Cephalosporin compounds and their use in the preparation of antibacterial medicaments

Abstract

The application belongs to the field of pharmaceutical chemistry and medicine, and relates to a cephalosporin compound and its use in preparing antibacterial drugs. Specifically disclosed are the use of a cephalosporin compound, or a pharmaceutically acceptable salt thereof, or a medicine composition taking the cephalosporin compound as an effective active ingredient in preparing a medicine for preventing and treating gram-negative drug-resistant bacterial infections. The compound can irreversibly covalently bind to metal beta-lactamase, inhibit the hydrolysis of beta-lactam antibiotics, effectively reduce the minimum inhibitory concentration (MIC) value and the blood bacterial load of gram-negative drug-resistant bacteria in a mouse with abdominal infection, and can be combined with beta-lactam antibiotics such as meropenem to prepare a compound preparation for treating diseases such as infections caused by gram-negative drug-resistant bacteria.

Description

Technical Field

[0001] This invention belongs to the fields of medicinal chemistry and pharmaceutical technology, and discloses cephalosporin compounds and their use in the preparation of antibacterial drugs. Specifically, it relates to the use of cephalosporin compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, or pharmaceutical compositions thereof with medically acceptable carriers in the preparation of inhibitors of metallo-β-lactamases including NDM-1, fluorescent probes with labeling function for metallo-β-lactamases, and in the preparation of drugs for the prevention and / or treatment of Gram-negative drug-resistant bacterial infections. Background Technology

[0002] Since Fleming's discovery of penicillin, β-lactam antibiotics have made significant contributions to combating bacterial infections over more than 90 years of development. Due to their superior tolerability and efficacy, β-lactam antibiotics have become the cornerstone of antibacterial drugs. However, in recent years, due to antibiotic overuse and other reasons, bacterial resistance to these life-saving drugs has become a major public health problem worldwide. The mechanisms by which Gram-negative bacteria develop resistance to β-lactams mainly include changes or mutations in the target site, decreased bacterial membrane permeability, the influence of efflux pumps, and the production of hydrolases. A key reason for Gram-negative bacteria's resistance to β-lactams is the production of β-lactamases. According to the Ambler classification scheme, β-lactamases are mainly divided into serine β-lactamases (SBLs, with three classes: A, C, and D) and metallo-β-lactamases (MBLs, only class B). Class B metallo-β-lactamases can be further subdivided into three classes: B1, B2, and B3. Class B1 and B3 have two zinc ion active sites, while Class B2 has only one. MBLs can hydrolyze carbapenem antibiotics. NDM-1, VIM-2, and IMP-1 belong to class B1, while GOB-18 and L1 belong to class B3. New Delhi metallo-β-lactamase 1 (NDM-1) is a Zn(II)-containing enzyme. It activates nucleophilic water or hydroxyl radicals and inactivates antibiotics by cleaving the β-lactam ring. It is the most widespread and harmful metallo-β-lactamase discovered in recent years. It was first discovered in a patient infected with Klebsiella pneumoniae in 2008 and subsequently spread to more than a dozen countries within a month. Currently, a large number of serine β-lactamases are available on the market. Besides early inhibitors like clavulanate potassium and sulbactam, many other inhibitors have been introduced in recent years. For example, avibactam was approved by the FDA in 2015 as a novel antibiotic combination with ceftazidime for the treatment of Gram-negative bacterial infections, and vaborbactam was approved by the FDA in 2017 for use in combination with meropenem for the treatment of complicated urinary tract infections in adults (Antimicrob Agents Chemother: 2015, 59, 2299-2304; JMed Chem: 2015, 58, 3682-3692). However, to date, no metallo-β-lactamases have been marketed.Although both MBLs and SBLs are amide hydrolases, their structures and catalytic mechanisms differ. In particular, unlike SBLs, the active site of MBLs consists of two zinc ions and their chelated water molecules, lacking serine and thus lacking the necessary conditions for SBLs to form covalent bonds. Therefore, the commonly used design strategies for covalent inhibitors of hydrolases are not applicable to NDM-1. Consequently, developing highly effective and low-toxicity drugs against Gram-negative bacterial infections targeting the metallo-β-lactamases of the superbug NDM-1 presents a significant challenge (Chem Rev: 2021, 121, 7957-8094).

[0003] Most existing research on metallo-β-lactamase inhibitors is based on competitive binding to Zn. + Mechanism-guided NDM-1 inhibitors have been developed to combat resistance in Gram-negative bacteria, including thiol inhibitors, carboxylic acid inhibitors, phosphonic acid inhibitors, borate inhibitors, rhodanine inhibitors, peptide inhibitors, allosteric inhibitors, and covalent inhibitors obtained through high-throughput screening (Chembiochem 2014,15,2541-2548; Biochemistry 2019,58,2834-2843; Chem Commun (Camb) 2015,51,9543-9546; Bioorg Med Chem 2013,21,3138-3146; Eur J Pharm Sci 2020,142,105161). However, these nucleophiles have not been widely developed due to the ease with which the coordination bond between the electron-donating group and Zn(II) is dissociated, making it difficult to sustainably inhibit NDM-1 and inactivate it. This results in low inhibitory activity, poor water solubility, or high toxicity due to lack of selectivity. Therefore, it is imperative to develop effective inhibitors of metallo-β-lactamases targeting the superbug NDM-1 for the treatment of Gram-negative bacterial infections. Summary of the Invention

[0004] Based on the deficiencies of the prior art, this application provides cephalosporin compounds and their use in the preparation of antibacterial drugs, specifically relating to the use of cephalosporin compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, or pharmaceutical compositions thereof with medically acceptable carriers in the preparation of inhibitors of metallo-β-lactamases including NDM-1, fluorescent probes having labeling function against metallo-β-lactamases, and in the preparation of drugs for the prevention and / or treatment of Gram-negative drug-resistant bacterial infections.

[0005] NDM-1 is a zinc(II) metallo-β-lactamase. Due to its ability to activate nucleophilic water or hydroxide ions, it can cleave almost all β-lactam antibiotics, including meropenem, ultimately leading to their inactivation. Therefore, to overcome the bottleneck of the lack of a serine residue at the NDM-1 catalytic site, this invention innovatively involves an irreversible covalent inhibitor of NDM-1 targeting the conserved Lys224 at the bottom of the active site, achieving safe and highly effective anti-drug-resistant bacterial activity when used in combination with meropenem. This lysine-targeting irreversible covalent inhibitor of NDM-1 provides a new strategy for the clinical treatment of Gram-negative bacteria.

[0006] The first objective of this invention is to provide a cephalosporin compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof; said cephalosporin compound being a compound of formula (I).

[0007]

[0008] in,

[0009] R 1 Selected from methyl, 2-thienyl, benzyl, ethynyl or (4-(4-((5-(dimethylamino)naphth-1-yl)sulfonyl)piperazin-1-yl)-4-oxobutyryl;

[0010] n is 0, 1, or 2;

[0011] X is a linking group, selected from:

[0012]

[0013] R 2 Selected from one or more of the structures 1), 2), 3), 4), and 5):

[0014] 1) The substituted phenoxy group has the following structure:

[0015] Among them, R 3 -R 7 The substituent is selected arbitrarily from one or more of the following: hydrogen, halogen, methoxy, trifluoromethoxy, methyl, nitro, cyano, formyl or morpholine;

[0016] 2) meta-pyridinephenol, its structure is:

[0017]

[0018] 3) Halogen-substituted alkoxy groups, such as hexafluoroisopropoxy or tetrafluoropropoxy groups substituted by halogens (F, Cl, Br, I).

[0019] 4) N-hydroxysuccinimide, its structure is:

[0020]

[0021] 5) Various substituted amines

[0022] Methyl or hydroxyl.

[0023] In this invention, the specific structures of some cephalosporin compounds represented by formula (I) are further provided:

[0024]

[0025]

[0026]

[0027] In this invention, pharmaceutically acceptable salts refer to compounds that, within the scope of reliable pharmaceutical evaluation, are suitable for contact with human or lower animal tissues without undue toxicity, irritation, or allergic reactions, have a reasonably reasonable benefit-risk ratio, are typically water- or oil-soluble or dispersible, and can be effectively used for their intended purpose.

[0028] Some compounds or stereoisomers of this invention contain basic groups such as amines that can form salts with acids, and can form salts with inorganic and / or organic acids or bases. These include zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkyl ammonium salts. These salts can be obtained directly during the final separation and purification of the compound or its stereoisomers. Alternatively, they can be obtained by mixing the compound or its stereoisomers with an appropriate amount (e.g., an equimolar amount) of acid. These salts may precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or be prepared by freeze-drying after reaction in an aqueous medium.

[0029] The pharmaceutically acceptable salts described in this 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, hydrofluoric acid, hydroiodide, hydrochloride, phosphate, etc.; or salts that can form glutamate or aspartate salts with amino acids such as glutamic acid or aspartic acid; sodium salts, potassium salts, calcium salts, etc. formed with inorganic bases; and salts formed with organic bases such as triethylamine, methylamine, ethylenediamine, etc.

[0030] The solvates of cephalosporin compounds of this invention are also within the scope of protection of this invention. The solvents are preferably water, alcohol or alcohol-water mixtures, where alcohol refers to methanol or ethanol.

[0031] The present invention further provides a pharmaceutical composition consisting of the cephalosporin compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, and a medically acceptable carrier.

[0032] A second object of the present invention is to provide the use of a cephalosporin compound of formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a pharmaceutical composition thereof consisting of a cephalosporin compound of formula (I) and a medically acceptable carrier, in the preparation of a metallo-β-lactamase inhibitor.

[0033] Enzyme inhibition activity tests showed that the cephalosporin compounds had micromolar to submicromolar inhibitory activity against NDM-1 and other metallo-β-lactamases, as shown in Tables 1 and 2.

[0034] A further object of the present invention is to provide the use of a pharmaceutical composition consisting of a cephalosporin compound of formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a medically acceptable carrier thereof, in the preparation of a fluorescent probe having labeling function against metallo-β-lactamases.

[0035] Fluorescent labeling experiments of NDM-1 based on SDS-PAGE showed that the compounds could covalently bind to metallo-β-lactamases such as NDM-1. The covalent binding results of the representative compound 13a are shown below. Figure 2 As shown.

[0036] A further object of the present invention is to provide the use of a pharmaceutical composition consisting of a cephalosporin compound of formula (I), an isomer thereof, a salt thereof, a solvate thereof, or a medically acceptable carrier thereof, in the preparation of an antibacterial medicament, wherein the antibacterial medicament is a medicament for the prevention and / or treatment of infections caused by Gram-negative drug-resistant bacteria.

[0037] In vitro antibacterial experiments against various bacteria using cephalosporin in combination with meropenem showed that the cephalosporin compounds exhibited good synergistic antibacterial activity with meropenem, as shown in Table 3. Figure 1 As shown, compound 6a, in particular, when used in combination with meropenem, significantly inhibited the activity of NDM-1-producing Escherichia coli and Klebsiella pneumoniae. Especially in a mouse model of intraperitoneal infection, compound 6a effectively improved mouse survival and reduced bacterial load in mouse blood, as shown in the activity results. Figure 3 As shown.

[0038] The aforementioned drugs may also contain one or more pharmaceutically acceptable carriers, including conventional pharmaceutical diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorbents, lubricants, etc., and flavoring agents, sweeteners, etc. may be added if necessary.

[0039] The present invention also provides an antimicrobial composition that exerts its antibacterial effect by acting as a metallo-β-lactamase inhibitor, wherein the antimicrobial composition is a tablet, capsule, pill, injection, sustained-release formulation, spray, or nano-drug delivery system.

[0040] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0041] The cephalosporin compound structural design strategy provided by this invention targets exogenous bacterial proteins and utilizes irreversible covalent bonding technology to avoid inhibitor dissociation from the target and achieve sustained protein inactivation. It innovatively overcomes the challenge of the lack of serine at the NDM-1 catalytic site, rationally designing an irreversible covalent inhibitor of NDM-1 that specifically targets the active pocket lysine Lys224, using a cephalosporin as the backbone and an active ester as the warhead. This discovery opens up a breakthrough in the challenging field of lysine-targeting NDM-1 covalent inhibitors, moving from enzyme inhibition activity to the bacterial level.

[0042] The beneficial effects of this invention lie in the fact that the provided cephalosporin compounds are a novel class of covalent inhibitors of metallo-β-lactamases, possessing not only micromolar and submicromolar NDM-1 inhibitory effects, but also exhibiting significant inhibitory activity against NDM-1-producing Escherichia coli and Klebsiella pneumoniae when used in combination with meropenem. In in vivo experiments, the combination of the compound and meropenem improved the survival rate of mouse models infected with E. coli BAA 2452 in the peritoneum, demonstrating good potential and application prospects. It can be further developed into a therapeutic antibacterial drug against infections caused by drug-resistant Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter, Pseudomonas aeruginosa, Enterobacter cloacae, Proteus, Citrobacter freundii, Klebsiella pneumoniae, Morganella morganii, and Providencia. Preferably, it belongs to the category of anti-Escherichia coli and anti-Klebsiella pneumoniae drugs.

[0043] The cephalosporin compounds and their pharmaceutically acceptable salts provided in this invention have not been reported in Scifinder. They represent a novel class of covalent inhibitors of NDM-1 and other metallo-β-lactamases. These compounds, through irreversible covalent binding to metallo-β-lactamases, inhibit the hydrolysis of β-lactam antibiotics, effectively reducing their minimum inhibitory concentration (MIC) and the bacterial load in the blood of mice infected with Gram-negative drug-resistant bacteria in the peritoneum. They can be used in combination with β-lactam antibiotics such as meropenem to formulate compound preparations for treating infections and other diseases caused by Gram-negative drug-resistant bacteria. This invention solves the problem of the lack of safe and effective irreversible covalently bound NDM-1 small molecule inhibitors for treating infections caused by drug-resistant Gram-negative bacteria in the prior art. It is original and innovative, providing a new treatment option and medication regimen for clinical use against infections caused by Gram-negative drug-resistant bacteria. Attached Figure Description

[0044] Figure 1 Compound 6a in combination with meropenem is effective against (A)E. coli BL21(NDM-1) + (B)E.coli BAA2452(bla) NDM-1 The in vitro antibacterial activity of two types of Escherichia coli.

[0045] Figure 2 Compounds 13a, 6a+13a, and captopril+13a were used to fluorescently label NDM-1. The staining methods were: A) Coomassie brilliant blue staining; and B) UV staining.

[0046] Figure 3 The therapeutic effect of compound 6a in combination with meropenem on E. coli BAA2452 intraperitoneal infection model mice. A. Mouse survival curve; B. Bacterial load in mouse blood. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0048] Example 1 Preparation of 8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)thio)methyl)-7-(2-phenylacetamido)-5-thio-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1a).

