A cephalosporin derivative and its use for ndm-1 inhibitors

Abstract

The application belongs to the field of pharmacy and relates to a cephalosporin derivative and use thereof for NDM-1 inhibitor. The structure of the cephalosporin derivative is shown in general formula I: (I); the application is based on the open-loop structure of Ebselen and the structure of cephalosporin, a new cephalosporin derivative containing Se atoms is prepared, the beta-lactam ring of cephalosporin is hydrolyzed and opened by NDM-1, the Ebselen fragment containing Se atoms is released, and the thiol of cysteine in the active region of NDM-1 forms a Se-S covalent bond, so that the activity of NDM-1 is improved, the targeting of NDM-1 is enhanced, the growth of drug-resistant bacteria is effectively inhibited, and the application can be applied to bacterial infectious diseases caused by NDM-1 drug resistance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and relates to a class of compounds for New Delhi metallo-beta-lactamase-1 (NDM-1) drug resistance and medical uses thereof, in particular to a class of selenium-containing cephalosporin derivatives and the use thereof for preparing NDM-1 inhibitors, and treatment of bacterial infections caused by NDM-1 drug resistance. BACKGROUND

[0002] The emergence of antibiotic resistance poses a serious threat to global public health, according to the investigation of Mohsen et al., about 4.95 million people died worldwide in 2019 due to drug-resistant bacteria, of which 1.27 million people died directly due to drug-resistant bacteria (Murray C J L, Ikuta K S, Sharara F, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis [J]. The lancet, 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. NDM-1 can pass through the inner membrane to the periplasm with the help of the signal peptide on its N-terminus. The active region of NDM-1 interacts with penicillin and cephalosporin antibiotics, breaks the C-N bond in the β-lactam ring structure of the antibiotic, and releases the hydrolysis product, leading to 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 various serine β-lactamase inhibitors in clinical use, such as avibactam, sulbactam, etc., but there is no significant inhibitory activity on metallo-β-lactamase NDM-1 (Marrs E C L, Day K M, Perry J D. In vitro activity of mecillinam against Enterobacteriaceae with NDM-1 carbapenemase. J. Antimicrob. Chemother. 2014, 10, 2873-2875.). Due to the rapid spread of NDM-1 worldwide, it is urgent to find NDM-1 inhibitors with good activity and strong specificity.

[0004] Ebselen (Ki = 0.38 mM) is a potent 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 thiol group of cysteine at position 208, thus inhibiting the activity of NDM-1 (Chiou J, Wan S, Chan K F, et al. Ebselen as a Potent Covalent Inhibitor of New Delhi Metallo-ß-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 NDM-1 high-expression engineering strains of Escherichia coli by up to 512-fold, but no significant inhibitory effect was achieved in clinical NDM-1 resistant isolates. Ebselen is prone to form Se-S covalent bonds with the thiol groups of cysteine, glutathione and thiol proteins due to its unstable Se-N structure, which promotes its various biological activities such as anti-inflammatory, anti-viral and anti-bacterial activities. However, Ebselen can also form Se-S covalent bonds with proteins containing thiol groups in the body, thus producing off-target effects and failing to effectively reach the active area of NDM-1 to exert its effect.

[0005] Another class of cephalosporin derivative NDM-1 covalent inhibitor Cefaclor (Ki = 2.4 mM) also has relatively weak inhibitory activity against NDM-1. In addition, cephalosporin drugs can be hydrolyzed by NDM-1. After cefuroxime is co-incubated with NDM-1, the β-lactam ring of cefuroxime is attacked by OH- ions, breaking the C-N bond in the β-lactam ring to form a carboxyl group; at the same time, the lone pair of electrons on the N atom is transferred to the C3 position, causing the C-O bond at the C17 position to break and release a 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 and ring-opened by NDM-1, causing the key active structure to be destroyed and thus losing antibacterial activity, which is not suitable for treating infections caused by NDM-1 resistant bacteria. SUMMARY

[0006] In view of the above problems, the present application aims to provide a cephalosporin derivative with novel structure, high NDM-1 inhibitory activity and Ebselen fragment targeted drug delivery, and use thereof in NDM-1 inhibitors for treating bacterial infectious diseases caused by NDM-1 drug resistance.

[0007] The present application adopts the strategy of introducing Se atom to construct novel cephalosporin derivatives, introduces NDM-1 inhibitor at C17 of cephalosporin to construct a "Trojan horse" molecule, introduces different structures of cephalosporin after ring opening of NDM-1 inhibitor Ebselen, and forms a C-Se bond which is broken after hydrolysis of NDM-1, so as to realize the site-specific release of Ebselen in the active center, realize the targeted drug delivery of Ebselen fragment, form Se-S covalent bond with the thiol group of the active region cysteine of NDM-1, and improve the inhibition of NDM-1 activity and realize the targeted drug delivery.

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

[0009]

[0010] In 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 with (C1-C6)alkyl, halogen, hydroxyl, trifluoromethyl, cyano, methoxy, amino or nitro.

[0011] Further, in general formula I, R is selected from acetyl, 2-cyanoacetyl, benzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl, 3,5-dimethoxybenzyl, 3,5-dimethylbenzyl, 3,5-di(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, tetrazolylacyl, thienoyl, methylsulfonyl, phenylsulfonyl, benzylsulfonyl, thienylsulfonyl, cyclopropylsulfonyl.

