Process for preparing PSMA conjugates

Abstract

The present disclosure relates to methods of preparing compounds for the treatment of mammalian diseases, such as cancer. In particular, the invention described herein relates to methods of preparing compounds capable of targeting cells expressing PSMA, and useful in the treatment of diseases caused by cells expressing PSMA, such as prostate cancer.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080050827.7, whose application date is May 19, 2020 and whose invention name is “Method for Preparing PSMA Conjugate”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 850,119, filed on May 20, 2019, U.S. Provisional Patent Application No. 62 / 910,777, filed on October 4, 2019, and U.S. Provisional Patent Application No. 62 / 912,353, filed on October 8, 2019, the entire disclosures of which are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to methods for preparing compounds for treating mammalian diseases such as cancer. In particular, the invention described herein relates to methods for preparing compounds that are capable of targeting cells expressing PSMA and can be used to treat diseases caused by cells expressing PSMA, such as prostate cancer. Background Art

[0005] The prostate is a male reproductive organ whose function is to produce and store semen, which provides nutrients and fluid for the survival of sperm introduced into the vagina during reproduction. Like other tissues, the prostate may develop malignant (cancerous) or benign (non-cancerous) tumors. In fact, prostate cancer is one of the most common male cancers in Western societies and the second most common malignancy in American men. Current treatments for prostate cancer include hormone therapy, radiation therapy, surgery, chemotherapy, photodynamic therapy, and combination therapies. However, many of these treatments can affect the patient's quality of life, especially for men over the age of 50 who are diagnosed with prostate cancer. For example, the use of hormonal drugs is often accompanied by side effects such as osteoporosis and liver damage. Such side effects can be mitigated by using treatments that are more selective or specific for the tissue that causes the disease state and avoid non-target tissues (such as bone or liver).

[0006] Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed in prostate cancer. PSMA is overexpressed in malignant prostate tissue compared to other organs in the human body, such as the kidneys, proximal small intestine, and salivary glands. PSMA is also expressed in the neovasculature of many non-prostate solid tumors, including lung cancer, colon cancer, breast cancer, kidney cancer, liver cancer, and pancreatic cancer, but not in the normal vasculature. PSMA is also minimally expressed in the brain. PSMA is a type II cell surface membrane-bound glycoprotein with a molecular weight of approximately 110 kD, including an intracellular fragment (amino acids 1-18), a transmembrane domain (amino acids 19-43), and a broad extracellular domain (amino acids 44-750). Although the functions of the intracellular fragment and the transmembrane domain are currently believed to be insignificant, the extracellular domain is involved in several different activities. For example, PSMA plays a role in the central nervous system, where PSMA metabolizes N-acetyl-aspartylglutamate (NAAG) to glutamate and N-acetylaspartate. PSMA also exerts its effects in the proximal small intestine, where it removes γ-linked glutamic acid from poly-γ-glutamylated folate and α-linked glutamic acid from peptides and small molecules. However, the specific function of PSMA on prostate cancer cells remains unanswered.

[0007] Unlike many other membrane-bound proteins, PSMA is rapidly internalized into cells in a manner similar to cell surface-bound receptors such as vitamin receptors. PSMA is internalized through clathrin-coated pits and can subsequently circulate to the cell surface or enter lysosomes. Therefore, diagnostic agents, imaging agents, and therapeutic agents can target PSMA for delivery to cells expressing PSMA, such as prostate cancer cells.

[0008] Described herein are methods of making compounds capable of binding to PSMA. Also described herein are methods of making compounds capable of targeting PSMA for the delivery of diagnostic, imaging, and therapeutic agents.

[0009] Unexpectedly, it was found that the conjugates prepared by the methods described herein exhibit high affinity for PSMA. It was also found that the compounds prepared by the methods described herein are effective in treating diseases caused by pathogenic cells expressing PSMA, such as prostate cancer cells.

[0010] Certain conjugates have been disclosed to exhibit high affinity for PSMA. The conjugates have also been disclosed to be effective in treating diseases caused by pathogenic cells expressing PSMA, such as prostate cancer cells.

[0011] One such conjugate is

[0012]

[0013] (aka PSMA-617) is described in WO 2015 / 055318 A1.

[0014] Due to the demand for pharmaceutical products such as PSMA-617, there is a need to provide synthetic methods that can deliver large quantities of such products at low cost and high purity.Described herein are methods for preparing compounds that can bind PSMA such as PSMA-617. Summary of the invention

[0015] In one aspect, the present disclosure provides a method for preparing a compound that can be used to treat disease, particularly cancer, in mammals, including humans.

[0016] In one embodiment, the present disclosure provides a method of preparing a compound for treating abnormal cell growth in a mammal, including a human, in need of such treatment.

[0017] In another embodiment, the abnormal cell growth is cancer. In another embodiment, the cancer is prostate cancer, metastatic prostate cancer, and metastatic castration-resistant prostate cancer.

[0018] In another aspect, the present disclosure provides a method for preparing a compound of formula I

[0019]

[0020] According to the following specific embodiments and and by the practice of this disclosure, the additional embodiments, features and advantages of this disclosure will be clear. The compounds of this disclosure can be described as embodiments in any of the following clauses. It should be understood that any embodiment described herein can be used in combination with any other embodiment described herein, as long as these embodiments do not contradict each other.

[0021] 1. A method for preparing a compound of formula I

[0022]

[0023] The method comprises

[0024] a. In a polar aprotic solvent, a resin-based compound of formula A

[0025]

[0026] contacting with a first deprotecting agent to provide a resin-based compound of formula A'

[0027] or

[0028] b. in the presence of an organic solvent and a base, contacting the resin-based compound of formula A' with a compound of formula B

[0029]

[0030] To provide a resin-based compound of formula C

[0031] or

[0032] c. in the presence of an organic solvent, contacting the resin-based compound of formula C with a second deprotecting agent to provide a resin-based compound of formula C'

[0033] or

[0034] d. in the presence of a polar aprotic solvent, a coupling agent and a base, the resin-based compound of formula C 'with PG 1 -3-(2-naphthyl)-L-alanine to provide a resin-based compound of formula D

[0035] or

[0036] e. contacting the resin-based compound of formula D with a first deprotecting agent in a polar aprotic solvent to provide a resin-based compound of formula D'

[0037] or

[0038] f. in the presence of a polar aprotic solvent, a coupling agent and a base, so that the resin-based compound of formula D 'with PG 1 - tranexamic acid to provide a resin-based compound of formula E

[0039] or

[0040] g. contacting the resin-based compound of formula E with a first deprotecting agent in a polar aprotic solvent to provide a resin-based compound of formula E'

[0041] or

[0042] h. in the presence of a polar aprotic solvent, a coupling agent and a base, the resin-based compound of formula E' and the 2’ Contact with compounds

[0043]

[0044] To provide a resin-based compound of formula F

[0045] or

[0046] i. contacting the resin-based compound of formula F with a third deprotecting agent and / or a cleavage agent in an organic solvent,

[0047] Among them PG 1 and PG 2 is an amine protecting group, LG is a leaving group, and R 1 C 1 -C 4 Alkyl or cyclohexyl.

[0048] 2. The method according to clause 1, wherein the first deprotecting agent of step (a) is selected from the group consisting of piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine and collidine.

[0049] 3. The process according to any one of the preceding clauses, wherein the first deprotecting agent of step (e) is selected from the group consisting of piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine and collidine.

[0050] 4. The process according to any one of the preceding clauses, wherein the first deprotecting agent of step (g) is selected from the group consisting of piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine and collidine.

[0051] 5. The process according to any one of the preceding clauses, wherein the first deprotecting agent of step (a) is piperidine.

[0052] 6. The process according to any one of the preceding clauses, wherein the first deprotecting agent of step (e) is piperidine.

[0053] 7. The process according to any one of the preceding clauses, wherein the first deprotecting agent of step (g) is piperidine.

[0054] 8. A process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (a) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0055] 9. A process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (d) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0056] 10. The process according to any of the preceding clauses, wherein the polar aprotic solvent of step (e) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0057] 11. A process according to any of the preceding clauses, wherein the polar aprotic solvent of step (f) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0058] 12. A process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (g) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0059] 13. A process according to any of the preceding clauses, wherein the polar aprotic solvent of step (h) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0060] 14. The process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (a) is dimethylformamide (DMF).

[0061] 15. The process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (d) is dimethylformamide (DMF).

[0062] 16. The process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (e) is dimethylformamide (DMF).

[0063] 17. The process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (f) is dimethylformamide (DMF).

[0064] 18. The process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (g) is dimethylformamide (DMF).

[0065] 19. The process according to any one of the preceding clauses, wherein the polar aprotic solvent of step (h) is dimethylformamide (DMF).

[0066] 20. A process according to any one of the preceding clauses, wherein the base of step (b) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

[0067] 21. A process according to any one of the preceding clauses, wherein the base of step (d) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

[0068] 22. A process according to any one of the preceding clauses, wherein the base of step (f) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

[0069] 23. A process according to any one of the preceding clauses, wherein the base of step (h) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

[0070] 24. A process according to any one of the preceding clauses, wherein the base in step (b) is N,N-diisopropylethylamine ( i Pr 2 NEt).

[0071] 25. A process according to any one of the preceding clauses, wherein the base in step (d) is N,N-diisopropylethylamine ( i Pr 2 NEt).

[0072] 26. A process according to any one of the preceding clauses, wherein the base in step (f) is N,N-diisopropylethylamine ( i Pr 2 NEt).

[0073] 27. A process according to any one of the preceding clauses, wherein the base of step (h) is N,N-diisopropylethylamine ( i Pr 2 NEt).

[0074] 28. The method according to any of the preceding clauses, wherein the second deprotecting agent of step (c) is selected from the group consisting of trifluoroacetic acid (TFA), acetic acid, trifluoroethanol, hexafluoroisopropanol, dichloromethane and combinations thereof.

[0075] 29. The method according to any one of the preceding clauses, wherein the second deprotecting agent of step (c) is trifluoroacetic acid (TFA) or a mixture of acetic acid and trifluoroethanol.

[0076] 30. A process according to any of the preceding clauses, wherein the coupling agent of step (d) is selected from the group consisting of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxy-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU),

[0013] N,N,N',N'-tetramethyl-S-(1-oxo-2-pyridyl)thiouronium tetrafluoroborate (TOTT), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

[0077] 31. A method according to any of the preceding clauses, wherein the coupling agent of step (f) is selected from the group consisting of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxy-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU),

[0013] N,N,N',N'-tetramethyl-S-(1-oxo-2-pyridyl)thiouronium tetrafluoroborate (TOTT), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

[0078] 32. A process according to any of the preceding clauses, wherein the coupling agent of step (h) is selected from the group consisting of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxy-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU),

[0013] N,N,N',N'-tetramethyl-S-(1-oxo-2-pyridyl)thiouronium tetrafluoroborate (TOTT), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

[0079] 33. The process according to any one of the preceding clauses, wherein the coupling reagent of step (d) is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).

