A c-3 tetrahydrofuran cephalosporin derivative and use thereof

Abstract

The application discloses a C-3 tetrahydrofuran cephalosporin derivative and application, and is characterized by a structural formula: on the basis of the research on the anti-infective drug cefovecin sodium for pets, the 3-position tetrahydrofuran ring structure of the cefovecin sodium is retained, the 7-position side chain thereof is selected to be modified, different structure side chains are introduced, the antibacterial activity and the antibacterial spectrum of the cefovecin sodium are changed, and in particular, the activity on common drug-resistant bacteria in clinic is improved, so that new antibiotic products with clinical value are obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a C-3 position tetrahydrofuran cephalosporin derivative and application. BACKGROUND

[0002] Antimicrobial drugs (including antibiotics, antiviral drugs, antifungal drugs and antiparasitic drugs) are drugs used to prevent and treat infections in humans, animals and plants. When bacteria, viruses, fungi and parasites change over time and no longer respond to drugs, antimicrobial drug resistance occurs. Antimicrobial drug resistance makes more and more infections more difficult to treat. Misuse and overuse of antimicrobial drugs are the main drivers of the development of drug-resistant pathogens.

[0003] Antimicrobial drug resistance occurs when bacteria, viruses, fungi and parasites no longer respond to antimicrobial drugs. Because of drug resistance, antibiotics and other antimicrobial treatments fail, making it more difficult, and even impossible, to treat infections. This could lead to the emergence of superbugs that are not inhibited by the preferred drugs used to treat these pathogens, increasing the risk of disease transmission, disability and death. This problem is considered one of the top ten global public health threats.

[0004] In recent years, with the extensive use of antibiotics, especially misuse, many pathogenic bacteria have developed serious resistance to clinical antibiotics, especially the problem of resistance to gram-negative bacteria, which has become a serious problem threatening human public health. However, the development of new antibiotics against negative drug-resistant bacterial infections is slow. Therefore, it is urgent to find new antibiotics and new antibacterial strategies to cope with the "antibiotic crisis" facing the world.

[0005] Cefovecin sodium is a new third-generation cephalosporin animal-specific antibiotic. Its structural feature is that a tetrahydrofuran heterocycle is linked to the C-position of the cephalosporin nucleus through a C-C bond, thereby endowing the product with broad-spectrum antibacterial activity and stability to β-lactamase. Cefovecin sodium, as an antibacterial agent, has a broad antibacterial spectrum, fast absorption, slow elimination, high bioavailability, good safety, high therapeutic index and other advantages in dogs and cats, and is an ideal long-acting antibiotic for dogs and cats, which has shown unique application prospects in veterinary clinics. It is mainly used for the treatment of skin, digestive tract, urogenital tract, bone and soft tissue infections caused by sensitive bacteria and other infections in dogs and cats, and pyoderma caused by staphylococci carrying the mecA gene in dogs, and can also be used as a drug to prevent postoperative infection after surgical operation in dogs and cats.

[0006]

[0007] The number of C-3 tetrahydrofuran heterocyclic cephalosporin derivatives reported in the literature is limited, and no further research and development of new derivatives with similar structures has been carried out except for cefovecin. SUMMARY

[0008] To solve the above technical problems, the first object of the present application is to provide a C-3 tetrahydrofuran cephalosporin derivative, and the second object is to provide its application.

[0009] To achieve the above first object, the present application is implemented by the following technical scheme: a C-3 tetrahydrofuran cephalosporin derivative, characterized in that the structural formula is:

[0010] ,

[0011] wherein R is:

[0012] , , , , , , , , , , , , , .

[0013] In the above scheme: X is one of C, N, C-Cl, Y is C1-C2 alkyl, H, CH2F, CR1R2COOH

[0014] wherein R1=R2=H or CH3;

[0015] or R1=H, R2=CH3, CF3, isopropyl, cyclohexyl, 4-methylcyclohexyl, 4-trifluoromethylcyclohexyl, 4,4-difluorocyclohexyl; or R1=CH3, R2=CF3.

[0016] In the above scheme: the structural formula is:

[0017] , ,

[0018] , ,

[0019] , ,

[0020] , .

[0021] Use of the C-3 tetrahydrofuran containing cephalosporin derivative in the preparation of a medicament for treating diseases caused by bacteria, viruses, fungi and parasites in humans and animals.

[0022] The bacteria are gram-negative bacteria or gram-positive bacteria.

[0023] The bacteria are Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus.

[0024] The selection of the R segment of the present application has the following characteristics:

[0025] 1. Amino thiazole oxime and amino thiazole oxime side chains, covering all side chain segments of the third generation cephalosporin to the fifth generation cephalosporin used clinically. The development of such cephalosporin drugs has witnessed the shift of the antibacterial spectrum of the drug from broad-spectrum to narrow-spectrum, especially the development and clinical use of special drug-resistant bacteria. The purpose of selecting such side chains aims to expand the antibacterial spectrum of cefovecin or improve the activity against drug-resistant bacteria, especially multiple drug-resistant bacteria.

[0026] 2. Selecting the unique side chain segments of broad-spectrum cephalosporins such as cefuroxime, cefcapene, cefamandole, cefotiam and cefoperazone for structural modification in order to improve the biological activity of cefovecin and obtain inhibitory effect on drug-resistant strains.

[0027] 3. Selecting phenylglycine and p-hydroxyphenylglycine side chains at the 7-position for modification to increase the dosage form of the drug molecule in order to improve the in vivo metabolic behavior of the drug.

[0028] 4. Selecting the side chains of narrow-spectrum first and second generation cephalosporins such as cephalothin, cephapirin and cefonicid for structural modification of new derivatives in order to obtain changes in antibacterial activity.

[0029] 5. Selecting the isoxazolyl-containing semisynthetic penicillin side chain effective against gram-positive bacteria for the synthesis of new derivatives in order to change its biological activity.

[0030] Advantages

[0031] The present application is based on the research of cefovecin sodium, an anti-infective drug for pets, and retains the 3-tetrahydrofuran ring structure. The 7-position side chain is selected for modification by introducing different structures to change the antibacterial activity and spectrum of cefovecin sodium, especially to improve the activity against common drug-resistant bacteria in the clinic, in order to obtain new antibiotic products with clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The MIC values of the three drugs against Escherichia coli are distributed.

[0033] Figure 2 MIC values distribution of 6 test compounds for E. coli.

[0034] Figure 3 MIC values distribution of 15 test compounds for E. coli.

[0035] Figure 4 MIC values distribution of 3 drugs for Staphylococcus.

[0036] Figure 5 MIC values distribution of 8 test compounds for Staphylococcus.

[0037] Figure 6 MIC values distribution of 13 test compounds for Staphylococcus. DETAILED DESCRIPTION

[0038] The present application will be further described in connection with the accompanying drawings and examples.

[0039] The C-3 tetrahydrofuran cephalosporin derivatives have the following structural formula:

[0040]

[0041] Each derivative can be made into a sodium salt or an ester.

[0042] wherein R is the following structural fragment:

[0043]

[0044] When the first structural fragment, X is one of C, N, C-Cl, Y is C1-C2 alkyl, H, CH2F, CR1R2COOH

[0045] wherein R1=R2=H or CH3;

[0046] or R1=H, R2=CH3, CF3, isopropyl, cyclohexyl, 4-methylcyclohexyl, 4-trifluoromethylcyclohexyl, 4,4-difluorocyclohexyl; or R1=CH3, R2=CF3.

[0047] ​​​​​​​​​​​​​​​​​The synthesis method of C-4 tetrahydrofuran cephalosporin mother nucleus (TBWX-Z10) is the prior art, which can be commercially purchased.

[0048] General synthesis method of new derivatives

[0049] (1) General synthesis formula of new derivatives of 7-position 2-aminothiazole oxime or 2-aminothiadiazole oxime side chain

[0050]

[0051]

[0052] Q = C, N, C-Cl;

[0053] Y = C1-C2 alkyl, CH2F, CR1R2COOH, wherein R1 = R2 = H, CH3; R1 = H, R2 = CH3, CF3, isopropyl, cyclohexyl, 4-methylcyclohexyl, 4-trifluoromethylcyclohexyl, 4, 4-difluorocyclohexyl; R1 = CH3, R2 = CF3

[0054] Z = COOH, Cl, OSO2CH3, 2-mercaptobenzothiazole, etc.

[0055] PG1 = an amino protecting group, such as Boc, trityl, formyl, etc.

[0056] R4 = H or p-nitrobenzyl;

[0057] M = Na, K or ester.

[0058] (2) General synthesis formula of new derivatives of 7-position other types of side chain

[0059]

[0060]

[0061] R is the structural fragment in Table 1 except the serial number; R4 = H or p-nitrobenzyl; M = Na, K or ester.

