Macrocyclic peptides

Abstract

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

Description

[0001] MACROCYCLIC PEPTIDES CROSS REFERENCE This application claims the benefit of U.S. Provisional Application No. 63 / 564,718 filed March 13, 2024 which is incorporated herein in its entirety. BACKGROUND The present disclosure provides novel macrocyclic peptides which inhibit the mouse LAG-3 / MHC Class II protein / protein interaction and are thus useful for the amelioration of various diseases, including cancer and infectious diseases. Lymphocyte activation gene-3 (LAG-3; LAG3; CD223) is a 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-like 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 II and its function is unknown. LAG-3 is composed of the intracellular signalling domain, a transmembrane domain and 4 extracellular domains, designated Dl to D4 (Huard 1997 Proc. Natl. Acad. Sci.94:5744-9). Domain 1-2 associates with MHC class Il ligand and it has been shown that the tip of domain 1 (extra loop) forms the binding site (Huard 1997 Proc. Natl. Acad. Sci.94:5744-9). LAG-3 can also associate with alternative ligands, Galectin-3 and LSECtin, which induce its inhibitory signalling (Kouo 2015 Cancer Immunol Res.3(4):412-23; Xu 2014 Cancer Res 74(13):3418-28). Association with Galectin-3 on cells or within the extracellular matrix could downregulate T cells that would not normally engage with MHC class II, such as CD8+ T cells. Therefore blockade of this ligand could serve as a mechanism for enhancing broad T cell function. A role of LAG-3 on T cells is to regulate T cell activation (Huard 1994 Eur. J. Immunol.24:3216- 21). LAG-3 engages with MHC class Il and this leads to down regulation of CD4+ T cells (Huard 1996 Eur. J. Immunol.26:1180-6). Upon T cell activation, LAG-3 surface expression increases. The engagement of LAG-3 dimer with ligand induces signalling through an intracellular KIEELE domain (Workman 2002 J. Immunol 169:5392-5) leading to downregulation of the T cell activity. Therefore, LAG-3 serves to modulate responses to antigens, preventing over-stimulation and maintaining immune homeostasis. It has been reported that LAG-3 plays an important role in promoting regulatory T cell (Treg) activity and in negatively regulating T cell activation and proliferation (Workman CJ, et al., J. Tmmunok 2005; 174:688-695). Both natural and induced Treg express increased LAG-3, which is required for their maximal suppressive function (Camisaschi C, et al., J. Tmmunok 2010; 184:6545-6551 and Huang CT, et al, Immunity. 2004; 21:503-513). Furthermore, ectopic expression of LAG-3 on CD4+ effector T cells reduced their proliferative capacity and conferred on them regulatory potential against third party T cells (Huang CT, et al, Immunity.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 (Blackburn SD, et ak, Nat. Tmmunok 2009; 10:29-37 and Grosso JF, et ak, J. Clin. Invest.2007; 117:3383-3392). In fact, LAG-3 maintained tolerance to self and tumor antigens via direct effects on CD8+T cells in 2 murine models (Grosso JF, et ak, J. Clin. Invest.2007; 117:3383-3392). Epstein-Barr virus infection is yet another factor to consider in the potential induction of T cell exhaustion in hematological malignancies. It is known that EBVassociated CLL, Richter’s syndrome, and lymphoma cases are usually more aggressive than their EBV(-) counterpart (Tsimberidou AM, et al., Leuk Lymphoma 2006;47:827; Ansell SM, et al., Am J Hematol 1999;60:99.; Dolcetti R, et al., Infectious Agents and Cancer 2010;5:22; Kanakry JA, et al., Blood 2013;121:3547). Interestingly, the expression of checkpoint inhibitors like PD-Ll 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 (Blackburn 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;1 l(Suppl 9):S5; Kotaskova J, et al., J Mol Diagn 2010;12(3):328— 334). LAG-3 expression on tumor-infiltrating lymphocytes (TILs) and peripheral blood also mediates T cell exhaustion in hematological malignancies (Dickinson JD, et al., Leuk Lymphoma 2006;47(2):231-44). Moreover, LAG-3 blockade with specific antibodies has shown antitumor activity in leukemia (Berrien-Elliott, M, et al., Cancer Research 2013; 73(2):605-616) and solid tumor models (Woo, S-R, et al., Cancer Research 2011; 72(4):917-927; Coding, S. R., et al., Journal of Immunology, Baltimore, Md.1950; 190(9):4899-909). Therefore, LAG-3 is a potential therapeutic target in hematological malignancies. Recent preclinical studies have documented a role for LAG-3 in CD8 T cell exhaustion, and blockade of the LAG-3 / MHC Class II interaction using LAG-3 blocking antibodies or LAG-3-Ig fusion proteins is being evaluated in a number of clinical trials in cancer patients. Additional background information can be found in WO2015 / 042246 A1, WP2015 / 116539 A1, and WO2014 / 008218 A1. LAG-3 blockade with macrocyclic peptide inhibitors, alone and in combination with standard of care (e.g., nivolumab, imatinib, lenalidomide) or with other checkpoint inhibitors deserves further exploration. The molecules described herein demonstrate the ability to block the interaction of LAG-3 with MHC Class II, in both biochemical and cell-based experimental systems. These results are consistent with a potential for therapeutic administration to enhance immunity in cancer or chronic infection, including therapeutic vaccine. The macrocyclic peptides described herein are capable of inhibiting the interaction of mouse Lag-3 with MHC class II. These compounds have demonstrated highly efficacious binding to mouse LAG-3, blockade of the interaction of mouse LAG-3 with MHC Class II, and are capable of promoting enhanced T cell functional activity, thus making them candidates for proof of concept studies in murine animal models. The present disclosure provides the compounds exemplified herein. In one embodiment the present disclosure provides a method blocking the interaction of mouse 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. Definitions The definitions provided herein apply, without limitation, to the terms as used throughout this specification, unless otherwise limited in specific instances. Those of ordinary