[0012] Still further, the present application provides the following compounds, or pharmaceutically acceptable salts thereof, for use in the field of treating bacterial infectious diseases caused by NDM-1 drug 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))acetamido)-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-(methylsulfonamido)-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- sulfinamido)-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)sulfinamido)- 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-l- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C17);

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

[0025] 7-(benzylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-l- 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-l-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-l-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-l-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- l-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-l-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- l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

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

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

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

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

[0036] 7-((3,5-di(trifluoromethyl))benzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl) seleno yl)methyl)-5-thia-l-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 yl)methyl)-5-thia-l-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 yl)methyl)-5-thia-l-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 yl)methyl)-5-thia-l-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 yl)methyl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C34).

[0041] The pharmaceutically acceptable salt of the compound of the present application is a salt formed by a pharmaceutically acceptable inorganic acid or organic acid, 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 application provides a pharmaceutical composition comprising a therapeutically effective amount of the cephalosporin derivative of the present application or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition can be in the form of a conventional tablet or capsule, sustained-release tablet, controlled-release tablet, granule, oral solution, syrup, suppository, transdermal preparation, injection, etc.

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

[0044] Further, the use of the compound of general formula I or a pharmaceutically acceptable salt thereof for the preparation of an antibacterial drug for treating bacterial infectious diseases caused by NDM-1 drug resistance.

[0045] Still further, the use of the above antibacterial drug in combination with other drugs for the preparation of a drug for treating bacterial infectious diseases caused by NDM-1 drug resistance.

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

[0047] Based on the open-loop structure of Ebselen and the structure of cephalosporin, the present application prepares a new cephalosporin derivative containing Se atom. The β-lactam ring of cephalosporin is hydrolyzed and opened by NDM-1, releasing the Ebselen fragment containing Se atom to form 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, enhancing the targeting of NDM-1 and effectively inhibiting the growth of drug-resistant bacteria. The present application can be applied to bacterial infectious diseases caused by NDM-1 drug resistance. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 is a cell viability test chart of the synthesized compounds C19 and C21 of the present application in Hek-239T cells and THP-1 cells.

[0049] (A) is the cell viability test result of compounds C19 and C21 in Hek-239T; (B) is the cell viability test result of compounds C19 and C21 in THP-1.

[0050] Figure 2 is a metabolic stability test chart of the synthesized compounds C19 and C21 of the present application 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 Figure is the effect of the synthetic compound C21 of the present application combined with meropenem (Mem) on the target organ colonization of infected mice. DETAILED DESCRIPTION

[0053] The specific embodiments of the present application are described in further detail below in conjunction with specific examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present application.

[0054] The novel cephalosporin compound described in the present application can be prepared by the following method:

[0055]

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

[0057] (1) Dissolve o-aminobenzoic acid in an aqueous hydrochloric acid solution, slowly add an aqueous sodium nitrite solution to the reaction solution under ice bath conditions to obtain a diazonium salt solution, and then react with freshly prepared Na2Se2 under ice bath conditions to generate diselenide compound C2;

[0058] (2) Dissolve compound C2 in dry dichloromethane and stir in an ice bath, then add 2 drops of N,N-dimethylformamide and oxalyl chloride in sequence to obtain a yellowish solid acyl chloride intermediate. Subsequently, the intermediate is dissolved in dry dichloromethane with aniline to generate acylamide compound C3 through acylation reaction;

[0059] (3) C3 is reduced to selenol compound C4 under the action of zinc powder and dilute hydrochloric acid;

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

[0061] (5) Intermediate C6 first introduces an acetyl group at N through acylation reaction, and then reacts with trifluoroacetic acid to remove the p-methoxybenzyl group to obtain the target compound C7.

[0062] The following novel cephalosporin derivatives are prepared according to the synthesis route of Example C7:

[0063] 7-acetamido-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)selenyl)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)selenyl)methyl)-5-thia- 1 - azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid (C8);

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

[0066] 7-(cyclopropanecarboxamide)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)selenyl)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)selenyl)methyl)-5-thia- 1 - azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid (C11);

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

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

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

[0071] 8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)selenyl)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)selenyl)methyl)-7-((phenylmethyl)sulfonamido)- 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C16);

[0073] 7-(cyclopropanesulfonamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)selenyl)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-((2- thienylmethyl)amino)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C23);

[0075] 7-((3-chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

[0076] 7-((3-chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

[0077] 7-((3-chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

[0078] 7-((3-chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

[0079] 7-((3-chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

[0080] 7-((3-chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

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

[0082] 7-((3-chlorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C25);

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

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

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

[0086] 7-((3,5-di(trifluoromethyl))benzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno yl)methyl)-5-thia-l-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 yl)methyl)-5-thia-l-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 yl)methyl)-5-thia-l-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 yl)methyl)-5-thia-l-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 yl)methyl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C34).

[0091] The application is further described in the following specific examples. It should be understood that these are not intended to limit the scope of the application. Any simple substitution and modification within the scope of the application should be covered by the scope of the application.

