Cephalosporin derivative and application thereof in NDM-1 inhibitor

Abstract

The invention belongs to the field of pharmacy, and relates to a cephalosporin derivative and application of the cephalosporin derivative in an NDM-1 inhibitor. The structure of the cephalosporin derivative is as shown in a general formula I: # imgabs0 # (I), a novel cephalosporin derivative containing Se atoms is prepared on the basis of an Ebseen open-loop structure and a cephalosporin structure, a beta-lactam ring of cephalosporin is subjected to NDM-1 hydrolysis ring opening, an Ebseen fragment containing Se atoms is released, and the Ebseen fragment and sulfydryl of cysteine in an NDM-1 active area form a Se-S covalent bond, so that the activity of inhibiting NDM-1 is improved, the targeting property of NDM-1 is enhanced, and the novel cephalosporin derivative containing Se atoms is prepared. Growth of drug-resistant bacteria is effectively inhibited, and the compound can be applied to bacterial infectious diseases caused by NDM-1 drug resistance.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, and relates to a class of compounds for treating New Delhi metallo-β-lactamase-1 (NDM-1) resistance and their medical uses, and specifically to a class of selenium-containing cephalosporin derivatives and their use in preparing NDM-1 inhibitors to treat bacterial infections caused by NDM-1 resistance. Background Art

[0002] The emergence of antibiotic resistance poses a serious threat to global public health. According to a survey conducted by Mohsen et al., approximately 4.95 million deaths worldwide in 2019 were related to drug-resistant bacteria, of which 1.27 million deaths were directly caused by drug-resistant bacteria (Murray CJL, Ikuta KS, Sharara F, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis [J]. Thelancet, 2022, 399(10325): 629-655.).

[0003] NDM-1 is a long-chain monomer protein composed of 269 amino acids with a relative molecular mass of about 27.5 kDa. With the signal peptide on its N-terminus, NDM-1 can pass through the intracellular membrane from the bacteria to the periplasm. The active region of NDM-1 interacts with penicillin and cephalosporin antibiotics, breaking the CN bond in the β-lactam ring structure of the antibiotics and releasing the hydrolysis products through ring opening, leading to the generation of drug resistance (Linciano P, Cendron L, Gianquinto E, et al. Ten Years with New Delhi Metallo-β-lactamase-1 (NDM-1): From Structural Insights to Inhibitor Design. ACS Infect. Dis. 2019, 1, 9-34.). NDM-1 can hydrolyze all β-lactam antibiotics except aztreonam. Although there are many serine β-lactamase inhibitors in clinical practice, such as avibactam and sulbactam, they have no obvious inhibitory activity against metallo-β-lactamase NDM-1 (Marrs ECL, Day KM, Perry JD. Invitro activity of mecillinam against Enterobacteriaceae with NDM-1carbapenemase. J. Antimicrob. Chemother. 2014, 10, 2873-2875.). Due to the rapid spread of NDM-1 around the world, it is urgent to find NDM-1 inhibitors with good activity and strong specificity.

[0004] Ebselen (Ki = 0.38 μM) is an effective covalent inhibitor of NDM-1. Ebselen breaks the unstable Se-N bond in the active site and forms a Se-S covalent bond with the sulfhydryl group in the cysteine ​​at position 208, thereby inhibiting the activity of NDM-1 (Chiou J, Wan S, Chan KF, et al. Ebselen as a Potent Covalent Inhibitor of New Delhi Metallo-β-Lactamase (NDM-1). Chem. Commun. 2015, 46, 9543-9546.). In vitro studies have shown that the combination of meropenem (MEM) and Ebselen can reduce the MIC value of MEM in the engineered strain of Escherichia coli with high expression of NDM-1, up to 512 times, but no significant inhibitory effect was achieved in clinical NDM-1 resistant isolates. Because of its unstable Se-N structure, Ebselen easily forms Se-S covalent bonds with cysteine, glutathione, and thiol groups in thiol proteins, which makes it have multiple biological activities such as anti-inflammatory, antiviral, and antibacterial. However, Ebselen also forms Se-S covalent bonds with thiol-containing proteins in the body, resulting in off-target effects and being unable to effectively reach the active area of ​​NDM-1 to exert its effect.

[0005] Another cephalosporin derivative, NDM-1 covalent inhibitor cefaclor (Cefaclor, Ki = 2.4 mM), also has relatively weak inhibitory activity against NDM-1. In addition, cephalosporin drugs can be hydrolyzed with NDM-1. After co-incubation of cefuroxime with NDM-1, the β-lactam ring of cefuroxime is attacked by the nucleophilic OH- ion, breaking the CN bond in the β-lactam to form a carboxyl group; at the same time, the lone pair of electrons on the N atom is transferred to the C3 position, resulting in the cleavage of the CO bond at the C17 position to release the methylamine fragment (Feng H, Ding J, Zhu D, et al. Structural and Mechanistic Insights into NDM-1 Catalyzed Hydrolysis of Cephalosporins. J. Am. Chem. Soc. 2014, 42, 14694-14697.). Therefore, antibiotics with β-lactam rings, such as penicillins, cephalosporins, cephamycins, and carbapenems, can be hydrolyzed by NDM-1 to open the ring, causing the key active structure to be destroyed, thereby losing antibacterial activity and are not suitable for the treatment of infections caused by NDM-1-resistant bacteria. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to provide a class of cephalosporin derivatives with novel structure, high NDM-1 inhibitory activity and Ebselen fragment targeted administration, and to use them as NDM-1 inhibitors to treat bacterial infectious diseases caused by NDM-1 resistance.

[0007] The present invention adopts the strategy of introducing Se atoms to construct a novel cephalosporin derivative, introduces an NDM-1 inhibitor at the C17 position of the cephalosporin to construct a "Trojan horse" molecule, and introduces the NDM-1 inhibitor Ebselen into cephalosporins with different structures after ring opening, so as to form a C-Se bond that is broken only after NDM-1 hydrolysis, so as to realize the point-by-point release of Ebselen at the active center, realize the targeted drug delivery of the Ebselen fragment, form a Se-S covalent bond with the thiol group of cysteine ​​in the active region of NDM-1, thereby improving the inhibition of NDM-1 activity and realizing targeted drug delivery.

[0008] In the first aspect, the present invention provides a compound as shown in the general formula I, or a pharmaceutically acceptable salt thereof:

[0009]

[0010] In the general formula I, R is selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkylalkyl, alkylacyl, arylalkylacyl, heteroarylacyl, heteroarylalkylacyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl; the alkyl is (C1-C6)alkyl; the alkyl, aryl or heteroaryl is optionally substituted by (C1-C6)alkyl, halogen, hydroxy, trifluoromethyl, cyano, methoxy, amino, or nitro.

[0011] Further, in the general formula I, R is selected from one of acetyl, 2-cyanoacetyl, benzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl, 3,5-dimethoxybenzyl, 3,5-dimethylbenzyl, 3,5-bis(trifluoromethyl)benzyl, 3-chloro-5-fluorobenzyl, 3-chloro-5-cyanobenzyl, 3-chloro-5-methylbenzyl, 3-chloro-5-methoxybenzyl, 2,6-dichloro-4-pyridyl, 2-thienylmethyl, cyclopropylmethyl, benzoyl, phenylacetyl, cyclopropylacyl, tetrazolyl, thiopheneacyl, methylsulfonyl, phenylsulfonyl, phenylmethylsulfonyl, thiophenesulfonyl and cyclopropylsulfonyl.

