CN110734456A

CN110734456A - compounds, preparation method and medical application thereof

Info

Publication number: CN110734456A
Application number: CN201911076481.3A
Authority: CN
Inventors: 梅岩
Original assignee: Nanjing Rui Rui Biotechnology Co Ltd
Current assignee: Nanjing Rui Rui Biotechnology Co Ltd
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2020-01-31

Abstract

The invention provides compounds, a preparation method and medical application thereof, wherein the compounds have a structure shown in a general formula (I), and the compounds also have the structure shown in the general formula (I)Can be in the form of a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, of the compound. The compound with the structure of the general formula (I) provided by the invention can be used as an arginase inhibitor by selecting a specific main chain structure and a corresponding substituent thereof, has high activity and has potential treatment prospects in various diseases.

Description

compounds, preparation method and medical application thereof

Technical Field

The invention relates to the technical field of antitumor drug synthesis, in particular to compounds, a preparation method and medical application thereof.

Background

Because tumor immune pathways and mechanisms can only be regulated by micromolecular drugs, the application range of the tumor immunotherapy can be expanded by regulating immune-related targets by the micromolecular drugs in the tumor microenvironment, and opportunities are also found for the therapy combining tumor-targeted drugs and biological immunomodulators.

The regulation of MDSCs, DCs and TAMs can be mediated by indoleamine 2, 3-dioxygenase 1(IDO1), arginase 1(ARG1), inducible nitric oxide synthase (iNOS), phosphodiesterase-5 (PDE5), and the regulation of purinergic signal transduction can be mediated by ATP, CD39, CD73, adenosine, and elevated cAMP, thus, these targets are likely to be sites of action of small molecule drugs.

The action mechanism of the arginase inhibitor is to improve the proliferation of cytotoxic T cells and Natural Killer (NK) of an immune system by regulating a tumor microenvironment for steps, and exert the immunosuppressive effect of the tumor microenvironment to kill tumor cells, NO has various positive cardiovascular physiological effects, such as vasodilation, local blood flow regulation, vascular smooth muscle cell proliferation inhibition, platelet adhesion aggregation inhibition, thrombosis prevention and the like.

Studies have shown that arginase inhibitors can inhibit tumor growth in immunocompromised mice, with a rapid increase in local concentration of arginine leading to an increase in the number of CD3+ T cells within the tumor, following inhibition of tumor growth, similar to when

indoleamine

2,3 dioxygenase (IDO) inhibitors block tryptophan degradation by IDO, leading to restoration of tumor and activated tryptophan levels in tumor-associated T cells.

Besides the function of regulating the local concentration of arginine, the arginase inhibitor can also be combined with other immune tumor therapeutic drugs targeting T cell activation, such as CTLA-4 and PD-1 antibodies, the small molecule Arg inhibitor has -wide application prospect in the treatment of renal cell carcinoma, breast cancer, non-small cell lung cancer, acute granulocyte leukemia and Arg-mediated bone marrow-derived suppressor cell-related tumors.

In addition, study on Journal of Clinical Endocrinology and Metabolism in 2016 shows that arginase inhibitor can improve the endothelial function of T2DM microvascular complication effectively, and that arginase inhibitor can improve the endothelial relaxation function of patient obviously, promote the increase of local microvascular blood flow and delay the occurrence and development of T2DM microvascular complication effectively.

The studies of animals published in American Journal of physiology in 2015 show that arginase preparations can effectively delay the progress of diabetic nephropathy, arginase inhibitors can increase renal medullary blood flow of diabetic mice and reduce urine protein amount, and renal pathological biopsy shows that arginase inhibitors can obviously improve the progress of diabetic nephropathy.

Disclosure of Invention

The invention aims to provide compounds, and a preparation method, a screening and evaluating method and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

kinds of compounds having a general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein the substituents have the following definitions:

R₁and R₂Selected from hydrogen atoms;

or, R₁And R₂Form a 3-to 8-membered cycloalkyl group with the attached carbon atom , preferably R₁And R₂Taken together with the attached carbon atom to form a cyclopropyl group;

R₃and R₅Selected from hydrogen atoms;

or R₃And R₅Form a 3-to 8-membered cycloalkyl group with the attached carbon atom , preferably R₃And R₅Taken together with the attached C atom to form a cyclopropyl group;

provided that when R is₁And R₂When forming a 3-to 8-membered cycloalkyl group with the attached carbon atom , or R₃And R₅When R forms a 3-to 8-membered cycloalkyl group with the carbon atom attached thereto₁、R₂And the carbon atom to which it is attached, with R₃、R₅And the carbon atom to which they are bonded, which are not simultaneously cyclized;

R₄selected from the group consisting of-C (O) R₆Heteroaryl or fused ring, wherein said heteroaryl or fused ring is optionally substituted by or more substituents selected from alkyl, amino, aminoalkyl, hydroxyalkyl, phenyl, heterocyclyl or 3-to 8-membered cycloalkyl at step , wherein said alkyl, phenyl, heterocyclyl or cycloalkyl is optionally substituted by or more substituents selected from halogen, alkyl, alkoxy, hydroxyalkyl, haloalkyl, haloalkoxy, amino or aminoalkyl at step , wherein said fused ring is a heteroaryl or fused ringFused rings of radicals with cycloalkyl or heterocyclyl radicals;

or, R₄And R₅(ii) forms a heteroaryl group with atom to which they are attached, wherein the heteroaryl group is optionally substituted further with or more substituents selected from halogen, alkyl, alkoxy, hydroxyalkyl, haloalkyl, haloalkoxy, amino or aminoalkyl;

R₆selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted at with a halogen, alkyl, alkoxy, hydroxyalkyl, haloalkyl, haloalkoxy, amino or aminoalkyl substituent.

Step wherein R₄Selected from 5-6 membered heteroaryl, preferably selected from pyrazolyl, imidazolyl, pyrimidinyl, thiazolyl or pyridyl, wherein said heteroaryl is optionally substituted in step with or more substituents selected from alkyl, amino, aminoalkyl, hydroxyalkyl, phenyl, heterocyclyl or 3-8 membered cycloalkyl, wherein said alkyl, phenyl, heterocyclyl or cycloalkyl is optionally substituted in step with or more substituents selected from halogen, alkyl, alkoxy, hydroxyalkyl, haloalkyl, haloalkoxy, amino or aminoalkyl.

Or, wherein R₄Selected from:

wherein:

R₇、R₈and R₉Is selected from hydrogen atom, alkyl, phenyl or 3-8 membered cycloalkyl, wherein the alkyl, phenyl or 3-8 membered cycloalkyl is optionally substituted by steps of alkyl, amino, hydroxyl, halogen, aminoalkyl, hydroxyalkyl, haloalkyl or 3-8 membered cycloalkyl substituents.

Step wherein R₆Selected from:

wherein:

the alkyl group is a C1-C8 alkyl group. Alkoxy is 1 to 8 membered alkoxy. Cycloalkoxy is a 1-to 8-membered cycloalkoxy. Cycloalkyl is 1 to 8 membered cycloalkyl.

The heterocyclic group is a 4-7 membered monocyclic aliphatic heterocyclic group containing 1-2 heteroatoms independently selected from the group consisting of oxygen atom, sulfur atom or nitrogen atom. The heteroaryl group is a 5-6 membered monocyclic heteroaryl group containing 1-4 heteroatoms independently selected from the group consisting of oxygen atom, sulfur atom or nitrogen atom.

Haloalkyl is a haloalkyl of 1 to 8 carbons, aminoalkyl is a lower aminoalkyl having 1 to 8 carbons and or more amino groups, hydroxyalkyl is a lower hydroxyalkyl having 1 to 8 carbons and or more hydroxy groups, haloalkoxy is a1 to 8-membered haloalkoxy.

More particularly, wherein said compound is selected from the group consisting of the structural formulas shown in table 1:

table 1 Compound structural formula

Synthetic routes to the compounds:

(1) when R is₄is-C (O) R₆、

When the synthesis route is shown in the specification,

(2) when the structural formula of the compound is

When the synthesis route is shown in the specification,

(3) when the structural formula of the compound is

When the synthesis route is shown in the specification,

wherein R is₁₀Is composed of

(4) When the structural formula of the compound isWhen the synthesis route is shown in the specification,

(5) when the structural formula of the compound is

When the synthesis route is shown in the specification,

the arginase inhibitor prepared by the compound can be applied to preparing tumor medicaments for inhibiting the immunity of renal cell carcinoma, breast cancer, non-small cell lung cancer, acute myelogenous leukemia and arginase secreting MDSCs. In addition, arginase activity is up-regulated in many diseases such as ischemia-reperfusion injury (heart, lung, kidney), hypertension, atherosclerosis, diabetes, erectile dysfunction, pulmonary hypertension, and the like.

Drawings

FIG. 1 is an optimization experiment of optimal absorption wavelength;

FIG. 2 shows an enzyme, a substrate and Mn²⁺Optimizing experiment of optimal concentration;

FIG. 3 is a diagram of the pLVX plasmid map and the arginase I insertion site;

FIG. 4 shows the measurement of the amount of ARG1 in stable cell lines;

FIG. 5 shows the investigation of the number of inoculated cells and the detection of Arginase activity.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only partial embodiments of of the present invention, rather than all embodiments.

The synthesis process steps of the compounds shown in table 1 are specifically as follows:

6-oxa-3-azabicyclo [3.1.0] hexane-3-carboxylic acid tert-butyl ester

Dissolving 90g (0.533mol) of N-Boc-3-pyrroline in 1.2L of dichloromethane, cooling to 0 ℃, adding 118g (0.586mo) of m-chloroperoxybenzoic acid in batches, controlling the temperature to be not higher than 10 ℃, finishing the addition, naturally heating for reaction overnight, concentrating most dichloromethane at 30 ℃ under reduced pressure, adding 1L of petroleum ether into the residue, stirring for 5 minutes, filtering to remove solids, adding 300ml of ethyl acetate into the filtrate, washing twice with saturated sodium carbonate solution, washing with water for times, washing with saturated salt for times, drying, filtering, concentrating, and purifying by crude product column chromatography to obtain 58.8g of a product.

¹H NMR(300M，CDCl3)：δ1.44(s，9H)，3.26-3.39(m，2H)，3.66-3.83(m，4H)。

3-allyl-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

620ml (0.62mol) of allyl magnesium bromide (1mol/L) is added into an anhydrous three-necked bottle filled with argon, the temperature is reduced to 0 ℃, 58g (0.31mol) of tetrahydrofuran solution of 6-oxa-3-azabicyclo [3.1.0] hexane-3-carboxylic acid tert-butyl ester is dripped, the reaction temperature is controlled to be not higher than 5 ℃, the dripping is finished, the reaction is naturally heated, the TLC detects the reaction end point, saturated ammonium chloride is quenched, ethyl acetate is extracted, saturated saline is washed twice, and the crude product is subjected to rapid column chromatography to obtain 60.3g of the product.