[0092] Example 1:

[0093]

[0094] Compound C6 (600 mg, 1 mmol) was dissolved in 20 mL of tetrahydrofuran, pyridine (160 mg, 2 mmol) and formyl chloride (120 mg, 2 mmol) were added successively, and stirred at room temperature for 1 h. TLC detection showed that the reaction was complete. The reaction solution was washed with saturated copper sulfate, and the organic phase was collected. The organic phase was purified by column chromatography to obtain a white solid. A solution of trifluoroacetic acid and anisole was prepared in a ratio of 5:1 by volume, and 18 mL of the solution was stirred at -10 °C for 20 min. The obtained white solid was added to the solution, and the reaction was maintained at -10 °C for 2 h. TLC detection showed that the reaction was complete. The reaction solution was poured into 50 mL of ice water, and the organic phase was collected. 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 to precipitate a large amount of white solid, which was collected by filtration. The white solid was dissolved in 20 mL of acetone solution, and 5 g of sodium iso-octanoate was added to the solution. After stirring for 10 min, the reaction solution was poured into 50 mL of water, washed with ethyl acetate (10 x 3 mL), and the aqueous phase was collected. The aqueous phase was adjusted to pH 4 with trifluoroacetic acid, extracted with ethyl acetate (15 x 3 mL), and the organic phase was collected. The organic phase was dried over anhydrous magnesium sulfate, and concentrated to obtain a semi-solid. The semi-solid was dissolved in 5 mL of ethyl acetate solution, and added dropwise to a mixture of 20 mL of diethyl ether and 20 mL of n-hexane to precipitate a large amount of white solid, which was filtered to obtain 180 mg of white solid; melting point: 112.3-114.2 °C; yield: 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 (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.77 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 7.9 Hz, 2H), 7.63 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.32 (q, J = 8.1 Hz, 3H), 7.07 (t, J = 7.4 Hz, 1H), 5.56 (dd, J = 8.4, 4.7 Hz, 1H), 5.01 (d, J = 4.7 Hz, 1H), 4.02 (d, J = 11.3 Hz, 2H), 3.67 (d, J = 17.3 Hz, 1H), 3.41 (d, J = 17.7 Hz, 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] Example 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, found 607.0672.

[0099] Example 3:

[0100]

[0101] 7-(cyclopropanecarboxamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)- 5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C10), prepared according to the procedure of Example 1, white solid; m.p. 137.6-139.9 °C; yield 25%. 1 H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.34 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 7.4 Hz, 2H), 7.70 - 7.59 (m, 4H), 7.53 (t, J = 7.1 Hz, 1H), 7.45 - 7.39 (m, 3H), 7.36 - (m, 3H), 7.07 (t, J = 7.3 Hz, 1H), 5.82 - 5.69 (m, 1H), 5.12 (d, J = 4.4 Hz, 1H), 4.04 (s, 2H), 3.66 (d, J = 17.3 Hz, 1H), 3.46 (d, J = 17.6 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 177.47, 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] Example 4:

[0103]

[0104] 7-(cyclopropanecarboxamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)- 5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C10), prepared according to the procedure of Example 1, white solid; m.p. 137.6-139.9 °C; yield 25%.1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.94 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.63 - 7.58 (m, 2H), 7.39 (t, J = 7.6 Hz, 1H), 7.31 (t, J = 7.9 Hz, 3H), 7.06 (t, J = 7.4 Hz, 1H), 5.54 (dd, J = 8.5, 4.7 Hz, 1H), 4.94 (d, J = 4.7 Hz, 1H), 4.08 (d, J = 11.4 Hz, 2H), 3.62 (d, J = 17.5 Hz, 1H), 3.36 (d, J = 17.8 Hz, 1H), 1.68 (q, J = 6.3 Hz, 1H), 0.67 (d, J = 7.0 Hz, 4H). 13 C NMR (100 MHz, DMSO-d6) δ 174.12, 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] Example 5:

[0106]

[0107] 7-(2-cyanoacetamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)- 5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C11), prepared according to the procedure of Example 1, white solid; mp 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] Example 6:

[0109]

[0110] 7-(2-(1H-tetrazol-1-yl)acetylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)selenomethyl)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 (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.45 (d, J = 8.4 Hz, 1H), 9.32 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.61 (dd, J = 19.4, 7.7 Hz, 2H), 7.41 (t, J = 7.5 Hz, 1H), 7.33 (q, J = 8.3 Hz, 3H), 7.07 (t, J = 7.4 Hz, 1H), 5.63 (dd, J = 8.4, 4.8 Hz, 1H), 5.39 - 5.25 (m, 2H), 5.06 (d, J = 4.9 Hz, 1H), 4.04 (d, J = 11.2 Hz, 2H), 3.71 (d, J = 17.5 Hz, 1H), 3.35 (d, J = 17.3 Hz, 1H); 13 C NMR (100 MHz, DMSO-d6) δ 167.27, 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 calcd for C 23 H 19 N7O5SSe[M+H] + :585.0334, found 585.0339.

