Mercaptopropanamide compound and application thereof in preparation of antibacterial drugs

Abstract

The invention relates to a sulfydryl propanamide compound and application thereof in preparation of antibacterial drugs. Specifically, the invention discloses a sulfydryl propionamide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer or a solvate thereof, or a pharmaceutical composition composed of the sulfydryl propionamide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer or a solvate thereof and a pharmaceutically acceptable carrier, and application of the sulfydryl propionamide compound, the pharmaceutically acceptable salt, the stereoisomer or the solvate thereof in preparation of an inhibitor for metal beta-lactamase NDM-1. The invention further discloses application of the compound in preparation of drugs for preventing and / or treating gram-negative drug-resistant bacterium infection. The compound provided by the invention can target metal matrix protease NDM-1, inhibit hydrolysis of beta-lactam antibiotics and effectively reduce the inhibitory concentration of gram-negative bacteria such as escherichia coli or klebsiella pneumoniae; the compound can be combined with beta-lactam antibiotics such as meropenem to prepare a compound preparation for treating diseases such as infection caused by gram-negative drug-resistant bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry and biomedical technology, and in particular, relates to a mercaptopropionamide compound and its use in the preparation of antibacterial drugs. Specifically, the present application relates to a mercaptopropionamide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a solvate thereof, or a pharmaceutical composition composed of them and a medically acceptable carrier, and the use of these mercaptopropionamide compounds, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a solvate thereof, or a pharmaceutical composition composed of them and a medically acceptable carrier in the preparation of an inhibitor of metallo-β-lactamase NDM-1, and in the preparation of a drug for preventing and / or treating Gram-negative drug-resistant bacterial infections. Background Art

[0002] Bacterial infections seriously endanger human health. According to statistics from the World Health Organization, three of the top ten causes of death in 2019 were caused by bacterial infections, namely lower respiratory tract infections, neonatal diseases and diarrhea. Although a large number of antibiotics have been developed for the treatment of bacterial infections, the speed of antibiotic development is far behind the speed of bacterial resistance (Ventola C L. The antibiotic resistance crisis: part 1: causes and threats [J]. P&T: a peer-reviewed journal for formulary management, 2015, 40 (4): 277-283). According to the 2020 bacterial resistance monitoring data (Sara E. Boyd, David M. Livermore, David C. Happer, William W. Hope. Metallo-β-Lactamases: Structure, Function, Epidemiology, Treatment Options, and the Development Pipeline [J]. Antimicrobial Agents and Chemotherapy, 2020. 64 (10): e00397-20), Gram-negative bacteria accounted for 65.7%–73.0% of the bacteria isolated from clinical hospital infections, among which Escherichia coli and Klebsiella pneumoniae accounted for the highest proportion. Moreover, Escherichia coli (E. Coli) mainly produces NDM (74.8%), and Klebsiella pneumoniae (K. Pneumoniae) mainly produces KPC (77.1%). Although carbapenem antibiotics can show good antibacterial effects against resistant Gram-negative bacteria and have always been considered the last line of defense for antimicrobial drugs, carbapenem-resistant bacteria have now become widespread, so that newborns, the elderly and patients in intensive care are extremely susceptible to infection by carbapenem-resistant bacteria, which has become a major problem in clinical anti-infection treatment.

[0003] There are four mechanisms by which bacteria develop drug resistance, including limiting antibiotic absorption, active antibiotic efflux, modifying antibiotic targets, and inactivating antibiotics. Among them, the production of β-lactamases during antibiotic inactivation is the main cause of 80% bacterial resistance (Tooke CL, Hinchliffe P, Bragginton EC, et al. β-Lactamases: a focus on current challenges [J]. Cold Spring Harb Perspect Med, 2017.7 (1)). β-lactamases can hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, carbapenems, etc. β-lactamases are divided into serine β-lactamases and metallo-β-lactamases. At present, there are corresponding inhibitors for serine β-lactamases in clinical practice, such as clavulanic acid, tazobactam, sulbactam, avibactam, etc., but there are currently no clinically used inhibitors for metallo-β-lactamases. Serine β-lactamase is divided into ACD type, and metallo β-lactamase is type B (type B is divided into B1, B2 and B3 subtypes). Among them, the subtype NDM-1 of the B1 subtype NDM is the most difficult β-lactamase. Since the discovery of bacteria containing the NDM-1 gene in India in 2008, it has been called a superbug and people are terrified. Superbug NDM-1 has two major characteristics: it is easy to spread and mutate, it can replicate and move freely in bacteria, and it can hydrolyze almost all antibiotics, even the "last line of defense" carbapenem antibiotics. So far, no metallo β-lactamase inhibitors have been listed. Therefore, it is urgent to develop NDM-1 inhibitors to resist infectious diseases caused by superbugs.

[0004] The key factor in the key active domain of NDM-1 hydrolyzing antibiotics is that it contains two zinc ions, which are connected by hydroxyl groups or water molecules to form a whole. Therefore, the key active domain of NDM-1 is an important target for the development of NDM-1 inhibitor drugs. Over the years, scientists have made unremitting efforts to overcome difficulties and have made certain preclinical research progress in the field of NDM-1 inhibitor research, such as metal chelators such as EDTA and aspergillus A, and metal replacement agents, involving metal inhibitors such as Bi(Ⅲ), Au(Ⅰ), Pt(Ⅱ), Pd(Ⅱ), and Ru(Ⅱ). These two types of molecules have good enzyme inhibition and antibacterial effects, but because they over-deplete Zn(Ⅱ) in the MBLs environment in the body, or completely replace Zn(Ⅱ) in MBLs, they are prone to cause great cytotoxicity, and their clinical applications are limited. In addition, zinc ion ligands are the most widely studied NDM-1 inhibitors to date, covering metal ligands with electron-rich functional groups and diverse structures such as thiol, carboxyl, phosphonic acid, semicarbazide and acylhydrazone, all of which show potential development prospects.

[0005] Chinese Patent CN110194731A discloses mercaptopropionamide compounds, their medicinal salts, their preparation methods and medicinal uses, especially their application in the preparation of drugs against Gram-negative drug-resistant bacteria. Some of the compounds provided in this application have relatively excellent NDM-1 enzyme inhibitory activity and can significantly reduce the minimum inhibitory concentration (MIC value) of meropenem; the compounds and their salts provided in this application can be used for the preparation of NDM-1 enzyme inhibitors and can be combined with meropenem to form a compound preparation for anti-Gram-negative drug-resistant bacteria. However, in this application, the structures of the compounds are all chain-like acids, lacking rigidity and having a weak ability to match the target. At the same time, the compounds in this application cannot be used to prepare quaternary ammonium salt compounds and cannot exhibit the advantageous characteristics (water solubility and quaternary ammonium salt characteristics) of the above antibacterial compounds. In addition, in this patent application, the mercapto groups of compounds 5a-5d, 6a-6b, and 7a-7c are all protected by acetyl groups, and they cannot complex with the zinc ions of NDM-1 functionally, so the enzyme inhibitory activity is weak. Summary of the Invention

[0006] Based on the deficiencies of the prior art, the present application provides a mercaptopropionamide compound with a novel structure and its use in the preparation of antibacterial drugs, specifically relating to mercaptopropionamide compounds, or their pharmaceutically acceptable salts, or their stereoisomers or their solvates, or pharmaceutical compositions composed of them and a medically acceptable carrier, and the use of these mercaptopropionamide compounds, or their pharmaceutically acceptable salts, or their stereoisomers or their solvates, or pharmaceutical compositions composed of them and a medically acceptable carrier in the preparation of inhibitors for metallo-β-lactamase NDM-1 and in the preparation of drugs for preventing and / or treating Gram-negative drug-resistant bacterial infections.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] NDM-1 is a zinc(II)-containing metallo-β-lactamase. The two zinc ions in its key active domain for hydrolyzing antibiotics have the characteristic of activating nucleophilic water or hydroxide ions, and can thus cleave almost all β-lactam antibiotics including meropenem and ultimately cause their inactivation.

