Activated Single-coupling procedure ” was followed with for (2S)-3- [4-(2-ethyl-4-hydroxyphenyl)phenyl]-2-({[(9H-fluoren-9- yl)methoxy (carbonyl } amino)propanoic acid;

“Symphony X Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH;

“Symphony X Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

“Symphony X Single-coupling procedure ” was followed with Fmoc-Gly-OH;

“Symphony X Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony X Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony X Single-coupling procedure ” was followed with Fmoc-Val-OH;

“Symphony X Pre-Activated Single-coupling procedure ” was followed with for (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert- butoxycarbonyl)phenyl)propanoic acid; ‘‘Symphony X Chloroacetic Anhydride coupling procedure ” “Symphony X Final rinse and dry procedure ” “Global Deprotection Method A ” was followed; “Cyclization Method A ” was followed.

The crude material was purified via preparative LC/MS. The yield of the product was

19.7 mg, and its estimated purity by LCMS analysis was 100%.

Analysis condition A: Retention time = 1.09 min; ESI-MS(+) m/z [M+2H]²⁺: 953.3.

Analysis condition B: Retention time = 1.44 min; ESI-MS(-) m/z [M-2H]²': 951.1.

Example 1535 was prepared, using Sieber resin on a 0.1 mmol scale, placed on a CEM microwave peptide synthesizer. The following procedures were then performed sequentially:

“CEM Method A: Resin-swelling procedure” was followed;

“CEM Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH;

“CEM Method A: Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH;

“CEM Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH;

“CEM Method A: Custom amino acids-coupling procedure ” was followed with Fmoc-N- Me-Tyr(tBu)-OH; “CEM Method A: After secondary amine coupling procedure” was followed with Fmoc-Asp(tBu)-OH; “CEM Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “CEM Method A: Custom amino acids-coupling procedure” was followed with Fmoc-Bip-OH; “CEM Method A: Single-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “CEM Method A: Custom amino acids-coupling procedure ” was followed with Fmoc- Bip-OH; “CEM Method A: Doublecoupling procedure ” was followed with Fmoc-Arg(Pbf)-OH; “CEM Method A: Singlecoupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “CEM Method A: Single coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “CEM Method A: Singlecoupling procedure ” was followed with Fmoc-Val-OH; ‘‘CEM Method A: Doublecoupling procedure ” was followed with Fmoc-Phe-OFT'Chloroacetic acid coupling procedure A ” was followed; “Global Deprotection Method A ” was followed; “Cyclization Method A ” was followed.

The crude material was purified via preparative LC/MS. The yield of the product was 55.3 mg. and its estimated purity by LCMS analysis was 94%.

Analysis condition A: Retention time = 1.45 min; ESI-MS(+) m/z [M+2H]²⁺: 1010.8. Analysis condition B: Retention time = 1.50 min; ESI-MS(+) m/z [M+2H]²⁺: 1011.3.

To a 25 mL polypropylene solid-phase reaction vessel was added Sieber resin (70 mg. 0.050 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:

“Symphony Method B: Resin-swelling procedure'’ was followed;

“Symphony Method B: Double-coupling procedure” was followed with Fmoc-Gly-OH; “Symphony Method B: Double-coupling procedure” was followed with Fmoc-Cys(Trt)- OH; “Symphony Method B: Double-coupling procedure” was followed with Fmoc-Gly- OH; “Symphony Method B: Double-coupling procedure” was followed with Fmoc- Tyr(tBu)-OH;“Symphony Method B: Double-coupling procedure” was followed with Fmoc-Asp(tBu)-OH;“Symphony Method B: Double-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH;“Symphony Method B: Double-coupling procedure” was followed with Fmoc-Bip-OH;“Symphony Method B: Double-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH;“Symphony Method B: Double-coupling procedure” was followed with Fmoc-Bip-OH;“Symphony Method B: Double-coupling procedure’' was followed with Fmoc-Arg(Pbf)-OH;“Symphony Method B: Double-coupling procedure” was followed with Fmoc-Asp(tBu)-OH;“Symphony Method B: Doublecoupling procedure” was followed with Fmoc-Tyr(tBu)-OH;

“Symphony Method B: Double-coupling procedure"’ was followed with Fmoc-Val-OH; “Symphony Method B: Double-coupling procedure” was followed with Fmoc-Phe-OH; “Chloroacetic acid coupling procedure B” was followed;“Deprotection Method B” was followed;“Cyclization Method B” was followed.

The crude material was purified via preparative LC/MS.The yield of the product was 7.5 mg, and its estimated purity by LCMS analysis was 95%.

Analysis condition A: Retention time = 1.48 min; ESI-MS(+) m/z [M+2H]²⁺: 1003.9. Analysis condition B: Retention time = 1.61 min; ESI-MS(+) m/z [M+2H]²⁺: 1004.7.