¹H NMR(300M，CDCl3)：δ1.45(s，9H)，2.01-2.22(m，3H)，3.03-3.26(m，2H)，3.51-3.61(m，2H)，4.03-4.05(m，1H)，5.03-5.09(m，2H)，5.71-5.83(m，1H)。

3-allyl-4-oxopyrrolidine-1-carboxylic acid tert-butyl ester

58g (0.26mol) of 3-allyl-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester is dissolved in dichloromethane, 120g (0.28mol) of dessimidine reagent is added in batches at 10-20 ℃, reaction is carried out at normal temperature, and TLC is used for detecting the reaction end point. The reaction was quenched by the addition of sodium thiosulfate solution and saturated sodium bicarbonate solution, separated, and the dichloromethane layer washed with water. The crude product is subjected to flash column chromatography to obtain 50g of product.

¹H NMR(300M，CDCl₃)：δ1.47(s，9H)，2.14-2.22(m，1H)，2.50-2.57(m，1H)，2.65-2.68(m，1H)，3.28-3.33(q，1H)，3.64-3.69(d，1H)，3.86-4.03(m，2H)，5.06-5.12(m，2H)，5.68-5.78(m，1H)；

(cis) -4-allyl-3- (trichloromethyl) -3- (trimethylsilyloxy) pyrrolidine-1-carboxylic acid tert-butyl ester

52.6m (0.41mol) of trimethylchlorosilane, 43ml (0.53mol) of chloroform and 500ml of tetrahydrofuran are added into a waterless three-necked bottle filled with argon flow, the temperature is reduced to-70 ℃, 400ml (0.4mol) of LHMDS is dripped, the temperature is controlled to be not higher than-60 ℃, and the temperature is kept for 30min after the dripping is finished at-60 to-70 ℃. Naturally heating to-30 ℃, dropwise adding 48g (0.21mol) of 3-allyl-4-oxopyrrolidine-1-carboxylic acid tert-butyl ester and 5.9g (0.02mol) of tetrabutylammonium acetate solution of DMF200ml, controlling the temperature between-20 ℃ and-30 ℃, and naturally heating overnight after dropwise adding. The reaction was quenched with saturated ammonium chloride, extracted with ethyl acetate, washed with brine, dried and concentrated to give 88g of crude product.

MS experimental value m/z: 416.1(M + 1).

(cis) -4-allyl-3- (trichloromethyl) -3-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

Dissolving 88g of crude tert-butyl (cis) -4-allyl-3- (trichloromethyl) -3- (trimethylsiloxy) pyrrolidine-1-carboxylate in 500ml of tetrahydrofuran, adding 12ml of acetic acid, cooling to 0-5 ℃, dropwise adding 220ml of TBAF, naturally heating, and detecting the reaction end point by TLC. Saturated sodium bicarbonate solution was added and stirred for 5 min. The mixture was extracted with ethyl acetate, washed with saturated brine and concentrated to give 72g of crude product.

MS experimental value m/z: 344.1(M + 1).

(trans) -4-allyl-3-azido-1- (tert-butoxycarbonyl) pyrrolidine-3-carboxylic acid

72g of crude (cis) -4-allyl-3- (trichloromethyl) -3-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester is dissolved in 350ml of dioxane, 36g (0.31mol) of trimethylsilyl azide is added, and the temperature is reduced in an ice bath. An aqueous solution of 25.2g (0.63mol) of sodium hydroxide (prepared using 350g of ice water) was prepared, and the mixture was added dropwise to a reaction flask and allowed to warm naturally overnight after dropping. Adding ammonium chloride and water, stirring for 10min, extracting with ethyl acetate, washing with saturated saline solution, drying, and concentrating to obtain crude product 58 g.

MS experimental value m/z: 297.2(M + 1).

(3R, 4S) -3-benzyl-1-tert-butyl-4-allyl-3-azidopyrrolidine-1, 3-dicarboxylic acid ester

58g of crude (trans) -4-allyl-3-azido-1- (tert-butoxycarbonyl) pyrrolidine-3-carboxylic acid was dissolved in 350ml of acetonitrile, and 158g (1.14mol) of potassium carbonate and 28ml (0.23mol) of benzyl bromide were added thereto and reacted at room temperature overnight. Most of acetonitrile is concentrated, saturated saline solution is added, ethyl acetate is used for extraction, saturated saline solution is used for washing, column chromatography is carried out to obtain 50g, and chiral preparative chromatography (SFC) is used for separation to obtain 21g of product.

1H NMR(300M，CDCl3)：δ1.44(s，9H)，1.76-1.86(m，1H)，2.11(br，1H)，2.42(br，1H)，3.23-3.64(m，3H)，3.83-3.97(m，1H)，4.93-4.05(m，2H)，5.20-5.31(m，2H)，5.56-5.70(m，1H)，7.38(s，5H)；

MS experimental value m/z: 387.2(M + 1).

(3R, 4S) -3-benzyl-1-tert-butyl-3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-1, 3-dicarboxylate

4.55g (11.8mmol) of (3R, 4S) -3-benzyl-1-tert-butyl-4-allyl-3-azidopyrrolidine-1, 3-dicarboxylic acid ester is dissolved in 50ml of dichloromethane, 256mg (0.38mmol) of iridium chloride and 309mg (0.78mmol) of DPPE are added, the temperature is reduced to 0 ℃, 2.21g (17.3mmol) of pinacolborane is added dropwise, the reaction is naturally heated after the addition, and the end point of the reaction is detected by TLC. Adding water to quench the reaction, separating an organic phase, extracting a water phase with dichloromethane, combining, washing with water, and performing column chromatography to obtain 5.1 g.

MS experimental value m/z: 515.3(M + 1).

(3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester

(3R, 4S) -3-benzyl-1-tert-butyl 3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-1, 3-dicarboxylate 5g (9.7mmol) was dissolved in dichloromethane (50ml), cooled to 10 ℃ and trifluoroacetic acid (10ml) was added to check the end of the reaction by TLC. Concentrating under reduced pressure, adding dichloromethane and saturated sodium bicarbonate into the residue to adjust the pH value to 7, separating liquid, extracting aqueous phase dichloromethane, combining, washing with water, drying and concentrating to obtain a crude product of 3.3 g.

MS experimental value m/z: 415.2(M + 1).

1- ((tert-butoxycarbonyl) aminomethyl) cyclopropanecarboxylic acid

1g (9mmol) of 1-cyanocyclopropanecarboxylic acid was dissolved in 20ml of methanol, 0.1g of palladium on carbon was added thereto, and the mixture was hydrogenated overnight. The reaction solution was concentrated to dryness, 20ml of methylene chloride and 2.5g (11mmol) of di-tert-butyl dicarbonate were added, reacted for 6 hours, and the product was directly subjected to column chromatography to obtain 0.86 g.

1H NMR(300M，CDCl3)：δ1.26-1.33(m，4H)，1.44(s，9H)，3.27-3.31(m，2H)

MS experimental value m/z: 216.1(M + 1).

Compound No. 1:

(3R, 4S) -3-amino-1- (1- (aminomethyl) cyclopropanecarbonyl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

Using a synthesis method using (3R, 4S) -3-amino-1- (1-aminocyclobutanecarbonyl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid, 103mg of the objective compound was prepared using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 1- ((tert-butoxycarbonyl) aminomethyl) cyclopropanecarboxylic acid as starting materials.

1H NMR(300M，CDCl3)：δ0.73-0.78(m，2H)，0.92-1.09(m，4H)，1.19-1.24(m，H)，1.35-1.42(m，2H)，1.57-1.64(m，1H)，2.47-2.61(m，1H)，2.86-2.89(m，1H)，3.03-3.26(m，1H)，3.39-3.42(m，1H)，3.54-3.57(m，1H)，3.999-4.02(m，1H)，4.15-4.20(m，1H)；

MS experimental value m/z: 296.1(M + 1-18).

(3R, 4S) -3-azido-1- (1- (tert-butoxycarbonyl) aminocyclobutanecarbonyl) -4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester

Boc-1-aminocyclobutanecarboxylic acid 0.26g (1.2mmol) was dissolved in 5ml of dichloromethane, HATU 0.46g (1.2mmol) was added, and after reaction at ordinary temperature for 30min, (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester 0.41g (1mmol), triethylamine 0.2g (2mmol) was added and the reaction was allowed to proceed overnight. The reaction solution was concentrated to dryness and subjected to column chromatography to obtain 0.56 g.

MS experimental value m/z: 612.4(M + 1).

(3R, 4S) -3-azido-1- (1- (tert-butoxycarbonyl) aminocyclobutanecarbonyl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid benzyl ester

(3R, 4S) -3-azido-1- (1- (tert-butoxycarbonyl) aminocyclobutanecarbonyl) -4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester 0.34g (0.56mmol) was dissolved in 5ml of water and 10ml of tetrahydrofuran, and 0.17g (2.2mmol) of amine acetate and 0.23g (1.1mmol) of sodium periodate were added and reacted overnight. Sodium thiosulfate solution is added to quench the reaction, and the reaction solution is extracted, washed and concentrated to obtain 0.3g of crude product.

MS experimental value m/z: 530.3(M + 1).

(3R, 4S) -3-azido-1- (1-aminocyclobutanecarbonyl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid benzyl ester

0.3g of crude benzyl (3R, 4S) -3-azido-1- (1- (tert-butoxycarbonyl) aminocyclobutanecarbonyl) -4- (3-boronopropyl) pyrrolidine-3-carboxylate was dissolved in 10ml of dichloromethane, and 2ml of trifluoroacetic acid was added thereto, followed by stirring for 4 hours and concentration to give 0.5g of crude product.

MS experimental value m/z: 430.2(M + 1).

Compound No. 2:

(3R, 4S) -3-amino-1- (1-aminocyclobutanecarbonyl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

The crude (3R, 4S) -3-azido-1- (1-aminocyclobutanecarbonyl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid benzyl ester trifluoroacetate was dissolved in 10ml of methanol, 0.1g of palladium on charcoal was added, and the reaction was hydrogenated overnight. Filtering, concentrating to dryness, separating, and lyophilizing to obtain target substance 45.3 mg.

1H NMR(300M，CDCl3)：δ0.76-0.80(m，2H)，1.24-1.49(m，3H)，1.60-1.67(m，1H)，2.05-2.13(m，1H)，2.22-2.30(m，1H)，2.32-2.41(m，2H)，2.64-2.70(m，1H)，2.82-2.97(m，2H)，3.65-3.72(m，2H)，3.93-3.97(m，1H)，4.10-4.13(m，1H)，4.20-4.24(m，1H)；

MS experimental value m/z: 296.1(M + 1-18).

Compound No. 3:

(3R, 4S) -3-amino-4- (3-boronopropyl) -1- (2- (pyrrolidin-1-yl) acetyl) pyrrolidine-3-carboxylic acid

Using a synthesis method using (3R, 4S) -3-amino-1- (1-aminocyclobutanecarbonyl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid, 77.5mg of the objective compound was prepared using (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester and fragment 2- (1-pyrrolidinyl) acetic acid as starting materials.

