SMAC mimetic for cancer treatment, its preparation process, and pharmaceutical composition

Abstract

The present invention relates to novel SMAC mimetic peptidomimetics useful for the treatment of proliferative diseases, including cancer, in mammals. The novel SMAC mimetics are prepared by incorporating (2S,5R)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylic acid, a novel unnatural amino acid that exclusively confers favorable transamide bond geometry for target protein binding. Here, the novel SMAC mimetic molecules not only show their efficacy in various cancer types, but also show in vitro and in vivo efficacy against therapy-resistant cancers as single agents. The novel SMAC mimetics disclosed in the present invention bind to the BIR-2 and BIR-3 domains of XIAP and show high anti-proliferative activity against a variety of mammalian cancer cell lines, including but not limited to chemotherapy- and TRAIL-resistant cell lines.

Description

[Technical Field]

[0001] This invention relates to SMAC (second mitochondrial caspase activator) mimic compounds useful for treating proliferative disorders, including cancer. [Background technology]

[0002] Avoiding apoptosis, or "programmed cell death," is one of the prominent features of human cancer. Therefore, restoring or inducing apoptosis in cancer cells is an attractive therapeutic strategy. Several endogenous cytotoxic proteins exist that regulate the complex balance between cell death and survival. One classic example of such reciprocal regulation within cells is the interaction between SMAC and IAP. SMAC is a pro-apoptotic protein that sensitizes cells to apoptosis in cancer cells by counteracting the activity of IAP. Thus, SMAC mimes have been found as a novel and targeted therapeutic approach for treating cancer (Abraha et.al, World J Gastrointest Oncol. 2016 Aug. 15;8(8):583-591). Currently, several clinical trials are underway for different SMAC mimes for various cancer types, demonstrating their significant importance in cancer treatment (Fulda et.al., Clinical Cancer Research. Volume 21, Issue 22, 2015. 5030-5036).

[0003] Inhibitory proteins (IAPs) are naturally occurring intracellular proteins that suppress caspase-dependent apoptosis. IAPs are key negative regulators that inhibit distinct caspases crucial for the initiation and execution of the apoptotic pathway. The mammalian IAP family comprises eight members. Among these, X-linked IAP (XIAP) is perhaps the best-characterized member of the IAP family known to play a direct role in regulating apoptosis. XIAP binds to caspase-3 and caspase-7, respectively, via its BIR2 domain and pre-linker region. In addition, XIAP also binds to caspase-9 via its BIR3 domain, thereby inhibiting caspase-9 dimerization and subsequent activation. Given that caspase-3 and caspase-7 play major roles in the execution of apoptosis in both the extrinsic and endogenous pathways, and that caspase-9 is a key inducible caspase in the endogenous pathway, XIAP is the most preferred target for reviving apoptosis. SMAC is a naturally available antagonist of the IAP protein. SMAC is released from mitochondria into the cytosol during apoptotic signaling and, in the case of matured SMAC proteins, is exposed at the amino terminus and binds to the BIR3 domain of XIAP via a preserved IAP-binding motif (IBM) containing four amino acid residues (AVPI) that interfere with the interaction of XIAP with XIAP caspases (Cong et.al., J.Med.Chem. 2019, 62, 5750-5772).

[0004] In addition, Smac also binds to the BIR3 domains of cIAP1 and cIAP2, thereby enhancing their E3 ligase activity, which promotes auto-ubiquitination and proteasomal degradation of cIAP1 and cIAP2. Several small molecule mimics of AVPIs, known as IAP inhibitors, are progressing in clinical trials for the treatment of cancer. LCL-161 and AT-406 are structurally monovalent, while Birinapant / TL32711 is bivalent and is one of the notable ones under development.

[0005] The primary approach used to design SMACs focuses on the synthesis of conformationally constrained compounds where the Xxx-Pro bond is preferably in trans configuration. Another area of ​​consideration has been the balance of lipophilic or divalent linkers at the C-terminus. Several efforts have been made in the past to evolve Smac AVPI tetrapeptides to develop bioavailable Smac mimics by systematically investigating the role of each amino acid in the AVPI(1) peptide and its tolerance to substitutions.

[0006] A library of tetrapeptides using the N-terminus of Smac was prepared using a novel strategy that controls the transgeometry around the proline residue, starting from the proline residue. These libraries replaced each of the four amino acid positions with any of the native amino acids. The alanine residue at position 1 of the tetrapeptide is critical for activity, and it has been shown that binding is significantly reduced when alanine is replaced with any native amino acid.

[0007] Therefore, in this field of technology, there is a need for novel compounds that can restore or induce apoptosis in cancer cells for the treatment of cancer. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Abraha et.al,World J Gastrointest Oncol.August 15, 2016;8(8):583-591 [Non-Patent Document 2] Fulda et.al.,Clinical Cancer Research.Volume21,Issue22,2015.5030-5036 [Non-Patent Document 3] Cong et.al.,J.Med.Chem.2019,62,5750-5772 [Overview of the project] [Problems that the invention aims to solve]

[0009] The main objective of the present invention is to develop peptide SMAC mimetic compounds that are useful as a safe and effective therapy for various types of cancer, both as monotherapy and in combination with available anticancer agents.

[0010] Another objective of the present invention is to develop a process for synthesizing SMAC mimics.

[0011] Another objective of the present invention is to develop a formulation of SMAC mimetic suitable for human application.

[0012] Another object of the present invention is the treatment of cancer using SMAC mimics.

[0013] Another object of the present invention is to provide targeted therapy for treatment-resistant cancer by using SMAC mimetic materials.

[0014] A further object of the present invention is to provide an SMAC mimetic, IAP antagonist, or IAP inhibitor that has the ability to strongly bind to both the BIR-2 and BIR-3 domains of XIAP / IAP and has significant bioavailability with robust in vitro and in vivo efficacy against treatment-resistant cancers. [Means for solving the problem]

[0015] Aspects of the present invention include SMAC mimic compounds of formula -I,

[0016] [ka]

[0017] Here, R 1 This includes hydrogen, and unsubstituted or substituted heteroaryl or C6-C10 selected from the group consisting of aryl; R 2 , R 3 and R 4 are each independently selected from the group consisting of H, C1-C6 alkyl, and C4-C8 cycloalkyl;[[ID=X]] A is selected from unsubstituted or substituted C1-C6 alkyl or C6-C 10 aryl; B is selected from C6-C 10 aryl, C(O)R 5 and C(O)N(R 6 )(R 7 ) from the group consisting of; R 5 is selected from the group consisting of OH, C1-C6 alkoxy, and C6 alkoxyaryl; R 6 and R 7 are each independently selected from the group consisting of hydrogen, C6-C 10 aryl, and C6-C 10 arylalkyl; or a pharmaceutically acceptable salt thereof.

[0018] Another aspect of the present invention provides a SMAC mimetic compound selected from the group consisting of the following.

[0019]

Chemical formula

[0020] Yet another aspect of the present invention is a process for preparing a SMAC mimetic compound of formula-I,[[ID=X]]

[0021]

Chemical formula

[0022] where, R 1 is hydrogen, and unsubstituted or substituted heteroaryl or C6-C 10Selected from the group consisting of aryls; R 2 , R 3 and R 4 Each is independently selected from the group consisting of H, C1-C6 alkyl, and C4-C8 cycloalkyl; A is unsubstituted or substituted C1-C6 alkyl or C6-C 10 Selected from the alphabet; B is C6-C 10 Aryl, C(O)R 5 and C(O)N(R 6 )(R 7 Selected from the group consisting of; R 5 It is selected from the group consisting of OH, C1-C6 alkoxy, or C6 alkoxyaryl; R 6 and R 7 These are, independently, hydrogen and C6-C 10 Aryl and C6-C 10 Selected from the group consisting of arylalkyl groups; a) The Boc group of 2-benzyl 1-(tert-butyl)(2S,5S)-5-(5-methylfuran-2-yl)pyrrolidine-1,2-dicarboxylate is removed by acid hydrolysis using TFA, followed by the conversion of the resulting amine into BocNHCH(R) in the presence of a peptide coupling reagent and a weak base. 2 The step of coupling with ')COOH to obtain the compound of formula P1;

[0023] [ka]

[0024] b) The Boc group is removed from the compound of formula P1 by acid hydrolysis using TFA, and the resulting amine is converted to the formula BocN(R) in the presence of a peptide coupling reagent and a weak base. 4 ′)CH(R 3 The step of coupling it with a compound of formula P2 by ')COOH;

[0025] [ka]

[0026] c) The step of catalytically hydrogenating the compound of formula P2 using a Pd catalyst in the presence of a solvent to obtain the free carboxylic acid of formula P3;

[0027] [ka]

[0028] d) A process is provided which includes the step of coupling the free carboxylic acid of formula P3 with NH2CH(A)(B) in the presence of a peptide coupling reagent and a weak base to obtain the compound of formula I.

[0029] Another aspect of the present invention provides a process comprising removing the Boc group 2-benzyl 1-(tert-butyl)(2S,5S)-5-(5-methylfuran-2-yl)pyrrolidine-1,2-dicarboxylate, then coupling the resulting amine with Boc-Val-OH to obtain compound II in which the Boc group is deprotected, then coupling it with Boc-Ala-OH to provide compound III in which the ester is saponified to a carboxylic acid, then coupling it with either H-Ile-OBn or a benzhydrylamine, and then acid-decomposing it to give compounds 2 and 3, respectively.

[0030] [ka]

[0031] Another aspect of the present invention provides a process comprising removing a Boc group from compound II, subsequently coupling it with Boc-N-Me-Ala-OH to provide compound V in which the ester is saponified to a carboxylic acid, subsequently coupling it with either H-Ile-OBn, benzhydrylamine, or H-Ile-benzhydrylamide, and subsequently acid-decomposing it to produce compounds 4, 5, and 6, respectively.

[0032] [ka]

[0033] Aspects of the present invention provide a process comprising removing the Boc group from intermediate I, then coupling the resulting amine with Boc-Chg-OH to obtain compound VII in which the Boc group has been deprotected, then coupling it with Boc-N-Me-Ala-OH to obtain compound VIII in which the ester has been converted to a carboxylic acid, and then coupling it with either H-Ile-OBn or a benzhydrylamine, followed by acid decomposition of the same to obtain compounds 7, 8, and 9, respectively.