[0049]

[0050] 1.1 Preparation of intermediate 3-(((2-(((4-methoxybenzyl)oxy)carbonyl)-5-oxo-8-oxo-7-(2-phenylacetyl)-5-thio-1-azabicyclo[4.2.0]octyl)-2-en-3-yl)methyl)thio)propionic acid (1a-3)

[0051] To a solution of chlorcephalosporin-p-methoxybenzyl ester 1a-1 (5 g, 10.27 mmol) in anhydrous DCM (70 mL), m-chloroperbenzoic acid (m-CPBA, 70% purity, 2.53 g, 10.27 mmol) was added. The reaction mixture was stirred at this temperature for 30 minutes, then raised to room temperature and stirred for another 3 hours. A white solid was filtered off and washed with cold DCM (3 x 20 mL) and diethyl ether (3 x 10 mL). The filtrate was concentrated under reduced pressure to give crude product 1a-2, dried under vacuum, and used for the next step without further purification. To a solution of compound 1a-2 (2 g, 3.98 mmol) in anhydrous DMF (20 mL), 3-mercaptopropionic acid (MPA, 0.69 mL, 7.95 mmol) and potassium carbonate (1.10 g, 7.95 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours. After quenching the reaction with H2O (200 mL), the mixture was adjusted to pH 3 with 4 M HCl (resulting in a large white solid precipitate). The precipitate was collected by filtration, dried by forced air drying, and further purified by silica gel column chromatography using DCM:CH3OH (48:2) as eluent to give 1a-3 as a white solid. Yield: 1.74 g (76.5%). 1 H NMR (400MHz, DMSO-d6) δ12.24(s,1H),8.42(t,J=8.6Hz,1H),7.36(d,J=8.6Hz,2H),7.32–7.27(m,4H),7.26–7.20(m,1H),6.93(t,J=5.8Hz,2H),5.7 8(dd,J=8.3,4.7Hz,1H),5.20(dd,J=27.2,11.9Hz,2H),4.92(d,J=3.7Hz, 1H),3.98(d,J=18.2Hz,1H),3.75(s,3H),3.68(d,J=14.2Hz,3H),3.55(dd ,J=13.8,11.3Hz,2H),2.64–2.54(m,1H),2.49–2.45(m,1H),2.41(dd,J=10.4,4.3Hz,2H).13CNMR(151MHz,DMSO-d6)δ172.9,171.0,164.2,161.0,1 59.4,135.8,130.4,129.1,128.3,126.9,126.5,123.4,122.3,113.8,67.3,66.6,58.1,55.1,46.3,41.5,34.2,32.6,25.2.MS(ESI,positive):m / z 595.0[M+Na] + .

[0052] 1.2 Preparation of intermediate 4-methoxybenzyl 8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)thio)methyl)-7-(2-phenylacetamido)-5-thio-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 5-oxide (1a-4).

[0053] EDCI (0.17 g, 0.91 mmol) was added fractionally to an ice-cold solution of 1a-3 (0.4 g, 0.70 mmol) and pentafluorophenol (PFP, 0.17 g, 0.91 mmol) in acetonitrile (10 mL) with stirring. The reaction mixture was slowly heated to room temperature and stirred for 6 hours. The solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography using dichloromethane:acetone (49:1) as the eluent to give 1a-4 as a white solid. Yield: 0.24 g (45.7%). 1 H NMR (400MHz, CDCl3) δ7.34(d,J=7.4Hz,5H),7.27(d,J=7.7Hz,2H),6.89(d,J=7.3Hz,2 H),6.73(d,J=9.7Hz,1H),6.02(d,J=7.1Hz,1H),5.28–5.15(m,2H),4.46(s,1H),4.10 (d,J=18.0Hz,1H),3.89(d,J=14.4Hz,1H),3.80(s,3H),3.68–3.55(m,2H),3.50(d,J= 13.9Hz,1H),3.20(d,J=18.2Hz,1H),2.93(s,2H),2.78(s,2H).MS(ESI,positive):m / z 761.0[M+Na] + .

[0054] 1.3 Preparation of the final product (1a)

[0055] Compound 1a-4 (200 mg, 270.75 μmol) was dissolved in a mixture of trifluoroacetic acid (TFA, 1.00 mL) and anisole (0.20 mL). The reaction mixture was stirred at room temperature for 1 hour, then diluted with cold diethyl ether (5 mL). The precipitate was collected by filtration, washed with cold diethyl ether (2 × 5 mL), and further dried under vacuum overnight to give compound 1a as a white solid. Yield: 112 mg (65.7%). 1H NMR (600MHz, DMSO-d6) δ8.33 (d, J=8.4Hz, 1H), 7.34–7.19 (m, 4H), 5.76 (dd, J= 8.3,4.7Hz,1H),4.91(d,J=3.5Hz,1H),3.99(d,J=18.2Hz,1H),3.78(d,J=13.7 Hz,1H),3.70(d,J=7.3Hz,1H),3.66(d,J=5.5Hz,2H),3.54(d,J=14.1Hz,1H), 3.11–3.08(m,2H),2.84(dt,J=13.8,6.9Hz,1H),2.76(dt,J=13.8,6.8Hz,1H). 13 C NMR (101MHz, DMSO-d6) δ173.0,171.1,164.06(s),162.5,135.9,129.2,128.4,126.7,124.6,121.3,66.6,58.1,46.2,41.6,34.3,32.6,25.2. 19 F NMR (376MHz, DMSO) δ-161.80 (dd, J=20.4, 6.5Hz), -165.43 (dd, J=23.5, 20.4Hz), -171.83 (tt, J=23.5, 6.5Hz). MS (ESI, positive): m / z619.0 [M+H] + 636.0 [M+NH4] + 641.0 [M+Na] + .

[0056] Example 2 Preparation of (6R,7R)-8-oxo-3-((((3-oxo-3-(3,4,5-trifluorophenoxy)propyl)thio)methyl)-7-(2-phenylacetamide)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1b).

[0057] 2.1 Preparation of intermediate 1b-4

[0058] To a solution of 1a-3 (0.4 g, 0.70 mmol) and 3,4,5-trifluorophenol (0.10 g, 0.70 mmol) in DCM (10 mL), EDCI (0.17 g, 0.91 mmol) and 4-dimethylaminopyridine (DMAP, 9 mg, 0.07 mmol) were added with stirring. The resulting mixture was stirred at room temperature for 8 hours, then evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using dichloromethane:acetone (49:1) as the eluent to give 1b-4, a white solid.1 H NMR (400MHz, CDCl3) δ7.38–7.30(m,5H),7.27(dd,J=7.5,1.9Hz,2H),6.92–6.86(m,2H),6.86–6.79(m, 2H),6.76(d,J=10.0Hz,1H),6.02(dd,J=10.0,4.7Hz,1H),5.32–5.10(m,2H),4.47(dd,J=4.8,1.7Hz,1H ),4.12(d,J=18.3Hz,1H),4.01–3.87(m,1H),3.79(d,J=3.1Hz,3H),3.69–3.54(m,2H),3.44(t,J=16.2H z,1H),3.19(d,J=18.3Hz,1H),2.91(dd,J=29.5,3.9Hz,1H),2.82–2.74(m,3H).MS(ESI,positive):m / z 701.60[M+H] + .MS(ESI,negative):m / z 701.90[MH] - .

[0059] 2.2 Preparation of final product 1b

[0060] The final product 1b was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.86 (s, 1H), 8.49 (d, J = 8.0Hz, 1H), 7.44–7.19 (m, 7H), 5.78 (s, 1H), 4.93 (s, 1H), 4. 00(d,J=17.9Hz,1H),3.78–3.61(m,4H),3.54(d,J=13.7Hz,1H),2.88(s,2H),2.78(dd,J=22.1,16.1Hz,2H). 13 C NMR (151MHz, DMSO-d6) δ171.1,169.8,164.1,162.5,150.9(d,J=5.5Hz),149.3(d,J=5.3Hz),145.5(t,J=10.5Hz),135.9, 129.1,128.3,126.6,124.7,121.0,108.1,107.9,66.5,58.1,46.1,41.5,34.1,32.5,24.7.HRMS(ESI,positive)m / zcalcd for C 25 H 21 F3N2O7S2[M+H] +:583.0815,found:583.0788.

[0061] Example 3 (6R,7R)-3-(((3-(4-fluoro-2-methoxyphenoxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thio-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxide (1c)

[0062] 3.1 Preparation of intermediate 1c-4

[0063] Intermediate 1c-4 was synthesized according to the method in step 2.1 of Example 2. 1 H NMR (600MHz, DMSO-d6) δ8.44(d,J=8.4Hz,1H),7.34(d,J=8.6Hz,2H),7.32–7.27(m,4H),7.23( ddd,J=8.6,5.7,3.3Hz,1H),7.11–7.03(m,2H),6.91(d,J=8.6Hz,2H),6.78(td,J=8.5,2.8Hz,1 H),5.79(dd,J=8.3,4.7Hz,1H),5.27–5.14(m,2H),4.93(d,J=3.6Hz,1H),4.02(d,J=18.1Hz,1H ),3.79–3.66(m,10H),3.54(d,J=14.0Hz,1H),2.80–2.71(m,3H),2.64(dt,J=13.5,6.7Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ171.0,169.7,164.2,161.0,159.4,151.9,135.8,135.5,130.4,129.1,128.3,126 .9,126.5,123.4,122.2,113.8,106.4,101.3,67.3,66.6,58.1,56.3,55.1,46.2,41.5,33.8,32.6,25.1.

[0064] 3.2 Preparation of final product 1c.

[0065] The final product 1c was synthesized according to the synthesis method in step 1.3 of Example 1. 1H NMR(600MHz,DMSO-d6)δ13.79(s,1H),8.42(d,J=8.3Hz,1H),7.33–7.27(m,4H),7.25–7.21(m,1H),7 .10(dd,J=8.7,5.9Hz,1H),7.06(dd,J=10.7,2.8Hz,1H),6.78(td,J=8.5,2.8Hz,1H),5.76(dd,J=8. 3,4.7Hz,1H),4.91(d,J=3.6Hz,1H),3.98(d,J=18.1Hz,1H),3.78(d,J=7.5Hz,3H),3.74–3.64(m,4H ),3.55–3.51(m,1H),2.84(dd,J=10.4,4.4Hz,2H),2.81–2.75(m,1H),2.70(dt,J=13.7,6.9Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ171.0,169.7,164.0,162.5,161.2,159.6,152.0,151.9,135.8,129.1,128.3,126.53(s),124.6, 123.5,123.5,121.0,106.4,106.3,101.3,101.1,66.5,58.0,56.3,46.1,41.5,33.8,32.5,25.0.MS(ESI,positive):m / z 577.00[M+H]+,599.16[M+Na]+.

[0066] Example 4: Preparation of (6R,7R)-3-(((3-(4-fluorophenoxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thio-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1d).

[0067] 4.1 Preparation of intermediate 1d-4

[0068] Intermediate 1d-4 was synthesized according to the method in step 2.1 of Example 2. 1H NMR (400MHz, CDCl3) δ7.36–7.27(m,5H),7.23(d,J=14.0Hz,2H),7.03(d,J=5.5Hz ,4H),6.86(d,J=8.1Hz,2H),6.79(d,J=9.8Hz,1H),5.99(dd,J=9.8,4.4Hz,1H),5. 24–5.15(m,2H),4.44(d,J=4.4Hz,1H),4.00(d,J=18.4Hz,1H),3.76(t,J=8.1Hz, 4H), 3.57 (dd, J=13.8, 6.5Hz, 3H), 3.23 (d, J=18.5Hz, 1H), 2.75 (d, J=11.1Hz, 4H).

[0069] 4.2 Preparation of the final product 1d

[0070] The final product 1d was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR(400MHz,DMSO-d6)δ13.99–13.40(m,75H),8.45(d,J=8.0Hz,87H),7.36 –7.22(m,809H),7.18–7.13(m,191H),6.01(s,1H),5.98–5.96(m,1H),5.76( dd,J=7.9,4.7Hz,95H),4.91(d,J=4.0Hz,98H),3.97(d,J=18.1Hz,78H),3.6 8(dd,J=21.6,9.4Hz,437H),3.53(d,J=13.9Hz,107H),2.90–2.66(m,429H). 19 F NMR(376MHz,DMSO-d6)δ-116.26–-117.24(m).MS(ESI,positive):m / z 569.16[M+Na] + HRMS(ESI,positive)m / z calcd for C 25 H 23 FN₂O₇S₂[M+Na] + :569.0823,found:569.0820.

[0071] Example 5: Preparation of (6R,7R)-3-(((3-(4-nitrophenoxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1e).

[0072] 5.1 Preparation of intermediate 1e-4

[0073] Intermediate 1e-4 was synthesized according to the method in step 2.1 of Example 2. 1 H NMR (400MHz, DMSO-d6) δ8.47(d,J=7.7Hz,1H),8.32(d,J=8.0Hz,2H),7.42(d,J=8.1Hz,2 H),7.35(d,J=7.9Hz,2H),7.29(s,4H),7.23(s,1H),6.91(d,J=7.7Hz,2H),5.79(s,1H), 5.22(dd,J=28.7,11.9Hz,2H),4.93(s,1H),4.03(d,J=18.4Hz,1H),3.74–3.63(m,7H),3 .54(d,J=13.9Hz,1H),2.86(d,J=5.6Hz,2H),2.82–2.63(m,2H).HRMS(ESI,positive)m / z calcd for C 33 H 31 N3O 10 S2[M+Na] + :716.1343,found:716.1346.

[0074] 5.2 Preparation of final product 1e

[0075] The final product 1e was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR(400MHz,DMSO-d6)δ13.82(s,1H),8.44(d,J=8.3Hz,1H),8.35–8.28(m,2H),7.50–7.40 (m,2H),7.32–7.28(m,4H),7.27–7.21(m,1H),5.77(dd,J=8.3,4.7Hz,1H),4.92(dd,J=4.8 1.2Hz,1H),3.99(d,J=18.1Hz,1H),3.73(s,2H),3.70(d,J=3.8Hz,1H),3.66(d,J=7.9Hz,1H),3.53(d, J=14.0Hz,1H),2.94(td,J=6.8,2.6Hz,2H),2.82(dt,J=13.8,6.8Hz,1H),2.73(dt,J=13.7,7.0Hz,1H). 13C NMR(101MHz,DMSO-d6)δ171.0,169.7,164.0,162.5,155.2,145.1,135.8,129.1,1 28.3,126.5,125.3,124.7,123.1,120.9,66.5,58.0,46.0,41.5,34.2,32.4,24.8.

[0076] Example 6 Preparation of (6R,7R)-3-(((3-(2-cyanophenoxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1f).

[0077] 6.1 Preparation of intermediate 1f-4

[0078] Intermediate 1f-4 was synthesized according to the method in step 2.1 of Example 2. 1 H NMR (400MHz, DMSO-d6) δ8.48(d,J=8.2Hz,1H),7.95(d,J=7.7Hz,1H),7.81(t,J=7.9Hz,1H),7.50(t,J=7.7H z,1H),7.43(d,J=8.3Hz,1H),7.35(d,J=8.6Hz,2H),7.29(t,J=5.2Hz,4H),7.25–7.20(m,1H),6.91(d,J=8.6 Hz,2H),5.79(dd,J=8.0,4.8Hz,1H),5.21(dd,J=26.6,12.0Hz,2H),4.93(d,J=3.7Hz,1H),4.05(d,J=18.1Hz ,1H),3.74–3.65(m,7H),3.54(d,J=14.0Hz,1H),2.92(t,J=6.8Hz,2H),2.74(ddt,J=27.4,13.6,6.7Hz,2H). 13 C NMR (151MHz, DMSO) δ171.0,169.6,164.2,161.0,159.4,151.7,135.8,135.0,133.6,130.4,129.1,128.3,127.0,126.9, 126.5,123.5,115.1,113.8,106.1,67.3,66.7,58.1,55.1,41.5,34.1,32.7,24.9.MS(ESI,positive):m / z696.18[M+Na] + .HRMS(ESI,positive)m / z calcd forC34 H 31 N3O8S2[M+Na] + :696.1445,found:696.1448

[0079] 6.2 Preparation of final product 1f

[0080] The final product 1f was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR(600MHz,DMSO-d6)δ8.42(d,J=8.3Hz,1H),7.95(dd,J=7.7,1.5Hz,1H),7.81(td,J=8.2,1.6Hz,1H), 7.49(td,J=7.7,0.9Hz,1H),7.44(d,J=8.3Hz,1H),7.33–7.28(m,4H),7.27–7.20(m,1H),5.76(dd,J=8. 3,4.7Hz,1H),4.91(dd,J=4.6,1.3Hz,1H),4.01(d,J=18.1Hz,1H),3.71(ddd,J=23.4,22.5,11.1Hz,4H) ,3.54(d,J=14.0Hz,1H),3.02–2.97(m,2H),2.84(dt,J=13.9,7.0Hz,1H),2.77(dt,J=13.7,6.8Hz,1H).