[0080] 34. The process according to any one of the preceding clauses, wherein the coupling reagent of step (f) is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).

[0081] 35. The process according to any one of the preceding clauses, wherein the coupling reagent of step (h) is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).

[0082] 36. A method according to any of the preceding clauses, wherein the third deprotecting agent of step (i) is selected from the group consisting of trifluoroacetic acid (TFA), hydrofluoric acid (HF), trifluoromethanesulfonic acid (TfOH), a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture, a trifluoroacetic acid (TFA) / dichloromethane (DCM) mixture, triethylsilane (TES), indole, a phenol / anisole mixture and thioanisole.

[0083] 37. A process according to any one of the preceding clauses, wherein the third deprotecting agent of step (i) is a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture.

[0084] 38. A method according to any of the preceding clauses, wherein the cleavage agent of step (i) is trifluoroacetic acid (TFA) or a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture.

[0085] 39. A method according to any of the preceding clauses, wherein the cleavage agent of step (i) is a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture.

[0086] 40. The method according to any of the preceding clauses, wherein the organic solvent of step (b) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile.

[0087] 41. The method according to any of the preceding clauses, wherein the organic solvent of step (c) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile.

[0088] 42. The method according to any one of the preceding clauses, wherein the organic solvent of step (i) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile.

[0089] 43. A method according to any one of the preceding clauses, wherein the organic solvent of step (b) is CH 2 Cl 2 .

[0090] 44. A method according to any of the preceding clauses, wherein the organic solvent of step (c) is CH 2 Cl 2 .

[0091] 45. A method according to any one of the preceding clauses, wherein the organic solvent of step (i) is CH 2 Cl 2 .

[0092] 46. ​​A method according to any of the preceding clauses, wherein PG 1 It is 9-fluorenylmethyl-carbonyl (Fmoc) or tert-butylcarbonyl (Boc).

[0093] 47. A method according to any of the preceding clauses, wherein PG1 It is 9-fluorenylmethyl-carbonyl (Fmoc).

[0094] 48. A method according to any of the preceding clauses, wherein PG 2 It is monomethoxytrityl (MMt) or 4-methyltrityl (Mtt).

[0095] 49. A method according to any of the preceding clauses, wherein PG 2 It is 4-methyltrityl (Mtt).

[0096] 50. A method according to any of the preceding clauses, wherein LG is

[0097]

[0098] where * represents the point of attachment to the rest of the compound.

[0099] 51. A method according to any of the preceding clauses, wherein R 1 It is tert-butyl.

[0100] 52. A method for preparing a compound of formula 1

[0101]

[0102] The method comprises one or more of the following steps:

[0103] a. in the presence of a solvent, the resin-based compound of formula 1

[0104]

[0105] contacting with a first deprotecting agent to provide a resin-based compound of Formula 2

[0106] or

[0107] b. in the presence of an organic solvent and a base, contacting the resin-based compound of Formula 2 with the compound of Formula 3

[0108]

[0109] To provide a resin-based compound of formula 4

[0110] or

[0111] c. contacting the resin-based compound of Formula 4 with a second deprotecting agent in the presence of an organic solvent to provide a resin-based compound of Formula 5

[0112] or

[0113] d. contacting the resin-based compound of Formula 5 with the compound of Formula 6 in the presence of a solvent, a coupling agent and optionally a base and / or an additive

[0114]

[0115] To provide a resin-based compound of Formula 7

[0116] or

[0117] e. contacting the resin-based compound of Formula 7 with a third deprotecting agent in the presence of a solvent to provide a resin-based compound of Formula 8

[0118] or

[0119] f. contacting the resin-based compound of Formula 8 with the compound of Formula 9 in the presence of a solvent, a coupling agent and optionally a base and / or an additive

[0120]

[0121] To provide a resin-based compound of formula 10

[0122] or

[0123] g. contacting the resin-based compound of formula 10 with at least one third deprotecting agent and / or cleaving agent in the presence of an organic solvent to provide a compound of formula I,

[0124] Among them, PG, PG 1 and PG 4 is an amine protecting group, PG 2 PG 3 and PG 5 is a carboxyl protecting group, and LG in compound C is a leaving group.

[0125] 53. The method according to clause 52, wherein PG, PG 1 and PG 4 Selected from the group consisting of 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt).

[0126] 54. The method according to clause 52 or 53, wherein PG is 9-fluorenylmethyl-carbonyl.

[0127] 55. A method according to any one of clauses 52 to 54, wherein PG 1 It is monomethoxytrityl (MMt) or 4-methyltrityl (Mtt).

[0128] 56. A method according to any one of clauses 52 to 55, wherein PG 1 It is 4-methyltrityl (Mtt).

[0129] 57. A method according to any one of clauses 52 to 56, wherein PG 4 It is 9-fluorenylmethyl-carbonyl.

[0130] 58. A method according to any one of clauses 52 to 57, wherein PG 2 PG 3 and PG 5 Each is tert-butyl.

[0131] 59. The method according to any one of clauses 52 to 58, wherein LG in compound C is selected from imidazolyl, 4-nitrophenoxy and phenoxy.

[0132] 60. A method according to any one of clauses 52 to 59, wherein the first deprotecting agent of step (a) comprises a reagent or a mixture of reagents capable of removing a protecting group selected from the group consisting of 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt).

[0133] 61. A process according to any one of clauses 52 to 60, wherein the first deprotecting agent of step (a) comprises a reagent or mixture of reagents capable of removing a 9-fluorenylmethyl-carbonyl group.

[0134] 62. A process according to any one of clauses 52 to 61, wherein the first deprotecting agent of step (a) is selected from the group consisting of piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine and collidine.

[0135] 63. The process according to any one of clauses 52 to 62, wherein the first deprotecting agent of step (a) is piperidine.

[0136] 64. The process according to any one of clauses 52 to 63, wherein the solvent of step (a) is a polar aprotic solvent.

[0137] 65. A process according to any one of clauses 52 to 64, wherein the solvent of step (a) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0138] 66. The process according to any one of clauses 52 to 65, wherein the base of step (b) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

[0139] 67. The method according to any one of clauses 52 to 66, wherein the organic solvent of step (b) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, and acetonitrile.

[0140] 68. A method according to any one of clauses 52 to 67, wherein the second deprotecting agent of step (c) comprises a reagent or a mixture of reagents capable of removing a protecting group selected from the group consisting of 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt).

[0141] 69. A method according to any one of clauses 52 to 68, wherein the second deprotecting agent of step (c) comprises a reagent or mixture of reagents capable of removing 4-methyltrityl (Mtt).

[0142] 70. The process according to any one of clauses 52 to 69, wherein the second deprotecting agent of step (c) is selected from the group consisting of trifluoroacetic acid (TFA), acetic acid, 2,2,2-trifluoroethanol, hexafluoroisopropanol, and combinations thereof.

[0143] 71. The process according to any one of clauses 52 to 70, wherein the second deprotecting agent of step (c) is a mixture of acetic acid and 2,2,2-trifluoroethanol.

[0144] 72. The method according to any one of clauses 52 to 71, wherein the organic solvent of step (c) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, and acetonitrile.

[0145] 73. A process according to any one of clauses 52 to 72, wherein the coupling agent of step (d) is selected from the group consisting of benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-aza-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyAOP), ethyl cyano(hydroxyimino)acetate-0 2 )-tris-(1-pyrrolidinyl)-phosphonium hexafluorophosphate (PyOxim), bromo-tripyrrolidine-phosphonium hexafluorophosphate (PyBrOP), 3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazine-4(3H)-one (DEPBT), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1, 1,3,3-Tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylammonium hexafluorophosphate (HCTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxido-1H-benzotriazol-1-yl)-4-morpholine [1-(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethylammonium tetrafluoroborate (TATU), ... (TFFH), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDAC) and 1,1'-carbonyldiimide (CDI).

[0146] 74. The process according to any one of clauses 52 to 73, wherein the base of step (d) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

[0147] 75. A process according to any one of clauses 52 to 74, wherein the additive in step (d), when present, is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), 1-hydroxybenzotriazole-6-sulfonamidomethyl resin HCl (HOBt-6-sulfonamidomethyl resin HCl), hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt), N-hydroxysuccinimide (NHS), 1-hydroxy-7-aza-1H-benzotriazole (HOAt), ethyl 2-cyano-2-(hydroxyimino)acetate and 4-(N,N-dimethylamino)pyridine (DMAP).

[0148] 76. The process according to any one of clauses 52 to 75, wherein the solvent of step (d) is a polar aprotic solvent.

[0149] 77. A process according to any one of clauses 52 to 76, wherein the solvent of step (d) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0150] 78. A method according to any one of clauses 52 to 77, wherein the third deprotecting agent of step (e) comprises a reagent or a mixture of reagents capable of removing a protecting group selected from the group consisting of 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt).

[0151] 79. A process according to any one of clauses 52 to 78, wherein the third deprotecting agent of step (e) comprises a reagent or mixture of reagents capable of removing a 9-fluorenylmethyl-carbonyl group.

[0152] 80. A process according to any one of clauses 52 to 79, wherein the first deprotecting agent of step (e) is selected from the group consisting of piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine and collidine.

[0153] 81. The process according to any one of clauses 52 to 80, wherein the first deprotecting agent of step (e) is piperidine.

[0154] 82. The process according to any one of clauses 52 to 81, wherein the solvent of step (e) is a polar aprotic solvent.

[0155] 83. A process according to any one of clauses 52 to 82, wherein the solvent of step (e) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0156] 84. A process according to any one of clauses 52 to 83, wherein the coupling agent of step (f) is selected from the group consisting of benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-aza-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyAOP), ethyl cyano(hydroxyimino)acetate-0 2)-tris-(1-pyrrolidinyl)-phosphonium hexafluorophosphate (PyOxim), bromo-tripyrrolidine-phosphonium hexafluorophosphate (PyBrOP), 3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazine-4(3H)-one (DEPBT), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1, 1,3,3-Tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylammonium hexafluorophosphate (HCTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxido-1H-benzotriazol-1-yl)-4-morpholine [1-(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethylammonium tetrafluoroborate (TATU), ... (TFFH), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDAC) and 1,1'-carbonyldiimide (CDI).

[0157] 85. A process according to any one of clauses 52 to 84, wherein the base of step (f), when present, is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

[0158] 86. A process according to any one of clauses 52 to 85, wherein the additive in step (f), when present, is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), 1-hydroxybenzotriazole-6-sulfonamidomethyl resin HCl (HOBt-6-sulfonamidomethyl resin HCl), hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt), N-hydroxysuccinimide (NHS), 1-hydroxy-7-aza-1H-benzotriazole (HOAt), ethyl 2-cyano-2-(hydroxyimino)acetate and 4-(N,N-dimethylamino)pyridine (DMAP).

[0159] 87. The process according to any one of clauses 52 to 86, wherein the solvent of step (f) is a polar aprotic solvent.