[0009] The strategy of designing a series of novel mercaptopropionamide zinc ion ligand inhibitors of the present invention is to target NDM-1 in MBLs, aim to improve the enzyme inhibition and antibacterial activity, select the old drug captopril used clinically for angiotensin converting enzyme inhibitors (ACEI) as a guide, and focus on the structure of the methyl and proline parts in its structure for separate or simultaneous transformation and optimization. On the one hand, focus on the hydrophobic groove in the protease domain of NDM-1, introduce variable and more hydrophobic structural fragments to replace the methyl group; on the other hand, use the five-membered tetrahydropyrrole ring to straighten the chain, and select other amino acids for replacement. The modified compound focuses on chelating two zinc ions, making it impossible to bind to the hydroxyl or water molecules that hydrolyze the β-lactam ring, fundamentally blocking the function of NDM-1 to hydrolyze the hydroxyl or water molecules of the β-lactam ring of the antibiotic, thereby inhibiting the enzyme activity of NDM-1, and realizing a safe and efficient anti-resistant bacteria effect after combined use with meropenem.

[0010] The first object of the present invention , provides a mercaptopropionamide compound, or a pharmaceutically acceptable salt, a stereoisomer or a solvate thereof; the mercaptopropionamide compound is a compound represented by formula (I),

[0011]

[0012] in,

[0013] R 1 Selected from C 1-6 Alkyl, aryl, substituted aryl or heteroaryl, preferably, R 1 Selected from:

[0014]

[0015] R 2 Selected from natural or synthetic amino acids, preferably, R 2 Selected from:

[0016]

[0017] In some embodiments of the present invention, the mercaptopropionamide compound is specifically selected from the compounds with the following structures:

[0018]

[0019] In the present invention, pharmaceutically acceptable salts refer to salts of compounds that are suitable for contact with human or lower animal tissues without undue toxicity, irritation, and allergic reactions within the scope of reliable medical evaluation, have a fairly reasonable benefit-risk ratio, are usually water- or oil-soluble or dispersible, and can be effectively used for their intended purpose.

[0020] Some compounds of the present invention or their stereoisomers contain basic groups such as amine groups, etc., which can form salts with acids, and can form salts with inorganic and / or organic acids or bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts can be directly obtained in the final separation and purification of the compound, or its stereoisomer. It can also be obtained by mixing the compound, or its stereoisomer, with a certain amount of acid appropriately (e.g., equivalent). These salts may form precipitation in the solution and be collected by filtering, or be recovered after solvent evaporation, or be obtained by freeze drying after reaction in an aqueous medium.

[0021] The pharmaceutically acceptable salts described in the present invention include organic acid salts such as citrate, benzenesulfonate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate; inorganic acid salts such as hydrochloride, sulfate, hydrobromide, hydrofluoride, hydroiodide, hydrochloride, phosphate, etc.; or glutamate or aspartate formed with amino acids such as glutamic acid or aspartic acid; sodium salt, potassium salt, calcium salt, etc. formed with inorganic bases; salts formed with organic bases such as triethylamine, methylamine, ethylenediamine, etc.

[0022] The solvates of the mercaptopropionamide compounds of the present invention also fall within the protection scope of the present invention, and the solvents thereof are preferably water, alcohol or an alcohol-water mixture, wherein the alcohol refers to methanol or ethanol.

[0023] The present invention further provides a pharmaceutical composition consisting of the mercaptopropionamide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof and a medically acceptable carrier.

[0024] The second object of the present invention is to , providing the use of a mercaptopropionamide compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a pharmaceutical composition composed of a mercaptopropionamide compound represented by formula (I) and a medically acceptable carrier in the preparation of an inhibitor targeting matrix metalloproteinase NDM-1.

[0025] The results of the enzyme inhibition activity test indicate that the mercaptopropionamide compounds have micromolar to submicromolar NDM-1 inhibitory activity, and the activity results are shown in Table 1.

[0026] The third object of the present invention is to , providing a method for preparing the following mercaptopropionamide compounds:

[0027] 1. Synthesis route of mercaptopropionamide compounds 6a-6q (see Scheme 1)

[0028]

[0029] The preparation method of Scheme 1 comprises the following steps:

[0030] (1) General method for synthesizing intermediate 2: dissolve ethyl 2-bromomethylacrylate and different substituted boric acids in water, add a strong base such as sodium hydroxide, potassium hydroxide, etc., and then add a metal catalyst such as palladium trifluoroacetate, tetrakistriphenylphosphine palladium, etc., stir and react at 90°C until the raw materials react completely, and separate by chromatographic column to obtain intermediate 2.

[0031] (2) General method for synthesizing intermediate 3: Dissolve intermediate 2 in an organic solvent such as tetrahydrofuran, methanol, ethanol, toluene, etc., then add sodium hydroxide or potassium hydroxide, stir at room temperature until the reaction is complete, and separate by chromatographic column to obtain intermediate 3.

[0032] (3) General method for synthesizing intermediate 4: Dissolve intermediate 3 in an anhydrous organic solvent such as dichloromethane, tetrahydrofuran, methanol, etc., then add thioacetic acid dissolved in the same anhydrous organic solvent under stirring at room temperature, react at room temperature until the reaction of the raw materials is complete, and separate by chromatographic column to obtain intermediate 4.

[0033] (4) General method for synthesizing intermediate 5: intermediate 4 is dissolved in an anhydrous organic solvent such as dichloromethane, tetrahydrofuran, etc., and an acylation catalyst such as N,N-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), etc. is added, and then a catalyst such as 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HoBt), etc. is added, and then a catalytic amount of an organic base such as triethylamine, N-methylmorpholine, N,N-diisopropylethylamine, etc. is added, and then different substituted amines or carboxyl protected amino acids such as 4-N-butylbenzylamine, 4-tert-butylbenzylamine, L-phenylalanine tert-butyl ester hydrochloride, L-homophenylalanine ethyl ester hydrochloride, etc. dissolved in the same anhydrous organic solvent are added dropwise under ice bath, and then reacted at room temperature until the reaction of the raw materials is complete, and separated by chromatographic column to obtain intermediate 5.

[0034] (5) General method for synthesizing target compounds 6a-6q: Dissolve intermediate 5 in an organic solvent such as dichloromethane, tetrahydrofuran, methanol, etc., add trifluoroacetic acid under ice bath conditions, and then stir at room temperature until the reaction is complete. After evaporating the organic solvent and trifluoroacetic acid, dissolve it in an organic solvent such as tetrahydrofuran, methanol, ethanol, etc., then add an inorganic strong base such as sodium hydroxide, potassium hydroxide, etc., stir at room temperature until the reaction is complete, and separate it by chromatographic column to obtain target compounds 6a-6q.

[0035] 2. Synthesis route of mercaptopropionamide compound 7o (quaternary ammonium salt of 6o) (see Scheme 2)

[0036]

[0037] The preparation method of Scheme 2 comprises the following steps:

[0038] The general method for synthesizing 7o is as follows: 6o is dissolved in methanol, potassium iodide is added, and the reaction is carried out at 80°C for 10 hours until the reaction is complete. The target compound 7o is obtained by recrystallization from ethyl acetate and methanol.

[0039] The fourth object of the present invention is to , providing the use of a pharmaceutical composition composed of a mercaptopropionamide compound shown in formula (I), or its isomers, or its salts or solvates, or its medically acceptable carriers, in the preparation of an anti-bacterial infection drug, wherein the anti-bacterial infection drug is a drug for preventing and / or treating Gram-negative resistant bacterial infection.

[0040] In vitro antibacterial experiments on two Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) in combination with meropenem showed that the mercaptopropionamide compounds and meropenem had good synergistic antibacterial activity, as shown in Table 2. In particular, some compounds of the present invention (such as (S,S)-6o and quaternary ammonium salt 7o) in combination with meropenem can significantly reduce the MIC value of meropenem. Therefore, the compounds of the present invention and their salts can be used in combination with other antibiotics to prepare compound preparations for anti-Gram-negative bacteria.

[0041] The above-mentioned drugs may also contain one or more pharmaceutically acceptable carriers, including conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and flavoring agents, sweeteners, etc. may also be added if necessary.

[0042] The present invention also provides an antibacterial drug composition that exerts an antibacterial effect by acting as a metallo-β-lactamase inhibitor, wherein the antibacterial drug composition comprises a mercaptopropionamide compound represented by formula (I), or an isomer thereof, or a salt or solvate thereof, or a medically acceptable carrier thereof, and the antibacterial drug composition is in the form of a tablet, capsule, pill, injection, sustained-release preparation, spray, or nano drug delivery system.

