Boric acid fused ring derivative, and pharmaceutical composition and application thereof

Abstract

The invention discloses a boric acid fused ring derivative as well as a pharmaceutical composition and application thereof, and particularly discloses a compound as shown in a formula I, a stereoisomer and pharmaceutically acceptable salt thereof. In order to solve the technical problem that a broad-spectrum beta-lactamase inhibitor is lacked clinically at present, the invention provides the boric acid fused ring derivative as well as the pharmaceutical composition and the application thereof. The compound provided by the invention can inhibit various SBLs and MBLs at the same time, and has relatively high inhibitory activity on CRAB. In addition, the compounds of the present invention can be used in combination with beta-lactam antibiotics for the treatment of bacterial infections.

Description

TECHNICAL FIELD

[0001] The present application relates to boronic acid and cyclic derivatives, pharmaceutical compositions thereof and uses thereof. BACKGROUND

[0002] Due to the overuse of antibiotics, bacterial drug resistance has become a major challenge in the field of global public health in the 21st century.

[0003] β-lactam antibiotics are the most widely used antibacterial drugs in clinical practice, including penicillins and their derivatives, cephalosporins, monobactams, carbapenems, and penemase inhibitors, accounting for more than 50% of the market. The mechanism of action of β-lactam antibiotics is to bind to penicillin-binding proteins (PBPs), inhibit the synthesis of bacterial cell wall components peptidoglycan, and destroy the cell wall structure to achieve bactericidal effect.

[0004] Bacteria can express β-lactamase, which can hydrolyze the amide bond of β-lactam antibiotics, resulting in inactivation, thereby enabling bacteria to resist the killing effect of antibacterial drugs. The expression of β-lactamase is one of the most important drug resistance strategies of bacteria. To date, more than 1400 β-lactamases have been discovered and identified. According to Ambler classification, β-lactamases can be divided into serine enzymes (SBLs, including classes A, C and D) and metalloenzymes (MBLs, class B). In clinical practice, carbapenem antibiotics are a class of potent antibacterial drugs with broad antibacterial spectrum, strong antibacterial activity, and good stability to many β-lactamases, and are the "last line of defense" for drug-resistant bacterial infections. Currently, the rapid spread of carbapenem-resistant bacteria has become a serious challenge in the treatment of drug-resistant bacteria in clinical practice. Carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are the most common carbapenem-resistant bacteria in China, which are mainly mediated by carbapenem resistance through the enzymes of class A KPC, class B NDM, VIM, and class D OXA. Among them, due to the complex drug resistance mechanism, the lack of effective treatment drugs, and the high mortality rate, CRAB has been listed as a "key priority" pathogen by the World Health Organization. In addition, carbapenem-resistant bacteria carrying MBLs have spread widely around the world, and there is currently no effective treatment drug approved. According to the 2023 monitoring results reported by the China Bacterial Drug Resistance Monitoring Network (CHINET), the resistance rates of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii to carbapenem antibiotics meropenem were 26%, 17.4%, and 73.7%, respectively.

[0005] Beta-lactamase inhibitors can enhance the antibacterial activity of beta-lactam antibiotics and have been used in combination in the treatment of clinically resistant bacterial infections. Currently, the OXA-23 type enzyme commonly found in MBLs and CRAB is still an important challenge in the clinical treatment of resistant bacteria. MBLs can hydrolyze almost all beta-lactam antibiotics except monobactams, and are distributed in CRE, CRPA and even CRAB. OXA-23 type carbapenemases are commonly found in CRAB. Both of them are highly dangerous, and there is currently no approved drug that can effectively inhibit MBLs and OXA-23 type enzymes at the same time. In recent years, Zoliflodacin (sulbactam / dorobactam) can effectively inhibit OXA-23 and has good inhibitory effect on CRAB, but it has no inhibitory activity on MBLs. Studies have found that CRAB resistant strains (such as ST164 clone) carrying MBLs have developed resistance to Zoliflodacin. QPX7728 is the fastest clinically advanced drug that can simultaneously inhibit a variety of SBLs and MBLs and has good inhibitory activity on CRAB (can effectively inhibit the OXA-23 type carbapenemase commonly found in CRAB). It has a wide inhibitory spectrum (can simultaneously inhibit a variety of enzymes of classes A, B, C and D) and high activity, and is still in clinical phase I.

[0006] In summary, for carbapenem-resistant bacteria, especially CRAB-resistant bacteria, the existing beta-lactamase inhibitors cannot meet the clinical needs. There is an urgent need for new broad-spectrum beta-lactamase inhibitors that can simultaneously inhibit a variety of SBLs and MBLs and have high inhibitory activity on CRAB to cope with the increasingly severe situation of clinically resistant bacterial infections. SUMMARY

[0007] The technical problem to be solved by the present application is the lack of broad-spectrum beta-lactamase inhibitors in current clinical practice. The present application provides boronic acid and cyclic derivatives, pharmaceutical compositions thereof and applications thereof. The compounds of the present application can simultaneously inhibit a variety of SBLs and MBLs and have high inhibitory activity on CRAB. In addition, the compounds of the present application can be used in combination with beta-lactam antibiotics for the treatment of bacterial infections.

[0008] The present application solves the above technical problems through the following technical solutions.

[0009] The present application provides a compound as shown in formula I, stereoisomers and pharmaceutically acceptable salts thereof.

[0010]

[0011] wherein,

[0012] ​Ring A is "5-6 membered heterocyclenyl having 1, 2, or 3 heteroatoms selected from N, O, and S, and having 1, 2, or 3 double bonds" or C 5-6 Cycloalkenyl;

[0013] R is NR 1 R 2 , S(O)2NR 3 R 4 , or C(O)NR 5 R 6 ;

[0014] n1 is 0 or 1;

[0015] R 0 is D;

[0016] n2 is 0, 1, 2, 3, or 4;

[0017] R 1 is H, D, C 1-6 deuteroalkyl, or C 1-6 alkyl;

[0018] R 2 is H, D, -C(O)R 2-1 , -S(O)R 2-1 , S(O)2R 2-1 , C 1-6 alkyl, C 1-6 deuteroalkyl, C 2-4 substituted with one or more R 1-6 deuteroalkyl, or C 2-4 substituted with one or more R 1-6 alkyl;

[0019] R 2-1 is C 1-6 alkyl, C 1-6 deuteroalkyl, C 2-1a substituted with one or more R 1-6 deuteroalkyl, or C 2-1a substituted with one or more R 1-6 alkyl;

[0020] R 2-1a is independently OH, OD, NR a R b , C 1-6 alkoxy, or C 1-6 deuteroalkoxy;

[0021] R 2-2 is H, D, C 1-6 deuteroalkyl, or C 1-6 alkyl;

[0022] R 2-3 is C 1-6 alkyl, C 1-6 deuteroalkyl, NR a R b , C 2-3a deuteroalkyl or C 1-6 deuteroalkyl substituted with one or more R 2-3a ; and R 1-6 is alkyl;

[0023] R 2-3a is independently OH, OD, NR a R b , C 1-6 deuteroalkyl or C 1-6 deuteroalkyl;

[0024] R 2-4 is independently OH, OD, NR a R b , C 1-6 deuteroalkyl or C 1-6 deuteroalkyl;

[0025] R a and R b are independently H, D, C 1-6 deuteroalkyl or C 1-6 alkyl;

[0026] R 3 and R 5 are independently H, D, C 1-6 deuteroalkyl or C 1-6 alkyl;

[0027] R 4 and R 6 are independently C 1-6 alkyl, C 1-6 deuteroalkyl, C 2-1a deuteroalkyl or C 1-6 deuteroalkyl substituted with one or more R 2-1a ; and R 1-6 is alkyl;

[0028] R 7 is independently H, D, C 1-6 deuteroalkyl or C 1-6 alkyl.

[0029] In certain preferred embodiments of the application, the definitions of certain groups in the compounds of Formula I, stereoisomers and pharmaceutically acceptable salts thereof, can be as follows, while the definitions of other groups can be as described in any embodiment of the application (hereinafter "in some embodiments"): R is NR 1 R2 .

[0030] In some embodiments, n1 is 0 or 1, for example n1 is 0, or for example n1 is 1.

[0031] In some embodiments, n2 is 0.

[0032] In some embodiments, R 1 is H.

[0033] In some embodiments, R 2 is H, -C(O)R 2-1 , or C 2-4 alkyl substituted with one or more R 1-6 . Preferably, R 2 is H or C 1-6 alkyl substituted with one or more R 2-4 .

[0034] In some embodiments, R 2-1 is C 1-6 alkyl substituted with one or more R 2-1a .

[0035] In some embodiments, R 2-1a is independently OH, NR a R b .