[0012] Furthermore, the present invention provides the following compounds, or pharmaceutically acceptable salts thereof, for use in the field of treating bacterial infectious diseases caused by NDM-1 resistance:

[0013] 7-Acetamido-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C7);

[0014] 8-Oxo-7-(2-phenylacetamido)-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C8);

[0015] 7-Benzamido-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C9);

[0016] 7-(Cyclopropanecarboxamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C10);

[0017] 7-(2-Cyanoacetamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C11);

[0018] 7-(2-(1H-tetrazol-1-yl))acetylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C12);

[0019] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(thiophene-2-carboxamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C13);

[0020] 7-(Methanesulfonamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C14);

[0021] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(thiophene-2-sulfonamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C15);

[0022] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-((phenylmethyl)sulfonamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C16);

[0023] 7-(Cyclopropylsulfonamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C17);

[0024] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(phenylsulfonamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C18);

[0025] 7-(Benzylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C19);

[0026] 7-((3-chloro-5-methoxybenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C20);

[0027] 7-((3,5-Dichlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C21);

[0028] 7-((3,5-Difluorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C22);

[0029] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-((2-thienylmethyl)amino)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C23);

[0030] 7-((Cyclopropylmethyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C24);

[0031] 7-((3-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

[0032] 7-((4-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C26);

[0033] 7-((2-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C27);

[0034] 7-((3,5-Dimethoxybenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C28);

[0035] 7-((3,5-Dimethylbenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C29);

[0036] 7-((3,5-bis(trifluoromethyl))benzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C30);

[0037] 7-((2,6-dichloro-4-pyridinyl)methyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C31);

[0038] 7-((3-chloro-5-fluorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C32);

[0039] 7-((3-chloro-5-cyanobenzyl)amino)-8-oxo-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C33);

[0040] 7-((3-chloro-5-methylbenzyl)amino)-8-oxo-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C34).

[0041] The pharmaceutically acceptable salts of the compounds of the present invention are salts formed from pharmaceutically acceptable inorganic or organic acids, preferably hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, maleic acid, fumaric acid, citric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, tartaric acid or acetic acid.

[0042] In a second aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of the cephalosporin derivative of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition can be in the form of conventional pharmaceutical preparations such as ordinary tablets or capsules, sustained-release tablets, controlled-release tablets, granules, oral liquids, syrups, suppositories, transdermal preparations, injections, etc.

[0043] In a third aspect, the present invention provides use of a compound of formula I or a pharmaceutically acceptable salt thereof for preparing an NDM-1 inhibitor.

[0044] Furthermore, the compound of formula I or a pharmaceutically acceptable salt thereof is used for preparing an antibacterial drug for treating bacterial infectious diseases caused by NDM-1 resistance.

[0045] Furthermore, the above antibacterial drugs are combined with other drugs to prepare drugs for treating bacterial infectious diseases caused by NDM-1 resistance.

[0046] The other drugs include beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, and carbapenems.

[0047] The invention prepares a novel cephalosporin derivative containing Se atoms based on the Ebselen ring-opening structure and the cephalosporin structure. The beta-lactam ring of the cephalosporin is ring-opened by NDM-1 hydrolysis, and the Ebselen fragment containing the Se atom is released to form a Se-S covalent bond with the thiol group of the cysteine ​​in the active region of NDM-1, thereby improving the inhibition of NDM-1 activity, enhancing the targeting of NDM-1, effectively inhibiting the growth of drug-resistant bacteria, and can be applied to bacterial infectious diseases caused by NDM-1 resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a graph showing the cell viability test of the compounds C19 and C21 synthesized by the present invention in Hek-239T cells and THP-1 cells.

[0049] (A) Cell viability test results of compounds C19 and C21 in Hek-239T; (B) Cell viability test results of compounds C19 and C21 in THP-1.

[0050] Figure 2 It is a metabolic stability test chart of the compounds C19 and C21 synthesized by the present invention in rat plasma and rat liver homogenate.

[0051] (A) Stability test results of compounds C19 and C21 in plasma; (B) Stability test results of compounds C19 and C21 in rat liver homogenate.

[0052] Figure 3 This is a diagram showing the effect of the combined use of the compound C21 synthesized by the present invention and meropenem (Mem) on the colonization of target organs of infected mice. DETAILED DESCRIPTION

[0053] The following examples are used for illustrative purposes only and are not intended to limit the scope of protection of the present application.

[0054] The novel cephalosporin compound of the present invention can be prepared by the following method:

[0055]

[0056] The synthesis method of compound C7 is as follows:

[0057] (1) dissolving anthranilic acid in an aqueous hydrochloric acid solution, slowly dropping an aqueous sodium nitrite solution into the reaction solution under ice bath conditions to obtain a diazonium salt solution, which is then reacted with newly prepared Na2Se2 under ice bath conditions to generate a diselenide compound C2;

[0058] (2) Compound C2 is dissolved in dry dichloromethane, stirred in an ice bath, and then 2 drops of N,N-dimethylformamide and oxalyl chloride are added in sequence to obtain a light yellow solid acyl chloride intermediate. The intermediate is then dissolved in dry dichloromethane with aniline to undergo an acylation reaction to produce a benzamide compound C3;

[0059] (3) Under the action of zinc powder and dilute hydrochloric acid, C3 is reduced to selenophene phenol compound C4;

[0060] (4) Under room temperature conditions, sodium bicarbonate is used as an acid-binding agent to undergo a substitution reaction between compounds C4 and C5 to obtain the key intermediate C6;

[0061] (5) The intermediate C6 is first subjected to an acylation reaction to introduce an acetyl group on the N-group, and then reacted with trifluoroacetic acid to remove the p-methoxybenzyl group to obtain the target compound C7.

[0062] The following novel cephalosporin derivatives were prepared by referring to the synthetic route of Example C7:

[0063] 7-Acetamido-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C7);

[0064] 8-Oxo-7-(2-phenylacetamido)-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C8);

[0065] 7-Benzamido-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C9);

[0066] 7-(Cyclopropanecarboxamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C10);

[0067] 7-(2-Cyanoacetamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C11);

[0068] 7-(2-(1H-tetrazol-1-yl))acetylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C12);

[0069] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(thiophene-2-carboxamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C13);

[0070] 7-(Methanesulfonamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C14);

[0071] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(thiophene-2-sulfonamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C15);

[0072] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-((phenylmethyl)sulfonamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C16);

[0073] 7-(Cyclopropylsulfonamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C17);

[0074] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(phenylsulfonamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C18);

[0075] 7-(Benzylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C19);

[0076] 7-((3-chloro-5-methoxybenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C20);

[0077] 7-((3,5-Dichlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C21);

[0078] 7-((3,5-Difluorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C22);

[0079] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-((2-thienylmethyl)amino)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C23);

[0080] 7-((Cyclopropylmethyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C24);

[0081] 7-((3-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

[0082] 7-((4-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C26);

[0083] 7-((2-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C27);

[0084] 7-((3,5-Dimethoxybenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C28);

[0085] 7-((3,5-Dimethylbenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C29);

[0086] 7-((3,5-bis(trifluoromethyl))benzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C30);

[0087] 7-((2,6-dichloro-4-pyridinyl)methyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C31);

[0088] 7-((3-chloro-5-fluorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C32);

[0089] 7-((3-chloro-5-cyanobenzyl)amino)-8-oxo-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C33);

[0090] 7-((3-chloro-5-methylbenzyl)amino)-8-oxo-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C34).

[0091] The present invention is further described in detail by the following specific examples. It should be understood that they are not limitations on the protection scope of the present invention. Any simple replacement and improvement within the protection scope of the present invention should be included in the protection scope of the present invention.