[0043] In one embodiment of the present invention, the anti-drug composition further contains an antibiotic, and the antibiotic can be selected as meropenem.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] The design strategy of the novel mercaptopropionamide compounds provided by the present invention is to select captopril, an old drug clinically used as an angiotensin-converting enzyme inhibitor (ACEI), as the lead compound. Based on fragment-based drug design and anti-NDM-1 enzyme activity as the guidance, chemical synthesis and structure-activity relationship screening were carried out at three levels: modifying its methyl part, proline part, and simultaneously modifying both the methyl and proline parts. Finally, mercaptopropionamide compounds with good targeted inhibition of NDM-1 activity were obtained. In particular, the design strategy of quaternary ammonium cephalosporins was utilized to solve the problem of their poor water solubility, achieving a breakthrough from enzyme inhibition activity to the bacterial level for targeted NDM-1 inhibitors. In combination with meropenem, they showed excellent antibacterial activity, low cytotoxicity, good water solubility, and relatively stable in vitro metabolic properties.

[0046] Neither the structures of the mercaptopropionamide compounds provided by the present invention nor their pharmaceutically acceptable salts have been reported in Scifinder. Moreover, their inhibitory activity against NDM-1 and their antibacterial activity in combination with meropenem against Gram-negative drug-resistant bacteria have not been reported. They are a class of novel NDM-1 metallo-β-lactamase inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Effects of compounds (S,S)-6o and 7o on the viability of HEK293 cells. DETAILED DESCRIPTION OF THE INVENTION

[0048] The following further clarifies the data basis of the present invention in combination with specific embodiments. However, these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0049] Example 1: Preparation of methyl (3-mercapto-2-(naphthalen-2-ylmethyl)propanoyl)-L-prolinate (6a)

[0050]

[0051] 1.1 Preparation of ethyl 2-(naphthalen-2-ylmethyl)acrylate (2a)

[0052] 2-Naphthylboronic acid (5 g, 29 mmol), ethyl 2-bromomethylacrylate (3.65 mL, 26 mmol), 0.1 mol% palladium trifluoroacetate, and potassium hydroxide (2.22 g, 40 mmol) were dissolved in 80 mL of water in a 500 mL round-bottom flask and stirred at 90 °C for 10 min. After the reaction was completed, it was filtered through diatomaceous earth, extracted with 80 mL of EA, the organic phase was collected, extracted with saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate. Finally, a colorless transparent oil (3.354 g, yield 75%) was obtained by column chromatography (PE / EA = 10:1). 1 H NMR (400 MHz, CDCl3 )δ7.80-7.76(m,3H),7.64(s,1H),7.45-7.41(m,2H),7.34-7.30(m,1H),6.27(s,1H),5.48 (d,J=1.2Hz,1H),4.17(q,J=7.1Hz,2H),3.79(s,2H),1.25(t,J=7.1Hz,3H)ppm; ESI-MS:m / z 240.1[M+H] + ,C 16 H 16 O 2 .

[0053] 1.2 Preparation of intermediate 3-(acetylthio)-2-(naphthalen-2-ylmethyl)propionic acid (4a)

[0054] In a 250mL round-bottom flask, add ethyl 2-(naphthalene-2-ylmethyl)acrylate (3.35g, 14mmol), dissolve with 20mL tetrahydrofuran, add 120mL 3M sodium hydroxide solution, stir at 90℃ for 1h, after the reaction is completed, add 3M hydrochloric acid solution dropwise, adjust the pH to 2-3, extract with 100mL EA, collect the organic phase and extract it again with saturated sodium chloride solution, dry the organic phase with sodium sulfate solid. Directly proceed to the next step without purification.

[0055] The untreated crude product was dissolved in 45 mL of DCM, and thioacetic acid (3.03 g, 42 mmol) and 0.1 mol% of triethylamine were added. 2 The reaction was carried out at room temperature under protection for 20 hours, and the progress of the reaction was monitored by TLC. After the reaction was complete, DCM was evaporated under reduced pressure, and EA or DCM was repeatedly added and then evaporated to dryness three times, and most of the thioacetic acid was evaporated with the solvent. After column chromatography (DCM / MeOH=20:1), 3.3 g of colorless oil was obtained, with a two-step yield of 80%. ESI-MS: m / z 288.1 [M+H] + ,C 16 H 16 O 3 S. Proceed directly to the next step.

[0056] 1.3 Intermediate (3-(acetylthio)-2-(naphthalen-2-ylmethyl)propionyl)-L-proline methyl ester (5a)

[0057] In a 100 mL round-bottom flask, 3-(acetylthio)-2-(naphthalen-2-ylmethyl)propanoic acid (500 mg, 1.74 mmol) was added, dissolved in 30 mL of DCM, and SOCl was added. 2(287 μL, 3.95 mmol and 0.1 mol% DMF, stirred at room temperature for 3 h, dried by rotation, dissolved in MeCN, added L-proline methyl ester hydrochloride (261 mg, 1.58 mmol), potassium carbonate (346 mg, 3.95 mmol), N 2 After the reaction was complete, 577 mg of yellow oil was obtained by column chromatography (PE:EA=2:1) ​​with a yield of 91%. 1 H NMR (400 MHz, CDCl 3 )δ7.85-7.81(m,1H),7.75(s,1H),7.63(s,1H),7.44(s,3H),7.34-7.30(m,1H),3.70(d,J=19.3Hz,3H),3.50(d,J=17.2Hz,1H),3. 28-3.18(m,2H),3.08(dt,J=17.0,9.6Hz,5H),2.36(d,J=13.4Hz,3H),1.87(dd,J=16.6,10.6Hz,2H),1.75(s,2H)ppm; ESI-MS:m / z 400.2[M+H] + ,C 22 H 25 NO 4 S.

[0058] 1.4 Preparation of the final product (3-mercapto-2-(naphthalen-2-ylmethyl)propionyl)-L-proline methyl ester (6a)

[0059]

[0060] Weigh 230 mg of (3-(acetylthio)-2-(naphthalen-2-ylmethyl)propionyl)-L-proline methyl ester and place it in a 50 mL eggplant-shaped bottle. Add 5 mL of tetrahydrofuran to dissolve it. Then, under nitrogen protection, add 30 mL of evacuated 3M sodium hydroxide aqueous solution. After reacting at room temperature for 6 h, continue to add evacuated 3M sulfuric acid under nitrogen protection, adjust the pH to 2-3, extract with dichloromethane three times, combine the organic layers, dry over anhydrous sodium sulfate, and separate by column chromatography to obtain a colorless oil with a yield of 88%. 1 H NMR (400 MHz, CDCl 3)δ7.80-7.76(m,2H),7.62(s,2H),7.48-7.44(m,2H),7.33-7.29(m,1H),4.42(d,J=6.0Hz,1H),3.47-3.27(m ,1H),3.13-3.00(m,6H),2.19(s,1H),1.76(d,J=6.5Hz,2H),1.54(d,J=8.6Hz,1H),1.27(t,J=7.1Hz,1H)ppm; 13 C NMR (150 MHz, CDCl 3 )δ175.0(174.5),173.9(173.4),136.0(135.7),133.6(132.3),128.5(128.3),127.7(127.7),127.5(127.4),127.2(127.2),126.4(126 .3),125.8(125.8),59.7(59.6),51.1(50.9),47.8(47.7),39.2(39.1),28.1(28.0),27.1(26.3),24.7(24.4)ppm; HRMS(ESI):m / z[M+Na] + Calculate for C 19 H 21 NO 3 S:366.1134,found:366.1135.

[0061] Example 2: Preparation of (3-mercapto-2-(naphthalen-2-ylmethyl)propionyl)-D-proline methyl ester (6b)

[0062]

[0063] Referring to the preparation method of Scheme 1, R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The other preparation routes and conditions were the same as those in Example 1 to obtain a colorless transparent oily liquid with a yield of 79%. 1 H NMR (400 MHz, CDCl 3)δ7.79-7.77(m,2H),7.62(s,2H),7.49-7.44(m,2H),7.34-7.30(m,1H),4.42(d,J=6.0Hz,1H),3.47-3.27(m ,1H),3.13-3.00(m,6H),2.19(s,1H),1.76(d,J=6.5Hz,2H),1.54(d,J=8.6Hz,1H),1.27(t,J=7.1Hz,1H)ppm; 13 C NMR (150 MHz, CDCl 3 )δ175.0(174.5),173.9(173.4),136.0(135.7),133.6(132.3),128.5(128.3),127.7(127.7),127.5(127.4),127.2(127.2),126.4(126 .3),125.8(125.8),59.7(59.6),51.1(50.9),47.8(47.7),39.2(39.1),28.1(28.0),27.1(26.3),24.7(24.4)ppm; HRMS(ESI):m / z[M+Na] + Calculate for C 8 H 13 NO 3 S:366.1134,found:366.1135.