[0111] Example 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 according to the procedure of Example 1, white solid; mp: 103.2-104.1 °C; yield 34%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.33 (d, J = 8.1 Hz, 1H), 7.94 (s, 1H), 7.78 (d, J = 4.9 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.61 (t, J = 7.3 Hz, 2H), 7.40 (t, J = 7.5 Hz, 1H), 7.31 (q, J = 7.6, 7.2 Hz, 3H), 7.12 (s, 1H), 7.06 (t, J = 7.5 Hz, 1H), 5.66 (t, J = 6.4 Hz, 1H), 5.04 (d, J = 4.6 Hz, 1H), 4.04 (q, J = 10.8 Hz, 2H), 3.62 (d, J = 17.3 Hz, 1H), 3.39 (d, J = 17.4 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.35, 164.23, 163.96, 162.09, 139.63, 138.76, 138.20, 132.66, 132.53, 131.71, 131.41, 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, found 599.0095.

[0114] Example 8:

[0115]

[0116] 7-(methylsulfonamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia- 1 -aza bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C14), prepared according to the procedure of Example 1, white solid; mp: 188.3-188.9 °C; yield 37%. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 8.42 (d, J = 9.6 Hz, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.63 (d, J = 7.4 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.3 Hz, 1H), 7.32 (q, J = 8.2 Hz, 3H), 7.07 (t, J = 7.4 Hz, 1H), 5.29 (dd, J = 9.2, 5.0 Hz, 1H), 5.05 (d, J = 4.6 Hz, 1H), 4.08 (d, J = 11.0 Hz, 1H), 3.99 (d, J = 11.0 Hz, 1H), 3.68 (d, J = 17.7 Hz, 1H), 3.44 (d, J = 17.6 Hz, 1H), 2.96 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.26, 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] Example 9:

[0118]

[0119] 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), prepared according to the procedure described in Example 1, white solid; m.p. 163.4-165.1 °C; yield 18%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.24 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 4.0 Hz, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.59 (dd, J = 16.9, 8.4 Hz, 3H), 7.38 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 7.6 Hz, 3H), 7.14 (t, J = 4.4 Hz, 1H), 7.07 (t, J = 7.2 Hz, 1H), 5.24 (s, 1H), 4.89 (s, 1H), 4.06 (d, J = 10.8 Hz, 1H), 3.98 (d, J = 10.8 Hz, 1H), 3.55 (d, J = 17.6 Hz, 1H), 3.34 (d, J = 17.6 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.32, 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, found 634.9767.

[0120] Example 10:

[0121]

[0122] 8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-((phenylmethyl)sulfenamido)-5-thia-1- azonia bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C16), prepared according to the procedure described in Example 1, white solid; m.p. 101.5-103.6 °C; yield 23%. 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 8.56 (d, J = 9.1 Hz, 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.62 - 7.57 (m, 2H), 7.43 - 7.36 (m, 3H), 7.35 - 7.28 (m, 6H), 7.07 (t, J = 7.4 Hz, 1H), 5.18 (s, 1H), 4.95 (s, 1H), 4.38 (s, 2H), 4.11 (d, J = 11.2 Hz, 1H), 4.00 (d, J = 10.9 Hz, 1H), 3.64 (d, J = 17.7 Hz, 1H), 3.43 (d, J = 17.5 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.32, 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 for C 28 H 25 N3O6S2Se[M+H] + : 643.0350, found 643.0357.

[0123] Example 11:

[0124]

[0125] 7-(cyclopropylsulfonamido)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C17), prepared according to the procedure of Example 1, white solid; mp 107.3-109.5 °C; yield 29%. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.45 (d, J = 9.7 Hz, 1H), 7.69 (d, J = 7.9 Hz, 2H), 7.59 (d, J = 6.1 Hz, 2H), 7.39 (t, J = 7.3 Hz, 1H), 7.30 (d, J = 7.7 Hz, 3H), 7.06 (t, J = 7.3 Hz, 1H), 5.21 (s, 1H), 4.95 (d, J = 4.0 Hz, 1H), 4.10 (d, J = 10.9 Hz, 1H), 4.01 (d, J = 10.9 Hz, 1H), 3.62 (d, J = 17.0 Hz, 1H), 3.42 (d, J = 17.2 Hz, 1H), 2.57 - 2.49 (m, 1H), 0.92 (t, J = 6.8 Hz, 4H). 13 C NMR (100 MHz, DMSO-d6) δ 167.34, 164.30, 139.63, 138.25, 132.42, 131.80, 131.39, 129.23, 128.62, 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, found 593.0121.

[0126] Example 12:

[0127]

[0128] 8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-(phenylsulfonamido)-5-thia- 1 -aza bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C18), prepared according to the procedure of Example 1, white solid; mp: 173.8-175.4 °C; yield 38%. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.99 (d, J = 9.2 Hz, 1H), 7.80 (d, J = 7.4 Hz, 2H), 7.68 (d, J = 7.8 Hz, 2H), 7.61 - 7.52 (m, 5H), 7.37 (t, J = 7.4 Hz, 1H), 7.30 (t, J = 7.5 Hz, 3H), 7.07 (t, J = 7.2 Hz, 1H), 5.19 (s, 1H), 4.83 (s, 1H), 4.06 (d, J = 11.1 Hz, 1H), 3.98 (d, J = 11.0 Hz, 1H), 3.52 (d, J = 17.5 Hz, 1H), 3.36 (d, J = 17.8 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.36, 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] Example 13:

[0130]