1H NMR(300M，CDCl3)：δ0.69-0.83(m，2H)，1.21-1.29(m，1H)，1.33-1.44(m，2H)，1.59-1.66(m，1H)，1.99-2.02(m，2H)，2.07-2.15(m，2H)，2.49-2.70(m，1H)，3.10-3.16(m，2H)，3.25-3.39(m，1H)，3.55-3.67(m，1H)，3.70-3.74(m，2H)，3.88-4.03(m，1H)，4.06-4.13(m，1H)，4.17-4.29(m，2H)；

MS experimental value m/z: 310.2(M + 1-18).

(3R, 4S) -3-azido-1- (3-nitropyridin-2-yl) -4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester

(3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester 0.41g (1mmol) was dissolved in dioxane, and 2-chloro-3-nitropyridine 0.16g (1mmol) and triethylamine 0.2g (2mmol) were added and reacted at 80 ℃ overnight. The reaction solution is directly subjected to column chromatography to obtain 0.38g of product.

1H NMR(300M，CDCl3)：δ0.66-0.72(m，2H)，1.07-1.14(m，1H)，1.21-1.22(d，12H)，1.42-1.45(m，3H)，1.57(s，3H)，2.47-2.54(m，1H)，3.38-3.43(q，1H)，3.48-3.51(d，1H)，3.72-3.77(q，1H)，3.79-3.82(d，1H)，5.18-5.25(q，2H)，6.71-6.74(q，1H)，8.09-8.12(dd，1H)，8.31-8.33(dd，1H)；

MS experimental value m/z: 537.3(M + 1).

Compound No. 4:

(3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

0.37g of (3R, 4S) -3-azido-1- (3-nitropyridin-2-yl) -4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester was dissolved in 10ml of methanol, and 0.4g of palladium on charcoal and 0.2g of acetic acid were added to conduct hydrogenation overnight. Filtering to remove palladium carbon, concentrating to dryness, adding 15ml3M HCl into the crude product, reacting at 55 deg.C for 6h, concentrating to dryness, and separating to obtain 50.8mg of target product.

1H NMR(300M，CDCl3)：δ0.71-0.82(m，2H)，1.29-1.46(m，3H)，1.64-1.70(m，1H)，2.67-2.74(m，1H)，3.58-3.67(m，1H)，3.97-4.13(m，2H)，4.25-4.30(m，1H)，6.97-7.09(m，1H)，7.42-7.48(m，1H)，7.93-7.96(m，1H)

MS experimental value m/z: 291.2(M + 1-18).

Compound No. 5:

(3R, 4S) -3-amino-1- (3-amino-4-methylpyridin-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

Using a synthetic method using (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-boranopropyl) pyrrolidine-3-carboxylic acid, the desired 112mg was prepared using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and the fragment 2-chloro-3-nitro-4-methylpyridine as starting materials.

1H NMR(300M，CDCl3)：δ0.76-0.83(m，2H)，1.36-1.50(m，3H)，1.65-1.73(m，1H)，2.31(s，3H)，2.70-2.79(m，1H)，3.51-3.57(m，1H)，4.01-4.12(m，3H)，7.08-7.11(d，1H)，7.49-7.51(d，1H)

MS experimental value m/z: 305.2(M + 1-18).

Compound No. 6:

(3R, 4S) -3-amino-1- (3-amino-5-methylpyridin-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

A desired compound (89 mg) was prepared by a synthesis method using (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-boranopropyl) pyrrolidine-3-carboxylic acid, and using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 2-chloro-3-nitro-5-methylpyridine as starting materials.

1H NMR(300M，CDCl3)：δ0.74-0.84(m，2H)，1.32-1.51(m，3H)，1.66-1.74(m，1H)，2.36(s，3H)，2.73-2.80(m，1H)，3.54-3.60(m，1H)，4.01-4.25(m，3H)，7.38(s，1H)，7.83(s，1H)

MS experimental value m/z: 305.2(M + 1-18).

Compound No. 7:

(3R, 4S) -3-amino-1- (3-amino-5-cyclopropylpyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid

The target compound was prepared by a method of synthesizing (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 2-chloro-3-nitro-5-cyclopropylpyridine as starting materials.

1H NMR(300M，CDCl3)：δ0.68-0.72(m，2H)，0.81-0.87(m，2H)，0.99-1.04(m，2H)，1.38-1.55(m，3H)，1.69-1.76(m，1H)，1.87-1.93(m，1H)，2.31(s，3H)，2.72-2.80(m，1H)，3.57-3.62(m，1H)，4.06-4.25(m，3H)，7.25(s，1H)，7.39(s，1H)

MS experimental value m/z: 331.2(M + 1-18).

Compound No. 8:

a target compound was prepared by a synthesis method using (3R, 4S) -3-amino-1- (3-amino-5, 6-dimethylpyridin-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid, and using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 2-chloro-3-nitro-5, 6-dimethylpyridine as starting materials.

1H NMR(300M，CDCl3)：δ0.74-0.80(m，2H)，1.31-1.47(m，3H)，1.62-1.68(m，1H)，2.16(s，3H)，2.40(s，3H)，2.65-2.72(m，1H)，3.50-3.54(t，1H)，3.96-4.15(m，3H)，7.42(s，1H)

MS experimental value m/z: 319.2(M + 1-18).

Compound No. 9:

(3R, 4S) -3-amino-1- (3-amino-6, 7-dihydro-5H-cyclopenta [ b ] pyridin-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

The target compound was prepared by a synthesis method using (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid, starting with benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 2-chloro-3-amino-5- (2, 3-dihydro-1H-inden-5-yl) pyridine.

1H NMR(300M，CDCl3)：δ0.69-0.75(m，2H)，1.30-1.43(m，3H)，1.59-1.65(m，1H)，2.07-2.14(m，2H)，2.68-2.75(m，1H)，2.78-2.82(t，2H)，2.92-3.03(m，2H)，3.52-3.56(t，1H)，3.96-4.00(m，1H)，4.02-4.13(q，2H)，7.60(s，1H)

MS experimental value m/z: 331.2(M + 1-18).

Compound No. 10:

(3R, 4S) -3-amino-1- (3-amino-5-isopropylpyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid

The target compound was prepared by a method of synthesizing (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 2-chloro-3-nitro-5- (prop-1-en-2-yl) pyridine as starting materials.

1H NMR(300M，CDCl3)：δ0.71-0.81(m，2H)，1.12-1.14(d，6H)，1.28-1.49(m，3H)，1.62-1.69(m，1H)，2.70-2.85(m，2H)，3.52-3.57(t，1H)，4.00-4.22(m，3H)，7.37-7.46(m，2H)；

MS experimental value m/z: 319.2(M + 1-18).

Compound No. 11:

(3R, 4S) -3-amino-1- (3-amino-6-isobutylpyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid

Using a synthesis method using (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid, 41mg of the objective compound was prepared using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 2-chloro-3-amino-6-isobutylpyridine as starting materials.

1H NMR(300M，CDCl3)：δ0.69-0.86(m，8H)，1.32-1.47(m，3H)，1.62-1.69(m，1H)，1.84-1.94(m，1H)，2.58-2.72(m，3H)，3.60-3.65(t，1H)，4.01-4.06(t，1H)，4.08-4.24(dd，2H)，6.88-6.91(d，1H)，7.47-7.49(d，1H)

MS experimental value m/z: 347.2(M + 1-18).

Compound No. 12:

(3R, 4S) -4-amino-1- (3-aminopyridin-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

A synthesis method using (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-boranopropyl) pyrrolidine-3-carboxylic acid was used, and 23mg of the objective compound was prepared using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 2-chloro-4-nitropyridine as starting materials.

1H NMR(300M，CDCl3)：δ

MS experimental value m/z: 291.2(M + 1-18).

Compound No. 13:

(3R, 4S) -3-amino-1- (3-amino-5-phenylpyridin-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

In the process of synthesizing (3R, 4S) -3-amino-1- (3-amino-5- (4-chlorphenyl) pyridine-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid, a hydrogenation byproduct (dechlorination) is hydrolyzed to prepare the target product.

1H NMR(300M，CDCl3)：δ0.81-0.90(m，2H)，1.37-1.54(m，3H)，1.71-1.79(m，1H)，2.75-2.83(m，1H)，3.65-3.70(m，1H)，4.01-4.11(m，1H)，4.18-4.36(m，2H)，7.47-7.68(m，7H)；

MS experimental value m/z: 367.2(M + 1-18).

Compound No. 14:

(3R, 4S) -3-amino-1- (3-amino-5- (4-chlorophenyl) pyridin-2-yl) -4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

The target compound was prepared by a method of synthesizing (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 2-chloro-3-nitro-5- (4-chlorophenyl) pyridine as starting materials.

1H NMR(300M，CDCl3)：δ0.80-0.89(m，2H)，1.37-1.55(m，3H)，1.73-1.79(m，1H)，2.77-2.85(m，1H)，3.65-3.70(m，1H)，4.04-4.09(m，1H)，4.20-4.23(d，1H)，4.35-4.38(d，1H)，7.26-7.50(m，6H)；

MS experimental value m/z: 401.2(M + 1-18).

1- (3-methoxy-3-oxopropanamido) cyclopropylcarboxylic acid methyl ester

37.5g (0.248mol) of methyl 1-aminocyclopropylcarboxylate hydrochloride was dissolved in methanol (400mL) and DMF (40mL), and 50.2g (0.496mol) of triethylamine and 25.6g (0.298mol) of methyl acrylate were added thereto at room temperature to conduct a reaction at room temperature for 48 hours. The reaction was checked by TLC. Most of the solvent was spin dried, extracted with ethyl acetate, filtered and concentrated. And performing column chromatography to obtain 33g of product.

¹H NMR(300M，CDCl3)：δ0.93-1.05(m，2H)，1.22-1.33(m，2H)，2.44(t，2H)，3.01(t，2H)，3.66-3.72(m，6H)

MS experimental value m/z: 202.1(M + 1).

1- ((4-methoxybenzyl) (3-methoxy-3-oxopropanamido)) cyclopropylcarboxylic acid methyl ester

Methyl 1- (3-methoxy-3-oxopropanamido) cyclopropylcarboxylate (31 g, 0.154mol) was dissolved in methanol (300mL) and DMF (300mL), and a catalytic amount of acetic acid (2 mL), p-methoxybenzaldehyde (62.9 g, 0.463mol) was added thereto at room temperature to react at room temperature for 12 hours. Then, 29.2g (0.463mol) of sodium cyanoborohydride was added in portions to the reaction solution, and the reaction was carried out for 12 hours. The reaction was monitored by TLC. Extracting with ethyl acetate, spin-drying the solvent, and performing column chromatography to obtain a crude product 28 g.

MS experimental value m/z: 322.2(M + 1).

