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WO2025193573A1 - Peptides macrocycliques utiles comme immunomodulateurs - Google Patents

Peptides macrocycliques utiles comme immunomodulateurs

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Publication number
WO2025193573A1
WO2025193573A1 PCT/US2025/019111 US2025019111W WO2025193573A1 WO 2025193573 A1 WO2025193573 A1 WO 2025193573A1 US 2025019111 W US2025019111 W US 2025019111W WO 2025193573 A1 WO2025193573 A1 WO 2025193573A1
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WIPO (PCT)
Prior art keywords
phe
lag
amino acid
symphony
procedure
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English (en)
Inventor
Jennifer X. Qiao
Tammy C. Wang
Michael A. Poss
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure provides novel macrocyclic peptides which inhibit the LAG-3/MHC Class II protein/protein interaction, and are thus useful for the amelioration of various diseases, including cancer and infectious diseases.
  • Lymphocyte activation gene-3 (LAG-3; LAG3; CD223) is a 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..
  • 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 II and its function is unknown.
  • MHC major histocompatibility complex
  • LAG-3 is composed of the intracellular signalling domain, a transmembrane domain and 4 extracellular domains, designated DI to D4 (Huard 1997 Proc. Natl. Acad. Sci. 94:5744-9).
  • Domain 1-2 associates with MHC class II ligand and it has been shown that the tip of domain 1 (extra loop) forms the binding site (Huard 1997 Proc. Natl. Acad. Sci. 94:5744-9).
  • LAG-3 can also associate with alternative ligands. Galectin-3 and LSECtin. which induce its inhibitory signalling (Kouo 2015 Cancer Immunol Res. 3(4):412-23; Xu 2014 Cancer Res 74(13):3418-28). Association with Galectin-3 on cells or within the extracellular matrix could downregulate T cells that would not normally engage with MHC class II, such as CD8+ T cells. Therefore blockade of this ligand could serve as a mechanism for enhancing broad T cell function.
  • LAG-3 plays an important role in promoting regulatory T cell (Treg) activity and in negatively regulating T cell activation and proliferation (Workman CJ, et al., J. Tmmunok 2005; 174:688-695). Both natural and induced Treg express increased LAG-3, which is required for their maximal suppressive function (Camisaschi C. et al., J. Tmmunok 2010; 184:6545-6551 and Huang CT, et al, Immunity. 2004: 21 :503-513). Furthermore, ectopic expression of LAG-3 on CD4+ effector T cells reduced their proliferative capacity and conferred on them regulatory potential against third part) 7 T cells (Huang CT, et al, Immunity 7 .
  • 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).
  • LAG-3 expression 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).
  • 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.
  • 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.
  • standard of care e.g., nivolumab. imatinib, lenalidomide
  • 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.
  • 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.
  • R b , R d , and R k are each independently hydrogen or methyl
  • R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and R lj are independently selected from a natural amino acid side chain and an unnatural amino acid side chain or R 6 , R 7 , R 9 , and R 1 ’ form a ring with the corresponding vicinal R group as described below;
  • R f is hydrogen or methyl, or R f and R 6 together with the atoms to which they are attached may form a ring selected from either the D- 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 mino, cyano, methyl, halo, and hydroxy;
  • R g is hydrogen or methyl, or R g and R 7 together with the atoms to which they are attached may 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;
  • R 1 is hydrogen or methyl, or R 1 and R 9 . together with the atoms to which they are attached, may 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;
  • R m is hydrogen or Ci-6 alky l, or R m and R 13 , together with the atoms to which they are attached, may form a ring selected from 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, methy l, halo, hydroxy , and phenyl; and wherein the pyrrolidine and the piperidine ring are optionally fused to a cyclohexyl, phenyl, or indole group;
  • R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 , R 12 and R 13 groups contains a fatty acid tail which is selected from a natural or unnatural amino acid side chain optionally substituted by the following: wherein:
  • X’ is a chain of between 8 and 60 atoms wherein the atoms are selected from carbon and oxygen and wherein the chain may contain one, two, three, or four groups selected from -NHC(O)-, -C(O)NH- and -NHC(O)NH-, embedded therein; and wherein the chain is optionally substituted with one to six groups independently selected from - CO 2 H, -C(O)NH 2 , and -(CH 2 )I- 2 CO 2 H, provided that X’ is other than unsubstituted PEG;
  • R 30 is selected from -CO2H, -C(O)NH2, -CH3, tetrazole, and lithocolic acid and analogs of lithocolic acid;
  • R 15 is selected from OH, 0-20 PEG units and NR 18 R 19 , wherein R 18 and R 19 can be independently selected from, hydrogen, C1-3 alkyl, (Ci-3)alkyl-C5-6-carbocycle or heterocycle wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy,
  • the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-Phe.