[0034] [ka]

[0035] Another aspect of the present invention is a process for preparing the SMAC mimetic compound (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-phenylpyrrolidine-2-carboxamide (10), a) Saponifying and coupling compound X with H-Ile-benzhydrylamide in a solvent in the presence of a peptide coupling reagent and a weak base to obtain compound XI;

[0036] [ka]

[0037] b) The step of removing the Boc group from compound XI by acid hydrolysis using TFA, followed by coupling the resulting amine with Boc-Val-COOH in the presence of a peptide coupling reagent and a weak base to obtain the compound of formula XII;

[0038] [ka]

[0039] c) The Boc group is removed from compound XII by acid hydrolysis using TFA, and then the resulting amine is coupled with Boc-N-Me-Ala-OH in the presence of a peptide coupling reagent and a weak base to obtain the compound of formula XIII;

[0040] [ka]

[0041] d) A process is provided comprising the step of removing the Boc group from the compound of formula XIII by acid hydrolysis to obtain compound 10.

[0042] [ka]

[0043] Another aspect of the present invention provides a compound of formula -I that inhibits the binding of SMAC proteins to apoptosis inhibitory proteins (IAPs) and is useful for treating proliferative diseases, including cancer.

[0044] Yet another aspect of the present invention is a SMAC mimic compound of formula-I,

[0045] [ka]

[0046] Here, R 1 This includes hydrogen, and unsubstituted or substituted heteroaryl or C6-C 10 Selected from the group consisting of aryls; R 2 , R 3 and R 4 Each is independently selected from the group consisting of H, C1-C6 alkyl, and C4-C8 cycloalkyl; A is unsubstituted or substituted C1-C6 alkyl or C6-C 10 Selected from the alphabet; B is C6-C 10 Aryl, C(O)R 5 and C(O)N(R 6 )(R 7 Selected from the group consisting of; R 5 It is selected from the group consisting of OH, C1-C6 alkoxy, and C6 alkoxyaryl; R 6 and R 7These are, independently, hydrogen and C6-C 10 Aryl and C6-C 10 Selected from the group consisting of arylalkyl groups; The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0047] Yet another aspect of the present invention is a SMAC mimic compound of formula-I,

[0048] [ka]

[0049] Here, R 1 This includes hydrogen, and unsubstituted or substituted heteroaryl or C6-C 10 Selected from the group consisting of aryls; R 2 , R 3 and R 4 Each is independently selected from the group consisting of H, C1-C6 alkyl, and C4-C8 cycloalkyl; A is unsubstituted or substituted C1-C6 alkyl or C6-C 10 Selected from the alphabet; B is C6-C 10 Aryl, C(O)R 5 and C(O)N(R 6 )(R 7 Selected from the group consisting of; R 5 It is selected from the group consisting of OH, C1-C6 alkoxy, and C6 alkoxyaryl; R 6 and R 7 These are, independently, hydrogen and C6-C 10 Aryl and C6-C 10 Selected from the group consisting of arylalkyl groups; The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable salt thereof, at least one anticancer agent, and a pharmaceutically acceptable excipient.

[0050] In aspects of the present invention, the SMAC-mimetic compound of formula I has potent antiproliferative activity against various mammalian cancer cell lines selected from the group consisting of colon, breast, kidney, prostate, brain, ovary, pancreas, melanoma, liver, leukemia, and lymphoma.

[0051] In another aspect of the present invention, SMAC-mimicking compounds are useful for treating treatment-resistant, refractory, and metastatic cancers in mammals.

[0052] In yet another aspect of the present invention, SMAC-mimicking compounds are useful in combination therapy with other antiproliferative agents selected from the group consisting of TRAIL agonists / MAbs, aromatase inhibitors, epigenetic modulators, kinase inhibitors, alkylating agents, microtubule disruptors, topoisomerase inhibitors, anti-angiogenic compounds, Hsp90 inhibitors, mTOR inhibitors, estrogen and androgen antagonists, MMP inhibitors, and biological response modifiers.

[0053] Another aspect of the present invention provides a method for treating cancer using a SMAC mimetic compound of formula I.

[0054] A further aspect of the present invention provides a method for treating cancer using a formula-I SMAC mimetic compound that has the ability to bind to both the BIR-2 and BIR-3 domains of XIAP / IAP and has significant bioavailability with robust in vitro and in vivo efficacy against treatment-resistant cancers. [Brief explanation of the drawing]

[0055] [Figure 1] This figure shows the in silico molecular docking analysis of C6. [Figure 2] This figure shows the modes of cytotoxicity in C6. [Figure 3] This diagram illustrates the target engagement of C6. [Figure 4] This figure shows the synergistic cytotoxic function of C-6 with the DR5 ligand TRAIL. [Figure 5] This figure illustrates the contribution of target engagement to its cytotoxic function. [Figure 6] This figure shows the results of the stability test for C6. [Figure 7] This diagram illustrates the in vivo antitumor effect of C6, which cisplatin was unable to deliver its effects to. [Figure 8] This figure shows the C6 treatment, which provides robust in vivo efficacy through subcutaneous and oral administration routes. [Figure 9] This figure shows the in vivo target engagement and tissue distribution of C6. [Modes for carrying out the invention]

[0056] abbreviation SMAC: Second mitochondrial-derived caspase activator TRAIL: Tumor necrosis factor-related apoptosis-inducing ligand

[0057] Herein, the present invention will be described in detail in relation to certain preferred and optional embodiments so that its various aspects may be more fully understood and recognized.

[0058] For convenience, prior to further description of this disclosure, certain terms and examples used herein are hereby described. These definitions shall be understood by those skilled in the art when read in light of the remainder of this disclosure. The terms used herein have meanings that are recognized and known to those skilled in the art, but for convenience and completeness, certain terms and their meanings are set forth below.

[0059] The articles "a," "an," and "the" are used to refer to one or more of the grammatical objects of the article (i.e., at least one).

[0060] The terms "comprise" and "comprising" are used in an inclusive, open sense, meaning that additional elements may be included. They are not intended to be interpreted as "consisting of only."

[0061] Throughout this specification, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” shall be understood to mean that they include the elements or steps or groups of elements or steps described, and not to exclude any other elements or steps or groups of elements or steps.

[0062] The present invention relates to a SMAC mimetic of formula-I that exhibits potent anti-cancer potential in vitro and in vivo via the apoptotic pathway.

[0063] This invention relates to a SMAC mimic compound of formula-I,

[0064] [ka]

[0065] Here, R 1 This includes hydrogen, and unsubstituted or substituted heteroaryl or C6-C 10 Selected from the group consisting of aryls; R 2 , R 3 and R 4 Each is independently selected from the group consisting of H, C1-C6 alkyl, and C4-C8 cycloalkyl; A is unsubstituted or substituted C1-C6 alkyl or C6-C 10 Selected from the alphabet; B is C6-C 10 Aryl, C(O)R 5 and C(O)N(R 6 )(R 7Selected from the group consisting of; R 5 It is selected from the group consisting of OH, C1-C6 alkoxy, and C6 alkoxyaryl; R 6 and R 7 These are, independently, hydrogen and C6-C 10 Aryl and C6-C 10 Selected from the group consisting of arylalkyl groups; The subject is either a pharmaceutically acceptable salt thereof.

[0066] In embodiments of the present invention, L-alanyl-L-valyl-L-prolyl-L-isoleucine (compound 1), Benzyl((2S,5R)-1-(L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carbonyl)-L-isoleucineate (compound 2), (2S,5R)-1-(L-alanyl-L-valyl)-N-benzhydryl-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 3), (2S,3S)-benzyl 3-methyl-2-((2S,5R)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)pentanoate (compound 4), (2S,5R)-N-benzhydryl-1-(methyl-L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 5), (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-(methyl-L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 6), (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate (compound 7), (2S,5R)-N-benzhydryl-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (Compound 8), (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide((2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide)(compound 9); and (2S,5R)-N-((2S,3S)-1-(Diphenylmethylamino)-3-methyl-1-oxopentan-2-yl)-1-(methyl-L-alanyl-L-alanyl)-5-phenylpyrrolidine-2-carboxamide (Compound 10) There are provided SMAC mimetic compounds of formula I selected from the group consisting of

[0067] Another embodiment of the present invention is a process for preparing a SMAC mimetic compound of formula -I,

[0068]

Chemical formula

[0069] where R 1 is selected from the group consisting of hydrogen, and unsubstituted or substituted heteroaryl or C6-C 10 aryl; R 2 , R 3 and R 4 are each independently selected from the group consisting of H, C1-C6 alkyl and C4-C8 cycloalkyl; A is selected from unsubstituted or substituted C1-C6 alkyl or C6-C 10 aryl; B is selected from the group consisting of C6-C 10 aryl, C(O)R 5 and C(O)N(R 6 )(R 7 ); R 5 is selected from the group consisting of OH, C1-C6 alkoxy and C6 alkoxyaryl; R 6 and R 7 are each independently selected from the group consisting of hydrogen, C6-C 10 aryl and C6-C 10 arylalkyl; i. The Boc group of 2-benzyl 1-(tert-butyl)(2S,5S)-5-(5-methylfuran-2-yl)pyrrolidine-1,2-dicarboxylate is removed by acid hydrolysis using TFA, followed by the conversion of the resulting amine into BocNHCH(R) in the presence of a peptide coupling reagent and a weak base. 2 The step of coupling with ')COOH to obtain the compound of formula P1;

[0070] [ka]

[0071] ii. The Boc group is removed from the compound of formula P1 by acid hydrolysis using TFA, and the resulting amine is converted to the formula BocN(R) in the presence of a peptide coupling reagent and a weak base. 4 ′)CH(R 3 The step of coupling it with a compound of formula P2 by ')COOH;

[0072] [ka]

[0073] iii. The step of catalytically hydrogenating the compound of formula P2 using a Pd catalyst in the presence of a solvent to obtain the free carboxylic acid of formula P3;

[0074] [ka]

[0075] The present invention provides a process comprising the step of coupling the free carboxylic acid of formula P3 with NH2CH(A)(B) in the presence of a peptide coupling reagent and a weak base to obtain the compound of formula I.

[0076] In embodiments of the present invention, a process is provided for preparing a SMAC-mimicking compound of formula -I, wherein the peptide coupling reagent is selected from the group consisting of HOBt, EDCI, and HBTU.

[0077] Another embodiment of the present invention provides a process for preparing an SMAC-like compound of formula -I, wherein the weak base is diethylisopropylamine.

[0078] In yet another embodiment of the present invention, a process is provided for preparing an SMAC-mimicking compound of formula -I, wherein the Pd catalyst is selected from Pd / C or Pd(OH)2 / C.