[0160] 88. A process according to any one of clauses 52 to 87, wherein the solvent of step (f) is a polar aprotic solvent selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

[0161] 89. A method according to any one of clauses 52 to 88, wherein the third deprotecting agent of step (g) is a reagent or a mixture of reagents capable of removing a protecting group selected from the group consisting of tert-butyl, benzyl, 2-chlorotrityl (2-Cl-Trt), 2,4-dimethoxybenzyl (Dmb), allyl, 1,1-dimethylallyl (Dma) and p-nitrobenzyl (pNB).

[0162] 90. A method according to any one of clauses 52 to 89, wherein the third deprotecting agent of step (g) is selected from the group consisting of trifluoroacetic acid (TFA), hydrofluoric acid (HF), trifluoromethanesulfonic acid (TfOH), a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture, a trifluoroacetic acid (TFA) / dichloromethane (DCM) mixture, triethylsilane (TES), indole, a phenol / anisole mixture and thioanisole.

[0163] 91. The process according to any one of clauses 52 to 90, wherein the third deprotecting agent of step (g) is a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture.

[0164] 92. The process according to any one of clauses 52 to 91, wherein the cleavage agent of step (g) is trifluoroacetic acid (TFA) or a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture.

[0165] 93. The process according to any one of clauses 52 to 92, wherein the cleavage agent of step (g) is a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture.

[0166] 94. The method according to any one of clauses 52 to 93, wherein the organic solvent of step (g) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, and acetonitrile.

[0167] 95. A method according to any one of clauses 52 to 94, wherein the compound of formula 6, a solvent, a coupling agent and optionally a base and / or an additive are combined to form a mixture comprising an activated compound of formula 6′ prior to contacting with the resin-based compound of formula 5.

[0168]

[0169] Wherein LG in compound 6' is a leaving group.

[0170] 96. A method according to any one of clauses 52 to 94, wherein the resin-based compound of formula 5, the compound of formula 6, a solvent, a coupling agent and optionally a base and / or an additive are contacted together before or simultaneously with the in situ formation of the activated compound of formula 6'.

[0171]

[0172] Wherein LG in compound 6' is a leaving group.

[0173] 97. The method according to clause 95 or 96, wherein LG in compound 6' is selected from the group consisting of: 1-(λ 1 -oxyalkyl)-1H-benzo[d][1,2,3], 3-(λ 1 -oxyalkyl)-3H-[1,2,3]triazolo[4,5-b]pyridine, ethyl cyano(hydroxyimino)acetate (Oxyma), bromide, 3-(λ 1 -oxyalkyl)benzo[d][1,2,3]triazine-4(3H)-one, 6-chloro-1-(λ 1 -oxyalkyl)-1H-benzo[d][1,2,3]triazole, 2,4-dimethoxy-6-(λ 1 -oxyalkyl)-1,3,5-triazine, etabonic acid, propylphosphonic acid, dicyclohexylurea, 1-(3-(dimethyl-λ 4 -azolyl)propyl)-3-ethylurea and imidazolyl.

[0174] 98. The method according to any one of clauses 95 to 97, wherein LG in compound 6' is imidazolyl.

[0175] 99. A method according to any of the preceding clauses, wherein prior to contacting with the resin-based compound of formula H, the compound of formula 9, a solvent, a coupling agent and optionally a base and / or an additive are combined to form a mixture comprising an activated compound of formula 9'

[0176]

[0177] Wherein LG in compound 9' is a leaving group.

[0178] 100. A method according to any one of clauses 52 to 98, wherein the resin-based compound of formula 8, the compound of formula 9, a solvent, a coupling agent and optionally a base and / or an additive are contacted together before or simultaneously with the in situ formation of the activated compound of formula 9'

[0179]

[0180] Wherein LG in compound 9' is a leaving group.

[0181] 101. The method according to clause 99 or 100, wherein LG in compound 9' is selected from the group consisting of: 1-(λ 1 -oxyalkyl)-1H-benzo[d][1,2,3], 3-(λ 1 -oxyalkyl)-3H-[1,2,3]triazolo[4,5-b]pyridine, ethyl cyano(hydroxyimino)acetate (Oxyma), bromide, 3-(λ 1 -oxyalkyl)benzo[d][1,2,3]triazine-4(3H)-one, 6-chloro-1-(λ 1 -oxyalkyl)-1H-benzo[d][1,2,3]triazole, 2,4-dimethoxy-6-(λ 1 -oxyalkyl)-1,3,5-triazine, etabonate, propylphosphonic acid, dicyclohexylurea, 1-(3-(dimethyl-λ 4 -azolyl)propyl)-3-ethylurea and imidazolyl.

[0182] 102. The method according to any one of clauses 99 to 101, wherein LG in compound 9' is imidazolyl.

[0183] 103. A resin-based compound of formula 4

[0184]

[0185] Among them PG 1 is an amine protecting group, and PG 2and PG 3 Each is independently a carboxyl protecting group.

[0186] 104. A resin-based compound of formula 5

[0187]

[0188] Among them PG 2 and PG 3 Each is independently a carboxyl protecting group.

[0189] 105. A resin-based compound of formula 7

[0190]

[0191] Among them PG 2 and PG 3 Each is independently a carboxyl protecting group, and PG 4 Amine protecting group.

[0192] 106. A resin-based compound of formula 8

[0193]

[0194] Among them PG 2 and PG 3 Each is independently a carboxyl protecting group.

[0195] 107. A resin-based compound of formula 10

[0196]

[0197] Among them PG 2 PG 3 and PG 5 Each is independently a carboxyl protecting group.

[0198] 108. A resin-based compound according to any one of clauses 103 to 107, wherein PG 1 and PG 4 , when present, is independently selected from the group consisting of 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt).

[0199] 109. A resin-based compound according to any one of clauses 103 to 108, wherein PG 1 , when present, is monomethoxytrityl (MMt) or 4-methyltrityl (Mtt).

[0200] 110. A resin-based compound according to any one of clauses 103 to 109, wherein PG 1 , when present, is 4-methyltrityl (Mtt).

[0201] 111. A resin-based compound according to any one of clauses 103 to 110, wherein PG 4 , when present, is 9-fluorenylmethyl-carbonyl.

[0202] 112. A resin-based compound according to any one of clauses 103 to 111, wherein PG 2 PG 3 and PG 5 , when present, are each tert-butyl.

[0203] 113. A resin-based compound of formula C

[0204]

[0205] Among them PG 2 is an amine protecting group, and each R 1 C 1 -C 4 114. A resin-based compound of formula C'

[0206]

[0207] Each R 1 C 1 -C 4 Alkyl or cyclohexyl.

[0208] 115. A resin-based compound of formula D

[0209]

[0210] Among them PG 1 is an amine protecting group, and each R 1 C 1 -C 4 116. A resin-based compound of formula D'

[0211]

[0212] Each R 1 C 1 -C 4 Alkyl or cyclohexyl.

[0213] 117. A resin-based compound of formula E

[0214]

[0215] Among them PG 1 is an amine protecting group, and each R 1 C 1 -C 4 118. A resin-based compound of formula E'

[0216]

[0217] Each R 1 C 1 -C 4 Alkyl or cyclohexyl.

[0218] 119. A resin-based compound of formula F

[0219]

[0220] Each R 1 C 1 -C 4 Alkyl or cyclohexyl.

[0221] 120. The resin-based compound according to clause 115 or 117, wherein PG 1 It is 9-fluorenylmethyl-carbonyl (Fmoc) or tert-butylcarbonyl (Boc).

[0222] 121. A resin-based compound according to any one of clauses 115, 117 or 120, wherein PG 1 It is 9-fluorenylmethyl-carbonyl (Fmoc).

[0223] 122. The resin-based compound according to clause 113, wherein PG 2 It is monomethoxytrityl (MMt) or 4-methyltrityl (Mtt).

[0224] 123. The resin-based compound according to clause 113 or 122, wherein PG 2 It is 4-methyltrityl (Mtt).

[0225] 124. A resin-based compound according to any one of clauses 113 to 119, wherein R 1 It is tert-butyl.

[0226] definition

[0227] As used herein, the term "alkyl" includes a carbon atom chain, which is optionally branched and contains 1 to 4 carbon atoms, etc., which may be referred to as "lower alkyl". Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0228] As used herein, and unless otherwise indicated, the term "abnormal cell growth" refers to cell growth that is independent of normal regulatory mechanisms (eg, loss of contact inhibition).

[0229] The term "subject" refers to a mammalian patient, such as a human, in need of such treatment.

[0230] As used herein, the term "protecting group" or "PG" refers to any group known to those of ordinary skill in the art, which can be introduced into a molecule by chemical modification of a functional group such as an amine or hydroxyl group to obtain chemical selectivity in a subsequent chemical reaction. It should be understood that such a protecting group can be removed from a functional group at a later time of the synthesis to provide a further opportunity for reaction at such a functional group, or in the case of a final product, to expose such a functional group. Protecting groups have been described in, for example, Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups in organic synthesis. [Greene's protective groups in organic synthesis] Hoboken, NJ: Wiley-Interscience [Wiley International Science]. Those skilled in the art will readily understand the chemical process conditions under which such a protecting group can be installed on a functional group. Suitable amine protecting groups relevant to the present disclosure include, but are not limited to, 9-fluorenylmethyl-carbonyl (Fmoc) and tert-butylcarbonyl (Boc).

[0231] As used herein, the term "leaving group" of "LG" refers to any group known to those of ordinary skill in the art that takes a pair of electrons away in the breakage of a heterolytic bond. The leaving group can be an anion or a neutral molecule, but in either case, it is critical that the leaving group is able to stabilize the additional electron density generated by the heterolytic bond. DETAILED DESCRIPTION

[0232] Before further describing the present disclosure, it should be understood that the present disclosure is not limited to the particular embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, as the scope of the present disclosure will be limited only by the appended claims.

[0233] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications cited herein are incorporated by reference in their entirety. If the definitions set forth in this section are contrary to or inconsistent with the definitions set forth in the patents, applications, or other publications incorporated herein by reference, the definitions set forth in this section take precedence over the definitions incorporated herein by reference.

