CN110028508B

CN110028508B - Antitumor diazo bicyclic apoptosis protein inhibitor

Info

Publication number: CN110028508B
Application number: CN201910408971.2A
Authority: CN
Inventors: 宋志春; 张崇光; 何东伟; 包金远; 张孝清
Original assignee: Nanjing Huawe Medicine Technology Group Co Ltd
Current assignee: Nanjing Huawe Medicine Technology Group Co Ltd
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2021-05-28
Anticipated expiration: 2039-05-16
Also published as: WO2020228478A1; CN110028508A

Abstract

The invention provides a novel apoptosis protein inhibitor or pharmaceutically acceptable salt and isomer thereof, a preparation method thereof and a pharmaceutical composition, wherein the definition of each group is shown in the specification; the invention also provides the application of the compound and the pharmaceutically acceptable salts and isomers thereof in preparing medicines for treating diseases related to IAP protein, the compound has better binding affinity with XIAP, cIAP1 and cIAP2 proteins, has better inhibition effect on cell growth in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines, and has higher medicinal value and wide market prospect.

Description

Antitumor diazo bicyclic apoptosis protein inhibitor

Technical Field

The field belongs to the field of medicinal chemistry, and particularly relates to an apoptosis protein inhibitor, and a preparation method and application thereof.

Background

Apoptosis or programmed cell death is a regulated mechanism in gene and biochemistry that plays an important role in invertebrate and vertebrate development and homeostasis. Apoptotic abnormalities that lead to premature cell death have been associated with a variety of developmental disorders. Defects in apoptosis leading to a lack of cell death have been associated with cancer and chronic viral infections.

Current cancer therapies, including chemotherapeutic agents, radiation, and immunotherapy, indirectly induce apoptosis in cancer cells. Thus, the inability of cancer cells to perform apoptotic programs due to defects in normal apoptotic mechanisms is often associated with increased resistance to chemotherapy, radiation, or immunotherapy-induced apoptosis. Such primary or acquired resistance of cancer to current therapies due to apoptosis defects is a major problem in current cancer therapies. One class of central negative regulators of apoptosis are Inhibitors of Apoptosis Proteins (IAPs). This class includes proteins such as XIAP, cIAP1, cIAP2, ML-IAP, HIAP, KIAP, TSIAP, NAIP, survivin, livin, ILP-2, apollon and BRUCE. IAP proteins, which are effective in inhibiting apoptosis in cancer cells induced by a wide variety of apoptotic stimuli, including chemotherapeutic agents, radiation, and immunotherapy, are novel targets for the apoptotic pathway.

Patent applications for inventions relating to IAP inhibitors are WO2011018474a1, WO2008016893a1, WO2014047024a1, CN101484151A, etc.

Among the IAP inhibitors currently under development are LCL161, birinapag, BV6, GDC-0152, AZD5582, AT406, etc., wherein AT-406 is a potent, orally available Smac mimetic that is an antagonist of IAPs and inhibits XIAP, cIAP1 and cIAP2 proteins having the structural formulae shown below:

currently, the effect of the immunodetection point inhibitor alone on solid tumors is weak clinically, the IAP inhibitor and the immunodetection point inhibitor are combined to treat the solid tumors to obtain a better treatment effect, the IAP inhibitor has the effect of cooperating with the immunodetection point inhibitor to treat cancers, and has a very good application prospect, but the development of the variety still has more important challenges. In order to meet the clinical requirement of the apoptosis protein inhibitor at present and achieve better treatment effect of tumor diseases, the study and development of drug design and the like of a series of high-efficiency and low-toxicity IAP inhibitors are dedicated, and the method has great significance for the field of medicine.

Disclosure of Invention

The invention aims to provide a novel apoptosis protein inhibitor or pharmaceutically acceptable salt and isomer thereof, which is characterized in that the compound has the following general formula:

wherein X represents a C or N atom, and when X is a C atom, Y is CONHR₃Z represents a H atom; when X is an N atom, Z is CONHR₃Y is absent;

said R₁And R₂Each independently is selected from C_1～20Alkylamino radical, C_5～8A cycloalkylamino group,

C_1～20Alkyl, aryl, heteroaryl, and heteroaryl,

Or an alpha-amino acid residue, or a pharmaceutically acceptable salt thereof,

wherein the alpha-amino acid residue is a substituent formed after the carboxyl on alpha-carbon in the alpha-amino acid is deleted, the Ar group represents aryl or heteroaryl with the atomic number of 5-8, and m and n are respectively and independently selected from any integer of 1-3;

R₃is selected from substituted or unsubstituted benzyl, and the substituent is selected from phenyl, halogen and C_1～4Alkyl or p-fluorophenyl;

further, a compound, or a pharmaceutically acceptable salt or isomer thereof, said compound having the general formula:

or

Wherein, in the formula I or II,

said R₁Is selected from C_1～20An alkylamino group,

C_1～20Alkyl, aryl, heteroaryl, and heteroaryl,

Or any one of alpha-amino acid residues, R₂Is selected from C_1～20Alkylamino radical, C_5～8Cycloalkylamino radical, C_1～20Alkyl, aryl, heteroaryl, and heteroaryl,

Or an alpha-amino acid residue,

wherein the alpha-amino acid residue is a substitution residue formed after the carboxyl on alpha-carbon in the alpha-amino acid is deleted, the Ar group represents aryl and heteroaryl with the atomic number of 5-8, m and n are respectively and independently selected from any integer of 1-3,

in one embodiment, the alkylamino group in formula I or II is

And isomers thereof;

in one embodiment, C is as described in formula I or II_5～6Cycloalkylamino radical being

In one embodiment, the α -amino acid residue is

Or

In one embodiment, R in formula I or II₂Is selected from C_1～13An alkyl group;

in one embodiment, aryl as described in formula I or II is phenyl;

in one embodiment, the heteroaryl group described in formula I or II is imidazolyl;

preferably, R₃In the group, the halogen is F.

Illustrative, non-limiting specific examples of the compounds of the present invention are shown below:

and salts or isomers thereof.

The invention also provides a preparation method of the compounds shown in the formula I and the formula II and salts or isomers thereof, but is not limited to the method described below. All starting materials are prepared or purchased directly according to the general rules of the target molecule and by protocols in these routes, methods well known to those of ordinary skill in the art of organic chemistry. The compounds of the invention can be synthesized by combining the methods described below with synthetic methods known in the art of synthetic organic chemistry or variations thereon as recognized by those skilled in the art.

In one embodiment, the compounds of the invention are prepared as follows:

scheme 1 comprises the following steps:

taking N-Boc pyrrolidine-2-methyl formate as a starting material, reacting with allyl bromide under the action of lithium bis-trimethylsilyl amide and the like to generate a compound 1, wherein the reaction temperature is-80 ℃ to-20 ℃, and the reaction solvent is selected from inert aprotic solvents such as tetrahydrofuran, toluene and the like;

removing the Boc protecting group of the compound 1 under an acidic condition to obtain a compound 2, wherein the acid can be hydrochloric acid, p-toluenesulfonic acid and the like, and the reaction solvent is methanol, ethanol, tetrahydrofuran and the like;

the compound 2 reacts with the condensation agent

Reaction to give the compound 3, P₁、P₂As the protecting group for the amino group, any suitable protecting group such as t-butyloxycarbonyl group (Boc), 9-fluorenylmethylalkenyloxycarbonyl group (Fmoc), benzyloxycarbonyl group (Cbz), allyloxycarbonyl group (Alloc), triphenylmethyl group (Trt), etc., P₁Is not equal to P₂(in embodiments, P₁And P₂Is an orthogonal protecting group). The condensing agent may be selected from N, N' -diisopropylcarbodiimide, benzoAny one of condensing agents such as triazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and the like;

under the condition of ozone, oxidizing carbon-carbon double bonds of the compound 3 into aldehyde with at least one carbon to obtain a compound 4, wherein the reaction temperature is selected from-80 ℃ to-20 ℃, the reaction solvent is selected from tetrahydrofuran, dichloromethane and methanol, and the reducing agent is selected from triphenylphosphine, sodium sulfite, triethylamine and the like;

compound 4 is acidic (P)₂Boc protecting group) or palladium on carbon (P)₂When it is a Cbz protecting group) or piperidine (P)₂Fmoc protecting group) under the same conditions₂Further carrying out reductive amination to obtain a macrocyclic compound 5, wherein a reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene and the like, and a reducing agent is sodium borohydride, sodium cyanoborohydride and the like;

compound 5 is prepared by reacting compound with a condensing agent in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide₁Performing condensation reaction on carboxylic acid compounds of the groups to obtain a compound 6, wherein the temperature is 25-120 ℃, and the condensing agent is N, N ' -diisopropylcarbodiimide, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, 1H-benzotriazole-1-oxytripyrrolidinyl hexafluorophosphate and the like;

saponifying the compound 6 under an alkaline condition, and then controlling the pH value under an acidic condition to obtain a compound 7, wherein the organic solvent is water-soluble solvents such as methanol and ethanol, and the alkali is inorganic alkali such as lithium hydroxide and sodium hydroxide;

compound 7 is prepared by reacting compound with a condensing agent in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide₃Performing condensation reaction on amino compounds of the groups to obtain a compound 8, wherein the temperature is 25-120 ℃, and the condensing agent is N, N ' -diisopropylcarbodiimide, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, 1H-benzotriazolAzol-1-yloxytripyrrolidinyl hexafluorophosphate and the like;

compound 8 is acidic (P)₁Boc protecting group) or palladium on carbon (P)₁When it is a Cbz protecting group) or piperidine (P)₁Fmoc protecting group) under the same conditions₁To obtain an amino compound 9, wherein a reaction solvent is selected from tetrahydrofuran, dichloromethane, methanol, toluene and the like;

compound 9 is prepared by reacting compound with a condensing agent and a solvent containing R in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide₂And (2) carrying out condensation reaction on carboxylic acid compounds of the groups to obtain a compound I, wherein the temperature is 25-120 ℃, and the condensing agent is N, N ' -diisopropylcarbodiimide, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, 1H-benzotriazole-1-oxytripyrrolidinyl hexafluorophosphate and the like.