[0079] In yet another embodiment of the present invention, a process is provided for preparing an SMAC-mimicking compound of formula-I, wherein the solvent is selected from DCM or DMF for peptide coupling.

[0080] In embodiments of the present invention, a process is provided for preparing an SMAC-like compound of formula -I, wherein the solvent is selected from MeOH or SiO for catalytic hydrogenation.

[0081] In another embodiment of the present invention, a process for preparing an SMAC-mimicking compound 10, (a) In a solvent, in the presence of a peptide coupling reagent and a weak base, compound X is saponified and coupled with H-Ile-benzhydrylamide to obtain compound XI;

[0082] [ka]

[0083] (b) The step of removing the Boc group from compound XI by acid hydrolysis using TFA, followed by coupling the resulting amine with Boc-Val-COOH in the presence of a peptide coupling reagent and a weak base to obtain the compound of formula XII;

[0084] [ka]

[0085] (c) The step of removing the Boc group from compound XII by acid hydrolysis using TFA, followed by coupling the resulting amine with Boc-N-Me-Ala-OH in the presence of a peptide coupling reagent and a weak base to obtain the compound of formula XIII;

[0086] [ka]

[0087] (d) A process is provided which includes the step of removing the Boc group from the compound of formula XIII by acid hydrolysis to obtain compound 10.

[0088] [ka]

[0089] In yet another embodiment of the present invention, a process is provided for preparing a SMAC-mimicking compound 10 in which the peptide coupling reagent is selected from the group consisting of HOBt, EDCI, and HBTU.

[0090] In yet another embodiment of the present invention, a process for preparing an SMAC-mimicking compound 10 is provided, wherein the solvent is selected from DCM or DMF.

[0091] Another embodiment of the present invention provides a process for preparing an SMAC-mimicking compound 10 in which the weak base is diethylisopropylamine.

[0092] In yet another embodiment of the present invention, a process is provided for preparing an SMAC-mimicking compound 10 in which the reagent for acid hydrolysis is TFA.

[0093] Another embodiment of the present invention provides a SMAC mimetic compound of formula I, wherein the compound inhibits the binding of Smac proteins to apoptosis inhibitory proteins (IAPs) and is useful for treating proliferative diseases, including cancer.

[0094] Yet another embodiment of the present invention is a SMAC mimetic compound of formula - I,

[0095]

Chemical formula

[0096] where, R 1 is selected from the group consisting of hydrogen, and unsubstituted or substituted heteroaryl or C6 - C 10 aryl; R 2 , R 3 and R 4 are each independently selected from the group consisting of H, C₁ - C₆ alkyl and C₄ - C₈ cycloalkyl; A is selected from unsubstituted or substituted C₁ - C₆ alkyl or C₆ - C 10 aryl; [[ID=--]]B is selected from the group consisting of C₆ - C 10 aryl, C(O)R 5 and C(O)N(R 6 )(R 7 ); R 5 is selected from the group consisting of OH, C₁ - C₆ alkoxy and C₆ alkoxyaryl; R 6 and R 7 are each independently selected from the group consisting of hydrogen, C₆ - C 10 aryl and C₆ - C 10 arylalkyl; or a pharmaceutical composition comprising a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient thereof is provided.

[0097] Yet another embodiment of the present invention provides a pharmaceutical composition comprising a SMAC mimetic compound of formula - I,

[0098]

Chemical formula

[0099] It should be noted that there seems to be a formatting or some content issue in the original text around line 34 where it says "C6 - C " without a complete number or proper continuation. I've translated it as best as possible with the available information. If this is a crucial part, it might need further clarification in the original source.Here, R 1 This includes hydrogen, and unsubstituted or substituted heteroaryl or C6-C 10 Selected from the group consisting of aryls; R 2 , R 3 and R 4 Each is independently selected from the group consisting of H, C1-C6 alkyl, and C4-C8 cycloalkyl; A is unsubstituted or substituted C1-C6 alkyl or C6-C 10 Selected from the alphabet; B is C6-C 10 Aryl, C(O)R 5 and C(O)N(R 6 )(R 7 Selected from the group consisting of; R 5 It is selected from the group consisting of OH, C1-C6 alkoxy, and C6 alkoxyaryl; R 6 and R 7 These are, independently, hydrogen and C6-C 10 Aryl and C6-C 10 Selected from the group consisting of arylalkyl groups.

[0100] Another embodiment of the present invention provides an SMAC-mimicking compound of formula I that has potent antiproliferative activity against mammalian cancer cell lines selected from the group consisting of colon, breast, kidney, prostate, brain, ovary, pancreas, melanoma, liver, leukemia, and lymphoma.

[0101] In yet another embodiment of the present invention, a SMAC-mimicking compound of formula I is provided that is useful for treating treatment-resistant, refractory, and metastatic cancers in mammals.

[0102] In yet another embodiment of the present invention, a SMAC mimetic compound of formula I is provided, which is useful in combination therapy with other antiproliferative agents selected from the group consisting of TRAIL agonists / MAbs, aromatase inhibitors, epigenetic modulators, kinase inhibitors, alkylating agents, microtubule disruptors, topoisomerase inhibitors, anti-angiogenic compounds, Hsp90 inhibitors, mTOR inhibitors, estrogen and androgen antagonists, MMP inhibitors, and biological response modifiers.

[0103] Yet another embodiment of the present invention provides a method for treating cancer using SMAC-mimicking compounds.

[0104] Therefore, the present invention relates to formula-I SMAC-mimicking peptide mimetic compounds useful for the treatment of cancer as monotherapy and combination therapy in which chemotherapy cannot produce its effects. Compounds 2 to 9 are prepared by incorporating (2S,5R)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylic acid, and compound 10 is prepared by incorporating a (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid residue.

[0105] The present invention provides a process for preparing SMAC-like compounds of formula-I as described in schemes A and B.

[0106] [ka]

[0107] (Reagents and conditions: (a) 20% TFA / DCM; (b) BocNHCH(R) 2 ′)-OH, EDCI.HCl, HOBt, DIPEA, DCM / DMF(1:1);(c)BocN(R 4 ′)CH(R 3(g) ')-OH, EDCI, HCl, HOBt, DIPEA, DCM / DMF (1:1); (d) 10 mol% Pd-C, H2, MeOH by balloon; (e) NH2-Ile-OBn, EDCI, HCl, HOBt, DIPEA, DCM / DMF (1:1); (f) Benzhydrylamine, IBCF, NMM, THF, -15℃; (g) NH2-Ile-Benzhydrylamide, HBTU, DIPEA, DMF)

[0108] [ka]

[0109] (Reagents and conditions: (a) 20% TFA / DCM; (b) Boc-Val-OH, EDCI, HCl, HOBt, DIPEA, DCM / DMF (1:1); (c) Boc-N-Me-Ala-OH, EDCI, HCl, HOBt, DIPEA, DCM / DMF (1:1); (g) H-Ile-benzhydrylamide, HBTU, DIPEA, DMF; (h) LiOH, THF, MeOH, Water;)

[0110] Compounds 2-9 are prepared according to the process described in Scheme A; and compound 10 is prepared according to the process described in Scheme B by the following method:

[0111] (i) The Boc group is removed from the main intermediate I, and the resulting amine is then coupled with Boc-Val-OH to obtain compound II (Scheme 1); the Boc group is removed from compound II, and the resulting amine is coupled with Boc-Ala-OH to obtain compound III (Scheme 1); free carboxylic acid IV is obtained by catalytic hydrogenation of compound III, which is then coupled with either H-Ile-OBn or benzhydrylamine, followed by acid decomposition to produce compound 2 or 3, respectively (Scheme 1).

[0112] [ka]

[0113] (Reagents and conditions: (a) 20% TFA / DCM; (b) Boc-Val-OH, EDCI.HCl, HOBt, DIPEA, DCM / DMF (1:1); (c) Boc-Ala-OH, EDCI.HCl, HOBt, DIPEA, DCM / DMF (1:1); (d) 10 mol% Pd-C, H2 by balloon; (e) H-Ile-OBn, EDCI.HCl, HOBt, DIPEA, DCM / DMF (1:1); (f) Benzhydrylamine, IBCF, NMM, THF, -15℃)

[0114] (ii) Remove the Boc group from compound-II, and couple the resulting amine with Boc-N-Me-Ala-OH to obtain compound V; catalytic hydrogenation of compound V yields compound VI having a free carboxylic acid, which is then coupled with either H-Ile-OBn, benzhydrylamine, or H-Ile-benzhydrylamide, followed by acid decomposition to produce compounds 4, 5, or 6, respectively (Scheme 2).

[0115] [ka]

[0116] (Reagents and conditions: (a) 20% TFA / DCM; (b) Boc-Val-OH, HBTU, DIPEA, DMF; (c) Boc-N-Me-Ala-OH, HBTU, DIPEA, DMF; (d) 10 mol% Pd-C, H2, MeOH by balloon; (e) H-Ile-OBn, HBTU, DIPEA, DMF; (f) Benzhydrylamine, IBCF, NMM, THF, -15℃; (g) NH2-Ile-Benzhydrylamide, HBTU, DIPEA, DMF)

[0117] (iii) Remove the Boc group of Compound-I and couple the resulting amine with Boc-Chg-OH to obtain Compound VII; acidolytic cleavage of the Boc-group of Compound VII, followed by coupling with Boc-N-Me-Ala-OH to form Compound VIII, and obtain Compound IX by catalytic hydrogenation of Compound VIII, which is coupled with either H-Ile-OBn, benzhydrylamine or Ile-benzhydrylamide, followed by acidolysis to yield Compound 7, 8 or 9, respectively (Scheme 3).

[0118]

Chem.

[0119] (Reagents and conditions: (a) 20% TFA / DCM; (b) Boc-Chg-OH, HBTU, DIPEA, DMF; (c) Boc-N(Me)-Ala-OH, HBTU, DIPEA, DMF; (d) 10 mol% Pd-C, H2 by balloon, MeOH; (e) NH2-Ile-OBn, HBTU, DIPEA, DMF; (f) benzhydrylamine, IBCF, NMM, THF, -15 °C; (g) NH2-Ile-benzhydrylamide, HBTU, DIPEA, DMF)

[0120] (iv) Saponify Compound X and then couple with H-Ile-benzhydrylamide to obtain Compound XI; Boc deprotection of Compound XI, followed by coupling with Boc-Val-OH to obtain Compound XII; Boc deprotection of Compound XII, followed by coupling with Boc-N-Me-Ala-OH to obtain Compound XIII; Boc deprotection of Compound XIII to obtain Compound 10 (Scheme 4).