[0062] Example 1

[0063] Synthesis of F50-001

[0064] (6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(((2-carboxypropan-2-yl)oxy)imino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0065] 1. Chemical reaction formula

[0066]

[0067]

[0068] 2. Operation steps

[0069] First step:

[0070] Into a 250 mL three-necked flask was added (Z)-2-(((1-(tert-butoxy)-2-methyl-1- oxoprop-2-yl)oxy)imino)-2-(2-((tert-butoxycarbonyl)amino)thiazol-4-yl)acetic acid 5.83 g, dichloromethane 150 mL, and the mixture was stirred under N2atmosphere and cooled to -20 °C. Methylsulfonyl chloride 1.56 g and triethylamine 1.75 g were added, and the mixture was stirred at -20 °C for 4 h to obtain a mixed anhydride solution.

[0071] Into another 500 mL three-necked flask was added (6R,7R)-7-amino-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4- nitrobenzyl ester hydrochloride 5.0 g and dichloromethane 50 mL, and the mixture was cooled to -20 °C under N2protection. Triethylamine 2.52 g was added, and the mixture was stirred for 10 min. The mixed anhydride solution obtained above was added, and the mixture was stirred at -15 °C to -10 °C until the reaction of (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester was completed, as monitored by TLC (PE:EA = 1:3). Water 200 mL was added, and the mixture was stirred for 15 min. The mixture was allowed to stand to separate into two phases, and the dichloromethane phase was collected. The mixture was concentrated under reduced pressure to obtain a crude product 12.63 g. The crude product was purified by silica gel column chromatography (EA:PE = 0:100 to EA:PE = 75:25) to obtain the product 8.11 g.

[0072] Second step:

[0073] Into a 100 mL flask was placed (6R,7R)-7-((Z)-2-((((1-(tert-butoxy)-2-methyl-1- oxoprop-2-yl)oxy)imino)-2-(2-((tert-butoxycarbonyl)amino)thiazol-4-yl)acetamido)-8- oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4- nitrobenzyl ester 5.0 g and dichloromethane 25 mL, and the solution was stirred at room temperature. Then trifluoroacetic acid 25 mL was added, and the reaction was stirred at room temperature for 24 hours, and the reaction was monitored by TLC. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure to obtain a crude product. The crude product was purified by silica gel column normal phase (MeOH:DCM=0:100~MeOH:DCM=20:80) to obtain a yellow solid 2.56 g.

[0074] Step 3:

[0075] Into a 50 mL flask was placed (6R,7R)-7-((Z)-2-((((2-carbonyloxypropan-2-yl)oxy)imino)-2-(2- (aminothiazol-4-yl)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid-4-nitrobenzyl ester 1.0 g, acetone 7 mL, and the solution was stirred at room temperature. Then water 5 mL was added, and the pH was adjusted to 7~8 with ammonia water, and then sodium hydrosulfite 1.11 g was added, and the reaction was stirred at room temperature for 1.5 hours. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove acetone. The remaining solution was adjusted to pH=2 with 1M HCl solution, and a yellow solid was precipitated, which was filtered to obtain a crude product. The crude product was purified by reverse phase flash chromatography, and the specific conditions were as follows: the chromatographic column was C18 silica gel; the mobile phase was a mixture of acetonitrile and pure water, which was changed at a gradient of 5% to 50% in 30 minutes; and the detection wavelength was 254 nm. The purified fraction was freeze-dried to obtain a white solid 260 mg.

[0076] 1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 7.21 (s, 2H), 6.73 (d, J =5.3 Hz, 1H), 5.64 (dt, J = 12.3, 5.8 Hz, 1H), 5.07 - 4.98 (m, 1H), 4.81 (dd, J = 9.0, 6.6 Hz, 1H), 3.80 (q, J = 6.9 Hz, 2H), 3.63 (dtd, J = 15.9, 7.5, 5.5 Hz, 3H), 3.37 (dd, J = 17.1, 5.7 Hz, 2H), 3.22 (dd, J = 27.7, 17.1 Hz, 2H), 2.12 - 2.00 (m, 1H), 1.82 (dddd, J = 23.1, 19.4, 9.1, 6.0 Hz, 2H), 1.66 - 1.52 (m, 1H), 1.47 (d, J = 3.2 Hz, 3H), 1.40 (s, 3H).

[0077] LCMS (ESI(-)): 524.0.

[0078] Example 2

[0079] Synthesis of F50-002

[0080] (6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-((carboxymethoxy)imino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0081] 1. Chemical Reaction Formula

[0082]

[0083]

[0084] 2. Operating Steps

[0085] First Step:

[0086] In a 250 mL three-necked flask, 80 mL of dichloromethane, 12 mL of ethanol and 1.2 mL of water were added, and the mixture was stirred and cooled to 0-2°C. Then, 4.78 g of (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid and 8.0 g of S-2-benzothiazolyl-(Z)-2-(2- aminothiazol-4-yl)-2-methoxycarbonylmethoxyiminothioacetic acid ester were added, and 3.0 mL of triethylamine was added dropwise at 0-5°C. The mixture was stirred at the same temperature for 8-10 hours. Then, 60 mL of water was added, and the mixture was stirred for 5 minutes, allowed to stand, and the layers were separated. The organic layer was added to 60 mL of water, stirred for 5 minutes, allowed to stand, and the layers were separated. The water layers were combined, and 1 g of activated carbon, 0.1 g of ferrous sulfate and 0.04 g of EDTA-2Na were added to the water layer. The mixture was concentrated under reduced pressure at 15-20°C for 30 minutes, filtered, and the filter cake was washed with water.

[0087] Second Step:

[0088] The mixture was cooled to 0-2°C, and 60 mL of water and 2.48 g of a sodium hydroxide aqueous solution pre-cooled to 0°C were added. The mixture was stirred for 10 minutes, and 4% hydrochloric acid was rapidly added at 5-8°C to adjust the pH to 5.0-5.5. The pH was further adjusted to 1.9-2.0, and the mixture was cooled to 0-5°C and stirred for 30 minutes. The mixture was filtered, washed with water, and dried under reduced pressure at 35-40°C for 6 hours to obtain 7.48 g of (6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-((carboxymethoxy)imino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid with a yield of 85%.

[0089] 1 H NMR (400 MHz, D2O)δ:1.69-2.18(4 H,m),3.32 and 3.51(2H,ABq,J=18 Hz),3.74-3.93(2H,m),4.52(2H,s),5.19(1H,d,J=5 Hz),5.77(1H,d,J=5 Hz)and 7.01(IH,s).

[0090] LCMS (ESI(-)): 495.9.

[0091] Example 3

[0092] Synthesis of F50-003

[0093] (6R,7R)-7-((Z)-2-(2-amino-5-chlorothiazol-4-yl)-2-(methoxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0094] 1. Chemical reaction

[0095]

[0096] 2. Operating steps

[0097] Into a 50 mL three-necked flask was placed (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 1.0 g, S-(benzo[d]thiazol-2-yl)-2-(2-amino-5-chlorothiazol-4-yl)-2-(methoxyimino)ethanethioate 1.76 g, tetrahydrofuran 12 mL, water 3 mL, and stirred at room temperature. A mixture of triethylamine 0.56 g and tetrahydrofuran 1 mL was added dropwise. The reaction was stirred at room temperature for 18 hours, and monitored by TLC.

[0098] After the reaction was completed, dichloromethane 6 mL was added to the reaction system, stirred for 10 minutes, and allowed to stand to separate the phases. The aqueous phase was retained. Dichloromethane 12 mL was added to the aqueous phase, stirred for 10 minutes, and allowed to stand to separate the phases. The aqueous phase was retained, and the organic phases were combined. Dichloromethane 12 mL was added to the aqueous phase, stirred for 10 minutes, and allowed to stand to separate the phases. The aqueous phase was retained, and the organic phases were combined. Water 18 mL was added to the aqueous phase, stirred for 10 minutes, and allowed to stand to separate the phases. The aqueous phases were combined. The aqueous phases were adjusted to pH = 2 with 1 M HCl, and a large amount of solid precipitated. The mixture was stirred for 10 minutes, filtered, and the crude product was obtained.

[0099] The crude product was purified by reverse-phase flash chromatography using the following conditions: a C18 silica gel column; a mobile phase of acetonitrile-water mixture, with a gradient of 5% to 50% over 30 minutes; and a detection wavelength of 254 nm. The purified fractions were freeze-dried to obtain (6R,7R)-7-{(Z)-2-[2-amino-5-chlorothiazol-4-yl]-2-(methoxyimino)propanamido}-8-oxo-3-[(S)-tetrahydrofuran-2-yl]-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (260 mg) as a white solid.

[0100] 1H NMR (400 MHz, DMSO-d6) δ 9.54 (dd, J = 9.8, 8.6 Hz, 1H), 7.41 (s,2H), 5.72 (td, J = 8.5, 4.8 Hz, 1H), 5.11 (dd, J = 7.6, 4.8 Hz, 1H), 4.73(dd, J = 8.9, 6.7 Hz, 1H), 3.86 (d, J = 1.4 Hz, 3H), 3.83 (d, J = 8.1 Hz,1H), 3.74 – 3.66 (m, 1H), 3.57 – 3.51 (m, 1H), 3.44 (s, 1H), 2.11 (dtd, J =10.5, 7.4, 5.3 Hz, 1H), 1.99 – 1.80 (m, 2H), 1.67 (ddq, J = 29.9, 12.0, 8.7Hz, 1H).