In another embodiment, the compounds of the invention are prepared as follows:

scheme 2 includes the following steps:

taking a compound 10 as an initial raw material, and generating a compound 11 by ring closure under the action of inorganic bases such as potassium carbonate, cesium carbonate and the like, wherein the reaction temperature is 25-120 ℃, and the reaction solvent is an inert aprotic solvent such as acetonitrile, tetrahydrofuran, N-dimethylformamide and the like;

the compound 11 is reacted with a condensing agent

Reaction to give the compound 12, P₁，P₂As the protecting group for the amino group, t-butyloxycarbonyl group (Boc), 9-fluorenylmethylalkenyloxycarbonyl group (Fmoc), benzyloxycarbonyl group (Cbz), allyloxycarbonyl group (Alloc), triphenylmethyl group (Trt), etc., P, can be selected₁Is not equal to P₂(in embodiments, P₁And P₂Is an orthogonal protecting group). The condensing agent is selected from N, N ' -diisopropylcarbodiimide, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate and the like;

under the condition of ozone, oxidizing a carbon-carbon double bond of the compound 12 into aldehyde with at least one carbon to obtain a compound 13, wherein the reaction temperature is selected from-80 ℃ to-20 ℃, the reaction solvent is selected from tetrahydrofuran, dichloromethane and methanol, and the reducing agent is selected from triphenylphosphine, sodium sulfite, triethylamine and the like;

compound 13 is acidic (P)₂Boc protecting group) or palladium on carbon (P)₂When it is a Cbz protecting group) or piperidine (P)₂Fmoc protecting group) under the same conditions₂Further carrying out reductive amination to obtain a macrocyclic compound 14, wherein a reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene and the like, and a reducing agent is sodium borohydride, sodium cyanoborohydride and the like;

compound 14 is prepared by reacting compound 14 with a condensing agent in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide and a solvent containing R₁Carrying out condensation reaction on carboxylic acid compounds of the groups to obtain a compound 15, wherein the temperature is 25-120 ℃, and the condensing agent is N, N ' -diisopropylcarbodiimide, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, 1H-benzotriazole-1-oxytripyrrolidinyl hexafluorophosphate and the like;

saponifying the compound 15 under an alkaline condition, and then controlling the pH value under an acidic condition to obtain a compound 16, wherein the organic solvent is water-soluble solvents such as methanol and ethanol, and the alkali is inorganic alkali such as lithium hydroxide and sodium hydroxide;

compound 16 is prepared by reacting compound 16 with a condensing agent in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide₃The amino compounds of the group are subjected to condensation reaction to obtain a compound 17, the temperature is selected from 25 ℃ to 120 ℃, and the condensing agent is selected from N, N '-diisopropylcarbodiimide, benzotriazole-N, N, N', N '-tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N', N-Tetramethyluronium hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate, and the like;

compound 17 is acidic (P)₁Boc protecting group) or palladium on carbon (P)₁When it is a Cbz protecting group) or piperidine (P)₁Fmoc protecting group) under the same conditions₁To obtain amino compound 18, wherein the reaction solvent is selected from tetrahydrofuran, dichloromethane, methanol, toluene and the like;

compound 18 is prepared by reacting compound 18 with a condensing agent in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide₂And (2) carrying out condensation reaction on carboxylic acid compounds of the groups to obtain a compound II, wherein the temperature is 25-120 ℃, and the condensing agent is N, N ' -diisopropylcarbodiimide, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, 1H-benzotriazole-1-oxytripyrrolidinyl hexafluorophosphate and the like.

Further, in the above schemes 1 and 2, P₁，P₂The radical being a protecting group for amino, R₁、R₂And R₃The groups are defined as described above in the specification.

The preparation of the compounds of formula I' can be carried out with reference to the methods of scheme 1 or scheme 2.

Among the above-mentioned preparation methods, methods for preparing compounds such as those involving protecting and deprotecting various chemical groups, and those skilled in the art can easily select an appropriate protecting group according to the requirements of protection and deprotection. The features mentioned above in the description, or the features mentioned in the examples, may be combined in any combination in accordance with the principles of medicine, and each feature disclosed in the description may be replaced by any alternative feature serving the same, equivalent or similar purpose. Unless specifically stated otherwise, the disclosed features are merely generic examples of similar or analogous features.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any compound selected from the group consisting of compounds of formula I' or formula I or formula II, or pharmaceutically acceptable salts or isomers thereof, as an active ingredient, in combination with one or more pharmaceutically acceptable carriers, diluents and/or excipients.

The pharmaceutical composition preferably contains 1-90% by weight of the compound of formula I' or formula I or formula II or a pharmaceutically acceptable salt or isomer thereof as an active ingredient, more preferably 10-80% by weight of the active ingredient.

Unless otherwise indicated, the following terms used in the claims and the specification have the following meanings or characteristics:

the term "C_1～20Alkyl "means a saturated aliphatic radical of 1 to 20 carbon atoms and includes straight and branched chain radicals (a numerical range referred to in this application, e.g.," 1 to 20 ", means that the radical, in this case alkyl, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). The alkyl group in the present invention includes an "alkylene group". Alkyl groups having 1 to 6 carbon atoms are referred to as lower alkyl groups. When a lower alkyl group has no substituent, it is referred to as unsubstituted lower alkyl. More preferably, the alkyl group is a medium size alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, ethylene, propyl, propylene, 2-propyl, n-butyl, isobutyl, butylene, t-butyl, pentyl, and the like. Preferably, the alkyl group is a lower alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, butylene, isobutyl, tert-butyl, etc. Alkyl groups may be substituted or unsubstituted.

The term "C_1～20Alkylamino represents a compound of the R-amino type, R being as defined for C_1～20Alkyl groups are as described above.

The term "C_5～8Cycloalkylamino "denotes C_5～8Amines in which one carbon of the cycloalkane is replaced by an amino group, e.g.

And the like.

The term "α -amino acid residue" denotes a substituted residue formed by deletion of the carboxyl group at the α -carbon in an amino acid.

The term "benzyl" denotes "benzeneMethyl ", structure formula C₆H₅CH₂-, or by Bn (abbreviation for Benzyl).

The term "aryl" denotes an all-carbon monocyclic or fused polycyclic group of 5 to 12 carbon atoms with a completely conjugated pi-electron system. Non-limiting examples of aryl groups are phenyl, naphthyl and anthracenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. The aryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more, more preferably one, two or three, even more preferably one or two, independently selected from the group consisting of lower alkyl, trihaloalkyl, halogen, hydroxy, lower alkoxy, mercapto, (lower alkyl) thio, cyano, acyl, thioacyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, nitro, N-sulphonylamino, S-sulphonylamino. Preferably, aryl is 5-membered monocyclic aryl, 6-membered monocyclic aryl.

The term "heteroaryl" denotes a monocyclic or fused ring group of 5 to 12 ring atoms, containing one, two, three or four ring heteroatoms selected from N, O or S, the remaining ring atoms being C, and additionally having a completely conjugated pi-electron system. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring joined to the parent structure is a heteroaryl ring. Heteroaryl groups may be substituted or unsubstituted. When substituted, the substituents are preferably one or more, more preferably one, two or three, and still more preferably one or two. Non-limiting examples of unsubstituted heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine, and carbazole; preferably, the heteroaryl is a nitrogen-containing 5-membered monocyclic heteroaryl, a nitrogen-containing 6-membered monocyclic heteroaryl.

The term "isomer" is selected from the cis, trans or a mixture of cis and trans isomers thereof.

The term "halogen" denotes fluorine, chlorine, bromine or iodine.

The term "pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:

(1) salts with acids are obtained by reaction of the free base of the parent compound with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, and the like, or with organic acids such as, but not limited to, acetic acid, malic acid, fumaric acid, maleic acid, p-toluenesulfonic acid, tartaric acid, citric acid, lactic acid, succinic acid, malonic acid, and the like. The salt has safety, effectiveness and bioactivity in mammalian body.

"pharmaceutical composition" refers to a mixture of one or more compounds described herein or their pharmaceutically acceptable salts, isomers, prodrugs, etc. with other chemical components, such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism.

By "pharmaceutically acceptable carrier" is meant a carrier or diluent that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the compound being administered.

"excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the compound. Examples of excipients include, without limitation, lactose, sucrose, microcrystalline cellulose, sorbitol, polyvinylpyrrolidone, cellulose, water, and methylcellulose, and the like.