[0121]

Chem.

[0122] (Reagents and conditions: (h) LiOH, THF, MeOH, water; (g) H-Ile-benzhydrylamide, EDCI, HOBt, DIPEA, DCM; (a) 30% TFA / DCM; (b) Boc-Val-OH, EDCI, HOBt, DIPEA, DCM; (c) Boc-N-Me-Ala-OH, EDCI, HOBt, DIPEA, DCM;

[0123] The SMAC mimetic peptidomimetics of formula -I of the present invention show strong binding affinity for the BIR-2 domain and BIR-3 domain of XIAP. The binding affinity is measured by in silico experiments (Figure 1) and by protein binding assays using the fluorescence polarization assay shown in Table 1. After confirmation of binding, the cytotoxic ability of the SMAC mimetic compound was evaluated against cancer cell lines relative to a non-human monkey kidney (VERO) cell line. All compounds showed cytotoxic activity against all cancer cells. Most importantly, among a series of Smac mimetic compounds, compound 6 shows strong cytotoxic activity against all cancer cells but has limited or minimal toxicity against non-human VERO cells that demonstrate its tumor cell selectivity (Table 1). Due to its tumor cell-selective cytotoxic nature and potential binding properties, compound 6 (C6) was selected as a potent molecule for further experimental analysis.

[0124]

Table 1

[0125] Furthermore, the IC 50 concentration of C6 against various cancer cell lines was determined, and it was observed that the SMAC mimetic peptidomimetics of formula -I show strong activity against various cancer cell lines including, but not limited to, colon, breast, kidney, prostate, brain, ovary, pancreas, liver, melanoma, leukemia and lymphoma (Table 2).

[0126]

Table 2

[0127] SMAC mimetic C6 promotes cell death in cancer cells by activating prominent SMAC-driven apoptotic features such as caspase cleavage and cIAP1 degradation, as shown in Figures 2 and 3. Furthermore, its apoptotic function is highly dependent on target engagement, and it promotes apoptosis in TRAIL-resistant cells, as shown in Figures 4 and 5. Stability and pharmacokinetic analyses of C6 show that it is highly stable in SIF, SGF, and microsomes (Figure 6) and has significant bioavailability via subcutaneous and oral administration routes, as shown in Table 3, suggesting the druggable potential of the SMAC mimetic disclosed in this invention.

[0128] [Table 3]

[0129] As shown in Figures 7 and 8, C6 exhibits robust in vivo antitumor activity via intraperitoneal, subcutaneous, and oral administration routes. It is also in vivo active against cisplatin-resistant colon cancer. C6 has been shown to be well-tolerated and non-toxic at doses in which no weight loss was observed in animals during the course of treatment, as shown in Figures 7 and 8. Furthermore, C6 reaches tumor sites via the oral administration route and is involved in its targets such as XIAP / IAP degradation and caspase cleavage (Figure 9).

[0130] (example) The following examples are given to support the present invention, but are not limited thereto.

[0131] Example 1 Synthesis of benzyl((2S,5R)-1-(L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carbonyl)-L-isoleucineate (compound 2) as mentioned in Scheme 1

[0132] (2S,5S)-2-benzyl 1-tert-butyl 5-(5-methylfuran-2-yl)pyrrolidine-1,2-dicarboxylate (850 mg, 2.2 mmol) was stirred in 20% TFA / DCM for 1 hour. The reaction mixture was then concentrated under reduced pressure and dried. Next, this amine was dissolved in anhydrous DMF (2 mL) and added to a solution of Boc-Val-OH (956 mg, 4.4 mmol) and HBTU (1.7 gm, 4.4 mmol) pre-stirred in dry DMF (3 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (1.2 mL, 6.6 mmol). The reaction mixture was stirred further at room temperature for an additional 4-6 hours. After the reaction was complete, water (30-40 mL) was added. The aqueous solution was extracted with ethyl acetate (3 × 60 mL). The combined organic layers were washed with 10% citric acid (aqueous solution), 10% NaHCO3 (aqueous solution), and finally brine. The organic layers were dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. This was purified by column chromatography using 18% ethyl acetate / hexane as the eluent to give the intermediate compound (2S,5R)-benzyl 1-((S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylate(II) as a brownish, rubbery oil in 56.8% yield (605.6 mg, 1.25 mmol HRMS(ESI)(M+H)). + Calculated for C 27 H 37 N2O6 + =485.2646, found 485.2645.

[0133] Intermediate compound II (484.6 mg, 1 mmol) was stirred in 20% TFA / DCM for 1 hour. The reaction mixture was then concentrated under reduced pressure and dried. Next, this amine was dissolved in anhydrous DMF (2 mL) and added to a pre-stirred solution of Boc-Ala-OH (378.4 mg, 2 mmol) and HBTU (758.5 ​​mg, 2 mmol) in dry DMF (3 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (0.56 mL, 3 mmol). The reaction mixture was further stirred at room temperature for an additional 4–6 hours. After the reaction and normal workup were completed, the intermediate compound (2S,5R)-benzyl 1-((S)-2-((S)-2-(tert-butoxycarbonylamino)propanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylate (III) was obtained as a yellowish, rubbery oil in 65% yield (361 mg, 0.65 mmol). HRMS(ESI)(M+H) + Calculate for C 30 H 42 N3O7 + =556.3017, found 556.3022.

[0134] A solution of compound III (1 mmol) was stirred in methanol (5 mL), to which 10 mol% Pd-C (0.1 mmol) was added and subjected to hydrogenation by purging with hydrogen gas using a balloon for 30 minutes. Subsequently, Pd was filtered using a Celite pad, and the filtrate was concentrated under vacuum to give the free acid (2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonylamino)propanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylic acid (IV), which was used in the next step without further purification. Subsequently, isoleucine benzyl ester (13.3 mg, 0.06 mmol) was dissolved in anhydrous DMF (1 mL) and added to a pre-mixed solution of compound IV (28 mg, 0.06 mmol) and HBTU (22.7 mg, 0.06 mmol) in dry DMF (1 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (0.034 mL, 0.18 mmol). The reaction mixture was further stirred at room temperature for an additional 4-6 hours. After the reaction and routine work-up were completed, the protected tetrapeptide (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonylamino)propanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate was obtained in 86% yield as a yellowish, rubbery oil (34.8 mg, 0.05 mmol). HRMS(ESI)(M+H) + Calculate for C36 H 53 N4O8 + =669.3858, found 669.3865.

[0135] The protected peptide (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonylamino)propanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate) (67 mg, 0.1 mmol) was stirred in 20% TFA / DCM for 30 minutes. The reaction mixture was then concentrated under reduced pressure and dried. The crude peptide was purified by reverse-phase HPLC (RP-HPLC) using a C-18 column, and the sample was then lyophilized to give the desired peptide compound (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-aminopropanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate(2)) as a white powder in 65% yield (37 mg, 0.065 mmol). Compound 2 was found to be 97% pure at 220 nm by analytical RP-HPLC. 1H NMR (500MHz, DMSO-d6): δ / ppm=8.67(d,1H,J=8.2Hz),8.08(br,2H),7.93(d,1H,J=8.2Hz),7. 38-7.31(m,5H),6.53(br,1H,J=3.1Hz),6.04(brdd,1H,J=1.0,3.1Hz),5.41(m,1H),5.13(m,2 H),4.41-4.22(m,3H),3.88(br,1H),2.24(s,3H),2.12-1.76(m,6H),1.42-1.34(m,1H),1.31( d,3H,J=7.0Hz),1.23-1.12(m,1H),0.86-0.77(m,9H),0.61(d,3H,J=6.7Hz);HRMS(ESI)(M+H) + Calculated for C 31 H 45 N4O6 + = 569.3334, found 569.3335.

[0136] Example 2 Synthesis of (2S,5R)-1-(L-alanyl-L-valyl)-N-benzhydryl-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 3) as mentioned in Scheme 1.

[0137] To a solution of compound IV (50 mg, 0.12 mmol) in tetrahydrofuran (1 mL) at -15°C, N-methylmorpholine (17.5 μL, 0.16 mmol, 1.5 equivalents) was added. After 5 minutes, isobutylchloroformate (17.5 μL, 0.144 mmol, 1.2 equivalents) was added to the reaction mixture. Then, after 5 minutes, benzhydrylamine (19 μL, 0.144 mmol, 1.2 equivalents) was added, and the reaction mixture was stirred at -15°C for a further 1 hour. After the reaction and normal work-up were completed, the compound-protected peptide tert-butyl(S)-1-((S)-1-((2S,5R)-2-(benzhydrylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropane-2-ylcarbamate (Boc-Ala-Val-Fro-benzhydrylamide) was obtained in 71% yield (53 mg, 0.085 mmol HRMS(ESI)(M+H)). + Calculate for C 36 H 47 N4O6 + =631.3490, found 631.3485.

[0138] The compound tert-butyl(S)-1-((S)-1-((2S,5R)-2-(benzhydrylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropane-2-ylcarbamate (Boc-Ala-Val-Fro-benzhydrylamide) (53 mg, 0.085 mmol) was stirred in 20% TFA / DCM for 30 minutes. The reaction mixture was then concentrated under reduced pressure and dried. The crude peptide was purified by reverse-phase HPLC (RP-HPLC) using a C-18 column, and the sample was then lyophilized to give the desired peptide (2S,5R)-1-((S)-2-((S)-2-aminopropanamido)-3-methylbutanoyl)-N-benzhydryl-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (3)) as a white powder in 70% yield (32 mg, 0.06 mmol). Compound 3 was found to be 96% pure at 220 nm by analytical RP-HPLC. 1 H NMR(500MHz,DMSO-d6):δ / ppm=8.69(d,1H,J=8.2Hz),8.42(d,1H,J=8.3Hz),8.08(br d,2H,J=3.7Hz),7.35-7.21(m,10H),6.52(d,1H,J=2.9Hz),6.09(d,1H,J=8.2Hz),6.0(br d,1H,J=2.9Hz),5.44(br d,1H,J=7.4Hz),4.45(m,1H),4.22(m,1H),3.87(m,1H),2.20-2.14(m,1H),2.15(s,3H),2.10-1.98(m,2H),1.96-1 .89(m,1H),1.88-1.80(m,1H),1.30(d,3H,J=6.9Hz),0.77(d,3H,J=6.7Hz),0.55(d,3H,J=6.7Hz);HRMS(ESI)(M+H) + Calculate for C 31 H 39N4O4 + = 531.2966, found 531.2958.