1- (4-methoxybenzyl) -5-cyclopropyl-4-oxopyrrolidine-3-carboxylic acid methyl ester

21g (0.065mol) of methyl 1- ((4-methoxybenzyl) (3-methoxy-3-oxopropanamido)) cyclopropylcarboxylate is dissolved in 200ml tetrahydrofuran, and LHMDS solution (0.196mol, 196ml) is slowly added dropwise at 0 ℃ and after the addition is complete, the reaction is continued for 1 hour. TLC showed the starting material was reacted. Slowly adding saturated ammonium chloride aqueous solution at 0 ℃ to quench the reaction, extracting with ethyl acetate, spin-drying the solvent, and carrying out column chromatography to obtain 18.7g of a product.

MS experimental value m/z: 290.1(M + 1).

1- (4-methoxybenzyl) -5-cyclopropyl-4-oxopyrrolidine-3-allyl-3-carboxylic acid methyl ester

Methyl 1- (4-methoxybenzyl) -5-cyclopropyl-4-oxopyrrolidine-3-carboxylate (18.7 g, 0.065mol) was dissolved in DMF (200ml), NaH (2.59 g, 0.065mol, 60%) was added portionwise at 0 ℃ and after stirring for half an hour, allyl bromide (7.83 g, 0.065mol) was added and after half an hour, the reaction was complete by TLC. Slowly adding saturated ammonium chloride aqueous solution at 0 ℃ to quench the reaction, extracting with ethyl acetate, and performing spin-drying solvent column chromatography to obtain 15.6g of a product.

¹H NMR(300M，CDCl3)：δ0.85-1.22(m，4H)，2.45-2.60(m，1H)，2.65-2.80(m，1H)，2.80-2.90(m，1H)，3.36(s，2H)，3.40-3.45(m，1H)，3.75(s，3H)，3.81(s，3H)，5.00-5.15(m，2H)，5.57-5.75(m，1H)，6.87(d，2H)，7.25(d，2H)。

MS experimental value m/z: 330.2(M + 1).

1- (4-methoxybenzyl) -5-cyclopropyl-3-allyl-4-oxopyrrolidine

Methyl 1- (4-methoxybenzyl) -5-cyclopropyl-4-oxopyrrolidine-3-allyl-3-carboxylate (18.8 g, 0.057mol) was dissolved in ethanol (395ml) and water (132ml), lithium hydroxide hydrate 4.82(0.115mol) was added at room temperature, the reaction was carried out at 80 ℃ for 1 hour, TLC showed completion of the reaction of the starting materials, the solvent was dried by spinning, and column chromatography gave 7.2 g.

¹H NMR(300M，CDCl3)：δ0.85-1.22(m，4H)，2.05-2.09(m，1H)，2.44-2.59(m，1H)，2.64-2.79(m，1H)，2.78-2.88(m，1H)，3.36(s，2H)，3.39-3.44(m，1H)，3.80(s，3H)，4.98-5.13(m，2H)，5.55-5.73(m，1H)，6.87(d，2H)，7.25(d，2H)。

MS experimental value m/z: 272.2(M + 1).

5-cyclopropyl-3-allyl-4-oxopyrrolidine

Dissolving 7.2g (0.026mol) of 5-cyclopropyl-3-allyl-4-oxopyrrolidine in dichloromethane (80ml), slowly dropwise adding 7.5g (0.053mol) of 1-chloroethyl chloroformate at 0 ℃, after dropwise adding, continuing to react for 2 hours, rotatably drying the solvent, adding 50ml of methanol, reacting at 50 ℃ for half an hour, rotatably drying the solvent to obtain 4.9g of a crude product, and directly feeding into steps.

MS experimental value m/z: 152.1(M + 1).

1-Boc-5-cyclopropyl-3-allyl-4-oxopyrrolidine

5-cyclopropyl-3-allyl-4-oxopyrrolidine hydrochloride 4.9g (0.026mol) was dissolved in dichloromethane (100mL), and triethylamine 5.3g (0.026mol) and (Boc) were added at room temperature₂O6.2 g (0.029mol), and reacting at room temperature for 12 h. TLC monitored the starting material reaction was complete. Extracting with dichloromethane, spin-drying solvent, performing column chromatography to obtain 8.5g crude product, and performing column chromatography to obtain 6.6g crude product.

¹H NMR(300M，CDCl3)：δ0.84-1.23(m，4H)，1.43(s，9H)2.05-2.09(m，1H)，2.45-2.60(m，1H)，2.64-2.79(m，1H)，2.99-3.44(m，2H)，4.98-5.13(m，2H)，5.55-5.73(m，1H)。

5-cyclopropyl-3-allyl-4- ((acetylamino) (tert-butylformyl)) 1-tert-butoxycarbonyl-pyrrolidine

1.6g (6.4mmol) of 1-tert-butoxycarbonyl-5-cyclopropyl-3-allyl-4-oxopyrrolidine was dissolved in trifluoroethanol (10ml), and 4.9g (64mmol) of ammonium acetate and 1.59g (19.2mmol) of tert-butyl isocyanate were added thereto at room temperature, followed by reaction at room temperature overnight. TLC monitored the reaction progress. Adding 50ml water, extracting with ethyl acetate, and carrying out column chromatography to obtain 0.5g of product.

¹H NMR(300M，CDCl₃)：δ0.84-1.23(m，4H)，1.42-1.45(m，18H)，2.02(s，3H)，2.04-2.09(m，1H)，2.45-2.60(m，1H)，2.65-2.80(m，1H)，2.78-2.88(m，1H)，3.41-3.46(m，1H)，4.98-5.13(m，2H)，5.55-5.73(m，1H)。

MS experimental value m/z: 394.2(M +1)

1-Boc-5-cyclopropyl-3-propylpinacol boronate-4- ((acetylamino) (tert-butylformyl)) pyrrolidine

450mg (1.14mmol) of 1-tert-butoxycarbonyl-5-cyclopropyl-3-allyl-4- ((acetylamino) (tert-butylformyl)) pyrrolidine was dissolved in 15ml of dichloromethane, 60mg of bis (1, 5-cyclooctadiene) iridium chloride dimer and 60mg of 1, 2-bis (diphenylphosphino) ethane were added at room temperature, 292mg (2.28mmol) of pinacolborane was slowly added dropwise at 0 ℃ and the reaction was carried out at room temperature for 12 hours. Adding water, extracting with ethyl acetate, and performing column chromatography to obtain 0.3g of product.

MS experimental value m/z: 522.3(M +1)

5-cyclopropyl-3-propylpinacol boronate-4- ((acetylamino) (tert-butylformyl)) pyrrolidine

0.29g of 1-tert-butoxycarbonyl-5-cyclopropyl-3-propylpinacol boronate-4- ((acetylamino) (tert-butylformyl)) pyrrolidine was dissolved in 5ml of dichloromethane, and 1ml of trifluoroacetic acid was added to stir the mixture for 2 hours. Concentrate to dryness and add dichloromethane and sodium carbonate solution. The mixture was subjected to liquid separation and extraction, and concentrated to obtain 0.21g of crude product.

MS experimental value m/z: 422.3(M +1)

1- (2-tert-Butoxycarbonylamino) propionyl-5-cyclopropyl-3-propylpinacol boronate-4- ((acetylamino) (tert-butylformyl)) pyrrolidine

Boc-1-aminocyclobutanecarboxylic acid 0.13g (0.6mmol) was dissolved in 5ml of dichloromethane, and HATU 0.23g (0.6mmol) was added to the solution, followed by reaction at room temperature for 30min, followed by addition of 5-cyclopropyl-3-propylpinacolato borate-4- ((acetylamino) (tert-butylformyl)) pyrrolidine 0.21g (0.5mmol) and triethylamine 0.1g (1mmol) to the solution, and the reaction was allowed to proceed overnight. The reaction solution was concentrated to dryness and subjected to column chromatography to obtain 0.22 g.

MS experimental value m/z: 593.4(M +1)

Compound No. 15:

3-amino-4- (3-boronopropyl) -1- (2-aminopropionyl) -pyrrolidine-3-carboxylic acid

1- (2-tert-Butoxycarbonylamino) propionyl-5-cyclopropyl-3-propylpinacolboronic acid ester-4- ((acetylamino) (tert-butylformyl)) pyrrolidine 0.2g, 6M HCl 10ml, refluxed for 8h, and concentrated to dryness. HPLC-Pre gave 36.7 mg.

¹H NMR(300M，CDCl3)：δ0.82-1.22(m，6H)，1.26-1.53(m，6H)，1.64-1.70(m，1H)，2.61-2.81(m，1H)，3.87-3.99(m，1H)，4.25-4.39(m，2H)。

MS experimental value m/z: 296.2(M +1-18)

2- (4-methoxybenzyloxy) ethanol

Ethylene glycol (10ml) was added to 2g (12.8mmol) of p-methoxybenzyl chloride, and 2.2g (38.4mmol) of potassium hydroxide was added in portions at room temperature, followed by reaction overnight. TLC monitored the progress of the reaction. Extracting with ethyl acetate, and spin-drying the solvent to obtain 2.5g of the product.

¹H NMR(300M，CDCl3)：δ3.56(t，2H)，3.68-3.76(m，2H)，3.80(s，3H)，4.49(s，2H)，6.88(d，2H)，7.27(d，2H)

2- (4-Methoxybenzyloxy) acetaldehyde

3.7ml of oxalyl chloride was dissolved in dichloromethane (75ml), the temperature was controlled at-70 ℃, dimethyl sulfoxide (6.4ml) was dissolved in dichloromethane (10ml), and the solution was slowly added dropwise, and after completion of the addition, the reaction was carried out for 1 hour. 6g (33.0mmol) of 2- (4-methoxybenzyloxy) ethanol is dissolved in dichloromethane (5ml) and slowly added, triethylamine (12.5ml) is slowly dropped after two hours of reaction, and after thirty minutes of reaction, a cooling device is removed, and the temperature is raised to zero. 3N HCl was added slowly, extracted and the organic layers combined. The mixture was washed with 1N HCl, saturated sodium bicarbonate solution and saturated brine, dried and the organic solvent was spin-dried to obtain 5g of the product.

¹H NMR(300M，CDCl3)：δ3.81(s，3H)，4.06(d，2H)，4.56(s，2H)，4.49(s，2H)，6.89(d，2H)，7.28(d，2H)，9.7(s，1H)。

2- (2- (4-methoxybenzyloxy) -1-hydroxyethyl) pentenenitrile

Pentenenitrile 1.8g (22.2mmol) was dissolved in tetrahydrofuran (20ml), LDA12.2ml (24.4mmol) was slowly added dropwise at 78 deg.C, and after the addition was completed, the reaction was carried out for 1 hour. 2- (4-methoxybenzyloxy) acetaldehyde 4g (22.2mmol) in tetrahydrofuran (30ml) was slowly added dropwise, and after completion of the addition, the reaction was allowed to proceed overnight at room temperature. Quenching by saturated ammonium chloride solution, extracting by ethyl acetate, spin-drying the solvent, and carrying out column chromatography to obtain 3g of a product.