[0101] LCMS (ESI(-)): 486.0, 488.0.

[0102] Example 4

[0103] Synthesis of F50-004

[0104] (6R,7R)-7-((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(((2-carboxypropan-2-yl)oxy)imino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0105] 1. Chemical Reaction Formula

[0106]

[0107]

[0108]

[0109] 2. Procedure

[0110] First Step:

[0111] Into a 50 mL single neck flask was placed (Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxy)imino)acetic acid 3.74 g, DMF 25 mL, HATU 4.73 g, N,N-diisopropylethylamine 4.39 g, stirred at room temperature for 15 min, then added (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester hydrochloride 5.0 g, stirred at room temperature for 16 h. TLC monitored the reaction.

[0112] After the reaction was completed, the reaction solution was poured into 100 mL of ethyl acetate, 100 mL of water was added, stirred for 10 min, and then allowed to stand to separate the liquid. The organic phase was retained, and the aqueous phase was extracted twice with 50 mL of ethyl acetate. The combined organic phases were washed once with 100 mL of saturated brine, and the organic phase was concentrated under reduced pressure to dryness to obtain a yellow solid crude product 9.33 g. The crude product was purified by silica gel column chromatography using petroleum ether containing 0% to 75% ethyl acetate as the eluent. The purified fractions were evaporated to dryness to obtain (6R,7R)-7-((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 7.16 g as a white solid.

[0113] Second step:

[0114] Into a 250 mL single neck flask was placed (6R,7R)-7-((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 4.0 g, dichloromethane 40 mL, stirred to dissolve at room temperature, then added trifluoroacetic acid 40 mL, stirred at room temperature for 16 h, TLC monitored the reaction.

[0115] After the reaction was completed, it was concentrated to dryness under reduced pressure, and (6R,7R)-7-((Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2-((2-carboxypropan-2- yl)oxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-l- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 2.7 g was obtained as a yellow solid.

[0116] Step 3:

[0117] To a 50 mL single necked flask was added (6R,7R)-7-((Z)-2-(5-amino-l,2,4- thiadiazol-3-yl)-2-((2-carboxypropan-2-yl)oxyimino)acetamido)-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4- nitrobenzyl ester 1.0 g, acetone 7 mL, and the solution was stirred clear. Ammonia was added to adjust the pH to 8, and sodium dithionite 3.74 g was added, followed by water 2 mL. The reaction was stirred at room temperature for 1.5 hours while maintaining the pH at 8 with ammonia. The reaction was monitored by TLC.

[0118] After the reaction was completed, it was concentrated to dryness under reduced pressure, and (6R,7R)-7-((Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2-((2-carboxypropan-2- yl)oxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-l- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 2.7 g was obtained as a yellow solid.

[0119] 1H NMR (400 MHz, DMSO-d6) δ 9.49 (t, J = 7.5 Hz, 1H), 8.20 (s, 2H),5.78 (ddd, J = 10.4, 8.5, 4.8 Hz, 1H), 5.14 (dd, J = 6.5, 4.8 Hz, 1H), 4.75(dd, J = 8.9, 6.7 Hz, 1H), 3.85 (dq, J = 14.1, 7.2 Hz, 1H), 3.70 (td, J =7.8, 5.1 Hz, 1H), 3.58 – 3.49 (m, 2H), 2.10 (ddd, J = 11.8, 7.2, 4.1 Hz, 1H),1.90 (ddq, J = 28.0, 12.2, 5.7, 4.2 Hz, 2H), 1.67 (ddq, J = 26.5, 12.1, 8.6Hz, 1H), 1.47 (d, J = 2.4 Hz, 6H).

[0120] LCMS (ESI(-)): 525.0.

[0121] Example 5

[0122] F50-012 (6R,7R)-7-((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(methoxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0123] 1. Chemical Reaction Formula

[0124]

[0125] 2. Procedure

[0126] Add 30 mL of tetrahydrofuran and 30 mL of purified water to a 250 mL three-necked flask, cool to 0-5 °C, add 5.0 g of (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid and 7.5 g of (Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(methoxyimino)thioacetic acid (S-2-benzothiazole) ester, and adjust the pH to 7.8-8.4 by adding a mixture of 2.9 mL of triethylamine and 4 mL of tetrahydrofuran dropwise at 0-5 °C, stirring for 5 hours. Add 50 mL of dichloromethane to the aqueous layer below 5 °C, stir for 5 minutes, and allow to separate into layers. Add 50 mL of dichloromethane to the aqueous layer, stir for 5 minutes, and allow to separate into layers. Combine the organic layers, add 15 mL of purified water, stir for 2 minutes, and allow to separate into layers. Combine the aqueous layers, adjust the pH to 2.6-3.0 by adding 15% hydrochloric acid dropwise at 0-10 °C, precipitate the solid, stir for 1 hour, filter, and wash with cold water. Dry under reduced pressure at 35-45 °C to obtain 4.0 g of product, with a yield of 85%.

[0127] 1 H NMR (400 MHz, DMSO-d6) δ 9.54 (t, J = 8.1 Hz, 1H), 8.12 (s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 5.17 – 5.05 (m, 1H), 4.75 (t, J = 7.9 Hz, 1H), 3.91(d, J = 1.4 Hz, 3H), 3.83 (dt, J = 15.2, 7.2 Hz, 1H), 3.69 (d, J = 5.5 Hz,1H), 3.56 – 3.47 (m, 1H), 3.43 (s, 1H), 2.11 (d, J = 6.7 Hz, 1H), 1.97 – 1.77(m, 2H), 1.75 – 1.55 (m, 1H).

[0128] LCMS (ESI(-)):453.0.

[0129] Example 6

[0130] Synthesis of F50-013 (6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0131] 1. Chemical reaction formula

[0132]

[0133]

[0134] 2. Operation steps

[0135] First step:

[0136] Into a 250 mL three-necked flask, add 40 mL tetrahydrofuran and 16 mL water, stir and cool to 15-20°C, add 4.78 g (6R, 7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid and 7.2 g (Z)-2-(2-aminothiazol-4-yl)-2- acetoxyiminothioacetic acid (S-2-benzothiazole) ester, drop 2.64 mL triethylamine and 4 mL tetrahydrofuran, control the pH value of the reaction solution to be 7.5-7.8, react at 15-20°C for about 4 hours, add 40 mL dichloromethane and 20 mL water, stir for 5 minutes, stand and separate the layers, wash the organic layer with 15 mL water once, combine the water layers, cool to 10-15°C, drop a solution of 2.8 g potassium carbonate and 12 mL water, adjust the pH value to 8.0-8.5, add 2.7 g ammonium chloride, at the same time, drop the potassium carbonate aqueous solution, adjust the pH value to 8.0-8.5, continue to stir for 30 minutes, add 8.2 g potassium acetate, pH ester 9.0-9.5, slowly cool to about 0°C, filter, and wash with cold acetone. Weigh the wet product.

[0137] Second step:

[0138] Into the reaction flask, add the wet product and 240 mL purified water, warm to 30-35°C, stir and dissolve, add 1.2 g activated carbon and stir for 10 minutes, filter; at 30-35°C, drop 5% sulfuric acid, adjust the pH value to 2.4-2.5, filter, wash with purified water, and dry under reduced pressure at 40-45°C to obtain 5.8 g of product, with a yield of 75%.

[0139] 1H NMR (400 MHz, DMSO-d6) δ 13.42 (s, 1H), 11.29 (d, J = 3.1 Hz, 1H), 9.43 (t, J = 8.6 Hz, 1H), 7.12 (s, 2H), 6.67 (d, J = 8.0 Hz, 1H), 5.72 (ddd, J = 10.3, 8.1, 4.7 Hz, 1H), 5.13 (dd, J = 7.2, 4.7 Hz, 1H), 4.99 - 4.72 (m, 1H), 3.85 (dq, J = 14.2, 7.2 Hz, 1H), 3.70 (tt, J = 7.9, 4.8 Hz, 1H), 3.55 - 3.43 (m, 2H), 2.16 - 1.80 (m, 3H), 1.66 (ddq, J = 26.4, 12.1, 8.7 Hz, 1H).

[0140] LCMS (ESI(-)): 438.0.

[0141] Example 7

[0142] Synthesis of F50-019

[0143] (6R,7R)-7-((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(hydroxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0144] 1. Chemical Reaction Formula

[0145]

[0146]

[0147] 2. Operating Steps

[0148] First Step:

[0149] In a 250 mL three-necked flask, 30 mL of tetrahydrofuran was added, and the temperature was lowered to 0°C. 2.32 g of (6R, 7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid and 5.0 g of (Z)-2-(2-aminothiazol-4-yl)- 2-trityloxyimino thioacetic acid (S-2-benzothiazole) ester were added with stirring, and a mixture of 4.2 mL of triethylamine and 8 mL of tetrahydrofuran was added dropwise while controlling the pH of the reaction solution to 7.5-7.8 at 15-20°C for about 48 hours. After concentration under reduced pressure, ethyl acetate was added for dissolution, and the solution was washed with dilute hydrochloric acid aqueous solution and then with brine. After drying over anhydrous sodium sulfate, the solution was filtered and concentrated under reduced pressure. The obtained product was used in the next reaction without purification.