[0139] Example 3 Synthesis of (2S,3S)-benzyl 3-methyl-2-((2S,5R)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)pentanoate (Compound 4) referred to in Scheme 2

[0140] Intermediate compound II (484.6 mg, 1 mmol) (obtained in Scheme 1) was stirred in 20% TFA / DCM for 1 hour. The reaction mixture was then concentrated under reduced pressure and dried. This amine was then dissolved in anhydrous DMF (2 mL) and added to a pre-stirred solution of Boc-N-Me-Ala-OH (406 mg, 2 mmol) and HBTU (758.5 ​​mg, 2 mmol) in dry DMF (3 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (0.56 mL, 3 mmol). The reaction mixture was further stirred at room temperature for an additional 4–6 hours. After the reaction and normal workup were completed, the compound (2S,5R)-benzyl 1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylate (V) was obtained in 62% yield as a brownish, rubbery oil (353 mg, 0.62 mmol). HRMS(ESI)(M+H) + Calculate for C 31 H 44 N3O7 + =570.3174, found 570.3407.

[0141] A solution of compound V (570 mg, 1 mmol) was stirred in methanol / tetrahydrofuran (5 mL, 1:1), to which 10 mol% Pd-C (0.1 mmol) was added, and the solution was subjected to hydrogenation by purging with hydrogen gas using a balloon for 30 minutes. Subsequently, Pd was filtered using a Celite pad, and the filtrate was concentrated under vacuum to give the free acid (2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylic acid (VI), which was used in the next step without further purification.

[0142] Isoleucine benzyl ester (55.5 mg, 0.25 mmol) was dissolved in anhydrous DMF (1 mL) and added to a pre-mixed solution of compound VI (120 mg, 0.25 mmol) and HBTU (95 mg, 0.25 mmol) in dry DMF (2 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (0.13 mL, 0.75 mmol). After the reaction and the usual workup of the crude product, it is purified by column chromatography using 5% methanol / dichloromethane as the eluent to obtain the compound Boc-protected tetrapeptide (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamide)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide)-3-methylpentanoate((2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate)(Boc-N(Me)-Ala-Val-Fro-Ile-OBn) was given as a brownish, rubbery oil in 80% yield (136 mg, 0.2 mmol). HRMS(ESI)(M+H) + Calculate for C 37 H 55 N4O8 + =683.4014, found 683.4011.

[0143] The compound (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-3-methylbutanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate) (53.5 mg, 0.08 mmol) was stirred in 20% TFA / DCM for 30 minutes. The reaction mixture was then concentrated under reduced pressure and dried. The crude peptide was purified by reverse-phase HPLC (RP-HPLC) using a C-18 column, and the sample was then lyophilized to give the desired compound (2S,3S)-benzyl 3-methyl-2-((2S,5R)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)pentanoate (4)) as a white powder. Yield (28 mg, 62%). Compound 4 was found to be 99% pure at 220 nm by analytical RP-HPLC. 1H NMR(500MHz,DMSO-d6):δ / ppm=8.96(br,1H),8.85(d,1H,J=8.7Hz),7.93(d,1H,J=8.5Hz),7.38-7.30(m,5H),6.54(br d,1H,J=3.2Hz),6.03(brdd,1H,J=1.0,3.2Hz),5.36(br d,1H,J=7.7Hz),5.16-5.08(m,2H),4.42-4.24(m,3H),3.82(br,1H),2.51(s,3H),2.23(s,3H),2.11-1.94(m,3H),1.92-1.75( m,3H),1.43-1.34(m,1H),1.33(d,3H,J=7.1Hz),1.22-1.13(m,1H),0.86-0.76(m,9H),0.62(d,3H,J=6.9Hz);HRMS(ESI)(M+H) + Calculate for C 32 H 47 N4O6 + = 583.3490, found 583.3482.

[0144] Example 4 Synthesis of (2S,5R)-N-benzhydryl-1-(methyl-L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 5) as mentioned in Scheme 2.

[0145] To a solution of intermediate compound VI (58 mg, 0.12 mmol) in tetrahydrofuran (1 mL) at -15°C, N-methylmorpholine (17.5 μL, 0.16 mmol, 1.5 equivalents) was added. After 5 minutes, isobutyl chloroformate (17.5 μL, 0.144 mmol, 1.2 equivalents) was added to the reaction mixture. Then, after 5 minutes, benzhydrylamine (19 μL, 0.144 mmol, 1.2 equivalents) was added, and the reaction mixture was stirred at -15°C for a further 1 hour. After the reaction and routine work-up, the crude product was obtained and purified with 4% methanol / dichloromethane to give the compound Boc-protected peptide tert-butyl(S)-1-((S)-1-((2S,5R)-2-(benzhydrylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-yl(methyl)carbamate (Boc-N(Me)-Ala-Val-Fro-benzhydrylamide) in 75% yield (57 mg, 0.09 mmol). HRMS(ESI)(M+H) + Calculate for C 37 H 49 N4O6 + =645.3647, found 645.3646.

[0146] The compound tert-butyl(S)-1-((S)-1-((2S,5R)-2-(benzhydrylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-yl(methyl)carbamate (Boc-N(Me)-Ala-Val-Fro-benzhydrylamide) (57 mg, 0.08 mmol) was stirred in 20% TFA / DCM for 30 minutes. The reaction mixture was then concentrated under reduced pressure and dried. The crude peptide was purified by reverse-phase HPLC (RP-HPLC) using a C-18 column, and the sample was then lyophilized to give the desired compound (2S,5R)-N-benzhydryl-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide(5)) as a white powder in 68% yield (30 mg, 0.055 mmol).

[0147] Compound 5 was found to be over 99% pure at 220 nm by analytical RP-HPLC. 1 H NMR(500MHz,DMSO-d6):δ / ppm=8.87(br,1H),8.85(d,1H,J=8.9Hz),8.41(d,1H,J=8.2Hz),7.35-7.21(m,10H),6.54(br d,1H,J=3.0Hz),6.10(d,1H,J=8.5Hz),5.99(brdd,1H,J=1.0,3.0Hz),5.40(br d,1H,J=6.1Hz),4.47-4.43(m,1H),4.27(t,1H,J=8.7Hz),3.83(m,1H),2.51(s,3H),2.21-2.13(m,1H),2.15( s,3H),2.12-1.81(m,4H),1.34(d,3H,J=6.9Hz),0.77(d,3H,J=6.7Hz),0.57(d,3H,J=6.7Hz);HRMS(ESI)(M+H)+ Calculate for C 32 H 41 N4O4 + = 545.3122, found 545.3116.

[0148] Example 5 Synthesis of (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-(methyl-L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 6) as mentioned in Scheme 2.

[0149] Isoleucine benzhdrylamide (178 mg, 0.6 mmol) was dissolved in anhydrous DMF (1 mL) and added to a pre-mixed solution of compound VI (288 mg, 0.6 mmol) and HBTU (227.5 mg, 0.6 mmol) in dry DMF (3 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (0.34 mL, 1.8 mmol). The reaction mixture was further stirred at room temperature for an additional 4-6 hours. After the reaction and normal workup were completed, the crude product thus obtained was purified by column chromatography using 5% methanol / dichloromethane as the eluent to give compound Boc-protected tert-butyl(S)-1-((2S,5R)-2-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentane-2-ylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-yl(methyl)carbamate as a brownish rubbery oil in 83% yield (379 mg, 0.5 mmol). HRMS(ESI)(M+H) + Calculate for C 43 H 60 N5O7 + =758.4487, found 758.4483.

[0150] The compound tert-butyl(S)-1-((S)-1-((2S,5R)-2-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentane-2-ylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-yl(methyl)carbamate (250 mg, 0.32 mmol) was stirred in 20% TFA / DCM for 30 minutes. The reaction mixture was then concentrated under reduced pressure and dried. The crude peptide was purified by reverse-phase HPLC (RP-HPLC) using a C-18 column, and then the sample was lyophilized to obtain the desired compound (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide(6)((2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (6)) was given as a white powder in a yield of 56% (119 mg, 0.18 mmol). Compound 6 was found to be 99% pure at 220 nm by analytical RP-HPLC. 1H NMR (500MHz, DMSO-d6): δ / ppm=8.93(d,1H,J=8.8Hz),8.86(br,1H),8.83(d,1H,J=8.4Hz),7.56(d,1H,J=8.9Hz),7.33-7.21(m,10H),6.52(br d,1H,J=3.0Hz),6.11(d,1H,J=8.7Hz),6.03(br d,1H,J=2.5Hz),5.35(m,1H),4.40-4.30(m,3H),3.8(br,1H),2.50(s,3H),2.25(s,3H),2.13-1.68(m,6H),1.45- 1.37(m,1H),1.33(d,3H,J=6.9Hz),1.09-0.98(m,1H),0.80-0.74(m,9H),0.59(d,3H,J=6.7Hz);HRMS(ESI)(M+H) + Calculate for C 38 H 52 N5O5 + =658.3963, found 658.3953.

[0151] Example 6 Synthesis of (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate (compound 7) as mentioned in Scheme 3.

[0152] 850 mg, 2.2 mmol of 2-benzyl-1-(tert-butyl)(2S,5S)-5-(5-methylfuran-2-yl)pyrrolidine-1,2-dicarboxylate was stirred in 20% TFA / DCM for 1 hour. The reaction mixture was then concentrated under reduced pressure and dried. This amine was then dissolved in 2 mL of anhydrous DMF and added to a pre-stirred solution of Boc-Chg-OH (1.13 gm, 4.4 mmol) and HBTU (1.7 gm, 4.4 mmol) in 3 mL of dry DMF under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (1.2 mL, 6.6 mmol). The reaction mixture was further stirred at room temperature for an additional 4–6 hours. After the reaction and the completion of normal post-processing and purification, the intermediate compound (2S,5R)-benzyl 1-((S)-2-(tert-butoxycarbonylamino)-2-cyclohexylacetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylate (VII) (629 mg, 1.2 mmol) was obtained in a yield of 54.5% as a yellowish, rubbery oil. HRMS(ESI)(M+H) + Calculate for C 30 H 41 N2O6 + = 525.2959, found 525.2952.