[0081] Example 7 Preparation of (6R,7R)-3-(((3-(3-morpholinophenoxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1 g).

[0082] 7.1 Preparation of intermediate 1g-4

[0083] Intermediate 1g-4 was synthesized according to the method in step 2.1 of Example 2. 1H NMR(400MHz, DMSO-d6)δ8.45(d,J=8.4Hz,1H),7.36(d,J=8.6Hz,2H),7.33–7.29(m,4H),7.28–7.23( m,2H),6.93(d,J=8.7Hz,2H),6.85(dd,J=8.3,2.0Hz,1H),6.66(dd,J=6.3,4.2Hz,1H),6.54(dd,J=7. 9,1.7Hz,1H),5.80(dd,J=8.3,4.7Hz,1H),5.22(dd,J=26.7,12.0Hz,2H),4.94(d,J=3.8Hz,1H),4.03 (d,J=18.3Hz,1H),3.76–3.67(m,11H),3.55(d,J=14.0Hz,1H),3.13–3.08(m,4H),2.82–2.63(m,4H).

[0084] 7.2 Preparation of 1g of final product

[0085] 1g of the final product was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (600MHz, DMSO-d6) δ8.42(d,J=8.3Hz,1H),7.33–7.27(m,4H),7.24–7.22(m,1H),6.83(dd,J=8. 4,2.1Hz,1H),6.65(t,J=2.1Hz,1H),6.54(dd,J=7.9,1.6Hz,1H),5.76(dd,J=8.3,4.7Hz,1H),4.91 (d,J=3.5Hz,1H),3.98(d,J=18.1Hz,1H),3.73–3.71(m,5H),3.69(d,J=4.6Hz,2H),3.67(d,J=9.0H z,1H),3.54(d,J=14.0Hz,1H),3.13–3.05(m,4H),2.87–2.75(m,3H),2.70(dt,J=13.5,6.9Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ171.0,170.3,164.0,162.5,152.2,151.4,135.8,129.6,129.1,128.3, 126.5,124.7,121.0,112.2,112.1,108.2,66.5,66.0,58.0,48.1,46.1,41.5,34.2,32.4,25.0.

[0086] Example 8 Preparation of (6R,7R)-3-(((3-(3-formylphenoxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1h).

[0087] 8.1 Preparation of the final product over 1 hour

[0088] The intermediate was synthesized for 1 hour and 4 hours according to step 2.1 of Example 2. It was then proceeded directly to the next step without purification. The final product was synthesized for 1 hour according to step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ10.02(s,2H),8.41(d,J=8.3Hz,2H),7.84(d,J=7.6Hz,2H),7.67(d d,J=13.3,5.0Hz,4H),7.48(dd,J=12.0,4.3Hz,2H),7.30(t,J=5.1Hz,10H),7.26–7.22(m,3 H),5.77(dd,J=8.2,4.7Hz,2H),4.91(t,J=5.4Hz,2H),3.99(d,J=18.2Hz,2H),3.76–3.64(m ,9H),3.53(d,J=14.0Hz,3H),2.97–2.88(m,5H),2.87–2.70(m,5H).MS(ESI,positive):m / z 579.16[M+Na] + .MS(ESI,negative):m / z 555.07[MH]-

[0089] Example 9 Preparation of (6R,7R)-3-(((3-(2-fluoro-4-nitrophenoxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1i).

[0090] 9.1 Preparation of intermediate 1i-4

[0091] Intermediate 1i-4 was synthesized according to the method in step 2.1 of Example 2. 1H NMR(400MHz, CDCl3)δ8.14(dd,J=9.2,5.5Hz,1H),7.36–7.32(m,2H),7.31(t,J=2.9Hz,2H),7.28(s, 2H),7.26–7.24(m,1H),7.15–7.06(m,1H),6.99(dd,J=8.3,2.7Hz,1H),6.89–6.82(m,3H),5.99(dd,J =9.9,4.7Hz,1H),5.29–5.15(m,2H),4.48(d,J=3.9Hz,1H),4.03(d,J=18.4Hz,1H),3.80–3.74(m,4H) ,3.60(q,J=16.0Hz,3H),3.27(d,J=18.4Hz,1H),2.90(dd,J=10.3,4.5Hz,2H),2.77(t,J=6.8Hz,2H).

[0092] 9.2 Preparation of final product 1i

[0093] The final product 1i was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.90(s,1H),8.55(d,J=8.1Hz,1H),8.37(d,J=6.3Hz,1H),7.62–7.49(m,2H),7.33(d,J=23.7Hz,5H),5.8 3(s,1H),4.98(s,1H),4.07(d,J=17.9Hz,1H),3.87–3.68(m,4H),3.59(d,J=13.9Hz,1H),3.01(d,J=6.0Hz,2H),2.92–2.74(m,2H).

[0094] Example 10 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-(4-(trifluoromethoxy)phenoxy)propyl)thio)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (1j).

[0095] 10.1 Intermediate 1j-4 was synthesized according to step 2.1 of Example 2. It was then proceeded directly to the next step without purification. Final product 1j was synthesized according to step 1.3 of Example 1. 1H NMR(600MHz,DMSO-d6)δ13.80(s,1H),8.42(d,J=8.3Hz,1H),7.44(d,J=8.6Hz,2H),7 .30(d,J=4.5Hz,4H),7.29–7.25(m,2H),7.23(dt,J=8.8,4.3Hz,1H),5.77(dd,J=8.3, 4.7Hz,1H),4.95–4.89(m,1H),3.97(t,J=16.0Hz,1H),3.75–3.65(m,4H),3.56–3.52( m,1H),2.92–2.85(m,2H),2.80(dd,J=13.8,7.0Hz,1H),2.72(dt,J=13.7,6.9Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ170.4,169.6,163.4,161.9,148.5,145.1,135.2,128.5,1 27.7,125.9,124.1,123.1,121.8,120.3,65.9,57.4,45.5,40.9,33.6,31.8,24.3.

[0096] Example 11 Preparation of (6R,7R)3-((3-(((-2-carboxyl-5-oxide-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methyl)thio)propionyl)oxy)pyridine-1-onium 2,2,2-trifluoroacetate (2a).

[0097] 11.1 Preparation of intermediate 2a-4. Intermediate 2a-4 was synthesized according to the method in step 2.1 of Example 2. 1H NMR (400MHz, DMSO-d6) δ8.48(d,J=8.0Hz,1H),8.13(d,J=2.2Hz,1H),8.02(d,J=4.4Hz,1H),7.36(d,J=8.4H z,2H),7.30(d,J=3.5Hz,4H),7.23(s,1H),7.20(dd,J=8.3,4.4Hz,2H),6.94(d,J=8.4Hz,2H),5.78(dd,J=8 .1,4.5Hz,1H),5.21(dd,J=26.8,11.9Hz,2H),4.92(d,J=4.5Hz,1H),4.00(dd,J=16.0,9.9Hz,1H),3.71(dd ,J=24.2,11.4Hz,7H),3.54(d,J=13.9Hz,2H),2.93–2.84(m,1H),2.67–2.55(m,2H),2.41(t,J=7.1Hz,1H). 13 C NMR (151MHz, DMSO-d6) δ172.3,170.4,163.6,160.4,158.8,153.1,139.6,137.4,135.2,129.8,128.5,127. 7,125.9,123.5,121.4,113.2,66.7,66.0,57.5,54.5,45.7,40.9,33.6,32.0,24.6.MS(ESI,positive):m / z 650.19[M+H] + .

[0098] 11.2 Preparation of final product 2a. Final product 2a was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ11.05(s,2H),8.45(d,J=7.9Hz,3H),8.30(s,4H),8.22(s,4H),7.57(d,J=12.8Hz,8H),7.30(s,15H),7.24(d,J=4.2Hz ,4H),5.82–5.72(m,3H),4.91(d,J=4.1Hz,3H),4.01–3.90(m,3H),3.76 –3.58(m,13H),3.53(d,J=13.9Hz,4H),2.68–2.54(m,8H),2.47(s,6H). 13C NMR (151MHz, DMSO-d6) δ172.9,171.0,164.0,162.4,155.2,136.4,135.9,133.8,129.1,128.3,127.3,126.5,126.1,124.5 121.1,66.5,58.0,46.1,41.5,34.2,32.5,25.1.

[0099] Example 12 Preparation of (6R,7R)-3-(((3-((1,1,1,3,3,3-hexafluoropropane-2-yl)oxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxide (3a).

[0100] 12.1 Preparation of intermediate 3a-4. Intermediate 3a-4 was synthesized according to the method in step 2.1 of Example 2. 1 H NMR (400MHz, CDCl3) δ7.34(td,J=10.9,5.9Hz,5H),7.27(d,J=7.5Hz,2H),6.89(d,J=8.1Hz, 2H),6.75(d,J=10.1Hz,1H),6.02(dd,J=10.0,4.6Hz,1H),5.77(dt,J=11.7,5.8Hz,1H),5.2 7–5.12(m,2H),4.46(d,J=4.5Hz,1H),4.10(d,J=18.4Hz,1H),3.90(d,J=14.1Hz,1H),3.81( s,3H),3.67–3.54(m,2H),3.38(d,J=14.1Hz,1H),3.16(d,J=18.2Hz,1H),2.86-2.65(m,4H).

[0101] 12.2 Preparation of final product 3a. Final product 3a was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, CDCl3) δ8.50(d,J=7.3Hz,1H),7.28(d,J=22.4Hz,5H),6.83(s,1H),5.76(s,1H),4.91(s,1H),3.97(d,J=18 .3Hz,1H),3.77–3.71(m,1H),3.64(dd,J=19.5,11.7Hz,3H),3.53(d,J=13.5Hz,1H),2.91(s,2H),2.71(d,J=20.4Hz,2H). 1H NMR (400MHz, DMSO-d6) δ8.50(d,J=7.3Hz,1H),7.28(d,J=22.4Hz,5H),6.83(s,1H),5.76(s,1H),4.91(s,1H), 3.97(d,J=18.3Hz,1H),3.72(dd,J=19.1,14.8Hz,2H),3.66–3.46(m,4H),2.91(s,2H),2.71(d,J=20.4Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ129.6,128.7,127.0,67.0,66.4(dt,J=67.2,33.6Hz),58.5,46.3,42.0,39.9,33.8,32.7,24.9.MS(ESI,positive):m / z602.98[M+H] + ;MS(ESI,positive):m / z 625.13[M+Na] + .

[0102] Example 13 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-(2,2,3,3-tetrafluoropropoxy)propyl)thio)methyl)-7-(2-phenylacetamide)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxygen compound (3b).

[0103] 13.1 Preparation of intermediate 3b-4. Intermediate 3b-4 was synthesized according to the method in step 2.1 of Example 2. 1 H NMR (400MHz, CDCl3) δ7.40–7.19(m,7H),6.88(d,J=8.3Hz,2H),6.82(d,J=10.0 Hz,1H),6.05–5.96(m,1H),5.28–5.13(m,2H),4.47(t,J=12.9Hz,3H),4.05(d,J =18.3Hz,1H),3.84(d,J=14.2Hz,1H),3.80(s,3H),3.60(s,2H),3.42(d,J=14. 1Hz,1H),3.18(d,J=18.4Hz,1H),2.66(d,J=7.9Hz,4H).MS(ESI,positive):m / z 709.17[M+Na] + .MS(ESI,negative):m / z685.22[MH]-.

[0104] 13.2 Preparation of final product 3b. Final product 3b was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.79(s,2H),8.45(d,J=7.9Hz,3H),7.27(d,J=25.3Hz,16H),6.59(t,J=51.8Hz,3H),5.74(d,J=5.8Hz,3 H),4.90(s,3H),4.61(t,J=14.1Hz,6H),3.95(d,J=18.1Hz,3H),3.73–3.60(m,13H),3.53(d,J=14.0Hz,4H),2.78–2.56(m,13H). 19 F NMR (376MHz, DMSO-) d6 )δ-124.6,-139.0.MS(ESI,positive):m / z 567.01[M+H] + .MS(ESI,positive):m / z 589.16[M+Na] + .MS(ESI,negative):m / z 565.22[MH] -

[0105] Example 14 Preparation of (6R,7R)-3-(((3-((2,5-dioxopyrrolidine-1-yl)oxy)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxide (4a).

[0106] 14.1 Preparation of intermediate 4a-4. Intermediate 4a-4 was synthesized according to the method in step 1.1 of Example 1. 1 H NMR(400MHz, DMSO-d6)δ8.48(d,J=8.3Hz,2H),7.38(d,J=8.7Hz,4H),7.34–7.29(m,8H), 7.27–7.22(m,2H),6.98–6.94(m,4H),5.79(dt,J=9.8,4.9Hz,2H),5.29–5.16(m,4H),4.9 3(d,J=3.4Hz,2H),4.04(d,J=18.1Hz,2H),3.76(s,6H),3.72–3.65(m,7H),3.55(d,J=13. 9Hz,2H),2.92(t,J=6.7Hz,4H),2.83(s,7H),2.79–2.60(m,5H).HRMS(ESI,positive)m / z calcd for C31 H 31 N3O 10 S2[M+Na] + :692.1343,found:692.1334.

[0107] 14.2 Preparation of final product 4a. Final product 4a was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.45(d,J=8.3Hz,1H),7.36–7.21(m,5H),5.77(dd,J=8.3,4.7Hz,1H),4.91(d,J=3.5Hz,1H),4.01(d,J=18.1Hz,1H),3.78(d ,J=13.8Hz,1H),3.69(d,J=14.1Hz,2H),3.67–3.61(m,2H),3.54(d,J=14. 0Hz, 2H), 2.99 (t, J=6.9Hz, 2H), 2.81 (d, J=5.5Hz, 4H), 2.80–2.68 (m, 2H).

[0108] Example 15 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-(((S)-2-oxotetrahydrothiophene-3-yl)amino)propyl)thio)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5a).

[0109] 15.1 Preparation of intermediate 5a-4. EDCI (0.17 g, 0.91 mmol) and 1-hydroxyphenyltriazole (HOBt, 123 mg, 0.91 mmol) were added to a solution of 1a-3 (0.4 g, 0.70 mmol) and (S) homocysteine ​​thiolactone hydrochloride (0.11 g, 0.70 mmol) in DCM (10 mL) under stirring. The resulting mixture was stirred at room temperature for 8 hours, then evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using dichloromethane:acetone (49:1) as the eluent to give 5a-4 as a white solid. 1HNMR(400MHz, CDCl3)δ7.53(dd,J=20.0,10.2Hz,1H),7.39–7.28(m,1H),6.93–6.86(m,2H),6.74–6.62(m,3H),6.13–6 .02(m,1H),5.29–5.17(m,1H),4.95(t,J=11.7Hz,1H),4.76(dd,J=13.1,6.8Hz,1H),4.50(d,J=4.9Hz,1H),4.45(dd,J =14.7,3.9Hz,1H),3.82(d,J=3.1Hz,1H),3.66(t,J=3.5Hz,1H),3.40(dt,J=11.4,5.8Hz,1H),3.32–3.24(m,1H),3.13 (d,J=14.6Hz,1H),3.02(d,J=19.4Hz,1H),2.74(t,J=12.9Hz,1H),2.31(d,J=5.4Hz,1H),1.88(dd,J=13.0,6.7Hz,1H).

[0110] 15.2 Preparation of final product 5a: Final product 5a was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6)δ. 8.46(d,J=8.3Hz,1H),8.22(d,J=8.3Hz,1H),7.30(s,4H),7.24(s,1H),5.86– 5.68(m,1H),4.91(d,J=4.4Hz,1H),4.69–4.59(m,1H),4.02(d,J=18.3Hz,1H), 3.67(d,J=7.5Hz,4H),3.53(s,1H),3.40(td,J=11.4,5.2Hz,1H),3.33–3.26( m,1H),2.67(t,J=6.6Hz,2H),2.48–2.30(m,4H),2.03(dd,J=12.3,7.0Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ205.6,171.0,170.6,163.9,162.4,135.8,129.1,128.3,12 6.5,124.3,121.70(s),66.5,58.2,58.0,45.9,41.5,35.4,33.0,30.5,26.8,26.4.

