Macrocyclic peptides useful as immunomodulators

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 5 diseases.

Description

[0001] MACROCYCLIC PEPTIDES USEFUL AS IMMUNOMODULATORS

[0002] CROSS REFERENCE

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 563,622 filed March 11, 2024 which is incorporated herein in its entirety.

[0004] BACKGROUND

[0005] 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.

[0006] Lymphocyte activation gene-3 (LAG-3; LAG3; CD223) is a type 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 histocompatibility complex (MHC) class II, for their functional activity. 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 Il and its function is unknown.

[0007] 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).

[0008] 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.

[0009] 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.

[0010] 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)7T 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).

[0011] 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.

[0012] 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.

[0013] Additional background information can be found in WO2015 / 042246 Al, WP2015 / 116539 Al, and W02014 / 008218 AL

[0014] LAG-3 blockade with macrocylic peptide inhibitors, alone and in combination with standard of care (e.g., nivolumab. imatinib, lenalidomide) or with other checkpoint inhibitors deserves further exploration.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] In its first embodiment the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0019] A is selected from a bond, wherein: denotes the point of attachment to the carbonyl group and r denotes the point of attachment to the nitrogen atom; n is 0 or 1;

[0020] R14and R15are independently selected from hydrogen and methyl;

[0021] R16is selected from OH, NR17R18, wherein R17and R18can 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,

[0022] -CHR19C(O)NR17R18, -CHR19C(O)OH,

[0023] -CHR19C(O)NHCHR20C(O)NR17R18, -CHR19C(O)NHCHR20C(O)OH,

[0024] -CHR19C(O)NHCHR20C(O)NHCHR21C(O)NR17R18,

[0025] -CHR19C(O)NHCHR20C(O)NHCHR21C(O)OH;

[0026] -CHR19C(O)NHCHR20C(O)NHCHR21C(O)NHCHR22C(O)NR17R18,

[0027] -CHR19C(O)NHCHR20C(O)NHCHR21C(O)NHCHR22C(O)OH; wherein R19is selected from hydrogen and sidechain of a natural or unnatural amino acid and wherein R20is selected from hydrogen and sidechain of a natural or unnatural amino acid and wherein R21is selected from hydrogen and sidechain of a natural or unnatural amino acid and wherein R22is 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

[0028] X is bond, S, O, CHR23, NR23wherein R23is hydrogen or 1-3 alkyl;

[0029] R'. Rb, Rd, Re, Rk, and R1, are each independently selected from hydrogen or methyl;

[0030] R1, R2, R1, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13are 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; Rfis hydrogen, or methyl, or Rfand R6together with the atoms to which they are attached can form a ring selected from either the D enatiomer or the L enantiomer of azetidine, pyrollidine, 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;

[0031] Rgis hydrogen, or methyl, or Rgand R7, together with the atoms to which they are attached, can form a ring selected from azetidine, pyrollidine. 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;

[0032] R1is hydrogen, or 1-6 akyl, or (CH2)I-2COOH, or R1and R9, together with the atoms to which they are attached, can form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, hydroxy, and phenyl or fused with another aromatic ring optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, hydroxy, and phenyl; and wherein the pyrrolidine and the piperidine ring are optionally fused to a cyclohexyl, phenyl, or indole group;

[0033] Rmis hydrogen or 1-6 alkyl or (CH2)I-2COOH or Rmand R13, together with the atoms to which they are attached, can form a ring selected from D and L enantiomers of azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, benzyl optionally substituted with a halo group, benzyloxy, cyano, cyclohexyl, methyl, halo, hydroxy, isoquinolinyloxy optionally substituted with a methoxy group, quinolinyloxy optionally substituted with a halo group, and tetrazolyl; and wherein the pyrrolidine and the piperidine ring are optionally fused to a cyclohexyl, phenyl, or indole group; and

[0034] In another embodiment the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein A is n = 0, 1,

[0035] X is bond, S, O, CHR23, NR23wherein R23is 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;

[0036] X = S, CH2, NH; R3is the side chain of L-Asp:

[0037] Rais hydrogen;

[0038] R1is hydrogen;

[0039] In another embodiment the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein A is

[0040] X = S, CH2, and NH;

[0041] In another embodiment the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein A is

[0042] X = S, CH2, and NH;

[0043] When Reis hydrogen, R5is the sidechain of D-Ala; when Reis methyl, R5groups are selected from the sidechains of Phe, Phe(2-F), Phe(4-F). Trp, Tyr. Tyr(CH2phenyl), and D-Phe;

[0044] When Rris hydrogen, R6is the sidechain of D-Ala, D-Asp, D-Asn, D-Glu, D-Gln, D-Leu, D-Trp, D-Tyr or Lys.

[0045] When Rfis methyl, R6is the sidechain of Gly, D-Ala, D-Leu, Ala or Leu, or alternatively, Rfand R6together with the atoms to which they are attached can form a ring selected from the D enatiomer of azetidine, pyrollidine, morpholine, and piperidine; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy, and wherein each ring can be fused with a six-membered aromatic or heteroaromatic ring;

[0046] R10groups are selected from the sidechains of Bzt, Trp, Trp(7-F), Trp(7-F), and Trp (1 -Me);

[0047] R12groups are selected from the sidechains of Bzt, Phe, Trp, and Tyr.

[0048] 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 macrocyclic 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] In compounds of formula (I), preferred R1groups are side chains of the following amino acids: phenylalanine, tyrosine, tryptophan, leucine, 2-fluorophenylalanine, 3- fluorophenyl alanine, 4-fluorophenylalanine. 3,4-difluorophenylalanine. 3,5- difluorophenylalanine, 3,4,5-difluorophenylalanine, pentafluorophenylalanine, 3- methylphenylalanine, 4-methylphenylalanine, 4-chlorophenylalanine, 3- methoxyphenylalananie, 4-methoxyphenylalanine, 1 -naphthylalanine, 2-naphthylalanine, 4-cyanophenylalanine, 4-difluoromethylphenylalanine, biphenylalanine, 3-(2-thienyl)- alanine, 3-(3-thienyl)-alanine, 3-benzothienylalanine (Bzt), 4-pyridinylalanine. 4- bromophenylalanine, 3-pyridinylalanine, 4-trifluoromethylphenylalanine, 4- benzoylphenylalanine, 3 -chlorophenyl alanine, 4-aminophenylalanine, 4- aminomethylphenylalanine, 4-carbamoylphenylalanine, 4-carboxyphenylalaine, 3-(4- thiazolylj-alanine, 4-acetamide-phenylalanine, 3-cyclohexylalanine, 2-pentylglycine. 0- phenyl-phenylalanine, 4-phenoxyphenylalanine, 4-cyclohexyloxyphenylalanine, and histidine;.

[0054] In compounds of formula (I) where R2is not part of a ring, preferred R2groups are side chains of the following amino acids: phenylalanine, tyrosine, tryptophan, 2- fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, 2- methylphenylalanine, 3-methylphenylalanine, 4-methylphenylalanine, 3- chlorophenylalanine. 4-chlorophenylalanine, 3-methoxyphenylalananie, 4- methoxyphenylalanine, 2-naphthylalanine, 3-cyanophenylalanine, 4-cyanophenylalanine, 4-difluoromethylphenylalanine, biphenylalanine, 3-(2-thienyl)-alanine, 3-(3-thienyl)- alanine, 3-benzothienylalanine, 2-pyridinylalanine, 3-pyridinylalanine, 4- pyridinylalanine, 4-bromophenylalanine, 4-trifluoromethylphenylalanine. 4- benzoylphenylalanine. 4-aminophenylalanine. 3-aminomethylphenylalanine, 4- aminomethylphenylalanine, 4-carbamoylphenylalanine, 4-carboxyphenylalaine, 3-(4- thiazolyl)-alanine, 4-acetamide-phenylalanine, 4-(prop-2-yn-l-yloxy)phenylalanine, 3- hydroxylphenylalanine, 3-carboxyphenylalanine, 4-benzoxyphenylalanine, 4- carboxymethoxyphenylalanine, 3-(6-(o-toly1)pyridinylalanine, 4-allyloxyphenylalanine, lysine, tert-butylglycine, valine, and glycine;.

[0055] In compounds of formula (I), preferred R3is side chain of aspartic acid.

[0056] In compounds of formula (I) where R4is not part of a ring, preferred R4groups are side chains of alanine, aspartic acid, histidine, asparagine, glutamic acid, homoglutamic acid, 2,3-diaminopropionic acid (Dap), phenylalanine. 4-carboxyphenylalamine, valine, tyrosine, glutamine, arginine, serine, glycine, 2-amino-4-aminobutyric acid (Dab), ornithine, threonine, lysine, lysine (COCH3), propargylglycine, 4-(prop-2-yn-l- yloxy)phenylalanine, 4-carboxymethoxyphenylalanine, tryptophan, tryptophan (1 -acetic aci d). and 4-pyridinylalanine.

[0057] In compounds of formula (I), where R5is not part of a ring, preferred R5groups are side chains of D-alanine. tryptophan, tyrosine, phenylalanine. 2-F-phenylalanine. 3-F- phenylalanine, 4-F-phenylalanine, leucine, 4-benzyltyrosine. 3-methoxyphenylalanine, threonine, and allo-threonine.

[0058] In compounds of formula (I), where R6is not part of a ring, preferred R6groups are side chains are: glycine, alanine, leucine, aspartic acid, asparagine, glutamic acid. glutamine, serine, lysine, tryptophan, tyrosine, phenylalanine, threonine, and allothreonine.

[0059] In compounds of formula (I) where R7is not part of a ring, preferred R7geoups are side chains of glycine, t-butylglycine, aspartic acid, asparagine, glutamic acid, 2,3- diaminopropionic acid (Dap), 2-amino-4-aminobutyric acid (Dab), ornithine, threonine, lysine, lysine (COCH3), serine, homo-serine, arginine, ornithine, histidine, glutamine, alanine, propargylglycine, 4-(prop-2-yn-l-yloxy)phenylalanine. tryptophan (1 -acetic acid), 4-trifluoromethylphenylalanine, 4-pyridinylphenylalanine, and 3- carboxy phenyl al anine.

[0060] In compounds of formula (I) preferred R8groups are side chains of glycine, aspartic acid, asparagine, glutamic acid, 2,3-diaminopropionic acid (Dap), 2-amino-4- aminobutyric acid (Dab), ornithine, threonine, lysine, serine, homo-serine, methyl-homo- serine, arginine, ornithine, histidine, glutamine, alanine, tryptophan (1 -acetic acid), 3- carboxyphenylalanine, 4-pyridinylphenylalanine, and 3-(2-tetrazolyl)alanine.