[0234] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Note further that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a reference basis for the use of the terminology "solely," "only," and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0235] Synthesis method

[0236] Method 1:

[0237] In some embodiments, the present disclosure provides a method for preparing a compound of formula I

[0238]

[0239] The method comprises

[0240] a. In a polar aprotic solvent, a resin-based compound of formula A

[0241]

[0242] contacting with a first deprotecting agent to provide a resin-based compound of formula A'

[0243] or

[0244] b. in the presence of an organic solvent and a base, contacting the resin-based compound of formula A' with a compound of formula B

[0245]

[0246]

[0247] To provide a resin-based compound of formula C

[0248] or

[0249] c. in the presence of an organic solvent, contacting the resin-based compound of formula C with a second deprotecting agent to provide a resin-based compound of formula C'

[0250] or

[0251] d. in the presence of a polar aprotic solvent, a coupling agent and a base, the resin-based compound of formula C 'with PG 1 -3-(2-naphthyl)-L-alanine to provide a resin-based compound of formula D

[0252] or

[0253] e. contacting the resin-based compound of formula D with a first deprotecting agent in a polar aprotic solvent to provide a resin-based compound of formula D'

[0254] or

[0255] f. in the presence of a polar aprotic solvent, a coupling agent and a base, so that the resin-based compound of formula D 'with PG 1 - tranexamic acid to provide a resin-based compound of formula E

[0256] or

[0257] g. contacting the resin-based compound of formula E with a first deprotecting agent in a polar aprotic solvent to provide a resin-based compound of formula E'

[0258] or

[0259] h. in the presence of a polar aprotic solvent, a coupling agent and a base, the resin-based compound of formula E' and the 2’ Contact with compounds

[0260]

[0261] To provide a resin-based compound of formula F

[0262] or

[0263] i. contacting the resin-based compound of formula F with a third deprotecting agent and / or a cleavage agent in an organic solvent,

[0264] Among them PG 1 and PG 2 is an amine protecting group, LG is a leaving group, and R 1 C 1 -C 4 Alkyl or cyclohexyl.

[0265] Method 2:

[0266] Alternatively, in some embodiments, the present disclosure provides a method for preparing a compound of Formula 1

[0267]

[0268] The method comprises one or more of the following steps:

[0269] a. in the presence of a solvent, the resin-based compound of formula 1

[0270]

[0271] contacting with a first deprotecting agent to provide a resin-based compound of Formula 2

[0272] or

[0273] b. in the presence of an organic solvent and a base, contacting the resin-based compound of Formula 2 with the compound of Formula 3

[0274]

[0275] To provide a resin-based compound of formula 4

[0276] or

[0277] c. contacting the resin-based compound of Formula 4 with a second deprotecting agent in the presence of an organic solvent to provide a resin-based compound of Formula 5

[0278] or

[0279] d. contacting the resin-based compound of Formula 5 with the compound of Formula 6 in the presence of a solvent, a coupling agent and optionally a base and / or an additive

[0280]

[0281] To provide a resin-based compound of Formula 7

[0282] or

[0283] e. contacting the resin-based compound of Formula 7 with a third deprotecting agent in the presence of a solvent to provide a resin-based compound of Formula 8

[0284] or

[0285] f. contacting the resin-based compound of Formula 8 with the compound of Formula 9 in the presence of a solvent, a coupling agent and optionally a base and / or an additive

[0286]

[0287] To provide a resin-based compound of formula 10

[0288] or

[0289] g. contacting the resin-based compound of formula 10 with at least one third deprotecting agent and / or cleaving agent in the presence of an organic solvent to provide a compound of formula I,

[0290] Among them, PG, PG 1 and PG 4 is an amine protecting group, PG 2 PG 3 and PG 5 is a carboxyl protecting group, and LG in compound C is a leaving group.

[0291] It should be understood that the present disclosure provides a method for preparing a compound of formula I described in the above paragraph (by method 1 or method 2), including more than one step listed in the alternative. Therefore, the present disclosure provides a method for preparing a compound of formula I, including steps (a) and (b). Alternatively, the present disclosure provides a method for preparing a compound of formula I, including steps (b) and (c). Alternatively, the present disclosure provides a method for preparing a compound of formula I, including steps (c) and (d). Alternatively, the present disclosure provides a method for preparing a compound of formula I, including steps (d) and (e). Alternatively, the present disclosure provides a method for preparing a compound of formula I, including steps (e) and (f). Alternatively, the present disclosure provides a method for preparing a compound of formula I, including steps (f) and (g). Alternatively, the present disclosure provides a method for preparing a compound of formula I, including steps (g) and (h). Alternatively, the present disclosure provides a method for preparing a compound of formula I, including steps (h) and (i). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (a), (b) and (c). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (b), (c) and (d). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (c), (d) and (e). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (d), (e) and (f). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (e), (f) and (g). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (d), (e) and (f). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (f), (g) and (h). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (d), (e) and (f). Alternatively, the present disclosure provides a method for preparing a compound of formula I, comprising steps (g), (h) and (i). Alternatively, the present disclosure provides a method for preparing a compound of Formula I, comprising steps (a), (b), (c), (d), (e), (f), (g) and (h).

[0292] In various embodiments described herein, it will be appreciated by those of ordinary skill in the art that the selection of the protecting group for preparing compound (I) can be selected from various alternatives known in the art. It will be further appreciated that any protecting group scheme will be selected so that the protecting group used is an orthogonal protection strategy. As used herein, "orthogonal protection" is a strategy that allows one or more protecting groups in a plurality of protecting groups to be deprotected, wherein a protecting group is removed each time using a set of dedicated reaction conditions without affecting other protecting groups, or a set of dedicated reaction conditions is used together as a subset of all protecting groups used in the synthesis without affecting other protecting groups.

[0293] In step (a) of method 1, the PG on the resin-based compound of formula A 1 It can be a protecting group, such as 9-fluorenylmethylcarbonyl (Fmoc) or tert-butylcarbonyl (Boc), PG 2 It can be an orthogonal protecting group, such as 4-methyltrityl (Mtt) or monomethoxytrityl (MMt). The first deprotecting agent in step (a) of method 1 can be an acidic or basic deprotecting agent, depending on the PG 1 and / or PG 2 In particular, the first deprotecting agent can be a basic deprotecting agent, such as piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine or collidine. In particular, the first deprotecting agent can be an acidic deprotecting agent, such as TFA, HCl or H 3 PO 4. Suitable deprotecting agents and conditions for step (a) of method 1 can be found, for example, in Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups in organic synthesis. [Greene's protective groups in organic synthesis] Hoboken, NJ: Wiley-Interscience [Wiley International Science]. In step (a) of method 1, the base used to remove the Fmoc group is piperidine. The polar aprotic solvent of step (a) of method 1 can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO) and the like. It should be understood that step (a) of method 1 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling, or heating conditions. The deprotection step (a) of method 1 can be carried out for a period of time, such as from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After the deprotection step (a) of method 1, the resin-based compound of formula A' produced in step (a) can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0294] In step (b) of method 1, R 1 Can be C 1 -C 4 alkyl, preferably tert-butyl, or cyclohexyl, and LG is a leaving group, such as an active ester. Suitable active esters known in the art include, but are not limited to, 4-nitrophenyl, 2,4,5-trichlorophenyl, NHS ester, benzotriazolyl, and the like. In some embodiments, LG is 4-nitrophenyl ester. It should be understood that R 1 The choice will be with PG 1 PG 2 The selection of the resin beads to be connected is orthogonal. In step (b) of method 1, the base can be any base known in the art for urea formation reactions. Suitable bases include, but are not limited to, N,N-diisopropylethylamine ( i Pr 2NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine. Step (b) of method 1 can be carried out in a solvent (e.g., an organic solvent). The organic solvent can be any solvent known in the art for resin-based synthesis. Suitable organic solvents include, but are not limited to, CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile, etc. In some embodiments, the solvent is CH 2 Cl 2. It should be understood that step (b) of method 1 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The deprotection step (b) of method 1 can be carried out for a period of time, such as from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After the deprotection step (a) of method 1, the resin-based compound of formula C produced in step (b) can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0295] In step (c) of method 1, according to PG 1 and / or PG 2 The second deprotecting agent may be an acidic deprotecting agent, a basic deprotecting agent or other deprotecting agent. In some embodiments, the second deprotecting agent may be an acidic deprotecting agent. Suitable acidic deprotecting agents include, but are not limited to, TFA, HCl or H 3 PO 4 In some embodiments, the second deprotecting agent of step (c) of method 1 is selected from the group consisting of trifluoroacetic acid (TFA), acetic acid, trifluoroethanol, hexafluoroisopropanol, dichloromethane, and combinations thereof. In some embodiments, the second deprotecting agent is TFA, for example, when PG 2 In some embodiments, the second deprotecting agent is a mixture of acetic acid and trifluoroethanol, for example, when PG 2 In some embodiments, the second deprotecting agent is a palladium deprotecting agent, for example, when PG 2is alloc. Suitable deprotecting agents and conditions for step (c) of method 1 can be found, for example, in Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups in organic synthesis. [Greene's protective groups in organic synthesis] Hoboken, NJ: Wiley-Interscience [Wiley International Science]. Acidic deprotecting agents (TFA) are used to cleave Mtt. Other suitable acidic deprotecting agents include, but are not limited to, acetic acid, trifluoroethanol, dichloromethane, hexafluoroisopropanol, dichloromethane, and combinations thereof. Step (c) of method 1 can be carried out in a solvent such as an organic solvent. The organic solvent can be any solvent known in the art for resin-based synthesis. Suitable organic solvents include, but are not limited to, CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile, etc. In some embodiments, the solvent is CH 2 Cl 2 .

[0296] In step (d), PG 1 -PG on 3-(2-naphthyl)-L-alanine 1

[0297]

[0298] It can be a protecting group, such as 9-fluorenylmethyl-carbonyl (Fmoc) or tert-butylcarbonyl (Boc). 1 For the selection of, please refer to step (a) of method 1 above. In step (d) of method 1, the polar aprotic solvent can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO). In some embodiments, the polar aprotic solvent is DMF. Suitable bases include, but are not limited to, triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine, and collidine. In some embodiments, the base in step (d) of method 1 is N,N-diisopropylethylamine ( i Pr 2NEt). The coupling agent can be any coupling agent known in the art for resin-based synthesis. Suitable coupling agents include, but are not limited to, HATU, TBTU, HBTU, CDMT, HDMC, COMU, TATU, TOTT, T3P, DMTMM. In some embodiments, the coupling agent in step (d) of method 1 is (PyBOP). It should be understood that step (d) of method 1 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The coupling step (d) of method 1 can be carried out for a period of time, for example, from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After the coupling step (d) of method 1, the resin-based compound of formula D produced in step (d) of method 1 can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0299] In step (e) of method 1, the PG on the resin-based compound of formula D 1 can be a protecting group, such as 9-fluorenylmethylcarbonyl (Fmoc) or tert-butylcarbonyl (Boc), and PG 2 It can be an orthogonal protecting group, such as 4-methyltrityl (Mtt) or monomethoxytrityl (MMt). The first deprotecting agent in step (e) of method 1 can be an acidic or basic protecting agent, depending on the PG 1 and / or PG 2 In particular, the first deprotecting agent can be a basic deprotecting agent, such as piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine or collidine. In particular, the first deprotecting agent can be an acidic deprotecting agent, such as TFA, HCl or H 3 PO 4. Suitable deprotecting agents and conditions for step (e) of method 1 can be found, for example, in Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups in organic synthesis. [Greene's protective groups in organic synthesis] Hoboken, NJ: Wiley-Interscience [Wiley International Science]. The polar aprotic solvent of step (e) of method 1 can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO), etc. It should be understood that step (e) of method 1 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling, or heating conditions. The deprotection step (e) of method 1 can be carried out for a period of time, such as from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 45 minutes, or about 60 minutes, or about 90 minutes. After the deprotection step (e) of method 1, the resin-based compound of formula D' produced in step (e) of method 1 can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0300] In step (f) of method 1, PG 1 -PG on Tranexamic Acid Reagent 1