[0036] In some embodiments, R 2-2 is H.

[0037] In some embodiments, R 2-3 is C 1-6 alkyl or NR a R b .

[0038] In some embodiments, R 2-4 is independently NR a R b .

[0039] In some embodiments, R a and R b are independently H.

[0040] In some embodiments, R 7 is H.

[0041] In some embodiments, the 5-6 membered heterocyclenyl is a 5 membered heterocyclenyl.

[0042] In some embodiments, the 5-6 membered heterocyclenyl is a 5 membered heterocyclenyl having one heteroatom, for example

[0043] In some embodiments, each of said C 5-6 Cycloalkenyl is independently C5cycloalkenyl or C6cycloalkenyl, i.e.

[0044] In some embodiments, each of said C 1-6 Alkyl and said substituted C 1-6 C in said substituted C 1-6 Alkyl is independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl or t-butyl, e.g. methyl or ethyl.

[0045] In some embodiments, each of said C 1-6 Alkoxy is independently methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy or t-butoxy.

[0046] In some embodiments, ring A is

[0047] In some embodiments, R is -NH2,

[0048] In some embodiments, R 7 is H.

[0049] In some embodiments, the compound of formula I is a compound of formula I-A:

[0050]

[0051] A, R, R 0 , R 7 , n1 and n2 are independently as defined in any of the aspects of the present application.

[0052] In some embodiments, the compound of formula I-A is a compound of formula I-A1 and / or I-A2:

[0053]

[0054] n1 is 1, A, R, R 0 , R 7 and n2 are independently as defined in any of the aspects of the present application.

[0055] In some embodiments, the compound of formula I is a compound of formula I-B, I-C or I-D:

[0056]

[0057]

[0058] In formulae I-B, I-C and I-D, R and n1 are independently defined as in any of the aspects of the application.

[0059] In some embodiments, in formula I-B, n1 is 0, i.e. the hydrogen on the N to which R is attached is unsubstituted, i.e. is

[0060]

[0061] In some embodiments, in formulae I-C and I-D, R is NR 1 R 2 and n1 is 1.

[0062] In some embodiments, the compound of formula I-C is a compound of formula I-C1 and / or I-C2:

[0063]

[0064] In some embodiments, the compound of formula I-D is a compound of formula I-D1 and / or I-D2:

[0065]

[0066] In some embodiments, the compound of formula I is any one of the following:

[0067]

[0068] The present application also provides a pharmaceutical composition comprising a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of the aspects of the application, and a pharmaceutically acceptable carrier.

[0069] The present application also provides a pharmaceutical composition comprising a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of the aspects of the application, and a beta-lactam antibiotic.

[0070] In some embodiments, the beta-lactam antibiotic is meropenem.

[0071] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0072] The present application also provides the use of a compound of formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of the aspects of the application, for the manufacture of a serine beta-lactamase (SBLs) and / or metallo-beta-lactamase (MBLs) inhibitor.

[0073] The present application also provides the use of a compound of Formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of the present application in combination with a beta-lactam antibiotic in the manufacture of a medicament for the treatment of drug-resistant bacteria.

[0074] In some embodiments, the medicament for the treatment of drug-resistant bacteria is a medicament for the treatment of beta-lactam antibiotic-resistant bacteria.

[0075] Definitions:

[0076] The term "stereoisomer" refers to a cis-trans isomer or an optical isomer, a cis-trans isomer is an isomer resulting from the inability of a double bond or a single bond of a ring-forming carbon atom to rotate freely, an optical isomer is a stereoisomer having different optical properties resulting from the absence of an inversion axis of symmetry in the molecule.

[0077] The term "heterocycloalkenyl" refers to a cyclic, unsaturated, monovalent hydrocarbon group having a specified number of ring atoms (e.g., 5-6 membered), a specified number of heteroatoms (e.g., 1, 2, or 3), a specified kind of heteroatoms (one or more of N, O, and S, wherein N and S are optionally oxidized, i.e., NO, S(O), or S(O)2), having one) carbon-carbon sp 2 double bond, which is monocyclic and non-aromatic. Heterocycloalkenyl groups are attached to the rest of the molecule by a carbon atom or a heteroatom. Heterocycloalkenyl groups include, but are not limited to:

[0078] The term "cycloalkenyl" refers to a cyclic, unsaturated, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C5-C6), having one) carbon-carbon sp 2 double bond, which is monocyclic and non-aromatic. Cycloalkenyl groups include, but are not limited to: and the like.

[0079] The term "alkyl" refers to a saturated aliphatic hydrocarbon group; for example, an alkyl group of 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, s-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, and the like, as well as various branched isomers thereof, and the like.

[0080] The term "deuterated alkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by D, wherein the definition of "alkyl" is the same as the term "alkyl".

[0081] The term "alkoxy" refers to the group R X -O-, R X is the same as the term "alkyl". Alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propyloxy, i-propyloxy, and the like.

[0082] The term "deuteroalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by D, wherein the definition of "alkoxy" is the same as the term "alkoxy".

[0083] The term "alkyl" as used herein refers to a straight-chain or branched-chain saturated hydrocarbon group having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably 1 to 3 carbon atoms. The term "alkyl" includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, and the like. The term "alkyl" includes both "C1-C6 alkyl" and "C1-C4 alkyl". " " refers to the corresponding group is connected to other fragments, groups in the compound through the site.

[0084] In addition, it should be noted that, unless otherwise explicitly stated, the description manner "… independently" used in the present application should be interpreted broadly, which means that each individual described is independent of each other and can be independently the same or different specific group. More specifically, the description manner "… independently" can mean that in different groups, the specific options expressed between the same symbols do not affect each other; or it can mean that in the same group, the specific options expressed between the same symbols do not affect each other.

[0085] "Pharmaceutically acceptable salt" refers to a salt of a compound obtained by reacting the compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, the base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, the acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0086] The term "pharmaceutically acceptable carrier" refers to excipients and additives used in the production of pharmaceutical products and the dispensing of prescriptions, which are all substances contained in pharmaceutical preparations in addition to active ingredients. For details, see the People's Republic of China Pharmacopoeia (2020 Edition) or Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009).

[0087] On the basis of common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present application.

[0088] The reagents and raw materials used in the present application are commercially available.

[0089] The present application provides boronic acid and cyclic derivatives, pharmaceutical compositions thereof, and uses thereof. The compounds of the present application can simultaneously inhibit multiple SBLs and MBLs, and have high inhibitory activity against CRAB. In addition, the compounds of the present application can be used in combination with beta-lactam antibiotics for the treatment of bacterial infections. DETAILED DESCRIPTION

[0090] The present application is further described in connection with the following specific examples. It is to be understood that these examples are merely by way of illustration and are not intended to limit the scope of the application. Unless otherwise indicated, the experimental procedures in the following examples were carried out according to conventional conditions for such reactions, or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages by weight and parts by weight. Unless otherwise indicated, the ratios of liquids are volume ratios.

[0091] The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

[0092] The following abbreviations are used in the present application: ACN represents acetonitrile; Boc2O represents di-tert-butyl dicarbonate; DCM represents dichloromethane; DIC represents N,N'-diisopropylcarbodiimide; DIPEA represents N,N-diisopropylethylamine; DMAP represents 4-dimethylaminopyridine; DMF represents N,N-dimethylformamide; Et3N represents triethylamine; Et2Zn represents diethylzinc; HATU represents 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBT represents 1-hydroxybenzotriazole; IrPPy3 represents tris(2-phenylpyridine)iridium; LDA represents lithium diisopropylamide; LiHMDS represents lithium bis(trimethylsilyl)amide; NaSH represents sodium hydrosulfide; Pd2(dba)3 represents tris(dibenzylideneacetone)dipalladium; Pd(OAc)2 represents palladium acetate; PhNTf2 represents N-phenylbis(trifluoromethanesulfonyl)imide; Sphos represents 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl; TFA represents trifluoroacetic acid; THF represents tetrahydrofuran; Xantphos represents 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene.

[0093] Example 1: Synthesis of Compound 1

[0094]

[0095] Step 1: Preparation of Compound 1B

[0096] Compound 1A (111 g, 581.2 mmol) and Boc20 (152.21 g, 697.4 mmol) were dissolved in DCM (1.2 L), and DMAP (3.55 g, 29 mmol) was added to the reaction system. The reaction solution was stirred at room temperature for 0.5 h. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography (5 / 1 petroleum ether / ethyl acetate) to obtain compound 1B (162 g, 96%) in the form of yellow oil. 1 HNMR (CDC13, 400 MHz) δ 7.55 (dd, J = 8.8, 5.6 Hz, 1H), 7.00 (dd, J = 8.8, 2.8 Hz, 1H), 6.91-6.86 (m, 1H), 1.57 (s, 9H).