[0061] In compounds of formula (I) where R9is not part of a ring, preferred R9side chains are: serine, homo-serine, methyl-homo-serine, propargylglycine. 4-(prop-2-yn-l- yloxy)phenylalanine. lysine, glycine, alanine, tyrosine, arginine, threonine, glutamine, glutamic acid, valine, aspartic acid, and tyrosine.

[0062] In compounds of formula (I) preferred R10geoups are side chains of tryptophan, 7-methyltry ptophan, benzothienylalanine, 2-napththylalanine, 7-fluorotry ptophan, 7- methyltry ptophan, tryptophan (1 -acetic acid), 3-methylphenylalanine, and N- methyltryptophan.

[0063] In compounds of formula (I) where R11is not part of a ring, preferred R11groups are side chains of tryptophan, tyrosine, 4-(prop-2-yn-l-yloxy)phenylalanine, propargylglycine, 3-hydroxyphenylalanine, N-methyltryptophan, 7-methyltryptophan, 3- pyridinylalanine, phenylalanine, 4-carboxyphenylalanine, 4-benzoxyphenylalanine, 1- napththylalanine. 3-carboxyphenylalanine, 4-aminomethylphenylalanine. 4- methoxyphenylalanine, 5-cyanotryptophan. 2-mehylphenyl alanine, 2-methyltryptophan, 2-napththylalanine, 4-fluorophenylalanine, glutamine, arginine, valine, tert-butylglycine, glycine, lysine, 1-acetic acidtryptophan. 3-methylphenylalanine, biphenylalanine, biphenylalanine, 3-benzothienylalanine, 4-(4-pyridinyl)phenylalanine, 4'-carboxy-4- biphenylalanine, 3 '-carboxy-4-bi phenylalanine, 3-quinolinylalanine, 6-quinolinylalanine, 6-isoquinolinylalanine and isotryptophan.

[0064] In compounds of formula (I) preferred R12groups are side chains of tryptophan, tyrosine, phenylalanine, 7-fluorotryptophan, 2-methyltryptophan, 1 -napththylalanine, 2- naphthylalanine, 4-aminomethylphenylalanine, and 4-benzoxyphenylalanine.

[0065] In compounds of formula (I) where R13is not part of a ring, preferred R13groups are side chains of glycine, propargylglycine, alanine, aspartic acid, asparigine, arginine, glutamic acid, glutamine, noevaline. serine, lysine, threonine. 4-carboxyphenylalanine. 1- acetic acid-tryptophan, 3-carboxyphenylalanine, 4-carbamoylphenylalanine, tetrazolylalanine, methyl-homo-serine, 4-carboxymethoxy phenylalanine, 4- pyridinylal anine, 3-py ri dinylalanine. benzoxy phenyl alanine, 4-(prop-2-yn- 1 - yloxy)phenylalanine. 2,3-diaminopropionic acid (Dap). 2-amino-4-aminobutyric acid (Dab), cyclopropylalanine; and ethyl.

[0066] As shown below in Tables 1-10, the following compounds of the invention show activity at less than or equal to 0.25 nM in the assay included later in the application.

[0067] Table 1

[0068]

[0069] Table 3

[0070] Table 4

[0071] Table 5

[0072] Table 6

[0073] Table 7 Table 8

[0074] Table 9

[0075] HO

[0076] Table 10

[0077]

[0078] Definitions

[0079] The definitions provided herein apply, without limitation, to the terms as used throughout this specification, unless otherwise limited in specific instances.

[0080] 5 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: where R and R' are as discussed herein.

[0081] Unless otherwise indicated, the term "amino acid" as employed herein, alone or as 0 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.

[0082] 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, try ptophan, ty rosine, and valine) usually in the S -configuration (i.e., the L-amino acid).

[0083] 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).

[0084] The term "treating" refers to: (i) preventing a disease, disorder, or condition from occurring in a patient that may be predisposed to the disease, disorder, and / or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder, or condition, i.e., arresting its development; and (iii) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and / or condition and / or symptoms associated with the disease, disorder, and / or condition.

[0085] The "inhibitory 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 ICso value. It is understood that another designation for IC50 is the half-maximal inhibitory' concentration.

[0086] 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.

[0087] 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.

[0088] 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 modifying an immune response, treating diseases such as cancer or infectious disease, stimulating a protective autoimmune response or to stimulate antigen-specific immune responses (<?.g., by coadministration of lag03 blocking peptides with an antigen of interest).

[0089] 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.

[0090] 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.

[0091] 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.

[0092] The term "immune response" refers to the action of, for example, lymphocytes, 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.

[0093] 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.

[0094] 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..

[0095] 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 ICso 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.

[0096] 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.

[0097] 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, i. 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.

[0098] 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

[0099] 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.

[0100] 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:

[0101] (a) the macrocyclic peptide binds to human LAG-3 with an IC50 of 200 nM or less;

[0102] (b) the macrocyclic peptide does not substantially bind to human CD4;

[0103] (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;

[0104] (d) the macrocyclic peptide inhibits the binding of LAG-3 to MHC Class II moleucules;

[0105] (e) the macrocyclic peptide inhibits tumor cell growth in a cellular assay and / or in vivo assay; and / or

[0106] 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 (z.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.

[0107] 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.

[0108] The percent identity' between two amino acid sequences can be determined using the algorithm of Meyers E. et al. , (Comput. Appl. Biosci.. 4: 11-17 (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.

[0109] Macrocyclic Peptides with Conservative Modifications

[0110] 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.

[0111] 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:

[0112] (a) the macrocyclic peptide binds to human LAG-3 with an IC50 of 200 nM or less;

[0113] (b) the macrocyclic peptide does not substantially bind to human CD4;

[0114] (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;

[0115] (d) the macrocyclic peptide inhibits the binding of LAG-3 to MHC Class II moleucules;

[0116] (e) the macrocyclic peptide inhibits tumor cell growth in a cellular assay and / or in vivo assay; and / or

[0117] 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, tryptophan, 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.

[0118] Pharmaceutical Compositions

[0119] The disclosure further relates to the polypeptides described herein wherein the sequence comprises one or more amino acid deletions from either the C-terminus and / or the N-terminus.

[0120] In preferred embodiments, the following N-terminal Compound No. 99 deletion polypeptides are encompassed by the present disclosure: X1-X13, X2-X13. X3-X13. X4- X13, X5-X13, X6-X13, X7-X13, X8-X13, X9-X13, X10-X13, X11-X13, and / or X12- XI 3 of Compound No. 99, wherein each X is representative of an amino acid at the indicated position for each peptide as outlined herein. The present disclosure also encompasses cyclic forms of these deletion mutants using the linking chemistries described elsewhere herein.

[0121] In preferred embodiments, the following C-terminal Compound No. 99 deletion polypeptides are encompassed by the present disclosure: X1-X13, X1-X12, Xl-Xl l, XI- XI 0, X1-X9, X1-X8, X1-X7, X1-X6, X1-X5, X1-X4 and / or X1-X3 of Compound No. 99, wherein each X is representative of an amino acid at the indicated position for each peptide as outlined herein. The present disclosure also encompasses cyclic forms of these deletion mutants using the linking chemistries described elsewhere herein.

[0122] In preferred embodiments, the following N-terminal Compound No. 1 deletion polypeptides are encompassed by the present disclosure: X1-X15, X2-X15. X3-X15, X4- X15, X5-X15, X6-X15, X7-X15, X8-X15, X9-X15. XI0-XI5, XI 1-X15. and / or X12- XI 5 of Compound No. 1, wherein each 1 is representative of an amino acid at the indicated position for each peptide as outlined herein. The present disclosure also encompasses cyclic forms of these deletion mutants using the linking chemistries described elsewhere herein.

[0123] In preferred embodiments, the following C-terminal Compound No. 1 deletion polypeptides are encompassed by the present disclosure: X1-X15, X1-X14, X1-X13, XI- X12, Xl-Xl l, X1-X10, XI -X9, X1-X8, X1-X7, X1-X6, X1-X5, X1-X4 and / or X1-X3 of Compound No. 1, wherein each X is representative of an amino acid at the indicated position for each peptide as outlined herein. The present disclosure also encompasses cyclic forms of these deletion mutants using the linking chemistries described elsewhere herein.

[0124] In preferred embodiments, the following N-terminal Compound No. 71 deletion polypeptides are encompassed by the present disclosure: X1-X14, X2-X14, X3-X14, X4- X14, X5-X14, X6-X14, X7-X14, X8-X14, X9-X14. X10-X14, X11-X14. and / or X12- X14 of Compound No. 71, wherein each X is representative of an amino acid at the indicated position for each peptide as outlined herein. The present disclosure also encompasses cyclic forms of these deletion mutants using the linking chemistries described elsewhere herein.

[0125] In preferred embodiments, the following C-terminal Compound No. 71 deletion polypeptides are encompassed by the present disclosure: X1-X14, X1-X13, X1-X12, XI- XI 1, X1-X10, X1-X9, X1-X8, X1-X7, X1-X6, X1-X5, X1-X4 and / or X1-X3 of Compound No. 71, wherein each X is representative of an amino acid at the indicated position for each peptide as outlined herein. The present disclosure also encompasses cyclic forms of these deletion mutants using the linking chemistries described elsewhere herein.

[0126] In another aspect, the present disclosure provides a composition, e.g, a pharmaceutical composition, containing one or a combination of macrocyclic peptides, or antigen-binding portion(s) thereof, of the present disclosure, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g, two or more different) macrocyclic peptides, or immunoconjugates or bispecific molecules of the disclosure. For example, a pharmaceutical composition of the disclosure can comprise a combination of macrocyclic peptides (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or that have complementary activities.

[0127] Pharmaceutical compositions of the disclosure also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a macrocyclic peptide combined with at least one other antiinflammatory or immunosuppressant agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the macrocyclic peptides of the disclosure.

[0128] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g.. by injection or infusion). Depending on the route of administration, the active compound, i.e., a macrocyclic peptide, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0129] The pharmaceutical compounds of the disclosure may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M. et al., J. Pharm. Set., 66:1-19 (1977)). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenylsubstituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0130] A pharmaceutical composition of the disclosure also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oilsoluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA). sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0131] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0132] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0133] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0134] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0135] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety -nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

[0136] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary' dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0137] For administration of the macrocyclic peptide, the dosage ranges from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg, of the host body weight. For example dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg bodyweight, 5 mg / kg body weight or 10 mg / kg body weight or within the range of 1-10 mg / kg. An exemplary treatment regime entails administration once per day, bi-weekly, tri-weekly, weekly, once every two weeks, once every three weeks, once every four weeks, once a month, once every- 3 months or once every three to 6 months. Preferred dosage regimens for a macrocyclic peptide of the disclosure include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every- four weeks for six dosages, then every three months; (ii) every- three weeks; (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks.