skill in the art of amino acid and peptide chemistry are aware that an amino acid includes a compound represented by the general structure: COOH COOH R w Unless otherwise indicated, the term "amino acid" as employed herein, alone or as part of another group, includes, without limitation, an amino group and a carboxyl group linked to the same carbon, referred to as "α" carbon, where R and / or R′ can be a natural or an un-natural side chain, including hydrogen. The absolute "S" configuration at the "α" carbon is commonly referred to as the "L" or "natural" configuration. In the case where both the "R" and the "R′"(prime) substituents equal hydrogen, the amino acid is glycine and is not chiral. The term “naturally occurring amino acid side chain,” as used herein, refers to side chain of any of the naturally occurring amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,-histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) usually in the S-configuration (i.e., the L-amino acid). The term “non-naturally occurring amino acid side chain,” as used herein, refers to a side chain of any naturally occurring amino acid usually in the R-configuration (i.e., the D-amino acid) or to a group other than a naturally occurring amino acid side chain in R- or S-configuration (i.e., the D- or L-amino acid, respectively). 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 IC50to IC90, and are preferably, IC50, IC60, IC70, IC80, or IC90, which represent 50%, 60%, 70%, 80% or 90% reduction in LAG-3 / MHC Class II molecules binding activity, respectively. More preferably, the "inhibitory concentration" is measured as the IC50 value. It is understood that another designation for IC50is the half-maximal inhibitory concentration. Binding of the macrocyclic peptides to LAG-3 can be measured, for example, by methods such as homogeneous time-resolved fluorescence (HTRF), Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC), nuclear magnetic resonance spectroscopy (NMR), and the like. Further, binding of the macrocyclic peptides to LAG- 3 expressed on the surface of cells can be measured as described herein in cellular binding assays. Peptide Synthesis The description of the present disclosure herein should be construed in congruity with the laws and principals of chemical bonding. It should be understood that the compounds encompassed by the present disclosure are those that are suitably stable for use as pharmaceutical agent. One of skill in the art will know what compounds would and would not be stable based on the general principles of chemical bonding and stability. 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, semi- synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. A preferred method to synthesize the macrocyclic peptides and analogs thereof described herein is chemical synthesis using various solid-phase techniques such as those described in Chan, W.C. et al, eds., Fmoc Solid Phase Synthesis, Oxford 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-fluorenylmethyloxycarbonyl) group (Fmoc) for temporary protection of the ^-amino group, in combination with the tert-butyl group (tBu) for temporary protection of the amino acid side chains (see for example Atherton, E. et al, "The Fluorenylmethoxycarbonyl Amino Protecting Group", in The Peptides: Analysis, Synthesis, Biology, Vol.9 : "Special Methods in Peptide Synthesis, Part C", pp.1-38, Undenfriend, S. et al, eds., Academic Press, San Diego (1987). The peptides can be synthesized in a stepwise manner on an insoluble polymer support (also referred to as "resin") starting from the C-terminus of the peptide. A synthesis is begun by appending the C-terminal amino acid of the peptide to the resin through formation of an amide or ester linkage. This allows the eventual release of the resulting peptide as a C-terminal amide or carboxylic acid, respectively. The C-terminal amino acid and all other amino acids used in the synthesis are required to have their ^-amino groups and side chain functionalities (if present) differentially protected such that the ^-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 ^-amino group of the N-terminal amino acid appended to the resin. The sequence of ^-amino group deprotection and coupling is repeated until the entire peptide sequence is assembled. The peptide is then released from the resin with concomitant deprotection of the side chain functionalities, usually in the presence of appropriate scavengers to limit side reactions. The resulting peptide is finally purified by reverse phase HPLC. The synthesis of the peptidyl-resins required as precursors to the final peptides utilizes commercially available cross-linked polystyrene polymer resins (Novabiochem, San Diego, CA; Applied Biosystems, Foster City, CA). Preferred solid supports are: 4- (2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methyl benzhydrylamine resin (Rink amide MBHA resin); 9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin); 4-(9-Fmoc)aminomethyl-3,5- dimethoxyphenoxy)valerylaminomethyl-Merrifield resin (PAL resin), for C-terminal carboxamides. Coupling of first and subsequent amino acids can be accomplished using HOBt, 6-Cl-HOBt or HOAt active esters produced from DIC / HOBt, HBTU / HOBt, BOP, PyBOP, or from DIC / 6-C1-HOBt, HCTU, DIC / HOAt or HATU, respectively. Preferred solid supports are: 2-chlorotrityl chloride resin and 9-Fmoc-amino-xanthen-3-yloxy- Merrifield resin (Sieber amide resin) for protected peptide fragments. Loading of the first amino acid onto the 2-chlorotrityl chloride resin is best achieved by reacting the Fmoc- protected amino acid with the resin in dichloromethane and DIEA. If necessary, a small amount of DMF may be added to solubilize the amino acid. The syntheses of the peptide analogs described herein can be carried out by using a single or multi-channel peptide synthesizer, such as an CEM Liberty Microwave synthesizer, or a Protein Technologies, Inc. Prelude (6 channels) or Symphony (12 channels) or Symphony X (24 channels) synthesizer. Useful Fmoc amino acids derivatives are shown below. Examples of Orthogonally Protected Amino Acids used in Solid Phase Synthesis