[0131] 7-(Benzylamino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5- thia- 1 -aza bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C19), prepared according to the procedure described in Example 1, white solid; m.p. 102.7-105.0 °C; yield 36%. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 7.68 (d, J = 7.6 Hz, 2H), 7.63 (d, J = 7.1 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.2 Hz, 1H), 7.37 - 7.27 (m, 7H), 7.25 (d, J = 5.7 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H), 4.96 (s, 1H), 4.72 (s, 1H), 4.08 (d, J = 11.0 Hz, 1H), 3.97 (d, J = 11.1 Hz, 1H), 3.87 (s, 2H), 3.64 (d, J = 17.5 Hz, 1H), 3.41 (d, J = 17.6 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.24, 163.73, 139.57, 138.14, 132.40, 131.66, 131.44, 129.23, 129.08, 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] Example 14:

[0133]

[0134] 7-((3-chloro-5-methoxybenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1- azonia bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C20), prepared according to the procedure described in Example 1, white solid; m.p. 148.9-149.5 °C; yield 40%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.63 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.3 Hz, 1H), 7.35 - 7.28 (m, 3H), 7.07 (t, J = 7.2 Hz, 1H), 6.97 (s, 1H), 6.89 (s, 2H), 4.95 (d, J = 4.1 Hz, 1H), 4.72 (s, 1H), 4.07 (d, J = 11.1 Hz, 1H), 3.97 (d, J = 11.3 Hz, 1H), 3.84 (s, 2H), 3.73 (s, 3H), 3.65 (d, J = 17.6 Hz, 1H), 3.42 (d, J = 17.5 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.25, 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 calcd for C 29 H 26 ClN3O5SSe[M+H] + : 643.0447, found 643.0445.

[0135] Example 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 according to the procedure described in Example 1, white solid; m.p. 130.8-133.1 °C; yield 33%. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 7.68 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.39 (d, J = 16.3 Hz, 4H), 7.34 - 7.28 (m, 3H), 7.06 (t, J = 7.3 Hz, 1H), 4.87 (d, J = 4.2 Hz, 1H), 4.64 (s, 1H), 4.06 (d, J = 11.1 Hz, 1H), 3.94 (d, J = 11.0 Hz, 1H), 3.78 (s, 2H), 3.64 (d, J = 17.5 Hz, 1H), 3.34 (d, J = 17.4 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.96, 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, found 646.9961.

[0138] Example 16:

[0139]

[0140] 7-((3,5-difluorobenzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia- 1 -aza bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C22), prepared according to the procedure of Example 1, white solid; mp: 109.3-111.2 °C; yield 21%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.62 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.2 Hz, 1H), 7.32 (q, J = 7.9 Hz, 3H), 7.05 (dd, J = 15.5, 7.8 Hz, 4H), 4.89 (d, J = 4.3 Hz, 1H), 4.65 (s, 1H), 4.05 (d, J = 11.0 Hz, 1H), 3.94 (d, J = 11.0 Hz, 1H), 3.80 (s, 2H), 3.65 (d, J = 17.8 Hz, 1H), 3.35 (d, J = 17.5 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 168.02, 167.26, 164.15 (d, J = 12.9 Hz), 163.89, 161.71 (d, J = 12.9 Hz), 145.98, 139.56, 138.24, 132.37, 131.68 (d, J = 26.3 Hz), 129.22, 128.69, 128.53, 126.64, 124.70, 124.29, 120.44, 111.35 (d, J = 24.9 Hz), 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] Example 17:

[0142]

[0143] 8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-7-((2- thienylmethyl)amino)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C23), prepared according to the procedure of Example 1, white solid; mp: 133.1-134.2 °C; yield 38%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.41 - 7.28 (m, 5H), 7.06 (t, J = 7.3 Hz, 1H), 6.96 (s, 1H), 6.93 (d, J = 3.9 Hz, 1H), 4.91 (d, J = 4.4 Hz, 1H), 4.65 (d, J = 4.3 Hz, 1H), 4.06 (d, J = 11.2 Hz, 1H), 3.97 (s, 2H), 3.94 (d, J = 11.0 Hz, 1H), 3.64 (d, J = 17.9 Hz, 1H), 3.37 (d, J = 17.6 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.67, 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 for C 26 H 23 N3O4S2Se[M+H] + : 585.0295, found 585.0299.

[0144] Example 18:

[0145]

[0146] 7-((cyclopropylmethyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno yl)methyl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C24), prepared according to the procedure described in Example 1, white solid; m.p. 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] Example 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 as described 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] Example 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 as described in Example 1, white solid; melting point: 108.4-109.6°C; yield 29%. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 7.73 - 7.65 (m, 2H), 7.64 (dd, J = 7.5, 1.6 Hz, 1H), 7.57 (dd, J = 7.8, 1.3 Hz, 1H), 7.41 (dd, J = 7.5, 1.6 Hz, 1H), 7.38 (s, 4H), 7.34 - 7.30 (m, 3H), 7.10 - 7.04 (m, 1H), 4.99 (d, J = 4.7 Hz, 1H), 4.78 (d, J = 4.6 Hz, 1H), 4.09 (d, J = 11.1 Hz, 1H), 3.99 (d, J = 11.1 Hz, 1H), 3.92 (d, J = 4.3 Hz, 2H), 3.65 (d, J = 17.5 Hz, 1H), 3.45 (d, J = 17.4 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.24, 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, found 613.0344.