[0138] In some methods, two or more macrocyclic peptides with different binding specificities are administered simultaneously, in which case the dosage of each compound administered falls within the ranges indicated. The compounds are usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of macrocyclic peptide to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1- 1000 .mu.g / ml and in some methods about 25-300 .mu.g / ml.

[0139] Alternatively, the macrocyclic peptide can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the macrocyclic peptide in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0140] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history7of the patient being treated, and like factors well known in the medical arts.

[0141] A "therapeutically effective dosage" of a macrocyclic peptide of the disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of tumors, a "therapeutically effective dosage" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit tumor grow th and / or HIV can be evaluated in an animal model system predictive of efficacy in human tumors or viral efficacy. Alternatively, this property of a composition can be evaluated by examining the ability7of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, decrease viral load, or otherw ise ameliorate symptoms in a subject. One of ordinary7skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. A composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety- of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Preferred routes of administration for macrocyclic peptides of the disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

[0142] Alternatively, a macrocyclic peptide of the disclosure can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0143] The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g. , Robinson, J.R., ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York (1978).

[0144] Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate: U.S. Patent No. 4.486,194, which discloses a therapeutic device for administering medication through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4.447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0145] In certain embodiments, the macrocyclic peptides of the disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that therapeutic compounds of the disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g.. U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade, V.V., J. Clin. Pharmacol., 29:685 (1989)). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun., 153: 1038 (1988)); macrocyclic peptides (Bloeman, P.G. et al., FEBS Lett., 357: 140 (1995); Owais, M. et al., Antimicrob. Agents Chemother., 39:180 (1995)); surfactant protein A receptor (Briscoe et al., Am. J. Physiol., 1233: 134 (1995)); pl20 (Schreier et al., J. Biol. C em.. 269:9090 (1994)); see also Keinanen, K. et al., FEBS Lett.. 346: 123 (1994); Killion, J. J. et al., Immunomethods 4:273 (1994).

[0146] Peptide Synthesis

[0147] 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 stability.

[0148] 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, enzy matic 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 University 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- or p-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).

[0149] 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.

[0150] The C-terminal amino acid and all other amino acids used in the synthesis are required to have their a- or p-amino groups and side chain functionalities (if present) differentially protected such that the a- or P-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- or P-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.

[0151] 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 Biosy stems, Foster City7, 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.

[0152] 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.

[0153] Useful Fmoc amino acids derivatives are shown below.

[0154] Examples of Orthogonally Protected Amino Acids used in Solid Phase Synthesis

[0155]

[0156] 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, Ini. 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 / TIS / DTT (96:3: 1), v:v:w; 1 mL / 100 mg of peptidyl resin) for 1-3 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 217 or 220 nm. The structures of the purified peptides can be confirmed by electro-spray MS analysis.

[0157] Analytical Data:

[0158] Mass Spectrometry: “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 “»rz” unit designation. Compounds with exact masses greater than 1000 were often detected as double-charged or triple-charged ions.

[0159] The crude material was purified via preparative LC / MS. Fractions containing the desired product were combined and dried via centrifugal evaporation.

[0160] Analytical LC / MS Condition A:

[0161] 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: 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.

[0162] Analytical LC / MS Condition B:

[0163] Column: Waters Acquity UPLC BEH C18, 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.

[0164] Analytical LCMS Condition C:

[0165] 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.

[0166] Analytical LC / MS Condition D:

[0167] Column: Waters Acquity UPLC BEH C18, 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.

[0168] Analytical LC / MS Condition E:

[0169] Column: Kinetex XB Cl 8, 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 Wateracetonitrile (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.

[0170] Analytical LC / MS Condition F:

[0171] Column: Ascentis Express C18, 2.1 x 50 mm, 2.7-pm particles; Mobile Phase A: 10 mM ammonium acetate in water: acetonitrile (95:5); Mobile Phase B: 10 mM ammonium acetate in Wateracetonitrile (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:

[0172] Column: X Bridge C18, 4.6 x 50 mm, 5-pm 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.

[0173] The following abbreviations are employed in the Examples and elsewhere herein:

[0174] General Procedures:

[0175] 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).

[0176] Prelude: Resin-swelling procedure, Single-coupling procedure. Single-coupling extended time procedure. Chloroacetic Anhydride coupling, Single-Coupling Manual Addition Procedure A, Single-Coupling Manual Addition Procedure B, Manual removal ofFmoc group procedure:

[0177] Symphony: Resin-swelling procedure, Single-coupling procedure, Single-coupling extended time procedure. Double-coupling extended time procedure, Chloroacetic Anhydride coupling:

[0178] 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.

[0179] 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, Fa Uy acid chain coupling procedure B, General Purification Procedures.

[0180] Unnatural Fmoc-Amino Acid Synthesis amd were fatty acid tails were prepared according to the published methods (e.g., WO 2023 / 225661).

[0181] Preparation of Example 1001

[0182] To a 45-mL polypropylene solid-phase reaction vessel was added Sieber resin (70 mg, 0.050 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;

[0183] “Symphony X Single-coupling procedure” was followed with Fmoc-Gly-OH;

[0184] “Symphony X Single-coupling procedure” was followed with Fmoc-Cys(Trt)-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;

[0185] “ Symphony X Pre-Activated Single-coupling procedure” was followed with for (2S)-3- {l-[(tert-butoxy)carbonyl]-7-methyl-lH-indol-3-yl}-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)propanoic acid (Fmoc-Trp(Boc,7-Me)-OH);

[0186] “Symphony X Single coupling 3 deprotections procedure” was followed with Fmoc- Trp(Boc)-OH; “ Symphony X Single-coupling procedure ” was followed with Fmoc-Pro- OH; “Symphony X Single- coupling 3 deprotections extended time procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony X Single- coupling 3 deprotections procedure ” was followed with Symphony X Single-coupling extended time procedure ” was followed with Fmoc-D-Pro-OH; “ Symphony X Singlecoupling extended time procedure ” was followed with Fmoc-N-Me-Phe-OH;

[0187] “ Symphony X Single-coupling 3 deprotections extended time 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 ” w as follow ed with Fmoc- Tyr(tBu)-OH; “ Symphony X Single-coupling procedure ” was follow ed with (2S)-2- ({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(3,4,5-trifluorophenyl)propanoic acid (Fmoc-Phe(3,4,5-triF)-OH); “Symphony X Chloroacetic anhydride coupling procedure “ Symphony X Final rinse and dry procedure “Global Deprotection

[0188] Method” was followed; “ Cyclization Method” was followed.

[0189] The crude material was purified via preparative LC / MS with the following conditions: Column: XBridge C18, 30 x 200 mm, 5-μm 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: 5-55% B over 20 minutes, then a 2- minute hold at 100% B; Flow: 45 mL / min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 10 mg, and its estimated purity by LCMS analysis was 86%. Analysis condition A: Retention time = 1.12 min; ESI-MS(-) m / z [M-2H]2": 1041.22. Analysis condition B: Retention time = 1.59 min; ESI-MS(+) m / z [M+2H]2+; 1043.24.

[0190] The following examples were prepared, using Sieber resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001, composed of the general procedures such as "Symphony X Resin-swelling procedure ”, “Symphony X Single-coupling procedure ”, “Symphony X Pre-Activated Single-coupling procedure ” with (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(l-methyl-lH- indol-3-yl)propanoic acid, Fmoc-Trp(l-Me)-OH, “ Symphony X Single-coupling extended time procedure ”, “Symphony X Single-coupling 3 deprotections time procedure ”, “Symphony X Single-coupling 3 deprotections extended time procedure ”, “Symphony X Chloroacetic Anhydride coupling procedure ”, “Symphony X Final rinse and dry procedure ”, “Global Deprotection Method” was followed, “Cyclization Method”. The crude material was purified via preparative LC / MS. Fractions containing the desired product were combined and dried via centrifugal evaporation.

[0191] Preparation of Example 1002

[0192] Preparation of Example 1003

[0193] Preparation of Example 1004 Preparation of Example 1005

[0194] Preparation of Example 1006

[0195] Preparation of Example 1007

[0196] Preparation of Example 1008

[0197] Example 1008 was prepared, using Sieber resin on a 50 umol scale, and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:

[0198] “Symphony Resin-swelling procedure ” was followed;

[0199] “Symphony Single-coupling procedure ” was followed with Fmoc-D-Ala-OH;

[0200] “Symphony Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH; “Symphony Single-coupling procedure " was followed with Fmoc-Pro-OH;

[0201] “Symphony Double-coupling extended time procedure ” was followed with Fmoc- Asp(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc- Asp(tBu)-OH; “Symphony Single-coupling extended time procedure ” was followed with Fmoc-D-Pro-OH; “Symphony Double extended-coupling extended time procedure " w as followed with Fmoc-Nme-Phe-OH; “Symphony Double extended-coupling extended time procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Single-coupling procedure" was followed coupling procedure ” was followed with (2S)-2-( { [(9H-fluoren-9- yl)methoxy]carbonyl}amino)-3-(3,4,5-trifluorophenyl)propanoic acid;

[0202] “Symphony X Chloroacetic Anhydride coupling procedure

[0203] “Symphony X Final rinse and dry procedure “Global Deprotection Method” was followed; “ Cyclization Method” was followed. The crude material was purified via preparative LC / MS with the following conditions: Column: XBridge C18. 19 x 200 mm, 5-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; Gradient: 20-60% B over 20 minutes, then a 5- minute hold at 100% B; Flow: 20 mL / min. The yield of the product was 9 mg, and its estimated purity by LCMS analysis was 93.3%. Analysis condition A: Retention time = 1.66 min: [M+2H]2+: 1043.1. Analysis condition B: Retention time = 1.19 min; ESI- MS(+) m / z [M+2H]2+: 1043.1. The following examples were prepared, using Sieber resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1008.

[0204] Preparation of Example 1009

[0205] Preparation of Example 1010

[0206] The following examples were prepared, using Sieber resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001. Preparation of Example 1011

[0207] Preparation of Example 1012 The following examples were prepared, using Sieber resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1008.