[0002] deprotected using any standard procedure (see, for example, King, D.S. et al, Int. J. Peptide Protein Res., 36:255-266 (1990)). A desired method is the use of TFA in the presence of water, TIS as scavenger, and DTT or TCEP as the disulfide reducing agent. Typically, the peptidyl-resin is stirred in TFA / water / TIS / DTT (94:3:3:1), v:v:v:w; 1 mL / 100 mg of peptidyl resin) for 1.5-3 hrs at room temperature. The spent resin is then filtered off and the TFA solution was cooled and Et2O solution was added. The precipitates were collected by centrifuging and decanting the ether layer (3 x). The resulting crude peptide is either redissolved directly into DMF or DMSO or CH3CN / H2O for purification by preparative HPLC or used directly in the next step. Peptides with the desired purity can be obtained by purification using preparative HPLC, for example, on a Waters Model 4000 or a Shimadzu Model LC-8A liquid chromatography. The solution of crude peptide is injected into a YMC S5 ODS (20 x 100 mm) column and eluted with a linear gradient of MeCN in water, both buffered with 0.1% TFA, using a flow rate of 14-20 mL / min with effluent monitoring by UV absorbance at 220 nm. The structures of the purified peptides can be confirmed by electro-spray MS analysis. Analytical Data: Mass Spectrometry: “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 “m / z” unit designation. Compounds with exact masses greater than 1000 were often detected as double-charged or triple-charged ions. The crude material was purified via preparative LC / MS. Fractions containing the desired product were combined and dried via centrifugal evaporation. Analytical LC / MS Condition A: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7-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; Temperature: 50 °C; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL / min; Detection: UV at 220 nm. Analytical LC / MS Condition B: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50 °C; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL / min; Detection: UV at 220 nm. Analytical LC / MS Condition C: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7-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; Temperature: 70 °C; Gradient: 0-100% B over 3 minutes, then a 2.0-minute hold at 100% B; Flow: 0.75 mL / min; Detection: UV at 220 nm. .Analytical LC / MS Condition D: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 70 °C; Gradient: 0-100% B over 3 minutes, then a 2.0-minute hold at 100% B; Flow: 0.75 mL / min; Detection: UV at 220 nm. Analytical LC / MS Condition E: Column: Kinetex XB C18, 3.0 x 75 mm, 2.6-μm particles; Mobile Phase A: 10 mM ammonium formate in water:acetonitrile (98:2); Mobile Phase B: 10 mM ammonium formate in Water:acetonitrile (02:98); Gradient: 20- 100% B over 4 minutes, then a 0.6-minute hold at 100% B; Flow: 1.0 mL / min; Detection: UV at 254 nm. Analytical LC / MS Condition F: Column: Ascentis Express C18, 2.1 x 50 mm, 2.7-μm particles; Mobile Phase A: 10 mM ammonium acetate in water:acetonitrile (95:5); Mobile Phase B: 10 mM ammonium acetate in Water:acetonitrile (05:95), Temperature: 50oC; Gradient: 0-100% B over 3 minutes; Flow: 1.0 mL / min; Detection: UV at 220 nm. Analytical LC / MS Condition G: Column: X Bridge C18, 4.6 x 50 mm, 5-μm particles; Mobile Phase A: 0.1% TFA in water; Mobile Phase B: acetonitrile, Temperature: 35oC; Gradient: 5-95% B over 4 minutes; Flow: 4.0 mL / min; Detection: UV at 220 nm. The following abbreviations may be employed in the Examples and elsewhere herein: Ph = phenyl Bn = benzyl i-Bu = iso-butyl i-Pr = iso-propyl Me = methyl Et = ethyl Pr = n-propyl Bu = n-butyl t-Bu = tert-butyl Trt = trityl TMS = trimethylsilyl TIS =triisopropylsilane Et2O = diethyl ether HOAc or AcOH = acetic acid MeCN or AcCN = acetonitrile DMF = N,N-dimethylformamide EtOAc = ethyl acetate THF = tetrahydrofuran