[0064] Example 3: Preparation of (3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)-L-proline methyl ester (6c)

[0065]

[0066] Referring to the preparation method of Scheme 1, the structure of R in (3-(dibenzo[b,d]thiophene-2-yl)-2-(mercaptomethyl)propionyl)-L-proline methyl ester (6c) was combined with 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The structure, other preparation routes and conditions were the same as those in Example 1. A colorless, transparent, oily liquid was obtained by a method similar to that in Example 1 with a yield of 86%. 1 H NMR (400 MHz, CDCl 3)δ9.84(s,1H),8.02(s,1H),7.86-7.82(m,1H),7.72(s,1H),7.62(s,1H),7.32(s,2H),7.16(s,1H),4.38(d,J=76.0Hz,1H), 3.54(d,J=47.2Hz,1H),3.10(s,1H),2.99-2.66(m,3H),2.43(s,1H),1.88(dt,J=143.2,69.2Hz,5H),1.30-1.06(m,1H)ppm; 13 C NMR (150 MHz, CDCl 3 )δ174.0,173.6,139.8(139.8),137.74,135.9(135.8),135.3(135.2),135. 0,128.0(127.8),126.9(126.8),124.6(124.5),123.0(122.9),122.8(122. 8),122.0(121.9),121.8(121.6),60.2(53.6),51.1(50.9),47.8(47.6),38 .9(38.6),28.7(28.7),27.2(26.1),24.9(24.5)ppm; HRMS(ESI):m / z[M+Na] + Calculate for C 21 H 21 NO 3 S 2 :422.0855,found:422.0872.

[0067] Example 4: Preparation of (3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)-D-proline methyl ester (6d)

[0068]

[0069] Referring to the preparation method of Scheme 1, combined with the structure of (3-(dibenzo[b, d]thiophene-2-yl)-2-(mercaptomethyl)propionyl)-D-proline methyl ester (6d), R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The same method as in Example 1 was used to obtain a colorless oil with a yield of 81%. 1 H NMR (400 MHz, CDCl 3)δ9.36(s,1H),8.02(s,1H),7.87-7.85(m,1H),7.72(s,1H),7.62-7.58(m,1H),7.32(s,2H),7.17(s,1H),4.58-3.97(m,1H),3.81-3.3 8(m,1H),3.17(s,2H),2.92(d,J=55.7Hz,2H),2.77(s,1H),2.58(s,1H),2.26-1.61(m,2H),1.53(s,1H),1.22(s,1H),1.20(s,1H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ174.9(173.1),139.9(138.1),136.09(136.0),135.3(135.2),134.8( 134.7),127.9(127.8),127.1(127.0),124.7(124.6),123.1(122.9),12 2.1(122.0),121.8(121.7),60.2(60.0),48.0(47.7),46.7(46.3),39.8 (38.6),29.8(29.6),27.8(27.6),24.8(24.4)ppm; HRMS(ESI):m / z[M+Na] + Calculate for C 21 H 21 NO 3 S 2 :422.0855,found:422.0872.

[0070] Example 5: Preparation of (2-((1H-indol-5-yl)methyl)-3-mercaptopropionyl)-L-proline methyl ester (6e)

[0071]

[0072] Referring to the preparation method of Scheme 1, combined with the structure of (2-((1H-indol-5-yl)methyl)-3-mercaptopropionyl)-L-proline methyl ester (6e), R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The same method as in Example 1 was used to obtain a colorless oil with a yield of 71%. 1 H NMR (400 MHz, CDCl 3)δ8.17(s,1H),7.41(s,1H),7.33-7.29(m,1H),7.24-7.20(m,1H),6.99-6 .96(m,1H),6.49(s,1H),4.55(dd,J=85.9,7.9Hz,1H),3.39(dd,J=41.7,7. 4Hz,1H),3.03(dd,J=22.7,14.1Hz,4H),2.65(d,J=9.8Hz,1H),2.45(d,J= 26.6Hz,1H),2.25(s,1H),1.73(s,1H),1.56-1.51(m,1H),1.26(s,1H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ174.3,173.0(172.8),135.2(135.1),126.0(126.0),122.8(121.7),121.1(120.7),118.4(118.2),117.4(116.8),111.3(110.6) ,110.4(110.0),58.7(58.5),49.3(48.4),46.7(46.6),27.5(27.3),27.0(26.9),26.0(25.5),23.6(23.3)ppm; HRMS(ESI):m / z[M+H] + Calculate for C 17 H 20 N 2 O 3 S:333.1267,found:333.1266.

[0073] Example 6: Preparation of (2-((1H-indol-5-yl)methyl)-3-mercaptopropionyl)-D-proline methyl ester (6f)

[0074]

[0075] Referring to the preparation method of Scheme 1, combined with the structure of (2-((1H-indol-5-yl)methyl)-3-mercaptopropionyl)-D-proline methyl ester (6f), R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The same method as in Example 1 was used to obtain a colorless oil with a yield of 65%. 1 H NMR (400 MHz, CDCl 3)δ8.68(s,0H),8.51(s,1H),7.58-7.54(m,1H),7.38-7.30(m,1H),7.21-7.06(m ,2H),7.00(s,1H),4.66-4.27(m,1H),3.46(ddd,J=25.0,14.9,8.6Hz,1H),3.23 -3.11(m,1H),3.12-3.02(m,2H),2.99-2.89(m,1H),2.68-2.56(m,1H),1.98(dd ,J=9.9,6.7Hz,1H),1.85-1.62(m,2H),1.58-1.45(m,1H),1.30-1.25(m,1H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ175.5,174.1(173.9),136.3(136.3),127.2(127.1),123.9(122.9),122.2(121.8),119.5(119.3),118.5(118.0),112.4(111.7) ,111.5(111.1),59.8(59.6),50.4(49.5),47.8(47.7),28.6(28.4),28.1(28.0),27.1(26.6),24.7(24.4)ppm; HRMS(ESI):m / z[M+H] + Calculate for C 17 H 20 N 2 O 3 S:333.1267,found:333.1266.

[0076] Example 7: Preparation of 2-(3-mercapto-2-methylpropionamido)benzoic acid (6 g)

[0077]

[0078] Referring to the preparation method of Scheme 1, combined with the structure of R in 2-(3-mercapto-2-methylpropionamido)benzoic acid (6 g), 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The structure was prepared by a method similar to that in Example 1 to obtain a white powdery solid with a yield of 67%. 1 H NMR (400 MHz, CDCl 3)δ11.21(s,1H),9.34(s,1H),8.75-8.79(m,1H),8.15-8.19(m,1H),7.65-7.61(m,1H),7.18-7.22(m, 1H),2.96-2.98(m,1H),2.71-2.75(m,2H),1.62(t,J=8.4Hz,1H),1.42(s,3H),1.27-1.31(m,1H)ppm; 13 C NMR (150 MHz, CDCl 3 )δ174.2,172.3,141.6,135.6,131.9,123.2 120.8,114.7,47.0,28.1,17.2ppm; HRMS(ESI):m / z[M+Na] + Calculate for C 11 H 13 NO 3 S:262.0508,found:262.0521.

[0079] Example 8: Preparation of (3S)-1-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)pyrrolidine-3-carboxylic acid (6h)

[0080]

[0081] Referring to the preparation method of Scheme 1, combined with the structure of (3S)-1-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)pyrrolidine-3-carboxylic acid (6h), R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The structure was prepared by the same method as in Example 1 with a yield of 88%. The upper point was a white solid with a melting point of 201-203°C. 1 H NMR (400 MHz, CDCl 3:MeOD=3:1)δ8.08(s,1H),7.88(s,1H),7.76(s,1H),7.70-7.66(m,1H),7.38(s,2H),7.22-7.18(m,1H),3.85-3.54(m,1H),3.44(d,J=27.3 Hz,2H),2.98(t,J=56.4Hz,5H),2.58(d,J=24.2Hz,2H),1.93(d,J=26.5Hz,1H),1.67(dd,J=32.9,11.9Hz,1H),1.23(d,J=46.3Hz,1H)ppm; 13 C NMR (100 MHz, CDCl 3 :MeOD=3:1)δ172.6,172.6,139.6,137.5,135.6,134.9,134.7,127.4,126.6,124.2,122.6,122.5,121.4,121.3,50.8,45.9,44.9,38.6,28.5,27.1,26.2ppm; the lower point is a white solid with a melting point of 183-184°C. 1 H NMR (400 MHz, CDCl 3 :MeOD=3:1)δ8.08(s,1H),7.90-7.86(m,1H),7.76(s,1H),7.69-7.65(m,1H),7.38(s,2H),7.26-7.05(m,1H),3.67(s,1H),3.40(s,1H),3 .25(d,J=23.2Hz,2H),2.95(dd,J=40.8,24.9Hz,4H),2.65(dd,J=38.5,11.9Hz,2H),1.91(s,1H),1.66(s,1H),1.19(d,J=12.7Hz,1H)ppm; 13 C NMR (100 MHz, CDCl 3 :MeOD=3:1)δ171.6,171.5,138.9,136.9,134.9,134.2,134.0,126.7,126.0,123.6,121.9 ,121.8,120.8,120.6,45.5,45.2,37.7,37.5,28.7,28.1,26.4ppm.HRMS(ESI):m / z[M+Na] + Calculate for C 21 H 21 NO 3 S 2:422.0855,found:422.0833.