[0150] Second step:

[0151] To the concentrated solution of the previous step, 30 mL of dichloromethane and 8 mL of triethylsilane were added, and the temperature was lowered to 0°C with stirring. 15 mL of trifluoroacetic acid was added, and the temperature was raised to room temperature with stirring for 2 hours. After concentration under reduced pressure, the obtained residue was dispersed with petroleum ether, filtered, and dried to obtain 2.5 g of the product.

[0152] 1 H NMR (400 MHz, DMSO-d6) δ 13.42 (s, 1H), 11.29 (d, J = 3.1 Hz, 1H), 9.43 (t, J = 8.6 Hz, 1H), 7.12 (s, 2H), 5.72 (ddd, J = 10.3, 8.1, 4.7 Hz, 1H), 5.13 (dd, J = 7.2, 4.7 Hz, 1H), 4.99 – 4.72 (m, 1H), 3.85 (dq, J = 14.2, 7.2 Hz, 1H), 3.70 (tt, J = 7.9, 4.8 Hz, 1H), 3.55 – 3.43 (m, 2H), 2.16 – 1.80 (m, 3H), 1.66 (ddq, J = 26.4, 12.1, 8.7 Hz, 1H).

[0153] LCMS (ESI(-)): 439.0.

[0154] Example 8

[0155] Synthesis of F50-035

[0156] 6R,7R)-7-((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(ethoxyimino)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0157] 1. Chemical reaction formula

[0158]

[0159] 2. Operating Procedures

[0160] first step:

[0161] Add 50 mL of dichloromethane to a 250 mL three-necked flask, cool under nitrogen protection to below -20 °C, add 5.5 g of phosphorus pentachloride, and add 5.4 g of 2-ethoxyimino-2-(5-amino-1,2,4-thiadiazol-3-yl)acetic acid while stirring. Stir at -15 to -12 °C for 30 minutes, then at -5 °C for 2 hours. Add 50 mL of isopropyl ether at -5 °C, and stir at -5 to 10 °C for 30 minutes. Filter, wash with isopropyl ether, and dry to obtain 6.02 g of 2-ethoxyimino-2-(5-amino-1,2,4-thiadiazol-3-yl)acetyl chloride hydrochloride, yield 89.1%.

[0162] Step Two:

[0163] Add 2.7 g of (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid and 50 mL of dichloromethane to a 100 mL reaction flask. Add 10 g of BSA (N,O-bistrimethylsilylacetamide) while stirring. Heat to 35 °C and stir until dissolved. Cool to -20 °C under nitrogen protection and add 2.8 g of... 2-Ethoxyimino-2-(5-amino-1,2,4-thiadiazol-3-yl)acetyl chloride hydrochloride was stirred at -18 to -12°C for 25 minutes, then stirred at -12 to -3°C for 20 minutes. A solution of 4g sodium bicarbonate and 30mL water was added, and the mixture was allowed to stand to separate the aqueous layer. The pH of the aqueous layer was adjusted to 2.6 to 3.0 with 10% hydrochloric acid, and crystals precipitated. The crystals were filtered, washed with water, and dried to obtain 3.2g of the product.

[0164] 1H NMR (400 MHz, DMSO-d6) δ 9.52 (t, J = 8.6 Hz, 1H), 8.13 (s, 2H), 5.74 (td, J = 9.5, 9.0, 4.7 Hz, 1H), 5.11 (dd, J = 7.1, 4.8 Hz, 1H), 4.75 (dd, J = 8.9, 6.7 Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 3.85 (dq, J = 14.7, 7.2 Hz, 2H), 3.69 (td, J = 7.9, 5.3 Hz, 2H), 2.11 (dtd, J = 12.0, 7.3, 3.6 Hz, 1H), 1.88 (dddd, J = 27.7, 17.1, 8.5, 4.7 Hz, 2H), 1.74 - 1.56 (m, 1H), 1.25 (t, J = 7.0 Hz, 3H).

[0165] LCMS (ESI(-)): 467.0.

[0166] Example 9

[0167] Synthesis of F50-057

[0168] (6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)pent-2-enamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0169] 1. Chemical Reaction Formula

[0170]

[0171] 2. Operation Steps

[0172] First Step:

[0173] Into a 100 mL three-necked flask was added dichloromethane 40 mL and (Z)-2-(2-((tert-butoxycarbonyl)amino)thiazol-4-yl)pent-2-enoic acid 6.6 g, stirred, and cooled to -20 °C under nitrogen protection. Methylsulfonyl chloride 2.43 g and triethylamine 2.8 g were added dropwise, and the reaction was continued for 3 hours to obtain a mixed anhydride, which was ready for use.

[0174] Second Step:

[0175] (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5.0 g was dissolved in a mixed solution of methanol 30 mL and dichloromethane 10 mL, cooled to -20 °C, TEA 4.12 g was added, maintained at -20 °C, dropwise added mixed acid anhydride solution, reacted at 0-5 °C for 8-10 hours, after concentration under reduced pressure, dispersed with ethyl acetate, filtered, and dried to obtain about 9.0 g of product.

[0176] Third step:

[0177] Take 500 mg of the dried product (6R,7R)-7-((Z)-2-(2-((tert- butoxycarbonyl)amino)thiazol-4-yl)pent-2-enamido)-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid in a 50 mL single-neck flask, dissolve in dichloromethane 1 mL, add trifluoroacetic acid 1 mL under ice bath, stir for 1 hour. Dilute with dichloromethane 5 mL, solid precipitates. Filter, rinse the filter cake with dichloromethane, and dry to obtain 120 mg of white solid.

[0178] 1 H NMR (400 MHz, DMSO-d6) δ 13.38 (s, 1H), 9.32 (dd, J = 7.9, 5.3 Hz,1H), 7.87 (s, 1H), 7.42 – 6.86 (m, 1H), 6.36 (d, J = 8.7 Hz, 1H), 6.25 (td, J= 7.7, 4.0 Hz, 1H), 5.73 (ddd, J = 23.5, 7.9, 4.7 Hz, 1H), 5.17 (dd, J = 7.1,4.7 Hz, 1H), 4.95 (s, 1H), 3.85 (dq, J = 14.5, 7.3 Hz, 1H), 3.72 (td, J =8.0, 4.6 Hz, 1H), 3.59 – 3.41 (m, 2H), 2.21 (m, J = 7.5, 4.2 Hz, 2H), 2.13 –1.96 (m, 1H), 1.94 – 1.77 (m, 2H), 1.76 – 1.60 (m, 1H), 1.02 (td, J = 7.5,1.7 Hz, 3H).

[0179] LCMS (ESI(-)): 449.0.

[0180] Example 10

[0181] Synthesis of F50-093

[0182] (6R,7R)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0183] 1. Chemical Reaction Formula

[0184]

[0185] 2. Operation Steps

[0186] Into a 50 mL three-necked flask, was added (6R,7R)-7-amino-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 1.5 g, dichloromethane 10 mL and water 0.6 mL, and stirred to cool to 0-5 °C, and then added dropwise a 10% NaOH solution to completely dissolve the solid, while controlling the pH to be 8. Then, 2-(thiophen-2-yl)acetyl chloride 0.98 g was added dropwise while controlling the pH to be 8. After the addition was completed, the reaction was allowed to proceed at 0-5 °C for 2 hours while maintaining the pH at 8, and the reaction was monitored by TLC. After the reaction was completed, the mixture was allowed to stand to separate into two layers, and the dichloromethane layer was concentrated under reduced pressure to dryness. Then, 9 mL of water was added, and the mixture was filtered. The filter cake was washed twice with water to obtain a crude product.

[0187] The crude product was purified by reverse-phase flash chromatography under the following conditions: a C18 silica gel column, a mobile phase of a mixture of acetonitrile in pure water, a gradient change of 5% to 50% over 30 minutes, and a detection wavelength of 254 nm. The purified fractions were subjected to freeze-drying to obtain (6R,7R)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-7-(2-(thiophen-2-yl)acetamido)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 210 mg as a white solid.

[0188] 1H NMR (400 MHz, DMSO-d6) δ 9.11 (d, J = 8.2 Hz, 1H), 7.36 (dd, J = 5.1, 1.3 Hz, 1H), 6.97 - 6.90 (m, 2H), 5.63 (ddd, J = 15.2, 8.3, 4.7 Hz, 1H), 5.09 (dd, J = 9.0, 4.7 Hz, 1H), 4.74 (dd, J = 8.9, 6.7 Hz, 1H), 3.86 (m, J = 7.6 Hz, 1H), 3.81 - 3.73 (m, 2H), 3.70 (dt, J = 7.9, 4.1 Hz, 1H), 3.59 - 3.40 (m, 2H), 2.24 - 2.06 (m, 1H), 2.01 - 1.75 (m, 2H), 1.73 - 1.54 (m, 1H).