[0092] Embodiment 1:

[0093]

[0094] Compound C6 (600 mg, 1 mmol) was dissolved in 20 mL of tetrahydrofuran, and pyridine (160 mg, 2 mmol) and formyl chloride (120 mg, 2 mmol) were added in sequence, and stirred at room temperature for 1 h. The reaction was complete by TLC detection. After the reaction solution was washed with saturated copper sulfate, the organic phase was collected, and the organic phase was purified by column chromatography to obtain a white solid. Trifluoroacetic acid and anisole were prepared in a volume ratio of 5:1, 18 mL of solution was prepared and placed at -10 ° C and stirred for 20 min, and then the obtained white solid was added to the solution, and kept at -10 ° C for 2 h. The reaction was complete by TLC detection, the reaction solution was poured into 50 mL of ice water, the organic phase was collected, and the organic phase was dried over anhydrous magnesium sulfate and concentrated to a semi-solid. The semi-solid was dissolved in 5 mL of ethyl acetate and added dropwise to 100 mL of petroleum ether. A large amount of white solid was precipitated, and the white solid was collected by filtration. Dissolve the white solid in 20 mL of acetone solution, add 5 g of sodium isooctanoate to the solution, stir for 10 min, pour the reaction solution into 50 mL of water, wash with ethyl acetate (10×3 mL) several times, and collect the aqueous phase. Adjust the pH value of the aqueous phase to 4 with trifluoroacetic acid, extract with ethyl acetate (15×3 mL), collect the organic phase, dry the organic phase over anhydrous magnesium sulfate, and concentrate to obtain a semisolid. Dissolve the semisolid in 5 mL of ethyl acetate solution, add dropwise to a mixed solution of 20 mL of ether and 20 mL of n-hexane, precipitate a large amount of white solid, filter, and obtain 180 mg of white solid; melting point: 112.3-114.2°C; yield is 34%.

[0095] 7-Acetamido-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C7). 1 H NMR (400MHz, DMSO-d6) δ10.35(s,1H),8.77(d,J=8.5Hz,1H),7.69(d,J=7.9Hz,2H) ,7.63(d,J=7.5Hz,1H),7.58(d,J=7.8Hz,1H),7.40(t,J=7.7Hz,1H),7.32(q,J=8.1 Hz,3H),7.07(t,J=7.4Hz,1H),5.56(dd,J=8.4,4.7Hz,1H),5.01(d,J=4.7Hz,1H),4 .02(d,J=11.3Hz,2H),3.67(d,J=17.3Hz,1H),3.41(d,J=17.7Hz,1H),1.85(s,3H). 13C NMR(100MHz,DMSO--d6)δ170.53,167.25,165.28,163.70,139.58,138.14,131.62,131.46, 129.24,128.73,126.63,124.30,120.44,59.41,58.25,29.12,28.34,22.58.HRMS(ESI)m / z calcd for C 23 H 21 N3O5SSe[M+H] + :531.0367,found531.0374.

[0096] Embodiment 2:

[0097]

[0098] 8-Oxo-7-(2-phenylacetamido)-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C8), prepared by the same method as in Example 1, white solid; melting point: 108.9-110.2°C; yield 29%. 1 H NMR (400MHz, DMSO-d6) δ10.43(s,1H),8.99(d,J=8.4Hz,1H),7.70(d,J=8.0Hz,2H ),7.60(t,J=8.9Hz,2H),7.38(t,J=7.6Hz,1H),7.31(t,J=7.8Hz,3H),7.26–7.15 (m,5H),7.06(t,J=7.5Hz,1H),5.54–5.40(m,1H),4.90(d,J=4.6Hz,1H),4.07(d, J=11.6Hz,2H),3.64–3.52(m,2H),3.47(d,J=17.4Hz,1H),3.39(d,J=17.3Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ171.49,167.40,164.38,139.69,138.39,136.42,132.56,131.88,131.31,129.56, 129.21,128.74,128.58,127.00,126.44,124.21,120.41,59.18,58.22,42.14,29.80,28.06.HRMS(ESI)m / z calcd for C 29 H 25 N3O5SSe[M+H] +:607.0680,found607.0672.

[0099] Embodiment 3:

[0100]

[0101] 7-Benzamido-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C9), prepared by the same method as in Example 1, white solid; melting point: 141.7-142.3°C; yield 37%. 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),9.34(d,J=8.0Hz,1H),7.87(d,J=7.4Hz,2H),7.70–7.59(m,4H),7.53(t,J=7.1Hz,1H),7.45–7.39(m,3H ),7.36–(m,3H),7.07(t,J=7.3Hz,1H),5.82–5.69(m,1H),5.12(d,J=4.4Hz,1H),4.04(s,2H),3.66(d,J=17.3Hz,1H),3.46(d,J=17.6Hz,1H). 13 C NMR (100MHz, DMSO-d6) δ177.47,167.47,167.26,164.45,163.73,139.58,138.13,133.57,132.58,132.40,131.61,131. 47,130.47,129.25,128.87,128.72,128.30,126.62,124.89,124.30,120.45,60.29,58.71,29.68,28.45.HRMS(ESI)m / z calcd for C 28 H 23 N3O5SSe[M+H] + :593.0524, found 593.0529.

[0102] Embodiment 4:

[0103]

[0104] 7-(Cyclopropanecarboxamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C10), prepared by the same method as in Example 1, white solid; melting point: 137.6-139.9°C; yield 25%.1 H NMR (400MHz, DMSO-d6) δ10.39(s,1H),8.94(d,J=8.6Hz,1H),7.69(d,J=8.0Hz,2H),7 .63–7.58(m,2H),7.39(t,J=7.6Hz,1H),7.31(t,J=7.9Hz,3H),7.06(t,J=7.4Hz,1H) ,5.54(dd,J=8.5,4.7Hz,1H),4.94(d,J=4.7Hz,1H),4.08(d,J=11.4Hz,2H),3.62(d, J=17.5Hz,1H),3.36(d,J=17.8Hz,1H),1.68(q,J=6.3Hz,1H),0.67(d,J=7.0Hz,4H). 13 C NMR(100MHz,DMSO-d6)δ174.12,167.36,165.49,139.65,138.34,132.50,131.85,131.33 ,129.23,128.58,124.23,120.40,59.16,58.26,28.10,13.64,7.68,7.48.HRMS(ESI)m / z calcd for C 25 H 23 N3O5SSe[M+H] + :557.0524, found 557.0531.

[0105] Embodiment 5:

[0106]

[0107] 7-(2-Cyanoacetamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C11), prepared by the same method as in Example 1, white solid; melting point: 127.1-127.8°C; yield 25%. 1H NMR (400MHz, DMSO-d6) δ10.39(s,1H),9.19(d,J=8.2Hz,1H),7.69(d,J=8.0Hz,2H ),7.60(d,J=7.7Hz,2H),7.39(t,J=7.6Hz,1H),7.31(dd,J=9.5,6.2Hz,3H),7.06 (t,J=7.4Hz,1H),5.51(dd,J=8.3,4.7Hz,1H),4.99(d,J=4.8Hz,1H),4.07(d,J=1 1.1Hz, 2H), 3.73 (d, J = 5.0Hz, 2H), 3.63 (d, J = 17.4Hz, 1H), 3.38 (d, J = 17.1Hz, 1H); 13 CNMR(100MHz,DMSO-d6)δ167.36,163.84,163.71,158.83,158.52,139.63,138.26,132.42,131.83,131.37, 129.23,128.61,126.53,124.25,120.42,119.30,116.23,59.45,57.86,29.45,28.22,25.59.HRMS(ESI)m / z calcd for C 24 H 20 N4O5SSe[M+H] + :556.0320,found556.0326.

[0108] Embodiment 6:

[0109]

[0110] 7-(2-(1H-tetrazol-1-yl))acetylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C12), prepared by the same method as in Example 1, white solid; melting point: 105.3-106.1°C; yield 21%. 1H NMR (400MHz, DMSO-d6) δ10.34(s,1H),9.45(d,J=8.4Hz,1H),9.32(s,1H),7.68(d,J =8.0Hz,2H),7.61(dd,J=19.4,7.7Hz,2H),7.41(t,J=7.5Hz,1H),7.33(q,J=8.3Hz,3 H),7.07(t,J=7.4Hz,1H),5.63(dd,J=8.4,4.8Hz,1H),5.39–5.25(m,2H),5.06(d,J =4.9Hz,1H),4.04(d,J=11.2Hz,2H),3.71(d,J=17.5Hz,1H),3.35(d,J=17.3Hz,1H); 13 C NMR(100MHz,DMSO-d6)δ167.27,166.16,164.42,163.59,158.89,158.57,145.71,139.56,138.17,132.35,131.68, 131.47,130.26,129.24,128.72,126.68,124.57,124.31,120.45,65.45,59.41,57.93,49.63,31.21.HRMS(ESI)m / z calcdfor C 23 H 19 N7O5SSe[M+H] + :585.0334, found 585.0339.