The pharmaceutical composition may further comprise: lubricants such as talc, magnesium stearate, mineral oil, and the like; a humectant; emulsifying and suspending agents; preservatives such as methyl benzoate and the like; sweetening agents and flavoring agents.

The compositions of the present invention are formulated, dosed (dosed) and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the location of the agent being delivered, the method of administration, the dosing regimen, and other factors known to medical practitioners.

The invention also provides the use of a compound of formula I' or formula I or formula II:

(1) for inducing apoptosis in a cell, or for sensitizing a cell, particularly a cancer cell, to apoptotic signals.

(2) For inducing apoptosis in a cell overexpressing an IAP protein (e.g., c-IAP1, c-IAP2, X-IAP, or ML-IAP).

(3) Preparing a medicament for treating diseases related to IAP proteins; more broadly, the compounds are useful for the treatment of cancer.

(4) Further, the compounds of the present invention may be used in the treatment of all cancer type drugs that fail to undergo apoptosis. Examples of such cancer types include neuroblastoma, intestinal cancers such as rectal, colon, familial adenomatous polyposis and hereditary nonpolyposis colorectal, esophageal, lip, larynx, hypopharynx, tongue, salivary gland, stomach, adenocarcinoma, medullary thyroid, papillary thyroid, kidney, renal parenchymal, ovarian, cervix, corpus, endometrium, choriocarcinoma, pancreas, prostate, testicular, breast, urinary, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, hodgkin's lymphoma, non-hodgkin's lymphoma, burkitt's lymphoma, Acute Lymphatic Leukemia (ALL), Chronic Lymphatic Leukemia (CLL), Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), colon carcinoma of the larynx, and the body, Adult T-cell leukemia lymphoma, hepatocellular carcinoma, gallbladder carcinoma, bronchial carcinoma, small-cell lung carcinoma, non-small-cell lung carcinoma, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, ewing's sarcoma, and plasmacytoma. Can be used for treating solid tumor. Can also be used for treating various major diseases such as breast cancer, pancreatic cancer or malignant melanoma.

The positive control compound AT-406 of the compound is a cIAP1 and cIAP2 inhibitor, and has weaker inhibition effect on XIAP than cIAP1 and cIAP2, while the compound of the invention has stronger inhibition activity on three proteins of XIAP, cIAP1 and cIAP2, so the compound of the invention has stronger inhibition effect on apoptosis proteins on the whole;

the cell pharmacological data show that the compound has strong inhibition effect on MDA-MB-231 breast cancer and PC-3 pancreatic cancer cells, while the positive control compound AT-406 only has strong effect on MDA-MB-231 breast cancer and has relatively weak inhibition effect on PC-3 pancreatic cancer cells;

the compound has better IAP (platelet activating protein) inhibition activity, can be developed into a cancer treatment drug which is taken alone in the later period, and can also be used for treating cancer by combining with other targeted drugs;

in conclusion, the compound has good binding affinity with XIAP, cIAP1 and cIAP2 proteins, has good inhibition effect on cell growth in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines, and has great medicinal value and wide market prospect.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following specific examples are intended to further illustrate the invention, but the invention is in no way limited to these examples (all materials are commercially available unless otherwise indicated).

(all compounds related to chirality in this example were prepared, separated and purified by chiral column)

Example 1: preparation of pyrrolidine-2-carboxylic acid methyl ester

40g (0.35mol) of proline and 250mL of methanol are taken to be placed in a 500mL single-mouth reaction bottle and cooled to 0 ℃, 45g (0.38mol) of thionyl chloride is taken to be placed in a 250mL constant-pressure dropping funnel and slowly dropped into the solution system, heat is released along with the dropping system, the internal temperature is controlled to be less than 5 ℃, and the dropping is finished within about 20 minutes. The temperature is raised to 70 ℃ to obtain a clear solution, and the clear solution is refluxed for 3 hours. After cooling to room temperature, the solvent was evaporated under reduced pressure to give 43g of a pale yellow oily liquid, and the crude product was used directly in the next reaction.

Example 2: preparation of N-Boc pyrrolidine-2-carboxylic acid methyl ester

A2L single-neck reaction flask was charged with 43g (0.33mol) of methyl pyrrolidine-2-carboxylate and 400mL of methylene chloride, and 88g (0.87mol) of triethylamine was added to the flask to obtain a white emulsion, which was stirred at room temperature for 15 minutes and cooled to 0 ℃.

152g (0.69mol) of Boc anhydride is dissolved in 600mL of dichloromethane and added into the system dropwise in a constant pressure funnel, a large amount of bubbles are released along with dropwise addition, heat is released, the dropwise addition speed is controlled, and the temperature in the system is kept to be less than 10 ℃. After the dropwise addition, a white suspension was obtained and stirred at room temperature for 12 hours. Filtration was carried out to remove a white precipitate, the filtrate was subjected to evaporation of the solvent under reduced pressure to give a white suspension, which was washed with 300mL of a citric acid solution (0.5M), ether-extracted (3 × 500mL), the organic layers were combined, washed with saturated brine, dried over sodium sulfate, filtered, and subjected to evaporation of the solvent under reduced pressure, and the residue was subjected to column chromatography (eluent: ethyl acetate: petroleum ether: 1:5) to give 75g of a clear colorless oily liquid with a yield of 94%.

Example 3: preparation of N-Boc-2-propenyl-pyrrolidine-2-carboxylic acid methyl ester

20g (87.3mmol) of methyl N-Boc-pyrrolidine-2-carboxylate and 200mL of tetrahydrofuran were taken in a 500mL three-necked flask, protected with nitrogen and cooled to-78 ℃.

And dropping 105mL (105mmol, 1M) of lithium bis (trimethylsilyl) amide into the system in a constant-pressure dropping funnel, controlling the dropping speed, and maintaining the temperature in the system to be lower than-78 ℃ to obtain a tan solution. After the addition, the mixture was stirred at-78 ℃ for 1.5 hours.

Dissolving 15.9g (131mmol) of allyl bromide in 100mL of tetrahydrofuran, dropwise adding the mixture into the system, controlling the dropwise adding speed, maintaining the temperature in the system to be lower than-78 ℃, and stirring for 1 hour at-78 ℃ after the dropwise adding is finished. The temperature was returned to room temperature and stirred for 1 hour to obtain a reddish brown solution. The reaction was complete by TLC and quenched with 50mL of water. Ethyl acetate extraction (3 × 200mL) combined organic layers, washed with saturated brine, dried over sodium sulfate, filtered, evaporated in vacuo and purified by column chromatography (eluent ethyl acetate: petroleum ether: 1:10) to give 18.7g of a colorless oily liquid in 82% yield.

Example 4: preparation of methyl 2-propenyl-1- ((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino) propionyl) pyrrolidine-2-carboxylate

18g (66.9mmol) of N-Boc-2-propenyl-pyrrolidine-2-carboxylic acid methyl ester and 50mL of absolute ethanol are put into a 500mL single-mouth reaction bottle, 100mL of hydrochloric ethanol is added, the mixture is stirred for 2 hours at room temperature, and TLC detection is carried out to complete the reaction. The solvent and excess hydrogen chloride were distilled off under reduced pressure to give 14g of a colorless oily liquid dissolved in 50mL of dichloromethane.

In a 500mL single-neck reaction flask were further taken 27g (80.3mmol) of (S) -2- (Boc-amino) -3- (Cbz-amino) propionic acid and 200mL of methylene chloride, 38g of HATU (100.35mmol) and 17g of DIEA (133.8mmol) were added, and the mixture was stirred at room temperature for 1 hour to obtain a yellow clear solution.

And (3) dropwise adding the dichloromethane solution prepared in the first step into the yellow clear liquid, stirring at room temperature for 6 hours to obtain a reddish brown solution, and detecting by TLC to complete the reaction. The solvent was distilled off under reduced pressure, the residue was dissolved in 400mL of ethyl acetate, washed with 1m hcl solution, saturated sodium bicarbonate solution, saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent ethyl acetate: petroleum ether: 1:2) to give 21.2g of a white solid in 65% yield.

Example 5: preparation of methyl 2-glyoxyl-1- ((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino) propionyl) pyrrolidine-2-carboxylate

20g (40.9mmol) of methyl 2-propenyl-1- ((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino) propionyl) pyrrolidine-2-carboxylate and 200mL of methylene chloride were taken in a 500mL three-necked reaction flask, cooled to-78 ℃, and ozone was introduced for about 30 minutes until the system became blue, and the mixture was stirred at-78 ℃ for 30 minutes, and then the introduction of ozone was stopped. And introducing nitrogen to bubble so as to remove excessive ozone until blue disappears, obtaining colorless solution, adding 100mL of triethylamine, returning to room temperature, stirring for 2 hours, and detecting the reaction completion by TLC. Washing with 1M citric acid, washing with saturated aqueous sodium chloride solution, drying the organic layer with sodium sulfate, filtering, evaporating the solvent under reduced pressure, and purifying the residue by column chromatography (eluent ethyl acetate: petroleum ether: 1:2) to obtain 16.8g of a white solid with a yield of 84%.