[0111] Example 16 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-(((R)-2-oxotetrahydrothiophene-3-yl)amino)propyl)thio)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5b).

[0112] 16.1 Preparation of intermediate 5b-4. Intermediate 5b-4 was synthesized according to the method in step 14.1 of Example 14. 1 HNMR(400MHz, CDCl3)δ7.43–7.27(m,23H),6.96–6.85(m,12H),6.04(dd,J=9.8,4.6Hz,3H),5.3 0(d,J=3.0Hz,2H),5.22(t,J=9.2Hz,6H),4.52(dd,J=5.0,1.4Hz,3H),4.48–4.33(m,6H),4.30– 4.21(m,3H),3.81(s,10H),3.65(t,J=2.3Hz,6H),3.36(td,J=11.7,5.1Hz,4H),3.31–3.22(m,4 H),3.12(d,J=18.4Hz,3H),3.02–2.79(m,14H),2.43(dt,J=11.7,4.5Hz,6H),2.13–1.97(m,4H).

[0113] 16.2 Preparation of final product 5b. Final product 5b was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.74(s,1H),8.46(d,J=8.3Hz,1H),8.20(d,J=8.2Hz,1H),7.29(d,J=4.0Hz, 4H),7.25–7.20(m,1H),5.74(dd,J=8.0,4.7Hz,1H),4.89(d,J=4.3Hz,1H),4.60(dt,J=15.0,7.7Hz,1 H),3.93(d,J=18.0Hz,1H),3.72–3.58(m,4H),3.51(d,J=13.9Hz,1H),3.43–3.38(m,1H),3.27(d,J=1 0.1Hz,1H),2.70–2.57(m,2H),2.46–2.39(m,1H),2.35(t,J=7.0Hz,2H),2.04(qd,J=12.1,7.0Hz,1H). 13C NMR(151MHz,DMSO-d6)δ205.4,171.0,170.5,163.9,162.4,135.8,129.1, 128.3,126.5,66.5,58.1,58.0,46.1,41.5,35.3,33.0,30.3,26.8,26.2.

[0114] Example 17 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-((4-(trifluoromethoxy)phenyl)amino)propyl)thio)methyl)-7-(2-phenylacetamide)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5c).

[0115] 17.1 Preparation of intermediate 5c-4. Intermediate 5c-4 was synthesized according to the method in step 15.1 of Example 15. 1 HNMR(400MHz,DMSO-d6)δ9.94(s,1H),8.50(d,J=8.2Hz,1H),7.43(s,3H),7.38(t,J=7.3Hz,2H),7.34(d,J=8.4Hz,3H),7 .30(d,J=4.2Hz,4H),7.25–7.22(m,1H),7.20(d,J=8.1Hz,1H),7.12(d,J=8.1Hz,1H),6.89(d,J=8.3Hz,2H),6.71(d,J=7 .9Hz,1H),5.84–5.72(m,1H),5.21(dd,J=34.0,11.9Hz,2H),5.06(s,2H),4.94(d,J=4.3Hz,1H),4.00(d,J=18.3Hz,1H), 3.77–3.69(m,5H),3.66(s,1H),3.60(d,J=13.8Hz,1H),3.53(d,J=13.9Hz,1H),2.83–2.56(m,2H),2.53(d,J=6.1Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ171.0,169.6,164.3,161.0,159.4,143.5,138.3,135.8,130.4,129.1,128.3,126.9,126.6 ,123.3,122.5,121.6,120.4,113.8,67.3,66.6,58.1,55.1,46.3,41.5,36.4,32.9,25.9.HRMS(ESI,positive)m / z calcd for C 34 H 32F3N3O8S2[M+H] + :754.1475,found:754.1482.

[0116] 17.2 Preparation of final product 5c. Final product 5c was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ10.14(d,J=8.8Hz,1H),8.47(d,J=8.1Hz,1H),7.70(d,J=8 .8Hz,2H),7.31(d,J=4.3Hz,6H),7.24(dd,J=8.4,4.2Hz,1H),5.77(dd,J=8.1,4.7H z,1H),4.93(d,J=4.2Hz,1H),3.98(d,J=18.1Hz,1H),3.72(t,J=8.4Hz,3H),3.67( d,J=10.4Hz,1H),3.53(d,J=13.9Hz,1H),2.82–2.66(m,2H),2.59(t,J=6.8Hz,2H). 13 C NMR (151MHz, DMSO-d6) δ171.0,169.7,164.0,162.4,143.4,138.3,135.8,129.1,128. 1,126.5,124.4,121.6,121.4,120.3,113.8,66.5,58.1,46.2,41.5,36.5,32.9,26.1. 19 F NMR (376MHz, DMSO-d6) δ -57.1.

[0117] Example 18 Preparation of (6R,7R)-3-(((3-((3,4-dimethoxyphenyl)amino)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5d).

[0118] 18.1 Preparation of intermediate 5d-4. Intermediate 5d-4 was synthesized according to the method in step 14.1 of Example 14. 1HNMR (400MHz, DMSO-d6) δ9.78 (s, 1H), 8.45 (d, J = 8.3Hz, 1H), 7.34 (d, J = 8.5Hz, 3H), 7.30 (d, J = 4.5Hz, 4H), 7. 26–7.21(m,1H),7.09(dd,J=8.7,2.2Hz,1H),6.93–6.85(m,3H),5.79(dd,J=8.3,4.7Hz,1H),5.21(dd,J=35. 5,11.9Hz,2H),4.94(d,J=3.8Hz,1H),4.00(d,J=18.3Hz,1H),3.73(dd,J=9.9,5.0Hz,11H),3.67(d,J=7.9Hz ,1H),3.59(d,J=13.8Hz,1H),3.54(d,J=13.9Hz,1H),2.67(ddd,J=19.9,13.1,6.2Hz,2H),2.49–2.40(m,2H).

[0119] 18.2 Preparation of the final product 5d

[0120] The final product was synthesized 5 days according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.77(s,1H),9.21(s,1H),8.42(dd,J=12.6,8.3Hz,1H),7.33–7.26(m,5H),7.23(td, J=4.6,2.2Hz,1H),7.08(dd,J=8.4,5.5Hz,2H),6.89(d,J=5.7Hz,1H),6.81(d,J=8.6Hz,2H),6.72(d,J=8.3Hz, 1H),5.82–5.69(m,1H),4.91(dd,J=8.7,4.1Hz,1H),3.92(d,J=18.5Hz,1H),3.80(s,1H),3.71(d,J=1.4Hz,2H ),3.69(d,J=3.5Hz,6H),3.66(s,4H),3.53(d,J=10.7Hz,1H),2.76–2.67(m,2H),2.54(dd,J=12.7,5.2Hz,2H).

[0121] Example 19 Preparation of (6R,7R)-3-(((3-(4-((5-(dimethylamino)naphth-1-yl)sulfonyl)piperazin-1-yl)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5e).

[0122] 19.1 Preparation of intermediate 5e-4. Intermediate 5e-4 was synthesized according to the method in step 14.1 of Example 14. 1 HNMR (600MHz, DMSO-d6) δ8.52(d,J=8.5Hz,1H),8.34(d,J=8.4Hz,1H),8.31(d,J=8.7Hz,1H),8.14(d,J=7.3Hz,1H),7.68–7.64(m,1H),7.64–7.58( m,1H),7.33–7.28(m,6H),7.26(d,J=7.5Hz,1H),7.24–7.21(m,1H),6.89 (t,J=5.7Hz,2H),5.79(dd,J=8.4,4.7Hz,1H),5.13(dd,J=42.8,12.0Hz,2 H),4.90(d,J=3.9Hz,1H),3.96(d,J=18.2Hz,1H),3.76–3.68(m,4H),3.66(d,J=6.7Hz,1H),3.56(dd,J=21.0,14.1Hz,3H),3.49(dd,J=11.4,5.9Hz ,2H),3.41(d,J=4.6Hz,2H),3.16–3.02(m,4H),2.82(d,J=4.5Hz,6H),2.50(dt,J=8.7,4.4Hz,2H),2.45(q,J=5.5Hz,2H).HRMS(ESI,positive)m / z calcd for C 43 H 47 N5O9S3[M+H] + :874.2609,found:874.2571.

[0123] 19.2 Preparation of final product 5e. Final product 5e was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.53(d,J=8.2Hz,1H),8.32(dd,J=19.0,8.4Hz,2H),8.13(d,J=7.0Hz,1H),7.70–7.55(m,2H),7.29(s,5H),7.24(s,1H),5 .75(s,1H),4.88(s,1H),3.92(d,J=18.0Hz,1H),3.68(d,J=14.2Hz,5H), 3.53(d,J=13.7Hz,2H),3.47(s,4H),3.10(d,J=18.2Hz,4H),2.83(s,6H). 13C NMR (101MHz, DMSO-d6) δ170.9,169.2,164.0,162.4,151.4,135.7,132.6,130.4,130.0,129.6,129.2,129.1,128.3,126. 5,124.3,123.7,121.6,118.9,115.4,66.4,58.0,46.0,45.5,45.2,45.0,44.4,41.5,32.9,25.6.HRMS(ESI,positive)m / z calcd for C 35 H 39 N5O8S3[M+H] + :874.2609,found:874.2571.

[0124] Example 20 Preparation of (6R,7R)-3-(((3-((4-chlorophenyl)amino)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide.

[0125] 20.1 Preparation of intermediate 5f-4. Intermediate 5e-4 was synthesized according to the method in step 14.1 of Example 14. 1 HNMR(400MHz,DMSO-d6)δ10.08(s,1H),8.48(d,J=8.1Hz,1H),7.63(d,J=8.6Hz,2H),7.38–7.32(m,4H ),7.28(t,J=12.6Hz,5H),6.90(d,J=8.4Hz,2H),5.86–5.74(m,1H),5.21(dd,J=33.5,11.9Hz,2H),4. 94(d,J=4.1Hz,1H),4.00(d,J=18.2Hz,1H),3.76–3.65(m,6H),3.59(d,J=13.9Hz,1H),3.53(d,J=13. 9Hz, 1H), 2.71 (dd, J=13.2, 6.8Hz, 1H), 2.61 (dd, J=13.1, 6.5Hz, 1H), 2.53 (s, 1H), 2.46–2.37 (m, 1H). 13C NMR(151MHz,DMSO-d6)δ171.0,169.5,164.2,161.0,159.4,138.0,135.8,130.4,129.1,128.6,128.3,126.9,126.7 ,126.5,123.3,122.5,120.6,113.8,67.3,66.6,58.1,55.1,46.3,41.5,36.4,32.9,25.9.HRMS(ESI,positive)m / z calcd for C 25 H 24 ClN3O6S2[M+H] + :562.0868,found:562.0864.

[0126] 20.2 Preparation of final product 5f. Final product 5f was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.77(s,1H),10.06(s,1H),8.47(d,J=8.3Hz,1H),7. 62(d,J=8.5Hz,2H),7.35(d,J=8.5Hz,2H),7.27(dd,J=26.3,3.9Hz,5H),5.76 (dd,J=7.7,4.6Hz,1H),4.92(d,J=4.2Hz,1H),3.97(d,J=18.2Hz,1H),3.74–3 .63(m,4H),3.53(d,J=13.8Hz,1H),2.83–2.64(m,2H),2.57(t,J=6.9Hz,2H). 13 C NMR (151MHz, DMSO-d) 6) δ171.0,169.6,164.0,162.4,138.0,135.8,129.1,128.6,128.,126.6 ,126.5,124.4,121.4,120.6,66.5,58.0,46.2,41.5,36.5,32.9,26.1.

[0127] Example 21 Preparation of (6R,7R)-3-(((3-((3-(benzyloxy)phenyl)amino)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5 g).

[0128] 21.1 Preparation of intermediate 5g-4. Intermediate 5g-4 was synthesized according to the method in step 14.1 of Example 14.1 HNMR(400MHz,DMSO-d6)δ9.94(s,1H),8.50(d,J=8.2Hz,1H),7.43(s,3H),7.38(t,J=7.3Hz,2H),7.34(d,J=8.4Hz,3H),7 .30(d,J=4.2Hz,4H),7.25–7.22(m,1H),7.20(d,J=8.1Hz,1H),7.12(d,J=8.1Hz,1H),6.89(d,J=8.3Hz,2H),6.71(d,J=7 .9Hz,1H),5.84–5.72(m,1H),5.21(dd,J=34.0,11.9Hz,2H),5.06(s,2H),4.94(d,J=4.3Hz,1H),4.00(d,J=18.3Hz,1H), 3.77–3.69(m,5H),3.66(s,1H),3.60(d,J=13.8Hz,1H),3.53(d,J=13.9Hz,1H),2.83–2.56(m,2H),2.53(d,J=6.1Hz,2H). 13 C NMR (151MHz, DMSO-d) 6) δ171.0,169.5,164.2,161.0,159.4,158.6,140.3,137.0,135.8,130.4,129.5,129.1,128.4,128.3,127.8, 127.6,126.9,126.5,113.8,111.6,109.4,105.8,69.1,67.3,66.6,58.1,55.1,46.3,41.5,36.5,32.9,26.0.

[0129] 21.2 Preparation of 5g of final product. 5g of final product was synthesized according to the synthesis method in step 1.3 of Example 1. 1H NMR (400MHz, DMSO-d6) δ13.78(s,1H),9.90(s,1H),8.46(t,J=10.8Hz,1H),7.44(d,J=7.3Hz,2H),7.42–7. 36(m,3H),7.32(dd,J=13.1,5.6Hz,5H),7.25–7.21(m,1H),7.19(d,J=8.1Hz,1H),7.12(d,J=8.0Hz,1H),6 .70(d,J=8.2Hz,1H),5.83–5.72(m,1H),5.05(d,J=12.7Hz,2H),4.92(d,J=4.4Hz,1H),3.97(d,J=18.2Hz, 1H), 3.71–3.63 (m, 4H), 3.53 (d, J=13.9Hz, 1H), 2.73 (ddt, J=19.3, 12.7, 6.4Hz, 2H), 2.56 (t, J=6.9Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ171.5,170.0,164.4,162.9,159.1,140.8,137.5,136.3,130.0,129.6,128.9,128.8,128.3,128.1 ,127.0,124.9,121.9,114.2,112.0,109.9,106.2,69.6,67.0,58.5,46.6,42.0,37.1,33.4,26.7.MS(ESI,positive):m / z 634.07[M+H] + .MS(ESI,negative):m / z 632.19[MH]-.HRMS(ESI,positive)m / z calcd for C 32 H 31 N3O7S2[M+Na] + :656.1496,found:656.1475.

[0130] Example 22 Preparation of (6R,7R)-3-(((3-((4-(morpholinomethyl)phenyl)amino)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5h).

[0131] 22.1 Preparation of intermediate 5h-4. Intermediate 5h-4 was synthesized according to the method in step 14.1 of Example 14. 1H NMR (400MHz, DMSO-d6) δ10.27(s,1H),8.44(d,J=8.3Hz,1H),7.69(d,J=7.7Hz,2H),7.53(d,J=7.1Hz,2 H),7.34(d,J=7.1Hz,2H),7.29(s,4H),7.23(s,1H),6.90(d,J=7.1Hz,2H),5.76(d,J=21.4Hz,1H),5.21 (dd,J=33.8,12.3Hz,2H),4.93(d,J=28.6Hz,1H),4.24(s,2H),4.00(d,J=18.0Hz,1H),3.89(s,2H),3.8 4–3.68(m,7H),3.67–3.57(m,2H),3.53(d,J=13.2Hz,1H),3.15(s,2H),3.03(s,2H),2.72-2.55(m,4H). 13 C NMR(151MHz,DMSO-d6)δ171.0,170.0,164.2,160.9,159.3,149.1,145.7,135.8,130.3,129.1,128.3,126.8,126.5,125.9, 123.6,122.4,117.1,113.8,67.2,66.5,58.3,55.3,55.1,53.8,47.9,45.8,45.0,41.5,27.0,26.6.HRMS(ESI,positive)m / z calcd for C 38 H 42 N4O8S2[M+H] + :747.2517,found:747.2511.