¹H NMR(300M，CDCl3)：δ2.18-2.43(m，2H)，2.86(m，1H)，3.50-3.77(m，3H)，3.82(s，3H)，4.55(s，2H)，5.03-5.09(m，2H)，5.85(m，1H)，6.88(d，2H)，7.26(d，2H)。

2- (2- (4-methoxybenzyloxy) -1- (tert-butyldimethylsilyloxy) ethylpentenenitrile

23g (0.0881mol) of 2- (2- (4-methoxybenzyloxy) -1-hydroxyethyl) pentenenitrile was dissolved in DMF (200ml), 30g (0.441mol) of imidazole was added at room temperature, and 15.9g (0.106mol) of t-butyldimethylsilyl chloride was slowly added overnight at room temperature. The reaction was monitored by TLC. Extracting with ethyl acetate, and performing column chromatography to obtain 28.7 g.

1- (1- (4-methoxybenzyloxy) -2- (tert-butyldimethylsilyloxy) hex-5-en-3-yl) cyclopropylamine

6.8g (0.018mol) of 2- (2- (4-methoxybenzyloxy) -1- (tert-butyldimethylsilyloxy) ethyl pentenenitrile is dissolved in 100ml of tetrahydrofuran, 6.18g (0.0218mol) of tetraisopropyl titanate is added at room temperature, 9ml (0.018mol) of ethyl magnesium bromide tetrahydrofuran solution is slowly added at 78 ℃, after the dropwise addition is finished, the temperature is slowly raised to the room temperature within hours, the room temperature is reacted for half an hour, boron trifluoride diethyl etherate is added for quenching reaction, stirring is carried out for half an hour, ethyl acetate is extracted, the solvent is dried, and column chromatography is carried out to obtain 4.7 g.

MS experimental value m/z: 406.3(M +1)

N-Boc-1- (1- (4-methoxybenzyloxy) -2- (tert-butyldimethylsiloxy) hex-5-en-3-yl) cyclopropylamine

0.52g (1.03mmol) of 1- (1- (4-methoxybenzyloxy) -2- (tert-butyldimethylsiloxy) hex-5-en-3-yl) cyclopropylamine was dissolved in 10ml of methylene chloride, and 0.28g (2.06mmol) of triethylamine and 0.45g (2.06mmol) of di-tert-butyl dicarbonate were added thereto at room temperature to conduct reaction overnight at room temperature. TLC monitored the reaction completion. Extraction with dichloromethane and column chromatography gave 0.56g of product.

¹H NMR(300M，CDCl3)：δ0.0-0.20(m，6H)，0.55-0.70(m，2H)，0.70-0.80(m，1H)，0.80-0.90(m，9H)，0.90-1.10(m，1H)，1.10-1.30(m，2H)，1.34(s，9H)，2.03-2.24(m，1H)，2.30-2.50(m，1H)，3.36-3.51(m，1H)，3.53-3.80(m，4H)，3.81-3.95(m，1H)，4.25-4.45(m，2H)，4.83-5.05(m，2H)，5.75-6.05(m，1H)，6.76-6.85(m，2H)，7.12-7.20(m，2H)。

N-Boc-1- (1-hydroxy-2- (tert-butyldimethylsilyloxy) hex-5-en-3-yl) cyclopropylamine

7g (13.8mmol) of N-Boc-1- (1- (4-methoxybenzyloxy) -2- (tert-butyldimethylsiloxy) hex-5-en-3-yl) cyclopropylamine was dissolved in 100ml of dichloromethane and 10ml of a pH7 buffer, and 3.77g (16.6mol) of 2, 3-dichloro-5, 6-dicyan-p-benzoquinone was added portionwise under ice. At room temperature overnight, TLC showed the reaction was complete. Extraction with saturated sodium bicarbonate and dichloromethane, combining organic layers, and spin-drying the solvent to obtain 7g of crude product.

MS experimental value m/z: 386.3(M +1)

N-Boc-1- (1- (methylsulfonyloxy) -2- (tert-butyldimethylsilyloxy) hex-5-en-3-yl) cyclopropylamine

7.0g (18.2mmol) of N-Boc-1- (1-hydroxy-2- (tert-butyldimethylsilyloxy) hex-5-en-3-yl) cyclopropylamine was dissolved in dichloromethane, 2.2g (21.8mmol) of triethylamine was added at room temperature, 2.3g (20.0mmol) of methanesulfonyl chloride was added at 0 ℃ and the reaction was carried out at room temperature for 3 hours, TLC monitored for completion of the reaction, dichloromethane extraction was carried out, the solvent was dried to give a crude product which was directly subjected to steps.

MS experimental value m/z: 464.2(M +1)

1-Boc-5-cyclopropyl-4-allyl-3-hydroxypyrrolidine

The crude product obtained in the previous step (18.2mmol in percentage yield) is dissolved in DMF (50ml), NaH 0.72g (18.2mmol) is added in portions in ice bath, the reaction is carried out for 12 hours at room temperature, TLC shows that the reaction is finished, saturated solution of ammonium chloride is quenched, ethyl acetate is extracted, and column chromatography is carried out to obtain 1.9g of product.

¹H NMR(300M，CDCl3)：δ0.86-1.21(m，4H)，1.46(s，9H)，1.96-2.01(m，1H)，2.45-2.60(m，1H)，2.65-2.80(m，1H)，2.89-2.99(m，1H)，3.41-3.50(m，2H)，4.98-5.13(m，2H)，5.55-5.73(m，1H)。

MS experimental value m/z: 254.2(M + 1).

1-Boc-5-cyclopropyl-4-allyl-3-oxopyrrolidine

1.8g (7.1mmol) of 1-tert-butoxycarbonyl-5-cyclopropyl-4-allyl-3-hydroxypyrrolidine was dissolved in methylene chloride (20ml), and 3.65g (8.6mmol) of DMP was added in portions at room temperature, followed by reaction at room temperature overnight. TLC showed the starting material was reacted. Extracting with dichloromethane, spin-drying solvent, and performing column chromatography to obtain 1.1g product.

MS experimental value m/z: 252.2(M + 1).

¹H NMR(300M，CDCl3)：δ0.84-1.23(m，4H)，1.42-1.45(m，18H)，2.02(s，3H)，2.04-2.09(m，1H)，2.45-2.60(m，1H)，2.63-2.78(m，1H)，2.86-2.96(m，1H)，3.49-3.54(m，1H)，4.98-5.13(m，2H)，5.53-5.71(m，1H)。

MS experimental value m/z: 394.2(M +1)

MS experimental value m/z: 522.3(M +1)

MS experimental value m/z: 422.3(M +1)

(s)1- (2-tert-Butoxycarbonylamino) propionyl-5-cyclopropyl-3-propylpinacol boronate-4- ((acetylamino) (tert-butylformyl)) pyrrolidine

MS experimental value m/z: 593.4(M +1)

Compound No. 16:

(s) -3-amino-4- (3-boronopropyl) -1- (2-aminopropionyl) -pyrrolidine-3-carboxylic acid

¹H NMR(300M，CDCl3)：δ0.83-1.21(m，6H)，1.25-1.52(m，6H)，1.64-1.70(m，1H)，2.60-2.80(m，1H)，3.87-3.99(m，1H)，4.22-4.36(m，2H)。

MS experimental value m/z: 296.2(M +1-18)

1-benzyl-L-4-hydroxyproline methyl ester

L-4-hydroxyproline methyl ester hydrochloride 8g (0.044mol) was dissolved in dichloromethane 80ml and methanol 10 ml. 4.6g (0.043mol) of benzaldehyde is added into the mixture, the mixture is subjected to ice-water bath, 3.6g (0.057mol) of sodium cyanoborohydride is added in batches, the ice-water bath is removed after the addition, and the reaction is carried out for 2 hours. Adding 80ml water, separating, extracting, washing, and performing flash column chromatography to obtain 11.8 g.

¹H NMR(300M，CDCl₃)：δ2.06-2.11(m，1H)，2.45-2.49(m，1H)，3.30-3.34(m，1H)，3.47(s，2H)，3.63-3.68(m，4H)，3.88-3.91(m，1H)，4.43-4.48(m，1H)，7.30-7.32(m，5H)

MS experimental value m/z: 236.1(M + 1).

1-benzyl-4- (tert-butyldiphenylsiloxy) pyrrolidine-2-carboxylic acid methyl ester

Dissolving 12.7g (0.054mol) of 1-benzyl-L-4-hydroxyproline methyl ester in 100ml of DMF, adding 5.51g (0.081mol) of imidazole, cooling in an ice-water bath, adding 17.82g (0.065mol) of tert-butyldiphenylchlorosilane, and naturally heating for reaction overnight. Tert-methyl ether and water are added, liquid separation, extraction and washing are carried out, and column chromatography is carried out to obtain 19.96g, wherein the yield is 78.1%.

MS experimental value m/z: 474.2(M + 1).

1-benzyl-4- (tert-butyldiphenylsiloxy) -2-hydroxymethylpyrrolidine

19g (0.04mol) of methyl 1-benzyl-4- (tert-butyldiphenylsiloxy) pyrrolidine-2-carboxylate was dissolved in 250ml of anhydrous tetrahydrofuran, and 3g (0.08mol) of lithium aluminum hydride was added in portions in an ice bath, and the mixture was incubated for 30min after completion of the addition. The ice bath was removed and the reaction was allowed to proceed for 2 h. And (3) performing ice bath, dropwise adding 10ml of ethyl acetate, stirring for 30min, removing the ice bath, and stirring for 30 min. Dropping water until no bubbles exist. And (5) filtering. The filtrate was concentrated and column chromatographed to obtain 12.6 g.

MS experimental value m/z: 446.2(M + 1).

4- (tert-butyldiphenylsiloxy) -2-hydroxymethylpyrrolidine

11.3g (0.025mol) of 1-benzyl-4- (tert-butyldiphenylsiloxy) -2-hydroxymethylpyrrolidine was dissolved in 150ml of methanol, 0.8g of palladium on carbon was added, the mixture was hydrogenated overnight, filtered and concentrated to give 8.66g of crude product.

¹H NMR(300M，CDCl₃：δ1.06(s，9H)，1.42-1.48(m，1H)，1.82-1.87(m，1H)，2.72-2.76(m，1H)，2.91-2.94(m，1H)，3.24-3.28(m，1H)，3.51-3.55(m，1H)，3.63-3.68(m，1H)，4.42-4.46(m，1H)，7.36-7.45(m，6H)，7.61-7.65(m，4H)

MS experimental value m/z: 356.2(M + 1).

1- (4- (tert-butyldiphenylsiloxy) -2- (hydroxymethyl) pyrrolidin-1-yl) -1-oxopropan-2-ylcarbamic acid benzyl ester

4.46g (0.02mol) of 2- (benzyloxycarbonyl) propionic acid, 6.1g (0.024mol) of BOP-Cl were added to methylene chloride, and the mixture was stirred for 20min, and 7.1g (0.02mol) of 4- (tert-butyldiphenylsilyloxy) -2-hydroxymethylpyrrolidine and 4g (0.04mol) of triethylamine were added thereto and reacted overnight. Washing with water, and performing column chromatography to obtain 4.96 g.