[0301]

[0302] It can be a protecting group, such as 9-fluorenylmethyl-carbonyl (Fmoc) or tert-butylcarbonyl (Boc). 1 For the selection of, please refer to step (a) of method 1 above. In step (f) of method 1, the polar aprotic solvent can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO). In some embodiments, the polar aprotic solvent is DMF. Suitable bases include, but are not limited to, triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine, and collidine. In some embodiments, the base in step (f) of method 1 is N,N-diisopropylethylamine ( i Pr2 NEt). The coupling agent can be any coupling agent known in the art for resin-based synthesis. Suitable coupling agents include, but are not limited to, HATU, TBTU, HBTU, CDMT, HDMC, COMU, TATU, TOTT, T3P, DMTMM. In some embodiments, the coupling agent in step (f) of method 1 is (PyBOP). It should be understood that step (f) of method 1 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The coupling step (f) of method 1 can be carried out for a period of time, for example, from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After the coupling step (f) of method 1, the resin-based compound of formula E produced in step (f) of method 1 can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0303] In step (g) of method 1, the PG on the resin-based compound of formula E 1 can be a protecting group, such as 9-fluorenylmethylcarbonyl (Fmoc) or tert-butylcarbonyl (Boc), and PG 2 It can be an orthogonal protecting group, such as 4-methyltrityl (Mtt) or monomethoxytrityl (MMt). The first deprotecting agent in step (g) of method 1 can be an acidic or basic protecting agent, depending on the PG 1 and / or PG 2 In particular, the first deprotecting agent can be a basic deprotecting agent, such as piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine or collidine. In particular, the first deprotecting agent can be an acidic deprotecting agent, such as TFA, HCl or H 3 PO 4. Suitable deprotecting agents and conditions for step (g) of method 1 can be found, for example, in Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups in organic synthesis. [Greene's protective groups in organic synthesis] Hoboken, NJ: Wiley-Interscience [Wiley International Science]. The polar aprotic solvent of step (g) of method 1 can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO), etc. It should be understood that step (g) of method 1 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling, or heating conditions. The deprotection step (g) of method 1 can be carried out for a period of time, such as from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 45 minutes, or about 60 minutes, or about 90 minutes. After the deprotection step (g) of method 1, the resin-based compound of formula E' produced in step (g) of method 1 can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0304] In step (h) of method 1, the polar aprotic solvent can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO). In some embodiments, the polar aprotic solvent is DMF. Suitable bases include, but are not limited to, triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine, and collidine. In some embodiments, the base in step (h) of method 1 is N,N-diisopropylethylamine ( i Pr 2NEt). The coupling agent can be any coupling agent known in the art for resin-based synthesis. Suitable coupling agents include, but are not limited to, HATU, TBTU, HBTU, CDMT, HDMC, COMU, TATU, TOTT, T3P, DMTMM. In some embodiments, the coupling agent in step (h) of method 1 is (PyBOP). It should be understood that step (h) of method 1 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The coupling step (h) of method 1 can be carried out for a period of time, for example, from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After the coupling step (h) of method 1, the resin-based compound of formula F produced in step (h) of method 1 can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0305] In step (i) of method 1, the third deprotecting agent may be the same as or different from the cleavage agent. 1 When R is cyclohexyl and the cleavage reagent is TFA, the third deprotection agent may be, for example, HF or TfOH. 1 When tert-butyl, the third deprotecting agent and the cleavage agent can be TFA at the same time. In certain embodiments, the third deprotecting agent and the cleavage agent can be a mixture of TFA, water and triisopropylsilane. Other suitable deprotecting agents include but are not limited to trifluoroacetic acid (TFA) / dichloromethane (DCM) mixtures, triethylsilane (TES), indole, phenol / anisole mixtures and thioanisole. Step (i) of method 1 can be carried out in a solvent (e.g., an organic solvent). The organic solvent can be any solvent known in the art for resin-based synthesis. Suitable organic solvents include but are not limited to CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile, etc. In some embodiments, the solvent is CH 2 Cl 2 .

[0306] In step (a) of method 2, PG on the resin-based compound of formula 1 may be a protecting group, in particular, an amine protecting group, such as 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) or 4-methyltrityl (Mtt). Also in step (a) of method 2, PG 1It can be a protecting group, especially an amine protecting group, such as 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butyloxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) or 4-methyltrityl (Mtt) orthogonal to PG. In some embodiments, PG can be 9-fluorenylmethyl-carbonyl (Fmoc) or tert-butylcarbonyl (Boc), and PG 1 It may be an orthogonal protecting group, such as 4-methyltrityl (Mtt) or monomethoxytrityl (MMt).

[0307] The first deprotecting agent in step (a) of method 2 can be a reagent or a mixture of reagents capable of removing amine protecting groups, such as 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) or 4-methyltrityl (Mtt). In some embodiments, the first deprotecting agent in step (a) of method 2 comprises a reagent or a mixture of reagents capable of removing 9-fluorenylmethyl-carbonyl. In some embodiments, the first deprotecting agent in step (a) of method 2 can be an acidic or basic deprotecting agent, depending on the PG and / or PG 1 In particular, the first deprotecting agent can be a basic deprotecting agent, such as piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine or collidine. In particular, the first deprotecting agent can be an acidic deprotecting agent, such as TFA, HCl or H 3 PO 4 Suitable deprotecting agents and conditions for step (a) of method 2 can be found, for example, in Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups in organic synthesis. Hoboken, NJ: Wiley-Interscience.

[0308] In some embodiments of step (a) of method 2, the base for removing the Fmoc group can be an amine base, such as piperidine. In some embodiments, the polar aprotic solvent of step (a) of method 2 can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include but are not limited to dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO) etc. It should be understood that step (a) can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The deprotection step (a) of method 2 can be carried out for a period of time, for example, from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After the deprotection step (a) of method 2, the resin-based compound of formula B produced in step (a) of method 2 can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0309] In step (b) of method 2, the PG on the compound of formula 3 2 and / or PG 3 Each of them can be independently a carboxyl protecting group, and LG is a leaving group, such as an active ester. Suitable active esters known in the art include, but are not limited to, imidazolyl, 4-nitrophenoxy, phenoxy, 2,4,5-trichlorophenyl, NHS ester, benzotriazolyl, etc. In some embodiments, LG in compound 3 is selected from the group consisting of: imidazolyl, 4-nitrophenoxy and phenoxy. In some embodiments, LG in compound 3 is 4-nitrophenyl ester. It should be understood that PG 2 and / or PG 3 The choice will be with PG and PG 1 In some embodiments, PG 2 and / or PG 3 In step (b) of method 2, the base may be any base known in the art for urea formation reactions. Suitable bases include, but are not limited to, N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

[0310] Step (b) of method 2 can be carried out in a solvent (e.g., an organic solvent). The organic solvent can be any solvent known in the art for resin-based synthesis. Suitable organic solvents include, but are not limited to, CH 2 Cl 2, THF, ether, MeOH, EtOH, acetone, acetonitrile, etc. In some embodiments, the solvent is CH 2 Cl 2 It should be understood that step (b) of method 2 can be performed at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The deprotection step (b) of method 2 can be performed for a period of time, such as from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After step (b) of method 2, the resin-based compound of formula 4 produced in step (b) can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0311] In step (c) of method 2, the second deprotecting agent can be a reagent or a mixture of reagents capable of removing a protecting group selected from the group consisting of 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt). In some embodiments, the second deprotecting agent can be a reagent or a mixture of reagents capable of removing 4-methyltrityl (Mtt). In some embodiments, according to PG 1 PG 2 and / or PG 3 The second deprotecting agent may be an acidic deprotecting agent, a basic deprotecting agent or other deprotecting agent. In some embodiments, the second deprotecting agent may be an acidic deprotecting agent. Suitable acidic deprotecting agents include, but are not limited to, TFA, HCl or H 3 PO 4 In some embodiments, the second deprotecting agent of step (c) of method 2 is selected from the group consisting of trifluoroacetic acid (TFA), acetic acid, trifluoroethanol, hexafluoroisopropanol, and combinations thereof. In some embodiments, the second deprotecting agent is TFA, for example, when PG 2 In some embodiments, the second deprotecting agent in step (c) of method 2 is a mixture of acetic acid and trifluoroethanol, for example, when PG 2is Mtt. Suitable deprotecting agents and conditions for step (c) of method 2 can be found, for example, in Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups in organic synthesis. Hoboken, NJ: Wiley-Interscience. Step (c) of method 2 can be carried out in a solvent such as an organic solvent. The organic solvent can be any solvent known in the art for resin-based synthesis. Suitable organic solvents include, but are not limited to, CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile, etc. In some embodiments, the solvent is CH 2 Cl 2 After the deprotection step (c) of method 2, the resin-based compound of formula 5 produced in step (c) of method 2 can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0312] In step (d) of method 2, PG 4 It can be a protecting group, in particular an amine protecting group, 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt). In some embodiments, PG 4 It can be 9-fluorenylmethyl-carbonyl (Fmoc) or tert-butylcarbonyl (Boc). 4 For the selection of, please refer to step (a) of method 2 above. In step (d) of method 2, the solvent can be a polar aprotic solvent. In some embodiments, the polar aprotic solvent can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO). In some embodiments, the polar aprotic solvent is DMF. Suitable bases used in step (d) of method 2 include, but are not limited to, N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and force. In some embodiments, the base in step (d) is N, N-diisopropylethylamine (i Pr 2 NEt).

[0313] The coupling agent can be any coupling agent known in the art for resin-based synthesis. Suitable coupling agents for use in step (d) of method 2 include, but are not limited to, benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-aza-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyAOP), ethyl cyano(hydroxyimino)acetate-O 2)-tris-(1-pyrrolidinyl)-phosphonium hexafluorophosphate (PyOxim), bromo-tripyrrolidine-phosphonium hexafluorophosphate (PyBrOP), 3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazine-4(3H)-one (DEPBT), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-( 1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylammonium hexafluorophosphate (HCTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxido-1H-benzotriazol-1-yl)-4-morpholinylmethylene]-N-methylmethanium hexafluorophosphate (HDMC), 1-cyano-2-ethoxy-2-oxoethylamino)dimethylamino- Morpholine-carbonium hexafluorophosphate (COMU), dimethylamino (triazolo [4,5-b] pyridin-3-yloxy) methylene]-dimethylammonium tetrafluoroborate (TATU), N,N,N',N'-tetramethyl-S-(1-oxido-2-pyridyl) thiouronium tetrafluoroborate (TOTT), tetramethylfluoroformamidine hexafluorophosphate (TFFH), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl In some embodiments, the coupling agent in step (d) of method 2 may be HATU, TBTU, HBTU, CDMT, HDMC, COMU, TATU, TOTT, T3P or DMTMM. In some embodiments, the coupling agent in step (d) of method 2 is (PyBOP).