Example 6: preparation of methyl (5R,10aR)5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate

15g (30.5mmol) of methyl 2-carboxaldehyde-1- ((3-N-benzyloxycarbonyl-2-N-tert-butoxycarbonyl) propionyl) pyrrolidine-2-carboxylate and 260mL of methanol were dissolved in a 500mL single-neck reaction flask to obtain a colorless clear solution, 1.5g (10%) of Pd/C was added, and the mixture was stirred at room temperature for 12 hours under a hydrogen atmosphere and was detected to be completely reacted by TLC. Pd/C was filtered off, the solvent was evaporated under reduced pressure, 200mL of tetrahydrofuran was added to dissolve it, 2.9g (45.8mmol) of sodium cyanoborohydride was added thereto, the mixture was stirred at room temperature, and the completion of the reaction was detected by TLC. The reaction was quenched with 20mL of water, filtered, the solvent was evaporated under reduced pressure, the residue was dissolved in 200mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 10:1) to give 7.8g of a white solid in 75% yield.

Example 7: preparation of methyl (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate

To a 250mL single-neck reaction flask were added 1.5g (6.5mmol) of N-Cbz-N-methyl-D-alanine and 60mL of methylene chloride, 3.4g of HATU (8.9mmol) and 1.5g of DIEA (11.8mmol) and the mixture was stirred at room temperature for 1 hour to obtain a yellow clear solution.

2g (5.9mmol) of methyl (5R,10aR)5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate was dissolved in 20mL of dichloromethane, added dropwise to the above system, stirred at room temperature for 6 hours and checked by TLC for completion of the reaction. The solvent was distilled off under reduced pressure, the residue was dissolved in 200mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 2.1g of a brown oil in 64% yield.

Example 8: preparation of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylic acid

2g (3.6mmol) of methyl (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butoxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate and 60mL of methanol were taken in a 250mL single-neck reaction flask, 0.4g (7.2mmol) of potassium hydroxide and 60mL of water were added, the mixture was heated to 50 ℃ and stirred for 3 hours, and the reaction was checked by TLC for completion. 60mL of water was added, methanol was distilled off under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2X 100mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, ethyl acetate (3X 100mL) was extracted, the organic layer was dried over sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to give 1.9g of oil which was used directly in the next reaction.

Example 9: preparation of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide

1.8g (3.3mmol) of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazocine ring-10 a-carboxylic acid and 2.6g of PyBop (5.0mmol) were taken in a 100mL single-neck reaction flask, 50mL of DMF was added, and 0.85g of DIEA (6.6mmol) was further taken and stirred at room temperature for 30 minutes. 1.2g (6.6mmol) of benzhydrylamine is added into the system, the temperature is heated to 90 ℃, the stirring is carried out for 12 hours, and the TLC detection reaction is complete. The reaction solution was poured into 300mL of water, extracted with ethyl acetate (3 × 100mL), washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 15:1) to give 1.7g of a pale yellow solid in 74% yield.

Example 10: preparation of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide

0.5g (0.7mmol) of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butoxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide and 10mL of absolute ethanol were taken in a 100mL single-neck reaction flask, 20mL of hydrochloric acid ethanol was added, the mixture was stirred at room temperature for 2 hours, and TLC detection was carried out to complete the reaction. The solvent and excess hydrogen chloride were distilled off under reduced pressure to give 0.5g of a colorless oily liquid which was used directly in the next reaction.

Example 11: preparation of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide

82mg (0.8mmol) of isovaleric acid and 10mL of dichloromethane were put into a 100mL single-neck reaction flask, and 0.42g of HATU (1.1mmol) and 0.18g of DIEA (1.4mmol) were added thereto, followed by stirring at room temperature for 30 minutes to obtain a pale yellow clear liquid.

Taking 0.5g of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5-amino-6-ketone-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -formamide, dissolving in 10mL of dichloromethane, adding into the system to obtain a yellow-green solution, stirring at room temperature for 3 hours, detecting by TLC, and completing the reaction. The solvent was distilled off under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 370mg of a pale yellow solid in 76% yield.

Example 12 preparation of (5R,10aR)3- ((R) -2-methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide) (I-11)

200mg (0.29mmol) of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -formamide and 50mL of methanol were dissolved in a 100mL single-neck reaction flask to obtain a colorless clear solution, 20mg (10%) of Pd/C was added, and the mixture was stirred at room temperature for 12 hours under a hydrogen atmosphere and then subjected to TLC to complete the reaction. Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 15:1) to give 138mg of a pale yellow solid in 85% yield.

¹H NMR(400MHz,d₆-DMSO):δ＝8.79-8.82(m,1H),7.90-8.10(m,1H),7.22-7.36(m,8H),7.13-7.15(m,2H),6.03-6.06(m,1H),4.75-4.90(m,1H),3.92-4.00(m,1H),3.65-3.80(m,1H),3.52(s,1H),3.40-3.43(m,2H),2.90-3.20(m,3H),2.10-2.30(m,5H),1.60-2.00(m,5H),1.24(s,2H),1.05-1.15(m,3H),0.84-0.89(m,6H)，[M+H]⁺:m/z＝562.3

Example 13: preparation of methyl (5R,10aR) 3-benzyloxycarbonyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate

5g (14.7mmol) of methyl (5R,10aR)5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate, 3.1g (36.8mmol) of sodium bicarbonate and 30mL of tetrahydrofuran were taken in a 100mL single-neck reaction flask, 30mL of water was added, cooling was carried out to 5 ℃ and 3.0g (17.6mmol) of Cbz-Cl was taken and dissolved in 15mL of tetrahydrofuran, and added dropwise thereto, after completion of the dropwise addition, stirring was carried out at room temperature for 6 hours and the reaction was checked by TLC to be complete. Filtration was carried out, the tetrahydrofuran was distilled off from the filtrate under reduced pressure, ethyl acetate (3 × 100mL) was extracted, a saturated sodium chloride solution was washed, the organic layer was dried over sodium sulfate, filtration was carried out, the solvent was distilled off under reduced pressure, and column chromatography of the residue (eluent dichloromethane: methanol ═ 20:1) was carried out to obtain 6.4g of a white solid in 92% yield.

Example 14: preparation of methyl (5R,10aR) 3-benzyloxycarbonyl-5-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate

6g (12.6mmol) (5R,10aR) 3-benzyloxycarbonyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylic acid methyl ester and 30mL of absolute ethanol were taken in a 100mL single-neck reaction flask, 30mL of hydrochloric acid ethanol was added, and the mixture was stirred at room temperature for 2 hours, checked by TLC and reacted completely. The solvent and excess hydrogen chloride were distilled off under reduced pressure to give 5.2g of a colorless oily liquid which was used directly in the next reaction.

Example 15: preparation of methyl (5R,10aR) 3-benzyloxycarbonyl-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate

2.8g (13.9mmol) of N-Boc-N-methyl-D-alanine and 60mL of dichloromethane were put in a 250mL single-neck reaction flask, and 7.2g of HATU (18.9mmol) and 3.3g of DIEA (25.2mmol) were added thereto, followed by stirring at room temperature for 1 hour to obtain a yellow clear solution.

5.2g of (5R,10aR) 3-benzyloxycarbonyl-5-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylic acid methyl ester was dissolved in 20mL of dichloromethane, and the solution was added dropwise to the system, stirred at room temperature for 6 hours, and the reaction was detected by TLC to completion. The solvent was distilled off under reduced pressure, the residue was dissolved in 300mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 5.1g of a reddish brown oil in a yield of 72%.

Example 16: preparation of (5R,10aR) 3-benzyloxycarbonyl-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylic acid

2g (3.6mmol) of methyl (5R,10aR) 3-benzyloxycarbonyl-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate and 60mL of methanol were taken in a 250mL single-neck reaction flask, 0.4g (7.2mmol) of potassium hydroxide and 60mL of water were added, the mixture was heated to 50 ℃ and stirred for 3 hours, and the reaction was checked by TLC for completion. 60mL of water was added, methanol was distilled off under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2X 100mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, ethyl acetate (3X 100mL) was extracted, the organic layer was dried over sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to give 1.9g of oil which was used directly in the next reaction.

Example 17: preparation of (5R,10aR) 3-benzyloxycarbonyl-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide

1.8g (3.3mmol) of (5R,10aR) 3-benzyloxycarbonyl-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazocine ring-10 a-carboxylic acid and 2.6g of PyBop (5.0mmol) were taken in a 100mL single-neck reaction flask, 50mL of DMF was added, and 0.85g of DIEA (6.6mmol) was further taken and stirred at room temperature for 30 minutes. 1.2g (6.6mmol) of benzhydrylamine is added into the system, the temperature is heated to 90 ℃, the stirring is carried out for 12 hours, and the TLC detection reaction is complete. The reaction solution was poured into 300mL of water, extracted with ethyl acetate (3 × 100mL), washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 15:1) to give 1.6g of a pale yellow solid in a yield of 72%.

Example 18: preparation of (5R,10aR) 3-amino-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide

Taking 0.5g (0.70mmol) of (5R,10aR) 3-benzyloxycarbonyl-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -formamide and 30mL of methanol in a 100mL single-mouth reaction bottle, dissolving to obtain a colorless clear solution, adding 50mg (10%) of Pd/C, stirring at room temperature for 12 hours under a hydrogen atmosphere, and detecting the reaction completion by TLC. Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 15:1) to give 344mg of a pale yellow solid in a yield of 85%.