[0097] Step 2: Preparation of compound 1C

[0098] Compound 1B (21.7 g, 74.5 mmol) was dissolved in THF (150 mL), and the system was cooled to -78 °C under nitrogen protection. An LDA solution (41 mL, 81.9 mmol, 2M in n-Hexane) was slowly added to the mixture. The reaction solution was stirred at -78 °C for 1 h, and the reaction system was slowly raised to room temperature for further reaction. After the reaction was completed, the reaction was quenched with 1M HC1 (200 mL), the reaction solution was concentrated, diluted with ethyl acetate (200 mL), and the organic phase was washed with water, saturated brine successively, combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1C (16.11 g, 74%) in the form of yellow oil. 1 H NMR (CDC13, 400 MHz) δ 7.55 (dd, J = 8.8, 5.6 Hz, 1H), 7.00 (dd, J = 8.8, 2.8 Hz, 1H), 6.91-6.86 (m, 1H), 1.57 (s, 9H).

[0099] Step 3: Preparation of compound 1D

[0100] Compound 1C (16 g, 55 mmol) was dissolved in DCM (200 mL), and TFA (100 mL) was slowly added. The reaction solution was stirred at room temperature for 1 h. After the reaction was completed, the mixture was concentrated, slurried with DCM (100 mL), and filtered to obtain compound 1D (12.75 g, 99%) in the form of white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 12.91 (br. s, 2H), 7.80 (dd, J = 8.8, 5.6 Hz, 1H), 6.77 (dd, J = 10.4, 8.8 Hz, 1H).

[0101] Step 4: Preparation of compound 1E

[0102] Compound 1D (8.0 g, 34 mmol) was dissolved in TFA (40 mL), trifluoroacetic anhydride (21.4 g, 102 mmol) was added, and acetone (9.87 g, 170 mmol) was slowly added dropwise to the mixture. The reaction system was heated to 100 °C, and the reaction was allowed to proceed for 48 h. After the starting material was completely reacted, the reaction solution was concentrated, and the residue was recrystallized with ethyl acetate / n-hexane mixed solvent (10 mL / 30 mL) to obtain white solid compound 1E (6.7 g, 38%). 1 H NMR (DMSO-d6, 400 MHz) δ 8.03 (dd, J = 9.2, 5.2 Hz, 1H), 7.08 (dd, J = 9.2, 9.2 Hz, 1H), 1.74 (s, 6H).

[0103] Step 5: Preparation of compound 1G

[0104] Compound 1E (6.7 g, 24.4 mmol), compound 1F (7.52 g, 48.8 mmol), Pd(OAc)2 (270 mg, 1.2 mmol), SPhos (1.0 g, 2.4 mmol) and Et3N (7.41 g, 73.2 mmol) were sequentially dissolved in 1,4-dioxane (120 mL), the system was heated to 85 °C, and the reaction was allowed to proceed for 48 h under nitrogen protection. After the reaction was completed, the mixture was filtered with diatomite, and the filtrate was concentrated to obtain yellow oily compound 1G crude product (8.5 g, 80%). LC-MS m / z = 349.0 [M+H] + .

[0105] Step 6: Preparation of compound 1I

[0106] Compound 1G (7.0 g, 20.1 mmol) and compound 1H (10.27 g, 60.3 mmol) were dissolved in THF (60 mL), and the reaction solution was heated to 65 °C and stirred for 4 h. After the reaction was completed, the reaction solution was concentrated, and purified and separated by column chromatography (10 / 1 petroleum ether / ethyl acetate) to obtain yellow solid compound 1I (6.75 g, 84%). LC-MS m / z = 401.4 [M+H] + .

[0107] Step 7: Preparation of compound 1J

[0108] Compound 1I (8.0 g, 20 mmol) and IrPPy3 (130 mg, 0.2 mmol) were dissolved in DCM (500 mL), and the reaction was stirred at room temperature under blue LED irradiation at a wavelength of 460-465 nm for 17 hours. After the reaction was completed, the reaction solution was concentrated, and the product was separated by column chromatography (10 / 1 petroleum ether / ethyl acetate) to obtain compound 1J (5.6 g, 63%) as a white solid. The recovered raw material was 2.1 g. 1 H NMR (CDC13, 400 MHz) δ 7.93 (dd, J = 8.8, 5.8 Hz, 1H), 7.27-7.24 (m, 1H), 6.78 (dd, J = 9.3, 9.3 Hz, 1H), 5.75 (d, J = 14.8 Hz, 1H), 4.31 (dd, J = 8.8, 2.0 Hz, 1H), 2.37-2.29 (m, 1H), 2.25-2.18 (m, 1H), 2.05-2.02 (m, 1H), 1.95-1.90 (m, 1H), 1.84-1.79 (m, 1H), 1.75 (s, 6H), 1.38 (s, 3H), 1.29 (s, 3H), 1.12 (d, J = 10.9 Hz, 1H), 0.84 (s, 3H). LC-MS m / z = 422.8 [M+Na] + .

[0109] Step 8: Preparation of compounds 1K and 1L

[0110] Et2Zn (12.8 mL, 25.6 mmol, 2M in Toluene) was dissolved in DCM (10 mL) under nitrogen protection, and diiodomethane (10.3 g, 38.4 mmol) was slowly added dropwise after the reaction system was cooled to -78°C. After the reaction solution was stirred at -78°C for 30 minutes, a DCM solution (10 mL) of compound 1J (1.3 g, 3.2 mmol) was slowly added dropwise. The reaction was slowly raised to room temperature, and the reaction was stirred at room temperature for 48 hours. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride, and extracted twice with ethyl acetate. The organic phases were combined and dried with anhydrous sodium sulfate, and the reaction solution was concentrated. The product was separated by column chromatography (3 / 1-1 / 1 petroleum ether / ethyl acetate) to obtain a mixture of two isomers 1K and 1L (1.2 g, 3:1 ratio) as a crude product. The mixture of the crude product was purified by preparative HPLC to obtain compound 1K (500 mg, 34%, HPLC retention time: 16.35 min) as a white solid and compound 1L (230 mg, 16%, HPLC retention time: 15.16 min) as a white solid.

[0111] HPLC purification conditions:

[0112] Column: XBridge C18, 50*150mm, 5pm; mobile phase: A: H2O (containing 0.1% NH4HCO3), B: ACN. Wavelength: 214 nm. Flow rate: 50 mL / min. Gradient elution procedure: linear gradient elution was performed by setting the volume ratio of the mobile phase A and the mobile phase B at 25:75 to 15:85 in 0-17 min; isocratic elution was performed by setting the volume ratio of the mobile phase A and the mobile phase B at 5:95 in 18-25 min.

[0113] Compound 1K: 1 H NMR (DMSO-d6, 300 MHz) δ 7.40 (dd, J = 8.7, 5.8 Hz, 1H), 6.96-6.86 (m, 1H), 4.08-4.01 (m, 1H), 2.29-2.08 (m, 2H), 1.74-1.69 (m, 9H), 1.52-1.44 (m, 1H), 1.39-1.31 (m, 1H), 1.19-1.08 (m, 7H), 0.70 (s, 3H), 0.49-0.33 (m, 2H). LC-MS m / z = 415.4 [M+H] + .

[0114] Compound 1L: 1 H NMR (DMSO-d6, 300 MHz) δ 7.49-7.42 (m, 1H), 6.91 (dd, J = 18.5, 9.2 Hz, 1H), 4.05 (d, J = 8.6 Hz, 1H), 2.28-2.18 (m, 1H), 2.11-1.90 (m, 2H), 1.80-1.60 (m, 8H), 1.19-1.08 (m, 6H), 1.01 (s, 3H), 0.68 (s, 3H), 0.58 (d, J = 10.4 Hz, 1H), 0.48-0.34 (m, 1H). LC-MS m / z = 415.4 [M+H] + .

[0115] Step 9: Preparation of compound 1M

[0116] Compound 1K (4.0 g, 9.66 mmol) was dissolved in DMF (60 mL) at room temperature under nitrogen protection, then 70% NaSH (2.0 g, 24.15 mmol) was added, the reaction system was warmed to 95 °C, and the reaction was stirred for 2 hours. After the reaction was completed, the system was cooled to 5 °C. Water (500 mL) was poured into the reaction solution to quench the reaction, extracted with ethyl acetate, the organic phase was collected and washed with saturated brine solution, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated. The crude compound 1M was purified by column chromatography to obtain white solid compound 1M (1.95 g, 47%). LC-MS m / z = 429.1 [M+H] + .