Example 2022 was prepared, using Sieber resin on a 0.05 mmol scale. The crude material was purified via preparative LC/MS. The yield of the product was 10.0 mg, and its estimated purity by LCMS analysis was 90%.

Analysis condition A: Retention time = 1.41 min; ESI-MS(+) m/z [M+2H]²⁺: 1000.4. Analysis condition B: Retention time = 1.37 min; ESI-MS(+) m/z [M+2H]²⁺: 1000.2. Example 2079 was prepared, using Sieber resin on a 0.10 mmol scale, placed on a CEM microwave peptide synthesizer. The following procedures were then performed sequentially:

“CEM Method A: Resin-swelling procedure” was followed;

“CEM Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH;

“CEM Method A: Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH;

“CEM Method A: Single-coupling procedure ” was followed with Fmoc-Gly-OH;

“CEM Method A: Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “CEM Method A: Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “CEM Method A: Single-coupling procedure ” was followed with Fmoc- Tyr(tBu)-OH; “CEM Method A: Custom amino acids -coupling procedure ” was followed with Fmoc- Bip-OH; “CEM Method A: Single-coupling procedure” was followed with Fmoc- Tyr(tBu)-OH; “CEM Method A: Custom amino acids-coupling procedure” was followed with Fmoc- Bip-OH; “CEM Method A: Double-coupling procedure ” was followed with Fmoc-Arg(Pbf)-OH; “CEM Method A: Single-coupling procedure ” was followed with Fmoc-Asp(lBu)-OH. “CEM Method A: Single-coupling procedure” was followedwith Fmoc-Tyr(tBu)-OH; “CEM Method A: Single-coupling procedure” was followedwith Fmoc-NmeVal-OH; “CEM Method A: After secondary amine coupling procedure” was followed with Fmoc-Phe-OH, “Chloroacetic acid coupling procedure B ” was followed;

“Deprotection Method B ” was followed; “Cyclization Method B ” was followed.

The crude material was purified via preparative LC/MS. The yield of the product was 2.5 mg, and its estimated purity by LCMS analysis was 94%.

Analysis condition A: Retention time = 1.44 min; ESI-MS(+) m/z [M+2H]²⁺: 1010.8. Analysis condition B: Retention time = 1.49 min; ESI-MS(+) m/z [M+2H]²⁺: 1010.8.

The following examples were prepared, using Sieber resin following the general synthetic sequence described for the preparation of the above Examples.

- Ill -

The following examples 2002-2022 were prepared following the general procedures described for Example 2001.

The following examples 2040-2046 were prepared following the general procedures

Method

LAG-3 cell binding assay: Human Raji cells expressing endogenous MHC Class II molecules were used for binding to either human LAG-3-mFc, mouse LAG-3, or cyno LAG-3-hFc proteins. Briefly Raji cells were plated in a 384-well plate (Coming 354663) at a density of 8000 cells/well. After 2 hour incubation at a 37 °C and 5% CO2 incubator, LAG-3 antigen (hLAG-3 -mFc, mLAG-3-mFc, or cLAG-3-hFc) were added to all wells at a final concentration of 0.088, 0.25, or 0.072 pg/ml and incubated for 30 minutes. Then, a corresponding detection antibody (R-Phycoerythrin conjugated anti-Mouse IgG, or anti -human IgG), Jackson Immuno Research Lab, PA) was added. The binding affinity of the LAG-3 antigen was quantified by reading the plate on a NXT High Content Reader (ThermoFisher). To assess the potency of LAG-3 compounds to block the binding of LAG-3 antigen to the MHCII molecules expressed on the Raji cell surface, compounds were serially diluted and added to the Raji cells prior to the addition of an appropriate LAG3 antigen.

A: IC50 < 0.005 pM, B: 0.005 pM <= IC50 < 0.05 pM; C: 0.05 pM <= IC50 < 0.1 pM.

Claims

CLAIMS We claim:

1. The compound selected from the compounds exemplified in the specification, or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 wherein the compound shows activity' less than or equal to 0. 1 pM in the LAG-3 cell binding assay.

3. A compound or a pharmaceutically acceptable salt thereof according to claim 2 showing activity at less than or equal to 1.0 nM in the LAG-3 cell binding assay in the following tables:

Table 1

Table 2

Table 3

Table 4 Table 5

Table 6

4. A pharmaceutical composition comprising one or more compounds 5 according to claim 1 in a pharmaceutically acceptable carrier.

5. A pharmaceutical composition comprising one or more compounds according to claim 2 in a pharmaceutically acceptable carrier.

6. A pharmaceutical composition comprising one or more compounds according to claim 3 in a pharmaceutically acceptable carrier.

10