[0082] Example 9: Preparation of (3R)-1-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)pyrrolidine-3-carboxylic acid (6i)

[0083]

[0084] Referring to the preparation method of Scheme 1, combined with the structure of (3R)-1-(3-(dibenzo[b,d]thiophene-2-yl)-2-(mercaptomethyl)propionyl)pyrrolidine-3-carboxylic acid (6i), R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The structure was prepared by the same method as in Example 1 with a yield of 89%. The upper point was a white solid with a melting point of 231-232°C. 1 H NMR (400 MHz, CDCl 3 :MeOD=3:1)δ8.06(s,1H),7.86(s,1H),7.74(s,1H),7.68-7.64(m,1H),7.36-7.32(m,2H),7.17(s,1H),3.87-3.54(m,1H),3.51 -3.17(m,2H),3.15-2.69(m,5H),2.55(d,J=34.7Hz,2H),1.92(d,J=29.4Hz,1H),1.79-1.51(m,1H),1.23(d,J=49.5Hz,1H)ppm; 13 C NMR (100 MHz, CDCl 3 :MeOD=3:1)δ172.6,172.5,139.6,137.6,135.6,134.9,134.8,127.4,126.7,124.3,122.7,122.5,121.5,121.3,50.9,46.0,45.0,38.8,28.7,27.3,26.3ppm; the lower point is a white solid with a melting point of 209-211°C. 1 H NMR (400 MHz, CDCl 3:MeOD=3:1)δ8.07(s,1H),7.86(m,1H),7.74(s,1H),7.66(m,1H),7.35(m,2H),7.25-7.02(m,1H),3.60(s,1H),3.41(s, 1H),3.23(dd,J=46.2,24.6Hz,2H),3.07-2.28(m,6H),1.92(d,J=32.5Hz,1H),1.74-1.46(m,1H),1.32-1.03(m,1H)ppm; 13 C NMR (100 MHz, CDCl 3 :MeOD=3:1)δ172.4,172.3,139.6,137.6,135.6,135.0,134.7,127.5,126.7,124.3,122.7 ,122.5,121.6,121.4,49.5,46.2,45.9,38.4,38.2,29.5,28.8ppm.HRMS(ESI):m / z[M+Na] + calcdfor C 21 H 21 NO 3 S 2 :422.0855,found:422.0849.

[0085] Example 10: Preparation of 1-(2-(((1H-indol-5-yl)methyl)-3-mercaptopropionyl)piperidine-4-carboxylic acid (6j)

[0086]

[0087] Referring to the preparation method of Scheme 1, combined with the structure of 1-(2-(((1H-indol-5-yl)methyl)-3-mercaptopropionyl)piperidine-4-carboxylic acid (6j), R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The same method as in Example 1 was used to obtain a colorless oil with a yield of 86%. 1H NMR(400MHz,DMSO)δ12.20(s,1H),10.85(s,1H),7.52-7.48(m,1H),7.33-7.29(m,1H),7.15-7.01(m,2H),6.98(s,1H),4.20(s,1H) ,3.25(s,1H),3.00-2.74(m,3H),2.64(dt,J=21.5,12.2Hz,4H),2.30(s,2H),1.66(s,1H),1.37(ddd,J=42.1,29.4,8.3Hz,2H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ178.9,178.7,173.1,173.0,136.4,136.3,128.6,128.2,127.3,127.2,124.6 ,124.1,122.7,122.7,122.3,122.2,119.7,119.2,118.7,118.4,112.9,112.7, 111.6,111.4,46.8,46.7,45.4,44.9,41.5,41.4,40.5,40.4,38.8,35.0,31.6, 30.3,29.8,29.1,28.9,28.2,27.8,27.7,27.5,27.2ppm; HRMS(ESI):m / z[M+Na] + Calculate for C 18 H 22 N 2 O 3 S:369.1243,found:369.1240.

[0088] Example 11: Preparation of 1-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)piperidine-4-carboxylic acid (6k)

[0089]

[0090] Referring to the preparation method of Scheme 1, combined with the structure of R in 1-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)piperidine-4-carboxylic acid (6k), 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The same method as in Example 1 was used to obtain a colorless oil with a yield of 85%. 1 H NMR (400 MHz, CDCl 3)δ8.07-8.01(m,1H),7.85(s,1H),7.81-7.60(m,2H),7.46-7.25(m,2H ),7.21-7.17(m,1H),4.33(dd,J=42.5,13.4Hz,1H),3.68-3.45(m,1H), 3.27-3.11(m,1H),3.03-2.87(m,3H),2.78-2.49(m,2H),2.29-2.16(m ,1H),1.69(t,J=14.3Hz,1H),1.57-1.34(m,3H),1.25-1.13(m,2H)ppm; 13 CNMR (150MHz, CDCl 3 )δ179.0(178.7),172.0,140.0(139.9),138.0(137.8),136.0(135.9),135.3(13 5.2),135.2,127.9(127.8),127.0(126.9),124.6(124.5),123.1(123.0),122.9 ,122.1(122.0),121.7(121.7),48.1,45.4(44.9),41.5(41.4),40.4(40.3),39. 5(39.5),29.8,28.2(27.8),27.6(27.6),27.4(27.2)ppm; HRMS(ESI):m / z[M+Na] + Calculate for C 22 H 23 NO 3 S 2 :414.1192,found:414.1195.

[0091] Example 12: Preparation of (2R)-1-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)piperidine-2-carboxylic acid (61)

[0092]

[0093] Referring to the preparation method of Scheme 1, combined with the structure of (2R)-1-(3-(dibenzo[b,d]thiophene-2-yl)-2-(mercaptomethyl)propionyl)piperidine-2-carboxylic acid (6l), R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The same method as in Example 1 was used to obtain a colorless oil with a yield of 78%. 1H NMR (400 MHz, CDCl 3 )δ7.94-7.90(m,2H),7.84-7.80(m,1H),7.75-7.71(m,1H),7.48-7.43(m,2H),7.24 (s,1H),5.37(d,J=15.9Hz,1H),3.73-3.51(m,1H),3.28(dt,J=11.2,7.2Hz,1H),3. 11-3.03(m,3H),2.94-2.82(m,1H),2.56-2.47(m,1H),2.27(d,J=13.0Hz,1H),2.16 -2.04(m,1H),1.71-1.61(m,2H),1.40(d,J=12.6Hz,1H),1.12(d,J=7.0Hz,2H)ppm; 13 C NMR (150 MHz, CDCl 3 )δ175.3(175.1),174.2(174.0),139.8(137.7),135.9(135.4),135.2(134 .8),128.0(127.8),126.9(126.8),124.5(124.4),122.9(122.9),122.1(12 2.0),121.8(121.6),52.5(52.3),48.1(47.8),44.1(43.8),39.2(38.4),2 7.4(26.7),26.5(26.2),25.4(24.5),20.9(20.5)ppm; HRMS(ESI):m / z[M+H] + Calculate for C 22 H 23 NO 3 S 2 :414.1192,found:414.1196.