[0111] Embodiment 7:

[0112]

[0113] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(thiophene-2-carboxamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C13), prepared by the same method as in Example 1, white solid; melting point: 103.2-104.1°C; yield 34%. 1H NMR (400MHz, DMSO-d6) δ10.39(s,1H),9.33(d,J=8.1Hz,1H),7.94(s,1H),7.78(d,J=4 .9Hz,1H),7.69(d,J=8.0Hz,2H),7.61(t,J=7.3Hz,2H),7.40(t,J=7.5Hz,1H),7.31(q ,J=7.6,7.2Hz,3H),7.12(s,1H),7.06(t,J=7.5Hz,1H),5.66(t,J=6.4Hz,1H),5.04(d ,J=4.6Hz,1H),4.04(q,J=10.8Hz,2H),3.62(d,J=17.3Hz,1H),3.39(d,J=17.4Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ167.35,164.23,163.96,162.09,139.63,138.76,138.20,132.66,132.53,131.71,1 31.41,130.18,129.23,128.71,128.63,126.49,124.26,120.43,59.90,58.54,29.51,28.21.HRMS(ESI)m / z calcd for C 26 H 21 N3O5S2Se[M+H] + :599.0088,found599.0095.

[0114] Embodiment 8:

[0115]

[0116] 7-(Methanesulfonylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C14), prepared by the same method as in Example 1, white solid; melting point: 188.3-188.9°C; yield 37%. 1H NMR (400MHz, DMSO-d6) δ10.34(s,1H),8.42(d,J=9.6Hz,1H),7.68(d,J=7.7Hz,2H),7.63 (d,J=7.4Hz,1H),7.58(d,J=7.8Hz,1H),7.40(t,J=7.3Hz,1H),7.32(q,J=8.2Hz,3H),7.0 7(t,J=7.4Hz,1H),5.29(dd,J=9.2,5.0Hz,1H),5.05(d,J=4.6Hz,1H),4.08(d,J=11.0Hz, 1H), 3.99 (d, J = 11.0Hz, 1H), 3.68 (d, J = 17.7Hz, 1H), 3.44 (d, J = 17.6Hz, 1H), 2.96 (s, 3H). 13 C NMR(100MHz,DMSO-d6)δ167.26,164.86,163.64,139.57,138.13,132.41,131.63,131.47,130.22, 129.25,128.74,126.65,124.57,124.31,120.45,62.44,58.45,41.65,29.06,28.43.HRMS(ESI)m / z calcd for C 22 H 24 N2O5SSe[M+H] + :567.0037,found 567.0046.

[0117] Embodiment 9:

[0118]

[0119] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(thiophene-2-sulfonylamino)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C15), prepared by the same method as in Example 1, white solid; melting point: 163.4-165.1°C; yield 18%. 1H NMR (400MHz, DMSO-d6) δ10.39(s,1H),9.24(d,J=8.1Hz,1H),7.90(d,J=4.0Hz,1H),7.6 8(d,J=7.9Hz,2H),7.59(dd,J=16.9,8.4Hz,3H),7.38(t,J=7.3Hz,1H),7.31(t,J=7.6H z,3H),7.14(t,J=4.4Hz,1H),7.07(t,J=7.2Hz,1H),5.24(s,1H),4.89(s,1H),4.06(d, J=10.8Hz,1H),3.98(d,J=10.8Hz,1H),3.55(d,J=17.6Hz,1H),3.34(d,J=17.6Hz,1H). 13 C NMR (100MHz, DMSO-d6) δ167.32,163.97,163.43,141.95,139.62,138.27,133.51,132.51,132.32,131. 81,131.36,129.23,128.62,128.29,126.56,124.25,120.42,62.35,58.40,29.35,28.32.HRMS(ESI)m / z calcd for C 25 H 21 N3O6S3Se[M+H] + :634.9758,found634.9767.

[0120] Embodiment 10:

[0121]

[0122] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-((phenylmethyl)sulfonamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C16), prepared by the same method as in Example 1, white solid; melting point: 101.5-103.6°C; yield 23%. 1H NMR (400MHz, DMSO-d6) δ10.36 (s, 1H), 8.56 (d, J = 9.1 Hz, 1H), 7.69 (d, J = 7. 5Hz,2H),7.62-7.57(m,2H),7.43-7.36(m,3H),7.35-7.28(m,6H),7.07(t, J=7.4Hz,1H),5.18(s,1H),4.95(s,1H),4.38(s,2H),4.11(d,J=11.2Hz,1 H), 4.00 (d, J = 10.9Hz, 1H), 3.64 (d, J = 17.7Hz, 1H), 3.43 (d, J = 17.5Hz, 1H). 13 C NMR (100MHz, DMSO-d6) δ167.32,164.53,164.03,139.59,138.25,132.33,131.78,131.53,130. 13,129.24,128.88,128.70,126.63,124.29,120.44,62.53,58.89,29.22,28.40.HRMS(ESI)m / z calcd forC 28 H 25 N3O6S2Se[M+H] + :643.0350,found 643.0357.

[0123] Embodiment 11:

[0124]

[0125] 7-(Cyclopropylsulfonylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C17), prepared by the same method as in Example 1, white solid; melting point: 107.3-109.5°C; yield 29%. 1H NMR (400MHz, DMSO-d6) δ10.40(s,1H),8.45(d,J=9.7Hz,1H),7.69(d,J=7.9Hz,2H),7. 59(d,J=6.1Hz,2H),7.39(t,J=7.3Hz,1H),7.30(d,J=7.7Hz,3H),7.06(t,J=7.3Hz,1H) ,5.21(s,1H),4.95(d,J=4.0Hz,1H),4.10(d,J=10.9Hz,1H),4.01(d,J=10.9Hz,1H),3. 62(d,J=17.0Hz,1H),3.42(d,J=17.2Hz,1H),2.57–2.49(m,1H),0.92(t,J=6.8Hz,4H). 13 C NMR(100MHz,DMSO-d6)δ167.34,164.30,139.63,138.25,132.42,131.80,131.39,129.23,12 8.62,126.53,124.25,120.42,62.64,58.83,31.30,29.43,28.29,5.90,5.61.HRMS(ESI)m / z calcd for C 24 H 23 N3O6S2Se[M+H] + :593.0193,found593.0121.

[0126] Embodiment 12:

[0127]

[0128] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(phenylsulfonamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C18), prepared by the same method as in Example 1, white solid; melting point: 173.8-175.4°C; yield 38%. 1H NMR (400MHz, DMSO-d6) δ10.40 (s, 1H), 8.99 (d, J = 9.2Hz, 1H), 7.80 (d, J = 7.4Hz, 2H),7.68(d,J=7.8Hz,2H),7.61-7.52(m,5H),7.37(t,J=7.4Hz,1H),7.30(t,J =7.5Hz,3H),7.07(t,J=7.2Hz,1H),5.19(s,1H),4.83(s,1H),4.06(d,J=11.1H z, 1H), 3.98 (d, J = 11.0Hz, 1H), 3.52 (d, J = 17.5Hz, 1H), 3.36 (d, J = 17.8Hz, 1H). 13 C NMR (100MHz, DMSO-d6) δ167.36,164.32,163.57,141.45,139.64,138.31,133.25,132.34,131.87,131. 34,129.72,129.23,128.58,126.95,126.51,124.24,120.41,62.26,58.44,29.45,28.21.HRMS(ESI)m / z calcd for C 27 H 23 N3O6S2Se[M+H] + :629.0193,found 629.0199.