  • R b is methyl, the R 2 group is selected from the sidechain of G L-Ala, L-Phe, and L-Tyr;
  • R 4 group is selected from the sidechain of Gly, L-Ala, L-Asp, L-Ser, and L-Tyr;
  • R 7 group is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-4-Pya, L-tertbutylGly, L-Asp, L-Glu, L- Dap, L-Dab, L-Phe(3-COOH), L-Lys, Lys(COMe), L-Asn, L-Om, L-Pra, L-Arg, L-Ser, L- Tyr(propargyl), and L-Trp(CH2COOH); alternatively when R s is methyl, the R 7 group is selected from the sidechain of Gly, L-Ala, L-Asp, L-Ser, and L-Tyr; alternatively, R 7 and R g together with the atoms attached form a pyrrolidine ring;
  • the R 9 group is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L- Bn Gly, D-Pip, Gly.
  • R 10 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-Trp, L-Trp(7-Me), L-Trp(7-F), and L-Trp(Me);
  • R 11 group is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-l-Nal.
  • L-2-Nal L-Ala(P-indol-4- yl).
  • L-3-Pya L-Bip, L-Bip(4 ? -Py).
  • L-Tyr L-Tyr(Me). L-Tyr(propargyl), L-Tyr(Bn), L-Tyr(CH2COOH); alternatively when R k is methyl, the R 11 group is selected from the sidechain of L- Tyr or L-Trp;
  • R 12 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting L-Phe, L-Trp, and L-Tyr;
  • R 13 group is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-4-Pya, L-3-Pya, L-Ala, L-Ala(CN), L-Abu, L-cyclopropylalanine, L-Asp, L-Dap, L-Dab, L-Glu, L-Phe(4-CONH2), L-Phe(4- COOH), L-Phe(3-COOH), L-His, L-tetrazolyl-alanine, L-homoSer(Me), L-Lys, L-Asn, L-Nva, L-Pra, L-Arg, L-Ser, L-Tic, L-Tyr(CH2COOH).
  • a naturally or nonnaturally occurring amino acid consisting of L-4-Pya, L-3-Pya, L-Ala, L-Ala(CN), L-Abu, L-cyclopropylalanine, L-Asp
  • R m is methyl
  • the R 13 group is selected from the sidechain of Gly, L-Ala, L-Glu
  • R m is n-butyl or -CH2COOH
  • the R 13 group is selected from the sidechain of Gly
  • R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 , R 12 and R 13 groups contains a fatty acid tail which is selected from a natural or unnatural amino acid side chain optionally substituted by the following: -(C(R 17a )2)i-3-phenyl-(C(R 17a )2)o-3-NHCO-X’-R 30 ,
  • R 18 , R 19 and R 20 are each independently selected from H, (CH2)I-4, sidechains of the following amino acid: Gly, L-Ala, L-Glu, L-Asp, L-Lys, D-Arg, L-Dap, L-Dab, L- Om, L-Asn, D-Ser, L-Pra, D-Dap, D-Dab, D-Om, L- m Leu, L-Pro, D-Pro, L-Trp, L- m Ala, L-Tic, D-Asp, L- m Phe, D-Arg, D-Lys, L-Phe, D-Asn, D-Om, L-Tic, and D-Ala; when NR 18 R 19 is at the C-terminal.
  • R 18 and R 19 are independently selected from H, C 1-6 alkyl, (CH2)i-4cycloalkyl, (CH2)I-4 5-6 carbocycles and heterocycles;
  • R 21 , R 22 and R 23 group are independently selected from H, sidechains of the following amino acid: L-y-Glu, L-Dab, L-Dap, D-Arg, L-Glu, Gly, L-Ser L-Om, L-Pra;
  • Preferred PEG spacer -(CH2CH20)I-IOCH2CH 2 OCH 2 CO-;
  • the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein
  • R 1 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-Phe, L-Phe(2-F), L-Phe(3-F), L-Phe(4-F), L-Phe(3,4,5-tri-F), L-Bzt, L- ⁇ -pheny 1 -pheny 1 al an ine ;
  • the R 4 group is selected from the sidechain of a naturally or nonnaturally occurnng amino acid consisting of L-Ala, L-Asp, D-Asp, L-Dap, L-Dab, L-Glu, L-Phe, L-Phe(3-COOH), L-Phe(4-COOH), L-His, L-homo-Glu, L-Lys, L-Lys(COCH3), L-Asn, L-Om, L-Pra, L-Arg, L-Trp(CH 2 COOH), L-Tyr(CH 2 COOH), and L-Tyr(propargyl); alternatively when R d is methyl, the R 4 group is selected from the sidechain of L- Ala.