[0208] Preparation of Example 1013 Preparation of Example 1017

[0209] Preparation of Example 1018

[0210] Preparation of Example 1019

[0211] Preparation of Example 1020 Preparation of Example 1021

[0212] Example 1021 was prepared, using 2-Chlorotrityl resin pre-loaded with Fmoc-Gly- OH on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001, composed of the following general procedures: “Symphony X Resin-swelling procedure ", “Symphony X Single-coupling procedure ", “Symphony X Pre-Activated Single-coupling procedure ” with (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-(3,4,5-trifluorophenyl)propanoic acid “Symphony X Single-coupling extended time procedure ’’, “ Symphony X Single-coupling 3 deprotections time procedure ”, “ Symphony X Single-coupling 3 deprotections extended time procedure “ Symphony X Chloroacetic Anhydride coupling procedure ”, “Symphony X Final rinse and dry procedure ”, “Global Deprotection Method” was followed, “Cyclization Method” was followed.

[0213] The crude material was purified via preparative LC / MS. The yield of the product was 1.5 mg, and its estimated purity by LCMS analysis was 98.3%.Analysis condition A: Retention time = 0.92 min: ESI-MS(+) m / z |M+2HJ2+: 1051.2. Analysis condition B: Retention time = 1.43 min; ESI-MS(+) m / z [M+2H]2+: 1051.2.

[0214] The following examples were prepared, using Sieber resin or 2-Chlorotrityl resin pre-loaded with Fmoc-Gly-OH on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0215] Preparation of Example 1022

[0216] Preparation of Example 1023

[0217] Preparation of Example 1024

[0218] Preparation of Example 1025

[0219] Preparation of Example 1026

[0220] Preparation of Example 1027

[0221] Preparation of Example 1028

[0222] Preparation of Example 1029

[0223] Preparation of Example 1030

[0224] To a 25-mL polypropylene solid-phase reaction vessel was added the PL-FMP resin preloaded with (5-methylpyrazin-2-yl)methanamine (ca. 100 mg, 0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:

[0225] “Symphony Resin-swelling procedure ” was followed;

[0226] “Symphony Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Ser(tBu)-OH;

[0227] “Symphony Double-coupling extended time procedure ” was followed with Fmoc- Asp(tBu)-OH;

[0228] “Symphony Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0229] “Symphony Single-coupling extended time procedure ” was followed with Fmoc-D-Pro- OH;

[0230] “Symphony Double extended-coupling extended time procedure ” was followed with Fmoc-Nme-Phe-OH;

[0231] “Symphony Double extended-coupling extended time procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0232] “Symphony Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Symphony Single-coupling procedure ” was followed with (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3,3-diphenylpropanoic acid; “Symphony Chloroacetic Anhydride coupling procedure’' ;

[0233] “Global Deprotection Method" was followed; “Cyclization Method” was followed.

[0234] The crude material was purified via preparative LC / MS. The yield of the product was 1.3 mg, and its estimated purity by LCMS analysis was 86.3%. Analysis condition A: Retention time = 0.92 min; ESI-MS(+) m / z [M+2H]2+: 1055.2.

[0235] Analysis condition B: Retention time = 1.48 min; ESI-MS(+) m / z [M+2H]2+: 1055.1.

[0236] Preparation of Example 1031 Example 1031 was prepared, using PL-FMP resin preloaded with (5- methylpyrazin-2-yl)methanamine (ca. 100 mg, 0.05 mmol), following the general synthetic sequence described for the preparation of Example 1030. The crude material was purified via preparative LC / MS. The yield of the product was 5 mg. and its estimated purity by LCMS analysis was 100%. Analysis condition A: Retention time = 1.18 min; ESI-MS(+) m / z [M+2H]2+: 1054.9. Analysis condition B: Retention time = 1.58 min; ESI-MS(+) m / z [M+2H]2+: 1054.9.

[0237] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0238] Preparation of Example 1032

[0239] Preparation of Example 1033

[0240] Preparation of Example 1034

[0241] Preparation of Example 1035

[0242] Preparation of Example 1036

[0243] Preparation of Example 1037

[0244] Preparation of Example 1038

[0245] Example 1038 was prepared, using Sieber resin on a 50 umol scale, and the reaction vessel was placed on the Prelude peptide synthesizer. The following procedures were then performed sequentially:

[0246] ‘Prelude Resin-swelling procedure ” was followed;

[0247] ‘Prelude Single-coupling procedure ” was followed with Fmoc-Gly-OH;

[0248] “Prelude Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH;

[0249] “Prelude Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0250] “Prelude Single-coupling procedure ” was follow ed with Fmoc-Trp(Boc)-OH;

[0251] “Prelude Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH;

[0252] “Prelude Single-coupling procedure” was followed with Fmoc-Trp(Boc)-OH;

[0253] “Symphony X Single-coupling procedure” was followed with Fmoc-Ser(tBu)-OH;

[0254] “Symphony X Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH; “Symphony X Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH;

[0255] “ Symphony X Pre-Activated Single-coupling procedure ” was followed with Fmoc-D- Hyp-OH;

[0256] “Symphony X Single-coupling extended time procedure” was followed with Fmoc-Nme- Phe-OH; “ Symphony X Single-coupling extended time 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- Tyr(tBu)-OH; “ Symphony X Single-coupling procedure” was followed with Fmoc-Phe- OH; “Symphony X Chloroacetic Anhydride coupling procedure “Symphony X Final rinse and dry procedure ” “Global Deprotection Method” was followed;

[0257] “Cyclization Method” was followed. The crude material was purified via preparative LC / MS. The yield of the product was 21.2 mg. and its estimated purity by LCMS analysis was 97. 1%. Analysis condition A: Retention time = 0.94 min; ESI-MS(-) m / z [M-2H]2’: 999.1. Analysis condition B: Retention time = 1.32 min; ESI-MS(+) m / z [M+2H]2+: 1000.9. The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1038.

[0258] Preparation of Example 1039

[0259] Preparation of Example 1040

[0260] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030.

[0261] Preparation of Example 1041

[0262] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001

[0263] Preparation of Example 1042

[0264] Preparation of Example 1043

[0265] Preparation of Example 1044

[0266] Preparation of Example 1045 The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1008.

[0267] Preparation of Example 1046

[0268] Preparation of Example 1047

[0269] Preparation of Example 1048

[0270] Preparation of Example 1049

[0271] Preparation of Example IOJO Preparation of Example 1051

[0272] Preparation of Example 1052

[0273] Preparation of Example 1053

[0274] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030 Preparation of Example 1054

[0275] Preparation of Example 1055

[0276] Preparation of Example 1056 Preparation of Example 1057

[0277] Preparation of Example 1058

[0278] Preparation of Example 1059

[0279] Preparation of Example 1060

[0280]

[0281] Preparation of Example 1061

[0282] Preparation of Example 1062

[0283] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0284] Preparation of Example 1063

[0285] Preparation of Example 1064

[0286] Example 1064 was prepared, using Sieber resin on a 50 umol scale, the following general procedures: “Prelude Resin-swelling procedure” was followed; “Prelude Single-coupling procedure ” was follow ed w ith Fmoc-Gly-OH;

[0287] “Prelude Single-coupling procedure” was followed with Fmoc-Cys(Trt)-OH;

[0288] “Prelude Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0289] “Prelude Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

[0290] “Prelude Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH;

[0291] “Prelude Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

[0292] “Prelude Single Single-coupling procedure ” was followed with Fmoc-Ser(tBu)-OH;

[0293] “Prelude Single Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH;

[0294] “Prelude Single Single-coupling procedure” was followed with Fmoc-Asp(tBu)-OH;

[0295] “Symphony X Pre-Activated Single -coupling procedure” was followed with Fmoc-D-Pro- OH; “Symphony X Single-coupling extended time procedure ”, was followed with Fmoc- Nme-Phe-OH; “ Symphony X Single-coupling extended time 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- Tyr(tBu)-OH; “ Symphony X Single-coupling procedure ” was followed with Fmoc-Phe- OH; "Symphony X Chloroacetic Anhydride coupling procedure ” "Symphony X Final rinse and dry procedure ” "Global Deprotection Method” was followed: "Cyclization Method” was followed. The crude material was purified via preparative LC / MS. The yield of the product was 21.8 mg, and its estimated purity by LCMS analysis was 86.4%. Analysis condition A: Retention time = 0.87 min; ESI-MS(+) m / z [M+2H]2+: 993.0. Analysis condition B: Retention time = 1.28 min; ESI-MS(+) m / z [M+2H]2+: 993.2.

[0296] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1064.

[0297] Preparation of Example 1065

[0298] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030.

[0299] Preparation of Example 1066 Preparation of Example 1067

[0300] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1064.

[0301] Preparation of Example 1068

[0302] Preparation of Example 1069 Preparation of Example 1070

[0303] Preparation of Example 1071

[0304] Preparation of Example 1073 Preparation of Example 1077

[0305] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1038.

[0306] Preparation of Example 1072

[0307] Preparation of Example 1074 Preparation of Example 1075

[0308] Preparation of Example 1076

[0309] Preparation of Example 1078

[0310] Preparation of Example 1079

[0311] Preparation of Example 1080 Example 1080 was prepared, using Sieber resin on a 50 umol scale, the following general procedures:

[0312] “Prelude Resin-swelling procedure ” was followed;

[0313] “Prelude Single-coupling procedure ” was followed with Fmoc-Gly-OH;

[0314] “Prelude Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH; “Prelude Single-coupling procedure ” w as followed with Fmoc- Asp(tBu)-OH;

[0315] “Prelude Single-coupling procedure ” w as follow ed with Fmoc-Trp(Boc)-OH;

[0316] “Prelude Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH;

[0317] “Prelude Single-coupling procedure " was followed with Fmoc-Trp(Boc)-OH;

[0318] “Prelude Single-coupling procedure ” was followed with Fmoc-Ser(tBu)-OH; “Prelude Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0319] “Prelude Single-coupling procedure ” was follow ed with Fmoc-Asp(tBu)-OH;

[0320] “Prelude Single-coupling procedure " was followed with Fmoc-Sar-OH;

[0321] “Prelude Single-coupling extended time procedure" was followed with Fmoc-Nme-Phe-

[0322] OH; “Prelude Single-coupling extended time procedure ” was followed with Fmoc- Asp(tBu)-OH; “Prelude Single-coupling procedure " was followed with Fmoc-Asp(tBu)- OH;

[0323] “Prelude Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH;

[0324] “Symphony X Single-coupling procedure ” was followed with (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-(3,4-difluorophenyl)propanoic acid;

[0325] “Symphony X Chloroacetic Anhydride coupling procedure "

[0326] “Symphony X Final rinse and dry procedure ” “Global Deprotection Method" was followed; “Cyclization Method” was followed.

[0327] The crude material was purified via preparative LC / MS with the following conditions: Column: XBridge Cl 8, 30 x 150 mm, 5-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; Gradient: 5-55% B over 20 minutes, then a 2-minute hold at 100% B; Flow: 40 mL / min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 7.7 mg, and purity by LCMS was 92.6%. Analysis condition A: Retention time = 0.87 min; ESI-MS(+) m / z |M+2H]2+: 998.2. Analysis condition B: Retention time = 1.35 min; ESI-MS(+) m / z [M+2H]2+: 998.1.