[0129] Embodiment 13:

[0130]

[0131] 7-(Benzylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C19), prepared by the same method as in Example 1, white solid; melting point: 102.7-105.0°C; yield: 36%. 1H NMR (400MHz, DMSO-d6) δ10.33(s,1H),7.68(d,J=7.6Hz,2H),7.63(d,J=7.1Hz,1H ),7.57(d,J=7.6Hz,1H),7.40(t,J=7.2Hz,1H),7.37–7.27(m,7H),7.25(d,J=5.7H z,1H),7.07(t,J=7.4Hz,1H),4.96(s,1H),4.72(s,1H),4.08(d,J=11.0Hz,1H),3. 97(d,J=11.1Hz,1H),3.87(s,2H),3.64(d,J=17.5Hz,1H),3.41(d,J=17.6Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ167.24,163.73,139.57,138.14,132.40,131.66,131.44,129.23,129.0 8,128.87,128.74,127.97,126.65,124.73,124.30,120.47,51.82,29.09,28.51.HRMS(ESI)m / z calcd for C 28 H 25 N3O4SSe[M+H] + :579.0731, found 579.0739.

[0132] Embodiment 14:

[0133]

[0134] 7-((3-Chloro-5-methoxybenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C20), prepared by the same method as in Example 1, white solid; melting point: 148.9-149.5°C; yield 40%. 1H NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.68(d,J=7.8Hz,2H),7.63(d,J=7.4Hz,1H),7.57 (d,J=7.6Hz,1H),7.40(t,J=7.3Hz,1H),7.35–7.28(m,3H),7.07(t,J=7.2Hz,1H),6.97( s,1H),6.89(s,2H),4.95(d,J=4.1Hz,1H),4.72(s,1H),4.07(d,J=11.1Hz,1H),3.97(d, J=11.3Hz,1H),3.84(s,2H),3.73(s,3H),3.65(d,J=17.6Hz,1H),3.42(d,J=17.5Hz,1H). 13 C NMR (100MHz, DMSO-d6) δ167.25,163.71,160.68,139.57,138.17,134.13,132.38,131.68,131.44,129.23,128. 72,126.66,124.67,124.30,120.99,120.46,113.69,113.54,67.62,56.09,51.21,29.11,28.51.HRMS(ESI)m / z calcdfor C 29 H 26 ClN3O5SSe[M+H] + :643.0447, found 643.0445.

[0135] Embodiment 15:

[0136]

[0137] 7-((3,5-Dichlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C21), prepared by the same method as in Example 1, white solid; melting point: 130.8-133.1°C; yield 33%. 1H NMR (400MHz, DMSO-d6) δ10.34(s,1H),7.68(d,J=7.6Hz,2H),7.61(d,J=7.2Hz, 1H),7.56(d,J=7.6Hz,1H),7.39(d,J=16.3Hz,4H),7.34-7.28(m,3H),7.06(t, J=7.3Hz,1H),4.87(d,J=4.2Hz,1H),4.64(s,1H),4.06(d,J=11.1Hz,1H),3.94 (d,J=11.0Hz,1H),3.78(s,2H),3.64(d,J=17.5Hz,1H),3.34(d,J=17.4Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ167.96,167.28,164.01,145.68,139.59,138.28,134.31,132.37,131.73,131.39, 129.22,128.67,127.12,126.85,126.62,124.27,120.44,68.93,59.33,50.95,29.32,28.19.HRMS(ESI)m / z calcd for C 28 H 23 Cl2N3O4SSe[M+H] + :646.9952,found646.9961.

[0138] Embodiment 16:

[0139]

[0140] 7-((3,5-Difluorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C22), prepared by the same method as in Example 1, white solid; melting point: 109.3-111.2°C; yield 21%. 1H NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.68(d,J=7.7Hz,2H),7.62(d,J=7.2Hz,1H ),7.56(d,J=7.6Hz,1H),7.39(t,J=7.2Hz,1H),7.32(q,J=7.9Hz,3H),7.05(dd,J =15.5,7.8Hz,4H),4.89(d,J=4.3Hz,1H),4.65(s,1H),4.05(d,J=11.0Hz,1H),3. 94(d,J=11.0Hz,1H),3.80(s,2H),3.65(d,J=17.8Hz,1H),3.35(d,J=17.5Hz,1H). 13 C NMR (100MHz, DMSO-d6) δ168.02, 167.26, 164.15 (d, J = 12.9Hz), 163.89, 161.71 (d, J = 12.9Hz), 145.98, 139.56, 138.24, 132.37, 131.68 (d, J = 26. 3Hz),129.22,128.69,128.53,126.64,124.70,124.29,120.44,111.35( d,J=24.9Hz),102.61,68.93,59.30,51.17,29.22,28.20.HRMS(ESI)m / z calcd for C 28 H 23 F2N3O4SSe[M+H] + :615.0543,found 615.0547.

[0141] Embodiment 17:

[0142]

[0143] 8-Oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-((2-thienylmethyl)amino)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C23), prepared by the same method as in Example 1, white solid; melting point: 133.1-134.2°C; yield 38%. 1H NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.68(d,J=8.0Hz,2H),7.62(d,J=7.4Hz,1H), 7.56(d,J=7.8Hz,1H),7.41-7.28(m,5H),7.06(t,J=7.3Hz,1H),6.96(s,1H),6.93(d ,J=3.9Hz,1H),4.91(d,J=4.4Hz,1H),4.65(d,J=4.3Hz,1H),4.06(d,J=11.2Hz,1H), 3.97(s,2H),3.94(d,J=11.0Hz,1H),3.64(d,J=17.9Hz,1H),3.37(d,J=17.6Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ167.67,167.26,163.86,143.85,139.56,138.18,132.39,131.65,131.43,129.22,128.83, 128.69,127.28,126.63,125.96,125.66,124.77,124.29,120.47,68.30,59.04,46.81,29.18,28.24.HRMS(ESI)m / z calcd forC 26 H 23 N3O4S2Se[M+H] + :585.0295,found585.0299.

[0144] Embodiment 18:

[0145]

[0146] 7-((Cyclopropylmethyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C24), prepared by the same method as in Example 1, white solid; melting point: 137.4-137.9°C; yield 27%. 1H NMR (400MHz, DMSO-d6) δ10.33 (s, 1H), 7.72–7.63 (m, 3H), 7.59 (d, J = 7.7Hz, 1H), 7. 41(t,J=7.5Hz,1H),7.36–7.29(m,3H),7.07(t,J=7.3Hz,1H),5.10(s,1H),5.00(s ,1H),4.11(d,J=10.8Hz,1H),4.03(d,J=10.7Hz,1H),3.69(d,J=17.0Hz,1H),3.53 (d,J=17.2Hz,1H),2.88(s,1H),2.63(s,1H)0.95(s,1H),0.50(s,2H),0.24(s,2H). 13 CNMR(100MHz,DMSO-d6)δ167.22,163.40,139.56,138.08,132.36,131.69,131.47,129.23,128.7 5,126.70,124.54,124.31,120.46,52.95,47.03,29.67,28.83,12.22,4.50,4.21.HRMS(ESI)m / z calcd for C 25 H 25 N3O4SSe[M+H] + :543.0731, found 543.0738.