[0153] Example 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 according to the procedure of Example 1, white solid; mp: 123.7-125.1 °C; yield 32%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.41 - 7.22 (m, 7H), 7.07 (t, J = 7.3 Hz, 1H), 4.92 (d, J = 4.2 Hz, 1H), 4.71 (s, 1H), 4.06 (d, J = 11.3 Hz, 1H), 3.94 (d, J = 11.3 Hz, 1H), 3.88 (s, 2H), 3.65 (d, J = 17.6 Hz, 1H), 3.38 (d, J = 17.7 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.75, 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 for C 28 H 24 ClN3O4SSe[M+H] + : 613.0341, found 613.0349.

[0156] Example 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 according to the procedure of Example 1, white solid; mp 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] Example 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 as described in Example 1, white solid; melting point: 131.2-134.1°C; yield 36%. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 7.76 - 7.61 (m, 3H), 7.58 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.1 Hz, 1H), 7.37 - 7.26 (m, 3H), 7.07 (t, J = 7.3 Hz, 1H), 6.95 (d, J = 13.5 Hz, 3H), 5.03 (s, 1H), 4.82 (s, 1H), 4.12 (d, J = 11.1 Hz, 1H), 4.01 (d, J = 10.8 Hz, 1H), 3.90 (s, 2H), 3.65 (d, J = 17.2 Hz, 1H), 3.49 (d, J = 17.2 Hz, 1H), 2.23 (s, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 167.23, 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] Example 24:

[0163]

[0164] 7-((3,5-Di(trifluoromethyl))benzyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl)seleno)methyl)-5-thia-1- azonia bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C30), prepared according to the procedure described in Example 1, white solid; m.p. 106.7-108.1 °C; yield 31%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 8.03 (d, J = 1.7 Hz, 2H), 7.91 (s, 1H), 7.71 - 7.64 (m, 2H), 7.62 (dd, J = 7.5, 1.7 Hz, 1H), 7.56 (dd, J = 7.8, 1.3 Hz, 1H), 7.39 (td, J = 7.6, 1.6 Hz, 1H), 7.35 - 7.27 (m, 3H), 7.10 - 7.02 (m, 1H), 4.90 (d, J = 4.7 Hz, 1H), 4.73 (d, J = 4.7 Hz, 1H), 4.05 (d, J = 11.2 Hz, 1H), 3.98 (s, 2H), 3.94 (d, J = 11.2 Hz, 1H), 3.64 (d, J = 17.8 Hz, 1H), 3.38 (d, J = 17.7 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.26, 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, found 715.0473.

[0165] Example 25:

[0166]

[0167] 7-((2,6-dichloro-4-pyridinyl)methyl)amino)-8-oxo-3-(((2-(phenylaminocarbonyl)phenyl) seleno)methyl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C31), prepared according to the procedure of Example 1, white solid; mp 109.4-111.8 °C; yield 38%. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.62 (d, J = 7.0 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.50 (s, 2H), 7.39 (t, J = 7.3 Hz, 1H), 7.32 (q, J = 7.8 Hz, 3H), 7.06 (t, J = 7.4 Hz, 1H), 4.89 (d, J = 4.4 Hz, 1H), 4.68 (s, 1H), 4.06 (d, J = 11.2 Hz, 1H), 3.94 (d, J = 11.2 Hz, 1H), 3.84 (s, 2H), 3.65 (d, J = 17.4 Hz, 1H), 3.35 (d, J = 17.6 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 168.02, 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] Example 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 according to the procedure of Example 1, white solid; mp: 103.8-105.5 °C; yield 26%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.71 - 7.65 (m, 2H), 7.63 (dd, J = 7.5, 1.7 Hz, 1H), 7.57 (dd, J = 7.8, 1.3 Hz, 1H), 7.39 (td, J = 7.6, 1.7 Hz, 1H), 7.36 - 7.23 (m, 5H), 7.17 (dt, J = 9.6, 2.2 Hz, 1H), 7.07 (ddt, J = 8.6, 7.3, 1.2 Hz, 1H), 4.92 (d, J = 4.7 Hz, 1H), 4.70 (d, J = 4.7 Hz, 1H), 4.06 (d, J = 11.2 Hz, 1H), 3.95 (d, J = 11.2 Hz, 1H), 3.84 (s, 2H), 3.65 (d, J = 17.7 Hz, 1H), 3.40 (d, J = 17.6 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 177.47, 167.25, 163.80, 161.41, 139.57, 138.22, 134.26 (d, J = 11.8 Hz), 132.37, 131.69 (d, J = 26.8 Hz), 129.22, 128.70, 126.64, 124.81, (d, J = 13.4 Hz), 124.28, 120.44, 115.18 (d, J = 25.4 Hz), 114.50 (d, J = 21.5 Hz), 68.35, 58.83, 50.92, 29.68, 28.34. HRMS (ESI) m / z calcd for C 28 H 23 ClFN3O4SSe[M+H] + : 631.0247, found 631.0245.