  • L-Asp. and L-Ser; R? is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of D-Ala.
  • R 7 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-4-Pya, L-tertbutylGly, L-Asp, L-Glu, L-Dap, L-Dab, L-Phe(3-COOH), L-Lys, Lys(COMe), L-Asn, L-Om, L-Pra, L-Arg, L- Ser, L-Tyr(propargyl), and L-Trp(CH2COOH); alternatively when R 8 is methyl.
  • R 7 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of Gly, L-Ala, L-Asp, L-Ser, and L-Tyr; alternatively, R7 and Rg together with the atoms attached form a pyrrolidine ring, selecting from the sidechain of L-Pro;
  • R 8 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-Ala, L-Asp, D-Asp, L-Glu, L-Phe(3-COOH), L-3-(2- tetrazolyl)alanine, L-homo-Ser, L-homoSer(Me), L-Asn, and L-Ser;
  • R 10 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-Trp and L-Trp(7-F);
  • R 11 group is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-l-Nal. L-2-Nal. L-Ala(0-indol-4- yl), L-3-Pya, L-Bip, L-Bip(4’-Py), L-Bip(4’-COOH), L-Bip(3’-COOH), L-Bzt, L-Phe(2- Me), L-Phe(3-OH), L-Phe(4-COOH), L-Phe(3-COOH), L-Phe(4-CH 2 NH 2 ), L-alanine(0- quinolin-3-yl), L-alanine(P-quinolin-6-yl).
  • R 12 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting L-Trp and L-Tyr;
  • R 13 is selected from the sidechain of a naturally or nonnaturally occurring amino acid consisting of L-Ala, L-Ala(CN).
  • L-Abu L- cyclopropylalanine, L-Asp, L-Dap, L-Dab, L-Glu.
  • L-Phe(4-COOH) L-Phe(3-C00H)
  • L-His L-tetrazolyl-alanine
  • L-homoSer(Me) L-Lys, L-Asn, L-Nva, L- Pra, L-Arg.
  • R 17 is selected from hydrogen, -(C(R 17a ) 2 )o-4-X’-R 31 and -(CH 2 )z-tnazolyl-X’-R 33 , wherein z is 1-3 and R 35 is selected from -COOH, CH3, tetrazole, -C(O)NH2, and lithocolic acid and analogs;
  • 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.
  • the method further comprises administering an additional agent prior to, after, or simultaneously with the macrocyclic peptide or peptides described herein.
  • the additional agent is an antimicrobial agent, an antiviral agent, a cytotoxic agent, and/or an immune response modifier.
  • 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.
  • 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.
  • NSCLC non-small cell lung cancer
  • colorectal cancer colorectal cancer
  • castration-resistant prostate cancer ovarian cancer
  • gastric cancer hepatocellular carcinoma
  • pancreatic carcinoma squamous cell carcinoma of the head and neck
  • 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.
  • the infectious disease is caused by a virus.
  • the virus is selected from HIV, Hepatitis A, Hepatitis B, Hepatitis C, herpes virus, and influenza.
  • 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.
  • 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.
  • an amino acid includes a compound represented by the general structure: where R and R' are as discussed herein.
  • amino acid as employed herein, alone or as part of another group, includes, without limitation, an amino group and a carboxyl group linked to the same carbon, referred to as "a" carbon, where R and/or R' can be a natural or an un-natural side chain, including hydrogen.
  • the absolute “S” configuration at the "a” carbon is commonly referred to as the “L” or “natural” configuration.
  • the amino acid is glycine and is not chiral.
  • naturally occurring ammo acid side chain 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).
  • 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
  • non-naturally occurring amino acid side chain,’ 7 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 IC50 to IC90, and are preferably, IC50, ICeo, IC70, ICso, or IC90, which represent 50%, 60%, 70%, 80% or 90% reduction in LAG-3/MHC Class II molecules binding activity, respectively. More preferably, the "inhibitory concentration” is measured as the IC50 value. It is understood that another designation for IC50 is the half-maximal inhibitor ⁇ ' 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.
  • HTRF homogeneous time-resolved fluorescence
  • SPR Surface Plasmon Resonance
  • ITC isothermal titration calorimetry
  • NMR nuclear magnetic resonance spectroscopy
  • Administration of a therapeutic agent described herein includes, without limitation, administration of a therapeutically effective amount of therapeutic agent.
  • therapeutically effective amount 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.
  • the disclosure pertains to methods of inhibiting growth of tumor cells in a subject using the macrocyclic peptides of the present disclosure.