[0147] Embodiment 19:

[0148]

[0149] 7-((3-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25), prepared by the same method as in Example 1, white solid; melting point: 118.6-120.9°C; yield 35%. 1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),7.72–7.65(m,2H),7.63(dd,J=7.5,1.6Hz,1H),7.5 7(dd,J=7.9,1.3Hz,1H),7.43(d,J=2.1Hz,1H),7.40(td,J=7.6,1.7Hz,1H),7.37–7.28(m ,6H),7.10–7.03(m,1H),4.97(d,J=4.7Hz,1H),4.76(d,J=4.7Hz,1H),4.09(d,J=11.2Hz, 1H), 3.98 (d, J = 11.1Hz, 1H), 3.91 (s, 2H), 3.65 (d, J = 17.5Hz, 1H), 3.44 (d, J = 17.4Hz, 1H). 13 C NMR (100MHz, DMSO-d6) δ167.24,163.64,139.56,138.15,133.56,132.36,131.68,131.45,130.75,129.23,129. 11,128.73,128.15,127.98,126.68,124.65,124.31,120.46,67.17,57.92,51.05,29.07,28.59.HRMS(ESI)m / z calcdfor C 28 H 24 ClN3O4SSe[M+H] + :613.0341, found 613.0347.

[0150] Embodiment 20:

[0151]

[0152] 7-((4-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C26), prepared by the same method as in Example 1, white solid; melting point: 108.4-109.6°C; yield 29%. 1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),7.73–7.65(m,2H),7.64(dd,J=7.5,1.6Hz,1H),7.5 7(dd,J=7.8,1.3Hz,1H),7.41(dd,J=7.5,1.6Hz,1H),7.38(s,4H),7.34–7.30(m,3H),7.1 0–7.04(m,1H),4.99(d,J=4.7Hz,1H),4.78(d,J=4.6Hz,1H),4.09(d,J=11.1Hz,1H),3.99 (d,J=11.1Hz,1H),3.92(d,J=4.3Hz,2H),3.65(d,J=17.5Hz,1H),3.45(d,J=17.4Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ167.24,163.57,139.55,138.11,133.09,132.36,131.67,131.43,129.23, 128.93,128.73,126.68,124.63,124.31,120.46,66.69,57.54,50.88,29.02,28.66.HRMS(ESI)m / z calcd for C 28 H 24 ClN3O4SSe[M+H] + :613.0341,found613.0344.

[0153] Embodiment 21:

[0154]

[0155] 7-((2-Chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C27), prepared by the same method as in Example 1, white solid; melting point: 123.7-125.1°C; yield 32%. 1H NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.68(d,J=7.8Hz,2H),7.62(d,J=7.2Hz, 1H),7.56(d,J=7.7Hz,1H),7.52(d,J=7.2Hz,1H),7.41–7.22(m,7H),7.07(t,J =7.3Hz,1H),4.92(d,J=4.2Hz,1H),4.71(s,1H),4.06(d,J=11.3Hz,1H),3.94( d,J=11.3Hz,1H),3.88(s,2H),3.65(d,J=17.6Hz,1H),3.38(d,J=17.7Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ167.75,167.26,163.89,139.57,138.23,137.76,133.03,132.39,131.67,131.42,130.28,129.59, 129.22,129.15,128.69,128.64,127.69,126.63,124.75,124.29,120.46,69.11,59.24,49.35,29.68,28.22.HRMS(ESI)m / z calcd forC 28 H 24 ClN3O4SSe[M+H] + :613.0341,found613.0349.

[0156] Embodiment 22:

[0157]

[0158] 7-((3,5-Dimethoxybenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C28), prepared by the same method as in Example 1, white solid; melting point: 131.3-133.1°C; yield 41%. 1H NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.68(d,J=7.6Hz,2H),7.63(d,J=7.2Hz,1H),7.5 7(d,J=7.6Hz,1H),7.39(t,J=7.3Hz,1H),7.32(q,J=8.0Hz,3H),7.07(t,J=7.0Hz,2H), 6.49(s,2H),6.33(s,1H),4.93(s,1H),4.65(s,1H),4.07(d,J=11.2Hz,1H),3.95(d,J= 11.1Hz,1H),3.75(s,2H),3.69(s,6H),3.63(d,J=17.4Hz,1H),3.38(d,J=17.3Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ167.25,163.84,160.95,139.57,138.21,132.39,131.67,131.42,129.23,128.70 ,126.64,124.73,124.30,120.45,106.44,99.59,68.37,59.01,52.06,47.04,29.68,28.33.HRMS(ESI)m / z calcd for C 30 H 29 N3O6SSe[M+H] + :639.0942,found639.0947.

[0159] Embodiment 23:

[0160]

[0161] 7-((3,5-Dimethylbenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C29), prepared by the same method as in Example 1, white solid; melting point: 131.2-134.1°C; yield 36%. 1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),7.76–7.61(m,3H),7.58(d,J=7.6Hz,1H) ,7.40(t,J=7.1Hz,1H),7.37–7.26(m,3H),7.07(t,J=7.3Hz,1H),6.95(d,J=13 .5Hz,3H),5.03(s,1H),4.82(s,1H),4.12(d,J=11.1Hz,1H),4.01(d,J=10.8Hz ,1H),3.90(s,2H),3.65(d,J=17.2Hz,1H),3.49(d,J=17.2Hz,1H),2.23(s,6H). 13 C NMR(100MHz,DMSO-d6)δ167.23,163.47,139.57,138.10,132.36,131.66,131.47,130.21,129.23, 128.76,127.65,126.68,124.65,124.31,120.47,65.72,56.95,51.48,28.91,21.37.HRMS(ESI)m / z calcd for C 30 H 29 N3O4SSe[M+H] + :607.1044, found 607.1047.

[0162] Embodiment 24:

[0163]

[0164] 7-((3,5-bis(trifluoromethyl))benzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C30), prepared by the same method as in Example 1, white solid; melting point: 106.7-108.1°C; yield 31%. 1H NMR(400MHz,DMSO-d6)δ10.32(s,1H),8.03(d,J=1.7Hz,2H),7.91(s,1H),7.71–7.64(m,2H) ,7.62(dd,J=7.5,1.7Hz,1H),7.56(dd,J=7.8,1.3Hz,1H),7.39(td,J=7.6,1.6Hz,1H),7.35– 7.27(m,3H),7.10–7.02(m,1H),4.90(d,J=4.7Hz,1H),4.73(d,J=4.7Hz,1H),4.05(d,J=11.2 Hz,1H),3.98(s,2H),3.94(d,J=11.2Hz,1H),3.64(d,J=17.8Hz,1H),3.38(d,J=17.7Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ167.26,167.09,163.79,159.00,158.65,143.74,139.57,138.25,132.33,131.70,131.40,130.69,130.37, 129.58,129.19,128.68,126.64,125.36,124.66,124.26,122.65,121.24,120.43,68.53,58.81,50.88,29.66,28.30.HRMS(ESI)m / z calcd for C 30 H 23 F6N3O4SSe[M+H] + :715.0479,found715.0473.

[0165] Embodiment 25:

[0166]

[0167] 7-((2,6-Dichloro-4-pyridinyl)methyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C31), prepared by the same method as in Example 1, white solid; melting point: 109.4-111.8°C; yield 38%. 1H NMR (400MHz, DMSO-d6) δ10.33(s,1H),7.68(d,J=7.7Hz,2H),7.62(d,J=7.0Hz,1H) ,7.56(d,J=7.6Hz,1H),7.50(s,2H),7.39(t,J=7.3Hz,1H),7.32(q,J=7.8Hz,3H),7 .06(t,J=7.4Hz,1H),4.89(d,J=4.4Hz,1H),4.68(s,1H),4.06(d,J=11.2Hz,1H),3 .94(d,J=11.2Hz,1H),3.84(s,2H),3.65(d,J=17.4Hz,1H),3.35(d,J=17.6Hz,1H). 13 C NMR (100MHz, DMSO-d6) δ168.02,167.27,163.95,158.33,149.54,139.58,138.27,132.37,131.71,131. 40,129.22,128.68,126.63,124.28,122.99,120.43,69.00,59.26,50.19,29.30,28.17.HRMS(ESI)m / z calcd for C 27 H 22 Cl2N4O4SSe[M+H] + :647.9904,found 647.9911.