[0132] 22.2 Preparation of final product 5h. Final product 5f was synthesized according to the synthesis method in step 1.3 of Example 1. 1H NMR (600MHz, DMSO-d6) δ10.13(s,1H),8.42(d,J=8.3Hz,1H),7.66(t,J=10.0Hz,2H),7.47(d,J= 8.5Hz,2H),7.36–7.29(m,4H),7.24(dt,J=6.0,3.8Hz,1H),5.76(dd,J=8.3,4.7Hz,1H),4.92(dd ,J=17.0,3.6Hz,1H),4.24(s,2H),3.97(d,J=18.2Hz,1H),3.86–3.65(m,7H),3.53(d,J=14.0Hz ,1H),3.44(dd,J=14.0,7.0Hz,1H),3.11(d,J=6.6Hz,4H),2.80–2.70(m,2H),2.63–2.56(m,2H). 13 C NMR(151MHz,DMSO-d6)δ171.0,169.8,164.0,162.4,140.2,135.8,131.9,129.1,128.3,126.6,124.4,121 .3,119.0,66.5,63.2,58.9,58.0,56.0,50.6,46.1,41.5,36.6,33.0,26.2,18.6.HRMS(ESI,positive)m / z calcd forC 30 H 34 N4O7S2[M+H] + :627.1942,found:627.1939.

[0133] Example 23 Preparation of (6R,7R)-3-(((3-methoxy-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5f).

[0134] 23.1 Preparation of intermediate 5i-4. Intermediate 5i-4 was synthesized according to the method in step 2.1 of Example 2. 1H NMR (600MHz, DMSO-d6) δ8.43(d,J=8.4Hz,1H),7.36(d,J=8.6Hz,2H),7.34–7.28(m,4H),7.26–7.20(m,1H ),6.94(d,J=8.6Hz,2H),5.79(dd,J=8.3,4.7Hz,1H),5.21(dd,J=41.3,12.0Hz,2H),4.92(d,J=3.6Hz,1H ),3.98(d,J=18.2Hz,1H),3.75(s,3H),3.68(d,J=5.3Hz,1H),3.67–3.62(m,2H),3.61–3.58(m,4H),3.54 (d,J=14.0Hz,1H),2.61(qd,J=8.4,5.6Hz,1H),2.54(dd,J=7.4,5.8Hz,1H),2.50(dd,J=5.7,4.0Hz,2H). 13 C NMR (151MHz, DMSO-d6) δ171.9,171.0,164.0,162.5,135.8,129.5,129.1,128.3,126.5 113.8,66.5,58.0,51.4,46.1,41.5,33.9,32.4,25.0.HRMS(ESI,positive)m / z calcd forC 28 H 30 N₂O₈S₂[M+Na] + :609.1336,found:609.1331.

[0135] 23.2 Preparation of final product 5i. Final product 5i was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.79(s,1H),8.46(d,J=8.1Hz,1H),7.30(s,4H),7.24(d,J=3.4Hz,1H),5.78–5.70(m,1H),4.90( d,J=3.9Hz,1H),3.94(d,J=18.1Hz,1H),3.76–3.66(m,3H),3.65–3.59(m,4H),3.53(d,J=13.9Hz,1H),2.71–2.55(m,4H). 13C NMR(151MHz,DMSO-d6)δ171.8,171.0,164.2,161.0,159.4,135.8,130.4,129.1,128.3,126 .9,126.5,123.4,122.2,113.8,67.3,66.6,58.1,55.2,51.5,46.2,41.5,33.9,32.6,25.1.

[0136] Example 24 Preparation of (6R,7R)-3-(((2-carboxyethyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (5 g).

[0137] 24.1 Preparation of final product 5j. 5g of final product was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.3Hz,1H),7.34–7.27(m,4H),7.27–7.20(m,1H),5.75(dd,J=8.2,4.7Hz,1H),4.90(d ,J=3.7Hz,1H),3.94(d,J=18.0Hz,1H),3.75-3.63(m,4H),3.53(d,J=14.0Hz,1H),2.69-2.53(m,2H),2.49-2.43(m,2H). 13 C NMR (151MHz, DMSO-d6) δ172.9,171.0,164.0,162.4,135.8,129.1,128.3,126.6,124.5,121.2,66.5,58.0,46.1,41.5,34.2,32.5,25.1.

[0138] Example 25 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)sulfinyl)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (6a).

[0139] 25.1 Preparation of intermediate 6a-5. (6R,7R)4-methoxybenzyl-8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)thio)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 5-oxide. At 0 °C, m-CPBA (70% content, 83 mg, 338 μmol) was added to 19 g (250 mg, 338 μmol) of dry DCM (10 mL). The reaction mixture was stirred at 0 °C for 30 min, then at room temperature for another 5 h. After removing the solvent by evaporation, the residue was treated with diethyl ether. The precipitate was collected by filtration to give 6a-5. 1 H NMR (400MHz, DMSO-d6) δ8.51–8.40(m,1H),7.36(d,J=8.5Hz,2H),7.30(s,4H),7.24(d,J=3.7Hz,1H),6.9 2(d,J=8.2Hz,2H),5.85(dd,J=8.2,4.5Hz,1H),5.20(dd,J=27.7,12.0Hz,2H),4.94(d,J=3.5Hz,1H),4.35 (t,J=16.6Hz,0.3H),4.15(t,J=14.0Hz,1H),3.93(t,J=16.8Hz,1H),3.83(dd,J=13.6,5.6Hz,0.7H),3.7 2(dd,J=21.2,10.3Hz,5H),3.55(d,J=13.9Hz,1H),3.20(d,J=10.3Hz,3H),3.00(dt,J=13.6,12.3Hz,1H).

[0140] 25.2 Preparation of final product 6a. Final product 6a was synthesized according to the synthesis method in step 1.3 of Example 1. Yield: 25 g (34.8%). 1 H NMR (400MHz, DMSO-d6) δ8.48–8.40(m,1H),7.30(s,4H),7.24(s,1H),5.81(s,1H),4.92(s,1H),4.39(t,J=15.6Hz,0.3H),4.27–4.07(m ,1H),3.96–3.87(m,1H),3.78(s,0.7H),3.70(d,J=13.9Hz,2H),3.54(d,J=14.0Hz,1H),3.18(d,J=65.9Hz,4H).MS(ESI,positive):m / z 844.0[M+NH4] + .

[0141] Example 26 Preparation of (6R,7R)-3-(((3-((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)sulfinyl)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxide (6b).

[0142] 26.1 Preparation of intermediate 6b-4. Intermediate 6b-4 was synthesized according to the method in step 1.1 of Example 1. 1 H NMR(400MHz, DMSO-d6)δ8.44(d,J=8.4Hz,1H),7.80(dd,J=8.0,2.9Hz,1H),7.40–7.34(m,2H),7.2 9(t,J=4.4Hz,4H),7.22(dt,J=9.0,5.6Hz,2H),6.93(t,J=9.2Hz,2H),5.79(dd,J=8.3,4.7Hz,1H) ,5.29–5.15(m,2H),4.94(d,J=3.7Hz,1H),4.09–3.99(m,1H),3.77–3.62(m,7H),3.54(d,J=14.1H z,1H),3.13–2.92(m,1H),2.86(d,J=6.5Hz,1H),2.84–2.77(m,1H),2.71(dt,J=13.4,8.0Hz,1H).

[0143] 26.2 Preparation of intermediate 6b-5. Intermediate 6b-5 was synthesized according to the method in step 25.2 of Example 25. 1 HNMR(400MHz, DMSO-d6)δ8.48–8.35(m,2H),7.36(dd,J=8.7,2.3Hz,4H),7.32–7.28(m,7H),7.24(dd, J=9.8,4.2Hz,2H),6.92(dd,J=8.8,7.3Hz,4H),5.89–5.77(m,2H),5.21(dt,J=25.2,7.7Hz,4H),4.93 (s,2H),4.33(t,J=13.3Hz,1H),4.17–4.06(m,2H),3.97–3.80(m,3H),3.75(s,6H),3.65(t,J=7.1Hz, 3H),3.55(d,J=14.1Hz,2H),3.18–3.07(m,2H),3.05–2.93(m,2H),2.83(d,J=4.2Hz,4H),2.59(s,7H).

[0144] 25.3 Preparation of final product 6b. Final product 6b was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (600MHz, DMSO-d6) δ8.46–8.36(m,1H),7.33–7.29(m,4H),7.25–7.22(m,1H),5.80(dd,J= 7.6, 4.6Hz, 1H), 4.91 (d, J = 3.0Hz, 1H), 4.37 (dd, J = 12.8, 3.7Hz, 0.5H), 4.18 (d, J = 12.8Hz, 0. 5H),4.11–4.08(m,0.5H),3.92-3.84(m,1.5H),3.75–3.67(m,2H),3.56-3.52(m,1H),3.17–3 .02(m,2H),2.96–2.85(m,1H),2.67–2.63(m,1H),2.63–2.52(m,4H).HRMS(ESI,positive)m / z calcd for C 23 H 23 N3O 10 S2[M+Na] + :588.0717,found:588.0679.

[0145] Example 26 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-((2-oxo-2H-chromene-7-yl)oxy)propyl)sulfinyl)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxide (6c).

[0146] 26.1 Preparation of intermediate 6c-4. EDCI (0.17 g, 0.91 mmol) and 4-dimethylaminopyridine (DMAP, 9 mg, 0.07 mmol) were added to a solution of 17 (0.4 g, 0.70 mmol) and 7-hydroxycoumarin (0.11 g, 0.70 mmol) in DCM (10 mL) under stirring. The resulting mixture was stirred at room temperature for 12 hours, then evaporated under reduced pressure. The crude residue was treated with anhydrous ethanol, the precipitate was collected by filtration and dried for the next step. 1H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.3Hz,1H),8.08(t,J=7.5Hz,1H),7.78(d,J=8.5Hz,1H),7.34(d,J=8.7Hz,2 H),7.29(d,J=4.3Hz,4H),7.23(td,J=4.7,2.4Hz,2H),7.17–7.09(m,1H),6.90(d,J=8.7Hz,2H),6.48(d,J=9.6 Hz,1H),5.79(dd,J=8.3,4.7Hz,1H),5.21(dd,J=28.0,12.0Hz,2H),4.93(d,J=3.5Hz,1H),4.02(d,J=18.2Hz,1 H),3.73(d,J=5.7Hz,4H),3.70–3.63(m,3H),3.53(d,J=13.9Hz,1H),2.84(t,J=6.4Hz,2H),2.81–2.63(m,2H). 13 C NMR (151MHz, DMSO-d6) δ171.0,169.9,164.3,161.1,159.7,159.4,154.1,152.7,143.8,135.8,130.4,129.4,129.1,128.3,126.9,12 6.5,123.5,122.2,118.5,116.8,115.6,113.8,110.0,67.30,66.7,58.1,55.1,46.2,41.5,34.2,32.7,25.0.HRMS(ESI,positive)m / z calcd for C 36 H 32 N2O 10 S2[M+H] + :717.1571,found:717.1561.

[0147] 26.2 Preparation of intermediate 6c-5. Intermediate 6c-5 was synthesized according to step 20.2 of Example 20. White solid. Yield: 159 mg (62.1%). 1H NMR (400MHz, DMSO-d6) δ8.47(d,J=8.0Hz,1H),8.09(d,J=9.5Hz,1H),7.79(d,J=8.6Hz,1H),7.34(d,J=7.5Hz,2 H),7.30(s,4H),7.24(s,1H),7.19(d,J=8.5Hz,1H),6.91(d,J=7.3Hz,2H),6.50(d,J=9.1Hz,1H),5.85(s,1H),5 .26–5.09(m,2H),4.94(s,1H),4.38(s,1H),4.20(d,J=12.5Hz,1H),3.97(d,J=18.5Hz,1H),3.89(d,J=12.4Hz, 1H), 3.79 (d, J = 18.4Hz, 1H), 3.70 (d, J = 20.2Hz, 4H), 3.54 (d, J = 13.3Hz, 1H), 3.18 (d, J = 7.7Hz, 1H), 3.03 (s, 2H). 3 C NMR(151MHz,DMSO-d6)δ171.1,169.8,169.7,164.3,164.2,160.9,160.9,159.7,159.4,159.3, 154.1,152.8,152.7,143.8,135.8,130.4,130.3,129.4,129.1,128.3,126.8,126.8,126.6,125 .9,125.5,118.5,117.1,117.0,116.8,115.7,113.8,113.,110.0,67.3,67.2,66.6,66.5,58.4, 58.3,55.3,55.1,55.1,53.9,48.6,47.9,45.8,45.0,41.5,27.1,26.7.HRMS(ESI,positive)m / z calcdfor C 36 H 32 N2O 11 S2[M+H] + :733.1520,found:733.1507.

[0148] 26.3 Preparation of final product 6c. Final product 6c was synthesized according to the synthetic method in step 1.3 of Example 1. It was a white solid. Yield: 62 mg (49.3%). 1H NMR (600MHz, DMSO-d6) δ13.85(s,1H),8.40(d,J=8.2Hz,1H),8.08(d,J=9.5Hz,1H),7.79(d ,J=8.3Hz,1H),7.30(s,4H),7.24(s,1H),7.19(d,J=7.9Hz,1H),6.49(d,J=9.5Hz,1H),5.81 (s,1H),4.92(s,1H),4.25(d,J=12.7Hz,1H),3.97–3.87(m,2H),3.77(d,J=18.2Hz,1H),3.7 0(d,J=13.9Hz,1H),3.55(d,J=14.0Hz,1H),3.27(d,J=11.4Hz,1H),3.08(d,J=10.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ171.0,169.8,163.9,162.4,159.7,154.1,152.8,143.8,135.8,129.4,129.1,128 .3,126.6,118.60,116.8,115.7,110.0,66.4,58.2,55.5,47.7,45.7,41.5,26.6.MS(ESI,positive):m / z 613.0[M+H]+,635.0[M+Na] + 651.0 [M+K] + .HRMS(ESI,positive)m / z calcd forC 28 H 24 N2O 10 S2[M+Na] + :635.0765,found:635.0749.

[0149] Example 27 Preparation of (6R,7R)-3-(((3-(2-fluoro-4-nitrophenoxy)-3-oxopropyl)sulfinyl)methyl)-8-oxo-7-(2-phenylacetamyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (6d).

[0150] 27.2 Preparation of intermediate 6d-4. 1H NMR(600MHz, DMSO-d6)δ8.46–8.37(m,2H),8.02(dd,J=9.2,6.2Hz,1H),7.59–7.50(m,1H),7.48–7.41(m,1H),7.39–7.3 4(m,2H),7.33–7.27(m,4H),7.24(d,J=6.2Hz,1H),6.93(dd,J=13.6,4.5Hz,2H),5.94–5.81(m,1H),5.28–5.14(m,2H), 4.97–4.89(m,1H),4.32(d,J=13.4Hz,0.3H),4.10(dd,J=26.6,15.6Hz,1H),4.01–3.90(m,1H),3.85(dd,J=12.2,5.6Hz ,0.7H),3.77–3.66(m,5H),3.55(d,J=14.1Hz,1H),3.13–2.92(m,2H),2.88–2.77(m,1H),2.60(dd,J=12.3,7.1Hz,1H).