  • 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.
  • 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 (e.g., by coadministration of lag03 blocking peptides with an antigen of interest).
  • treatment 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.
  • a condition e.g, a disease
  • 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.
  • 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.
  • 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:
  • the macrocyclic peptide binds to human LAG-3 with an IC50 of 200 nM or less;
  • the macrocyclic peptide binds to human LAG-3 and one or more of the following: cynomolgus monkey LAG-3, and/or mouse LAG-3; (d) the macrocyclic peptide inhibits the binding of LAG-3 to MHC Class II moleucules;
  • the macrocyclic peptide inhibits tumor cell growth in a cellular assay and/or in vivo assay
  • 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.
  • 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.
  • 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.
  • the percent homology 7 between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity 7 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 PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman et al. (J. Mol.
  • 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.
  • 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:
  • the macrocyclic peptide binds to human LAG-3 with an ICso of 200 nM or less;
  • the macrocyclic peptide binds to human LAG-3 and one or more of the following: cynomolgus monkey LAG-3, and/or mouse LAG-3;
  • 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
  • aromatic side chains e.g.
  • 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 (z.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.
  • 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.
  • 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.
  • compositions of the disclosure also can be administered in combination therapy, z.e., combined with other agents.
  • 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.
  • 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.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • 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.
  • 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. Sci., 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.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • 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.
  • a pharmaceutical composition of the disclosure also may include a pharmaceutically acceptable anti-oxidant.
  • 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.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oilsoluble antioxidants such as ascorbyl palmitate, butylated hydroxyani
  • aqueous and nonaqueous carriers examples 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.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • 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.
  • 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.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 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.
  • 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 by measuring 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.
  • 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.
  • 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.
  • 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.
  • 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 history of the patient being treated, and like factors well known in the medical arts.
  • 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.
  • 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 growth and/or HIV can be evaluated in an animal model system predictive of efficacy in human tumors or viral efficacy.
  • this property of a composition can be evaluated by examining the ability of 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 otherwise ameliorate symptoms in a subject.
  • One of ordinary skill 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.
  • routes 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.
  • a macrocyclic peptide of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • a non-parenteral route such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the active compounds can be prepared with earners that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • 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).
  • compositions can be administered w ith medical devices known in the art.
  • 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.
  • 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.
  • the macrocyclic peptides of the disclosure can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • therapeutic compounds of the disclosure cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • 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 7 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 a ⁇ ., Antimicrob. Agents Chemother., 39: 180 (1995)); surfactant protein A receptor (Briscoe et al.. Am. J.
  • 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.
  • the peptides can be synthesized in a stepwise manner on an insoluble polymer support (also referred to as "resin") starting from the C-terminus of the peptide.
  • a synthesis is begun by appending the C-terminal amino acid of the peptide to the resin through formation of an amide or ester linkage. This allows the eventual release of the resulting peptide as a C-terminal amide or carboxylic acid, respectively.
  • the C-terminal amino acid and all other amino acids used in the synthesis are required to have their a-amino groups and side chain functionalities (if present) d ifferentially protected such that the a-amino protecting group may be selectively removed during the synthesis.
  • the coupling of an amino acid is performed by activation of its carboxyl group as an active ester and reaction thereof with the unblocked a-amino group of the N-terminal amino acid appended to the resin.
  • the sequence of a-amino group deprotection and coupling is repeated until the entire peptide sequence is assembled.
  • the peptide is then released from the resin with concomitant deprotection of the side chain functionalities, usually in the presence of appropriate scavengers to limit side reactions.
  • the resulting peptide is finally purified by reverse phase HPLC.
  • peptidyl-resins required as precursors to the final peptides utilizes commercially available cross-linked polysty rene polymer resins (Novabiochem, San Diego, CA; Applied Biosystems, Foster City , CA).
  • Preferred solid supports are: 4- (2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methyl benzhydrylamine resin (Rink amide MBHA resin); 9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin); 4-(9-Fmoc)aminomethyl-3,5- dimethoxyphenoxy)valerylaminomethyl-Merrifield resin (PAL resin), for C-terminal carboxamides. Coupling of first and subsequent amino acids can be accomplished using HOBt, 6-Cl-HOBt or HO At active esters produced from DIC/HOBt, HBTU/HOBt.
  • 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.
  • 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.
  • the peptidyl-resin precursors for their respective peptides may be cleaved and deprotected using any standard procedure (see, for example, King, D.S. et al, Int. J. Peptide Protein Res.. 36:255-266 (1990)).
  • a desired method is the use of TFA in the presence of water, TIS as scavenger, and DTT or TCEP as the disulfide reducing agent.