[0117] Step 10: Preparation of compound 1O

[0118] LiHMDS (18 mL, 17.84 mmol, 1M in THF) was slowly added dropwise to a solution of 1-tert-butoxycarbonyl-3-pyrrolidinone (3 g, 16.22 mmol) in THF (40 mL) at -78 °C. After the mixture was stirred for 30 minutes, PhNTf2 (6.95 g, 19.46 mmol) was added to the system, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution, concentrated, diluted with ethyl acetate, and the organic phase was washed with saturated brine solution, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated. The mixture was purified by column chromatography (97 / 3 petroleum ether / ethyl acetate) to obtain compound 1N (3 g, 58%). 1 H NMR (CDCI3, 400 MHz) δ

[0119] 5.77-5.69 (m, 1H), 4.26-4.16 (m, 4H), 1.47 (s, 9H).

[0120] Compound 1M (100 mg, 0.23 mmol) and compound 1N (110 mg, 0.34 mmol), Pd(OAc)2 (5 mg, 0.02 mmol), XantPhos (12 mg, 0.02 mmol), DIPEA (89 mg, 0.69 mmol) were dissolved in DMF (3 mL) at room temperature under nitrogen protection, the reaction system was warmed to 100 °C, and the reaction was stirred for 4 hours. After the reaction was completed, the reaction was concentrated, and the crude compound 1O was purified by column chromatography to obtain white solid compound 1O (95 mg, 69%). LC-MS m / z = 496.2 [M-100+H] + .

[0121] Step 11: Preparation of compound 1

[0122] Intermediate 10 (95 mg, 0.16 mmol) was dissolved in 1,4-dioxane (2 mL) at room temperature, followed by the addition of 3 M NaOH solution (2 mL, 9.66 mmol), and the reaction was stirred at room temperature for 12 hours. The reaction was monitored by LC-MS. To the reaction mixture was added triethoxysilane (52 mg, 0.32 mmol), TFA (4 mL), and isobutylboronic acid (32 mg, 0.32 mmol) sequentially, and the mixture was stirred at room temperature for 1 hour. The mixture was filtered, and the pH of the filtrate was adjusted to 5-6 with saturated aqueous NaHC03solution. The mixture was concentrated to give compound 1 as a white solid, which was purified by preparative HPLC to give compound 1 (10.4 mg, 21%) as a white solid. 1 H NMR (1 drop of 3 M NaOH solution in D20, 400 MHz) δ 7.11 (d, J = 7.7 Hz, 1H), 6.76 (d, J = 7.7 Hz, 1H), 5.75-5.73 (m, 1H), 3.63-3.61 (m, 2H), 3.50-3.47 (m, 2H), 1.84-1.79 (m, 1H), 0.87-0.82 (m, 1H), 0.37-0.32 (m, 1H), 0.28-0.23 (m, 1H). LC-MS m / z = 304.1 [M+H] + .

[0123] Examples 2 and 3: Synthesis of compounds 2 and 3

[0124]

[0125] Step 1: Preparation of compound 2B

[0126] Prepared according to the synthesis protocol for compound 10 of Example 1. Compound 2B (3.0 g, 86%) was obtained as a yellowish oil from compound 1M (2.4 g, 5.6 mmol). LC-MS m / z = 646.6 [M+Na] + .

[0127] Step 2: Preparation of compounds 2C and 2D

[0128] Chiral resolution of compound 2B (3.0 g) gave compound 2C (1.3 g, 37%, HPLC retention time: 2.273 min) as a yellowish oil and compound 2D (1.3 g, 37%, HPLC retention time: 3.067 min) as a yellowish oil.

[0129] Chiral HPLC conditions for compounds 2C and 2D:

[0130] Column: OD, 100*3.0 mm, 3.0 μm; mobile phase: A: supercritical CO2, B: MeOH (containing 0.1% DEA); wavelength: 214 nm; flow rate: 1.5 mL / min. Elution program: isocratic elution with the volume ratio of supercritical CO2 and MeOH (containing 0.1% DEA) in the mobile phase being 75:25 for 0-8 min, column temperature being 35 °C.

[0131] Step 3: Preparation of compound 2

[0132] Compound 2C (1.3 g, 2.085 mmol) was dissolved in DCM (13 mL) at room temperature, and zinc bromide (3.75 g, 16.68 mmol) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was filtered, the filter cake was washed with DCM, and the filtrate was collected and concentrated. The crude product was purified by column chromatography to obtain a yellowish oil (750 mg, 69%). LC-MS m / z = 524.3 [M+H] + .

[0133] Compound 2 was prepared according to the synthesis scheme of compound 1 in Example 1. White solid compound 2 (261.31 mg, 55%) was obtained from the above yellowish oil (750 mg, 1.43 mmol). 1 H NMR (400 MHz, D2O with 2 drops of 3 M NaOH solution) δ 7.10 (d, J = 7.8 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 5.80-5.76 (m, 1H), 2.96-2.88 (m, 1H), 2.37-2.28 (m, 1H), 2.18-2.11 (m, 2H), 1.91-1.76 (m, 3H), 1.50-1.39 (m, 1H), 0.89-0.83 (m, 1H), 0.37-0.32 (m, 1H), 0.31-0.23 (m, 1H). LC-MS m / z = 332.1 [M+H] + .

[0134] Step 4: Preparation of compound 3

[0135] Compound 2D (1.3 g, 2.085 mmol) was dissolved in DCM (13 mL) at room temperature, and zinc bromide (3.75 g, 16.68 mmol) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was filtered, the filter cake was washed with DCM, and the filtrate was collected and concentrated. The crude product was purified by column chromatography to obtain a yellowish oil (750 mg, 69%). LC-MS m / z = 524.3 [M+H] + .

[0136] Compound 3 was prepared according to the synthetic protocol for Compound 1 of Example 1. Compound 3 was obtained as a white solid (10.42 mg, 27%) from the light yellow oil (60 mg, 0.114 mmol) described above. 1 H NMR (400 MHz, D20 with 2 drops of 3 M NaOH solution) δ 7.11 (d, J = 7.7 Hz, 1H), 6.71 (d, J = 7.7 Hz, 1H), 5.81 - 5.77 (m, 1H), 2.97 - 2.90 (m, 1H), 2.38 - 2.29 (m, 1H), 2.19 - 2.12 (m, 2H), 1.93 - 1.76 (m, 3H), 1.51 - 1.41 (m, 1H), 0.90 - 0.84 (m, 1H), 0.38 - 0.33 (m, 1H), 0.31 - 0.23 (m, 1H). LC-MS m / z = 332.1 [M+H] + .

[0137] Example 4: Synthesis of Compound 4

[0138]

[0139] Step 1: Preparation of Compound 4A

[0140] Compound 2B (497 mg, 0.79 mmol) was dissolved in DCM (3 mL) at room temperature, and zinc bromide (1.43 g, 6.38 mmol) was added. The reaction was stirred at room temperature for 16 hours. The reaction was filtered, the filter cake was washed with DCM, and the filtrate was collected and concentrated. The crude compound 4A was purified by column chromatography to give compound 4A as a white solid (388 mg, 93%). LC-MS m / z = 524.3 [M+H] + .

[0141] Step 2: Preparation of Compound 4C

[0142] Compound 4A (120 mg, 0.23 mmol) was dissolved in DMF (5 mL) at room temperature, followed by the addition of compound 4B (68 mg, 0.46 mmol) and DIPEA (60 mg, 0.46 mmol). The mixture was stirred at 70 °C overnight. After the reaction was completed, water and ethyl acetate were added to the reaction system, and the organic phase was collected. The organic phase was washed with saturated brine, and the combined organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated. The mixture was purified by column chromatography (9 / 1 DCM / MeOH) to give compound 4C as a yellow solid (85 mg, 25%). LC-MS m / z = 566.2 [M+H] + .

[0143] Step 3: Preparation of Compound 4

[0144] Prepared according to the synthesis protocol of compound 1 of Example 1. Compound 4 (10.87 mg, 19%) was obtained as a white solid from compound 4C (85 mg, 0.15 mmol). 1 H NMR (D20, 400 MHz) δ 7.11 (d, J = 7.7 Hz, 1H), 6.74 (d, J = 7.7 Hz, 1H), 5.72-5.70 (m, 1H), 3.70-3.68 (m, 1H), 2.49-2.39 (m, 1H), 2.22-2.13 (m, 2H), 2.11-2.01 (m, 1H), 1.89-1.79 (m, 2H), 1.77-1.67 (m, 1H), 0.89-0.82 (m, 1H), 0.38-0.32 (m, 1H), 0.31-0.23 (m, 1H). LC-MS m / z = 374.1 [M+H] + .