¹H NMR(300M，CDCl₃)：δ1.03(s，9H)，1.28-1.29(d，3H)，1.50-1.57(m，1H)，1.74-1.77(m，1H)，3.29-3.33(m，1H)，3.44-3.48(m，1H)，3.55-3.58(m，1H)，3.64-3.69(m，1H)，4.37-4.47(m，2H)，4.58-4.60(m，1H)，5.11(s，2H)，7.33-7.44(m，10H)，7.59-7.65(m，5H)

1- (4- (tert-butyldiphenylsilyloxy) -2-formylpyrrolidin-1-yl) -1-oxopropan-2-ylcarbamic acid benzyl ester

Benzyl 1- (4- (tert-butyldiphenylsiloxy) -2- (hydroxymethyl) pyrrolidin-1-yl) -1-oxopropan-2-ylcarbamate 2.8g (0.005mol) was dissolved in 50ml dichloromethane, and DMP 2.5g (0.006mol) was added and the reaction was carried out for 4 hours. Filtering, and performing flash column chromatography to obtain 2.22 g.

Benzyl 1- (6- (tert-butyldiphenylsiloxy) -6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamate

Benzyl 1- (4- (tert-butyldiphenylsilyloxy) -2-formylpyrrolidin-1-yl) -1-oxopropan-2-ylcarbamate 0.5g, ammonium acetate 2.5g, toluene 10ml, 100 ℃ reaction overnight. Adding water, extracting, and performing column chromatography to obtain 0.41 g.

¹H NMR(300M，CDCl₃)：δ1.06(s，9H)，1.48-1.49(d，3H)，2.75-2.90(m，2H)，3.83-3.96(m，2H)，4.80-4.84(m，1H)，4.91-4.94(m，1H)，5.06-5.15(m，2H)，6.54(s，1H)，7.30-7.46(m，10H)，7.62-7.66(m，5H)

1- (6-hydroxy-6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamic acid benzyl ester

0.4g of benzyl 1- (6- (tert-butyldiphenylsiloxy) -6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamate was dissolved in 10ml of tetrahydrofuran, and TBAF1ml was added thereto and reacted for 2 hours. Adding water and ethyl acetate, extracting, separating liquid, and performing column chromatography to obtain 0.21 g.

¹H NMR(300M，CDCl₃)：δ1.51-1.52(d，3H)，2.74-2.78(m，1H)，3.05-3.10(m，1H)，3.92-4.05(m，2H)，4.83-4.87(m，1H)，4.98(m，1H)，5.03-5.11(m，2H)，6.57(s，1H)，7.31-7.36(m，5H)

MS experimental value m/z: 302.1(M + 1).

1- (6-oxo-6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamic acid benzyl ester

0.2g (0.00067mol) of benzyl 1- (6-hydroxy-6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamate is dissolved in 5ml of DCM, 0.34g (0.0008mol) of DMP is added, stirring is carried out for 2H, filtering is carried out, and the filtrate is subjected to column chromatography to obtain 0.185 g.

MS experimental value m/z: 300.1(M + 1).

1- (7-allyl-6-oxo-6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamic acid benzyl ester

0.33g (0.0011mol) of benzyl 1- (6-oxo-6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamate is dissolved in 5ml of anhydrous tetrahydrofuran, cooled to-80 ℃, 1ml (1mmol) of LDA is added dropwise, and the mixture is kept warm for 10 min. 0.12g of allyl bromide is added dropwise, and the reaction is naturally warmed up. After 2 hours, the reaction was quenched, and then ethyl acetate and saturated brine were added, followed by liquid-separation extraction and column chromatography to obtain 0.23 g.

¹H NMR(300M，CDCl₃)：δ1.51-1.52(d，3H)，2.50-2.57(m，1H)，2.79-2.99(m，2H)，3.93-4.04(m，2H)，4.96-5.01(m，1H)，5.05-5.12(m，4H)，5.68-5.78(m，1H)，6.56(s，1H)，7.31-7.36(m，5H)

MS experimental value m/z: 340.2(M + 1).

Benzyl 1- (6-acetylamino-7-allyl-6- (tert-butylcarbamoyl) -6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamate

0.6g (0.002mol) of benzyl 1- (7-allyl-6-oxo-6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamate was dissolved in 5ml of trifluoroethanol, and 0.83g (0.01mol) of tert-butyl isocyanate and 1.54g (0.02mol) of ammonium acetate were added thereto and reacted overnight. Adding water, extracting with ethyl acetate, and performing column chromatography to obtain 0.53 g.

¹H NMR(300M，CDCl₃)：δ1.42(m，9H)，1.51-1.52(d，3H)，2.02(s，3H)，2.50-2.57(m，1H)，2.80-3.00(m，2H)，3.92-4.03(m，2H)，4.96-5.01(m，1H)，5.05-5.11(m，4H)，5.68-5.78(m，1H)，6.56(s，1H)，7.30-7.36(m，5H)

MS experimental value m/z: 482.3(M + 1).

Benzyl 1- (6-acetylamino-6- (tert-butylcarbamoyl) -7- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) -6,7 dihydro-5H-pyrrolo [1,2-E ] imidazol-3-yl) ethylcarbamic acid

0.39g (0.0008mol) of benzyl 1- (6-acetamido-7-allyl-6- (tert-butylcarbamoyl) -6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazol-3-yl) ethylcarbamate is dissolved in 5ml of dichloromethane, 26.9mg (0.04mmol) of bis (1, 5-cyclooctadiene) iridium chloride dimer and 32.4mg (0.82mmol) of DPPE are added, the temperature is reduced to 0 ℃, 0.2g (1.6mmol) of pinacol borane is added dropwise, the reaction is naturally warmed after the addition, and the end point of the reaction is detected by TLC. Adding water to quench the reaction, separating an organic phase, extracting a water phase with dichloromethane, combining, washing with water, and performing column chromatography to obtain 0.31 g.

MS experimental value m/z: 610.4(M + 1).

6-acetylamino-3- (1-aminoethyl) -N-tert-butyl-7- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) -6-, 7-dihydro-5H-pyrrolo [1,2-E ] imidazole-6-carboxamide

Benzyl 1- (6-acetylamino-6- (tert-butylcarbamoyl) -7- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) -6,7 dihydro-5H-pyrrolo [1,2-E ] imidazol-3-yl) ethylcarbamic acid 0.31g, methanol 5ml, palladium on charcoal 0.05g, hydrogenated overnight. Filtered and concentrated to obtain 0.25g of crude product.

MS experimental value m/z: 476.3(M + 1).

Compound No. 17:

6-amino-3- (1-aminoethyl) -7- (3-boropropyl) -6, 7-dihydro-5H-pyrrolo [1,2-e ] imidazole-6-carboxylic acid

6-acetylamino-3- (1-aminoethyl) -N-tert-butyl-7- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) -6-, 7-dihydro-5H-pyrrolo [1,2-E ] imidazole-6-carboxamide 0.25g crude product was reacted with 5ml of 4MHCl at 50 ℃ for 7 hours, concentrated to dryness, purified to give 33.5 mg.

¹H NMR(300M，D₂O)：δ0.80-0.89(m，2H)，1.36-1.55(m，6H)，1.73-1.79(m，1H)，2.89-2.92(m，1H)，3.92-4.08(m，3H)，6.55(s，1H)

MS experimental value m/z: 279.2(M + 1-18).

2-methyl-4-chlorothiazole

1g (6.5mmol) of 2, 4-dichlorothiazole, 6.5ml (6.5mmol, 1mol/L) of trimethylaluminum, 923mg (0.65mmol) of palladium tetratriphenylphosphine and 10ml of anhydrous 1, 4-dioxane were added into a microwave reaction tube. Argon gas replacement and microwave reaction at 100 deg.c for 20 min. Cooling, pouring the reaction solution into saturated sodium bicarbonate solution, extracting with ethyl acetate, washing with saturated saline solution, and performing column chromatography to obtain 450 mg.

¹H NMR(300M，CDCl3)：δ2.80(s，3H)，6.96(s，1H)

2-methyl-4-chloro-5-nitrothiazole

360mg (2.7mmol) of 2-methyl-4-chlorothiazole was dissolved in 5ml of concentrated sulfuric acid, and 0.32g (3.12mmol) of potassium nitrate was added thereto in an ice bath, followed by allowing the reaction to proceed overnight with natural warming. The reaction solution was poured into crushed ice to precipitate a solid, which was filtered and dried to obtain 322 mg.

¹H NMR(300M，CDCl3)：δ2.93(s，3H)

Compound No. 18:

(3R, 4S) -3-amino-1- (5-amino-2-methylthiazol-4-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid

Using a synthetic method of (3R, 4S) -3-amino-1- (3-aminopyridin-2-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid, 16mg of the objective compound was prepared using (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester and fragment 4-chloro-5-amino-2-methylthiazole as starting materials.

1H NMR(300M，CDCl3)：δ0.67-0.85(m，2H)，1.20-1.28(m，1H)，1.32-1.45(m，2H)，1.58-1.66(m，1H)，2.55-2.75(m，4H)，3.60-3.66(m，1H)，3.91-3.96(m，1H)，4.12-4.30(m，2H)

MS experimental value m/z: 311.1(M + 1-18).

3-iodo-1-methyl-1H-pyrazoles

0.97g (0.01mol) of 1-methyl-1H-pyrazole-3-amine is dissolved in 10ml of 4M HCl, the temperature is reduced to-10 ℃, and 0.76g (0.011mol) of aqueous solution (5ml) of sodium nitrite is dripped, and the temperature is controlled to be lower than 0 ℃. And preserving the temperature for 20 min.

3g (0.02mol) of sodium iodide are dissolved in 10ml of water and the temperature is reduced to below 0 ℃. The amine solution is added dropwise into sodium iodide, and the temperature is controlled to be not higher than 0 ℃. After dropping, naturally raising the temperature for reaction, and after half an hour, removing the ice bath for reaction for 4 hours. Adjusting the pH value to 8-9, extracting with dichloromethane and methanol, and concentrating to obtain 1.2g of crude product. The column chromatography gave 0.5 g.

1H NMR(300M，CDCl3)：δ3.92(s，3H)，6.39-6.40(d，1H)，7.19-7.20(d，1H)

3-iodo-1-methyl-4-nitro-1H-pyrazoles

0.42g (2mmol) of 3-iodo-1-methyl-1H-pyrazole was dissolved in 3ml of concentrated sulfuric acid, and 0.21g (2.2mmol) of concentrated nitric acid was added dropwise thereto, followed by completion of the addition at 60 ℃ for 2 hours. The reaction solution was poured into crushed ice to precipitate a white solid, which was then filtered and dried to obtain 0.4 g.

1H NMR(300M，CDCl3)：δ3.69(s，3H)，8.11(s，1H)

(3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester 0.82g (2mmol), 3-iodo-1-methyl-4-nitro-1H-pyrazole 0.5g (2mmol), palladium diacetate 22.5mg (0.1mmol), sodium tert-butoxide 0.38g (4mmol), toluene 20ml, 1,1' -bis (diphenylphosphino) ferrocene 110mg (0.2mol) were added to a reaction flask in this order, replaced with argon three times, and reacted at 80 ℃ for 4 hours. The reaction solution was extracted with water and ethyl acetate, washed and subjected to column chromatography to obtain 0.53g of crude product.