TFA = trifluoroacetic acid TFE = α,α,α-trifluoroethanol Et2NH = diethylamine NMM = 4-methylmorpholine NMP = N-methylpyrrolidone DCM = dichloromethane TEA = triethylamine min. = minute(s) h or hr = hour(s) L = liter mL or ml = milliliter ^L = microliter g = gram(s) mg = milligram(s) mol = mole(s) mmol = millimole(s) meq = milliequivalent rt or RT = room temperature sat or sat'd = saturated aq. = aqueous mp = melting point BOP reagent = benzotriazol-1-yloxy-tris-dimethylamino-phosphonium hexafluorophosphate (Castro's reagent) PyBOP reagent = benzotriazol-1-yloxy-tripyrrolidino phosphonium hexafluorophosphate HBTU = 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronim hexafluorophosphate HATU = O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronim hexafluorophosphate HCTU = 2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate T3P = 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide DMAP = 4-(dimethylamino)pyridine DIEA = diisopropylethylamine Fmoc or FMOC = fluorenylmethyloxycarbonyl Boc or BOC = tert-butyloxycarbonyl HOBT or HOBT^H2O = 1-hydroxybenzotriazole hydrate Cl-HOBt = 6-Chloro-benzotriazole HOAT = 1-hydroxy-7-azabenzotriazole HPLC = high performance liquid chromatography LC / MS = high performance liquid chromatography / mass spectrometry MS or Mass Spec = mass spectrometry NMR = nuclear magnetic resonance Sc or SC or SQ = sub-cutaneous IP or ip = intra-peritoneal General Procedures: All manipulations were performed under automation on a Prelude Prelude,or a Symphony, or Symphony X peptide synthesizer (Protein Technologies). All procedures were performed according to the published methods (e.g., WO 2023 / 225661). Prelude: Resin-swelling procedure, Single-coupling procedure, Single-coupling extended time procedure, Chloroacetic Anhydride coupling, Single-Coupling Manual Addition Procedure A, Single-Coupling Manual Addition Procedure B, Manual removal of Fmoc group procedure: Symphony: Resin-swelling procedure, Single-coupling procedure, Single-coupling extended time procedure, Double-coupling extended time procedure, Chloroacetic Anhydride coupling: Symphony X: Resin-swelling procedure, Single-coupling procedure, Single-coupling 3 deprotections procedure, Single-coupling extended time procedure, Single-coupling 3 deprotections extended time procedure, Pre-activated single-coupling procedure, Single- Coupling Manual Addition Procedure A, Single-Coupling Manual Addition Procedure B: Chloroacetic Anhydride coupling, Final rinse and dry procedure. The following procedures were performed according to the published methods (e.g., WO 2023 / 225661). Global Deprotection Method, Cyclization Method, N-Methylation on-Resin Method A, N- Methylation On-resin Method B (Turner, R.A. et al, Org. Lett., 15(19):5012-5015 (2013)), N-Alkylation On-resin Procedure Method A, N-Alkylation On-resin Procedure Method B, N-Nosylate Formation Procedure, N-Nosylate Removal Procedure, General Procedure for Preloading amines on the PL-FMP resin, General Procedure for Preloading Fmoc-Amino Acids on Cl-trityl resin, Click Reaction On-Resin Method A, Click Reaction On-Resin Method B, Suzuki Reaction On-resin Procedure, Fatty acid chain coupling procedure A, Fatty acid chain coupling procedure B, General Purification Procedures. Unnatural Fmoc-Amino Acid Synthesis amd fatty acid tails were prepared according to the published methods (e.g., WO 2023 / 225661). To a 45-mL polypropylene solid-phase reaction vessel was added chlorotrityl resin preloaded with FmocNH-(CH2)10COOH (0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially: “Symphony Method: Resin-swelling procedure” was followed; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Glu-OtBu; “Symphony Method: Single-coupling procedure” was followed with Fmoc-PEG9-OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Glu-OtBu; “Symphony Method: Single-coupling procedure” was