[0171] Example 27:

[0172]

[0173] 7-((3-chloro-5-cyanobenzyl)amino)-8-oxo-3-((2-(phenylaminocarbonyl)phenyl) seleno)methyl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C33), prepared according to the procedure described in Example 1, white solid; m.p. 106.9-107.5 °C; yield 43%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.87 (s, 1H), 7.75 (s, 2H), 7.68 (d, J = 7.7 Hz, 2H), 7.62 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.3 Hz, 1H), 7.32 (q, J = 8.0 Hz, 3H), 7.07 (t, J = 7.2 Hz, 1H), 4.89 (d, J = 4.3 Hz, 1H), 4.68 (d, J = 4.0 Hz, 1H), 4.05 (d, J = 10.9 Hz, 1H), 3.94 (d, J = 11.2 Hz, 1H), 3.84 (s, 2H), 3.62 (d, J = 17.5 Hz, 1H), 3.38 (d, J = 17.7 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 167.65, 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] Example 28:

[0175]

[0176] 7-((3-chloro-5-methylbenzyl)amino)-8-oxo-3-((2-(phenylaminocarbonyl)phenyl) seleno)methyl)-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (C34), prepared according to the procedure of Example 1, white solid; mp: 133.1-133.8 °C; yield 37%. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 7.69 - 7.62 (m, 3H), 7.58 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.4 Hz, 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 = 3.7 Hz, 1H), 4.79 (s, 1H), 4.10 (d, J = 11.2 Hz, 1H), 3.99 (d, J = 10.9 Hz, 1H), 3.88 (d, J = 14.1 Hz, 2H), 3.65 (d, J = 17.4 Hz, 1H), 3.46 (d, J = 17.3 Hz, 1H), 2.27 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.24, 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] Example 29:

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

[0179] First, the residual activity of some compounds of the present application on NDM-1 enzyme was tested.

[0180] (1) Experimental method

[0181] Cefalotin was selected as the substrate, and Ebselen was used as the positive control to determine the inhibitory activity of Ebselen derivatives on NDM-1. In a 96-well plate, 6 nmol / L of NDM-1 enzyme and a fixed concentration of 64 μg / mL of Ebselen derivative were sequentially added, and after incubation at 25°C for 15 min, cefalotin was added, and then the plate was placed in an enzyme marker, and the absorbance was recorded at a wavelength of 492 nM for more than 30 scanning cycles. The negative control was not added to the drug group, and the residual activity of NDM-1 at a concentration of 64 μg / mL of Ebselen derivative was calculated. The test was repeated at least 3 times in parallel, and finally the RA value was calculated by GraphPad Prism 8 software.

[0182] RA(%) = OD Drug / OD Blank x 100.

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

[0184] (2) Experimental results

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

[0186]

[0187]

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

[0189] Second, the inhibition rate of some compounds of the present application on NDM-1 enzyme

[0190] (1) Experimental method

[0191] Cefalotin was selected as the substrate, and Ebselen was used as the positive control to determine the inhibitory activity of Ebselen derivatives on NDM-1. In a 96-well plate, 6 nmol / L NDM-1 enzyme and different concentrations of Ebselen derivatives (concentration range 1-128 μg / mL) were sequentially added, and after incubation at 25°C for 15 min, cefalotin was added, and then placed in an enzyme marker, and monitored at a wavelength of 492 nM for more than 30 scanning cycles. The negative control was not added to the drug group, and the inhibition rate of Ebselen derivatives at different concentrations was calculated. The test was repeated at least 3 times, and finally the IC 50 value was calculated using GraphPad Prism 8 software.

[0192]

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

[0194] (2) Experimental results

[0195] Table 2 Half-inhibitory concentration IC 50

[0196]

[0197] The experimental results are shown in Table 2, and the results show that the IC 50 values of the preferred compounds on NDM-1 are between 7.03-150.27 μM, and the compound C21 exhibits the strongest NDM-1 inhibitory activity, with an IC 50 value of 7.03±0.96 μM, which is significantly better than the activity of the positive control Ebselen. 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 treating NDM-1 resistant bacteria for further treatment.

[0198] Third, the synergistic antibacterial activity test of some compounds of the present application combined with Mem in NDM-1 producing E. coli

[0199] (1) Experimental method

[0200] The minimum inhibitory concentration MIC of Ebselen derivatives combined with Mem was determined by broth dilution method according to CLSIM24 guidelines. Mem at a concentration of 64 μg / mL was diluted by broth and added to 96-well plates, and then bacterial suspension and compounds at a concentration range of 0-64 μg / mL were added in turn, and the plates were incubated in a constant temperature incubator at 37 °C for 24 h. The growth state of bacteria in each well was observed by naked eye, and whether there was bacterial growth was determined by enzyme-labeled instrument to determine the MIC of the compounds and Mem combination. In the selected NDM-1 expressing E. coli, E. coli BL21, K. pneumonia K-N-1 and A. baumanii aba were engineering bacteria, and E. coli ZJ487 was a clinical isolated bacteria.

[0201] (2) Experimental results

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

[0203]

[0204] Note: MIC results were determined at least 3 times (n≥3); MIC a The minimum inhibitory concentration is indicated; Mem alone b Mem alone is indicated; combination c The tested compound and Mem were mixed and then administered.