[0153] Compound VII (524.6 mg, 1 mmol) was stirred in 20% TFA / DCM for 1 hour. The reaction mixture was then concentrated under reduced pressure and dried. Next, this amine was dissolved in anhydrous DMF (2 mL) and added to a pre-stirred solution of N-Boc-N-methylalanine (406 mg, 2 mmol) and HBTU (758.5 ​​mg, 2 mmol) in dry DMF (3 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (0.56 mL, 3 mmol). The reaction mixture was stirred further at room temperature for an additional 4-6 hours. After the reaction was complete, water (10-20 mL) was added. The aqueous solution was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with 10% citric acid (aqueous solution), 10% NaHCO3 (aqueous solution), and finally brine. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. This was purified by column chromatography using 32% ethyl acetate / hexane as the eluent to give the intermediate compound (2S,5R)-benzyl 1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-2-cyclohexylacetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylate (VIII) in 62% yield as a brownish, rubbery oil (378 mg, 0.62 mmol). HRMS(ESI)(M+H) + Calculate for C 34 H 48 N3O7 + =610.3487, found 610.3378.

[0154] A solution of compound VIII (378 mg, 0.62 mmol) was stirred in methanol / tetrahydrofuran (5 mL, 1:1), to which 10 mol% Pd-C (0.06 mmol) was added, and the solution was subjected to hydrogenation by purging with hydrogen gas using a balloon for 30 minutes. Subsequently, Pd was filtered using a Celite pad, and the filtrate was concentrated under vacuum to give the free acid (2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-2-cyclohexylacetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylic acid (IX), which was used in the next step without further purification. Isoleucine benzyl ester (55.5 mg, 0.25 mmol) was dissolved in anhydrous DMF (1 mL) and added to a pre-mixed solution of Boc-N(Me)-Ala-Chg-Fro-OH (130 mg, 0.25 mmol) and HBTU (95 mg, 0.25 mmol) in dry DMF (2 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (0.13 mL, 0.75 mmol). The reaction mixture was further stirred at room temperature for an additional 4-6 hours.After the reaction and normal workup are complete, the compound Boc-protected tetrapeptide (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamide)-2-cyclohexylacetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide)-3-methylpentanoate((2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-2-cyclohexylacetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate)(Boc-N(Me)-Ala-Chg-Fro-Ile-OBn) was obtained as a yellowish, rubbery oil in 80% yield (144 mg, 0.2 mmol). HRMS(ESI)(M+H). + Calculate for C 40 H 59 N4O8 + =723.4327, found 723.4325.

[0155] Compound (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamide)-2-cyclohexylacetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide)-3-methylpentanoate((2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-2-cyclohexylacetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate)(Boc-N(Me)-Ala-Chg-Fro-Ile-OBn) (58 mg, 0.08 mmol) was stirred in 20% TFA / DCM for 30 minutes. The reaction mixture was then concentrated under reduced pressure and dried. The crude peptide was purified by reverse-phase HPLC (RP-HPLC) using a C-18 column, and the sample was then lyophilized to give the desired compound (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamido)-3-methylpentanoate 7) as a white powder in 62% yield (28 mg, 0.05 mmol). Compound 7 was found to be 98% pure at 220 nm by analytical RP-HPLC. 1H NMR(500MHz,DMSO-d6):δ / ppm=8.88(br,1H),8.79(d,1H,J=8.3Hz),7.86(d,1H,J=8.3Hz),7.37-7.32(m,5H),6.51(br d,1H,J=3.0Hz),6.05(brdd,1H,J=1.0,3.0Hz),5.34(m,1H),5.17-5.08(m,2H) ,4.42-4.32(m,3H),3.83-3.79(m,1H),2.50(s,3H),2.24(s,3H),2.09-1.86(m, 4H),1.83-1.76(m,1H),1.70-1.41(m,7H),1.33(d,3H,J=6.9Hz),1.20-1.12(m ,1H),1.06-0.92(m,3H),0.86-0.77(m,7H),0.55-0.46(m,1H);HRMS(ESI)(M+H) + Calculate for C 35 H 51 N4O6 + =623.3803, found 623.3800.

[0156] Example 7 Synthesis of (2S,5R)-N-benzhydryl-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 8) as mentioned in Scheme 3.

[0157] To a solution of intermediate compound IX (62.4 mg, 0.12 mmol) in tetrahydrofuran (1 mL) at -15°C, N-methylmorpholine (17.5 μL, 0.16 mmol, 1.5 equivalents) was added. After 5 minutes, isobutyl chloroformate (17.5 μL, 0.144 mmol, 1.2 equivalents) was added to the reaction mixture. Then, after 5 minutes, benzhydrylamine (19 μL, 0.144 mmol, 1.2 equivalents) was added, and the reaction mixture was stirred at -15°C for a further 1 hour. After the reaction and routine work-up, the compound Boc-protected tetrapeptide tert-butyl(S)-1-((S)-2-((2S,5R)-2-(benzhydrylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethylamino)-1-oxopropan-2-yl(methyl)carbamate was obtained in 67% yield (55 mg, 0.08 mmol). HRMS(ESI)(M+H) + Calculate for C 40 H 53 N4O6 + =685.3960, found 685.3939.

[0158] The compound tert-butyl(S)-1-((S)-2-((2S,5R)-2-(benzhydrylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethylamino)-1-oxopropan-2-yl(methyl)carbamate (55 mg, 0.08 mmol) was stirred in 20% TFA / DCM for 30 minutes. The reaction mixture was then concentrated under reduced pressure and dried. The crude peptide was purified by reverse-phase HPLC (RP-HPLC) using a C-18 column, and the sample was then lyophilized to give the desired compound (2S,5R)-N-benzhydryl-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (8)) as a white powder in 62.5% yield (29 mg, 0.05 mmol). Compound 8 was found to be 99% pure at 220 nm by analytical RP-HPLC. 1 H NMR(500MHz,DMSO-d6):δ / ppm=8.88(br,1H),8.81(d,1H,J=8.0Hz),8.30(d,1H,J=8.4Hz),7.36-7.21(m,10H),6.51(br d,1H,J=3.1Hz),6.10(d,1H,J=8.1Hz),6.01(brdd,1H,J=1.0,3.1Hz),5. 38(m,1H),4.46(m,1H),4.36(t,1H,J=8.5Hz),3.82(m,1H),2.50(s,3H),2 .21-1.89(m,4H),2.14(s,3H),1.70-1.39(m,5H),1.33(d,3H,J=7.0Hz), 1.13-0.89(m,4H),0.79-0.69(m,1H),0.51-0.42(m,1H);HRMS(ESI)(M+H) + Calculate for C 35 H 45 N4O4 += 585.3435, found 585.3427.

[0159] Example 8 Synthesis of (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 9)

[0160] (2S,3S)-2-amino-N-benzhydryl-3-methylpentanamide (60 mg, 0.2 mmol) is dissolved in anhydrous DMF (1 mL) and (2S,5R)-1-((S)-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamide)-2-cyclohexylacetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxylic acid To a pre-mixed solution of acid (104 mg, 0.2 mmol) and HBTU (75.8 mg, 0.2 mmol) in dry DMF (1 mL), the solutions were added at 0°C under a nitrogen atmosphere, followed by the addition of DIPEA (0.11 mL, 0.6 mmol). The reaction mixture was further stirred at room temperature for an additional 4-6 hours. After the reaction and normal work-up, the compound Boc-protected peptide tert-butyl(S)-1-((S)-2-((2S,5R)-2-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-ylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethylamino)-1-oxopropan-2-yl(methyl)carbamate was obtained in 85% yield as a brownish, rubbery oil (135 mg, 0.17 mmol). HRMS(ESI)(M+H) + Calculate for C 46 H 64 N5O7 + =798.4800, found 798.4817.

[0161] The compound tert-butyl(S)-1-((S)-2-((2S,5R)-2-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-ylcarbamoyl)-5-(5-methylfuran-2-yl)pyrrolidine-1-yl)-1-cyclohexyl-2-oxoethylamino)-1-oxopropan-2-yl(methyl)carbamate (80 mg, 0.1 mmol) was stirred in 20% TFA / DCM for 30 minutes. The reaction mixture was then concentrated under reduced pressure and dried. The crude peptide was purified by reverse-phase HPLC (RP-HPLC) using a C-18 column, and then the sample was lyophilized to obtain the desired compound (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide(9)((2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (9)) was given as a white powder in a yield of 60% (41 mg, 0.06 mmol). Compound 9 was found to be 98% pure at 220 nm by analytical RP-HPLC. 1H NMR (500MHz, DMSO-d6): δ / ppm=8.92(d,1H,J=8.6Hz),8.80(br,1H),8.75(d,1H,J=8.0Hz),7.52(br d,1H,J=9.1Hz),7.33-7.22(m,10H),6.48(br d,1H,J=3.0Hz),6.10(d,1H,J=8.4Hz),6.04(brdd,1H,J=1.0,3.0Hz),5.33(m,1H),4.43-4.38(m,3H),3.82(br m,1H),2.50(s,3H),2.26(s,3H),2.14-1.96(m,4H),1.76-1.37(m,8H),1.33(d,3H,J=6.8Hz) ,1.09-0.98(m,2H),0.97-0.86(m,2H),0.79-0.72(m,7H),0.54-0.44(m,1H);HRMS(ESI)(M+H) + Calculate for C 41 H 56 N5O5 + =698.4276, found 698.4245.

[0162] Example 9 Synthesis of (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-(methyl-L-alanyl-L-alanyl)-5-phenylpyrrolidine-2-carboxamide (compound 10) as mentioned in Scheme 4

[0163] In a round-bottom flush, compound 1-(tert-butyl)2-methyl(2S,5R)-5-phenylpyrrolidine-1,2-dicarboxylate (3.089 gm, 10.127 mmol) was added to 50 ml of methanol / water (10:1) mixture. Then, LiOH (890 mg, 20.255 mmol) was added to the reaction mixture at 0°C. The reaction reaction was monitored by TLC. After the reaction was complete, 50 ml of water was added to the reactants to evaporate the organic solvent from the reaction mixture. Then, ethyl acetate and water were added to separate the organic layer containing impurities. The aqueous layer was acidified with citric acid, and ethyl acetate was added to that layer. The organic layer was separated, washed with brine, and dried over anhydrous Na2SO4. The organic layer was evaporated under reduced pressure to obtain the free acid as a white solid. The crude acid was used directly in the next step without further purification. NH2-Ile-benzhydrylamide (4.42 g, 14.948 mmol) was dissolved in dry DCM (15 mL) and added to a pre-stirred solution of crude acid (2.9 g, 9.96 mmol), EDC.HCl (5.71 g, 29.896 mmol), and HOBt (4.035 g, 29.896 mmol) in dry DCM (35 mL) under 0°C and a nitrogen atmosphere. Subsequently, DIPEA (5.17 mL, 29.896 mmol) was added. The reaction mixture was stirred further at room temperature for an additional 4-6 hours. After the reaction and routine workup were completed, the crude product was purified by column chromatography using silica gel (40% ethyl acetate / hexane) to give the marked compound (2S,5R)-tert-butyl 2-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentane-2-ylcarbamoyl)-5-phenylpyrrolidine-1-carboxylate (XI) as a white solid (4.657 gm, 8.184 mmol, yield 82%).