Example 19: preparation of (5R,10aR)3- (isovaleryl) -amino-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide

To a 100mL single-neck reaction flask containing 72mg (0.7mmol) of isovaleric acid and 10mL of methylene chloride were added 342mg of HATU (0.9mmol) and 154mg of DIEA (1.2mmol), and the mixture was stirred at room temperature for 30 minutes to obtain a pale yellow clear solution.

344mg (0.6mmol) of (5R,10aR) 3-amino-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazocine ring-10 a- (N-benzhydryl) -formamide is dissolved in 10mL dichloromethane and added to the system to obtain a yellow-green solution, the solution is stirred at room temperature for 3 hours, TLC detection is carried out, and the reaction is completed. The solvent was distilled off under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 277mg of a white solid in 70% yield.

Example 20: preparation of (5R,10aR)3- (isovaleryl) -amino-5- ((R) -2-methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide) (I-12)

277mg (0.42mmol) of (5R,10aR)3- (isovaleryl) -amino-5- ((R) -2- (tert-butoxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-benzhydryl) -carboxamide and 10mL of absolute ethanol were taken in a 100mL single-neck reaction flask, 10mL of hydrochloric acid ethanol was added, stirring was carried out at room temperature for 2 hours, TLC detection was carried out, and the reaction was completed. The solvent and excess hydrogen chloride were distilled off under reduced pressure to give a colorless oily liquid, 100mL of ethyl acetate was added, the pH was adjusted to 7-8 with a saturated sodium bicarbonate solution, the layers were separated, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 15:1) to give 160mg of a white solid in a yield of 68%.

¹H NMR(400MHz,d₆-DMSO):δ＝8.82-8.79(m,1H),8.10-7.90(m,1H),7.36-7.22(m,8H),7.15-7.13(m,2H),6.06-6.03(m,1H),4.90-4.75(m,1H),4.00-3.92(m,1H),3.80-3.65(m,1H),3.52(s,1H),3.43-3.40(m,2H),3.20-2.90(m,3H),2.30-2.10(m,5H),2.00-1.60(m,5H),1.24(s,2H),1.15-1.05(m,3H),0.89-0.84(m,6H)，[M+H]⁺:m/z＝562.3

Example 21: preparation of methyl (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate

6g (10.7mmol) of methyl (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butoxycarbonyl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate and 30mL of absolute ethanol were taken in a 100mL single-neck reaction flask, 30mL of ethanol hydrochloride was added, the mixture was stirred at room temperature for 2 hours, and the reaction was completed by TLC detection. The solvent and excess hydrogen chloride were distilled off under reduced pressure to give 5.2g of a colorless oily liquid which was used directly in the next reaction.

Example 22: preparation of methyl (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate

A single-neck reaction flask of 100mL was charged with 1.2g (11.8mmol) of isovaleric acid and 20mL of dichloromethane, and then 6.1g of HATU (16.1mmol) and 2.8g of DIEA (21.4mmol) were added thereto, followed by stirring at room temperature for 30 minutes to obtain a yellow clear solution.

5.2g of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5-amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-methyl formate is dissolved in 20mL of dichloromethane and added to the system to obtain a yellow-green solution, the solution is stirred for 3 hours at room temperature, TLC detection is carried out, and the reaction is completed. The solvent was distilled off under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 4.7g of a white solid in a yield of 81%.

Example 23: preparation of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylic acid

470mg (0.86mmol) of methyl (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a-carboxylate and 20mL of methanol were taken in a 100mL single-neck reaction flask, 150mg of potassium hydroxide (2.59mmol) and 20mL of water were added, the mixture was heated to 50 ℃ and stirred for 3 hours, and the reaction was checked by TLC for completion. 20mL of water was added, methanol was distilled off under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2X 50mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, ethyl acetate (3X 60mL) was extracted, the organic layer was dried over sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to give 460mg of oil which was used directly in the next reaction.

Example 24: preparation of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-2-phenyl-benzyl) -carboxamide

460mg of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazocine ring-10 a-carboxylic acid and 670mg of PyBop (1.29mmol) were taken in a 100mL single-neck reaction flask, 20mLDMF was added, and 221mg of DIEA (1.72mmol) was further taken and stirred at room temperature for 30 minutes. 173mg (0.95mmol) of 2-benzyl aniline is added into the system, heated to 90 ℃, stirred for 12 hours, and the TLC detection reaction is complete. The reaction solution was poured into 300mL of water, extracted with ethyl acetate (3 × 100mL), washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 15:1) to give 358mg of a white solid with a yield of 60%.

Example 25: preparation of (5R,10aR)3- ((R) -2-methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-2-phenyl-benzyl) -carboxamide) (I-02)

358g (0.52mmol) of (5R,10aR)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrrolo [1,2-a ] [1,5] diazacyclo-10 a- (N-2-phenyl-benzyl) -carboxamide and 20mL of methanol were dissolved in a 100mL single-neck reaction flask to give a colorless clear solution, 36mg (10%) of Pd/C was added, and the mixture was stirred at room temperature for 12 hours under a hydrogen atmosphere and then subjected to TLC to complete the reaction. Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 15:1) to give 256mg of a pale yellow solid in 88% yield.

¹H NMR(400MHz,d₆-DMSO):δ＝8.43-8.42(m,1H),7.97-7.95(m,1H),7.44-7.41(m,2H),7.38-7.29(m,6H),7.19-7.18(m,1H),4.80-4.78(m,1H),4.60-4.54(m,1H),3.96-3.91(m,2H),2.30-2.02(m,6H),2.09-1.74(m,6H),1.72-1.62(m,1H),1.05-1.03(d,2H),0.98-0.96(m,3H),0.83-0.78(m,6H)，[M+H]⁺:m/z＝562.3

Example 26

Example 27

H-NMR(400MHz,d₆-DMSO):δ＝8.83-8.81(d,1H),7.88-7.86(d,1H),7.37-7.24(m,8H),7.16-7.10(m,2H),6.06-6.04(d,1H),4.99-4.95(m,1H),3.95-3.92(m,2H),3.73-3.70(m,1H),3.59-3.40(m,3H),2.42-2.21(m,6H),2.05-1.92(m,3H),1.79-1.73(m,2H),1.34-1.18(m,6H),0.89-0.85(m,6H).[M+H]⁺:m/z＝562.3

Example 28

¹H NMR(400MHz,d₆-DMSO):δ＝8.57(m,1H),8.03-7.95(m,1H),7.31-7.27(m,2H),7.22-7.21(m,3H),4.93-4.79(m,1H),4.65-4.52(m,1H),4.05-3.93(m,10H),2.89(s,2H),2.73(s,2H),2.07(s,2H),2.02-1.98(s,12H),1.19-1.15(s,3H),1.00-0.98(m,2H),0.90-0.88(m,6H)，[M+H]⁺:m/z＝486.3

Example 29

Example 30

H-NMR(400MHz,d₆-DMSO):δ＝8.77-8.75(d,1H),7.94-7.92(d,1H),7.37-7.34(m,2H),7.19-7.15(m,4H),7.11-7.06(m,2H),6.08-6.06(d,1H),4.94-4.92(m,1H),3.99-3.92(m,2H),3.72-3.52(m,1H),3.48-3.41(m,3H),2.50-2.41(m,1H),2.18-2.13(m,6H),1.95-1.92(m,4H),1.25(s,2H),1.05-0.98(m,3H),0.87-0.84(m,6H).[M+H]⁺:m/z＝598.4

Example 31

H-NMR(400MHz,d₆-DMSO):δ＝8.80-8.78(d,1H),7.90-7.88(d,1H),7.34-7.32(m,4H),7.27-7.23(m,4H),7.11-7.09(m,2H),6.03-6.01(d,1H),4.92-5.05(m,1H),3.98-3.92(m,1H),3.86-3.68(m,1H),3.52-3.40(m,1H),3.38-3.10(m,8H),2.42-2.21(m,2H),2.15-2.02(m,2H),1.79-1.73(m,2H),1.42-1.38(m,1H),1.34-1.18(m,28H),1.12-0.95(m,1H),0.89-0.85(m,3H).[M+H]⁺:m/z＝716.6

Example 32

H-NMR(400MHz,d₆-DMSO):δ＝8.82-8.80(d,1H),7.98-7.95(d,1H),7.33-7.30(m,4H),7.29-7.21(m,9H),7.18-7.13(m,1H),6.08-6.06(d,1H),4.99-4.96(m,1H),3.98-3.95(m,1H),3.79-3.75(m,1H),3.26-3.24(m,2H),3.21-3.12(m,2H),2.89-2.80(s,1H),2.79-2.70(m,3H),2.60-2.51(m,1H),2.32-2.26(m,2H),2.19-2.15(s,3H),1.86-1.68(m,2H),1.36-1.30(m,1H),1.26-1.24(m,1H),1.07-0.98(m,4H).[M+H]⁺:m/z＝610.4

Example 33

¹H-NMR(400MHz,d₆-DMSO):δ＝8.83-8.81(d,1H),8.17-8.15(m,1H),7.58-6.85(m,13H),6.07-6.05(d,1H),4.94-4.93(d,1H),4.16-4.14(m,2H),4.08-4.01(m,2H),3.95-3.92(d,3H),2.77-2.75(m,2H),2.39-1.854(m,10H),1.07-1.02(m,3H).[M+H]+:m/z＝600.2