[0205] The results are shown in Table 3. When Mem was used alone, the MIC in the four NDM-1 expressing E. coli was ≥32 μg / mL, indicating that the four E. coli were resistant to Mem. When Mem was combined with the preferred compounds, the MIC in the four resistant bacteria was reduced by 2-32 times compared with when Mem was used alone. Compound C21 showed the strongest synergistic antibacterial activity in the four resistant bacteria, and the MIC of the combination of C21 and Mem was reduced by 4-32 times, especially in E. coli ZJ487, the MIC of the combination was less than 1 μg / mL.

[0206] Four, cytotoxicity test of compounds C19 and C21 of the application on Hek-239T and THP-1

[0207] (1) Experimental method

[0208] 1 × 10 4HEK-239T cells (human embryonic kidney cells) and THP-1 cells (human monocytic leukemia cells) were seeded in 96-well plates in triplicate. After 12 hours, the culture medium was replaced with fresh medium containing various concentrations of compound (8, 32, and 128 μg / mL). DMSO was used as a control. To test cytotoxicity, cells were incubated for 48 hours, and cell viability was then assessed using a CCK8 assay.

[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 in Rat Liver Homogenate and Plasma

[0212] (1) Experimental methods

[0213] To a centrifuge tube, 135 μL of rat liver homogenate or plasma was added, followed by a 5 mg / mL ebselen derivative. The mixture was vortexed and incubated in a 37°C water bath. At 0, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours of incubation, the reaction was terminated by adding 600 μL of acetonitrile to the tube. After centrifugation for 15 minutes, the remaining ebselen derivative content was determined by high-performance liquid chromatography. A blank control of 15 μL of water, 600 μL of acetonitrile, and 135 μL of rat liver homogenate or plasma was used. The compound content at different time periods was calculated, with the remaining content at 0 hour of incubation as 100%.

[0214] (2) Experimental results

[0215] like Figure 2 As shown in Figure 2, after 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 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, 73.56% and 59.34% of compounds C19 and C21 remained, respectively, indicating that the preferred compounds have 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 method

[0218] BALB / c mice of 6-8 weeks old and weighing 18-22 g were selected and randomly divided into different groups. E. coli ZJ487 (dose of 5 x 107CFUs) was injected intraperitoneally into the mice. After 2 hours of bacterial challenge, DMSO, meropenem (10 mg / kg), compound (40 mg / kg), or meropenem (10 mg / kg) combined with compound (40 mg / kg) were injected subcutaneously every 12 hours to observe the therapeutic effect. The mice were sacrificed 96 hours after infection, and their liver and kidney tissues were further detected. The organ samples were homogenized in sterile PBS and diluted in suspension, and the number of CFUs was counted on LB plates to determine the bacterial load. 6 CFUs) was injected intraperitoneally into the mice. After 2 hours of bacterial challenge, DMSO, meropenem (10 mg / kg), compound (40 mg / kg), or meropenem (10 mg / kg) combined with compound (40 mg / kg) were injected subcutaneously every 12 hours to observe the therapeutic effect. The mice were sacrificed 96 hours after infection, and their liver and kidney tissues were further detected. The organ samples were homogenized in sterile PBS and diluted in suspension, and the number of CFUs was counted on LB plates to determine the bacterial load.

[0219] (2) Experimental results

[0220] After 96 hours of bacterial infection in mice, the number of bacterial colonization in the liver and kidney of mice in different treatment groups was as follows: Figure 3 When Mem alone was used at a concentration of 10 mg / kg, the number of colony colonization in the liver and kidney of mice was slightly lower than that of the control group, indicating that Mem had only weak inhibition on the growth of drug-resistant bacteria in mice. When compound C21 alone was used at a concentration of 10 mg / kg, there was no significant difference in the number of colony colonization in the liver and kidney compared with the control group, indicating that compound C21 had no significant antibacterial effect in mice. When 10 mg / kg of Mem was combined with 10 mg / kg of compound C21, the number of colony colonization in the liver and kidney of mice was significantly lower than that of the control group and Mem alone. The above results revealed that the combination of compound C21 and Mem could effectively inhibit the growth and reproduction of drug-resistant bacteria in mice, and could 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: a structure represented by the general formula I or a pharmaceutically acceptable salt thereof: I； In the general formula I, R is selected from one of 2-cyanoacetyl, benzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 3, 5-dichlorobenzyl, 3, 5-difluorobenzyl, 3, 5-dimethoxybenzyl, 3, 5-dimethylbenzyl, 3, 5-di(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, tetrazolylacyl, thienoyl, methylsulfonyl, phenylsulfonyl, benzylsulfonyl, thienylsulfonyl, cyclopropylsulfonyl.

2. The cephalosporin derivative according to claim 1, wherein, The pharmaceutically acceptable salt is a salt formed with 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.

3. A pharmaceutical composition, characterized by, A therapeutically effective amount of the cephem derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier.

4. Use of the cephem derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 for the manufacture of an NDM-1 inhibitor.

5. Use of the cephem derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 for the manufacture of a medicament for the treatment of a bacterial infectious disease caused by NDM-1 resistance.

6. Use of the cephem derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 in combination with other drugs for the manufacture of a medicament for the treatment of a bacterial infectious disease caused by NDM-1 resistance.

7. Use according to claim 6, characterized in that, The other drug is a β-lactam antibiotic.

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