[0094] Example 13: Preparation of (2S)-1-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)piperidine-2-carboxylic acid (6m)

[0095]

[0096] Referring to the preparation method of Scheme 1, combined with the structure of (2S)-1-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionyl)piperidine-2-carboxylic acid (6m), R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R2 The same method as in Example 1 was used to obtain a colorless oil with a yield of 76%. 1 H NMR (400 MHz, CDCl 3 )δ7.94(s,2H),7.86-7.78(m,2H),7.48-7.43(m,3H),3.64(d,J=12.6Hz,1H),3.38(s,1H),3.05(d,J=11.5Hz ,3H),2.60(dd,J=24.1,11.1Hz,2H),2.16-1.98(m,1H),1.71(dd,J=29.8,16.5Hz,3H),1.35-1.20(m,4H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ175.6(175.4),174.2(174.1),139.9(137.8),135.97(135.2),135.4(13 4.9),127.9(128.1),127.0(126.9),124.6(124.5),123.0(122.9),122.1(1 22.0),121.8(121.7),52.5(52.4),48.2(47.9),44.1(43.8),39.3(38.5),2 7.5(26.2),26.7(26.6),25.4(24.6),20.9(20.6)ppm; HRMS(ESI):m / z[M+H] + Calculate for C 22 H 23 NO 3 S 2 :414.1192,found:414.1193.

[0097] Example 14: Preparation of 2-(3-(dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propionamido)benzoic acid (6n)

[0098]

[0099] Referring to the preparation method of Scheme 1, combined with the structure of R in 2-(3-(dibenzo[b,d]thiophene-2-yl)-2-(mercaptomethyl)propionamido)benzoic acid (6n), 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The same method as in Example 1 was used to obtain a white powdery solid with a yield of 71%. 1H NMR (400 MHz, CDCl 3 )δ8.44-8.40(m,1H),8.04-8.00(m,2H),7.71-7.67(m,3H),7.46-7.42(m,1H ),7.32-7.28(m,2H),7.24-7.20(m,1H),6.98-6.94(m,1H),3.15(m,6H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ172.5,172.2,141.3,139.8,137.9,136.1,135.5,135.3,134.9,131.8,127.9,126.8,12 4.4,123.1,122.9,122.8,122.0,121.6,120.9,56.2,39.0,26.3ppm; HRMS(ESI):m / z[M+H] + Calculate for C 23 H 19 NO 3 S 2 :422.0879,found:422.0875.

[0100] Example 15: Preparation of (2-(4-(dimethylamino)benzyl)-3-mercaptopropionyl)-L-phenylalanine (6o)

[0101]

[0102] Referring to the preparation method of Scheme 1, the structure of (2-(4-(dimethylamino)benzyl)-3-mercaptopropionyl)-L-phenylalanine (6o) was combined with R 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The structure was prepared by the same method as in Example 1 with a yield of 86%. (S,S)-6o was a white solid with a melting point of 123-126°C. 1 H NMR (400 MHz, CDCl 3 )δ7.16(s,3H),7.16-7.11(m,2H),6.87-6.82(m,4H),6.26-6.22(m,1H),4.85(d,J=7.1Hz,1H),3.05( dd,J=13.6,5.2Hz,1H),2.98-2.91(m,1H),2.88(s,6H),2.85-2.67(m,3H),2.49(d,J=6.6Hz,2H)ppm; 13C NMR (100 MHz, CDCl 3 )δ174.5,173.5,148.7,136.1,130.0,129.6,128.6,127.1,114.7,53.8,53.2,41.8,37.6,37.4,26.4ppm; HRMS(ESI):m / z[M+Na] + calcdfor C 21 H 26 N 2 O 3 S:409.1556,found:409.1553.(R,S)-6o is a white solid with a melting point of 142-143°C. 1 HNMR (400MHz, CDCl 3 )δ7.28-7.24(m,5H),7.00(s,2H),6.73-6.69(m,2H),5.58(s,2H),4.80(s,1H), 3.24(s,1H),3.03(s,1H),2.89(s,6H),2.68(s,2H),2.48(d,J=25.9Hz,2H)ppm; 13 CNMR (100MHz, CDCl 3 )δ174.2,173.1,147.7,136.5,130.6,129.9,129.7,128.6,127.1,115.6,54.1,53.7,42.3,38.1,37.8,26.1ppm; HRMS(ESI):m / z[M+Na] + Calculate for C 21 H 26 N 2 O 3 S:409.1556,found:409.1553.

[0103] Example 16: Preparation of (3-mercapto-2-(4-morpholinobenzyl)propionyl)-L-phenylalanine (6q)

[0104]

[0105] Referring to the preparation method of Scheme 1, (3-mercapto-2-(4-morpholinobenzyl)propionyl)-L-phenylalanine (R in the structure of 6q) is combined with 1 , R 2 The choice of replacing R on the basis of Example 1 1 , R 2 The same method as in Example 1 was used to obtain a slightly yellow oil with a yield of 78%.1 H NMR(400MHz,CDCl 3 )δ7.28-7.13(m,4H),7.13-7.05(m,2H),7.01-6.83(m,3H),3.91(s,1H),3.43(s,4H),3.21-3.13(m,4H),3.07-2.90(m,2H),2.85(d02d,J=13.5,7.6Hz,1H),2.77(d,J=17.6Hz,2H),2.59-2.39(m,2H),1.59(d,J=8.9Hz,1H)ppm; 1 H NMR(400MHz,CDCl 3 )δ7.25(m,5H),7.11(m,2H),6.87(m,2H),5.58(s,2H),4.80(s,1H),3.86(s,4H),3.24(s,1H),3.13(s,4H),3.03(s,1H),2.89(s,6H),2.68(s,2H),2.48(d,J=25.9Hz,2H)ppm; 13 C NMR(100MHz,CDCl 3 )174.5,173.5(173.4),148.7(148.6),136.1(136.0),130.0(129.9),129.6(129.5),128.6(128.5),127.1(127.0),114.7(114,6),53.8(53.6),53.2(53.1),41.8(41.8),37.6(37.5),37.4(37.3),26.4(26.4)ppm; 13 CNMR(100MHz,CDCl 3 )δ173.1(173.0),172.8(172.8),136.4(136.2),129.9(129.8),129.7(129.6),128.8(128.3),128.4(128.4),127.4(127.3),127.0(127.0),126.9(126.9),116.7(116.1),66.9(66.6),53.6(53.4),53.1(52.8),50.3(49.7),37.9(37.6),37.5(37.4),32.0(30.4),29.7(29.7),26.4(25.9)ppm;HRMS(ESI):m / z[M+H] + calcd forC 23 H 28 N2 O 4 S:429.1843,found:429.1843.

[0106] Example 17: Preparation of 4-(3-((((S)-1-carboxy-2-phenylethyl)amino)-2-(mercaptomethyl)-3-oxypropyl)-N,N,N-trimethylbenzylamine (7o)

[0107]

[0108] Dissolve 300 mg (2-(4-(dimethylamino)benzyl)-3-mercaptopropionyl)-L-phenylalanine in 6 mL methanol and add it to a 100 mL round-bottom flask. Slowly drop iodomethane and place in an 80°C oil bath to react for 10 hours. After the reaction is completed, vacuum dry the solvent and recrystallize the product with MeOH / EA to obtain a white solid with a yield of 85%. The melting point is 57-77°C. 1 HNMR (400MHz, D 2 O)δ7.72-7.68(m,2H),7.42-7.22(m,5H),7.04-7.00(m,2H),4.64(dd,J=7.4,6.0Hz,1H),3.54(s,9H),3.00-2.80(m,5H),2.80-2.59(m,3H)ppm; 13 C NMR (100MHz, D 2 O)δ175.2,174.5,145.1,141.3,136.4,130.9,129.1,128.7,127.2,119.7,57.0,53.6,51.4,36.9,36.7,25.6ppm; HRMS(ESI):m / z[M] + Calculate for C 22 H 29 N 2 O 3 S + :401.1899,found:401.1897.

[0109] Example 18: Inhibition test of New Delhi metallo-β-lactamase activity by the compounds prepared in the above examples of the present invention

[0110] Experimental methods:

[0111] All compounds were selected for the New Delhi metallo-β-lactamase activity inhibition experiment, and the experimental method adopted the fluorescence detection method (such as J. Med. Chem. 2015, 56, 6945-6953).

[0112] (1) Preparation of buffer

[0113] 20 mM Tris (1.21 g), 200 mM NaCl (5.84 g), UP water were used to prepare a 500 mL solution, the pH was adjusted to 7.5, and filtered (add the corresponding volume of 0.01% Triton X-100 each time).