followed with Fmoc- PEG 9-OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc- Asp(OtBu)- OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc- Cys(Trt)-OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Bip(4,4’)-OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Bip(4,4’)-OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc- Tyr(OtBu)- OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Tyr(OtBu)- OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Asn(tBu)-OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-D-Pro-OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Asp(OtBu)- OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Asp(OtBu)- OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Trp(Boc)- OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Bip(3’- COOtBu)-OH; “Symphony Method: Single-coupling procedure” was followed with Fmoc-Phe-OH; Deprotecting reagent (DDT:TIS:H2O=1:1:1 ratio) in TFA was used to treat the resin for 2hs and the desired sequence was precipitated in Et2O (25 mLx3); The desire macrocyclic peptide was formed by dissovling in the above solids in DMF with DIEA (0.1M) and shaking at RT overnight. The crude material was purified via preparative LC / MS with the following conditions: Column: XBridge C18, 30 x 150 mm, 5-μm 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: 20-70% 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 material was further 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 13.0 mg, and its estimated purity by LCMS analysis was 100%. Analysis condition A: Retention time = 1.14 min; ESI-MS(+) m / z [M+2H]2+: 1412.2. Analysis condition B: Retention time = 1.73 min; ESI-MS(+) m / z [M+2H]2+: 1412.2. the Symphony peptide synthesizer. The crude product was obtained according to similar procedures for Example 1001. The crude material was purified via preparative LC / MS with the following conditions: Column: XBridge C18, 200 mm x 30 mm, 5-μm 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: a 0-minute hold at 24% B, 24-64% B over 20 minutes, then a 2-minute hold at 100% B; Flow Rate: 45 mL / min; Column Temperature: 25 C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 25.6 mg, and its estimated purity by LCMS analysis was 95%. Analysis condition A: Retention time = 1.21 min; ESI-MS(+) m / z [M+2H]2+: 1368.3. Analysis condition B: Retention time = 1.80 min; ESI-MS(+) m / z [M+2H]2+: 1368.0. Step 2: To a solution of (2S,5S,19S,33S)-2- ((3S,6S,9S,12S,15S,18R,24R,27S,30S,33S,36S,41aS)-30-((1H-indol-3-yl)methyl)-12,15- bis([1,1'-biphenyl]-4-ylmethyl)-3-(2-amino-2-oxoethyl)-24-benzyl-27-((4'-carboxy-[1,1'- biphenyl]-4-yl)methyl)-33,36-bis(carboxymethyl)-6,9-bis(4-hydroxybenzyl)- 1,4,7,10,13,16,22,25,28,31,34,37-dodecaoxooctatriacontahydro-1H,21H-pyrrolo[1,2- s][1]thia[4,7,10,13,16,19,22,25,28,31,34,37]dodecaazacyclononatriacontine-18- carboxamido)-3,8,17,22,31-pentaoxo-12,15,26,29-tetraoxa-4,9,18,23,32- pentaazahexatriacont-35-yne-1,5,19,33-tetracarboxylic acid (60 mg, 0.022 mmol) and 3- (2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (8.27 mg, 0.026 mmol) in DMF (1 mL) was added a mixture of sodium sodium (R)-2-((S)-1,2-dihydroxyethyl)-4- hydroxy-5-oxo-2,5-dihydrofuran-3-olate (14.78 mg, 0.075 mmol) andcopper(II) sulfate pentahydrate (6.57 mg, 0.026 mmol) in 0.3 mL H2O. The reaction mixture was stirred at rt for 1h. The solvent was removed, and the residue was dissolved in DMF (1 mL) and submitted to RP HPLC purification. The crude material was purified via preparative LC / MS with the following conditions: Column: XBridge C18, 200 mm x 30 mm, 5-μm 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: a 0-minute hold at 25% B, 25-65% B over 20 minutes, then a 2-mmute hold at 100% B; Flow Rate: 45 mL / min; Column Temperature: 25 C. Fracton collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC / MS with the following