[0145] Example 5: Synthesis of compound 5

[0146]

[0147] Step 1: Preparation of compound 5B

[0148] Compound 4A (140 mg, 0.27 mol) was dissolved in DMF (5 mL) at room temperature, then compound 5A (47 mg, 0.27 mmol), DIC (34 mg, 0.27 mmol) and HOBT (36 mg, 4.8 mmol) were added successively. After the reaction was completed, the reaction solution was quenched by pouring into water (5 mL), extracted with ethyl acetate, and the organic phase was collected and washed with saturated brine solution, and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated. The crude compound 5B was purified by column chromatography to obtain compound 5B (180 mg, 99%) as a white solid. LC-MS m / z = 681.1 [M+H] + .

[0149] Step 2: Preparation of compound 5C

[0150] Prepared according to the synthesis protocol of compound 4A of Example 4. Compound 5C (131 mg, 90%) was obtained as a white solid from compound 5B (170 mg, 0.25 mmol). LC-MS m / z = 581.2 [M+H] + .

[0151] Step 3: Preparation of compound 5

[0152] Prepared according to the synthesis protocol for compound 1 of Example 1. From compound 5C (130 mg, 0.22 mmol) as a off-white solid (14.0 mg, 16%). 1 H NMR (D20, 400 MHz) δ 7.11 (d, J = 7.8 Hz, 1H), 6.73 (d, J = 7.8 Hz, 1H), 5.79-5.75 (m, 1H), 3.99-3.92 (m, 1H), 3.27 (s, 2H), 2.46-2.37 (m, 1H), 2.21-2.13 (m, 2H), 2.10-2.00 (m, 1H), 1.86-1.76 (m, 2H), 1.76-1.63 (m, 1H), 0.89-0.83 (m, 1H), 0.39-0.32 (m, 1H), 0.31-0.23 (m, 1H). LC-MS m / z = 389.1 [M+H] + .

[0153] Example 6: Synthesis of compound 6

[0154]

[0155] Step 1: Preparation of compounds 6B and 6C

[0156] Prepared according to the synthesis protocol for compound 1N of Example 1. From compound 6A (3 g, 15 mmol) as a mixture of compound 6B and 6C (1 g, 20%) in light yellow oil. 1 H NMR (CDC13, 400 MHz) δ 5.61-5.56 (m, 1H), 4.85-4.76 (m, 1H), 4.41-4.31 (m, 1H), 2.98 (dd, J = 17.3, 8.0 Hz, 1H), 2.84 (dd, J = 17.3, 5.0 Hz, 1H), 2.48-2.41 (m, 1H), 2.29-2.21 (m, 1H), 1.44 (s, 9H).

[0157] Step 2: Preparation of compound 6D

[0158] Prepared according to the synthesis protocol for compound 1O of Example 1. From compound 1M (500 mg, 1.15 mmol) as a starting material, compound 6D (400 mg) was obtained as a white solid. LC-MS m / z = 632.3 [M+Na] + .

[0159] Step 3: Preparation of compound 6E

[0160] Prepared according to the synthesis protocol for compound 4A of Example 4. Compound 6E (63 mg, 0.12 mmol) gave compound 6 (7.3 mg, 19%) as a white solid. + .

[0161] Step 4: Preparation of compound 6

[0162] Prepared according to the synthesis protocol for compound 1 of Example 1. Compound 6E (63 mg, 0.12 mmol) gave compound 6 (7.3 mg, 19%) as a white solid. 1 H NMR (D20, 400 MHz) δ 7.25-7.22 (m, 1H), 6.98-6.94 (m, 1H), 5.59-5.52 (m, 1H), 4.03-3.96 (m, 1H), 2.92-2.79 (m, 2H), 2.50-2.41 (m, 1H), 2.39-2.29 (m, 1H), 2.12-2.04 (m, 1H), 1.17-1.09 (m, 1H), 0.50-0.39 (m, 2H). LC-MS m / z = 318.0 [M+H] + .

[0163] Example 7: Synthesis of compound 7

[0164]

[0165] Step 1: Preparation of compound 7A

[0166] Prepared according to the synthesis protocol for compound 1 of Example 1. Compound 4A (388 mg, 0.74 mmol) gave compound 7A (84 mg, 34%) as a white solid. 1 H NMR (D20, 400 MHz) δ 7.10 (d, J = 7.9 Hz, 1H), 6.70 (d, J = 7.9 Hz, 1H), 5.79-5.75 (m, 1H), 2.96-2.88 (m, 1H), 2.36-2.27 (m, 1H), 2.17-2.10 (m, 2H), 1.92-1.73 (m, 3H), 1.51-1.38 (m, 1H), 0.89-0.81 (m, 1H), 0.38-0.31 (m, 1H), 0.30-0.22 (m, 1H). LC-MS m / z = 332.0 [M+H] + .

[0167] Step 2: Preparation of compound 7

[0168] Compound 7A (30 mg, 0.091 mmol) and compound 7B (22 mg, 0.18 mmol) were dissolved in ACN (1.5 mL) at room temperature, K2CO3 (50 mg, 0.36 mmol) was added, and the reaction was stirred at room temperature for 16 h. After the reaction was completed, the reaction solution was filtered and concentrated, and the mixture was purified by preparative HPLC to obtain compound 7 (6.94 mg, 23%) as a light yellow solid. 1 H NMR (D2O, 400 MHz) δ

[0169] 7.05 (d, J = 7.7 Hz, 1H), 6.66 (d, J = 7.7 Hz, 1H), 5.75-5.71 (m, 1H), 3.61-3.54 (m, 1H), 2.38-2.30 (m, 1H), 2.14-2.07 (m, 2H), 1.94-1.91 (m, 1H), 1.88 (s, 3H), 1.79-1.71 (m, 2H), 1.61-1.50 (m, 1H), 0.84-0.77 (m, 1H), 0.31-0.26 (m, 1H), 0.25-0.18 (m, 1H). LC-MS m / z = 373.1 [M+H] + .

[0170] Example 8: Synthesis of compound 8

[0171]

[0172] Step 1: Preparation of compound 8B

[0173] Compound 4A (140 mg, 0.27 mmol) was dissolved in DMF (5 mL) at room temperature, followed by the addition of HATU (203 mg, 0.53 mmol), DIPEA (104 mg, 0.8 mmol) and compound 8A (110 mg, 0.53 mmol) in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, the mixture was partitioned with water and ethyl acetate, the aqueous phase was washed with ethyl acetate, the organic phases were combined and washed with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, filtered and concentrated. The mixture was purified by column chromatography (3 / 2 petroleum ether / ethyl acetate) to obtain compound 8B (105 mg, 55%) as a yellow solid. LC-MS m / z = 711.4 [M+H] + .

[0174] Step 2: Preparation of compound 8C

[0175] Prepared according to the compound 4A synthesis protocol of Example 4. Compound 8C (55 mg, 0.09 mmol) gave compound 8 (5.32 mg, 14%) as a white solid. + .

[0176] Step 3: Preparation of compound 8

[0177] Prepared according to the compound 1 synthesis protocol of Example 1. Compound 8C (55 mg, 0.09 mmol) gave compound 8 (5.32 mg, 14%) as a white solid. 1 H NMR (D20, 400 MHz) δ 7.22 (d, J = 7.9 Hz, 1H), 6.93-6.89 (m, 1H), 5.82-5.76 (m, 1H), 4.10-4.00 (m, 2H), 3.99-3.81 (m, 2H), 2.50-2.41 (m, 1H), 2.20-2.04 (m, 4H), 1.83-1.75 (m, 2H), 1.16-1.10 (m, 1H), 0.49-0.37 (m, 2H). LC-MS m / z = 419.2 [M+H] + .

[0178] Examples 9 and 10: Synthesis of compounds 9 and 10

[0179]

[0180] Step 1: Preparation of compound 9B

[0181] Compound 9A (20 g, 125 mmol) was dissolved in anhydrous THF (200 mL) at room temperature, replaced with nitrogen for three times, the mixture was cooled to -78 °C, LiHMDS (187.5 mL, 187.5 mmol, 1 M in THF) was added dropwise into the reaction solution. After the addition was completed, the mixture was stirred at room temperature for 0.5 hours. Then a solution containing N-phenyl bis(trifluoromethanesulfonyl) imide (68 g, 187.5 mmol) in THF (100 mL) was added dropwise into the system and stirred at low temperature for 1 hour. TLC showed that the raw material was completely reacted, the reaction solution was slowly raised to 0 °C, saturated aqueous ammonium chloride solution (150 mL) was added to quench the reaction, extracted with ethyl acetate for three times, the organic phase was washed with water and saturated aqueous sodium chloride solution in turn, the organic phase was collected and dried with anhydrous sodium sulfate, filtered and concentrated. Purified by column chromatography (47 / 3 petroleum ether / ethyl acetate) to give compound 9B (36 g, 99%) as a yellow oil. 1H NMR (CDC13, 400 MHz) δ 5.68-5.64 (m, 1H), 4.01-3.97 (m, 4H), 2.56-2.51 (m, 2H), 2.42-2.39 (m, 2H), 1.90 (t, J = 6.6 Hz, 2H).