[0151] 27.3 Preparation of intermediate 6d-4. 1 H NMR (400MHz, DMSO-d6) δ13.82(s,1H),8.44(t,J=8.5Hz,1H),8.38–8.26(m,1H),7.53(d,J=8.3Hz, 1H),7.47(t,J=8.0Hz,1H),7.29(s,4H),7.23(s,1H),5.80(d,J=3.7Hz,1H),4.91(s,1H),4.42(d, J=13.3Hz,0.3H),4.26(d,J=12.6Hz,0.4H),4.10(d,J=18.9Hz,0.4H),3.90(dd,J=24.5,11.7Hz,1 .5H),3.72(dd,J=28.8,17.0Hz,2H),3.53(d,J=14.0Hz,1H),3.25(s,1H),3.08(d,J=10.2Hz,3H). 19 F NMR(376MHz,DMSO-d6)δ-100.6.

[0152] Example 28 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-((3-oxo-3-(perfluorophenoxy)propyl)thio)propyl)thio)methyl)-7-(2-phenylacetamyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (7a).

[0153] 28.1 Preparation of intermediate 7a-4. Intermediate 7a-4 was synthesized according to the method in step 1.1 of Example 1. 1 H NMR(400MHz, DMSO-d6)δ8.46(d,J=8.3Hz,1H),7.42–7.34(m,2H),7.34–7.18(m,5H),6.98–6.90(m ,2H),5.80(m,1H),5.30–5.14(m,2H),4.93(m,1H),3.99(d,J=18.2Hz,1H),3.77(s,3H),3.71(d,J =3.3Hz,1H),3.67(s,1H),3.66–3.62(m,1H),3.61–3.52(m,2H),3.05(m,2H),2.88(dd,J=8.4,6.4 Hz,1H),2.80–2.74(m,1H),2.72–2.53(m,2H),2.43(dd,J=7.3,5.9Hz,1H).MS(ESI,positive):m / z 844.0[M+NH4] +

[0154] 28.2 Preparation of final product 7a. Final product 7a was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.81(s,1H),8.43(dd,J=8.1,5.7Hz,1H),7.35–7.27(m,4H),7.27–7.20(m,1H),5.79–5.72(m,1H),4.91(t,J=4.9H z,1H),3.97(t,J=18.1Hz,1H),3.79(d,J=13.7Hz,1H),3.66(t,J=14.7Hz,3H),3.53(d,J=14.0Hz,1H),3.26–2.93(m,4H),2.92–2.63(m,4H). 13 C NMR(151MHz,DMSO-d6)δ173.1,172.8,171.2,164.1,162.5,135.9,129.2,128.4,12 6.7,124.6,121.3,66.6,58.1,46.2,41.6,41.6,40.1,34.3,33.9,32.6,25.2,23.9. 19F NMR(376MHz, DMSO-d6)δ-153.0(d,J=20.3Hz),-157.9(t,J=23.3Hz),-162.6(t,J=22.5Hz).MS(ESI,positive):m / z 707.0[M+H] + 729.0 [M+Na] + 745.0 [M+K] + .HRMS(m / z):[C 28 H 23 F5N2O8S3+Na] + calcd:729.0429; found:729.0425

[0155] Example 29 Preparation of (6R,7R)-3-(((3-((3-(4-nitrophenoxy)-3-oxopropyl)thio)-3-oxopropyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (7b).

[0156] 29.1 Preparation of intermediate 7b-4. Intermediate 7b-4 was synthesized according to the method in step 1.1 of Example 1. 1 H NMR(400MHz, CDCl3)δ8.29(d,J=9.0Hz,2H),7.40-7.28(m,9H),6.91(dd,J=8.2,3.3Hz,2H),6.85 –6.75(m,1H),6.04(d,J=7.6Hz,1H),5.24(q,J=11.8Hz,2H),4.51(dd,J=9.3,4.5Hz,1H),4.23–4 .06(m,1H),4.06–3.94(m,1H),3.86–3.68(m,4H),3.63(d,J=2.6Hz,2H),3.55-3.43(m,1H),3.27 -3.24(m,1H),3.23-3.16(m,1H),2.98–2.85(m,4H),2.83-2.69(m,2H).HRMS(ESI,positive)m / z calcd for C 36 H 35 N3O 11 S3[M+Na] + :804.1326,found:804.1330.

[0157] 29.2 Preparation of final product 7b. Final product 7b was synthesized according to the synthesis method in step 1.3 of Example 1. 1H NMR (400MHz, DMSO-d6) δ8.48(s,1H),8.33(s,2H),7.44(s,2H),7.27(d,J=25.1Hz,5H),5.77(s,1H),4.91(s,1H),3.96(d,J=13.6Hz,1H),3.7 0(d,J=20.8Hz,4H),3.53(d,J=14.2Hz,1H),3.14(d,J=37.6Hz,2H),2.92(d,J=26.9Hz,4H),2.74(s,2H).MS(ESI,positive):m / z662.10[M+H] + .

[0158] Example 30 Preparation of (6R,7R)-8-oxo-3-(((2-oxo-2-(perfluorophenyl)ethyl)thio)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (8a).

[0159] 30.1 Preparation of intermediate 8a-4. Intermediate 8a-4 was synthesized according to the method in step 1.1 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.51(s,1H),7.38–7.22(m,7H),6.90(d,J=7.1Hz,2H),5.82(d,J=19.9Hz,1H),5.15(dt,J=22.3,11. 7Hz, 2H), 4.93 (s, 1H), 4.00 (d, J = 17.7Hz, 1H), 3.87 (d, J = 29.7Hz, 1H), 3.73 (dd, J = 33.0, 18.0Hz, 8H), 3.54 (d, J = 14.0Hz, 1H).

[0160] 30.2 Preparation of final product 8a. Final product 8a was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR(400MHz,DMSO-d6)δ8.63(s,1H),7.47–7.31(m,5H),5.94-5.90(m,1H),5.05(s,1H),4.10(d ,J=17.7Hz,1H),3.97(d,J=29.7Hz,1H),3.82(dd,J=33.0,18.0Hz,5H),3.64(d,J=14.0Hz,1H). 13C NMR(151MHz,DMSO-d6)δ171.0,171.0,164.00,162.3,137.9,135.8,129.1,128.3,12 6.6,124.9,120.2,66.5,58.1,46.3,41.5,33.2,32.6.HRMS(ESI,positive)m / zcalcd for C 24 H 17 F5N2O7S2[M+Na] + :627.0290,found:627.0294.

[0161] Example 31 Preparation of (6R,7R)-3-(((2-(4-nitrophenoxy)-2-oxoethyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (8b).

[0162] 31.1 Preparation of intermediate 8b-4. Intermediate 8b-4 was synthesized according to the method in step 1.2 of Example 1. 1 H NMR (400MHz, CDCl3) δ8.24(d,J=8.8Hz,2H),7.36(t,J=7.0Hz,2H),7.29(dd,J=14.2,6 .6Hz,7H),6.87(d,J=8.2Hz,2H),6.70(d,J=10.0Hz,1H),6.02(dd,J=9.7,4.4Hz,1H),5 .19(q,J=11.7Hz,2H),4.43(d,J=4.2Hz,1H),4.10(d,J=18.1Hz,1H),3.99(d,J=14.2H z,1H),3.79(s,3H),3.68–3.55(m,3H),3.47(t,J=16.0Hz,2H),3.26(d,J=18.3Hz,1H).

[0163] 32.2 Preparation of final product 8b. Final product 8b was synthesized according to the synthesis method in step 1.3 of Example 1. 1H NMR (400MHz, DMSO-d6) δ13.79(s,2H),8.46(d,J=8.0Hz,3H),8.28(d,J=8.8Hz,6H),7.43(d,J=9.0Hz,6H),7.24(t,J=12.4Hz,17H),5.72( s,3H),4.85(s,3H),3.93(d,J=18.2Hz,3H),3.72(s,6H),3.70–3.61(m,9H),3.59(s,4H),3.49(d,J=13.8Hz,4H).HRMS(ESI,positive)m / z calcd for C 24 H 21 N3O9S2[M+Na] + :582.0611,found:582.0616.

[0164] Example 33 Preparation of (6R,7R)-3-(((2-((4-fluorophenyl)amino)-2-oxoethyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (8c).

[0165] 33.1 Preparation of final product 8c. Intermediate 8c-4 was synthesized according to step 14.1 of Example 14. It was proceeded directly to the next step without purification. Final product 8c was synthesized according to step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.69(s,1H),8.51(s,1H),8.41(d,J=8.2Hz,1H),7.28( t,J=6.4Hz,6H),7.23(d,J=3.6Hz,1H),7.14(t,J=8.7Hz,2H),5.74(dd,J=7.9,4 .7Hz,1H),4.84(d,J=4.1Hz,1H),4.23(d,J=5.7Hz,2H),3.89(dd,J=15.8,8.6Hz ,2H),3.73–3.61(m,2H),3.54(t,J=14.2Hz,2H),3.14(dd,J=34.1,14.2Hz,2H). 13C NMR (151MHz, DMSO-d6) δ171.0,168.6,163.9,162.3,162.0,160.4,135.8,135.48(d,J=3.0Hz ),129.2,129.2,129.1,128.3,126.5,115.1,114.9,66.4,58.0,46.5,41.6,41.5,34.0,33.8.

[0166] Example 34 Preparation of (6R,7R)-3-(((3-((4-morpholinophenyl)amino)-2-oxoethyl)thio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (8d).

[0167] 34.1 Preparation of intermediate 8d-4. Intermediate 8d-4 was synthesized according to the method in step 14.1 of Example 14. 1 HNMR(400MHz,DMSO-d6)δ9.84(s,1H),8.43(d,J=8.0Hz,1H),7.45(d,J=8.7Hz,2H),7.3 5–7.29(m,6H),7.24(s,1H),6.95–6.84(m,4H),5.74(dd,J=7.9,4.9Hz,1H),5.24–5.08 (m,2H),4.92–4.75(m,1H),4.07(d,J=13.6Hz,1H),3.95(d,J=18.3Hz,1H),3.78–3.67( m,9H),3.59–3.48(m,2H),3.26(d,J=14.2Hz,1H),3.16(d,J=14.4Hz,1H),3.04(s,4H).

[0168] 34.2 Preparation of final product 8d. Final product 8d was synthesized according to the synthesis method in step 1.3 of Example 1. 1H NMR (600MHz, DMSO-d6) δ13.69(s,1H),9.83(s,1H),8.37(d,J=8.3Hz,1H),7.44(d,J=8.9Hz,2H),7.3 1(dd,J=7.9,5.5Hz,4H),7.27–7.19(m,1H),6.91(d,J=8.9Hz,2H),5.77–5.66(m,1H),4.82(d,J=3.7H z,1H),3.97(d,J=13.6Hz,1H),3.93(d,J=18.2Hz,1H),3.76–3.71(m,5H),3.71–3.66(m,2H),3.60(d, J=13.6Hz,1H),3.56–3.51(m,1H),3.29(d,J=14.1Hz,1H),3.22(d,J=14.1Hz,1H),3.09–3.01(m,4H). 13 C NMR (151MHz, DMSO-d6) δ171.0,166.8,163.9,162.3,147.1,135.8,131.4 129.1,128.3 126.6,124.9,120.7,120.4,115.6,66.4,66.0,58.0,49.1,46.6,41.5,35.1,33.8.HRMS(ESI,positive)m / z calcd for C 28 H 30 N4O7S2[M+H] + :599.1629,found:599.1625.

[0169] Example 35 Preparation of (6R,7R)-7-acetamido-8-oxo-3-(((3-oxo-3-(4-(trifluoromethyl)phenoxy)propyl)thio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (9a).

[0170] 35.1 Preparation of Intermediate 9a-1. Acetyl chloride (0.51 mL, 7.14 mmol) was added dropwise to an anhydrous acetonitrile (50 mL) solution of ACTH·HCl (2 g, 4.76 mmol), triethylamine (0.68 mL, 4.76 mmol), and 2,6-lutidine (1.11 mL g, 9.52 mmol) at 0 °C. After the addition was complete, the mixture was allowed to rise to room temperature for 4 h, and the reaction was monitored by TLC. After the reaction was complete, 50 mL of ethyl acetate was added for dilution, followed by washing three times with saturated NaHCO3 aqueous solution and saturated brine, respectively. The solution was dried over anhydrous sodium sulfate, and column chromatography was performed using PE:EA = 5:1 as the eluent to obtain a white solid.1 H NMR(400MHz, DMSO-d6)δ8.90(s,1H),7.36(d,J=3.2Hz,2H),6.94(d,J=3.3Hz,2H),5.73(d,J=4.3Hz,1H),5.24(d,J=12.1Hz,1H),5 .16(d,J=9.4Hz,2H),4.52(d,J=11.9Hz,1H),4.43(d,J=11.5Hz,1H),3.81–3.64(m,4H),3.54(d,J=18.0Hz,1H),2.00–1.81(m,3H).

[0171] Intermediate 9a-2 was synthesized according to the method for synthesizing 1a-2 in step 1.1 of Example 1. Intermediate 9a-3 was synthesized directly according to the method for synthesizing 1a-3 in Example 1 without purification. 1 H NMR (400MHz, DMSO-d6) δ12.29(s,1H),8.24(d,J=8.3Hz,1H),7.36(d,J=8.2Hz,2H),6. 94(d,J=8.2Hz,2H),5.78(dd,J=7.7,4.9Hz,1H),5.21(dd,J=29.0,11.9Hz,2H),4.94(d ,J=4.0Hz,1H),3.98(d,J=18.0Hz,1H),3.75(s,3H),3.72(d,J=10.8Hz,1H),3.68(d,J= 5.9Hz,1H),3.56(d,J=13.8Hz,1H),2.65–2.51(m,2H),2.48–2.37(m,2H),1.94(s,3H).

[0172] Preparation of intermediate 9a-4. Intermediate 9a-4 was synthesized according to the method in step 1.2 of Example 1. 1 H NMR (600MHz, DMSO-d6) δ8.23(d,J=8.3Hz,1H),7.82(d,J=8.6Hz,2H),7.36(dd,J=18. 1,10.4Hz,4H),6.92(t,J=5.8Hz,2H),5.80(dd,J=8.3,4.8Hz,1H),5.22(dd,J=43.2, 12.0Hz,2H),4.95(s,1H),4.03(d,J=18.1Hz,1H),3.76–3.67(m,6H),2.85(td,J=6.8 ,2.2Hz,2H),2.77(dt,J=13.9,6.9Hz,1H),2.68(dt,J=13.7,6.9Hz,1H),1.95(s,3H). 13CNMR(151MHz,DMSO-d6)δ170.0,164.5,161.1,159.4,153.3,130.4,128.56–126.40(m),124.9, 123.5,123.1,122.8,122.0,115.7,113.8,67.3,66.6,58.0,55.1,46.3,34.2,32.6,25.0,22.1.

[0173] 35.2 Preparation of final product 9a. Final product 9a was synthesized according to the synthesis method in step 1.3 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ13.84(s,1H),8.25(d,J=8.2Hz,1H),7.83(d,J=8.1Hz,2H),7.37(d,J=8.1Hz,2H),5.86–5.68(m,1H),4.9 4(d,J=4.0Hz,1H),4.00(d,J=18.1Hz,1H),3.71(d,J=20.4Hz,3H),2.92(s,2H),2.77(ddt,J=34.5,13.6,6.9Hz,2H),1.95(s,3H). 13 C NMR(151MHz,DMSO-d6)δ170.0,164.2,162.5,153.3,127.95–125.94(m),12 4.9,124.9,123.1,122.9,120.8,66.5,58.0,46.2,34.2,32.4,24.8,22.1. 19 F NMR (376MHz, DMSO-d6) δ-60.6.

[0174] Example 36 Preparation of (6R,7R)-8-oxo-3-((((3-oxo-3-(4-(trifluoromethyl)phenoxy)propyl)thio)methyl)-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (10a).