Example 34

H-NMR(400MHz,d₆-DMSO):δ＝8.89-8.86(d,1H),8.84-8.80(d,1H),7.38-7.18(m,8H),7.14-7.10(m,2H),6.06-6.04(d,1H),5.00-4.95(m,1H),4.10-3.75(m,2H),3.08-3.00(s,3H),2.52-2.10(m,6H),2.09-2.07(m,3H),1.11-1.38(m,3H),0.98-0.92(m,9H).[M+H]⁺:m/z＝562.3

Example 35

H-NMR(400MHz,d₆-DMSO):δ＝8.78-8.76(d,1H),8.03-8.02(d,1H),7.37-7.24(m,8H),7.16-7.10(m,2H),6.06-6.04(d,1H),4.99-4.95(m,1H),3.95-3.92(m,1H),3.73-3.70(m,3H),3.59-3.40(m,2H),2.42-2.21(m,6H),2.05-1.92(m,2H),1.79-1.73(m,2H),1.34-1.18(m,3H),0.89-0.85(m,3H).[M+H]⁺:m/z＝590.4

Example 36

¹H-NMR(400MHz,d6-DMSO):δ＝8.31-8.29(m,2H),8.02-8.00(m,1H),7.92-7.88(m,1H),7.29-7.18(m,10H),4.79-4.93(m,4H),4.52-4.65(m,1H),3.93-4.05(m,10H),2.89(s,2H),2.73(s,2H),2.07(s,2H),1.98-2.02(s,12H),1.15-1.19(s,3H),0.98-1.00(m,2H),0.88-0.90(m,6H)，[M+H]+:m/z＝500.4

Example 37

¹HNMR(400MHz,d₆-DMSO):δ＝8.70-9.00(m,1H),8.20-8.50(m,1H),7.18-7.40(m,5H),4.80-5.05(m,2H),3.80-4.20(m,2H),3.40-3.70(m,2H),2.98-3.18(m,1H),2.60-2.80(m,1H),2.30-2.40(m,1H),1.70-2.20(m,10H),1.30-1.60(m,7H),1.10-1.30(s,6H),0.85(s,6H)，[M+H]⁺:m/z＝500.4

Example 38

¹H NMR(400MHz,d₆-DMSO):δ＝9.58(s,1H),8.95(s,1H),8.47(s,1H),8.10-8.02(m,1H),7.78-7.68(m,2H),7.50(s,1H),7.44(s,1H),7.36-7.22(m,10H),7.15-7.13(m,2H),6.42-6.38(s,1H),6.10-6.01(s,1H),4.75-4.90(s,1H),4.11-3.92(m,8H),3.82-3.75(s,1H),3.72-3.58(s,1H),3.15-2.92(m,1H),2.42-2.38(m,1H),2.10-2.00(m,6H),1.96-1.89(s,2H),1.78-1.62(s,1H),1.60-1.53(s,1H),1.51-1.48(s,1H),1.32-1.18(m,6H),1.15-1.05(m,3H),0.86-0.75(m,2H)，[M+H]⁺:m/z＝868.2

Example 39: preparation of ethyl 4-p-toluenesulfonyloxybutyrate

50g (0.38mol) of ethyl 4-hydroxybutyrate and 300mL of dichloromethane are put into a 1L single-mouth reaction bottle, 76.8g (0.76mol) of triethylamine is added, the mixture is cooled to 5 ℃, 86.6g (0.46mol) of p-toluenesulfonyl chloride is dissolved in 200mL of dichloromethane and added into the system dropwise, after the dropwise addition is finished, the mixture is stirred for 6 hours at room temperature, TLC detection is carried out, and the reaction is completed. The resulting mixture was washed with 300mL of water, diluted hydrochloric acid (1M), saturated brine and dried over sodium sulfate, filtered and the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent ethyl acetate: petroleum ether: 1:20) to give 100g of a colorless oily liquid in 92% yield.

Example 40: preparation of ethyl 2- (tert-butoxycarbonyl) hydrazinobutyrate

50g (0.17mol) of ethyl 4-tosyloxybutyrate, 67g (0.51mol) of tert-butyloxycarbonyl hydrazine and 600ml of DMF are taken in a 1L single-mouth reaction bottle, heated to 90 ℃, stirred for 6 hours, detected by TLC and reacted completely. 2L of water was added, extracted with ethyl acetate (3X 300mL), washed with saturated brine, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent was ethyl acetate: petroleum ether: 1:10) to give 25g of a colorless oily liquid with a yield of 60%.

Example 41: preparation of 1-allyl-2- (tert-butoxycarbonyl) hydrazinobutyric acid ethyl ester

Taking 25g (0.10mol) of ethyl 2- (tert-butoxycarbonyl) hydrazinobutyrate and 300mL of DMMF (lithium bis-formamide) in a 500mL single-neck reaction bottle, adding 27.6g (0.20mol) of potassium carbonate, stirring at room temperature, adding 14.5g (0.12mol) of allyl bromide, heating to 90 ℃, stirring for 12 hours, detecting by TLC (thin layer chromatography), and completing the reaction. 1L of water was added, extracted with ethyl acetate (3X 300mL), washed with saturated brine, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent was ethyl acetate: petroleum ether: 1:15) to give 21.5g of a colorless oily liquid with a yield of 75%.

Example 42: preparation of 1-allyl-2- (tert-butoxycarbonyl) hydrazino-2-bromo-ethyl butyrate

Taking 20g (70mmol) of 1-allyl-2- (tert-butoxycarbonyl) ethyl hydrazinobutyrate and 200mL of predried tetrahydrofuran in a 500mL three-mouth reaction bottle, cooling to-60 ℃ under the protection of nitrogen, taking 140mL (1M in THF,0.14mol) of lithium diisopropylamide, dropwise adding the lithium diisopropylamide into the reaction bottle, stirring for 2 hours at the temperature of-60 ℃, cooling to-78 ℃, further taking 46g (0.14mol) of carbon tetrabromide, dissolving in 100mL of tetrahydrofuran, dropwise adding the mixture into the system, stirring for 2 hours at the temperature of-78 ℃, detecting by TLC, and completing the reaction. The reaction was quenched by addition of 20mL of a saturated ammonium chloride solution, washed with dilute hydrochloric acid (1M), washed with saturated brine, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent was ethyl acetate: petroleum ether: 1:15) to give 20.7g of a colorless oily liquid in a yield of 81%.

Example 43: preparation of 1-allyl-hydrazino-2-bromo-ethyl butyrate

20g (54.8mmol) of 1-allyl-2- (tert-butoxycarbonyl) hydrazino-2-bromo-ethyl butyrate and 100mL of absolute ethanol were put into a 500mL single-neck reaction flask, 100mL of ethanol hydrochloride was added, the mixture was stirred at room temperature for 2 hours, and the reaction was completed by TLC detection. The solvent and excess hydrogen chloride were distilled off under reduced pressure to give 16.2g of a colorless oily liquid which was used directly in the next reaction.

Example 44: preparation of 1-allyl-pyrazole-3-carboxylic acid ethyl ester

Taking 16.2g of 1-allyl-hydrazino-2-bromo-ethyl butyrate and 200mL of DMMF (lithium bis-bromoacetate) into a 500mL single-mouth reaction bottle, adding 18.9g (0.14mol) of potassium carbonate, heating to 90 ℃, stirring for 12 hours, detecting by TLC (thin layer chromatography), and completing the reaction. 600mL of water was added, extracted with ethyl acetate (3X 300mL), washed with saturated brine, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether: 1:10) to give 6.3g of a white solid in 63% yield.

Example 45: preparation of ethyl 1-allyl-2- ((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino) propionyl) -pyrazole-3-carboxylate

To a 500mL one-necked reaction flask were added 13.9g (41mmol) of (S) -2- (Boc-amino) -3- (Cbz-amino) propionic acid and 200mL of methylene chloride, 20.8g of HATU (54.7mmol) and 8.8g of DIEA (68.4mmol) and the mixture was stirred at room temperature for 1 hour to obtain a yellow clear solution.

6.3g (34.2mmol) of ethyl 1-allyl-pyrazole-3-carboxylate is dissolved in 100mL of dichloromethane, and is dripped into the yellow clear liquid, and the mixture is stirred for 6 hours at room temperature to obtain a reddish brown solution, and the reaction is detected completely by TLC. The solvent was distilled off under reduced pressure, the residue was dissolved in 400mL of ethyl acetate, washed with 1M HCl solution, washed with saturated sodium bicarbonate solution, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether: 1:2) to give 14.6g of a white solid in 85% yield.

Example 46: preparation of ethyl 1-glyoxyl-2- ((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino) propionyl) -pyrazole-3-carboxylate

14.6g (29mmol) of ethyl 1-allyl-2- ((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino) propionyl) -pyrazole-3-carboxylate and 200mL of methylene chloride were taken in a 500mL three-necked reaction flask, cooled to-78 ℃, and ozone was introduced for about 30 minutes until the system became blue, and the mixture was stirred at-78 ℃ for 30 minutes, and then the introduction of ozone was stopped. And introducing nitrogen to bubble so as to remove excessive ozone until blue disappears, obtaining colorless solution, adding 100mL of triethylamine, returning to room temperature, stirring for 2 hours, and detecting the reaction completion by TLC. Washing with 1M citric acid, washing with saturated aqueous sodium chloride solution, drying the organic layer with sodium sulfate, filtering, evaporating the solvent under reduced pressure, and purifying the residue by column chromatography (eluent ethyl acetate: petroleum ether: 1:2) to obtain 11.5g of a white solid with a yield of 78%.