[0182] Step 2: Preparation of compound 9C

[0183] Compound 1M (1 g, 2.33 mmol) was dissolved in 1,4-dioxane (10 mL) at room temperature, then Pd2(dba)3 (213 mg, 0.233 mmol), Xantphos (122 mg, 0.233 mmol), compound 9B (1 g, 3.495 mmol) and Et3N (235.33 mg, 3.5 mmol) were added into the system successively. The reaction was stirred at 100 °C for 2 hours under argon protection. LC-MS showed that the starting material was completely reacted. The reaction was concentrated, the crude product was diluted with ethyl acetate, and the organic phase was washed with water and saturated sodium chloride aqueous solution successively. The organic phase was collected and dried with anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography (41 / 9 petroleum ether / ethyl acetate) gave compound 9C (1.2 g, 91%) as a white solid. LC-MS m / z = 567.3 [M+H] + .

[0184] Step 3: Preparation of compound 9D

[0185] Compound 9C (3.4 g, 6 mmol) was dissolved in ACN (15 mL) at room temperature, and lithium tetrafluoroborate (558 mg, 6 mmol) was added into the system. Then the reaction was stirred at 80 °C for 16 hours. LC-MS detection showed that the starting material was completely reacted. The reaction was quenched by adding saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, and the organic phase was washed with saturated brine. The organic phase was collected and dried with anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography (22 / 3 petroleum ether / ethyl acetate) gave compound 9D (2.9 g, 94%) as a yellow solid. LC-MS m / z = 523.2 [M+H] + .

[0186] Step 4: Preparation of compound 9F

[0187] Compound 9D (650 mg, 1.24 mmol) was dissolved in MeOH (3 mL) at room temperature. Then Boc-ethylenediamine (398 mg, 2.49 mmol) was added into the system, and the mixture was stirred at room temperature for 1 h. Then sodium cyanoborohydride (253 mg, 3.72 mmol) was added into the system portionwise, and the reaction was stirred at 25 °C for another 3 h. After concentration under reduced pressure, the organic phase was diluted with ethyl acetate (30 mL), washed with water (10 mL) and saturated aqueous sodium chloride solution (10 mL) in turn, collected and dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified by column chromatography (48 / 52 petroleum ether / ethyl acetate) to give compound 9F (380 mg, 46%) as a white solid. LC-MS m / z = 667.3 [M+H] + .

[0188] Step 5: Preparation of compounds 9G and 9H

[0189] Compound 9F (380 mg) was subjected to chiral resolution to give compound 9G (106 mg, 28%, HPLC retention time: 3.843 min) and compound 9H (136 mg, 36%, HPLC retention time: 5.310 min).

[0190] Chiral HPLC detection conditions of compounds 9G and 9H:

[0191] Chromatographic column:OD, 100*3.0 mm, 3.0 μm; mobile phase: A: supercritical CO2, B: MeOH (containing 0.1% DEA); wavelength: 214 nm; flow rate: 1.5 mL / min. Elution program: isocratic elution was performed at 0-8 min with the volume ratio of supercritical CO2 and MeOH (containing 0.1% DEA) in the mobile phase being 80:20, and the column temperature was 35 °C.

[0192] Step 6: Preparation of compound 9

[0193] Compound 9G (100 mg, 0.15 mmol) was dissolved in DCM (5 mL) at room temperature, and zinc bromide (270 mg, 1.2 mmol) was added into the system. The reaction was stirred at room temperature for 16 h. After the reaction was completed, water (8 mL) was added into the mixture to quench the reaction, and the reaction was concentrated, extracted with DCM, and the organic phase was washed with saturated aqueous sodium chloride solution, collected, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (47 / 3 DCM / MeOH) to give compound 9 (80 mg, 94%) as a yellow solid. LC-MS m / z = 567.2 [M+H] + .

[0194] Compound 9 was prepared according to the synthetic protocol for compound 1 of Example 1. White solid compound 9 (10.32 mg, 19%) was obtained from the above yellow solid compound (80 mg, 0.14 mmol). 1 H NMR (400 MHz, D20 with 2 drops of 3 M NaOH solution) δ 7.08 (d, J = 7.8 Hz, 1H), 6.68 (d, J = 7.8 Hz, 1H), 5.80-5.75 (m, 1H), 2.76-2.59 (m, 5H), 2.39-2.31 (m, 1H), 2.17-2.07 (m, 2H), 1.91-1.77 (m, 3H), 1.48-1.38 (m, 1H), 0.88-0.80 (m, 1H), 0.38-0.30 (m, 1H), 0.29-0.22 (m, 1H). LC-MS m / z = 375.2 [M+H] + .

[0195] Step 7: Preparation of compound 10

[0196] Compound 9H (130 mg, 0.195 mmol) was dissolved in DCM (5 mL) at room temperature, and zinc bromide (270 mg, 1.2 mmol) was added to the system. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, water (8 mL) was added to the mixture to quench the reaction, and the reaction solution was concentrated, extracted with DCM, and the organic phase was washed with saturated aqueous sodium chloride solution. The organic phase was collected and dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by column chromatography (47 / 3 DCM / MeOH) to obtain a yellow solid compound (90 mg, 82%). LC-MS m / z = 567.2 [M+H] + .

[0197] Compound 10 was prepared according to the synthetic protocol for compound 1 of Example 1. White solid compound 10 (16.52 mg, 28%) was obtained from the above yellow solid compound (90 mg, 0.16 mmol). 1H NMR (400 MHz, D20 with 2 drops of 3 M NaOH solution) δ 7.08 (d, J = 7.8 Hz, 1H), 6.67 (d, J = 7.8 Hz, 1H), 5.79-5.75 (m, 1H), 2.77-2.69 (m, 1H), 2.68-2.58 (m, 4H), 2.38-2.31 (m, 1H), 2.15-2.07 (m, 2H), 1.92-1.83 (m, 2H), 1.82-1.76 (m, 1H), 1.47-1.36 (m, 1H), 0.86-0.80 (m, 1H), 0.35-0.30 (m, 1H), 0.28-0.21 (m, 1H). LC-MS m / z = 375.2 [M+H] + .

[0198] Test Example 1: Compounds against beta-lactamase IC 50 Test.

[0199] (1) Purpose of the experiment: to detect the inhibitory activity of selected compounds against KPC-2, NDM-1, AmpC and OXA-23.

[0200] (2) Experimental materials: test compounds QPX7728, compounds 2, 3, 9, 10 and selected beta-lactamases (see Table 1).

[0201] Table 1 Beta-lactamases for testing

[0202] Enzyme family Beta-lactamase Bacterial species Class A KPC-2 K. pneumoniae / K. oxytoca Class B NDM-1 K. pneumoniae Class C AmpC-PA P. aeruginosa Class D OXA-23 A. baumannii

[0203] (3) Experimental steps:

[0204] ①: Class A beta-lactamase KPC-2, class C beta-lactamase AmpC-PA and class D beta-lactamase OXA-23 use PBS buffer (pH 7.4 1xPBS, 0.1 mg / mL BSA) as reaction buffer, and nitrocefin as substrate to detect the activity of beta-lactamase.

[0205] ②: Class B beta-lactamase NDM-1 uses HEPES buffer (pH 7.0 50 mM HEPES) as reaction buffer, and imipenem as substrate to detect the activity of beta-lactamase.

[0206] ③: All test compounds are dissolved in DMSO to prepare a stock solution with a concentration of 10 mM. The final concentration of the test compounds is: 25000 nM, 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM and 0 nM, and avibactam as a positive control.

[0207] IV: Cefinone was dissolved in DMSO and prepared as 16 mM stock solution. Before use, it was diluted with buffer to 2x final concentration (100 μΜ final concentration of Cefinone substrate). Imipenem was dissolved in ultrapure water and prepared as 16 mM stock solution. Before use, it was diluted with buffer to 20x final concentration (200 μΜ final concentration of Imipenem substrate).

[0208] V: β-lactamases were diluted with corresponding reaction buffer to 2x (KPC-2, AmpC-PA, OXA-23) or 1.064x (NDM-1) of optimal reaction concentration.