[0328] The following examples were prepared using Sieber resin or on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1080.

[0329] Preparation of Example 1081 Preparation of Example 1082

[0330] Preparation of Example 1083

[0331] Preparation of Example 1084

[0332] Preparation of Example 1085 Preparation of Example 1086

[0333] Preparation of Example 1087

[0334] Preparation of Example 1088

[0335] The following examples were prepared using Sieber resin or PL-FMP resin preloaded with amines on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030. Preparation of Example 1089

[0336] Preparation of Example 1090

[0337] Preparation of Example 1091

[0338] Preparation of Example 1092

[0339] Preparation of Example 1093

[0340] Preparation of Example 1094

[0341] Preparation of Example 1095

[0342] Preparation of Example 1096

[0343] Preparation of Example 1097

[0344] Preparation of Example 1098

[0345] Preparation of Example 1099

[0346] Preparation of Example 1100 Preparation of Example 1101

[0347] Preparation of Example 1102

[0348] Preparation of Example 1103 Preparation of Example 1104

[0349] Preparation of Example 1105

[0350] Preparation of Example 1106 Preparation of Example 1107

[0351] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0352] Preparation of Example 1108

[0353] Preparation of Example 1109 Preparation of Example 1110

[0354] The following examples were prepared using Sieber resin or Cl-trilyl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030.

[0355] Preparation of Example 1111

[0356] Preparation of Example 1112 The following examples were prepared using Sieber resin or Cl -tri tyl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0357] Preparation of Example 1113

[0358] Preparation of Example 1114

[0359] Preparation of Example 1115

[0360] Preparation of Example 1116

[0361] Preparation of Example 1117 The following examples were prepared using Sieber resin or Cl -tri tyl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030.

[0362] Preparation of Example 1118

[0363] Preparation of Example 1119

[0364] Preparation of Example 1120 Preparation of Example 1121

[0365] Preparation of Example 1124

[0366] Preparation of Example 1125

[0367] Preparation of Example 1122 Example 1122 was prepared, using Sieber resin on a 50 umol scale, and the reaction vessel was placed on the Symphony X peptide synthesizer. The following procedures were then performed sequentially:

[0368] “Symphony Resin-swelling procedure ” was followed;

[0369] “Symphony Single-coupling procedure ” was followed with Fmoc-D-Ala-OH;

[0370] “Symphony Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH;

[0371] “Symphony Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Pro-OH;

[0372] “ Symphony Double-coupling extended time procedure ” was followed with Fmoc- Asp(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc- Asp(tBu)-OH; “Symphony Single-coupling extended time procedure ” was followed with Fmoc-D-Pro-OH; “ Symphony Double-coupling extended time procedure ” was followed with Fmoc-Nme-Phe-OH; “Symphony Double-coupling extended time procedure ” was followed with Fmoc-Asp(tBu)-OH; “Symphony Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0373] “Symphony Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH;

[0374] “Prelude Single-coupling procedure ” was followed with (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-(4-cyanophenyl)propanoic acid;

[0375] “Prelude Chloroacetic Anhydride coupling procedure

[0376] “Global Deprotection Method” was followed; “Cyclization Method” was followed.

[0377] The crude material was purified via preparative LC / MS. The yield of the product w as 15 mg, and purity by LCMS was 97.5%. Analysis condition A: Retention time = 0.81 min; ESI-MS(+) m / z [M+2H]2+: 991.7. Analysis condition B: Retention time = 1.25 min; ESI-MS(+) m / z [M+2H]2+: 992.1.

[0378] The following examples were prepared using Sieber resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1122. Preparation of Example 1123

[0379] Preparation of Example 1126

[0380] Preparation of Example 1127

[0381] Preparation of Example 1128

[0382] Preparation of Example 1129

[0383] Preparation of Example 1130

[0384] Preparation of Example 1131 Preparation of Example 1132

[0385] Preparation of Example 1133

[0386] Preparation of Example 1134

[0387] Preparation of Example 1135

[0388] Preparation of Example 1136

[0389] Preparation of Example 1137

[0390] Preparation of Example 1138

[0391] Preparation of Example 1139

[0392] Preparation of Example 1140

[0393] Preparation of Example 1141

[0394] Preparation of Example 1142

[0395] Preparation of Example 1143

[0396] Preparation of Example 1144

[0397] Preparation of Example 1145

[0398] Preparation of Example 1150

[0399] Preparation of Example 1153

[0400] Preparation of Example 1154

[0401] Preparation of Example 11 J 5

[0402] Preparation of Example 1156

[0403] Preparation of Example 1157

[0404] Preparation of Example 1158

[0405] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1064. Preparation of Example 1146

[0406] Preparation of Example 1147 Preparation of Example 1151 Preparation of Example 1152

[0407] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1122.

[0408] Preparation of Example 1148

[0409] Preparation of Example 1149 The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030. Preparation of Example 1159

[0410] Preparation of Example 1160

[0411] Preparation of Example 1161 Preparation of Example 1162

[0412] Preparation of Example 1163

[0413] Preparation of Example 1164

[0414] Preparation of Example 1166

[0415] Preparation of Example 1167

[0416] Preparation of Example 1168

[0417] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1064. Preparation of Example 1169

[0418] Preparation of Example 1170

[0419] Preparation of Example 1171

[0420] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001. Preparation of Example 1165

[0421] Preparation of Example 1172 Preparation of Example 1173

[0422] The following examples were prepared using Sieber resin or Cl-trilyl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030. Preparation of Example 1174

[0423] Preparation of Example 1175

[0424] Preparation of Example 1182 Preparation of Example 1176

[0425] Example 1176 was prepared, using Sieber resin on a 50 umol scale, and the reaction vessel was placed on the Symphony X peptide synthesizer. The following procedures were then performed sequentially:

[0426] ‘Prelude Resin-swelling procedure ” was followed;

[0427] “Prelude Single-coupling procedure ” was followed with Fmoc-Gly-OH;

[0428] “Prelude Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH;

[0429] “Prelude Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0430] “Prelude Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

[0431] “Prelude Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH;

[0432] “Prelude Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

[0433] “Prelude Single-coupling procedure ” was followed with Fmoc-Ser(tBu)-OH;

[0434] “Prelude Double extended-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0435] “Prelude Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0436] “Prelude Single-coupling extended time procedure ” was followed with Fmoc-Nme-Gly- OH; “Prelude Double extended-coupling extended time procedure ” was follow ed with Fmoc-Nme-Phe-OH; “Prelude Double extended-coupling extended time procedure ” was followed with Fmoc-Asp(tBu)-OH; “Prelude Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH; “Prelude Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “ Symphony X Single-coupling procedure” was followed with (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-methoxyphenyl)propanoic acid;

[0437] “Symphony X Chloroacetic Anhydride coupling procedure “ Symphony X Final rinse and dry procedure “Global Deprotection Method” was followed; “ Cyclization Method” was followed. 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 93.3%. Analysis condition A: Retention time = 1.09 min; ESI-MS(+) m / z [M+2H]2+: 994.1. Analysis condition B: Retention time = 1.38 min; ESI-MS(+) m / z [M+2H]2+: 994.2. The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1176.

[0438] Preparation of Example 1177

[0439] Preparation of Example 1178

[0440] Preparation of Example 1179 Preparation of Example 1180

[0441] Preparation of Example 1187

[0442] Preparation of Example 1188

[0443] Preparation of Example 1189

[0444] Preparation of Example 1181 To a 45-mL polypropylene solid-phase reaction vessel was added Rink resin (200 mg, 0.100 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;

[0445] “ Symphony X Single-coupling procedure " was followed with Fmoc-Gly-OH; “Symphony X Pre-Activated Single-coupling procedure ” was followed with Fmoc-

[0446] Glu(O-2-PhiPr)-OH; “ Symphony X Pre-Activated Single-coupling procedure ” was followed with Fmoc-Tic-OH; “Symphony X Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

[0447] “Symphony X Single-coupling procedure ” was followed with Fmoc-Tyr(tBu)-OH; “Symphony X Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

[0448] “Symphony X Single-coupling procedure ” was followed with Fmoc-Ser(tBu)-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;

[0449] “ Symphony X Single-coupling extended time procedure " was followed with Fmoc-D-Pro- OH; “Symphony X Single-coupling extended time procedure ” was followed with Fmoc-N- Me-Phe-OH; “Symphony X Single-coupling extended time procedure ” was followed with Fmoc-Asn(Trt)-OH; “ Symphony X Single-coupling procedure ” was followed with Fmoc- Asp(tBu)-OH; “ Symphony X Single-coupling procedure ” was followed with Fmoc- Tyr(tBu)-OH; '‘Symphony X Single-coupling procedure" was followed with Fmoc-Phe- OH; “Symphony X Chloroacetic anhydride coupling procedure “Symphony X Final rinse and dry procedure ”

[0450] The above resin containing the linear peptide was then transferred into a 10 mL Bio-Rad tube. The resins were then treated with 2 mL of a solution of 9.35: / : 1.5 DCM:TIS:TFA v:v:v 3 times for 2 minutes each time. After the final treatment the resins were washed with 5 mL DCM twice, 5 mL DMF twice and 5mL of 10% DIEA / DMF solution to remove all residual TFA. The resins were then suspended in 2 mL DMF to which was added the cyclization reagents: HATU (0.035 g, 0.100 mmol, N-Methylmorpholine (0.022 mL. 0.200 mmol), HOBt (17.0 mg. 0.100 mmol) and the reaction was gently shaken for 16 hours. The solutions were filtered through the frit and the resins were rinsed with DMF (3 X 5 mL) and DCM (3 X 5 mL). The peptides were the released from the solid support using the "Global Deprotection Method A" to provide the crude cyclized peptide as a solid.

[0451] The crude material was purified via preparative LC / MS. The yield of the product was 5.1 mg, and its estimated purity by LCMS analysis was 100%. Analysis condition A: Retention time = 1.16 min; ESI-MS(+) m / z [M+2H]2+: 998.5. Analysis condition B: Retention time = 1.43 min: ESI-MS(+) m / z [M+2H]2+: 998.3.

[0452] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1064. Preparation of Example 1183

[0453] Preparation of Example 1184

[0454] Preparation of Example 1185

[0455] Preparation of Example 1186

[0456] The following examples were prepared using Sieber resin or Cl -tri tyl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1176.

[0457] Preparation of Example 1190

[0458] Preparation of Example 1191 Preparation of Example 1198

[0459] Preparation of Example 1199

[0460] Preparation of Example 1200

[0461] Preparation of Example 1207

[0462] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030.