[0003] 5 conditions: Column: XBridge Cl 8, 200 mm x 30 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: a 0-minute hold at 24% B, 24-64% B over 20 minutes, then a 2 -minute hold at 100% B; Flow Rate: 45 mL / min; Column Temperature: 25 C. Fraction collection was triggered by MS and UV signals. Fractions

[0004] 10 containing the desired product were combined and dried via centrifugal evaporation.

[0005] The yield of the product was 10.0 mg, and its estimated purity by LCMS analysis was 93%.

[0006] Analysis condition A: Retention time 1.14 mm; ESI-MS(+) m / z | M+3H |3: 1017.0.

[0007] Analysis condition B: Retention time 1.75 mm; ESI-MS(+) m / z [M+2H]2+: 1016.9.

[0008] 15

[0009] The following examples were prepared using the procedures similar to those in the preparation of Examples 1001, 1002 and intermediates.

[0010] - 17 -

[0011] SUBSTITUTE SHEET (RULE 26)

[0012]

[0013] Method

[0014] Mouse LAG- 3 cell binding assay: 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 (Corning 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 µg / 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 an 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.

Claims

CLAIMS We claim:

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

2. A compound according to claim 1 wherein the compound shows activity less than or equal to 0.1 µM in the mouse LAG-3 cell binding assay.

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

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

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