[0189] LCMS (ESI(-)): 393.0.

[0190] Example 11

[0191] Synthesis of F50-95

[0192] (6R,7R)-7-(2-cyanoacetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0193] 1. Chemical Reaction Formula

[0194]

[0195]

[0196] 2. Operation Steps

[0197] First Step:

[0198] Dissolve (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 2.1 g in 30 mL DMF, add 3.2 g DIEA and dissolve. Add 2-cyanoacetic acid 2.1 g, add HATU 2.1 g, stir at room temperature for 1 hour. Add water and stir with ethyl acetate, separate the layers, concentrate the organic layer under reduced pressure, purify on a silica gel column to obtain 2 g of yellow solid.

[0199] Second step:

[0200] Dissolve (6R,7R)-7-(2-cyanoacetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 1g in 6 mL of acetone, stir to dissolve. Add sodium dithionite 1.9 g portionwise, and adjust the pH to 7-8 with aqueous ammonia. Stir at room temperature for 1 hour. TLC monitor the reaction to completion. Extract with dichloromethane, and purify the aqueous phase on a reverse phase silica gel column. Lyophilize to give a light yellow powder 0.2 g.

[0201] 1 H NMR (400 MHz, DMSO-d6) δ 13.43 (s, 1H), 9.28 (dd, J = 10.1, 8.2 Hz, 1H), 5.64 (ddd, J = 15.4, 8.1, 4.7 Hz, 1H), 5.12 (dd, J = 9.6, 4.7 Hz, 1H), 4.86 (ddd, J = 87.1, 8.6, 6.7 Hz, 1H), 3.91 - 3.82 (m, 1H), 3.78 (t, J = 5.7 Hz, 2H), 3.74 - 3.68 (m, 1H), 3.62 - 3.52 (m, 1H), 3.46 (dd, J = 17.8, 11.8 Hz, 1H), 2.19 - 1.89 (m, 2H), 1.89 - 1.56 (m, 2H).

[0202] LCMS (ESI(-)): 336.0.

[0203] Example 12

[0204] Synthesis of F50-096

[0205] (6R,7R)-8-oxo-7-(2-(pyridin-4-ylthio)acetamido)-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0206] 1. Chemical Reaction Formula

[0207]

[0208]

[0209] 2. Operation steps

[0210] First step:

[0211] Take (6R, 7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 1.0 g, dissolve in 10 mL DMF, add 1.6 g DIEA, stir to dissolve, add 2-(4-pyridylthio)acetic acid 0.83 g, add HATU 1.03 g, stir at room temperature for 1 hour. TLC plate monitor the reaction of raw materials is complete. Add water and stir with ethyl acetate, stand to separate layers, the organic layer is concentrated under reduced pressure, silica gel column purification (eluent petroleum ether / ethyl acetate), get yellow solid 1 g.

[0212] Second step:

[0213] Take (6R, 7R)-8-oxo-7-(2-(4-pyridylthio)acetamide)-3-((S)-tetrahydrofuran-2-yl)-5- thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrophenyl ester 1 g, dissolve in 6 mL acetone, stir to dissolve. Add sodium dithionite in batches, and adjust the pH to 7-8 with ammonia water. Stir at room temperature for 1 hour, TLC monitor the reaction of raw materials is complete. Extracted with dichloromethane, the aqueous phase was purified by reverse phase column, freeze-dried to get light yellow powder 0.2 g.

[0214] 1 H NMR (400 MHz, DMSO-d6) δ 13.41 (s, 1H), 9.28 (t, J = 8.3 Hz, 1H),8.63 – 8.23 (m, 2H), 7.30 (dt, J = 4.6, 1.3 Hz, 2H), 5.65 (ddd, J = 15.4,8.3, 4.7 Hz, 1H), 5.10 (dd, J = 9.5, 4.7 Hz, 1H), 4.85 (ddd, J = 82.8, 8.6,6.7 Hz, 1H), 4.00 – 3.78 (m, 3H), 3.72 (tdd, J = 7.8, 5.4, 1.5 Hz, 1H), 3.51(qd, J = 17.5, 10.7 Hz, 2H), 2.21 – 1.89 (m, 2H), 1.89 – 1.58 (m, 2H).

[0215] LCMS (ESI(-)): 420.0.

[0216] Example 13

[0217] Synthesis of F50-099

[0218] (6R,7R)-7-((Z)-2-(furan-2-yl)-2-(methoxyimino)acetamido)-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0219] 1. Chemical Reaction Formula

[0220]

[0221] 2. Operation Steps

[0222] First Step:

[0223] Into a 100 mL three-necked flask, add dry dichloromethane 36.5 mL, and then add phosphorus pentachloride 5.16 g under nitrogen atmosphere, and heat to 30 °C, and stir until most of the solid dissolves, and then cool to -25 °C, and drop in dimethyl carbonate 7.5 mL, and control the temperature to be no higher than -12 °C. After the drop is complete, stir at -25 °C for 30 minutes, and then add (Z)-2-(furan-2-yl)-2-(methoxyimino)acetamide 4.13 g, and incubate and stir at -15 ~ -12 °C for 1 hour, and then cool to -20 °C, and stir for 30 minutes. Measure 2 °C water 18 mL, and slowly drop into the reaction system, and control the temperature to be 0 ~ 1 °C. After the drop is complete, incubate and stir at 0 ~ 1 °C for 10 minutes, and then stand to separate, and wash the organic phase with 2 °C water 18 mL twice. Cool the organic phase to -5 °C for standby, and obtain a (Z)-2-(furan-2-yl)-2-(methoxyimino)acetyl chloride solution.

[0224] Second Step:

[0225] Into a 250 mL three-necked flask was placed (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5.0 g, water 21.5 mL, and the solution was cooled to 0-2 °C. A 10% NaOH solution was added slowly until the solid was completely dissolved and the pH was 8. The temperature was controlled to be no higher than 5 °C. A solution of (Z)-2-(furan-2-yl)-2-(methoxyimino)acetyl chloride was added dropwise to the reaction system. The temperature was controlled to be -2-6 °C and the pH was 6.5-7.0. After the addition was completed, the reaction was allowed to proceed for 2.5 hours. The reaction was monitored by HPLC. After the reaction was completed, the solution was allowed to stand and the organic phase was washed once with 5 mL of cold water at 2 °C. The water phases were combined and the pH was adjusted to 2 with 1 M HCl solution. A large amount of solid was precipitated and was filtered. The crude product was obtained as 7.9 g.

[0226] Step 3:

[0227] The crude product 1.0 g was purified by reverse phase flash chromatography using the following conditions: a C18 silica gel column; a mobile phase of acetonitrile in water with a gradient of 5% to 50% over 30 minutes; and a detection wavelength of 254 nm. The purified fractions were freeze-dried to obtain (6R,7R)-7-((Z)-2-(furan-2-yl)-2-(methoxyimino)acetylamino)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid as a white solid, 400 mg.

[0228] 1 H NMR (400 MHz, DMSO-d6) δ 9.75 (dd, J = 8.0, 5.9 Hz, 1H), 7.83 (d, J= 1.7 Hz, 1H), 6.74 – 6.61 (m, 2H), 5.71 (ddd, J = 15.3, 7.9, 4.7 Hz, 1H),5.15 (dd, J = 7.5, 4.7 Hz, 1H), 5.04 – 4.74 (m, 1H), 3.89 (d, J = 1.8 Hz,3H), 3.84 (dt, J = 14.3, 7.2 Hz, 1H), 3.70 (tt, J = 7.9, 4.2 Hz, 1H), 3.58 –3.52 (m, 1H), 3.44 – 3.38 (m, 1H), 2.16 – 2.06 (m, 1H), 2.00 – 1.78 (m, 2H),1.65 (ddq, J = 26.4, 12.1, 8.6 Hz, 1H).

[0229] LCMS (ESI(-)): 420.0.

[0230] Example 14

[0231] Synthesis of F50-100

[0232] (6R,7R)-7-((R)-2-(formyloxy)-2-phenylacetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0233] 1. Chemical Reaction Formula

[0234]

[0235]

[0236] 2. Operation Steps

[0237] First Step:

[0238] Dissolve (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 3g in 50 mL DCM, add 2.25g TEA. Stir and cool to below -5°C, add (R)-2-chloro-2-oxo-1- phenylethyl formate 2.2g, and stir at the same temperature for 30 minutes. After TLC monitoring shows that the reaction is complete, add water, separate the organic layer, dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify on a silica gel column (petroleum ether / ethyl acetate) to obtain 3.5g of a yellow solid.

[0239] Second Step:

[0240] Dissolve (6R,7R)-7-((R)-2-(formyloxy)-2-phenylacetamido)-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4- nitrobenzyl ester 1.0g in 6 mL acetone, and stir until clear. Add water to the reaction solution, which becomes light yellow and turbid. Add sodium hydrosulfite 1.38g in portions, and adjust the pH to 7-8 with ammonia water. Stir at room temperature for 1 hour. TLC monitoring shows that the starting material is completely reacted. Extract with dichloromethane, and retain the aqueous phase. Adjust the pH of the aqueous phase to about 2 with hydrochloric acid, and filter to obtain a solid. Purify the wet product by reverse phase (pure water system), and freeze-dry to obtain 0.2g of a light white solid.