  • 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 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.
  • Mass Spectrometry ‘'ESI-MS(+)” signifies electrospray ionization mass spectrometry 7 performed in positive ion mode; “ESI-MS(-)” signifies electrospray ionization mass spectrometry 7 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 7 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.
  • 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 ⁇
  • Step 1 Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid
  • Octadecanoic acid (7.5 g, 23.85 mmol) was suspended in toluene (42.6 mL) and the mixture was heated to reflux.
  • l,l-Di-tert-butoxy-N,N-dimethylmethanamine (15.33 mL, 63.9 mmol) was added drop-wise over 30 min. The mixture was reflux overnight.
  • the solvent was removed in vacuo at 50 °C and the crude material was suspended in CH2C12/EtOAc (110 mL. 1: 1) and stirred for 15 min. The solids were removed by filtration and washed with CH2CI2 (40 mL).
  • Step 3 Preparation of (S)-5-(tert-butoxy)-4-(l8-(tert-butoxy)-18-oxooctadecanamido)- 5-oxopentcmoic acid
  • Step 4 Preparation of (S) -tert-butyl 1 -azido-40-(tert-butoxycarbonyl)-37 ,42-dioxo-
  • 35- azido-3,6,9,12.15,18,21,24,27.30,33-undecaoxapentatriacontan-l-amine (300 mg, 0.526 mmol) was then added, and the solution stirred at rt overnight. The mixture was poured into water and extracted with CH2CI2 (3 x). The combined organic extracts were dried over MgSO-i.
  • Step 5 Preparation of 17- ⁇ [(lS)-3-[(35-azido-3,6, 9, 12,15, 18,21,24,27,30,33- undecaoxapentatriacontan-l-yl)carbamoyl]-l-carboxypropyl]carbamoyl ⁇ heptadecanoic acid
  • Example 1001 was prepared, using Sieber resin on a 50 pmol scale, following the general synthetic procedures describe previously. 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 w ere then performed sequentially:
  • 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: 20-60% 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 7.8 mg. and its estimated purity by LCMS analysis was 97.9%.
  • Example 1001 was prepared as follows: Example 1001A (83 mg, 40 pmol) and tert-buty l (S)-l-azido-40-(tert-butoxycarbonyl)-37,42-dioxo-
  • the material was further purified via preparative LC/MS with the following conditions: Column: XBridge Cl 8, 30 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: 13-53% 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 1.1 mg, and its estimated purity by LCMS analysis was 96.4%.
  • Example 1002 A was prepared, using Sieber resin on a 50 pmol scale, following the general synthetic procedures described previously. To a 25 -mL polypropylene solid- phase reaction vessel was added the Sieber resin (71 mg, 0.05 mmol), and the reaction vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
  • Example 1002 was prepared on a 35 pmol scale from Example 1002A and 1 -tertbutyl 2,3,4,5,6-pentafluorophenyl tetradecanedioate using the following general procedure: "Fatty acid chain coupling procedure B".
  • Example 1005 A 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 Single-coupling extended time procedure ” or “Symphony X Singlecoupling procedure” was followed with Fmoc-Tyr(tBu)-OH; “Symphony X Singlecoupling 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 extended time procedure ” was followed with Fmoc-D-Pro-
  • Example 1039A The following examples were prepared using exocyclic amines following the ‘Tatty acid chain coupling procedure A or B’ ? general procedure as shown in Example 1004. Preparation of Example 1008
  • Example 1029 was prepared as follows: The mixture of 2,2',2",2"',2"- ((3S,6S,9S,12S,15S,18S,21S,24R,30S,33S,36S,39S,42S,47aR)-12,15,18-tris((lH-indol-3- yl)methyl)-24-((2-amino-2-oxoethyl)carbamoyl)-42-benzy l-33-(4-hydroxybenzyl)-41- methyl- 1 ,4.7.10, 13, 16, 19,22,28,31 ,34.37,40,43-tetradecaoxo-9-(4-(prop-2-yn- 1 - yloxy)benzyl)-30-(3,4,5-trifluorobenzyl)tetratetracontahydro-lH,27H-pyrrolo[2,l- al ] [1 ]thia[4,7, 10,13,16,

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Abstract

Selon la présente divulgation, des composés macrocycliques qui inhibent l'interaction protéine/protéine LAG-3/CMH de classe II ont été découverts, ces composés pouvant être utiles pour améliorer diverses maladies, notamment le cancer et des maladies infectieuses.
PCT/US2025/019111 2024-03-11 2025-03-10 Peptides macrocycliques utiles comme immunomodulateurs Pending WO2025193573A1 (fr)

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