MS experimental value m/z: 540.3(M +1)

Compound No. 19:

(3R, 4S) -3-amino-1- (4-amino-1-methyl-1H-pyrazol-3-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid

(3R, 4S) -3-azido-1- (3-nitropyridin-2-yl) -4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester was dissolved in 10ml of methanol, 0.2g of palladium on charcoal and 0.2g of acetic acid were added, and hydrogenation was carried out overnight. Filtering to remove palladium carbon, concentrating to dryness, adding 15ml of 3M HCl into the crude product, reacting at 55 ℃ for 6h, concentrating to dryness, and preparing and separating to obtain 99.3mg of a target product.

1H NMR(300M，CDCl3)：δ0.72-0.78(m，2H)，1.30-1.48(m，3H)，1.63-1.69(m，1H)，2.63-2.69(m，1H)，3.49-3.53(m，1H)，3.39(s，3H)，3.93-4.13(m，3H)，7.38(s，1H)

MS experimental value m/z: 294.2(M + 1-18).

3-chloro-1- (2- (tetrahydro-2H-pyran-2-yloxy) ethyl) -1H-pyrazole

1.75g (0.017mol) of 3-chloro-1H-pyrazole, 4.1g (0.02mol) of 2- (2-bromoethoxy) -tetrahydro-2H-pyran, 5g (0.036mol) of potassium carbonate, 0.9g (2.8mmol) of tetrabutylammonium bromide and 40ml of DMF, reacting at normal temperature overnight, adding water, extracting with EA, washing, and carrying out column chromatography on a crude product to obtain 3.58 g.

1H NMR(300M，CDCl3)：δ1.47-1.57(m，4H)，1.64-1.76(m，2H)，3.43-3.49(m，1H)，3.61-3.68(m，1H)，3.71-3.77(m，1H)，4.01-4.06(m，1H)，4.24-4.27(m，2H)，4.52-4.54(m，1H)，6.14-6.15(d，1H)，7.42-7.43(d，1H)

Compound No. 20:

(3R, 4S) -3-amino-4- (3-dihydroxyboropropyl) -1- (1- (2-hydroxyethyl) -1H-pyrazol-3-yl) pyrrolidine-3-carboxylic acid

Synthesis of (3R, 4S) -3-amino-1- (4-amino-1-methyl-1H-pyrazol-3-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and fragment 3-chloro-1- (2- (tetrahydro-2H-pyran-2-yloxy) ethyl) -1H-pyrazole gave 36.1mg of the title compound.

1H NMR(300M，CDCl3)：δ0.69-0.84(m，2H)，1.28-1.45(m，3H)，1.65-1.69(m，1H)，2.66-2.74(m，1H)，3.58-3.75(m，2H)，3.96-4.14(m，3H)，4.22-4.30(m，3H)，6.12-6.13(d，1H)，7.41-7.42(d，1H)

MS experimental value m/z: 309.2(M + 1-18).

3, 5-dibromo-4-cyano-1-H-pyrazoles

6.76g (25.2mmol) of 4-cyano-1-H-pyrazole was dissolved in 150ml of ethanol and 225ml of water, and 29.1g (355mmol) of sodium acetate was added thereto. 10.75ml (209mmol) of bromine was added dropwise with stirring, and stirring was continued for 2 hours. Diluting with water, extracting with dichloromethane, and washing with sodium thiosulfate and water. Concentrating, dissolving the crude product with ethyl acetate, adding n-hexane under stirring to turn turbid, continuing stirring for half an hour, filtering off insoluble substances, and concentrating the filtrate to obtain 8.9 g.

¹H NMR(300M，DMSO-d6)：δ15.02(br，1H)

MS experimental value m/z: 249.9(M +1)

1-methyl-3, 5-dibromo-4-cyano-1-H-pyrazoles

Dissolving 1.85g (7.37mmol) of 3, 5-dibromo-4-cyano-1-H pyrazole in 5ml of DMF, cooling to 0 ℃ under the protection of argon, adding 0.32g (8mmol) of sodium hydrogen, dropwise adding 1.38ml (22mmol) of methyl iodide after 5 minutes, and naturally heating to react for 2 hours after dropwise adding. Adding saturated ammonium chloride for quenching, extracting by ethyl acetate and washing. The crude product is subjected to flash column chromatography to obtain 1.48 g.

MS experimental value m/z: 263.9(M +1)

1-methyl-3-bromo-4-cyano-5-amino-1-H-pyrazoles

1.5g (5.66mmol) of 1-methyl-3, 5-dibromo-4-cyano-1-H pyrazole, 1.23g (7.36mmol) of 2, 4-dimethoxybenzylamine and 4ml of N-methylpyrrolidone were added to the pot, and the reaction was carried out under sealed conditions at 190 ℃ for 3 hours. The reaction solution is subjected to column chromatography directly to obtain 286 mg.

MS experimental value m/z: 201(M +1)

1-methyl-3-bromo-4-cyano-1-H-pyrazoles

0.22g (1.1mmol) of 1-methyl-3-bromo-4-cyano-5-amino-1-H-pyrazole was dissolved in 10ml of tetrahydrofuran, and 0.26ml (2.2mmol) of tert-butyl nitrite was added. Reacting at 50 ℃ for 3 hours, pouring the reaction liquid into water, extracting by ethyl acetate and washing. Concentrate to give crude 210 mg.

¹H NMR(300M，DMSO-d6)：δ3.9(s，3H)，8.57(s，1H)

MS experimental value m/z: 186(M +1)

Compound No. 21:

(3R, 4S) -3-amino-1- (4- (aminomethyl) -1-methyl-1H-pyrazol-3-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid

Synthesis of (3R, 4S) -3-amino-1- (4-amino-1-methyl-1H-pyrazol-3-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid using benzyl (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylate and tert-butyl ester of the fragment (1-methyl-1H-pyrazol-4-yl) methylcarbamate as starting materials gave 62.8mg of the objective compound.

¹H NMR(300M，CDCl3)：δ0.68-0.79(m，2H)，1.30-1.48(m，3H)，1.63-1.68(m，1H)，2.63-2.69(m，1H)，3.48-3.53(m，1H)，3.39(s，3H)，3.91-4.12(m，3H)，7.36(s，1H)

MS experimental value m/z: 308.2(M + 1-18).

3-Hydroxyazetidine-1-carboxylic acid tert-butyl ester

2.5g (0.023mol) of azetidine-3-ol hydrochloride was added 50ml of methylene chloride, 3.6g (0.036mol) of triethylamine, and 5.5g (0.025mol) of di-tert-butyl dicarbonate, and the reaction was allowed to proceed overnight. The reaction solution is subjected to flash column chromatography to obtain 2.5g of a product.

3- (benzyloxy) azetidine-1-carboxylic acid tert-butyl ester

2.1g (0.012mol) of 3-hydroxyazetidine-1-carboxylic acid tert-butyl ester is dissolved in 30ml of DMF, the temperature is reduced to 0 ℃, 0.6g (0.015mol) of sodium hydride is added, the mixture is stirred for 10min, 2.1g (0.012mol) of benzyl bromide is added dropwise, and the mixture is naturally heated and reacted overnight. Cooling, adding saturated ammonium chloride to quench reaction, extracting with ethyl acetate, washing, and performing column chromatography to obtain 2.4g of product.

¹H NMR(300M，CDCl3)：δ1.43(s，9H)，3.84-3.88(m，2H)，4.03-4.07(m，2H)，4.28-4.33(m，1H)，4.45(s，2H)，7.31-7.38(m，5H)

3- (benzyloxy) azetidines

2.4g of tert-butyl 3- (benzyloxy) azetidine-1-carboxylate was dissolved in 50ml of methanol, and 0.25g of palladium on carbon was added thereto, followed by hydrogenation overnight. Filtered and concentrated to give 1.39g of crude product.

¹H NMR(300M，CDCl3)：δ3.69-3.73(m，2H)，3.96-4.00(m，2H)，4.25-4.30(m，1H)，4.44(s，2H)，7.31-7.39(m，5H)

MS experimental value m/z: 164.1(M + 1).

1-methyl-4-chloro-3-nitro-1H-pyrazoles

4.14g (0.033mol) of 1-methyl-3-nitro-1H-pyrazole was dissolved in DMF40ml, and NCS 7g (0.052mol) was added. Oil bath reaction at 100 ℃ for 7h, and column chromatography to obtain 2.63g of product.

¹H NMR(300M，CDCl3)：δ7.65(s，1H)

4- (3- (benzyloxy) azetidin-1-yl) -1-methyl-3-nitro-1H-pyrazole

1g (6.2mmol) of 1-methyl-4-chloro-3-nitro-1H-pyrazole, 1g (6.2mmol) of 3- (benzyloxy) azetidine, 67.5mg (0.3mmol) of palladium diacetate, 1.18g (12.4mmol) of sodium tert-butoxide, 20ml of toluene, 330mg (0.6mol) of 1,1' -bis (diphenylphosphino) ferrocene, 100 ℃ reaction for 8H. Diluting with ethyl acetate 20ml, washing with water, and performing column chromatography to obtain 1.16g of product.

MS experimental value m/z: 189.1(M + 1).

4- (3- (benzyloxy) azetidin-1-yl) -1-methyl-3-amino-1H-pyrazole

0.96g of 4- (3- (benzyloxy) azetidin-1-yl) -1-methyl-3-nitro-1H-pyrazole is dissolved in 15ml of acetic acid, 1g of iron powder is added, reflux reaction is carried out for 1H, filtration is carried out, filtrate is concentrated and dichloromethane is added for dilution, saturated sodium carbonate is added for adjusting the pH to 8-9, liquid separation is carried out, dichloromethane is washed, drying is carried out, 0.65g of crude product is concentrated, and the step is directly carried out for steps.

MS experimental value m/z: 259.1(M + 1).

4- (3- (benzyloxy) azetidin-1-yl) -3-iodo-1-methyl-1H-pyrazole

0.6g (1.6mmol) of 4- (3- (benzyloxy) azetidin-1-yl) -1-methyl-3-amino-1H-pyrazole was dissolved in 5ml of isopropanol, and 0.45g (3.2mmol) of iodomethane was added. 0.33g (3.2mmol) of tert-butyl nitrite is dripped, reaction is carried out for 3 hours at 50 ℃, concentration and direct column chromatography are carried out to obtain 0.49g of product.

MS experimental value m/z: 370.0(M + 1).

Compound No. 22:

(3R, 4S) -3-amino-4- (3-dihydroxyboropropyl) -1- (4- (3-hydroxypyrrolidin-1-yl) -1-methyl-1H-pyrazol-3-yl) pyrrolidine-3-carboxylic acid

Synthesis of (3R, 4S) -3-amino-1- (4-amino-1-methyl-1H-pyrazol-3-yl) -4- (3-dihydroxyboropropyl) pyrrolidine-3-carboxylic acid using (3R, 4S) -3-azido-4- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-3-carboxylic acid benzyl ester and fragment 4- (3- (benzyloxy) azetidin-1-yl) -3-iodo-1-methyl-1H-pyrazole gave 61.1mg of the title compound.