[0241] 1 H NMR (400 MHz, DMSO-d6) δ 13.46 (s, 1H), 9.38 (dd, J = 10.4, 8.2 Hz,1H), 8.41 – 8.27 (m, 1H), 7.50 (h, J = 3.4 Hz, 2H), 7.39 (qd, J = 5.2, 4.2,1.9 Hz, 3H), 6.13 – 6.03 (m, 1H), 5.69 – 5.51 (m, 1H), 4.99 (dd, J = 7.9, 4.7Hz, 1H), 4.77 – 4.66 (m, 1H), 3.84 (tt, J = 14.3, 7.4 Hz, 1H), 3.73 – 3.64(m, 1H), 3.55 – 3.38 (m, 2H), 2.14 – 1.79 (m, 3H), 1.71 – 1.55 (m, 1H).

[0242] LCMS (ESI(-)): 431.0.

[0243] Example 15

[0244] Synthesis of F50-101

[0245] (6R,7R)-7-(2-(2-aminothiazol-4-yl)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0246] 1. Chemical Reaction Formula

[0247]

[0248]

[0249] 2. Operating Steps

[0250] First Step:

[0251] Take (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 3.0 g dissolved in 50 mL DMF, after adding 4.8 g DIEA, the solution is clear. Add 2-(2- aminothiazol-4-yl)acetic acid 1.4 g and 3.4 g HATU, stir at room temperature for 1 hour, TLC monitor the complete reaction of raw materials, add water, stir, extract with ethyl acetate, concentrate the organic layer under reduced pressure to obtain white solid 4 g.

[0252] Second step:

[0253] Take (6R,7R)-7-(2-(2-aminothiazol-4-yl)acetamido)-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4- nitrobenzyl ester 1.0 g dissolved in acetone 6 mL, add 1.44 g sodium hydrosulfite in batches, and adjust the pH to 7-8 with ammonia water. Stir at room temperature for 1 hour. TLC monitor the complete reaction of raw materials. Filter out a small amount of solid residue, wash the filtrate with dichloromethane, and purify the aqueous phase on a reverse phase silica gel column (pure water system), then freeze dry to obtain a light white solid 0.2 g.

[0254] 1 H NMR (400 MHz, DMSO-d6) δ 8.87 (t, J = 7.8 Hz, 1H), 6.90 (d, J = 3.9Hz, 2H), 6.25 (d, J = 2.5 Hz, 1H), 5.63 (ddd, J = 13.1, 8.5, 4.7 Hz, 1H),5.08 (dd, J = 9.0, 4.8 Hz, 1H), 4.76 (dd, J = 8.9, 6.7 Hz, 1H), 3.86 (m, J =7.1 Hz, 1H), 3.70 (td, J = 7.9, 5.3 Hz, 1H), 3.60 – 3.48 (m, 1H), 3.43 (d, J= 18.0 Hz, 1H), 3.38 (t, J = 2.9 Hz, 2H), 2.19 – 2.06 (m, 1H), 1.90 (ddtd, J= 35.2, 15.3, 7.4, 3.8 Hz, 2H), 1.66 (ddq, J = 30.0, 12.2, 8.7 Hz, 1H).

[0255] LCMS (ESI(-)): 409.0.

[0256] Example 16

[0257] Synthesis of F50-103

[0258] (6R,7R)-7-((R)-2-(4-ethyl-2,3-dioxopiperazine-1-carboxamido)-2-(4- hydroxyphenyl)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0259] 1. Chemical Reaction Formula

[0260]

[0261]

[0262] 2. Operation Steps

[0263] First Step:

[0264] Dissolve (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 2.0 g in 35 mL DMF, add 3.2 g DIEA, stir to dissolve. Add (R)-2-(4-ethyl-2,3-dioxopiperazine-1- carboxamido)-2-(4-hydroxyphenyl)acetic acid 2.0 g and 2.25 g HATU, stir at room temperature for 1 hour. TLC monitor the reaction complete, stop the reaction. Add water and ethyl acetate, stand to separate the organic layer, the organic layer is concentrated under reduced pressure, the residue is purified by silica gel column normal phase (petroleum ether / ethyl acetate) system, get light yellow solid 2.8 g.

[0265] Second Step:

[0266] Dissolve (6R,7R)-7-((R)-2-(4-ethyl-2,3-dioxopiperazine-1-carboxamido)-2-(4- hydroxyphenyl)acetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 1.0 g in 6 mL acetone, stir to dissolve. Add water to the reaction solution, add 1.9 g sodium hydrosulfite in batches, and adjust the pH to 7-8 with ammonia water. Stir at room temperature for 1 hour, TLC monitor the reaction complete. Wash with dichloromethane, adjust the pH of the aqueous phase to about 2 with hydrochloric acid, solid precipitates, suction filtration, wet product is purified by reverse phase column chromatography (pure water system), freeze-dried to get light white solid 0.15 g.

[0267] 1 H NMR (400 MHz, DMSO-d6) δ 9.77 – 9.62 (m, 2H), 9.25 (dd, J = 24.4,8.5 Hz, 1H), 7.21 (ddd, J = 9.6, 4.8, 2.4 Hz, 2H), 6.78 – 6.65 (m, 2H), 5.48(dd, J = 8.1, 5.7 Hz, 2H), 4.91 – 4.82 (m, 1H), 3.98 – 3.84 (m, 2H), 3.84 –3.73 (m, 1H), 3.70 – 3.51 (m, 3H), 3.44 – 3.37 (m, 2H), 3.31 – 3.08 (m, 2H),2.10 (ddd, J = 24.1, 11.9, 6.1 Hz, 1H), 1.90 – 1.70 (m, 2H), 1.58 – 1.42 (m,1H), 1.08 (t, J = 7.2 Hz, 3H).

[0268] LCMS (ESI(-)):586.0.

[0269] Example 17

[0270] Synthesis of F50-105

[0271] (6R,7R)-7-(2,6-dimethoxybenzamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0272] 1. Chemical Reaction Formula

[0273]

[0274]

[0275] 2. Operating Steps

[0276] First Step:

[0277] Into a 250 mL flask was placed 2,6-dimethoxybenzoic acid 1.62 g, dichloromethane 100 mL, and the flask was purged with N2 three times. The flask was stirred and cooled to -20 °C under N2 atmosphere. Methylsulfonyl chloride 1.02 g and triethylamine 1.14 g were added, and the mixture was stirred at -20 °C for 4 h to give a mixed anhydride solution, which was used immediately.

[0278] Into another 250 mL flask was placed (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thioxo-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 3.26 g and dichloromethane 30 mL, and the flask was cooled to -20 °C. Triethylamine 1.65 g was added, and the mixture was stirred for 15 min. The mixed anhydride solution was added, and the mixture was stirred at -15 to -10 °C. The reaction was monitored by TLC (PE:EA = 1:3) until (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thioxo-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester was completely consumed. Water 130 mL was added, and the mixture was stirred for 15 min. The mixture was allowed to stand, and the dichloromethane layer was collected. The organic phase was concentrated under reduced pressure to give a crude product 3.30 g, which was purified by silica gel column chromatography (EA:PE = 0:100 to EA:PE = 75:25) to give a white solid 2.66 g.

[0279] Second Step:

[0280] Into a 50 mL flask was placed (6R,7R)-7-(2,6-dimethoxybenzamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5- thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 1.0 g, acetone 7 mL, and the mixture was stirred until the solution was clear. Water 5 mL was added, and the pH was adjusted to 8 with ammonia water. Sodium dithionite 1.29 g was added, and water 2 mL was added. The mixture was stirred at room temperature for 1.5 h while maintaining the pH at 8 with ammonia water. The reaction was monitored by TLC.

[0281] The reaction mixture was concentrated under reduced pressure until the acetone was completely removed. The pH was adjusted to 2 with 1 M HCl solution, and a large amount of solid precipitated. The mixture was stirred for 10 min, and the solid was collected by filtration to give a yellow solid crude product 0.77 g.

[0282] The crude product was purified by reverse-phase flash chromatography using the following conditions: a C18 silica gel column; a mobile phase of acetonitrile in pure water with a gradient of 5% to 50% over 30 min; and detection at 254 nm. The purified fractions were freeze-dried to give (6R,7R)-7-(2,6-dimethoxyanilino)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thioxo-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 540 mg as a white solid.

[0283] 1 H NMR (400 MHz, DMSO-d6) δ 9.09 (dd, J = 11.7, 8.7 Hz, 1H), 7.39 –7.21 (m, 1H), 6.67 (d, J = 3.1 Hz, 1H), 6.65 (d, J = 3.0 Hz, 1H), 5.80 (td, J= 8.9, 4.8 Hz, 1H), 5.13 (dd, J = 6.7, 4.8 Hz, 1H), 4.81 – 4.72 (m, 1H), 3.87(q, J = 7.4 Hz, 1H), 3.73 (s, 6H), 3.70 (dd, J = 7.9, 5.3 Hz, 1H), 3.53 (d, J= 18.1 Hz, 1H), 3.48 – 3.39 (m, 1H), 2.11 (td, J = 11.9, 7.3 Hz, 1H), 1.89(dtdd, J = 25.0, 20.1, 10.0, 5.4 Hz, 2H), 1.75 – 1.58 (m, 1H).