[0314] It should be understood that step (d) of method 2 can be carried out in the presence of an additive. Suitable additives include, but are not limited to, 1-hydroxybenzotriazole (HOBt), 1-hydroxybenzotriazole-6-sulfonamidomethyl resin HCl (HOBt-6-sulfonamidomethyl resin HCl), hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt), N-hydroxysuccinimide (NHS), 1-hydroxy-7-aza-1H-benzotriazole (HOAt), ethyl 2-cyano-2-(hydroxyimino)acetate or 4-(N,N-dimethylamino)pyridine (DMAP). It should be understood that step (d) of method 2 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The coupling step (d) of method 2 can be carried out for a period of time, for example, from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After the coupling step (d) of method 2, the resin-based compound of formula 7 produced in step (d) of method 2 can be quenched with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0315] In step (e) of method 2, the PG on the resin-based compound of formula 7 4 It can be an amine protecting group, such as 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt). The third deprotecting agent in step (e) of method 2 comprises a reagent or a mixture of reagents capable of removing a protecting group, wherein the protecting group is selected from the group consisting of 9-fluorenylmethyl-carbonyl (Fmoc), benzyl carbamate (Cbz), tert-butoxycarbonyl (Boc), acetamide, trifluoroacetamide, p-toluenesulfonamide, trityl, monomethoxytrityl (MMt) and 4-methyltrityl (Mtt). In some embodiments, PG 4 It can be 9-fluorenylmethyl-carbonyl (Fmoc). In some embodiments, the third deprotecting agent can be the third deprotecting agent of step (e) of method 2, which comprises a reagent or a mixture of reagents capable of removing 9-fluorenylmethyl-carbonyl. In some embodiments, the third deprotecting agent can be an acidic or basic protecting agent. In some embodiments, the third deprotecting agent can be a basic deprotecting agent, such as piperidine, morpholine, 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris (2-aminoethyl) amine, pyridine or collidine. In some embodiments, the third deprotecting agent can be an acidic deprotecting agent, such as TFA, HCl or H3 PO 4 Suitable deprotecting agents and conditions for step (e) of method 2 can be found, for example, in Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups in organic synthesis. Hoboken, NJ: Wiley-Interscience.

[0316] In some embodiments, the step (e) of method 2 can be carried out in a solvent (e.g., a polar aprotic solvent). The polar aprotic solvent of the step (e) of method 2 can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO) etc. It should be understood that the step (e) of method 2 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The deprotection step (e) of method 2 can be carried out for a period of time, for example, from about 5 minutes to about 200 minutes, or from about 10 minutes to about 100 minutes, or from about 40 to about 60 minutes to about 45 minutes, or about 60 minutes, or about 90 minutes. After the deprotection step (e) of method 2, the resin-based compound of formula 9 produced in step (e) of method 2 can be treated with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0317] In step (f) of method 2, PG 5 Can be a carboxyl protecting group. In some embodiments, PG 5 It can be tert-butyl. 5 For the selection of, please refer to step (b) of method 2 above. In step (f) of method 2, the solvent can be a polar aprotic solvent. In some embodiments, the polar aprotic solvent can be any polar aprotic solvent known in the art. Suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO). In some embodiments, the polar aprotic solvent is DMF. Suitable bases used in step (f) include, but are not limited to, N,N-diisopropylethylamine ( i Pr2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and force. In some embodiments, the base in step (f) of method 2 is N, N-diisopropylethylamine ( i Pr 2 NEt). The coupling agent of step (f) of method 2 can be any coupling agent known in the art for resin-based synthesis. Suitable coupling agents for step (f) of method 2 include, but are not limited to, benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-aza-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyAOP), cyano(hydroxyimino)ethyl-O-acetyl-P-hydroxy-1 ... 2)-tris-(1-pyrrolidinyl)-phosphonium hexafluorophosphate (PyOxim), bromo-tripyrrolidine-phosphonium hexafluorophosphate (PyBrOP), 3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazine-4(3H)-one (DEPBT), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-( 1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylammonium hexafluorophosphate (HCTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxido-1H-benzotriazol-1-yl)-4-morpholinylmethylene]-N-methylmethanium hexafluorophosphate (HDMC), 1-cyano-2-ethoxy-2-oxoethylamino)dimethylamino- Morpholine-carbonium hexafluorophosphate (COMU), dimethylamino (triazolo [4,5-b] pyridin-3-yloxy) methylene]-dimethylammonium tetrafluoroborate (TATU), N,N,N',N'-tetramethyl-S-(1-oxido-2-pyridyl) thiouronium tetrafluoroborate (TOTT), tetramethylfluoroformamidine hexafluorophosphate (TFFH), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl In some embodiments, the coupling agent in step (f) of method 2 may be HATU, TBTU, HBTU, CDMT, HDMC, COMU, TATU, TOTT, T3P or DMTMM. In some embodiments, the coupling agent in step (f) of method 2 is (PyBOP).

[0318] It should be understood that step (f) of method 2 can be carried out in the presence of an additive. Suitable additives include, but are not limited to, 1-hydroxybenzotriazole (HOBt), 1-hydroxybenzotriazole-6-sulfonamidomethyl resin HCl (HOBt-6-sulfonamidomethyl resin HCl), hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt), N-hydroxysuccinimide (NHS), 1-hydroxy-7-aza-1H-benzotriazole (HOAt), ethyl 2-cyano-2-(hydroxyimino)acetate or 4-(N,N-dimethylamino)pyridine (DMAP). It should be understood that step (f) of method 2 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The coupling step (f) of method 2 can be carried out for a period of time, for example, from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 15 minutes, or about 30 minutes, or about 90 minutes. After the coupling step (f) of method 2, the resin-based compound of formula 10 produced in step (f) of method 2 can be quenched with reagents known in the art, such as DMF, IPA, CH 2 Cl 2 Wait for washing.

[0319] In step (g) of method 2, the PG on the resin-based compound of formula 10 2 PG 3 and / or PG 5 Each of them may be a protecting group, such as a carboxyl protecting group. In some embodiments, the third deprotecting agent of step (g) of method 2 is a reagent or a mixture of reagents capable of removing a protecting group, wherein the protecting group is selected from the group consisting of tert-butyl, benzyl, 2-chlorotrityl (2-Cl-Trt), 2,4-dimethoxybenzyl (Dmb), allyl, 1,1-dimethylallyl (Dma) and p-nitrobenzyl (pNB). In some embodiments, PG 2 PG 3 and PG 5 The third deprotecting agent in step (g) of method 2 can be an acidic or basic protecting agent, depending on the PG 2 PG 3 and / or PG 5 In some embodiments, the third deprotecting agent can be an acidic deprotecting agent, such as TFA, HCl or H 3 PO 4. In some embodiments, the third deprotecting agent can be selected from the group consisting of trifluoroacetic acid (TFA), hydrofluoric acid (HF), trifluoromethanesulfonic acid (TfOH), trifluoroacetic acid (TFA) / water / triisopropylsilane mixture, trifluoroacetic acid (TFA) / dichloromethane (DCM) mixture, triethylsilane (TES), indole, phenol / anisole mixture and anisole. Suitable deprotecting agents and conditions for step (g) can be found in, for example, Wuts, PGM, Greene, TW, Greene, TW, & John Wiley & Sons. (2006). Greene's protective groups inorganic synthesis. [Greene's protective groups in organic synthesis] Hoboken, NJ: Wiley-Interscience [Wiley International Science]. In some embodiments, the third deprotecting agent of step (g) of method 2 is a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture. In some embodiments, the cleavage agent of step (g) of method 2 is a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture.

[0320] It should be understood that the organic solvent in step (g) of method 2 can be any organic solvent known in the art. Suitable organic solvents include, but are not limited to, CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile, etc. It should be understood that step (g) of method 2 can be carried out at any temperature commonly used in solid phase synthesis, such as room temperature, cooling or heating conditions. The deprotection step (g) of method 2 can be carried out for a period of time, such as from about 5 minutes to about 200 minutes, or about 10 minutes to about 100 minutes, or about 40 to about 60 minutes to about 45 minutes, or about 60 minutes, or about 90 minutes.

[0321] Method 2, specific embodiment:

[0322] PSMA 617(I) can be prepared using solid phase peptide synthesis techniques as shown in the following scheme.

[0323]

[0324]

[0325]

[0326] The following reagents and intermediates may be used.

[0327]

[0328]

[0329] Three chemical transformations are performed. The Fmoc protecting group is removed from Fmoc-Lys(Mtt)-Wang resin (2-1) by mixing the resin with a base in a polar aprotic solvent, and then the resin is washed with a solvent. L-glutamic acid di-tert-butyl ester hydrochloride (3') is converted into activated urea or carbamate (2-3) using, for example, a base and carbonyl diimidazole, p-nitrophenyl chloroformate, phenyl chloroformate, etc. in an organic solvent. The deprotected compound 2-2 is mixed with a solution of 2-3. When the conversion to 2-4 is complete, the resin is washed with an organic solvent.

[0330] The methyl trityl protecting group is removed from 2-4 by mixing the resin with a mixture of 2,2,2-trifluoroethanol, acetic acid, and dichloromethane. After removal of the trityl group, the resin is washed with a polar aprotic solvent. The deprotected compound 2-5 is mixed with (2S)-2-(9-fluorenylmethoxycarbonylamino)-3-naphthalen-2-yl-propionic acid (2-6), a base, and an activator such as, but not limited to, a phosphonium salt activator in a polar aprotic solvent. After conversion to 2-7, the resin is washed with a polar aprotic solvent.

[0331] The Fmoc protecting group is removed from 2-7 by mixing the resin with a base in a polar aprotic solvent and washing the resin with a solvent. The deprotected compound 2-8 is mixed with 2-9, a base and an activator (such as, but not limited to, a phosphonium salt activator in a polar aprotic solvent). After conversion to 2-10, the resin is washed with a polar aprotic solvent and an organic solvent.

[0332] The tert-butyl group is removed and the resin is cleaved by mixing with a mixture of trifluoroacetic acid, triisopropylsilane and water. The liquid is separated from the cleaved resin, added to an organic solvent and filtered. The crude PSMA 617 is purified by column chromatography and the material is desalted using a C18 column and an acetonitrile / water gradient eluent. Acetonitrile is evaporated or distilled from the aqueous solution under vacuum and PSMA 617 (I) is isolated from the solution by lyophilization.

[0333] Examples

[0334] The examples and preparations provided below further illustrate and illustrate certain aspects of the embodiments of the present disclosure.It should be understood that the scope of the present disclosure is not limited in any way by the scope of the following examples.