[0209] Table 2 β-lactamase reaction final concentration

[0210] Beta-lactamase Test final concentration (nM) KPC-2 2.49 AmpC-PA 0.82 OXA-23 0.6 NDM-1 14.98

[0211] VI: Reaction system was the same as Table 3. Compounds were dispensed by Echo liquid handler, followed by addition of diluted enzyme, incubation at corresponding temperature for 10 min, and then addition of diluted substrate. For KPC-2, AmpC-PA and OXA-23, Cefinone was used as reaction substrate, and the test wavelength was 490 nm, with 40 min reading. For NDM-1, Imipenem was used as reaction substrate, and the test wavelength was 294 nm, with 15 min reading.

[0212] Table 3 β-lactamase reaction system

[0213]

[0214] VII: OD values at different concentrations of each compound were linearly fitted with reaction time to calculate the substrate hydrolysis rate at that concentration.

[0215] VIII: Inhibition rate was calculated according to the following formula:

[0216] ZPE: (Zero Effect Control): Substrate hydrolysis rate in DMSO + β-lactamase + substrate system;

[0217] HPE: (Full Effect Control): Substrate hydrolysis rate in DMSO + Buffer + substrate system;

[0218] Test: Substrate hydrolysis rate in compound + β-lactamase + substrate system;

[0219] IX: The formula provided by GraphPad Prism was used to calculate IC 50 .

[0220] (4) IC 50 Test results

[0221] Table 4 IC of selected compounds 50 Values (nM)

[0222]

[0223]

[0224] As shown in Table 4, the marketed drug Avibactam has good inhibitory effect on KPC-2 (class A), AmpC-PA (class C) SBLs enzymes, has no activity on class B NDM-1 enzyme, and has poor inhibitory effect on OXA-23 (class D) enzyme commonly expressed in CRAB. At present, there is no marketed drug that can effectively inhibit class B MBLs and OXA-23 type enzymes at the same time. In the present application, compounds 2, 3, 9 and 10 can effectively inhibit KPC-2, NDM-1, AmpC-PA and OXA-23 four types of enzymes, which has a significant advantage compared with the marketed drug Avibactam. Compounds 2, 3, 9 and 10 have very high inhibitory activity on NDM-1 type metalloenzyme and OXA-23 enzyme commonly expressed in CRAB, which shows comparable or better inhibitory activity than the currently reported clinical phase 1 candidate drug QPX7728 with super broad-spectrum and high enzyme inhibitory activity.

[0225] Test Example 2: Minimum drug concentration (MIC) test of compounds on bacterial growth inhibition

[0226] (1) Purpose of the experiment: Test the in vitro antibacterial activity of meropenem combined with compounds.

[0227] (2) Main experimental instruments

[0228] Table 5 Experimental instruments

[0229] Name Model Fixed asset number Manufacturer Autoclave mvs-83 38301028 ALPHAVITA Biological safety cabinet BSC-1604IIA2 3551529 Suzhen Incubator ZXSD-B1270 2083348 Zhi Cheng Turbidimeter Harvard-80-2116-30 2622217 Harvard Analytical balance XS205 385414 Mettler Toledo

[0230] (3) Experimental materials and reagents

[0231] Table 6 Experimental consumables

[0232]

[0233] (4) Experimental strains

[0234] Table 7 Information of experimental strains

[0235]

[0236] (5) Experimental steps

[0237] ①Strain preparation: The strains in Table 7 were taken out from -80°C, streaked on CAMHA solid agar medium, and incubated in an incubator at 35±2°C for 18-24h.

[0238] ②Compound test plate preparation: Meropenem and compounds were dissolved in DMSO to prepare stock solutions, the concentration of meropenem stock solution was 12.8mg / mL, and the concentration of compound stock solution was 0.8mg / mL. The meropenem stock solution was diluted by 2-fold gradient in DMSO, a total of 10 times, to obtain 11 2-fold gradient dilutions. In 96-well plates 1-11 columns, 1 μL of meropenem gradient dilution was added in order of high to low concentration, and 1 μL of DMSO was added in column 12. In 96-well plates B-H rows, 1 μL of different compound stock solutions was added in order to obtain a compound test plate.

[0239] ③Inoculum preparation: Single colonies were picked from overnight solid agar plates, dissolved in sterile normal saline, and adjusted to 0.5 McFarland turbidity. Then the inoculum was diluted 200-fold in test medium to 5×105CFU / ml to obtain an inoculum, and 198 μL / well of the inoculum was transferred to the compound test plate.

[0240] ④MIC reading: After the test plate was incubated at 36.5°C for 20h, the MIC was read by naked eye, and the MIC was the minimum antibiotic concentration that could completely inhibit bacterial growth observed by naked eye.

[0241] (6) Test results

[0242] Table 8 MIC values of selected compounds combined with meropenem against different drug-resistant bacteria (μg / mL)

[0243]

[0244]

[0245]

[0246] The meaning of "*" indicates that the concentration of the selected compound is 4 μg / mL

[0247] As shown in Table 8, the compounds of the present application in combination with meropenem are effective against most of the strains. Compounds 2, 3, 9 and 10 in combination with meropenem show good antibacterial activity against the selected 4 drug-resistant strains (ATCC BAA-1705, ATCC BAA-2470, ARLG-1791, ATCC-1605) and 1 non-drug-resistant strain (ATCC BAA-2523), and have good inhibitory effect on drug-resistant strains carrying SBLs, MBLs and CRAB expressing OXA-23 type enzymes. Among them, compounds 2, 3, 9 and 10 in combination with meropenem show comparable bacteriostatic efficacy and antibacterial spectrum to QPX7728, a clinical phase 1 candidate drug with the highest inhibitory activity against drug-resistant strains reported so far, in combination with meropenem.

[0248] Test Example 3: Test of compound binding rate to plasma proteins (PPB) of different species

[0249] (1) Test drugs, reagents and instruments: compounds 2, 9 and 10, warfarin, quinidine, ranitidine, 96-well microbalance dialysis device (HT Dialysis Company): dialysis membrane 1101 (HT Dialysis Company): LC-MS / MS / TripleQuad 6500 (SCIEX Company).

[0250] (2) Test objects: CD-1 mouse plasma, SD rat plasma, beagle dog plasma, cynomolgus monkey plasma and human plasma.

[0251] (3) Test method: The equilibrium dialysis method was used to determine the binding rate of compounds 2, 9, 10 and QPX7728 to mouse, rat, beagle dog, cynomolgus monkey and human plasma proteins. The binding rates of warfarin, quinidine and ranitidine to mouse, rat, beagle dog, cynomolgus monkey and human plasma proteins were also determined as positive controls. Different species plasma samples containing 3, 9, 10 and QPX7728 were prepared, and the concentration was 10 μM. Different species plasma samples containing warfarin, quinidine and ranitidine were prepared, and the concentration was 1 μM.

[0252] Preparation of immediate (0 h) sample treatment: After the preparation of the plasma sample, 25 μL of the sample was added to 25 μL of blank PBS solution, 200 μL of internal standard solution was added, vortexed for 10 minutes (500 rpm), centrifuged for 10 minutes (4000 g). The supernatant was taken, dried by nitrogen flow, redissolved with 200 μL of water, and centrifuged for 10 minutes (4000 g), and finally detected by LC-MS / MS method.

[0253] Plasma protein binding test: 100 μL of plasma sample and 100 μL of blank PBS buffer were added to the plasma sample side and PBS side of the dialysis device respectively, 2 samples for each concentration, sealed, and shaken at 37°C for 5h (150rpm). After dialysis, 25 μL of the plasma side sample was taken and 25 μL of blank PBS solution was added, and 25 μL of the PBS side sample was taken and 25 μL of blank plasma was added. 200 μL of internal standard solution was added, vortexed for 10 minutes (500rpm), and centrifuged for 10 minutes (4000g). The supernatant was taken, dried with nitrogen flow, reconstituted with 200 μL of water, and centrifuged for 10 minutes (4000g). Finally, LC-MS / MS method was used for detection.

[0254] (4) Plasma protein binding rate (PPB) test results: The plasma protein binding rates of the positive control compounds warfarin, quinidine and ranitidine in mice, rats, beagle dogs, cynomolgus monkeys and human plasma all meet the data acceptance standard. The plasma protein binding rate data of compounds 3, 9 and 10 in mice, rats, beagle dogs, cynomolgus monkeys and humans are shown in Table 9 below.