[0168] Embodiment 26:

[0169]

[0170] 7-((3-Chloro-5-fluorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C32), prepared by the same method as in Example 1, white solid; melting point: 103.8-105.5°C; yield 26%. 1H NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.71–7.65(m,2H),7.63(dd,J=7.5,1.7Hz,1H),7.57(dd ,J=7.8,1.3Hz,1H),7.39(td,J=7.6,1.7Hz,1H),7.36–7.23(m,5H),7.17(dt,J=9.6,2.2Hz,1H) ,7.07(ddt,J=8.6,7.3,1.2Hz,1H),4.92(d,J=4.7Hz,1H),4.70(d,J=4.7Hz,1H),4.06(d,J=11 .2Hz,1H),3.95(d,J=11.2Hz,1H),3.84(s,2H),3.65(d,J=17.7Hz,1H),3.40(d,J=17.6Hz,1H). 13 C NMR (100MHz, DMSO-d6) δ177.47,167.25,163.80,161.41,139.57,138.22,134.26(d,J=11.8Hz),132.37,131.69(d,J=26.8Hz),129.22,128.70,12 6.64,124.81,(d,J=13.4Hz),124.28,120.44,115.18(d,J=25.4Hz),114.50(d,J=21.5Hz),68.35,58.83,50.92,29.68,28.34.HRMS(ESI)m / zcalcd for C 28 H 23 ClFN3O4SSe[M+H] + :631.0247,found 631.0245.

[0171] Embodiment 27:

[0172]

[0173] 7-((3-chloro-5-cyanobenzyl)amino)-8-oxo-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C33), prepared by the same method as in Example 1, white solid; melting point: 106.9-107.5°C; yield 43%. 1H NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.87(s,1H),7.75(s,2H),7.68(d,J=7.7Hz,2H),7 .62(d,J=7.2Hz,1H),7.56(d,J=7.6Hz,1H),7.39(t,J=7.3Hz,1H),7.32(q,J=8.0Hz,3H) ,7.07(t,J=7.2Hz,1H),4.89(d,J=4.3Hz,1H),4.68(d,J=4.0Hz,1H),4.05(d,J=10.9Hz, 1H), 3.94 (d, J = 11.2Hz, 1H), 3.84 (s, 2H), 3.62 (d, J = 17.5Hz, 1H), 3.38 (d, J = 17.7Hz, 1H). 13 C NMR (100MHz, DMSO-d6) δ167.65,167.24,163.84,139.58,138.25,134.33,133.52,132.37,131.69,131.39,131.09,130. 69,129.21,128.68,126.63,124.66,124.26,120.42,118.19,113.31,68.70,59.06,50.71,29.20,28.26.HRMS(ESI)m / z calcd for C 29 H 23 ClN4O4SSe[M+H] + :638.0294,found 638.0299.

[0174] Embodiment 28:

[0175]

[0176] 7-((3-chloro-5-methylbenzyl)amino)-8-oxo-3-((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C34), prepared by the same method as in Example 1, white solid; melting point: 133.1-133.8°C; yield 37%. 1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),7.69–7.62(M,3H),7.58(d,J=7.6Hz,1H),7.40(t ,J=7.4Hz,1H),7.35–7.28(M,3H),7.23(s,1H),7.17(s,1H),7.12(s,1H),7.07(t,J=7.3 Hz,1H),4.99(d,J=3.7Hz,1H),4.79(s,1H),4.10(d,J=11.2Hz,1H),3.99(d,J=10.9Hz,1 H), 3.88 (d, J = 14.1Hz, 2H), 3.65 (d, J = 17.4Hz, 1H), 3.46 (d, J = 17.3Hz, 1H), 2.27 (s, 3H). 13 C NMR (100MHz, DMSO-d6) δ167.24,163.55,159.09,158.73,140.63,139.57,138.12,133.35,132.36,131.68,131.45,129.22,129. 04,128.85,128.74,126.68,126.49,124.64,124.30,120.46,117.77,114.86,66.47,57.41,50.93,29.67,28.75.HRMS(ESI)m / z calcd for C 29 H 26 ClN3O4SSe[M+H] + :627.0498,found627.0493.

[0177] Embodiment 29:

[0178] The following are the pharmacological experiments and results of NDM-1 enzyme inhibition activity, stability and synergistic antibacterial activity of some compounds of the present invention.

[0179] 1. Test of residual activity of some compounds of the present invention on NDM-1 enzyme

[0180] (1) Experimental methods

[0181] Nitrocefin was selected as the substrate and Ebselen was used as the positive control to determine the inhibitory activity of Ebselen derivatives on NDM-1. NDM-1 enzyme at a concentration of 6nmol / L and Ebselen derivatives at a fixed concentration of 64μg / mL were added to a 96-well plate in sequence. Nitrocefin was added after incubation at 25℃ for 15min, and then placed in a microplate reader. The absorbance was recorded by monitoring more than 30 scanning cycles at a wavelength of 492nM. The group without drug addition was used as the negative control to calculate the residual activity of NDM-1 at a concentration of 64μg / mL of Ebselen derivatives. The experiment was repeated in parallel for no less than 3 times, and the RA value was calculated using GraphPad Prism 8 software.

[0182] RA(%)=OD Drug / OD Blank ×100.

[0183] In the formula, RA represents residual activity, OD represents Drug Represents the absorbance after adding Ebselen derivative, OD Blank Represents the absorbance after adding the solvent DMSO.

[0184] (2) Experimental results

[0185] Table 1 Effect of some compounds on residual activity of NDM-1 enzyme

[0186]

[0187]

[0188] The results are shown in Table 1. Compounds C8-C34 all showed different degrees of inhibition on NDM-1 enzyme. After co-incubation, the residual activity of NDM-1 was 6.53%-78.19%. Compounds C8, C9, C10, C14, C17, C19, C21, C23, C32, and C34 had obvious inhibitory effects on NDM-1 enzyme, and the residual activity of NDM-1 was 6.53%-48.94%. Among them, compounds C19 and C21 showed the strongest NDM-1 inhibitory activity, and the residual activities of NDM-1 were 17.83% and 6.53%, respectively, which could significantly inhibit the activity of NDM-1 enzyme.

[0189] II. Inhibition rate of some compounds of the present invention on NDM-1 enzyme

[0190] (1) Experimental methods

[0191] Nitrocefin was selected as the substrate and Ebselen was used as the positive control to determine the inhibitory activity of Ebselen derivatives on NDM-1. NDM-1 enzyme at a concentration of 6nmol / L and different concentrations of Ebselen derivatives (concentration range of 1-128μg / mL) were added to a 96-well plate in sequence. Nitrocefin was added after incubation at 25°C for 15 minutes, and then placed in an ELISA reader and monitored at a wavelength of 492nM for more than 30 scanning cycles. The group without drug addition was used as the negative control to calculate the inhibition rate of Ebselen derivatives at different concentrations. The experiment was repeated in parallel for no less than 3 times, and the IC was calculated using GraphPad Prism 8 software. 50 value.

[0192]

[0193] In the formula, IR represents the inhibition rate, Ve represents the enzyme reaction rate after adding the Ebselen derivative, and Vd represents the enzyme reaction rate after adding the solvent DMSO.

[0194] (2) Experimental results

[0195] Table 2 IC50 of some compounds against NDM-1 50

[0196]

[0197] The experimental results are shown in Table 2. The results show that the IC 50 The values ​​ranged from 7.03 to 150.27 μM, among which compound C21 exhibited the strongest NDM-1 inhibitory activity, with an IC 50 The value was 7.03±0.96μM, which was significantly better than the positive control Ebselen activity. The above results show that compound C21 has a strong inhibitory effect on NDM-1 enzyme and can be used as a potential drug for the treatment of NDM-1 resistant bacteria for further treatment.