[0175] 36.1 Preparation of intermediate 10a-1. 10a-1 was prepared according to the synthesis method of 9a-1 in Example 35. 1H NMR (400MHz, DMSO-d6) δ9.09(d,J=8.3Hz,1H),7.25(d,J=8.3Hz,3H),6.82(d,J=8.6Hz,4H),5.65(d,J=5.0Hz,1H),5.12– 5.00(m,2H),4.43(d,J=11.1Hz,1H),4.34(d,J=11.4Hz,1H),3.65(d,J=9.9Hz,6H),3.45(d,J=18.0Hz,1H),3.28(s,2H).

[0176] Intermediate 10a-2 was prepared according to the method described in Example 1a-2. 1 H NMR (400MHz, CDCl3) δ7.35(d,J=8.3Hz,9H),7.27(s,4H),7.06–6.96(m,8H),6.88(dd,J=19.1,9.5Hz,9H),6.08(dd,J=9.9,4.5Hz,3H),5.25(s,7 H),4.98(d,J=12.4Hz,3H),4.47(d,J=4.5Hz,4H),4.22(d,J=12.2Hz,4H ),3.86(s,8H),3.82(s,12H),3.56–3.41(m,2H),3.36(d,J=18.7Hz,3H).

[0177] Intermediate 10a-4 was synthesized according to steps 1a-4 in Example 1. 1 H NMR(400MHz, DMSO-d6)δ8.50(d,J=8.0Hz,1H),7.83(d,J=8.3Hz,2H),7.41–7.32( m,5H),6.98–6.88(m,4H),5.82(dd,J=7.9,4.9Hz,1H),5.21(dd,J=27.7,11.9Hz,2 H),4.96(d,J=3.9Hz,1H),4.03(d,J=18.3Hz,1H),3.90(d,J=15.3Hz,1H),3.80(d ,J=15.3Hz,1H),3.71(d,J=15.4Hz,6H),2.84(d,J=6.5Hz,2H),2.81–2.60(m,2H).

[0178] 34.2 Preparation of final product 10a. Final product 10a was synthesized according to the synthesis method in step 1.3 of Example 1. 1HNMR(600MHz,DMSO-d6)δ13.81(s,1H),8.43(t,J=10.6Hz,1H),7.82(d,J=8.5Hz,2H),7.3 7(dd,J=5.0,3.1Hz,3H),7.00–6.93(m,2H),5.85–5.70(m,1H),4.99–4.88(m,1H),4.01–3 .98(m,1H),3.90(d,J=15.4Hz,1H),3.82–3.79(m,1H),3.73(d,J=6.5Hz,2H),3.70(t,J=5 .2Hz,1H),2.95–2.89(m,2H),2.82(dt,J=13.9,7.0Hz,1H),2.74(dt,J=13.8,7.0Hz,1H). 13 C NMR (151MHz, DMSO-d6) δ170.0,164.0,162.5,157.8,153.3,136.8,129.5,127. 0,126.7,126.5,125.1,122.9,113.8,66.5,58.0,46.1,35.7,34.2,32.4,24.8. 19 F NMR(376MHz,DMSO-d6)δ-60.6.HRMS(ESI,positive)m / z calcd for C 24 H 21 F3N2O7S3[M+Na] + :625.0355,found:625.0353.

[0179] Example 37 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)thio)methyl)-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (10b).

[0180] 37.1 Preparation of intermediate 10b-4. Intermediate 10b-4 was synthesized according to the method in step 36.1 of Example 36. 1HNMR (400MHz, CDCl3) δ7.34(d,J=6.5Hz,2H),6.98(s,2H),6.92–6.82(m,3H),6.04(s,1H),5.28–5.16(m,2H),4.49(s,1H),4.12(d,J=1 7.9Hz,1H),3.90(d,J=14.7Hz,1H),3.85(s,2H),3.81(s,3H),3.51(d,J=13.5Hz,1H),3.23(d,J=18.4Hz,1H),2.94(s,2H),2.78(s,2H).

[0181] 35.2 Preparation of final product 10b. Final product 10b was synthesized according to the synthesis method in step 1.3 of Example 1. 1 HNMR(400MHz,DMSO-d6)δ8.48(d,J=8.3Hz,1H),7.43–7.34(m,1H),6.95(s,2H),5.79(dd,J=7.9,4.8Hz,1H),4.94(d,J=4.1Hz,1 H),4.00(d,J=18.3Hz,1H),3.90(d,J=15.4Hz,1H),3.83–3.75(m,2H),3.70–3.62(m,2H),3.17–3.04(m,2H),2.86–2.71(m,2H).

[0182] Example 38 Preparation of (6R,7R)-7-(hept-6-amino)-8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)sulfonyl)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5,5-dioxide (11a).

[0183] 38.1 Preparation of intermediate 11a-1

[0184]

[0185] At 0 °C, a dichloromethane solution (40 mL) of 6-heptyneic acid (0.6 g, 4.76 mmol) and ACTH·HCl (2 g, 4.76 mmol) was added to a round-bottom flask. EDCI (1.19 g, 6.19 mmol) and DMAP (0.06 g, 0.48 mmol) were then added. After reacting for 6 h, the mixture was washed three times successively with saturated sodium bicarbonate solution and saturated brine. The resulting organic phase was dried over anhydrous magnesium sulfate and precipitated by DCM:CH3OH = 99:1 column chromatography. After concentrating the organic phase, diethyl ether was added to replace the solvent. After a large amount of solid precipitated, the mixture was filtered and dried under vacuum to obtain a white powdery solid intermediate 11a-1, 1.97 g, with a yield of 87.1%. 1 H NMR (400MHz, CDCl3) δ7.35(d,J=8.4Hz,5H),6.91(d,J=8.3Hz,4H),5.86(dd,J=8.9,4.8Hz,2H ),5.23(s,4H),4.97(d,J=4.9Hz,2H),4.54(dd,J=11.8,2.3Hz,2H),4.45(d,J=11.8Hz,2H),3 .82(s,7H),3.66(d,J=18.3Hz,2H),3.49(d,J=18.3Hz,2H),2.31(t,J=7.5Hz,4H),2.23(td,J =6.9,2.3Hz,4H),1.97(s,2H),1.83–1.74(m,4H),1.63–1.53(m,4H).HRMS(ESI,positive)m / z calcd for C 23 H 25 ClN2O5S[M+Na] + :499.1065,found:499.1045.

[0186] 38.2 Preparation of intermediate 11a-2. Intermediate 11a-2 was synthesized according to the method for synthesizing 1a-2 in step 1.1 of Example 1. 1H NMR(400MHz, DMSO-d6)δ8.24(d,J=8.1Hz,1H),7.39(t,J=5.7Hz,2H),7.03–6.89(m,2H),5.85(dd,J=8 .0,4.8Hz,1H),5.27(dd,J=31.3,12.0Hz,2H),4.96(d,J=3.6Hz,1H),4.69(d,J=11.6Hz,1H),4.52(d,J =11.5Hz,1H),4.04–3.89(m,1H),3.80–3.69(m,4H),2.76(t,J=2.5Hz,1H),2.29(ddd,J=14.2,13.4,7 .2Hz,2H),2.18(td,J=7.0,2.6Hz,2H),1.68–1.55(m,2H),1.55–1.40(m,2H).HRMS(ESI,positive)m / z calcd forC 23 H 25 ClN2O6S[M+Na] + :515.1014,found:515.1003.

[0187] 38.3 Preparation of intermediate 11a-3. Intermediate 11a-3 was synthesized according to the method for synthesizing 1a-3 in step 1.1 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ12.32(s,1H),8.29–8.12(m,1H),7.38(d,J=8.5Hz,2H),6.95(d,J=8.5Hz,2H),5.88–5.72(m, 1H),5.32–5.16(m,2H),4.94(dd,J=13.7,3.8Hz,1H),4.00(d,J=18.3Hz,1H),3.75(d,J=11.1Hz,4H),3.68(d,J=13.3 Hz,1H),3.63–3.55(m,1H),2.96–2.90(m,1H),2.77(d,J=21.3Hz,2H),2.67–2.53(m,2H),2.43(t,J=6.8Hz,2H),2.33 –2.21(m,2H),2.20–2.14(m,2H),1.61(dt,J=14.5,7.2Hz,2H),1.46(dt,J=14.1,6.9Hz,2H).HRMS(ESI,positive)m / z calcd for C 26 H 30 N₂O₈S₂[M+Na] +:563.1516,found:563.1505.

[0188] 38.4 Preparation of intermediate 11a-4. Intermediate 11a-4 was synthesized according to the method in step 1.2 of Example 1. 1 HNMR (400MHz, DMSO-d6) δ8.26–8.17(m,1H),7.36(dd,J=8.6,3.5Hz,2H),6.93(dd,J=8.6,6.8Hz,2H),5.78(dt,J=8.3,4.2Hz, 1H),5.22(dt,J=28.9,7.4Hz,2H),4.95(t,J=5.1Hz,1H),4.01(t,J=18.7Hz,1H),3.77–3.71(m,4H),3.70–3.66(m,1H),3.65–3 .54(m,1H),3.02(t,J=7.0Hz,1H),2.84–2.65(m,2H),2.60(ddd,J=21.0,14.2,7.0Hz,1H),2.41(t,J=6.7Hz,1H),2.27(ddd,J= 14.1,13.2,7.3Hz,2H),2.16(td,J=6.9,2.4Hz,2H),1.65–1.54(m,2H),1.44(dt,J=14.3,7.0Hz,2H).HRMS(ESI,positive)m / z calcd for C 32 H 29 F5N2O8S2[M+H] + :729.1358,found:729.1347.

[0189] Preparation of intermediate 11a-5 (38.5). Intermediate 11a-5 was synthesized according to step 25.1 of Example 25. 1HNMR (400MHz, DMSO-d6) δ8.20(t,J=8.4Hz,2H),7.38(dd,J=8.7,2.6Hz,4H),6.95(dd,J=8.7,2.1Hz,4H),5.84(dt,J=8.4,4.3Hz,2H),5 .22(dt,J=28.1,7.4Hz,4H),4.97(d,J=3.0Hz,2H),4.33(d,J=12.5Hz,1H),4.12(t,J=16.2Hz,2H),3.96(d,J=18.4Hz,1H),3.89–3.81(m ,2H),3.77(s,7H),3.69(dd,J=31.0,7.0Hz,2H),2.99(td,J=12.9,7.6Hz,2H),2.83(dt,J=13.4,6.7Hz,2H),2.76(t,J=2.6Hz,1H),2.62 (dt,J=11.6,5.9Hz,4H),2.35–2.22(m,4H),2.18(tt,J=6.9,3.3Hz,4H),1.67–1.56(m,4H),1.50–1.41(m,4H).HRMS(ESI,positive)m / z calcd for C 32 H 29 F5N2O9S2[M+H] + :745.1307,found:745.1307.

[0190] 38.2 Preparation of final product 11a. Final product 11a was synthesized according to the synthesis method in step 1.3 of Example 1. 1 HNMR(400MHz,DMSO-d6)δ13.84(s,1H),8.21(s,1H),5.79(s,1H),4.93(s,1H),4.40 (d,J=13.4Hz,0.3H),4.28–4.05(m,1H),3.95-3.88(m,1H),3.80-3.74(m,1H),3.70- 3.61(m,1.6H),3.32-3.18(m,3H),3.15-3.02(m,1H),2.75(s,1H),2.37-2.20(m,2H ),2.20-2.09(m,2H),1.66-1.52(m,2H),1.48-1.36(m,2H).HRMS(ESI,positive)m / z calcd forC 24 H 21 F5N2O8S2[M+H] + :647.0552,found:647.0532

[0191] Example 39 Preparation of (6R,7R)-8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)sulfonyl)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5,5-dioxide (12a).

[0192] 39.1 Preparation of intermediate 12a-5. Intermediate 12a-5 was synthesized by increasing the amount of added m-CPBA from 1 equivalent to 3.1 equivalents according to step 25.1 in Example 25. 1 H NMR (600MHz, DMSO-d6) δ8.45(d,J=8.4Hz,1H),7.39–7.34(m,2H),7.33–7.27(m,4H),7.23(ddd,J=8.5,5. 7,2.5Hz,1H),6.97–6.88(m,2H),5.87(dd,J=8.3,4.9Hz,1H),5.26(s,1H),5.17(d,J=12.0Hz,1H),5.00(t ,J=5.1Hz,1H),4.80(d,J=13.8Hz,1H),4.35(d,J=13.9Hz,1H),4.08(d,J=18.3Hz,1H),3.79(d,J=18.2Hz, 1H),3.75(d,J=7.0Hz,3H),3.68(d,J=14.0Hz,1H),3.65–3.53(m,3H),3.32(s,1H),3.26(t,J=7.1Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ171.0,167.0,164.5,160.8,159.4,135.8,130.3,129.1,128.3,127.8,126.66( s),126.5,113.8,111.0,67.5,66.7,58.4,55.5,55.1,47.7,47.6,41.4,26.3.HRMS(ESI,positive)m / z calcd for C 33 H 27 F5N2O 11 S2[M+Na] + :809.0869,found:809.0873.

[0193] 39.2 Preparation of final product 12a. Final product 12a was synthesized according to the synthesis method in step 1.3 of Example 1. 1HNMR (600MHz, DMSO-d6) δ14.07 (s, 1H), 8.43 (d, J = 7.0 Hz, 1H), 7.27 (d, J = 38. 8Hz,5H),5.84(s,1H),4.98(s,1H),4.85(t,J=15.8Hz,1H),4.30(dd,J=63.9 ,13.4Hz,1H),4.03(t,J=14.7Hz,1H),3.77(d,J=17.9Hz,1H),3.69(d,J=13. 8Hz,1H),3.66–3.58(m,2H),3.55(d,J=14.0Hz,1H),3.38(d,J=32.1Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ171.0,167.1,164.2,162.2,135.8,129.1,128.9,128.3, 126.6,110.2,66.6,58.3,55.7,48.1,47.6,41.5,26.3.HRMS(ESI,positive)m / z calcd for C 25 H 19 F5N2O 10 S2[M+Na] + :689.0294,found:689.0286.

[0194] Example 40: Preparation of (6R,7R)-7-(4-(4-((5-(dimethylamino)naphth-1-yl)sulfonyl)piperazin-1-yl)-4-oxobutyramide)-8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)sulfinyl)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (13a).

[0195] 40.1 Preparation of intermediates

[0196]

[0197] 4-(4-((5-(dimethylamino)naphth-1-yl)sulfonyl)piperazin-1-yl)-4-oxobutyric acid (13a-2). Succinic anhydride (0.60 g, 6.01 mmol) was added dropwise to a DCM solution (20 mL) of 13a-1 (1.60 g, 5.01 mmol) and TEA (0.84 mL, 6.01 mmol) with stirring. The reaction mixture was first stirred at 0 °C for 30 min, then at room temperature for 8 h. The solvent was then evaporated under reduced pressure and removed by vacuum rotation. The resulting residue was then purified by silica gel chromatography on silica gel using DCM:CH3OH (19:1) as eluent to give 13a-2 as a yellow solid. Yield: 2.71 g (93.0%) 1 H NMR (400MHz, DMSO-d6) δ11.99(s,1H),8.54(d,J=8.5Hz,1H),8.33(d,J=8.7Hz,1H),8.16(dd,J=7.3,1.0Hz,1H),7.72–7.66(m,1H),7.66– 7.60(m,1H),7.28(d,J=7.6Hz,1H),3.54–3.45(m,4H),3.12(d,J=17.7Hz,4H),2.83(s,6H),2.48(t,J=6.4Hz,2H),2.38(t,J=6.3Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.8,169.7,151.5,132.5,130.4,130.1,129.7,129.2,128.3, 123.7,118.9,115.3,45.4,45.3,45.0,44.3,40.7,28.8,27.3.HRMS(ESI,positive)m / z calcd for C 20 H 25 N3O5S[M+H] + :420.1588,found:420.1584.