¹H NMR(300M，CDCl3)：δ0.72-0.77(m，2H)，1.30-1.48(m，3H)，1.63-1.68(m，1H)，2.62-2.68(m，1H)，3.39(s，3H)，3.45-3.50(m，1H)，3.69-3.73(m，2H)，3.93-4.13(m，5H)，4.25-4.30(m，1H)，7.38(s，1H)

MS experimental value m/z: 350.2(M + 1-18).

The compounds shown in table 1 were used as arginase inhibitors, and the evaluation test was as follows:

establishment and optimization of arginase activity detection method

Arginase can catalyze L-arginine to convert L-ornithine and urea, so that the activity level of the arginase can be judged by detecting the content of the urea in a reaction end point solution.

(1) Sources of materials

TABLE 2

All reagents are stored according to the requirements of the specification after being received, and are strictly sealed after being prepared each time.

(2) Solution preparation

Which comprises the following steps: 500mM L-Arginine (A600205-0100, Sangon); 250mM MnSO4(A601717-0250, Sangon); various concentrations of inhibitor (1000 ×); arginase I (AR1-H5228, ACROBIOSystems) at 200. mu.g/ml; reagent A (see Table 3 for composition); reagent B (see Table 3 for composition).

TABLE 3 composition of Buffer A and Buffer B and product-related information

Arginase I from human origin was dissolved in sterile deionized water containing 0.1% BSA at a stock concentration of 200 μ g/mL. ABH and all candidate inhibitors were dissolved in DMSO, and the stock concentrations were selected based on the weight of compound provided (50mM/100 mM). L-arginine (500mM) and magnesium sulfate (250mM) were prepared in sterile DMEM medium.

(3) Detection method

In the optimization process, different gradient conditions of L-arginine concentration (5mM/25mM), manganese ion concentration (2.5mM/5mM), arginase I dosage (2ng/5ng/50ng/100ng) and absorbance reading wavelength (450nm/490nm) are searched, as shown in FIG. 1-2, and the final optimization result is obtained.

TABLE 4 optimum concentrations and conditions of the different reagents in the reaction System

The method for detecting the activity of the arginase specifically comprises the following steps:

① A reaction mixture was prepared according to Table 5, and the mixture was added to a 96-well plate for enzyme-catalyzed reaction, each run was divided into 5 separate replicates, treated with 9 different inhibitor concentration gradients.

TABLE 5

② 37 ℃ for 2 hours;

③ stop buffer was prepared according to Table 6;

TABLE 6

Component name	Volume (μ l)
		Reagent A	75
Reagent B	75

In a 96-well plate, 150. mu.l of stop buffer was added to each reaction well.

④ was allowed to stand at room temperature for 1 hour.

The absorbance at 450nm was read in an ⑤ microplate reader.

⑥ read OD₄₅₀Analyzing the data, fitting and calculating IC₅₀。

By testing urea (2ng) and the inhibitor positive control ABH (IC)₅₀2.4 +/-1.2 mu M and the literature reports 1.6 +/-0.8 mu M), and the established method for detecting the arginase activity is high in sensitivity, repeatability and accuracy and meets the required standard.

II, screening of arginase I inhibitor candidate activity

Inhibitor candidates, DMSO dissolved and dispensed. Different concentration gradients were designed as in table 7.

TABLE 7

Inhibitor concentration (stock solution, mM)	Volume (μ l)	Final concentration (μ M)
			0	0.1	0
0.01	0.1	0.01
			0.05	0.1	0.05
0.1	0.1	0.1
			0.5	0.1	0.5
1	0.1	1
			2	0.1	2
5	0.1	5

Through literature reports and a large number of experiments, the DMSO concentration is not influenced by the cells when the DMSO dosage is less than or equal to 0.1 percent, so the dilution concentration is selected to be 0.1 percent.

For each inhibitor IC₅₀8 different concentration gradients and 4-6 independent replicates were designed. And each replicate was run in parallel with a positive control, ABH, for comparison of inhibitory activity.

Thirdly, construction of arginase I overexpression cell line

In order to confirm the inhibitory effect of the inhibitor on Arginase I in step , a cell line which stably overexpresses human Arginase I in CHO cells is constructed, and the expression quantity and the expression activity of the Arginase I are detected on the constructed cell line.

(1) Construction of cell line stably overexpressing human Arginase I in CHO cells

As shown in FIG. 3, hARGI fragment was amplified using cDNA from CHO cells as a template and constructed into lentiviral vector pLVX-IRES-Puro (Cat #632183, Clontech). pLVX-hARGI was co-transferred with the plasmid of the lentivirus system into 293T cells using transfection reagents and the venom was collected after 48 h. Adding the venom into CHO cells, and replacing with fresh culture medium after 8-12 h. After 2-3 days of infection, puromycin with a concentration of 10. mu.g/mL was used for drug resistance selection and stable over-expressing cell lines were obtained.

And (3) utilizing the constructed cell strain to select the inhibitor for primary screening as an inhibitor compound which is subjected to cell level further screening and cytological toxicity test of the inhibitor, and finally obtaining the high-efficiency and low-toxicity inhibitor compound.

(2) Detection of Arginase I expression level and Activity in stably overexpressing cell lines:

collecting over-expression cells, extracting RNA, performing RT-PCR detection after reverse transcription, and obtaining a quantitative detection result shown in figure 4.

Arginase activity assay was performed on the constructed cell lines and appropriate inoculum numbers were searched, and the results are shown in FIG. 5. According to the experimental results and observations, inoculation was 1.5X 10⁴The cells are suitably single/well cells and the over-expressing cell line has Arginase activity.

The experimental procedure was as follows:

(1) day 1: and (4) inoculating the target cells. 2X 10⁴Repeating for each hole, namely a 96-hole plate, and 3-5 holes;

and (4) taking target cells in logarithmic phase, digesting and collecting, and centrifuging to remove supernatant. Rinsing with PBS 1-2 times, resuspending the cells in DMEM/F12 basal medium, counting, diluting the cells to 2X 10⁵one/mL. And (3) inoculating 100 mu l of cell suspension into a 96-well plate, and repeating 3-5 wells of cells.

(2) Day 2: adding an arginase inhibitor to be tested to the cells;

the culture medium in the well plate was aspirated, and 100. mu.l/well of the following mixture was added to the well plate, followed by incubation at 37 ℃ for 2 hours.

The specific reaction system is shown in Table 8:

TABLE 8

Components	Concentration of	Volume of
			L-arginine	500mM	1μl
Inhibitors	Corresponding different concentrations	0.1μl
			DMEM/F12 air culture	1×	98.9μl

The experimental group is wells inoculated with cells, and the control group is wells not inoculated with cells, which are repeated in 3-5 wells.

(3) Day 3: the reaction was terminated and detected.

The well plate was removed, the supernatant was transferred to a new well plate, 150. mu.l of a stop solution (a mixture of 75. mu.l of solution A and 75. mu.l of solution B) was added to each well, and after incubation at room temperature for 1.5 hours, OD was read with a microplate reader₄₅₀Numerical, analysis of data, fitting and calculation of IC₅₀。

The final results are shown in table 9 below,

TABLE 9

Potency 1:0.1nM to 250nM,2:251nM to 1000nM,3:1001nM to 2000nM,4:2001nM to 5000nM,5: >5001 nM.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1, kinds of compounds, characterized in that, the compound is represented by the general formula (I),

wherein:

R₁and R₂Selected from hydrogen atoms;

R₃and R₅Selected from hydrogen atoms;

R₄selected from the group consisting of-C (O) R₆Heteroaryl or fused ring, wherein said heteroaryl or fused ring is optionally substituted more times with or more substituents selected from alkyl, amino, aminoalkyl, hydroxyalkyl, phenyl, heterocyclyl or 3-8 membered cycloalkyl, wherein said alkyl, phenyl, heterocyclyl or cycloalkyl is optionally more times with or more substituents selected from halogen, alkyl, alkoxy, hydroxyalkylSubstituted with a substituent of a group, haloalkyl, haloalkoxy, amino or aminoalkyl; wherein the fused ring is a fused ring of heteroaryl and cycloalkyl or heterocyclic group;

2. A compound according to claim 1, characterized in that: wherein R is₄Selected from 5-6 membered heteroaryl, preferably selected from pyrazolyl, imidazolyl, pyrimidinyl, thiazolyl or pyridyl, wherein said heteroaryl is optionally substituted in step with or more substituents selected from alkyl, amino, aminoalkyl, hydroxyalkyl, phenyl, heterocyclyl or 3-8 membered cycloalkyl, wherein said alkyl, phenyl, heterocyclyl or cycloalkyl is optionally substituted in step with or more substituents selected from halogen, alkyl, alkoxy, hydroxyalkyl, haloalkyl, haloalkoxy, amino or aminoalkyl.

3. A compound according to claim 1, characterized in that: wherein R is₄Selected from:

wherein:

R₇、R₈and R₉Selected from hydrogen atom, alkyl, phenyl or 3-8 membered cycloalkyl, wherein the alkyl, phenyl or 3-8 membered cycloalkyl is optionally further substituted by alkyl or aminoA hydroxyl group, a halogen group, an aminoalkyl group, a hydroxyalkyl group, a haloalkyl group or a 3-to 8-membered cycloalkyl group.

4. A compound according to claim 1, characterized in that: wherein R is₆Selected from:

5. a compound according to claims 1 to 4, characterized in that: wherein said compound is selected from:

6. the compound of according to any one of claims 1-5, which is further characterized by being a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

7. A process for the preparation of a compound according to claims 1 to 5, comprising the steps of:

reacting the compound of the general formula (IA) with … to obtain a compound of the general formula (IB), and hydrolyzing the compound of the general formula (IB) to obtain a compound of the general formula (I);

wherein: r₁～R₅Is as defined in claim 1.

8. A process for the preparation of a compound according to claims 1 to 5, comprising the steps of:

hydrolyzing the compound of formula (IC) at step to obtain a compound of formula (I);

wherein: r₁～R₅Is as defined in claim 1.

A pharmaceutical composition of comprising an effective amount of a compound of any of of claims 1-5 or a tautomer, mesomer, racemate, enantiomer, diastereomer, solvate, hydrate, pharmaceutically acceptable salt, or mixture thereof.

An arginase inhibitor comprising the compound of any of claims 1-5 or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 9.

An medicament for immunotherapy of tumors, comprising the compound of any of claims 1-5 or its tautomer, mesomer, racemate, enantiomer, or diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, or the pharmaceutical composition of claim 9, wherein said tumors are preferably selected from renal cell carcinoma, breast cancer, non-small cell lung cancer, acute myelogenous leukemia.

12, A therapeutic agent for ischemia reperfusion injury, hypertension, atherosclerosis, diabetes, erectile dysfunction or pulmonary hypertension, comprising the compound of any of claims 1-5 or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 9.