[0335] abbreviation

[0336] The materials used in the examples described herein include, but are not limited to, those described by the following abbreviations known to those skilled in the art:

[0337]

[0338]

[0339] Example 1a: Preparation of AB2

[0340] Compound AB2 was prepared according to the following synthetic scheme:

[0341]

[0342] Under argon, 403 mg of 4-nitrophenyl chloroformate (2.0 mmol, 0.95 eq.) was added to 621 mg of L-glutamic acid di-tert-butyl ester hydrochloride (2.1 mmol, 1.0 eq.) in 21 mL of dichloromethane (CH 2 Cl 2 ) was added to the stirred solution in . The solution was cooled to 0°C and then added dropwise using a pressure equalizing addition funnel in 5 mL of CH 2 Cl 2 0.730 mL of diisopropylethylamine ( i Pr 2 NEt) (4.2 mmol, 2.0 equiv). i Pr 2 After NEt was added, the reaction mixture was stirred for 30 min, warmed to room temperature, and stirred for another 30 min. The reaction mixture was then evaporated to give a crude product as an oily residue. The residue was used without further purification and was kept under high vacuum until needed.

[0343] Example 1b: Alternative preparation of AB2

[0344] 0.891 g of 4-nitrophenyl chloroformate (4.42 mmol) and 15 mL of dichloromethane (CH 2 Cl 2 ) was loaded into a reaction vessel and purged with argon. The reaction vessel was equipped with a temperature probe and the solution was cooled to ≤-10°C using an IPA / ice bath. In a separate reaction flask, 1.44 g of L-glutamic acid di-tert-butyl ester hydrochloride (4.87 mmol) was dissolved in 15 mL of dichloromethane (CH 2 Cl 2 ), then add 1.54 mL of diisopropylethylamine ( i Pr 2 NEt) (8.84 mmol). The L-glutamic acid di-tert-butyl ester hydrochloride solution was added dropwise to the cooled 4-nitrophenyl chloroformate solution while maintaining a temperature of -11°C to -5°C. After the addition was complete, the ice bath was removed and the reaction mixture was stirred at room temperature for 4 hours, at which time UPLC / MS showed that the reaction was complete. The reaction mixture was used without further purification.

[0345] Example 2: Preparation of PSMA-617

[0346] Starting from Fmoc-Lys(N-4-methoxytrityl)-2-chlorotrityl-resin, the compound of formula I was prepared by SPPS. The compound of formula I was prepared according to the following synthetic scheme:

[0347]

[0348]

[0349] Tables 1 and 2 below are summaries of the above synthetic schemes performed according to the methods described herein:

[0350]

[0351] Table 1. Reaction steps of the synthesis scheme of PSMA-617.

[0352]

[0353] Preprocessing steps:

[0354] Prior to use in the synthesis method, the peptide synthesis vessel was loaded with 2.00 g (1.08 mmol) of Fmoc-Lys(Mtt)-Wang resin (resin loading = 0.54 mmol / g). The resin was washed with 20 mL of dimethylformamide (DMF) for 5 minutes, and then the liquid was drained from the vessel. The washing step was repeated once more.

[0355] Step 1: Urea formation

[0356] Add lysine resin to peptide synthesis vessel. Add 20% piperidine solution in DMF (about 20 mL) for Fmoc deprotection. Bubble argon through the solution for 15 min and then drain. Add 20% piperidine in DMF (about 20 mL) and continue bubbling for 5 min before draining (repeat 2X). Resin is sequentially treated with DMF (about 20 mL x 3), IPA (about 20 mL x 3) and CH 2 Cl 2 The crude residue of AB2 was dissolved in 60 mL of CH 2 Cl 2 Then add half of the volume to the deprotected lysine resin and then add i Pr 2 NEt. Argon was bubbled through the solution for 45 min and then vented. The remaining crude AB2 solution and i Pr 2 NEt was added to the resin sequentially, and argon was bubbled through the solution again for 30 min. The reaction vessel was then evacuated and the resin was sequentially heated with CH 2 Cl 2(about 20mL x 3), IPA (about 20mL x 3) and CH 2 Cl 2 (about 20mL x 3) washing.

[0357] Alternative urea formation:

[0358] Add lysine resin to the peptide synthesis vessel. Mix the AB2 reaction mixture from Example 1b with 0.188 mL i Pr 2 NEt (1.08 mmol) was added to the resin. The reaction mixture was stirred overnight (about 18 hours). The resin was treated with CH 2 Cl 2 (about 20mL x 3) washing.

[0359] Step 2: Mtt deprotection

[0360] Join CH 2 Cl 2 (about 20 mL) and bubble argon through the solution for 5 min. If bubbling is vigorous, add a small amount of CH 2 Cl 2 to maintain the same volume. Then drain the yellow solution and repeat five times. 2 Cl 2 Wash until the filtrate remains clear. Add TFA in CH 2 Cl 2 If the solution remains clear, drain the reaction mixture and proceed to the next coupling step. If the solution turns yellow, replace with fresh CH 2 Cl 2 The resin was washed until clear and the process was repeated until a clear reaction solution was obtained. The resin was washed with DMF (about 20 mL x 3).

[0361] Alternative Mtt deprotection

[0362] Prepare 112 mL of CH 2 Cl 2, 32mL 2,2,2-trifluoroethanol (TFE) and 16mL acetic acid (AcOH) solution. Add 20mL AcOH-TFE solution to the resin in the peptide synthesis vessel. Stir the mixture for about 1 hour and drain the liquid from the peptide synthesis vessel. Repeat the procedure once more. Add 20mL AcOH-TFE solution to the resin in the peptide synthesis vessel and stir the mixture overnight. Drain the liquid from the peptide synthesis vessel. Add 20mL AcOH-TFE solution to the resin in the peptide synthesis vessel and stir the mixture for about 1 hour. Drain the liquid from the peptide synthesis vessel. Repeat the procedure once more. The resin was treated with 3xCH 2 Cl 2 , 3x 2-propanol (IPA) and 3x DMF washes.

[0363] Step 3: Amine coupling

[0364] The amino acid solution in DMF (about 20 mL) i Pr 2 NEt and PyBOP were added to the peptide synthesis vessel. Argon was bubbled through the solution for 1 h and then vented. The resin was washed with DMF (about 20 mL x 3), IPA (about 20 mL x 3), and DMF (about 20 mL x 3) in sequence.

[0365] Alternative amine coupling

[0366] The amino acid solution in DMF (about 20 mL), PyBOP in DMF (about 20 mL) and i Pr 2 NEt was added to the peptide synthesis vessel containing the resin. The mixture was stirred for about 4 hours, and then the liquid was drained from the peptide synthesis vessel. The resin was washed with DMF (about 20 mL x 3) and then with IPA (about 20 mL x 3).

[0367] Step 4: Fmoc deprotection

[0368] Deprotection steps were performed before each amino acid coupling step (except for Mtt deprotection, which used CH 2 Cl 2 2% TFA in DMF). A 20% piperidine solution in DMF (about 20 mL) was added for Fmoc deprotection. Argon was bubbled through the solution for 15 min and then drained. 20% piperidine in DMF (about 20 mL) was added and continued to bubble for 5 min (2X) before draining. The resin was washed with DMF (about 20 mL x 3), IPA (about 20 mL x 3), and DMF (about 20 mL x 3) in sequence.

[0369] Alternative FMOC deprotection

[0370] The alternative deprotection step was performed before each amino acid coupling step (except for the Mtt deprotection). A 20% piperidine solution in DMF (about 20 mL) was added to the resin. The reaction was stirred for about 10 minutes. The liquid was drained from the reaction vessel and the process was repeated two more times. The resin was washed with DMF (about 20 mL x 3) and then with IPA (about 20 mL x 3). In some cases, the resin was further washed with DMF (about 20 mL x 3) or alternatively with CH 2 Cl 2 (about 20mL x 3) washing.

[0371] Step 5: Resin cleavage

[0372] The resin was washed with CH 2 Cl 2 (about 20 mL x 3) and drain. Add 25 mL of lysis reagent (95% TFA, 2.5% H 2 0.2.5% triisopropylsilane) was added to the peptide synthesis vessel, bubbled with argon for 1 h, drained, and repeated twice with fresh cleavage agent (10 mL, 15 min). The filtrate was stirred overnight under argon and room temperature. The reaction mixture was concentrated under reduced pressure until 10 ml remained. The product was ground in 40 mL of ether and centrifuged. The solution was poured out from the resulting particles. The previous step was repeated twice by resuspending the particles in 50 mL of ether and centrifuging. The particles were dried over an argon stream and then dried under high vacuum.

[0373] Alternative resin cracking

[0374] Prepare a mixture of 42mL trifluoroacetic acid (TFA), 0.90mL water and 3.0mL triisopropylsilane (TIPS), and add it to the resin in the peptide synthesis container. The mixture was stirred for 6 hours (the last hour was heated to 30°C in a water bath). The resin was filtered and the solid was washed with 2mL TFA. The filtrate and washings containing the product were merged. Washed with 420mL methyl tert-butyl ether (MTBE) cooled in an ice-water bath. The solid product was collected by filtration. The solid was washed with 5mL MTBE. The solid was dried under vacuum to provide 1.19g of a thick compound of formula I.

[0375] Purification Procedure 1

[0376] The obtained powder was dissolved in a small amount of DMSO and loaded onto a C18 column. The desired product was purified by reverse phase chromatography (0-55% acetonitrile in 50mM ammonium carbonate buffer, pH 7.0). The acetonitrile was evaporated under reduced pressure, and the remaining buffered aqueous solution was frozen and removed by lyophilization. 256mg (49.2%) of the desired product was collected as a white powder.

[0377] Purification Procedure 2

[0378] The compound of Formula I was purified using a Biotage 60g C18 ULTRA column with mobile phase A = 0.1% TFA in water, mobile phase B = acetonitrile (ACN) (flow rate = 45 mL / min, 1CV = 90 mL, collection wavelength was 275 nm). Crude PSMA-617 in 20 mL of 0.1% TFA in water and 3 mL ACN were loaded onto the column. Purification was performed using 1CV 15% B, 7CV 15% B to 25% B held at 25% B. The product peak began to elute at approximately 5CV and fractions were collected. Fractions were checked using UPLC-MS, acceptable product fractions (≥98% at 275 nm) were combined, and the product solution was concentrated using a rotary evaporator to remove ACN (final volume = 59 mL). The compound of Formula I was desalted using a Biotage 30g C18 ULTRA column set to: mobile phase A = 0.1% AcOH in water, mobile phase B = ACN (flow rate = 25mL / min, 1CV = 45mL, collection wavelength of 275nm). 0.28g ammonium acetate, 0.36mL AcOH and 3mL ACN were added to the PSMA-617 aqueous solution (59mL). The PSMA-617 solution was loaded onto the column and the column was washed with 5CV 5% B. The product was eluted from the column using 3CV 39% B. The product peak was eluted at 6CV and 1 fraction was collected. The product solution was concentrated using a rotary evaporator to remove ACN. The solid was isolated by lyophilization to provide 0.675g of the compound of Formula I. Example 3: Characterization of PSMA-617 by NMR and high resolution MS ES+ (TOF):

[0379] 1 H NMR (D 6-DMSO):8.03(t,1H),7.88(t,1H),7.84(d,1H),7.69-7.80(m,2H),7.69(s,1H),7.43-7.47(m,2H), 7.39(dd,1H),6.29-6.33(m,2H),4.50-4.52(m,1H),4.08-4.11(m,1H),3.99-4.02(m,1H),3.46(br s,6H),3.13(br s,2H),3.10(d,1H),2.89-3.04(m,17H),2.68(s,4H),2.22-2.27(m,2H),2.07(s,1H),1.91(s,1H),1.57-1 .74(m,5H),1.46-1.50(m,2H),1.31-1.35(m,3H),1.22-1.26(m,3H),1.06-1.12(m,1H),0.80-0.82(m,2H).