[0463] Preparation of Example 1192

[0464] Preparation of Example 1193 Preparation of Example 1194

[0465] Preparation of Example 1195

[0466] Preparation of Example 1196

[0467] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1064. Preparation of Example 1197

[0468] Preparation of Example J 206

[0469] Preparation of Example 1226

[0470] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030. Preparation of Example 1201

[0471] Preparation of Example 1202

[0472] Preparation of Example 1203

[0473] Preparation of Example 1204 Preparation of Example 1205

[0474] Preparation of Example 1208

[0475] Preparation of Example 1210 Preparation of Example 1211

[0476] Preparation of Example 1212

[0477] Preparation of Example 1215

[0478] Preparation of Example 1216

[0479] Preparation of Example 1217

[0480] Preparation of Example 1218

[0481] Preparation of Example 1224

[0482] Preparation of Example 1225 Preparation of Example 1228

[0483] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001. Preparation of Example 1209

[0484] Preparation of Example 1213

[0485] Preparation of Example 1214 Preparation of Example 1222

[0486] Preparation of Example 1223 Preparation of Example 1229

[0487] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1122. Preparation of Example 1219

[0488] Preparation of Example 1220

[0489] Preparation of Example 1221

[0490] Preparation of Example 1227 The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0491] Preparation of Example 1230

[0492] Preparation of Example 1231

[0493] Preparation of Example 1235 Preparation of Example 1236

[0494] Preparation of Example 1237

[0495] Preparation of Example 1241 Preparation of Example 1261

[0496] Preparation of Example 1262

[0497] Preparation of Example 1263 Preparation of Example 1265

[0498] Preparation of Example 1266

[0499] Preparation of Example 1267 Preparation of Example 1268

[0500] Preparation of Example 1269

[0501] Preparation of Example 1270 The following examples were prepared using Sieber resin or Cl -tri tyl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1064

[0502] Preparation of Example 1232

[0503] Preparation of Example 1233

[0504] Preparation of Example 1238 Example 1238 was prepared, using Sieber resin on a 25 umol scale, following the general synthetic sequence described for the preparation of Example 1181. The resin containing the linear peptide was then transferred into a 20 mL syringe equiped with a frit. The resins were then treated with 2 mL of a solution of 9.35: / : 1.5 DCM:TIS:TFA v:v:v 3 times for 2 minutes each time. After the final treatment the resins were washed with 5 mL DCM twice, 5 mL DMF twice and 5mL of 10% DIEA / DMF solution to remove all residual TFA. The resins were then suspended in 2 mL DMF to which was added the cyclization reagents: HATU (0.035 g, 0.100 mmol, N-methylmorpholine (0.022 mL, 0.200 mmol), HOBt (17.0 mg, 0.100 mmol) and the reaction was gently shaken for 16 hours. The solutions were filtered through the frit and the resins were rinsed with 3 X 5 mL DMF and 3 X 5 mL DCM. The peptides were the released from the solid support using the "Global Deprotection Method A" to provide the crude cyclized peptide as a solid. The crude material was purified via preparative LC / MS. The yield of the product was 3.7 mg, and purity by LCMS was 97.2%. Analysis condition A: Retention time = 1.28 min: ESI-MS(+) m / z [M+2H]2+: 1042.0. Analysis condition B: Retention time = 1.63 min; ESI-MS(+) m / z [M+2H]2+: 1042.1.

[0505] The following examples were prepared using Sieber resin or preloaded chloro-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030.

[0506] Preparation of Example 1234 Preparation of Example 1239

[0507] Preparation of Example 1240

[0508] Preparation of Example 1242

[0509] Preparation of Example 1243

[0510] Preparation of Example 1244

[0511] Preparation of Example 1245 Preparation of Example 1246

[0512] Preparation of Example 1247

[0513] Preparation of Example 1249

[0514] Preparation of Example 1250

[0515] Preparation of Example 1251 Preparation of Example 1252

[0516] The following examples were prepared using Sieber resin or preloaded chloro-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1008. Preparation of Example 1248

[0517] Preparation of Example J 253

[0518] Preparation of Example 1255

[0519] Preparation of Example 1256

[0520] Preparation of Example 1257

[0521] Preparation of Example 1258 Preparation of Example 1259

[0522] Preparation of Example 1260 The following examples were prepared using Sieber resin or preloaded chloro-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1038.

[0523] Preparation of Example 1264 The following examples were prepared using Sieber resin or preloaded chloro-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0524] Preparation of Example 1271

[0525] Preparation of Example 1272

[0526] Preparation of Example 1273 Preparation of Example 1274

[0527] Preparation of Example 1275

[0528] Preparation of Example 1276

[0529] Preparation of Example 1277

[0530] Preparation of Example 1278

[0531] Preparation of Example 1279 Preparation of Example 1280

[0532] Preparation of Example 1281

[0533] Preparation of Example 1282

[0534] Preparation of Example 1283

[0535] Preparation of Example 1284

[0536] Preparation of Example 1285

[0537] Preparation of Example 1286

[0538] Preparation of Example 1287

[0539] Preparation of Example 1288

[0540] Preparation of Example 1289

[0541] Preparation of Example 1290

[0542] Preparation of Example 1291

[0543] Preparation of Example 1292

[0544] Preparation of Example 1293

[0545] Preparation of Example 1294

[0546] Preparation of Example 1295 Preparation of Example 1296

[0547] Preparation of Example 1297

[0548] Preparation of Example 1298

[0549] Preparation of Example 1299 Preparation of Example 1300

[0550] Preparation of Example 1301

[0551] Preparation of Example 1302

[0552] Preparation of Example 1303 Preparation of Example 1304

[0553] Preparation of Example 1305

[0554] Preparation of Example 1306

[0555] Preparation of Example 1307 Preparation of Example 1308

[0556] Preparation of Example 1309

[0557] Preparation of Example 1310

[0558] Preparation of Example 1311 Preparation of Example 1312

[0559] Preparation of Example 1313

[0560] Preparation of Example 1314

[0561] Preparation of Example 1315

[0562] Preparation of Example 1316

[0563] Preparation of Example 1317

[0564] Preparation of Example 1318

[0565] Preparation of Example 1319

[0566] Preparation of Example 1320

[0567] Preparation of Example 1321

[0568] Preparation of Example 1322

[0569] Preparation of Example 1323

[0570] Preparation of Example 1324

[0571] Preparation of Example 1325

[0572] Preparation of Example 1326

[0573] Preparation of Example 1327 Preparation of Example 1328

[0574] Preparation of Example 1329

[0575] Preparation of Example 1330

[0576] Preparation of Example 1331

[0577] Preparation of Example J 332

[0578] Preparation of Example 1333

[0579] Preparation of Example 1334

[0580] Preparation of Example 1335

[0581] Preparation of Example 1336

[0582] Preparation of Example 1337

[0583] Preparation of Example 1338

[0584] Preparation of Example 1339

[0585] Preparation of Example 1340

[0586] Preparation of Example 1341

[0587] Preparation of Example 1342

[0588] Preparation of Example 1343

[0589] Preparation of Example 1344

[0590] Preparation of Example 1345

[0591] Preparation of Example 1346

[0592] Preparation of Example 1347

[0593] Preparation of Example 1348

[0594] Preparation of Example 1349

[0595] Preparation of Example 1350

[0596] Preparation of Example 1351

[0597] Preparation of Example 1352 Preparation of Example 1353

[0598] Preparation of Example 1354

[0599] Preparation of Example 1355

[0600] Preparation of Example 1356 Preparation of Example 1367

[0601] Preparation of Example 1358

[0602] Preparation of Example 1360 Preparation of Example 1361

[0603] Preparation of Example 1362

[0604] Preparation of Example 1363

[0605] Preparation of Example 1364 Preparation of Example 1365

[0606] Preparation of Example 1366

[0607] Preparation of Example 1367

[0608] Preparation of Example 1368 Preparation of Example 1369

[0609] Preparation of Example 1370

[0610] Preparation of Example 1371

[0611] Preparation of Example 1372 Preparation of Example 1373

[0612] Preparation of Example 1374

[0613] Preparation of Example 1375

[0614] Preparation of Example 1376 Preparation of Example 1377

[0615] Preparation of Example 1378

[0616] Preparation of Example 1379

[0617] Preparation of Example 1380 Preparation of Example 1381

[0618] Preparation of Example 1382

[0619] Preparation of Example 1383

[0620] Preparation of Example 1384 Preparation of Example 1385

[0621] Preparation of Example 1386

[0622] Preparation of Example 1387

[0623] Preparation of Example 1388 Preparation of Example 1389

[0624] Preparation of Example 1390

[0625] Preparation of Example 1391

[0626] Preparation of Example 1392

[0627] Preparation of Example J 393

[0628] Preparation of Example 1394

[0629] Preparation of Example 1395 Preparation of Example 1396

[0630] Preparation of Example J 397

[0631] Preparation of Example 1398

[0632] Preparation of Example 1399 Preparation of Example 1400

[0633] Preparation of Example 1401

[0634] Preparation of Example 1402

[0635] Preparation of Example 1403 Preparation of Example 1404

[0636] Preparation of Example 1405

[0637] Preparation of Example 1406

[0638] Preparation of Example 1407 Preparation of Example 1408

[0639] Preparation of Example 1409

[0640] Preparation of Example 1410

[0641] Preparation of Example 1411 Preparation of Example 1412

[0642] Preparation of Example 1413

[0643] Preparation of Example 1414

[0644] Preparation of Example 1415 Preparation of Example 1416

[0645] Preparation of Example 1417

[0646] Preparation of Example 1418

[0647] Preparation of Example 1419 Preparation of Example 1420

[0648] Preparation of Example 1421

[0649] Preparation of Example 1422

[0650] Preparation of Example 1423

[0651] Preparation of Example 1424

[0652] Preparation of Example 1425

[0653] Preparation of Example 1426 Preparation of Example 1427

[0654] Preparation of Example 1428

[0655] Preparation of Example 1429

[0656] Preparation of Example 1430 Preparation of Example 1431

[0657] Preparation of Example 1432

[0658] Preparation of Example 1433

[0659] Preparation of Example 1434 Preparation of Example 1435

[0660] Preparation of Example 1436

[0661] Preparation of Example 1437

[0662] Preparation of Example 1438 Preparation of Example 1439

[0663] Preparation of Example 1440

[0664] Preparation of Example 1441

[0665] Preparation of Example 1442 Preparation of Example 1443

[0666] Preparation of Example 1444

[0667] Preparation of Example 1445

[0668] Preparation of Example 1446 Preparation of Example 1447

[0669] Preparation of Example 1448

[0670] Preparation of Example 1449

[0671] Preparation of Example 1450

[0672] Preparation of Example 1451

[0673] Preparation of Example 1452 Preparation of Example 1454

[0674] Preparation of Example 1455

[0675] Preparation of Example 1456

[0676] Preparation of Example 1457 Preparation of Example 1458

[0677] Preparation of Example 1459

[0678] Preparation of Example 1460

[0679] Preparation of Example 1461 Preparation of Example 1462

[0680] Preparation of Example 1463

[0681] Preparation of Example 1464

[0682] Preparation of Example 1465 Preparation of Example 1466

[0683] Preparation of Example 1467

[0684] Preparation of Example 1468

[0685] Preparation of Example 1469 Preparation of Example 1470

[0686] Preparation of Example 1471

[0687] Preparation of Example 1472

[0688] Preparation of Example 1473 Preparation of Example 1474

[0689] Preparation of Example 1475

[0690] Preparation of Example 1476

[0691] Preparation of Example 1477 Preparation of Example 1478

[0692] Preparation of Example 1479

[0693] Preparation of Example 1480

[0694] Preparation of Example 1481 Preparation of Example 1482

[0695] Preparation of Example 1483

[0696] Preparation of Example 1484

[0697] Preparation of Example 1485 Preparation of Example 1486

[0698] Preparation of Example 1487

[0699] Preparation of Example 1488

[0700] Preparation of Example 1489 Preparation of Example 1490

[0701] Preparation of Example 1491

[0702] Preparation of Example 1492

[0703] Preparation of Example 1495 Preparation of Example 1517

[0704] The following examples were prepared using Sieber resin or Cl -tri tyl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030.