[0198] (6R,7R)4-Methoxybenzyl 3-(chloromethyl)-7-(4-(4-(((5-(dimethylamino)naphth-1-yl)sulfonyl)piperazin-1-yl)-4-oxobutyramide)-8-oxo-5-thia-1-danza bicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxide (13a-4). N-methylmorpholine (2.41 mL g, 21.47 mmol) was added dropwise to a suspension of 13a-2 (2.00, 4.77 mmol), ACTH·HCl (1.84 g, 4.77 mmol), and phenyl dichlorophosphate (1.48 mL g, 7.16 mmol) in DCM (40 mL) at -40 °C. The resulting mixture was stirred at -40 °C for 4 hours, then thawed with aq. 0.2 mol / L HCl (93 mL), and then slowly heated to room temperature. The aqueous layer was extracted with DCM, and the combined extracts were washed with saturated aqueous solution. Sodium bicarbonate, water, and brine were dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography using DCM:acetone (49:1) as the eluent to give 13a-3 as a yellow solid. At 0 °C, m-CPBA (70% purity, 0.30 g, 1.23 mmol) was added to 25 (1.0 g, 1.30 mmol) of dried DCM (10 mL). The reaction mixture was stirred at 0 °C for 30 min, followed by stirring at room temperature for 6 h. The reaction mixture was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography using DCM:CH3OH (48:2) as the eluent to give 13a-4 as a yellow solid. Yield: 0.543 g (53.1%). 1H NMR (400MHz, CDCl3) δ8.61(d,J=6.8Hz,1H),8.39(d,J=8.4Hz,1H),8.20(d,J=7.3Hz,1H),7.55(t,J=8.4Hz,2H),7.34(d,J=8.4Hz ,2H),7.21(d,J=7.3Hz,1H),6.91(t,J=9.9Hz,3H),6.01(dd,J=9.9,4.7Hz,1H),5.28–5.21(m,2H),5.00(d,J=12.4Hz,1H),4.46( d,J=4.3Hz,1H),4.23(d,J=12.4Hz,1H),3.81(s,4H),3.76(s,1H),3.63(d,J=4.4Hz,2H),3.51(d,J=3.8Hz,2H),3.38(d,J=18.6H z,1H),3.20(t,J=4.7Hz,2H),3.15(t,J=4.7Hz,2H),2.91(s,6H),2.72–2.55(m,2H),2.55–2.42(m,2H).HRMS(ESI,positive)m / z calcd for C 36 H 40 ClN5O9S2[M+H] + :786.2029,found:786.2015.

[0199] (6R,7R)4-methoxybenzyl 7-(4-(4-((5-(dimethylamino)naphth-1-yl)sulfonyl)piperazin-1-yl)-4-oxobutyramide)-8-oxo-3-(((3-oxo-3-(perfluorophenoxy)propyl)sulfinyl)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxide (13a-6). Compound 13a-5 was prepared from 13a-4 using a procedure similar to that described for 1a-3. The reaction mixture was quenched with H2O and the pH was adjusted to 6.5 with 4M HCl. The resulting precipitate was collected by filtration and washed with cold water (3 x 10 mL). The crude product was air-dried and used in the next step without further purification. Compound 13a-6 was prepared from 13a-5 and PFP using a procedure similar to that described for 1a-4. Pale yellow solid. Yield: 0.24 g, 51.3%. 1H NMR (400MHz, DMSO-d6) δ8.53(d,J=8.5Hz,1H),8.31(d,J=8.7Hz,1H),8.14(d,J=7.3Hz,1H),8.13–8.06(m,1H),7.70–7.65(m,1H),7. 65–7.59(m,1H),7.36(dd,J=8.6,3.8Hz,2H),7.28(d,J=7.5Hz,1H)6.93(dd,J=8.6,6.7Hz,2H),5.79–5.70(m,1H),5.27-5.15(m,2H) ,4.93–4.85(m,1H),4.05–3.92(m,1H),3.78–3.70(m,4H),3.69–3.61(m,1H),3.61-3.54(m,1H),3.49(s,4H),3.15-3.06(m,4H),3.0 2(t,J=6.7Hz,1H),2.83(s,6H),2.80–2.69(m,1H),2.69–2.52(m,2H),2.46-2.42(m,2H),2.44–2.40(m,2H).HRMS(ESI,positive)m / z calcd for C 45 H 44 F5N5O 11 S3[M / 2+H] + :511.6133,found:511.6122.

[0200] (6R,7R)4-methoxybenzyl 7-(4-(4-((5-(dimethylamino)naphth-1-yl)sulfonyl)piperazin-1-yl)-4-oxobutyamido)-8-oxo-3-(((3-oxo-3-perfluorophenyl)propyl)sulfinyl)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid 5-oxide (13a-7). Compound 13a-7 was prepared from 13a-6 by a similar method as described in step 20.2. The resulting residue was purified by silica gel chromatography using DCM:methanol (48:2) as eluent to give 13a-7. 1H NMR (400MHz, DMSO-d6) δ8.54(d,J=8.5Hz,1H),8.31(d,J=8.7Hz,1H),8.15(d,J=6.8Hz,1H),8.11-8.06(m,1H),7.70–7.65(m,1H),7.65 –7.60(m,1H),7.40-7.33(m,2H),7.28(d,J=7.5Hz,1H),6.96-6.90(m,2H),5.84-5.75(m,1H),5.20(dd,J=26.0,12.0Hz,1H),4.92(d,J= 3.4Hz,1H),4.40-4.30(m,0.5H),4.22–4.05(m,1H),3.98–3.90(m,1H),3.88–3.79(m,1H),3.76-3.72(m,3.5H),3.50(s,4H),3.22(s,1 H),3.11(d,J=20.6Hz,4H),3.03-2.93(m,1H),2.84(s,6H),2.69–2.53(m,2H),2.48(s,2H),2.46–2.33(m,2H).HRMS(ESI,positive)m / z calcd for C 45 H 44 F5N5O 12 S3[M+Na] + :1060.1961,found:1060.1948.

[0201] 40.2 Preparation of final product 13a. Final product 13a was synthesized according to the synthesis method in step 1.2 of Example 1. 1HNMR (400MHz, DMSO-d6) δ8.55(d,J=8.5Hz,1H),8.34(d,J=8.7Hz,1H),8.15(d,J=7.3Hz,1H),8.09(t,J=9.0Hz,1H),7.70(t,J=7.9Hz,1 H),7.65(t,J=8.3Hz,1H),7.33(d,J=7.6Hz,1H),5.77(s,1H),4.90(d,J=4.6Hz,1H),4.39(d,J=13.2Hz,0.4H),4.20(d,J=12.9Hz,0.6H ),4.08(d,J=18.4Hz,0.4H),3.88(t,J=16.8Hz,1H),3.82–3.74(m,1H),3.73–3.65(m,0.6H),3.49(s,4H),3.13(s,2H),3.08(t,J=5.4H z,2H),3.04(d,J=6.6Hz,1H),2.91(t,J=6.7Hz,1H),2.88–2.84(m,6H),2.65(q,J=6.6Hz,2H),2.48(s,2H),2.42(q,J=7.4,6.2Hz,2H). 19 F NMR (376MHz, CDCl3) δ -71.2, -147.4, -152.7, -157.4 (d, J = 27.0Hz). 19 F NMR shows that it contains trifluoroacetic acid.HRMS(ESI,positive)m / z calcd for C 45 H 44 F5N5O 12 S3[M+H] + :918.1566,found:918.1547.

[0202] Example 41

[0203] The inhibitory activity of the cephalosporin compounds obtained in the above embodiments of the present invention against NDM-1 was tested. The inhibitory activity of compound 6a against other metallo-β-lactamases was also tested.

[0204] Experimental methods: The inhibitory activity of compound I on NDM-1 and compound 6a on other metallo-β-lactamases was determined by the kinetics of NDM-1 hydrolysis of fluorescent substrate FC5.

[0205] Main reagents used in the experiment: recombinant NDM-1 (expressed and purified from E. coli system), FC5, black 96-well plate, Biacore T200 multi-functional microplate reader.

[0206] Experimental procedure: All compounds were prepared as 100 mM DMSO stock solutions, and FC5 was prepared as a 1 mg·mL solution. -1 DMSO stock solution. The IC50 was determined by pre-incubating NDM-1 (final concentration 0.2 nM) with the desired compound (final concentrations between 0.030 and 600 μM in a 3-fold dilution series) in buffer (20 mM Tris-HCl, pH 7.5) for 2 h. 50 The reaction was initiated by adding substrate FC5 (final concentration 5 μM) to 200 mM NaCl and monitoring the fluorescence at λex (380 nm) and λem (460 nm) for 8 minutes. Each experiment was repeated three times. The IC50 was obtained by plotting the activity versus inhibitor concentration using GraphPad Prism software. 50 value.

[0207] The results are shown in Table 1.

[0208] The inhibitory effects of 6a on other metallo-β-lactamases are shown in Table 2.

[0209] Since all our compounds share similar cephalosporin structures, identical physicochemical properties, and the same mechanism of action, and all exhibit inhibitory activity against NDM-1, and other metallo-β-lactamases also share similar structures with NDM-1, these other compounds, like compound 6a, exhibit similar inhibitory activity against other metallo-β-lactamases.

[0210] Example 42

[0211] The inhibitory activity of the cephalosporin compounds described in this invention in combination with meropenem against Gram-negative bacteria that produce metallo-β-lactamases such as NDM-1 was tested.

[0212] Experimental method: The checkerboard method was used to determine the inhibitory activity of compound I against NDM-1-producing Gram-negative bacteria.

[0213] Main reagents used in the experiment: E. coli BL21 (NDM-1) + E. coli BAA2452 (bla) NDM-1 E. coli 1864 (bla) NDM-1 K. pneumoniae 1, K. pneumoniae 2, K. pneumoniae 3, K. pneumoniae 4, K. pneumoniae 5, MHB medium, constant temperature incubator, clean bench (Cytiva, USA).

[0214] Experimental steps: IC 50Values ​​were determined according to the standard broth microdilution method as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. Clinical isolates and engineered strains of *E. coli* and *Klebsiella pneumoniae* expressing blaNDM 1 were incubated overnight in Mueller-Hinton broth (MHB) at 37°C and 250 rpm, and the concentrations were measured at 600 nm (OD). 600 The OD value was measured at ( ). The bacterial density was then diluted to 1 × 10⁻⁶ in MHB. 6 CFU·mL -1 Meropenem (final concentrations in a 2-fold dilution series between 0.25 and 256 μg·mL⁻¹), the compound (final concentrations in a 2-fold dilution series between 4 and 128 μg·mL⁻¹), and the prepared bacterial suspension were then added to 96-well microtiter plates. Wells without inhibitors and without meropenem served as growth controls. Wells without added bacteria served as background controls. The plates were incubated at 37°C for 20 hours. The minimum inhibitory concentration (MIC) of the drug that could inhibit microbial growth was determined by visual readings and OD readings at 600 nm using a microtiter plate reader.

[0215] The results are as follows Figure 1 As shown in Table 3. Because these compounds all have similar cephalosporin structures and the same mechanism of action, other compounds also exhibit similar synergistic antibacterial activity against NDM-1-producing Gram-negative bacteria when used in combination with penem, as 6a.

[0216] Example 43

[0217] The labeling effect of the cephalosporin compounds of the present invention on NDM-1.

[0218] Experimental method: Fluorescently labeled proteins were identified by SDS-PAGE.

[0219] Main reagents and instruments used in the experiment: captopril (Jiuding Chemical), SDS-PAGE protein loading buffer (5X, Beyotime), 20mM Tris-HCl buffer (pH 7.5), color pre-stained protein molecular weight standards (15-120kD, Beyotime), SDS-PAGE rapid electrophoresis buffer (Tris-Gly, 20X, Beyotime), electrophoresis apparatus (Bio-Rad).

[0220] Experimental Procedure: NDM-1 (final concentration 45 μM) was added to a 20 mM Tris-HCl buffer (pH 7.5) solution at 25 °C, followed by solutions of compound 6a (final concentration 300 μM) and captopril (final concentration 1 mM). After 1 h, compound 15a (final concentration 300 μM) was added to pure NDM-1 and the two previously treated protein samples. 5×SDS gel loading buffer (100 mM Tris-HCl buffer (pH 6.8), 2.5% SDS, 20% glycerol) was added to the three treated protein samples, and separation was performed by SDS-PAGE. The gels were then photographed using a camera or by irradiating them with 254 nm and 365 nm UV light. The gels were stained with Coomassie Brilliant Blue, and images of the stained gels were captured using an imager.

[0221] The results are as follows Figure 2 As shown.

[0222] Example 44

[0223] The cephalosporin compound described in this invention, combined with meropenem, is effective in treating intraperitoneal infections in mice.

[0224] Experimental methods: A mouse model of intraperitoneal infection was established, and compound 6a was used in combination with meropenem for treatment.

[0225] Main reagents and instruments used in the experiment: Meropenem (Bide), Cyclophosphamide (Bide).

[0226] Experimental Procedure: Compound 6a was combined with meropenem to treat mice with intraperitoneal infection. Male BalB / c mice (18–22 g) aged 6–8 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. All mice were intraperitoneally injected with 150 mg / kg on days 1 and 4, respectively. -1 and 100 mg·kg -1 Cyclophosphamide was used for immunosuppression, and 8×10⁸ liters were injected intraperitoneally on day 5. 7 CFU of Escherichia coli BAA-2452 was administered to four groups of mice 4 hours after infection with 100 μL PBS, meropenem (10 mg·kg⁻¹), or 6a (20 mg·kg⁻¹) as monotherapy or in combination (n = 8, 8, 8, and 12). Treatment was administered once daily via intraperitoneal injection for five days.

[0227] The results are as follows Figure 3 As shown. Other compounds have similar cephalosporin structures, mechanisms of action, and properties to 6a, and therefore also have the effect of being used in combination with meropenem to treat intraperitoneal infections in mice.

[0228] Table 1. Inhibitory activity of compounds against NDM-1

[0229]

[0230] Half-maximal inhibitory concentration (IC50) 50 :*: >100μM, **: 10-100μM, ***: 1-10μM, ****: <1μM.

[0231] Table 2. Inhibitory effects of compound 6a on other metallo-β-lactamases

[0232]

[0233] Half-maximal inhibitory concentration (IC50) 50 :*: >100μM, **: 10-100μM, ***: 1-10μM, ****: <1μM.

[0234] Table 3. In vitro antibacterial activity of compound 6a in combination with meropenem against clinically isolated K. pneumoniae and E. coli expressing NDM-1.

[0235]

[0236] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope of the present invention.

Claims

1. Any of the following cephalosporin compounds, or pharmaceutically acceptable salts thereof, 、 、 、 、 、 、 、 。 2. A pharmaceutical composition consisting of a cephalosporin compound of claim 1, or a pharmaceutically acceptable salt thereof, and a medically acceptable carrier.

3. Use of the cephalosporin compound of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of metallo-β-lactamase inhibitors.

4. Use of the cephalosporin compound of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a fluorescent probe with labeling function against metallo-β-lactamases.

5. The use according to any one of claims 3 and 4, characterized in that, The metallo-β-lactamase mentioned is selected from NDM-1.

6. Use of the cephalosporin compound of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of an antibacterial medicament.

7. The use according to claim 6, characterized in that, The antibacterial drug is a drug for the prevention and / or treatment of infections caused by Gram-negative drug-resistant bacteria.

8. The use according to claim 7, characterized in that: The Gram-negative drug-resistant bacteria are selected from metallo-β-lactamase-producing Escherichia coli, Klebsiella pneumoniae, Acinetobacter bacillus, Pseudomonas aeruginosa, Enterobacter cloacae, Proteus, Citrobacter freundii, Klebsiella pneumoniae, Morganella morganii, or Providenella.

9. The use according to claim 8, characterized in that, The antibacterial drug mentioned is an antibacterial drug against Escherichia coli or Klebsiella pneumoniae that produce NDM-1 metallo-β-lactamase.

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