[0284] LCMS (ESI(-)):432.9.

[0285] Example 18

[0286] Synthesis of F50-106

[0287] (6R,7R)-7-(5-methyl-3-phenylisoxazole-4-carboxamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0288] 1. Chemical Reaction Formula

[0289]

[0290] 2. Procedure

[0291] In a 50 mL flask, add (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5- thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 3.0 g, water 15 mL, stir and cool to 0-5 °C, add 10% NaOH solution dropwise, make the solid completely dissolved, control pH = 8. Slowly add 5-methyl-3-phenylisoxazole-4-carbonyl chloride 2.71 g, and control pH = 8. After dropwise addition is complete, react at 0-5 °C for 2 hours, maintain pH = 8, TLC monitor the reaction. After the reaction is complete, adjust the pH of the reaction solution to 2 with 1 M HCl solution, the solid precipitates, filter, and wash the filter cake twice with 2 °C cold water 5 mL, dry, to obtain 3.55 g of crude product.

[0292] Take 1.0 g of crude product, purify by reverse phase flash chromatography, the specific conditions are as follows: the chromatographic column is C18 silica gel; the mobile phase is a mixture of acetonitrile in pure water, with a gradient of 5% to 50% over 30 minutes; the detection wavelength is 254 nm. The purified fractions are freeze-dried to obtain (6R,7R)-7-(5-methyl-3-phenylisoxazole-4-carboxamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 350 mg, white solid.

[0293] 1 H NMR (400 MHz, DMSO-d6) δ 9.50 (d, J = 8.0 Hz, 1H), 7.72 – 7.63 (m,2H), 7.54 – 7.45 (m, 3H), 5.78 (ddd, J = 23.1, 8.0, 4.7 Hz, 1H), 5.20 (dd, J= 8.8, 4.7 Hz, 1H), 4.88 (ddd, J = 93.1, 8.7, 6.7 Hz, 1H), 3.92 – 3.80 (m,1H), 3.71 (td, J = 7.9, 5.2 Hz, 1H), 3.61 – 3.45 (m, 2H), 2.57 (d, J = 4.3Hz, 3H), 2.16 – 2.06 (m, 1H), 2.01 – 1.78 (m, 2H), 1.76 – 1.56 (m, 1H).

[0294] LCMS (ESI(-)):454.0

[0295] Example 19

[0296] Synthesis of F50-107

[0297] (6R,7R)-7-(3-(2-chlorophenyl)-5-methylisoxazole-4-carboxamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid

[0298] 1. Chemical Reaction Formula

[0299]

[0300] 2. Operation Steps

[0301] Into a 50 mL flask was added (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 3.0 g and water 15 mL, and stirred to cool to 0-5 °C. A 10% NaOH solution was added dropwise to dissolve the solid completely, and the pH was controlled at 8. 3-(2-chlorophenyl)-5-methylisoxazole-4-carboxylic acid chloride 3.13 g was added slowly while controlling the pH at 8. After the dropwise addition was completed, the reaction was allowed to proceed at 0-5 °C for 2 hours while maintaining the pH at 8, and the reaction was monitored by TLC. After the reaction was completed, the reaction solution was adjusted to pH 2 with a 1 M HC1 solution, and a solid was precipitated. The solid was filtered, and the filter cake was washed twice with 2 °C cold water 5 mL, and dried to obtain a crude product 4.25 g.

[0302] The crude product 1.0 g was purified by reverse-phase flash chromatography using the following conditions: a C18 silica gel column, a mobile phase of acetonitrile in pure water with a gradient of 5% to 50% over 30 minutes, and a detection wavelength of 254 nm. The purified fractions were freeze-dried to obtain (6R,7R)-7-(3-(2-chlorophenyl)-5-methylisoxazole-4-carboxamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 100 mg as a white solid.

[0303] 1H NMR (400 MHz, DMSO-d6) δ 9.17 - 9.09 (m, 1H), 7.62 - 7.39 (m, 4H), 5.71 (s, 1H), 5.08 (dd, J = 11.2, 4.8 Hz, 1H), 4.77 (s, 1H), 3.84 (dq, J = 13.6, 7.2 Hz, 1H), 3.72 - 3.64 (m, 1H), 3.50 (d, J = 17.7 Hz, 1H), 3.41 (s, 1H), 2.64 (d, J = 4.4 Hz, 3H), 2.11 (s, 1H), 1.97 - 1.78 (m, 2H), 1.70 - 1.56 (m, 1H).

[0304] LCMS (ESI(-)): 488.0, 489.9.

[0305] Example 20

[0306] Synthesis of F50-108

[0307] (6R,7R)-7-(3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxamido)-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0308] 1. Chemical Reaction Formula

[0309]

[0310] 2. Procedure

[0311] In a 50 mL flask, (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 3.0 g and water 9 mL were added, and the temperature was reduced to 0-5 °C under stirring. Ammonia water was added dropwise until the solid was completely dissolved, and the pH was controlled at 8. 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxylic chloride 3.13 g was diluted with 15 mL of acetone, and the resulting solution was added dropwise to the reaction system while controlling the pH at 8 with ammonia water. After the addition was completed, the reaction was carried out at 0-5 °C for 2 hours while maintaining the pH at 8, and the reaction was monitored by TLC. After the reaction was completed, the reaction solution was adjusted to pH 2 with 1 M HCl solution, and a solid was precipitated. The solid was filtered, washed with 2 °C cold water 5 mL twice, and dried to obtain a crude product 4.11 g.

[0312] The crude product 1.0 g was purified by reverse flash chromatography with the following conditions: C18 silica gel as the column material; the mobile phase was a mixture of acetonitrile in pure water, with a gradient of 5% to 50% in 30 minutes; and the detection wavelength was 254 nm. The purified fraction was freeze-dried to obtain (6R,7R)-7-(3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxamido)-8-oxo-3-((S)- tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 120 mg as a white solid.

[0313] 1 H NMR (400 MHz, DMSO-d6) δ 9.16 (t, J = 8.1 Hz, 1H), 7.68 – 7.49 (m,3H), 5.67 (ddd, J = 19.2, 8.2, 4.7 Hz, 1H), 5.08 (dd, J = 7.9, 4.7 Hz, 1H),4.76 (dd, J = 8.9, 6.7 Hz, 1H), 3.87 (q, J = 7.3 Hz, 1H), 3.70 (td, J = 7.8,5.2 Hz, 1H), 3.50 (dd, J = 17.3, 14.8 Hz, 2H), 2.71 (d, J = 6.0 Hz, 3H), 2.11(ddd, J = 12.6, 9.9, 5.9 Hz, 1H), 2.00 – 1.78 (m, 2H), 1.65 (ddq, J = 26.5,12.1, 8.7 Hz, 1H).

[0314] LCMS (ESI(-)): 522.0, 524.0.

[0315] Example 21

[0316] Synthesis of F50-109

[0317] (6R,7R)-7-((R)-2-(4-ethyl-2,3-dioxopiperazine-1-carboxamido)-2-phenylacetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0318] 1. Chemical Reaction Formula

[0319]

[0320]

[0321] 2. Operation Steps

[0322] First Step:

[0323] Dissolve (6R,7R)-7-amino-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 3.0 g in 50 mL DMF, add 4.79 g DIEA and dissolve. Add (R)-2-(4-ethyl-2,3-dioxopiperazine-1- carboxamide)-2-phenylacetic acid, adjust pH with DIEA to make the reaction alkaline. Add 3.38 g HATU, stir at room temperature for 1 hour. Monitor the reaction completion by TLC, stop the reaction. Add water and ethyl acetate, separate the layers, concentrate the organic layer under reduced pressure, purify the residue on a silica gel column to obtain 4.3 g of a light yellow solid.

[0324] Second Step:

[0325] To (6R,7R)-7-((R)-2-(4-ethyl-2,3-dioxopiperazine-1-carboxamido)-2- phenylacetamido)-8-oxo-3-((S)-tetrahydrofuran-2-yl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-4-nitrobenzyl ester 1.0 g was dissolved in 6 mL of acetone, the solution was stirred, 6.0 mL of water was added to the reaction solution, which was light yellow turbid. 1.11 g of hyposulfite sodium was added in batches, and the pH was adjusted to 7-8 with ammonia water. Stir at room temperature for 1 hour. TLC monitoring of the reaction was complete. Dichloromethane was added to wash, the aqueous phase was separated, the pH was adjusted to about 2 with hydrochloric acid, the solid was precipitated, and the filter cake was washed with water. The wet product was purified by reverse phase silica gel column chromatography (pure water system), and then freeze-dried to obtain 0.2 g of light white solid.