[0705] Preparation of Example 1493

[0706] Preparation of Example 1494 Preparation of Example 1496

[0707] Preparation of Example 1497

[0708] Preparation of Example 1498

[0709] Preparation of Example 1499

[0710] Preparation of Example 1500

[0711] Preparation of Example 1501

[0712] Preparation of Example 1507

[0713] Preparation of Example 1508

[0714] Preparation of Example 1502

[0715] Preparation of Example 1503

[0716] Preparation of Example 1504

[0717] Preparation of Example 1505

[0718] Preparation of Example 1506

[0719] Preparation of Example 1509

[0720] Preparation of Example 1510 Preparation of Example 1511

[0721] Preparation of Example 1512

[0722] Preparation of Example 1513 Preparation of Example 1514

[0723] Preparation of Example 1515

[0724] Preparation of Example 1516

[0725] Preparation of Example 1518

[0726] To a 45-mL polypropylene solid-phase reaction vessel was added Sieber resin (70 mg, 0.050 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;

[0727] “Symphony X Single-coupling procedure " was followed with Fmoc-Gly-OH;

[0728] “Symphony X Single-coupling procedure ” was followed with Fmoc-Cys(Trt)-OH;

[0729] “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 Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

[0730] “Symphony X Single-coupling procedure ” was followed with Fmoc-Trp(Boc)-OH;

[0731] “Symphony X Single-coupling extended time procedure ” or “Symphony X Singlecoupling procedure” was followed with Fmoc-Tyr(tBu)-OH;

[0732] “Symphony X Single-coupling extended time procedure ” was followed with Fmoc- Asp(tBu)-OH;

[0733] “Symphony X Single-coupling procedure ” was followed with Fmoc-Asp(tBu)-OH;;

[0734] “Symphony X Single-coupling extended time procedure” was followed with Fmoc-D-Pro- OH; “ Symphony X Single-coupling extended time procedure ” was followed with Fmoc-N- Me-Phe-OH; “ Symphony X Double-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 extended time procedure” was followed with Fmoc-Tyr(tBu)-OH; “Symphony X Single -coupling procedure” was followed with (2S)-2- ({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(3,4,5-trifluorophenyl)propanoic acid; “Symphony X Chloroacetic Anhydride coupling procedure “Symphony X Final rinse and dry procedure ” “Global Deprotection Method” was followed; “Cyclization Method” was followed.

[0735] The crude material was purified via preparative LC / MS. The yield of the product was 29.1 mg, and purity7by LCMS was 85.9%. Analysis condition A: RT= 0.96 min;

[0736] [M+2H]2+: 1069.1. Analysis condition B: RT= 1.48 mm; [M+2H]2+: 1069.1.

[0737] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1518. Preparation of Example 1519

[0738] Preparation of Example 1520 Preparation of Example 1521

[0739] Preparation of Example 1522

[0740] Preparation of Example 1523 Preparation of Example 1524

[0741] Preparation of Example 1525

[0742] Preparation of Example 1526

[0743] Preparation of Example 1527

[0744] Preparation of Example 1528

[0745] Preparation of Example 1529 Preparation of Example 1530

[0746] Preparation of Example 1531 Preparation of Example 1533

[0747] Preparation of Example 1534 Preparation of Example 1536

[0748] Preparation of Example 1537

[0749] Preparation of Example 1538 Preparation of Example 1539

[0750] Preparation of Example 1540

[0751] Preparation of Example 1541 Preparation of Example 1542

[0752] Preparation of Example 1543

[0753] Preparation of Example 1544 Preparation of Example 1545

[0754] Preparation of Example 1046

[0755] Preparation of Example 1547 Preparation of Example 1548

[0756] Preparation of Example 1556

[0757] Preparation of Example 1557 Preparation of Example 1558

[0758] Preparation of Example 1559

[0759] Preparation of Example 1560 Preparation of Example 1561

[0760] Preparation of Example 1563 Preparation of Example 1564

[0761] Preparation of Example 1565

[0762] Preparation of Example 1566 Preparation of Example 1567

[0763] Preparation of Example 1568

[0764] Preparation of Example 1569 The following examples were prepared using Sieber resin or Cl -tri ty 1 resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0765] Preparation of Example 1549

[0766] Preparation of Example 1550

[0767] Preparation of Example 1551 Preparation of Example 1552

[0768] Preparation of Example 1553

[0769] Preparation of Example 1554

[0770] Preparation of Example 1555

[0771] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1030.

[0772] Preparation of Example 1571 Preparation of Example 1572 , Preparation of Example 1575

[0773] Preparation of Example 1576

[0774] Preparation of Example 1594 Preparation of Example 1595

[0775] The following examples were prepared using Sieber resin or Cl-trityl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1001.

[0776] Preparation of Example 1577

[0777] Preparation of Example 1578 Preparation of Example 1579

[0778] Preparation of Example 1580

[0779] Preparation of Example 1581

[0780] Preparation of Example 1582 Preparation of Example 1583

[0781] Preparation of Example 1584

[0782] Preparation of Example 1585

[0783] Preparation of Example 1586 Preparation of Example 1587

[0784] Preparation of Example J 588

[0785] Preparation of Example 1589

[0786] Preparation of Example 1590 Preparation of Example 1591

[0787] Preparation of Example 1592

[0788] Preparation of Example 1593

[0789] The yield of the product was 25 mg, and purity by LCMS was 100%. Analysis condition A: RT = 0.96 min; [M+2H]2+: 988.0. Analysis condition B: RT = 1.26 min; [M+2H]2+: 988.1. Preparation of Example 1596

[0790] Preparation of Example 1597

[0791] Preparation of Example 1598

[0792] Preparation of Example 1599

[0793] Preparation of Example 1600 Preparation of Example 1601

[0794] Preparation of Example 1602

[0795] Preparation of Example 1603 Preparation of Example 1604

[0796] Preparation of Example 1605

[0797] Preparation of Example 1606 Preparation of Example 1607

[0798] Preparation of Example 1608

[0799] Preparation of Example J 609

[0800] Preparation of Example 1610

[0801] Preparation of Example 1611

[0802] Preparation of Example 1612

[0803] Preparation of Example 1613 Preparation of Example 1614

[0804] Preparation of Example 1615

[0805] Preparation of Example 1616

[0806] Preparation of Example 1617 Preparation of Example 1618

[0807] Preparation of Example 1619

[0808] Preparation of Example 1620

[0809] Preparation of Example 1621

[0810] Preparation of Example 1622

[0811] Preparation of Example 1623

[0812] Preparation of Example 1624

[0813] Preparation of Example 1625

[0814] Preparation of Example 1626

[0815] Preparation of Example 1627

[0816] Preparation of Example 1628 Preparation of Example 1629

[0817] Preparation of Example 1630

[0818] Preparation of Example 1631

[0819] Preparation of Example 1632

[0820] Preparation of Example 1633

[0821] Preparation of Example 1634

[0822] To a 45-mL polypropylene solid-phase reaction vessel was added Sieber resin (70 mg, 0.050 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;

[0823] “Symphony X Single-coupling procedure ’' was followed with Fmoc-Gly-OH; “Symphony X Single-coupling procedure was followed with Fmoc-Gly-OH; “Symphony X Single-coupling procedure ” was followed with Fmoc-Ala-OH;

[0824] “Symphony X Single-coupling procedure” was followed with Fmoc-Cys(Trt)-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 Single-coupling 3 deprotections procedure ” was followed with Fmoc- Tyr(tBu)-OH;

[0825] “ Symphony X Single-coupling 3 deprotections procedure ” was followed with Fmoc- Trp(Boc)-OH;

[0826] “Symphony X Single-coupling procedure” was followed with Fmoc-Ser(tBu)-OH;

[0827] “Symphony X Single-coupling 3 deprotections extended time procedure ” was followed with Fmoc-Asp(tBu)-OH;

[0828] “ Symphony X Single-coupling 3 deprotections procedure” was followed with Fmoc- Asp(tBu)-OH;

[0829] “Symphony X Single-coupling procedure” was followed with Fmoc-D-Pro-OH;

[0830] “ Symphony X Single-coupling procedure " was followed with Fmoc-Nme-Phe-OH;

[0831] “ Symphony X Single-coupling 3 deprotections extended time procedure” was followed with Fmoc-Asn(Trt)-OH;

[0832] “Symphony X Single-coupling 3 deprotections procedure ” was followed with Fmoc- Asp(tBu)-OH;

[0833] “Symphony X Single-coupling procedure” was followed with Fmoc-Tyr(tBu)-OH;

[0834] “Symphony X Single-coupling procedure” was followed with Fmoc-Phe-OH;

[0835] “Symphony X Chloroacetic Anhydride coupling procedure

[0836] “Symphony X Final rinse and dry procedure ”

[0837] “Global Deprotection Method” was followed;

[0838] “Cyclization Method” was followed.

[0839] The crude material was purified via preparative LC / MS with the following conditions: Column: XBridge C18, 19 x 200 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: 5-45% 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.

[0840] The yield of the product was 35.6 mg, and purity' by LCMS was 94.4%. Analysis condition B: RT = 1.35 min; [M+2H]2+: 1056.1.