Example 47: preparation of methyl (5S,8S)5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8-carboxylate

11.5g (22.7mmol) of 1-carboxaldehyde-2- ((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino) propionyl) -pyrazole-3-carboxylic acid ethyl ester and 200mL of methanol were dissolved in a 500mL single-neck reaction flask to obtain a colorless clear solution, 1.2g (10%) of Pd/C was added, and the mixture was stirred at room temperature for 12 hours under a hydrogen atmosphere and was subjected to TLC to complete reaction. Pd/C was filtered off, the solvent was evaporated under reduced pressure, 200mL of tetrahydrofuran was added to dissolve it, 2.1g (34.1mmol) of sodium cyanoborohydride was added thereto, the mixture was stirred at room temperature, and the completion of the reaction was detected by TLC. The reaction was quenched with 20mL of water, filtered, the solvent was evaporated under reduced pressure, the residue was dissolved in 200mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 10:1) to give 6.2g of a white solid in 80% yield.

Example 48: preparation of methyl (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8-carboxylate

2.6g (10.9mmol) of N-Cbz-N-methyl-D-alanine and 80mL of methylene chloride were put in a 250mL single-neck reaction flask, 5.2g of HATU (13.6mmol) and 2.4g of DIEA (18.1mmol) were added, and the mixture was stirred at room temperature for 1 hour to obtain a yellow clear solution.

3.1g (9.1mmol) of methyl (5S,8S) -5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8-carboxylate is dissolved in 50mL of dichloromethane and added dropwise to the system, and the mixture is stirred at room temperature for 6 hours and the reaction is detected to be complete by TLC. The solvent was distilled off under reduced pressure, the residue was dissolved in 300mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 3.7g of a brown oil in a yield of 72%.

Example 49: preparation of (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclono-8-carboxylic acid

1g (1.8mmol) of methyl (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclonidine-8-carboxylate and 30mL of methanol were taken in a 250mL single-neck reaction flask, and 0.3g (5.4mmol) of potassium hydroxide and 30mL of water were added, the mixture was heated to 50 ℃ and stirred for 3 hours, and the reaction was detected to be complete by TLC. 60mL of water was added, methanol was distilled off under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2X 100mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, ethyl acetate (3X 100mL) was extracted, the organic layer was dried over sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to give 0.98g of oil which was used directly in the next reaction.

Example 50: preparation of (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -carboxamide

0.98g (1.8mmol) of (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8-carboxylic acid and 1.4g of PyBop (2.7mmol) were put in a 100mL single-neck reaction flask, 50mL of DMF was added thereto, 0.46g of DIEA (3.6mmol) was further added, and the mixture was stirred at room temperature for 30 minutes. 0.66g (3.6mmol) of benzhydrylamine is added into the system, the temperature is heated to 90 ℃, the stirring is carried out for 12 hours, and the TLC detection reaction is complete. The reaction solution was poured into 300mL of water, extracted with ethyl acetate (3 × 100mL), washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 15:1) to give 0.77g of a pale yellow solid with a yield of 60%.

Example 51: preparation of (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5-amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -carboxamide

0.5g (0.7mmol) of (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -carboxamide and 10mL of absolute ethanol are taken to be put into a 100mL single-neck reaction flask, 20mL of hydrochloric ethanol is added, the mixture is stirred for 2 hours at room temperature, TLC detection is carried out, and the reaction is completed. The solvent and excess hydrogen chloride were distilled off under reduced pressure to give 0.45g of a colorless oily liquid which was used directly in the next reaction.

Example 52: preparation of (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclono-8- (N-benzhydryl) -carboxamide

Dissolving 0.45g of (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5-amino-6-ketone-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -formamide in 10mL of dichloromethane, adding the dichloromethane into the system to obtain a yellow-green solution, stirring at room temperature for 3 hours, detecting by TLC, and completely reacting. The solvent was distilled off under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 345mg of a white solid with a yield of 71%.

Example 53: preparation of (5S,8S)3- ((R) -2-methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -carboxamide (I-13)

Taking 200mg (0.29mmol) of (5S,8S)3- ((R) -2- (benzyloxycarbonyl) methylamino) -propionyl-5- (isovaleryl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclonidine-8- (N-benzhydryl) -formamide and 50mL of methanol in a 100mL single-neck reaction flask, dissolving to obtain a colorless clear solution, adding 20mg (10%) of Pd/C, stirring at room temperature for 12 hours under a hydrogen atmosphere, and detecting by TLC to complete the reaction. Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 15:1) to give 138mg of a white solid in 85% yield.

¹H NMR(400MHz,d₆-DMSO):δ＝8.03-8.00(m,1H),7.49-7.32(m,2H),7.30-7.22(m,4H),7.19-7.10(m,4H),6.06-6.03(m,1H),5.05-4.99(m,1H),4.45-4.39(m,1H),3.80-3.62(m,1H),3.52(s,1H),3.43-3.40(m,2H),3.20-2.90(m,3H),2.78-2.47(m,2H),2.30-2.10(m,5H),2.00-1.60(m,5H),1.18-1.05(m,3H),0.89-0.82(m,6H)，[M+H]⁺:m/z＝563.3

Example 54: preparation of methyl (5S,8S)3- (isovaleryl) -5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8-carboxylate

To a 100mL single-neck reaction flask were added 1.1g (11mmol) of isovaleric acid and 30mL of dichloromethane, 5.7g of HATU (15mmol) and 2.6g of DIEA (20mmol) and the mixture was stirred at room temperature for 30 minutes to obtain a pale yellow clear solution.

3.4g (10mmol) of (5S,8S)5- (tert-butyloxycarbonyl) -amino-6-ketone-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclonidine-8-carboxylic acid methyl ester is dissolved in 30mL of dichloromethane and added into the system to obtain a yellow-green solution, the solution is stirred for 3 hours at room temperature, and the reaction is completed by TLC detection. The solvent was distilled off under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 2.8g of a white solid in 66% yield.

Example 55: preparation of (5S,8S)3- (isovaleryl) -5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8-carboxylic acid

1g (2.3mmol) of (5S,8S) methyl 3- (isovaleryl) -5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8-carboxylate and 30mL of methanol were taken in a 100mL single-neck reaction flask, and 0.3g (5.9mmol) of potassium hydroxide and 30mL of water were added, heated to 50 ℃, stirred for 3 hours and the reaction was detected to be complete by TLC. 30mL of water was added, methanol was distilled off under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2X 100mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, ethyl acetate (3X 100mL) was extracted, the organic layer was dried over sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to give 0.95g of oil which was used directly in the next reaction.

Example 56: preparation of (5S,8S)3- (isovaleryl) -5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -carboxamide

0.95g (2.3mmol) of (5S,8S)3- (isovaleryl) -5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclonidine-8-carboxylic acid and 1.8g of PyBop (3.6mmol) were put in a 100mL single-neck reaction flask, and 50mL of DMF was added thereto, and 0.60g of DIEA (4.6mmol) was taken and stirred at room temperature for 30 minutes. 0.84g (4.6mmol) of benzhydrylamine is added into the system, the temperature is heated to 90 ℃, the stirring is carried out for 12 hours, and the TLC detection reaction is complete. The reaction solution was poured into 300mL of water, extracted with ethyl acetate (3 × 100mL), washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 15:1) to give 0.70g of a pale yellow solid in 53% yield.

Example 57: preparation of (5S,8S)3- (isovaleryl) -5-amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -carboxamide

200mg (0.35mmol) of (5S,8S)3- (isovaleryl) -5- (tert-butyloxycarbonyl) -amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -formamide and 10mL of absolute ethanol are taken in a 100mL single-neck reaction flask, 20mL of ethanol hydrochloride is added, the mixture is stirred for 2 hours at room temperature, and TLC detection is carried out to complete the reaction. The solvent and excess hydrogen chloride were distilled off under reduced pressure to give 0.17g of a colorless oily liquid which was used directly in the next reaction.

Example 58: preparation of (5S,8S)3- (isovaleryl) -5- ((S) -2- (benzyloxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -carboxamide

99mg (0.42mmol) of N-Cbz-N-methyl-D-alanine and 20mL of methylene chloride were put in a 100mL single-neck reaction flask, and 0.24g of HATU (0.63mmol) and 0.12g of DIEA (0.88mmol) were added thereto, followed by stirring at room temperature for 1 hour to obtain a yellow clear solution.

Dissolving 0.17g of (5S,8S)3- (isovaleryl) -5-amino-6-ketone-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -formamide in 20mL of dichloromethane, dropwise adding the dichloromethane into the system, stirring at room temperature for 6 hours, and detecting the reaction by TLC. The solvent was distilled off under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, washed with a saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol ═ 20:1) to give 160mg of a brown oil in 66% yield.

Example 59: preparation of (5S,8S)3- (isovaleryl) -5- ((S) -2-methylamino) -propionyl-amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclon-8- (N-benzhydryl) -carboxamide (I-14)

Taking 160mg (0.23mmol) of (5S,8S)3- (isovaleryl) -5- ((S) -2- (benzyloxycarbonyl) methylamino) -propionyl-amino-6-one-dehydropyrazolo [1,2-a ] [1,2, 5] triazacyclonidine-8- (N-benzhydryl) -formamide and 50mL of methanol in a 100mL single-neck reaction flask, dissolving to obtain a colorless clear solution, adding 16mg (10%) of Pd/C, stirring at room temperature for 12 hours under a hydrogen atmosphere, and detecting by TLC to complete the reaction. Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent dichloromethane: methanol 15:1) to give 106mg of a white solid in 82% yield.