[0164] Compound XI (4.45 gm, 7.82 mmol) was mixed with 30% TFA / DCM (25 mL) [3 ml / mM, i.e., 1 ml TFA and 2 ml DCM / mM] at 0°C, and the reaction mixture was stirred at room temperature for a further 1 hour. The reaction mixture was then concentrated under reduced pressure and dried, to which DCM (150 mL) was added, and the mixture was washed with 10% Na2CO3 (aqueous solution). The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give the crude amine as a white solid (3.36 gm, 7.036 mmol, 90% yield). This crude amine (600 mg, 1.27 mmol) was dissolved in dry DCM (10 mL) and added to a pre-mixed solution of Boc-NH-valine (692 mg, 3.195 mmol), EDC.HCl (1.342 g, 7.034 mmol), and HOBt (950 mg, 7.034 mmol) in dry DCM (25 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (1.22 mL, 7.034 mmol). The reaction mixture was further stirred at room temperature for an additional 4-6 hours. After the reaction and routine workup were completed, the crude product was purified by column chromatography using silica gel (40% EA / hexane) to give the marked compound tert-butyl(S)-1-((2S,5R)-2-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentane-2-ylcarbamoyl)-5-phenylpyrrolidine-1-yl)-3-methyl-1-oxobutane-2-ylcarbamate (XII) as a white solid (723 gm, 1.082 mmol, yield 85%).

[0165] Compound XII (700 mg, 1.047 mmol) was mixed with 30% TFA / DCM (6 mL) [3 ml / mM, i.e., 1 ml TFA and 2 ml DCM / mM] at 0°C, and the reaction mixture was stirred at room temperature for a further 1 hour. The reaction mixture was then concentrated under reduced pressure and dried, to which DCM (30 mL) was added, and the mixture was washed with 10% Na2CO3 (aqueous solution). The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give the crude amine as a white solid (740 mg, 0.827 mmol, yield 80%). This crude amine (430 mg, 0.757 mmol) was dissolved in dry DCM (2 mL) and added to a pre-mixed solution of Boc-val-OH (385 mg, 1.892 mmol), EDC.HCl (1.05 g, 4.163 mmol), and HOBt (563 mg, 4.163 mmol) in dry DCM (8 mL) under a nitrogen atmosphere at 0°C, followed by the addition of DIPEA (0.97 mL, 4.163 mmol). The reaction mixture was further stirred at room temperature for an additional 4-6 hours. After the reaction and routine workup were completed, the crude product was purified by column chromatography using silica gel (40% EA / hexane) to give the marked compound tert-butyl(S)-1-((S)-1-((2S,5R)-2-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentane-2-ylcarbamoyl)-5-phenylpyrrolidine-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-yl(methyl)carbamate (XIII) as a white solid (477 gm, 0.633 mmol, yield 84%).

[0166] Compound XIII (430 mg, 0.571 mmol) was mixed with 30% TFA / DCM (6 mL) [3 ml / mM, i.e., 1 ml TFA and 2 ml DCM / mM] at 0°C, and the reaction mixture was stirred at room temperature for a further 1 hour. The reaction mixture was then concentrated under reduced pressure and dried, to which DCM (30 mL) was added, and the mixture was washed with 10% Na2CO3 (aqueous solution). The organic layer was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography using silica gel (8% MeOH / DCM) to obtain the marked compound (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-phenylpyrrolidine-2-carboxamide(10)((2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-5-phenylpyrrolidine-2-carboxamide (10)) was given as a white solid (326 mg, 0.499 mmol, yield 87%). ESIMS(M+H) + Calculate for C 39 H 52 N5O4 = 654.4, found 654.6

[0167] biological activity Example 10: Binding assay SMAC mimetic compounds 1-9 were tested for their ability to replace the fluorescently labeled peptide AVPIAQK(FAM)-OH with XIAP BIR2 or XIAP BIR3 protein. Dose-dependent binding experiments were then performed by sequentially increasing the concentrations of the SMAC mimetic compounds with a constant concentration of the fluorescently labeled peptide and XIAP BIR2 or XIAP BIR3 protein. 50The values ​​were determined from plots drawn using Prism software with nonlinear least squares analysis. The measured ICs were measured using a web-based program freely accessible at http: / / sw16.im.med.umich.edu / software / calc_ki / .In-silico. 50 The value of K between the tracer AVPIAQK(FAM)-OH and the XIAP BIR2 or XIAP BIR3 complex. d The values, as well as the Ki values ​​of the SMAC mimetic based on the concentrations of the protein and tracer in the competitive assay, were calculated.

[0168] The SMAC mimetic binds to the BIR2 and BIR3 domains of XIAP as determined by FPA. Binding isotherms were plotted between FP readings and the log values ​​of protein concentration in nM. Data were analyzed using GraphPad prism, and Boltzmann-sigmoid nonlinear regression curve fitting was used. d The values ​​were determined. First, the K values ​​for the binding of the fluorescently labeled peptide to XIAP BIR2 and XIAP BIR3 were determined. d Determine the value and the precise K for each molecule binding to the BIR2 / 3 domain. i The values ​​were evaluated and are shown in Table 1.

[0169] [Table 4]

[0170] Example 11: Cytotoxicity assay (SRB assay) The in vitro cytotoxic activity of different compounds was evaluated using standard SRB assays in different cell types. The absorbance of treated and untreated cells was measured at a wavelength of 510 nm using a multi-well scan spectrophotometer (Epoch Microplate Reader, Biotek, USA). Growth inhibition rates were calculated using the formula [100 - (absorbance of compound-treated cells / absorbance of untreated cells)] × 100.

[0171] SMAC mimetic enhances tumor cell-selective cytotoxicity. SW620, HT29, HCT116, and VERO cells were treated with a series of 10 μM Smac mimetics for 48 hours, and cytotoxicity was measured by SRB assay. Growth inhibition rates are summarized in Table 1 above. C6 showed robust in vitro cytotoxicity against tumor cells but not against VERO cells; therefore, we investigated the IC50 effect of C6 against different types of cancer cell lines. 50 The value was determined. IC 50 The values ​​are shown in Table 2. The inventors also evaluated in silico molecular docking analysis of C6 (Figure 1) and observed that the contributing residues involved in the stabilization of C-6 on BIR2 show active roles of S162, E163, and R166 via hydrogen bonding, and that Y161 and F229 form base stacking interactions. Furthermore, analysis of the binding pocket of BIR3 shows that Y324, R299, and G306 form hydrogen bonds, and that the W323 residue exhibits a base stacking interaction in the stabilization of the C6 compound.

[0172] [Table 5]

[0173] Example 12: Apoptosis antibody array analysis Apoptosis arrays were performed using the R&D Systems Proteome Profiler Human Apoptosis Array Kit (ARY009) according to the manufacturer's instructions. Detailed assay procedures were followed. Images were captured using a gel documentation system (Bio-Rad chemidoc XRS plus), and ImageJ software (NIH) was used for analysis and quantification. Plotly software was used to generate heatmaps (Montreal, Canada).

[0174] Compound C6 was observed to promote SMAC-driven apoptotic features, as seen by the right shift of the histogram overlay showing Annexin-V positive cells (Figure 2, left). Furthermore, C6 treatment drives SMAC-mediated Hallmark apoptotic features, as observed by the cleavage of PARP and caspase-3 and the degradation of cIAP-1 in treated cells compared to controls (Figure 2, right and Figure 3). In addition, C6 robustly sensitizes TRAIL-mediated tumor cell cytotoxicity responses in vitro (Figure 4). Apoptosis or caspase inhibition, or overexpression of its targets such as XIAP, or knockdown of DR5 rescues C6-mediated cell death.

[0175] Example 13: Colony formation assay Clonal colony formation assays were performed using single-cell suspensions. Briefly, cells were seeded in complete McCoy medium in 12-well plates and treated after 24 hours with different doses of different agents, either alone or in combination. Cells were cultured for 2 weeks, changing the media every 3 days. Plates were washed with PBS, fixed with ice-cold methanol, and then stained with 0.5% crystal violet in methanol for 30 minutes. Excess staining was removed by thorough washing with water, and the plates were dried. Representative images were captured using a gel documentation system (Bio-Rad chemidoc XRS plus), and ImageJ software (NIH) was used for analysis and quantification to monitor single-cell colony formation efficiency under different treatment combinations.

[0176] Inhibition of apoptosis by the Pan caspase inhibitor Z-VAD-FMK significantly rescued the C6 cytotoxic phenotype observed by colony formation assays, confirming that C6 induces apoptotic cell death in cancer cells (Figure 5, upper center panel). Similarly, colony formation assays in XIAP-overexpressing cells and DR5-knockdown cells showed significant resistance to C6-mediated apoptotic cell death, suggesting significant involvement of XIAP and DR5 in the overall apoptotic process (Figure 5, lower left and right panels).

[0177] Example 14: In vivo trials in xenograft tumor models All animals were maintained in pathogen-free facilities under a day-night cycle. According to our well-established colon cancer xenograft model, 2 × 10⁶ cells were administered in 100 μl of PBS. 6 Cells (SW620 and HCT116) or 0.5 × 10⁻⁶ 6 Cells (HCT116) were subcutaneously inoculated into the left and right hind limbs of nude Crl:CD1-Foxn1nu mice, each 4–6 weeks old. Mice were randomly assigned to groups by blinded independent investigators. Throughout the experiment, tumors were measured at regular intervals using electronic digital calipers, and tumor volume was calculated using the standard formula V = Π / 6 × a² × b, where "a" is the short tumor axis and "b" is the long tumor axis. At the end of the experiment, the mice were sacrificed, and the subcutaneous tumors were dissected for further examination. Portions of the harvested tumors were cut into small pieces with sterile forceps and scissors and homogenized for lysate preparation.