[0380] Note: six carboxylic acid protons are distributed throughout the spectrum; additional resonances from residual acetic acid (1.91 ppm) and acetonitrile (2.07 ppm) are present.

[0381] 13 C NMR (D 6 -DMSO):175.09,174.52,174.14,173.73,170.99,170.55,170.11,169.64,157.26,1 35.78,132.86,131.70,127.81,127.37,127.32,127.24,127.22,125.83,125.24,57 .80,55.34,54.86,53.85,52.33,51.78,51.24,50.52,49.96,49.84,44.54,43.77,38.14,37.86,36.81,31.65,30.05,29.61,29.50,28.63,28.56,28.38,27.69,22.41.

[0382] Note: there are additional resonances from residual acetic acid (171.88 ppm, 20.98 ppm) and acetonitrile (1.068 ppm).

[0383] High-resolution MS ES+(TOF): M+H + =1042.5067m / z (theoretical value 1042.5096m / z).

Claims

1. A method for preparing a compound of formula I The method comprises a) reacting a resin-based compound of formula A in a polar aprotic solvent contacting with a first deprotecting agent to provide a resin-based compound of formula A' b) contacting the resin-based compound of formula A' with the compound of formula B in the presence of an organic solvent and a base To provide a resin-based compound of formula C c) contacting the resin-based compound of formula C with a second deprotecting agent in the presence of an organic solvent to provide a resin-based compound of formula C' d) in the presence of a polar aprotic solvent, a coupling agent and a base, reacting the resin-based compound of formula C' with PG 1 -3-(2-naphthyl)-L-alanine to provide a resin-based compound of formula D e) contacting the resin-based compound of formula D with the first deprotecting agent in a polar aprotic solvent to provide a resin-based compound of formula D' f) in the presence of a polar aprotic solvent, a coupling agent and a base, reacting the resin-based compound of formula D' with PG 1 - tranexamic acid to provide a resin-based compound of formula E g) contacting the resin-based compound of formula E with the first deprotecting agent in a polar aprotic solvent to provide a resin-based compound of formula E' h) in the presence of a polar aprotic solvent, a coupling agent and a base, reacting the resin-based compound of formula E' with a 2’ Contact with compounds To provide a resin-based compound of formula F i) contacting the resin-based compound of formula F with a third deprotecting agent and / or cleaving agent in an organic solvent, Among them PG 1 and PG 2 is an amine protecting group, LG is a leaving group, and R 1 C 1 -C 4 Alkyl or cyclohexyl.

2. The method according to claim 1, wherein the first deprotecting agent of step (a) is selected from the group consisting of piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine and collidine.

3. The method according to claim 2, wherein the first deprotecting agent in step (a) is piperidine.

4. The method of claim 3, wherein the polar aprotic solvent of step (a) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

5. The method according to claim 4, wherein the polar aprotic solvent of step (a) is dimethylformamide (DMF).

6. The process according to claim 1, wherein the base of step (b) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

7. The method according to claim 6, wherein the base in step (b) is N,N-diisopropylethylamine ( i Pr 2 NEt).

8. The method according to claim 7, wherein the organic solvent of step (b) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile.

9. The method according to claim 8, wherein the organic solvent in step (b) is CH 2 Cl 2 .

10. The method of claim 1, wherein the second deprotecting agent of step (c) is selected from the group consisting of trifluoroacetic acid (TFA), acetic acid, trifluoroethanol, dichloromethane, hexafluoroisopropanol, dichloromethane, and combinations thereof.

11. The method according to claim 10, wherein the second deprotecting agent in step (c) is trifluoroacetic acid (TFA) or a mixture of acetic acid and trifluoroethanol.

12. The method according to claim 11, wherein the organic solvent of step (c) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile.

13. The method according to claim 12, wherein the organic solvent in step (c) is CH 2 Cl 2 .

14. The method of claim 1, wherein the polar aprotic solvent of step (d) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

15. The method according to claim 14, wherein the polar aprotic solvent of step (d) is dimethylformamide (DMF).

16. The method of claim 15, wherein the base of step (d) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

17. The method according to claim 16, wherein the base in step (d) is N,N-diisopropylethylamine ( i Pr 2 NEt).

18. The method of claim 17, wherein the coupling agent of step (d) is selected from the group consisting of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxy-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), [0013] N,N,N',N'-tetramethyl-S-(1-oxy-2-pyridyl)thiouronium tetrafluoroborate (TOTT), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

19. The method of claim 18, wherein the coupling agent of step (d) is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).

20. The method of claim 1, wherein the first deprotecting agent of step (e) is selected from the group consisting of piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine and collidine.

21. The method according to claim 20, wherein the first deprotecting agent in step (e) is piperidine.

22. The method of claim 21, wherein the polar aprotic solvent of step (e) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures and dimethyl sulfoxide (DMSO).

23. The method of claim 22, wherein the polar aprotic solvent of step (e) is dimethylformamide (DMF).

24. The process of claim 1, wherein the polar aprotic solvent of step (f) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO).

25. The process of claim 24, wherein the polar aprotic solvent of step (f) is dimethylformamide (DMF).

26. The process of claim 25, wherein the base of step (f) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

27. The method according to claim 26, wherein the base in step (f) is N,N-diisopropylethylamine ( i Pr 2 NEt).

28. The method of claim 27, wherein the coupling agent of step (f) is selected from the group consisting of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxy-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), [0013] N,N,N',N'-tetramethyl-S-(1-oxy-2-pyridyl)thiouronium tetrafluoroborate (TOTT), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

29. The method of claim 28, wherein the coupling agent of step (f) is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).

30. The method of claim 1, wherein the first deprotecting agent of step (g) is selected from the group consisting of piperidine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), dicyclohexylamine, 4-methylpiperidine (4MP), tris(2-aminoethyl)amine, pyridine and collidine.

31. The method of claim 30, wherein the first deprotecting agent in step (g) is piperidine.

32. The method of claim 31, wherein the polar aprotic solvent of step (g) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO).

33. The process of claim 32, wherein the polar aprotic solvent of step (g) is dimethylformamide (DMF).

34. The process of claim 1, wherein the polar aprotic solvent of step (h) is selected from the group consisting of dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), dichloromethane / dimethylformamide mixtures, acetonitrile (ACN), acetonitrile / dimethylformamide mixtures, and dimethyl sulfoxide (DMSO).

35. The process of claim 34, wherein the polar aprotic solvent of step (h) is dimethylformamide (DMF).

36. The process of claim 35, wherein the base of step (h) is selected from the group consisting of: N,N-diisopropylethylamine ( i Pr 2 NEt), triethylamine (TEA), 4-methylmorpholine (NMM), imidazole, pyridine and collidine.

37. The method according to claim 36, wherein the base in step (h) is N,N-diisopropylethylamine ( i Pr 2 NEt).

38. The method of claim 37, wherein the coupling agent of step (h) is selected from the group consisting of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-[(5-chloro-3-oxy-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), [0013] N,N,N',N'-tetramethyl-S-(1-oxy-2-pyridyl)thiouronium tetrafluoroborate (TOTT), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-propanephosphonic anhydride (T3P), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

39. The method of claim 38, wherein the coupling agent of step (h) is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).

40. The method of claim 1, wherein the third deprotecting agent of step (i) is selected from the group consisting of trifluoroacetic acid (TFA), hydrofluoric acid (HF), trifluoromethanesulfonic acid (TfOH), a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture, a trifluoroacetic acid (TFA) / dichloromethane (DCM) mixture, triethylsilane (TES), indole, a phenol / anisole mixture, and thioanisole.

41. The method of claim 40, wherein the third deprotecting agent of step (i) is a mixture of trifluoroacetic acid (TFA) / water / triisopropylsilane.

42. The method of claim 41, wherein the cleavage agent in step (i) is trifluoroacetic acid (TFA) or a mixture of trifluoroacetic acid (TFA) / water / triisopropylsilane.

43. The method of claim 42, wherein the organic solvent of step (i) is selected from the group consisting of: CH 2 Cl 2 , THF, ether, MeOH, EtOH, acetone, acetonitrile.

44. The method of claim 43, wherein the cleavage agent in step (i) is a trifluoroacetic acid (TFA) / water / triisopropylsilane mixture.

45. The method according to claim 44, wherein the organic solvent in step (i) is CH 2 Cl 2 .

46. ​​The method of claim 1, wherein PG 1 It is 9-fluorenylmethyl-carbonyl (Fmoc) or tert-butylcarbonyl (Boc).

47. The method of claim 46, wherein PG 1 It is 9-fluorenylmethyl-carbonyl (Fmoc).

48. The method of claim 1, wherein PG 2 It is monomethoxytrityl (MMt) or 4-methyltrityl (Mtt).

49. The method of claim 47, wherein PG 2 It is 4-methyltrityl (Mtt).

50. The method of claim 1, wherein LG is where * represents the point of attachment to the rest of the compound.

51. The method of claim 1, wherein R 1 It is tert-butyl.

52. A resin-based compound of formula C Among them PG 2 is an amine protecting group, and each R 1 C 1 -C 4 Alkyl or cyclohexyl.

53. A resin-based compound of formula C' Each R 1 C 1 -C 4 Alkyl or cyclohexyl.

54. A resin-based compound of formula D Among them PG 1 is an amine protecting group, and each R 1 C 1 -C 4 Alkyl or cyclohexyl.

55. A resin-based compound of formula D' Each R 1 C 1 -C 4 Alkyl or cyclohexyl.

56. A resin-based compound of formula E Among them PG 1 is an amine protecting group, and each R 1 C 1 -C 4 Alkyl or cyclohexyl.

57. A resin-based compound of formula E' Each R 1 C 1 -C 4 Alkyl or cyclohexyl.

58. A resin-based compound of formula F Each R 1 C 1 -C 4 Alkyl or cyclohexyl.

59. The resin-based compound according to claim 54 or 56, wherein PG 1 It is 9-fluorenylmethyl-carbonyl (Fmoc) or tert-butylcarbonyl (Boc).

60. The resin-based compound according to any one of claims 54, 56 or 59, wherein PG 1 It is 9-fluorenylmethyl-carbonyl (Fmoc).

61. The resin-based compound of claim 52, wherein PG 2 It is monomethoxytrityl (MMt) or 4-methyltrityl (Mtt).

62. The resin-based compound according to claim 52 or 61, wherein PG 2 It is 4-methyltrityl (Mtt).

63. A resin-based compound according to any one of claims 52 to 62, wherein R 1 It is tert-butyl.

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