[0326] 1 H NMR (400 MHz, DMSO-d6) δ 9.84 (dd, J = 7.3, 3.7 Hz, 1H), 9.38 (dd, J = 8.5, 4.1 Hz, 1H), 7.49 – 7.39 (m, 2H), 7.39 – 7.27 (m, 3H), 5.65 (dd, J = 12.4, 7.3 Hz, 1H), 5.53 (ddd, J = 22.3, 8.6, 4.7 Hz, 1H), 4.97 – 4.84 (m, 2H), 3.90 (td, J = 5.3, 2.7 Hz, 2H), 3.79 (m, J = 7.2 Hz, 1H), 3.68 – 3.59 (m, 1H), 3.56 (m, J = 4.4 Hz, 2H), 3.41 (d, J = 7.0 Hz, 2H), 3.28 – 3.14 (m, 2H), 2.09 (q, J = 6.5 Hz, 1H), 1.90 – 1.73 (m, 2H), 1.53 (dq, J = 12.1, 9.0 Hz, 1H), 1.08 (t, J = 7.1 Hz, 3H).

[0327] LCMS (ESI(-)): 570.0.

[0328] Example 22

[0329] Biological activity evaluation

[0330] Cefvitine belongs to the third-generation cephalosporin class of antibiotics. Its antibacterial activity is comparable to other cephalosporins, and it is mainly characterized by its long-lasting effect. Previous studies have found cross-resistance between cefvitine and other third-generation cephalosporins, and it exhibits resistance to strains producing extended-spectrum β-lactamases. To screen for cefvitine derivatives with superior antibacterial activity, in vitro drug susceptibility testing was performed on 21 compounds. The results are reported below.

[0331] 1 strain tested

[0332] 1) Escherichia coli resistant to cefavirenz and producing extended-spectrum β-lactamase;

[0333] 2) Carbapenemase-producing Escherichia coli resistant to cefavirenz;

[0334] 3) Standard strain of Escherichia coli;

[0335] 4) Pseudomonas intermedius that are sensitive to and resistant to ceftriaxone;

[0336] 5) Enzyme-producing Staphylococcus aureus;

[0337] 6) Standard strain of Staphylococcus aureus;

[0338] 7) Cefavirenz-resistant Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0339] 2 Experimental Methods

[0340] The minimum inhibitory concentrations (MICs) of 21 tested compounds, cefotaxime, cefotaxime, and ceftazidime against the tested bacterial strains were determined using the agar dilution method, with drug concentrations ranging from 0.0625 mg / L to 128 mg / L.

[0341] 3. Experimental Results

[0342] 3.1 Distribution of MIC values ​​for Escherichia coli

[0343] The statistical results of the distribution of MIC values ​​of cefotaxime, cefotaxime, and ceftazidime against Escherichia coli are as follows: Figure 1 As shown. By Figure 1 It can be seen that the MIC value of ceftazidime (CAZ) ranges from 0.125 to >128 μg / mL, and the number of strains with MIC values ​​in the range of 0.125 to 4 μg / mL accounts for 61.54% of the total number of strains; the MIC value of cefotaxime (CTX) ranges from 0.0625 to >128 μg / mL, and the number of strains with MIC values ​​in the range of 0.0625 to 4 μg / mL accounts for only 15.38% of the total number of strains; the MIC value of cefotaxime (INN) ranges from 0.5 to >128 μg / mL, and the number of strains with MIC values ​​in the range of 0.5 to 2 μg / mL accounts for only 7.69% of the total number of strains.

[0344] The statistical distribution of the MIC values ​​of 21 tested compounds against Escherichia coli is as follows: Figure 2 and Figure 3 As shown. By Figure 2 It can be seen that the six tested compounds have good antibacterial effects against Escherichia coli. The MIC values ​​are distributed as follows: F50-001 and F50-004 have MIC values ​​ranging from 0.5 to >128 μg / mL; F50-003 has MIC values ​​ranging from 16 to >128 μg / mL; F50-013 has MIC values ​​ranging from 0.0625 to >128 μg / mL; and F50-035 has MIC values ​​ranging from 4 to >128 μg / mL.

[0345] Among the tested compounds, F50-001 had the highest proportion of strains with a MIC value ≤ 2 μg / mL, with 6 strains accounting for 23.08% of the total. This was followed by F50-004 and F50-013, accounting for 7.69% of the total. F50-003 and F50-035 had none. Of these six tested compounds, F50-001 showed the best antibacterial effect against cefotaxime-resistant Escherichia coli, even surpassing cefotaxime and cefotaxime.

[0346] Depend on Figure 3 It can be seen that the proportion of strains with MIC values ​​>128 μg / mL for the remaining 15 tested compounds is relatively high, all exceeding 76%, indicating that these 15 compounds have poor antibacterial effects against cefvitamin-resistant Escherichia coli.

[0347] 3.2 Distribution of MIC values ​​for Staphylococcus aureus

[0348] The statistical results of the distribution of MIC values ​​of cefotaxime, cefotaxime, and ceftazidime against Staphylococcus aureus (Pseudomonas intermedius and Staphylococcus aureus) are as follows: Figure 4 As shown. By Figure 4 It can be seen that the MIC value of cefotaxime (INN) ranges from 0.25 to 32 μg / mL, and the number of strains with MIC values ​​< 8 μg / mL accounts for 80.00% of the total number of strains, indicating good antibacterial effect; the MIC value of cefotaxime (CTX) ranges from 0.25 to 32 μg / mL, and the number of strains with MIC values ​​< 8 μg / mL accounts for 73.33% of the total number of strains, indicating good antibacterial effect; the MIC value of ceftazidime (CAZ) ranges from 4 to 128 μg / mL, and the number of strains with MIC values ​​< 8 μg / mL accounts for 26.67% of the total number of strains.

[0349] The statistical distribution of the MIC values ​​of 21 tested compounds against Staphylococcus aureus is as follows: Figure 5 and Figure 6 As shown. By Figure 5The MIC values ​​of the eight tested compounds against Staphylococcus aureus were distributed as follows: F50-93 had an MIC value range of 0.0625–2 μg / mL, showing better antibacterial effects than cefotaxime, cefotaxime, and ceftazidime; F50-101 had an MIC value range of 0.0625–8 μg / mL, with 86.67% of the strains having MIC values ​​< 8 μg / mL, indicating better antibacterial effects than cefotaxime; F50-095 and F50-096 both had MIC values ​​range of 0.0625–32 μg / mL, with 93.33% of the strains having MIC values ​​< 8 μg / mL, indicating even better antibacterial effects than cefotaxime; F50-109 had an MIC value range of 0.125–32 μg / mL, with MIC values ​​< 8 μg / mL. The number of strains with MIC values ​​of 0.0625–8 μg / mL accounted for 93.33% of the total number of strains; the MIC value distribution range of F50-101 was 0.0625–8 μg / mL, with strains having MIC values ​​<8 μg / mL accounting for 86.67% of the total number of strains, and its antibacterial effect was better than that of cefotaxime; the MIC value distribution range of F50-013 was 0.0625–128 μg / mL, with strains having MIC values ​​<8 μg / mL accounting for 86.67% of the total number of strains; the MIC value distribution range of F50-100 was 0.125–64 μg / mL, with strains having MIC values ​​<8 μg / mL accounting for 86.67% of the total number of strains; and the MIC value distribution range of F50-103 was 0.25–64 μg / mL, with strains having MIC values ​​<8 μg / mL accounting for 80.00% of the total number of strains.

[0350] Overall, all eight compounds showed antibacterial effects against Staphylococcus aureus, with F50-93 exhibiting the best antibacterial effect.

[0351] Depend on Figure 6 It can be seen that the MIC values ​​of Staphylococcus against the other 13 tested compounds ranged from 0.125 to >128 μg / mL. The compounds with a concentrated distribution range of 0.125 to 4 μg / mL were: F50-099, F50-107, F50-106, F50-003, and F50-108. These 6 compounds had better antibacterial effects than ceftazidime.

[0352] 3.3 Distribution of MIC values ​​for Klebsiella pneumoniae and Pseudomonas aeruginosa

[0353] Almost all of the compounds had MICs >128 μg / mL against both bacteria; only F50-93 and F50-107 had MICs of 0.0625 μg / mL and 0.5 μg / mL against Pseudomonas aeruginosa, respectively.

[0354] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary of the principles and application of the present application. Numerous modifications and adaptions can be effected without departing from the spirit and scope of the present application, which is not limited to the exact construction and arrangement described. It is intended, therefore, to cover all modifications and adaptions that fall within the scope of the claims and their equivalents.

Claims

1. A cephalosporin derivative containing tetrahydrofuran at the C-3 position, characterized in that, The structural formula is: ， The R group is: 、 、 、 、 、 、 、 。 2. The use of the C-3 position tetrahydrofuran cephalosporin derivative of claim 1 in the preparation of a medicament for treating diseases in humans and animals caused by bacteria.

3. The application according to claim 2, characterized in that: The bacteria are either Gram-negative or Gram-positive.

4. The application according to claim 3, characterized in that: The bacteria mentioned are Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus.

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