[0178] Using a cisplatin-resistant SW-620 xenograft model, the inventors determined the in vivo efficacy of C6 and observed that it possessed potent antitumor efficacy against the same model (Figure 7). Similarly, subcutaneous and oral administration of C6 was observed to significantly reduce HCT-116 xenograft tumor volume and weight compared to the respective controls (Figure 8). Immunoblots from tumor tissue revealed that C6-treated tumors showed reduced XIAP and cIAP1 protein expression compared to vehicle-treated tumors in vivo, confirming that C6 targets these proteins and reduces tumor volume (Figure 9, left panel). Furthermore, the inventors' organ distribution analysis observed significant amounts of C6 in tumor tissue, confirming molecular delivery to tumor sites (Figure 9, right panel).

[0179] Example 15: Stability and pharmacokinetic studies of C6 SIF, SGF, and other stability tests were performed according to the following standard protocols. LC-MS / MS was developed for C6: intravenous group (C6, 4 mg / kg), subcutaneous group (C6, 30 mg / kg), and oral group (C6, 30 mg / kg). Mice were administered the respective doses according to body weight intravenously (lateral vein), subcutaneously, and orally, respectively. Blood samples were collected at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, and 48 hours. Plasma was separated and processed for analysis. For pharmacokinetic analysis, plasma concentration versus time data were plotted and analyzed by a non-compartmental analysis method using WinNonlin (Pharsight, Mountain View, CA) software.

[0180] As shown in Figure 6, C6 is highly stable in SIF, SGF, plasma, MLM, and HLM. The pharmacokinetic profiles of C6 via IV, SC, and oral routes are shown in the figure, and the pharmacokinetic parameters are shown in Table 3. The absolute bioavailability of C6 via the subcutaneous and oral routes was found to be 56.61±7.21% and 55.93±11.15%, respectively, as shown in Table 3. The invention described in the original claims of this application is listed below. [1] SMAC mimic compound of formula-I,

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[10] A pharmaceutical composition comprising a SMAC mimetic compound of formula-I,

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[11] A SMAC-mimicking compound as described in [1] that has potent antiproliferative activity against mammalian cancer cell lines selected from the group consisting of colon, breast, kidney, prostate, brain, ovary, pancreas, melanoma, liver, leukemia, and lymphoma.

[12] A SMAC-mimicking compound described in [1] that is useful for the treatment of treatment-resistant, refractory, and metastatic cancers in mammals.

[13] A SMAC mimic compound described in [1] is useful in combination therapy with other antiproliferative agents selected from the group consisting of TRAIL agonists / MAbs, aromatase inhibitors, epigenetic modulators, kinase inhibitors, alkylating agents, microtubule disruptors, topoisomerase inhibitors, anti-angiogenic compounds, Hsp90 inhibitors, mTOR inhibitors, estrogen and androgen antagonists, MMP inhibitors, and biological response modifiers.

[14] A method for treating cancer using the SMAC mimetic compound described in [1].

[0181] Table 6

Claims

1. SMAC mimic compound of formula I, 【Chemistry 1】 Here, R １ is unsubstituted or substituted heteroaryl or C ６ -C １０ Selected from the group consisting of aryls; R ２ H, C １ -C ６ Alkyl and C ４ -C ８ Selected from the group consisting of cycloalkyl groups; R ３ and R ４ are each independently selected from the group consisting of H and C １ -C ６ alkyl; A is either non-substitutable or substituted C １ -C ６ Alkyl or C ６ -C １０ Selected from the alphabet; B is C ６ -C １０ Aryl, C(O)R ５ and C(O)N(R ６ ) (Caution ７ Selected from the group consisting of; R ５ is OH, C １ -C ６ Alkoxy and C ６ Selected from the group consisting of alkoxyaryls; R ６ and R ７ These are, independently, hydrogen and C ６ -C １０ Aryl and C ６ -C １０ Selected from the group consisting of arylalkyls; or a pharmaceutically acceptable salt thereof.

2. Benzyl((2S,5R)-1-(L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carbonyl)-L-isoleucineate (compound 2), (2S,5R)-1-(L-alanyl-L-valyl)-N-benzhydryl-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 3), (2S,3S)-benzyl 3-methyl-2-((2S,5R)-1-((S)-3-methyl-2-((S)-2-(methylamino)propanamide)butanoyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide)pentanoate (compound 4), (2S,5R)-N-benzhydryl-1-(methyl-L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 5), (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-(methyl-L-alanyl-L-valyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 6), (2S,3S)-benzyl 2-((2S,5R)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamide)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide)-3-methylpentanoate (compound 7), (2S,5R)-N-benzhydryl-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamide)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 8), (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamide)acetyl)-5-(5-methylfuran-2-yl)pyrrolidine-2-carboxamide (compound 9); and (2S,5R)-N-((2S,3S)-1-(benzhydrylamino)-3-methyl-1-oxopentan-2-yl)-1-(methyl-L-alanyl-L-alanyl)-5-phenylpyrrolidine-2-carboxamide (compound 10) A SMAC mimic compound according to claim 1, selected from the group consisting of the following.

3. A process for preparing an SMAC mimic compound of formula-I, 【Chemistry 2】 Here, R １ This is a 5-methylfuran-2-yl group; R ２ H, C １ -C ６ Alkyl and C ４ -C ８ Selected from the group consisting of cycloalkyl groups; R ３ and R ４ These are H and C, respectively, independently. １ -C ６ Selected from the group consisting of alkyl groups; A is either non-substitutable or substituted C １ -C ６ Alkyl or C ６ -C １０ Selected from the alphabet; B is C ６ -C １０ Aryl, C(O)R ５ and C(O)N(R ６ ) (Caution ７ Selected from the group consisting of; R ５ is OH, C １ -C ６ Alkoxy and C ６ Selected from the group consisting of alkoxyaryls; R ６ and R ７ These are, independently, hydrogen and C ６ -C １０ Aryl and C ６ -C １０ Selected from the group consisting of arylalkyls; i. The Boc group of 2-benzyl 1-(tert-butyl)(2S,5S)-5-(5-methylfuran-2-yl)pyrrolidine-1,2-dicarboxylate is removed by acid hydrolysis using TFA, and the resulting amine is subsequently converted to BocNHCH(R) in the presence of a peptide coupling reagent and a weak base. ２ The step of coupling with COOH to obtain the compound of formula P1; 【Transformation 3】 ii. The Boc group is removed from the compound of formula P1 by acid hydrolysis using TFA, and the obtained amine is converted to the compound of formula BocN(R) in the presence of a peptide coupling reagent and a weak base. ４ ')CH(R ３ The step of coupling it with a COOH compound to obtain the compound of formula P2; 【Chemistry 4】 iii. The step of catalytically hydrogenating the compound of formula P2 using a Pd catalyst in the presence of a solvent to obtain the free carboxylic acid of formula P3; 【Transformation 5】 iv. The free carboxylic acid of formula P3 is converted to NH in the presence of a peptide coupling reagent and a weak base. ２ The steps include coupling with CH(A)(B) to obtain the compound of formula I, A process that includes this.

4. The peptide coupling reagent is selected from the group consisting of HOBt, EDCI, and HBTU, the weak base is diethylisopropylamine, and the Pd catalyst is Pd / C or Pd(OH) ２ The process according to claim 3, selected from / C.

5. The process according to claim 3, wherein the solvent is selected from DCM or DMF for peptide coupling and MeOH or EtOAc for catalytic hydrogenation.

6. A process for preparing the SMAC mimic compound 10 described in claim 2, (a) Saponifying and coupling compound X with H-Ile-benzhydrylamide in a solvent in the presence of a peptide coupling reagent and a weak base to obtain compound XI; 【Transformation 6】 (b) The step of removing the Boc group from compound XI by acid hydrolysis using TFA, and then coupling the obtained amine with Boc-Val-COOH in the presence of a peptide coupling reagent and a weak base to obtain the compound of formula XII; 【Transformation 7】 (c) The step of removing the Boc group from compound XII by acid hydrolysis using TFA, and then coupling the obtained amine with Boc-N-Me-Ala-OH in the presence of a peptide coupling reagent and a weak base to obtain the compound of formula XIII; 【Transformation 8】 (d) The step of removing the Boc group from the compound of formula XIII by acid decomposition to obtain the compound 10, 【Chemistry 9】 A process that includes this.

7. The process according to claim 6, wherein the peptide coupling reagent is selected from the group consisting of HOBt, EDCI, and HBTU, the solvent is selected from DCM or DMF, the weak base is diethylisopropylamine, and the reagent is TFA for acid hydrolysis.

8. The SMAC mimetic compound according to claim 1, which inhibits the binding of SMAC protein to apoptosis inhibitory proteins (IAPs) and is useful for treating proliferative diseases, including cancer.

9. SMAC mimic compound of formula I, 【Chemistry 10】 Here, R １ is unsubstituted or substituted heteroaryl or C ６ -C １０ Selected from the group consisting of aryls; R ２ H, C １ -C ６ Alkyl and C ４ -C ８ Selected from the group consisting of cycloalkyl groups; R ３ and R ４ These are H and C, respectively, independently. １ -C ６ Selected from the group consisting of alkyl groups; A is either non-substitutable or substituted C １ -C ６ Alkyl or C ６ -C １０ Selected from the alphabet; B is C ６ -C １０ Aryl, C(O)R ５ and C(O)N(R ６ ) (Caution ７ Selected from the group consisting of; R ５ is selected from the group consisting of OH, C １ -C ６ alkoxy and C ６ alkoxyaryl; R ６ and R ７ These are, independently, hydrogen and C ６ -C １０ Aryl and C ６ -C １０ Selected from the group consisting of arylalkyls; or its pharmaceutically acceptable salts and pharmaceutically acceptable excipients A pharmaceutical composition containing the above.

10. The SMAC mimetic compound according to claim 1, which has potent antiproliferative activity against mammalian cancer cell lines selected from the group consisting of colon, breast, kidney, prostate, brain, ovary, pancreas, melanoma, liver, leukemia, and lymphoma.

11. The SMAC mimetic compound according to claim 1, which is useful for treating treatment-resistant, refractory, and metastatic cancers in mammals.

12. The SMAC mimetic compound according to claim 1, which is useful in combination therapy with other antiproliferative agents selected from the group consisting of TRAIL agonists / MAbs, aromatase inhibitors, epigenetic modulators, kinase inhibitors, alkylating agents, microtubule disruptors, topoisomerase inhibitors, anti-angiogenic compounds, Hsp90 inhibitors, mTOR inhibitors, estrogen and androgen antagonists, MMP inhibitors, and biological response modifiers.

13. The SMAC mimic compound according to claim 1, used for treating cancer.

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