[0114] (2) Drug preparation

[0115] 1) 6 mg of pyridine dicarboxylic acid was placed in a 15 mL centrifuge tube, dissolved with a small amount of Watson's distilled water, and buffer was added to make the final volume 10 mL. At this time, the concentration of the mother solution was 3.6 mM. Other drugs were prepared with reference to pyridine dicarboxylic acid to prepare a mother solution with a concentration of 3.6 mM;

[0116] 2) Take 1mL of the stock solution (use a 5mL round-bottom EP tube) and add 2mL of buffer to a concentration of 1.2mM. Then take 1mL from the newly prepared solution and add 2mL of buffer to a concentration of 0.4μM (3-fold dilution to 10 concentrations)

[0117] 3) Perform the same dilution and finally obtain 10 groups of drugs with different concentrations of 3.6 mM, 1.2 mM, 0.4 mM, 133 μM, 44.4 μM, 14.8 μM, 4.94 μM, 1.65 μM, 0.55 μM, and 0.18 μM;

[0118] 4) The preparation methods of other drugs can refer to the preparation method of pyridine dicarboxylic acid;

[0119] (3) Preparation of protein

[0120] 1) The initial concentration of NDM-1 enzyme is 10μM. After melting on ice, take 1μL of enzyme solution and add it to 8.332mL MBL buffer (using a 15mL centrifuge tube) to make the protein concentration 1.2nM; (Try not to touch the enzyme part of the EP tube during preparation, and the enzyme needs to be placed on ice to maintain activity)

[0121] (4) Preparation of fluorescent substrate

[0122] 1) Dissolve the substrate FC-5 (2 mM) in DMSO and store at -20°C. Shake well before use.

[0123] 2) Take 10 μL of substrate solution (1.5 mL EP tube) and use 657 μL MBL buffer to make a 30 μM solution (the final concentration of the substrate during the test is 5 μM)

[0124] (5) Test steps

[0125] 1) Take 10 μL of each diluted compound solution and add it to a black 96-well plate, with three replicate wells for each concentration;

[0126] 2) Add 30 μL of buffer to each well of the compound column, 40 μL of buffer to each well of the positive column, and 50 μL of buffer to each well of the negative column;

[0127] 3) Add enzyme, except for the negative column, 10 μL per well, place on a shaker, and incubate at room temperature for 10 min;

[0128] 4) Using the TECAN Infinite 200 instrument, set up the protocol, first set the kinetics, total time 8 min, interval 45 s, interval shaking, minimum shaking rate, then set the fluorescence, excitation light 380 nm, emission light 460 nm, temperature 25 ° C, and store;

[0129] 5) Quickly add 10 μL of substrate to each well, starting from the low concentration column, and measure immediately after addition;

[0130] 6) Export the data to Excel and calculate IC using GraphPad Prism software 50 , and obtain a curve relationship diagram between activity and inhibitor concentration.

[0131] The experimental results are shown in Table 1:

[0132] Table 1: Half-maximal inhibitory concentration of test compounds on NDM-1 enzyme (unit: μM)

[0133]

[0134]

[0135] The above experimental results show that all the compounds of the present invention have excellent NDM-1 enzyme inhibitory activity and can be used as NDM-1 enzyme inhibitors.

[0136] Example 19: In vitro anti-Gram-negative bacteria experiment of the compound of the present invention combined with meropenem

[0137] Experimental methods:

[0138] According to the recommendations of the relevant documents of the Clinical and Laboratory Standards Institute (CLSI), the MIC of the compound combined with meropenem against clinical isolates was determined by the microbroth dilution method.

[0139] (1) Preparation of antimicrobial drugs

[0140] L-captopril, compound and meropenem were dissolved and diluted with sterile water. The concentration of L-captopril and compound stock solutions was 1280 mg / L, and the concentration of meropenem stock solution was 2560 mg / L;

[0141] (2) Drug concentration range

[0142] a) The concentration range of meropenem is 256-0.06 mg / L;

[0143] b) For Klebsiella pneumoniae: the combined drug concentration of meropenem, L-captopril and compound ranged from 256 / 32 to 0.125 / 32 mg / L;

[0144] c) For Escherichia coli: the combined drug concentration of meropenem, L-captopril and compound ranged from 256 / 15 to 0.125 / 16 mg / L;

[0145] (3) Combination of meropenem and compounds

[0146] The meropenem stock solution was diluted to 1024 mg / L as the first tube working solution for the dilution ratio, and then a 1:1 dilution was performed to form a working solution of 1024-0.5 mg / L.

[0147] L-captopril and compounds, for Klebsiella pneumoniae: the compound stock solution was diluted to 128 mg / L as a fixed concentration working solution; after meropenem was mixed with the compound in equal volumes, the range was reduced to 512 / 64-0.25 / 64 mg / L, and the drug series concentration was mixed with an equal volume of bacterial solution to change the investigation multiple ratio range: 256 / 32-0.125 / 32 mg / L. For Escherichia coli: the compound stock solution was diluted to 64 mg / L as a fixed concentration working solution, and after meropenem was mixed with the compound in equal volumes, the range was reduced to 512 / 32-0.25 / 32 mg / L, and the drug series concentration was mixed with an equal volume of bacterial solution to change the investigation multiple ratio range: 256 / 16-0.125 / 16 mg / L.

[0148] (4) Meropenem alone

[0149] The meropenem stock solution was diluted to 512 mg / L as the first tube working solution for the dilution ratio, and then a 1:1 dilution was performed to form a working solution of 512-0.125 mg / L. After the drug series concentrations were mixed with an equal volume of bacterial solution, the investigation multiple range became: 256-0.06 mg / L.

[0150] (5) Culture medium: Cation-Adjusted Mueller-Hinton Broth (CAMHB), a product of BBL, USA, batch number: 0252334, was used.

[0151] (6) Inoculation volume: The test bacteria cultured overnight were prepared into a 0.5 McFarland turbidimeter using the direct colony suspension method, diluted 100-fold and added to the sample. After mixing with the drug, the final inoculation volume in the drug sensitivity plate was 105 CFU / mL.

[0152] (7) Culture conditions: Incubate at (35±2)℃ for 16-20 hours in air

[0153] (8) Reading and judging the results

[0154] The interpretation of drug sensitivity test results refers to the CLSI 2019 version M100 29th Edition standard

[0155] The experimental results are shown in the following table:

[0156] Table 2: Minimum inhibitory concentration (MIC, mg / L) of meropenem combined with test compounds (32 μg / mL for Klebsiella pneumoniae and 16 μg / mL for Escherichia coli)

[0157]

[0158] The results of in vitro antibacterial activity showed that the combination of meropenem and some compounds of the present invention (such as (S,S)-6o and 7o) can significantly reduce the MIC value of meropenem. Therefore, the compounds of the present invention and their salts can be combined with other antibiotics to prepare compound preparations for use against Gram-negative bacteria.

[0159] Example 20: Study on water solubility and LogP of the compounds of the present invention

[0160] Experimental methods:

[0161] (1) Determine HPLC test conditions

[0162] The liquid phase conditions of the compound were repeatedly measured to ensure that the compound was in the middle of the peak time. The injection conditions for 33h-1 were finally determined as follows: flow rate 0.6mL / min, injection volume 10μL, running for 20min, setting method water changed from 60% to 5%, methanol changed from 40% to 95%. The injection conditions for 33h-1′ were: flow rate 0.6mL / min, injection volume 10μL, running for 10min, water changed from 95% to 5%, methanol changed from 5% to 95%.

[0163] (2) Establishing a standard curve

[0164] Dissolve the sample in methanol to prepare different concentration gradients: 200 μg / mL, 100 μg / mL, 25 μg / mL, 6.25 μg / mL, 1.56 μg / mL, 0.39 μg / mL, and measure the peak area at each concentration. Calculate the data to obtain a standard curve with the drug concentration on the abscissa and the peak area on the ordinate.

[0165] (3) Test water solubility

[0166] Dissolve an excessive amount of the compound in 1 mL of water and sonicate for 10 h until there is undissolved compound. Filter through a membrane filter, and take 200 μL of the filtered aqueous solution to measure the peak area of the compound content. Substitute it into the standard curve obtained above to obtain the water solubility.

[0167] (4) Test the lipophilicity-hydrophilicity partition coefficient

[0168] Mix 1 mL of n-octanol and 1 mL of water, add 1 mg of the compound, vortex for 5 min, and sonicate for 24 h to allow the compound to be fully distributed and dissolved in the two phases. Take the upper n-octanol phase and the lower water phase respectively, measure the peak area of the compound content by HPLC, substitute it into the standard curve to calculate the concentration, and use the formula log(Co / Cw) to calculate the lipophilicity-hydrophilicity partition coefficient log P.