[0841] The following examples were prepared using Sieber resin or Cl-trilyl resin on a 50 umol scale, following the general synthetic sequence described for the preparation of Example 1634.

[0842] Preparation of Example 1635

[0843] Preparation of Example 1636

[0844] Preparation of Example 1637 Preparation of Example 1638

[0845] Preparation of Example 1639

[0846] Preparation of Example 1640 Preparation of Example 1641

[0847] Method

[0848] 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 < 0.001 pM, B: 0.001 pM <= IC50 < 0.01 pM; C: 0.01 pM <= IC5o < O. l pM; D: < 1 pM.

[0849] Table 1

[0850] Biological Data for Examples 1001-1633

Claims

CLAIMSWe claim:

1. A compound selected fromExample 1001, Example 1002, Example 1003, Example 1004. Example 1005, Example 1006, Example 1007, Example 1008, Example 1009, Example 1010, Example 1011, Example 1012, Example 1013, Example 1014, Example 1015, Example 1016, Example 1017, Example 1018, Example 1019, Example 1020, Example 1021, Example 1022, Example 1023, Example 1024, Example 1025, Example 1026. Example 1027, Example 1028, Example 1029. Example 1030. Example 1031. Example 1032, Example 1033, Example 1034, Example 1035, Example 1036, Example 1037, Example 1038, Example 1039, Example 1040, Example 1041, Example 1042, Example 1043, Example 1044, Example 1045, Example 1046, Example 1047, Example 1048, Example 1049, Example 1050, Example 1051, Example 1052. Example 1053. Example 1054, Example 1055, Example 1056, Example 1057, Example 1058, Example 1059, Example 1060, Example 1061, Example 1062, Example 1063, Example 1064, Example 1065, Example 1066, Example 1067, Example 1068, Example 1069, Example 1070, Example 1071. Example 1072, Example 1073, Example 1074. Example 1075. Example 1076, Example 1077, Example 1078, Example 1079, Example 1080, Example 1081, Example 1082, Example 1083, Example 1084, Example 1085, Example 1086, Example 1087, Example 1088, Example 1089, Example 1090, Example 1091, Example 1092, Example 1093, Example 1094, Example 1095, Example 1096, Example 1097. Example 1098, Example 1099, Example 1100, Example 1101, Example 1102. Example 1103. Example 1104, Example 1105, Example 1106, Example 1107, Example 1108, Example 1 109, Example 1110, Example 1111, Example 1112, Example 1113, Example 1114, Example 1115, Example 1116, Example 1117, Example 1118, Example 1119, Example 1120, Example 1121, Example 1122, Example 1123, Example 1124, Example 1125. Example 1126, Example 1127, Example 1128, Example 1129, Example 1130, Example 1 131, Example 1 132, Example 1133, Example 1134, Example 1135, Example 1136, Example 1137, Example 1138, Example 1139, Example 1140, Example 1141, Example 1142, Example 1143, Example 1144, Example 1145, Example 1146, Example 1147. Example 1148, Example 1149, Example 1150. Example 1151. Example 1152. Example 1 153, Example 1 154,Example 1155, Example 1156, Example 1157, Example 1158, Example 1159, Example 1160, Example 1161, Example 1162. Example 1163. Example 1164, Example 1165, Example 1166, Example 1167, Example 1168, Example 1169, Example 1170, Example 1171, Example 1172, Example 1173, Example 1174, Example 1175, Example 1176, Example 1177, Example 1178, Example 1179, Example 1180, Example 1181, Example 1182, Example 1183, Example 1184, Example 1185. Example 1186, Example 1187, Example 1188, Example 1189, Example 1190. Example 1191. Example 1192. Example 1193, Example 1194, Example 1195, Example 1196, Example 1 197, Example 1198, Example 1199, Example 1200, Example 1201, Example 1202, Example 1203, Example 1204, Example 1205, Example 1206, Example 1207. Example 1208, Example 1209, Example 1210, Example 1211, Example 1212, Example 1213. Example 1214, Example 1215, Example 1216, Example 1217, Example 1218, Example 1219, Example 1220, Example 1221, Example 1222, Example 1223, Example 1224, Example 1225, Example 1226, Example 1227, Example 1228, Example 1229, Example 1230, Example 1231, Example 1232, Example 1233, Example 1234, Example 1235. Example 1236, Example 1237, Example 1238. Example 1239. Example 1240. Example 1241, Example 1242, Example 1243, Example 1244, Example 1245, Example 1246, Example 1247, Example 1248, Example 1249, Example 1250, Example 1251, Example 1252, Example 1253, Example 1254, Example 1255, Example 1256, Example 1257, Example 1258, Example 1259, Example 1260, Example 1261. Example 1262. Example 1263, Example 1264, Example 1265, Example 1266, Example 1267, Example 1268, Example 1269, Example 1270, Example 1271, Example 1272, Example 1273, Example 1274, Example 1275, Example 1276, Example 1277, Example 1278, Example 1279, Example 1280. Example 1281, Example 1282, Example 1283. Example 1284. Example 1285, Example 1286, Example 1287, Example 1288, Example 1289, Example 1290, Example 1291, Example 1292, Example 1293, Example 1294, Example 1295, Example 1296, Example 1297, Example 1298, Example 1299, Example 1300, Example 1301, Example 1302, Example 1303, Example 1304, Example 1305, Example 1306. Example 1307, Example 1308, Example 1309, Example 1310, Example 1311. Example 1312. Example 1313. Example 1314, Example 1315, Example 1316, Example 1317, Example 1318, Example 1319, Example 1320, Example 1321, Example 1322, Example 1323, Example 1324, Example 1325, Example 1326, Example 1327, Example 1328, Example 1329, Example 1330,Example 1331, Example 1332, Example 1333, Example 1334, Example 1335, Example 1336, Example 1337, Example 1338. Example 1339. Example 1340, Example 1341, Example 1342, Example 1343, Example 1344, Example 1345, Example 1346, Example 1347, Example 1348, Example 1349, Example 1350, Example 1351, Example 1352, Example 1353, Example 1354, Example 1355, Example 1356, Example 1357, Example 1358, Example 1359, Example 1360, Example 1361. Example 1362, Example 1363, Example 1364, Example 1365, Example 1366. Example 1367. Example 1368. Example 1369, Example 1370, Example 1371, Example 1372, Example 1373, Example 1374, Example 1375, Example 1376, Example 1377, Example 1378, Example 1379, Example 1380, Example 1381, Example 1382, Example 1383. Example 1384, Example 1385, Example 1386, Example 1387, Example 1388, Example 1389. Example 1390, Example 1391, Example 1392, Example 1393, Example 1394, Example 1395, Example 1396, Example 1397, Example 1398, Example 1399, Example 1400, Example 1401, Example 1402, Example 1403, Example 1404, Example 1405, Example 1406, Example 1407, Example 1408, Example 1409, Example 1410, Example 1411. Example 1412, Example 1413, Example 1414. Example 1415. Example 1416. Example 1417, Example 1418, Example 1419, Example 1420, Example 1421, Example 1422, Example 1423, Example 1424, Example 1425, Example 1426, Example 1427, Example 1428, Example 1429, Example 1430, Example 1431, Example 1432, Example 1433, Example 1434, Example 1435, Example 1436, Example 1437. Example 1438. Example 1439, Example 1440, Example 1441 , Example 1442, Example 1443, Example 1444, Example 1445, Example 1446, Example 1447, Example 1448, Example 1449, Example 1450, Example 1451, Example 1452, Example 1453, Example 1454, Example 1455, Example 1456. Example 1457, Example 1458, Example 1459. Example 1460. Example 1461, Example 1462, Example 1463, Example 1464, Example 1465, Example 1466, Example 1467, Example 1468, Example 1469, Example 1470, Example 1471, Example 1472, Example 1473, Example 1474, Example 1475, Example 1476, Example 1477, Example 1478, Example 1479, Example 1480, Example 1481, Example 1482. Example 1483, Example 1484, Example 1485, Example 1486, Example 1487. Example 1488. Example 1489. Example 1490, Example 1491, Example 1492, Example 1493, Example 1494, Example 1495, Example 1496, Example 1497, Example 1498, Example 1499, Example 1500, Example 1501, Example 1502, Example 1503, Example 1504, Example 1505, Examplel506,Example 1507, Example 1508, Example 1509, Example 1510, Example 1511, Example 1512, Example 1513, Example 1514. Example 1515. Example 1516, Example 1517, Example 1518, Example 1519, Example 1520, Example 1521, Example 1522, Example 1523, Example 1524, Example 1525, Example 1526, Example 1527, Example 1528, Example 1529, Example 1530, Example 1531, Example 1532, Example 1533, Example 1534, Example 1535, Example 1536, Example 1537. Example 1538, Example 1539, Example 1540, Example 1541, Example 1542. Example 1543. Example 1544. Example 1545, Example 1546, Example 1547, Example 1548, Example 1549, Example 1550, Example 1551, Example 1552, Example 1553, Example 1554, Example 1555, Example 1556, Example 1557, Example 1558, Example 1559. Example 1560, Example 1561, Example 1562, Example 1563, Example 1564, Example 1565. Example 1566, Example 1567, Example 1568, Example 1569, Example 1570, Example 1571, Example 1572, Example 1573, Example 1574, Example 1575, Example 1576, Example 1577, Example 1578, Example 1579, Example 1580, Example 1581, Example 1582, Example 1583, Example 1584, Example 1585, Example 1586, Example 1587. Example 1588, Example 1589, Example 1590. Example 1591. Example 1592. Example 1593, Example 1594, Example 1595, Example 1596, Example 1597, Example 1598, Example 1599, Example 1600, Example 1601, Example 1602, Example 1603, Example 1604, Example 1605, Example 1606, Example 1607, Example 1608, Example 1609, Example 1610, Example 1611, Example 1612, Example 1613. Example 1614. Example 1615, Example 1616, Example 1617, Example 1618, Example 1619, Example 1620, Example 1621 , Examplel622, Example 1623, Example 1624, Example 1625, Example 1626, Example 1627, Example 1628, Example 1629, Examplel630, Example 1631, Example 1632, Example 1633, Example 1634, Example 1635, Example 1636. Example 1637, Example 1638, Example 1639, Example 1640, Example 1641, or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 wherein the compound shows activity less than or equal to 0.25 nM in the LAG-3 cell binding assay.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 2 as shown in the following tables:Table 2Table 3Table 4Table 7Table 8Table 9Table 104. A pharmaceutical composition comprising one or more compounds according to claim 1 in a pharmaceutically acceptable carrier.

5. A pharmaceutical composition comprising one or more compounds according to 5 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.

Images