¹H NMR(400MHz,d₆-DMSO):δ＝8.05-8.02(m,1H),7.47-7.29(m,2H),7.31-7.12(m,4H),7.18-7.08(m,4H),6.06-6.03(m,1H),5.00-4.93(m,1H),4.42-4.39(m,1H),3.92-3.73(m,1H),3.54(s,1H),3.41-3.39(m,2H),3.22-2.88(m,3H),2.70-2.33(m,2H),2.21-2.12(m,5H),2.05-1.78(m,5H),1.28-1.15(m,3H),0.92-0.85(m,6H)，[M+H]⁺:m/z＝563.3

Example 60:

example 61:

example 62:

¹HNMR(400MHz,d₆-DMSO):δ＝8.04-8.01(m,1H),7.52-7.36(m,1H),7.27-7.21(m,2H),7.16-7.05(m,2H),6.05-6.01(m,1H),5.04-4.97(m,1H),4.52-4.43(m,1H),4.00-3.92(m,3H),3.84-3.67(m,1H),3.58(s,1H),3.45-3.41(m,2H),3.27-2.93(m,3H),2.74-2.53(m,2H),2.35-2.18(m,5H),2.12-1.68(m,5H),1.21-1.09(m,3H),0.93-0.88(m,6H)，[M+H]⁺:m/z＝563.3

example 63:

¹H-NMR(400MHz,d₆-DMSO):δ＝8.12-8.05(m,1H),7.41-7.39(m,2H),7.32-7.21(m,6H),7.13-7.06(m,1H),6.11-6.07(m,1H),5.07-4.97(m,1H),4.46-4.37(m,1H),3.81-3.64(m,1H),3.55(s,1H),3.46-3.41(m,2H),3.25-2.96(m,3H),2.73-2.49(m,2H),2.33-2.17(m,5H),2.06-1.68(m,5H),1.12-1.09(m,3H),0.92-0.88(m,6H)，[M+H]⁺:m/z＝563.6

example 64:

¹H NMR(400MHz,d₆-DMSO):δ＝8.08-8.02(m,1H),7.44-7.38(m,1H),7.32-7.26(m,2H),7.19-7.15(m,4H),7.11-7.06(m,2H),6.06-6.03(m,1H),5.06-4.97(m,1H),4.44-4.35(m,1H),3.82-3.61(m,1H),3.58(s,1H),3.47-3.42(m,2H),3.23-2.96(m,3H),2.73-2.41(m,2H),2.38-2.12(m,5H),2.07-1.66(m,5H),1.11-1.08(m,3H),0.95-0.86(m,6H)，[M+H]⁺:m/z＝599.4

example 65: binding affinity assay for Compounds to XIAP, cIAP1, cIAP2

To test the binding affinity of the compounds of the invention to XIAP, cIAP1, cIAP2 proteins, assays were performed using a Fluorescence Polarization (FP) -based competitive assay, for cIAP1-BIR3, cIAP2-BIR3 assays, using a fluorescently labeled Smac mimetic (Smac-2F) as a fluorescent probe. K of Smac-2F with cIAP1-BIR3, cIAP2-BIR3_dValues were determined by monitoring the total fluorescence polarization of a mixture made up with solid concentrations of fluorescent probe and increasing concentrations of protein up to full saturation. Fluorescence polarization was measured in a Microfluor 296-well black round bottom plate (Fisher Scientific) using an Infinite M-1000 microplate reader (Tecan U.S., Research Triangle Park, NC). In 96-wells, 1nM Smac-2F and increasing concentrations of protein were added to assay buffer (100mM potassium phosphate, PH 7.5; 100ug/ml bovine gamma globulin; 0.02% sodium azide, Invitrogen) in a final volume of 125 ul. The plates were mixed on a shaker for 15 minutes and incubated at room temperature for 1-3 hours. The polarization values were measured in millipolarization units (millipolarization unit) at an excitation wavelength of 485nm and an emission wavelength of 530 nm. Equilibrium dissociation constants (K) were then calculated by fitting the sigmoidal dose-dependent FP increase as a function of protein concentration using GraphPad prism5.0 software_d)

K of the compound_iValues were determined by breath-and-me dose-dependent competitive binding experiments in which serial dilutions of the compound competed with a fixed concentration of fluorescent probe for binding to a fixed concentration of protein (typically 2-3 fold higher than the Kd value determined above). 5ul of test compound in DMSO was mixed with 120ul in assay buffer (100mM potassium phosphate, pH 7.5; 100 ug/m)l bovine gamma globulin; pre-incubated protein/tracer complex mixture in 0.02% sodium azide, Invitrogen) was added to the assay plate and incubated for 1-3 hours at room temperature. The final concentrations of protein and probe were 3nm and 1nm, 5nm and 1nm for the cIAP1-BIR3 and cIAP2-BIR3 assays, respectively. A negative control containing only protein/probe complexes (corresponding to 0% inhibition) and a positive control containing only free probes (corresponding to 100% inhibition) were included in each assay plate. EP values were measured as described above. IC50 values were determined by non-linear regression fitting of competition curves. Ki values for competitive inhibitors were calculated based on the measured IC50 values, Kd values of probes to different proteins, and concentrations of protein and probe in the purification assay using the deductive equation previously described.

The same procedure was used for the FP-based assay for the XIAP-BIR2-BIR3 protein. In this assay, a bivalent fluorescent-tagged peptide Smac mimetic (Smac-1F) was used as a fluorescent probe, which was determined similarly to the Kd value of XIAP-BIR2-BIR3 by saturation experiments. 0.01% Triton X-100 was added to the assay buffer to achieve fluorescence and polarization values for the stability of the dimeric fluorescent probe. The final protein and probe concentrations utilized in the competitive assay were 2nm and 1nm, respectively. The results of the compound testing of the present invention are shown in the following table.

The experimental result shows that the compound has better binding affinity with XIAP, cIAP1 and cIAP2 proteins and better IAP inhibition activity.

Example 67: cell growth inhibition assay in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines

The effect of the compounds of the invention on the growth of different cancer cell lines was tested. Cells were seeded at 3000 cells/well with test compounds on 96-well flat-bottomed cell culture plates and fine cells were platedCells were incubated at 37 ℃ in 95% air and 5% CO₂Cultured in the atmosphere of (3) for 4 days. The rate of inhibition of cell growth after treatment with different concentrations of compound was determined using the WST-8 kit and 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt. WST-8 was added to each well at a final concentration of 10%, and the plates were then incubated at 37 ℃ for 2-3 hours. The absorbance of the samples was measured at 450nm using an ULTRATecan reader (molecular device). The concentration of test compound that inhibits cell growth by 50% (IC) was calculated by comparing the absorbance in untreated cells and cells treated with test compound₅₀). The test results are shown in the following table.

The compound has better inhibition effect on cell growth in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that said compound has the following general formula:

C_1～20Alkyl, aryl, heteroaryl, and heteroaryl,

Or an alpha-amino acid residue, or a pharmaceutically acceptable salt thereof,

wherein the alpha-amino acid residue is

Or

The Ar group represents aryl or heteroaryl with the atomic number of 5-8, and m and n are independently selected from any integer of 1-3;

R₃is selected from substituted or unsubstituted benzyl, and the substituent is selected from phenyl, halogen and C_1～4Alkyl or p-fluorophenyl.

2. A compound or a pharmaceutically acceptable salt thereof, characterized in that said compound has the following general formula:

or

Wherein, in the formula I or II,

said R₁Is selected from C_1～20An alkylamino group,

C_1～20Alkyl, aryl, heteroaryl, and heteroaryl,

Or an alpha-amino acid residue,

R₂is selected from C_1～20Alkylamino radical, C_5～8Cycloalkylamino radical, C_1～20Alkyl, aryl, heteroaryl, and heteroaryl,

Or an alpha-amino acid residue,

wherein the alpha-amino acid residue is

Or

The Ar group represents aryl and heteroaryl with the atomic number of 5-8, m and n are independently selected from any integer of 1-3,

3. The compound of claim 2, wherein the alkylamino group in formula I or II is

4. The compound of claim 2, wherein C is as set forth in formula I or II, and pharmaceutically acceptable salts thereof_5～6Cycloalkylamino radical being

5. The compound of claim 2, wherein R in formula I or II is₂Is selected from C_1～13An alkyl group.

6. The compound of claim 2, wherein aryl in formula I or II is phenyl, and pharmaceutically acceptable salts thereof.

7. The compound of claim 2, wherein the heteroaryl group in formula I or II is imidazolyl, and pharmaceutically acceptable salts thereof.

8. A compound characterized by a structure selected from

And pharmaceutically acceptable salts thereof.

9. A pharmaceutical composition characterized by comprising as active ingredient a therapeutically effective amount of a compound as defined in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers, diluents and/or excipients.

10. Use of a compound as defined in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating a disease associated with IAP proteins.

11. Use of a compound as defined in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of all cancer types that fail to undergo apoptosis.