[0198] 3. Synergistic antibacterial activity test of some compounds of the present invention in combination with Mem in NDM-1 producing Escherichia coli

[0199] (1) Experimental methods

[0200] According to the CLSIM24 guidelines, the minimum inhibitory concentration (MIC) of Ebselen derivatives combined with Mem was determined by broth dilution method. Mem with a concentration of 64 μg / mL was diluted in broth and added to a 96-well plate. The bacterial suspension and compounds with a concentration range of 0-64 μg / mL were added in sequence and placed in a constant temperature incubator at 37°C for 24 hours. The bacterial growth status of each well was observed with the naked eye, and the presence of bacterial growth was determined by an ELISA instrument to determine the MIC of the compound and the combination with Mem. Among the selected E. coli expressing NDM-1, E. coli BL21, K. pneumonia KN-1 and A. baumanii aba were engineered bacteria, and E. coli ZJ487 was a clinical isolate.

[0201] (2) Experimental results

[0202] Table 3 Synergistic antibacterial activity of preferred compounds with Mem in NDM-1 producing Escherichia coli

[0203]

[0204] Note: MIC results were determined at least 3 times (n≥3); MIC a Indicates minimum inhibitory concentration; used alone b Indicates that Mem is administered alone; combined use c It represents that the test compound and Mem are mixed and then administered.

[0205] The results are shown in Table 3. When Mem was used alone, the MIC in four NDM-1 expressing E. coli was ≥32 μg / mL, indicating that these four E. coli were resistant to Mem. When Mem was used in combination with the preferred compound, the MIC in the four resistant bacteria decreased by 2-32 times compared with the single compound. Among them, compound C21 showed the strongest synergistic antibacterial activity in the four resistant bacteria. Its MIC decreased by 4-32 times after combined use with Mem, especially in E. coli ZJ487, the MIC after combined use with Mem was less than 1 μg / mL.

[0206] IV. Cytotoxicity test of compounds C19 and C21 of the present invention on Hek-239T and THP-1 cells

[0207] (1) Experimental methods

[0208] 1×10 4Hek-239T cells (human embryonic kidney cells) and THP-1 cells (human monocytic leukemia cells) were seeded in 96-well plates, and repeated 3 times. After 12 hours, fresh medium containing different concentrations (8, 32 and 128 μg / mL) of compounds was replaced, and DMSO was used as a control. To detect cytotoxicity, cells were incubated for 48 hours, and then cell viability was detected using CCK8 reagent.

[0209] (2) Experimental results

[0210] like Figure 1 As shown, when Hek-239T and THP-1 cells were treated with compound C19 at a concentration of 128 μg / mL, their survival rates exceeded 100%; when treated with compound C21 at a concentration of 128 μg / mL, the survival rate of Hek-239T cells exceeded 100%, and the survival rate of THP-1 cells was above 90%, indicating that compounds C19 and C21 had no obvious toxicity to the two cell types.

[0211] V. Stability test of compounds C19 and C21 of the present invention in rat liver homogenate and plasma

[0212] (1) Experimental methods

[0213] 135 μL of rat liver homogenate or plasma was added to the centrifuge tube in sequence, and then the Ebselen derivative at a concentration of 5 mg / mL was added, and the mixture was vortexed and placed in a 37°C constant temperature water bath for incubation. After 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h and 48h of incubation, 600 μL of acetonitrile was added to the centrifuge tube to terminate the reaction. After centrifugation for 15 min, the residual content of the Ebselen derivative was detected by high performance liquid chromatography. 15 μL of water, 600 μL of acetonitrile and 135 μL of rat liver homogenate or plasma were used as blank controls, and the residual content at 0h of incubation was taken as 100%, and the content of the compound at different time periods was calculated.

[0214] (2) Experimental results

[0215] like Figure 2 As shown in the figure, after the compounds C19 and C21 were co-incubated in rat plasma for 48 hours, the remaining content of the compounds was still higher than 80%, indicating that the compounds C19 and C21 were slowly metabolized in rat plasma and had good plasma stability. Figure 2 As shown in Figure B, after incubation in liver homogenate for 48 hours, compounds C19 and C21 remained 73.56% and 59.34%, respectively, indicating that the preferred compounds have a certain stability in liver homogenate and are metabolized relatively slowly in the liver after oral administration.

[0216] VI. Evaluation of the combined antibacterial activity of compound C21 of the present invention in vivo

[0217] (1) Experimental methods

[0218] Female BALB / c mice aged 6-8 weeks, weighing 18-22 g, were randomly divided into different groups and E. coli ZJ487 (at a dose of 5 × 10 6 CFUs) were injected intraperitoneally into mice. 2h after bacterial challenge, DMSO, meropenem (10mg / kg), compound (40mg / kg) or meropenem (10mg / kg) combined with compound (40mg / kg) were injected subcutaneously every 12 hours to observe the therapeutic effect. Mice were killed 96h after infection and their liver and kidney tissues were taken for further testing. Organ samples were homogenized in sterile PBS and the suspension was diluted. The number of CFU was counted on LB plates to determine the bacterial load.

[0219] (2) Experimental results

[0220] 96 hours after mice were infected with bacteria, the number of bacterial colonization in the liver and kidney of mice in different treatment groups was as follows Figure 3 . When Mem was used alone at a concentration of 10 mg / kg, the number of colonies in the liver and kidneys of mice was slightly lower than that of the control group, indicating that Mem had only a weak inhibitory effect on the growth of drug-resistant bacteria in mice; and when compound C21 was used alone at a concentration of 10 mg / kg, there was no significant difference in the number of colonies in the liver and kidneys compared with the control group, indicating that compound C21 had no significant antibacterial effect in mice. When 10 mg / kg of Mem was co-administered with 10 mg / kg of compound C21, the number of colonies in the liver and kidneys of mice was significantly lower than that of the control group and when Mem was used alone. The above results reveal that compound C21 can effectively inhibit the growth and reproduction of drug-resistant bacteria in mice when used in combination with Mem, and can be used as a potential drug for NDM-1 drug-resistant bacterial infection diseases for further application research.

Claims

1. A cephalosporin derivative, characterized in that: The structure is shown in general formula I or a pharmaceutically acceptable salt thereof: In the general formula I, R is selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkylalkyl, alkylacyl, arylalkylacyl, heteroarylacyl, heteroarylalkylacyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl; the alkyl is (C1-C6)alkyl; the alkyl, aryl or heteroaryl is optionally substituted by (C1-C6)alkyl, halogen, hydroxy, trifluoromethyl, cyano, methoxy, amino, or nitro.

2. The cephalosporin derivative according to claim 1, characterized in that R is selected from one of acetyl, 2-cyanoacetyl, benzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl, 3,5-dimethoxybenzyl, 3,5-dimethylbenzyl, 3,5-bis(trifluoromethyl)benzyl, 3-chloro-5-fluorobenzyl, 3-chloro-5-cyanobenzyl, 3-chloro-5-methylbenzyl, 3-chloro-5-methoxybenzyl, 2,6-dichloro-4-pyridyl, 2-thienylmethyl, cyclopropylmethyl, benzoyl, phenylacetyl, cyclopropylacyl, tetrazolyl, thiopheneacyl, methylsulfonyl, phenylsulfonyl, phenylmethylsulfonyl, thiophenesulfonyl and cyclopropylsulfonyl.

3. The cephalosporin derivative according to claim 1, characterized in that The pharmaceutically acceptable salt is a salt formed by hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, maleic acid, fumaric acid, citric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, tartaric acid or acetic acid.

4. A pharmaceutical composition, characterized in that A composition comprising a therapeutically effective dose of a cephalosporin derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. Use of the cephalosporin derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 for preparing an NDM-1 inhibitor.

6. Use of the cephalosporin derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 for preparing a medicament for treating bacterial infectious diseases caused by NDM-1 resistance.

7. Use of the cephalosporin derivative according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in combination with other drugs for preparing a drug for treating bacterial infectious diseases caused by NDM-1 resistance.

8. The use according to claim 7, characterized in that The other drugs are penicillins, cephalosporins, cephamycins, and carbapenem β-lactam antibiotics.

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