[0169] Test results: The water solubility of (S,S)-6o is 19.4 mg / mL, LogP = 0.7, the water solubility of 7o is 339 mg / mL, LogP = -1.5. The water solubilities of the two compounds are both good, and they have good drug-likeness.

[0170] Example 21: Cytotoxicity test of the compounds of the present invention

[0171] Experimental method:

[0172] (1) Cell culture

[0173] Inoculate HEK293 cells in the logarithmic growth phase with good growth into a 96-well plate, 1×104 cells per well, and culture in a constant temperature incubator at 5% CO2 and 37 °C for 24 h;

[0174] (2) Add the compound

[0175] Dissolve the compound in a small amount of DMSO (so that the final DMSO content in each well for testing is less than 1%), dilute it with water to prepare stock solutions of 1 mM and 2 mM, and add the two stock solutions to the above 96 wells respectively for 10-fold dilution. Continue the culture; after 24 h, aspirate the cell culture medium, add 10% (V / v) CCK8 (TaoSu Biochemical) reagent, and place it in the incubator for 1 h;

[0176] (3) Test

[0177] The absorbance at 450 nm was measured using an enzyme reader (Thermo Fisher), and the inhibition rate was calculated using GraphPad Prism 5 software.

[0178] Test results see Figure 1 The results showed that the IC50 of compounds (S,S)-6o and 7o for inhibiting cell proliferation was >200μM, indicating low cytotoxicity.

[0179] Example 22: Liver microsome metabolic stability experiment (performed by WuXi AppTec)

[0180] Experimental methods:

[0181] (1) Preparation before the experiment

[0182] 1) Solution preparation: The test compound and control compound testosterone, diclofenac and propafenone were prepared into a 10M stock solution with DMSO. 5 μL of the test compound and control stock solution was diluted 100 times with 495 μL of acetonitrile (ACN) to a solution with a concentration of 100 μM (99% ACN).

[0183] 2) Preparation of coenzyme NADPH: Weigh an appropriate amount of NADPH powder (NADPH·4Na, supplier: Chem-Impex International, catalog number 00616) and dilute to 10 mM MgCl 2 In solution (solution concentration: 10 units / mL; final concentration in the reaction system: 1 unit / mL);

[0184] 3) Preparation of liver microsomes: Prepare liver microsome working solution of appropriate concentration in 100 mM potassium phosphate buffer;

[0185] 4) Preparation of quenching solution: Cold (4° C.) acetonitrile (ACN) containing 200 ng / mL toluenesulfonamide and 200 ng / mL labetalol as an internal standard (IS) was used as a quenching solution.

[0186] (2) Experimental procedures

[0187] 1) Preheat empty culture plates T60 and NCF60 for 10 minutes.

[0188] 2) Dilute liver microsomes to 0.56 mg / mL in 100 mM phosphate buffer.

[0189] 3) Transfer 445uL of liver microsome working solution (0.56mg / mL) to preheated culture plates T60 and NCF60, and then pre-incubate the culture plates T60 and NCF60 for 10 minutes with constant shaking at 37°C. Transfer 54μL of liver microsomes to a blank plate, then add 6μL of NAPDH cofactor to the blank plate, and then add 180μL of quenching solution to the blank plate.

[0190] 4) Add 5 μL of compound working solution (100 μM) to the culture plates containing microsomes (T60 and NCF60) and mix thoroughly 3 times.

[0191] 5) For NCF60 plates, add 50 μL of buffer and mix well 3 times. Start timer; incubate the plate at 37°C for 60 minutes while shaking.

[0192] 6) In the quench plate T0, add 180 μL of quench solution and 6 μL of NAPDH cofactor, making sure to keep the plate cool to prevent evaporation.

[0193] 7) For the T60 plate, mix thoroughly 3 times and immediately transfer 54 μL of the mixture to the quench plate at time 0 minutes. Then add 44 μL of NAPDH cofactor to the culture plate (T60). Start the timer; incubate the plate at 37°C for 60 minutes while shaking.

[0194] 8) At 5, 10, 20, 30, and 60 minutes, add 180 μL of the quenching solution to the quenching plate, mix once, and then transfer 60 μL of the sample in the T60 plate to the quenching plate at each time point in sequence.

[0195] 9) For NCF60: Mix once and at the 60 minute time point transfer 60 μL of sample from the NCF60 incubation to the "Quench" plate containing the Quench Solution.

[0196] 10) All sample plates were shaken for 10 minutes and then centrifuged at 4000 rpm for 20 minutes at 4°C.

[0197] 11) Transfer 80 μL of supernatant to 240 μL of HPLC water and mix on a plate shaker for 10 minutes.

[0198] 12) Seal each bioassay plate and shake for 10 minutes before LC-MS / MS analysis

[0199] The results are shown in the following table:

[0200] Table 3: In vitro metabolic stability of compound (S,S)-6o on human and mouse liver microsomal enzymes

[0201]

[0202]

[0203] The results showed that compound (S,S)-6o had good liver microsomal metabolic stability in both humans and mice, indicating that it had certain drugability.

[0204] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A mercaptopropionamide compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, in, R 1 Selected from: R 2 Selected from:

2. The mercaptopropionamide compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, It is characterized in that The mercaptopropionamide compound is specifically selected from the compounds with the following structures:

3. A pharmaceutical composition consisting of a mercaptopropionamide compound of formula (I) as described in any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof and a medically acceptable carrier.

4. Use of the mercaptopropionamide compound of formula (I) as described in any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or the pharmaceutical composition as described in claim 3 in the preparation of an inhibitor targeting metal matrix protease NDM-1.

5. Use of the mercaptopropionamide compound of formula (I) as described in any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or the pharmaceutical composition as described in claim 3 in the preparation of a medicament for preventing and / or treating infectious diseases caused by Gram-negative resistant bacteria.

6. The use according to claim 5, Features: The Gram-negative drug-resistant bacteria are selected from Escherichia coli, Klebsiella pneumoniae, Acinetobacter, Pseudomonas aeruginosa, Enterobacter cloacae, Proteus, Citrobacter freundii, Klebsiella oxytoca, Morganella or Providencia that produce NDM-1 metallo-β-lactamase.

7. The use according to any one of claims 5 or 6, It is characterized in that The anti-bacterial infection drug is a drug for resisting Escherichia coli or Klebsiella pneumoniae producing NDM-1 metallo-β-lactamase.

8. The method for preparing the mercaptopropionamide compound according to claim 2, It is characterized in that The following synthetic route was used: The following steps are involved: (1) A method for synthesizing intermediate 2: dissolving ethyl 2-bromomethylacrylate and different substituted boric acids in water, adding a strong base, and then adding a metal catalyst, stirring the reaction until the raw materials react completely, and separating the intermediate 2 through a chromatographic column; (2) Method for synthesizing intermediate 3: Dissolve intermediate 2 in an organic solvent, then add sodium hydroxide or potassium hydroxide, stir at room temperature until the reaction is complete, and separate by chromatographic column to obtain intermediate 3; (3) A method for synthesizing intermediate 4: intermediate 3 is dissolved in an anhydrous organic solvent, and then thioacetic acid dissolved in the same anhydrous organic solvent is added under stirring at room temperature, and the reaction is carried out at room temperature until the reaction of the raw materials is complete, and intermediate 4 is obtained by separation through a chromatographic column; (4) General method for synthesizing intermediate 5: Dissolve intermediate 4 in an anhydrous organic solvent, add a catalyst, add dropwise different substituted amines or carboxyl protected amino acids dissolved in the same anhydrous organic solvent under an ice bath, and then react at room temperature until the reaction of the raw materials is complete, and separate by chromatographic column to obtain intermediate 5; (5) General method for synthesizing target compounds 6a-6q: Dissolve intermediate 5 in an organic solvent, add trifluoroacetic acid under ice bath conditions, and then stir at room temperature until the reaction is complete. After evaporating the organic solvent and trifluoroacetic acid, dissolve it in an organic solvent again, then add a strong inorganic base, stir at room temperature until the reaction is complete, and separate by chromatographic column to obtain target compounds 6a-6q.

9. A pharmaceutical composition for use against Gram-resistant negative bacteria, It is characterized in that It contains: A therapeutically effective amount of the mercaptopropionamide compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, Antibiotics; and / or, A pharmaceutically acceptable carrier.

10. A pharmaceutical composition for use against Gram-resistant negative bacteria according to claim 9, It is characterized in that The antibiotic of choice is meropenem.

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