[0255] Table 9 Plasma protein binding rate (PPB) of compounds in humans, beagle dogs and rats

[0256]

[0257] Table 9 shows that the plasma protein binding rates of the selected compounds 3, 9 and 10 in mice and rats are closer to the reference compound QPX7728, but the plasma protein binding rates in beagle dogs, cynomolgus monkeys and humans, especially in humans, are significantly lower than those of the reference compound QPX7728. At the same plasma concentration of the drug, a lower plasma protein binding rate can provide a higher plasma free concentration, thereby ensuring better drug efficacy. In addition, these compounds are mainly excreted out of the body through the kidneys via urine, and a lower plasma protein binding rate can more effectively be filtered through the glomerulus to be excreted out of the body, thereby avoiding long-term accumulation in the body and causing safety problems.

[0258] Test Example 4: In vivo pharmacokinetic test of compounds in mice

[0259] (1) Test drug: compounds 3, 9, 10 and QPX7728.

[0260] (2) Test animals: Balb / C male mice, weighing 18-30g, purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.

[0261] (3) Test scheme:

[0262] a) Preparation of drug solution: compounds 3, 9, 10, QPX7728 were prepared with 0.9% sodium chloride injection.

[0263] b) Dosing regimen: 3 male mice per group, fasted overnight before dosing, and allowed free access to food 4 h after dosing, and free access to water. The dosing was a single intravenous injection (about 1 min), the dose was 10 mg / kg, the concentration was 2 mg / mL, and the volume was 5 mL / kg.

[0264] c) Sample collection and processing: 30 μL blood was collected from the orbital plexus before dosing and at 0.083, 0.25, 0.5, 1, 2, 4, 8 h after dosing, and placed in EDTA-K2 anticoagulant tubes, centrifuged at 6000 g for 5 min (4°C), and the plasma was separated. All collected plasma samples were stored on dry ice or in a -70°C freezer.

[0265] d) Plasma sample determination method: LC-MS / MS method was used to determine the concentration of the test drug in the plasma of mice at different time points after dosing.

[0266] e) Data processing: The pharmacokinetic parameters of mice after dosing were calculated using 8.2 software of non-compartment model.

[0267] f) Test results

[0268] Table 10 Summary of pharmacokinetic parameters of mice after a single intravenous injection of 10 mg / kg of each test drug

[0269]

[0270] The results in Table 10 show that the compounds 3, 9 and 10 of the present application have more significant pharmacokinetic advantages compared with QPX7728. β-lactamase inhibitors generally have a short half-life and a low volume of distribution in vivo, and because they need to be used in combination with β-lactam antibiotics, the pharmacokinetic parameters need to match those of the β-lactam antibiotics. However, QPX7728 has a plasma half-life in humans of up to 30 h, while the plasma half-life of antibiotics in humans is short, such as 2 h for meropenem, and the pharmacokinetic characteristics of the two are very different, making it difficult to use them in combination. The plasma half-lives of the compounds 3, 9 and 10 of the present application in mice are lower than that of QPX7728, and they have the characteristics of high drug exposure, low volume of distribution, low clearance rate and short half-life, and have significant pharmacokinetic advantages.

Claims

1. A compound, a stereoisomer, and a pharmaceutically acceptable salt thereof, as shown in Formula I; in, Ring A is "a 5-6 membered heterocyclic alkenyl group selected from one, two, or three of N, O, and S, with one, two, or three heteroatoms" or C 5-6 Cycloalkenyl; R is NR 1 R 2 S(O)2NR 3 R 4 or C(O)NR 5 R 6 ; n1 is either 0 or 1; R 0 The answer is D; n2 is 0, 1, 2, 3 or 4; R 1 For H, D, C 1-6 Deuterated alkyl or C 1-6 alkyl; R 2 For H, D, -S(O)R 2-1 S(O)2R 2-1 -C(O)R 2-1 , C 1-6 Alkyl, C 1-6 Deuterated alkyl, with one or more R 2-4 Replacement C 1-6 Deuterated alkyl or with one or more R 2-4 Replacement C 1-6 alkyl; R 2-1 C 1-6 Alkyl, C 1-6 Deuterated alkyl, with one or more R 2-1a Replacement C 1-6 Deuterated alkyl or with one or more R 2-1a Replacement C 1-6 alkyl; R 2-1a Independently OH, OD, NR a R b C 1-6 Alkoxy or C 1-6 Deuterated alkoxy groups; R 2-2 For H, D, C 1-6 Deuterated alkyl or C 1-6 alkyl; R 2-3 C 1-6 Alkyl, C 1-6 Deuterated alkyl, NR a R b , by one or more R 2-3a Replacement C 1-6 Deuterated alkyl or with one or more R 2-3a Replacement C 1-6 alkyl; R 2-3a Independently OH, OD, NR a R b C 1-6 Alkoxy or C 1-6 Deuterated alkoxy groups; R 2-4 Independently OH, OD, NR a R b C 1-6 Alkoxy or C 1-6 Deuterated alkoxy groups; R a and R b H, D, C independently 1-6 Deuterated alkyl or C 1-6 alkyl; R 3 and R 5 H, D, C independently 1-6 Deuterated alkyl or C 1-6 alkyl; R 4 and R 6 Independently for C 1-6 Alkyl, C 1-6 Deuterated alkyl, with one or more R 2-1a Replacement C 1-6 Deuterated alkyl or with one or more R 2-1a Replacement C 1-6 alkyl; R 7 Independently defined as H, D, C 1-6 Deuterated alkyl or C 1-6 alkyl.

2. The compound, stereoisomer, and pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, It meets one or more of the following conditions: (1) R is NR 1 R 2 ; (2) n1 is 0 or 1, for example n1 is 0, or for example n1 is 1; (3) n2 is 0; (4)R 1 For H; (5)R 2 H, -C(O)R 2-1 , Or by one or more R 2-4 Replacement C 1-6 alkyl; (6)R 2-1 For one or more R 2-1a Replacement C 1-6 alkyl; (7)R 2-1a Independent of OH and NR a R b ; (8)R 2-2 For H; (9)R 2-3 C 1-6 Alkyl or NR a R b ; (10)R 2-4 Independent for NR a R b ; (11)R a and R b H is independent; (12)R 7 For H; and (13) The compound represented by Formula I is the same as the compound represented by Formula IA:

3. The compound, stereoisomer, and pharmaceutically acceptable salt thereof as described in claim 2, characterized in that, It meets one or more of the following conditions: (1)R 2 For H or by one or more R 2-4 Replacement C 1-6 Alkyl; and (2) When n1 is 1, the compound represented by formula IA is the compound represented by formula I-A1 and / or formula I-A2:

4. The compound, stereoisomer, and pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, It meets one or more of the following conditions: (1) The 5-6 membered heterocyclic alkenyl group is a 5 membered heterocyclic alkenyl group; (2) In the 5-6 membered heterocyclic alkenyl group, the heteroatom is N, and the number of heteroatoms is 1; preferably, the 5-6 membered heterocyclic alkenyl group is (3) The C 5-6 The cycloalkenyl group is independently either a C5 cycloalkenyl or a C6 cycloalkenyl; (4) Each of the C's 1-6 Alkyl groups and the substituted C 1-6 C in alkyl 1-6 The alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; for example, independently methyl or ethyl; and (5) Each of the C's descriptions 1-6 The alkoxy group is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy.

5. The compound, stereoisomer, and pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, It meets one or more of the following conditions: (1) Ring A is (2) R is -NH2, and (3)R 7 For H.

6. The compound, stereoisomer, and pharmaceutically acceptable salt thereof of Formula I as described in any one of claims 1-5, characterized in that, The compound represented by Formula I is a compound represented by Formula IB, IC, or ID: Preferably, in formula IB, n1 is 0; Preferably, in formulas IC and ID, R is NR. 1 R 2 n1 is 1; Preferably, the compound represented by formula C1 is a compound represented by formulas I-C1 and / or I-C2: Preferably, the compound represented by formula ID is a compound represented by formula I-D1 and / or formula I-D2:

7. The compound, stereoisomer, and pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, The compound represented by Formula I is any one of the following compounds:

8. A pharmaceutical composition comprising a compound of Formula I as described in any one of claims 1-7, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

9. A pharmaceutical composition comprising a compound of Formula I as described in any one of claims 1-7, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a β-lactam antibiotic; Preferably, the β-lactam antibiotic is meropenem; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

10. Use of the compound of Formula I as described in any one of claims 1-7, its stereoisomers, or pharmaceutically acceptable salts thereof, or the pharmaceutical composition as described in claim 8 or 9, wherein the use is selected from: (1) Preparation of serine β-lactamase (SBLs) and / or metallo-β-lactamase (MBLs) inhibitors; (2) Prepare a drug for anti-drug-resistant bacteria by using it in combination with β-lactam antibiotics; preferably, the drug for anti-drug-resistant bacteria is a drug for anti-β-lactam antibiotic-resistant bacteria.

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