CN109535180A

CN109535180A - The synthesis and application of the antibacterials ASC of a kind of targeted therapy Methicillin-resistant Staphylococcus aureus, S. aureus L-forms and superbacteria infection

Info

Publication number: CN109535180A
Application number: CN201910082057.3A
Authority: CN
Inventors: 杨科武; 许立伟; 张亦琳; 刘雅; 康鹏伟
Original assignee: Northwest University
Current assignee: Northwest University
Priority date: 2018-03-21
Filing date: 2019-01-28
Publication date: 2019-03-29
Anticipated expiration: 2039-01-28
Also published as: CN109535180B; CN108794508A

Abstract

The present invention relates to a kind of targeted therapy Methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus L-forms (SAU) infection and the synthesis and application of the antibacterials ASC for cooperateing with antibiotic treatment superbacteria to infect.The effective concentration that ASC treats MRSA infection is 2 μ g/mL, 4 times stronger than the effect of standard drug oxacillin；The dosage specific treatment mouse core of 35 mg/kg, lung MRSA infection, and significantly reduce the colonization amount of MRSA in its liver,spleen,kidney tissue.The broad spectrum inhibitors of ASC or a kind of antibiotics resistance target protein metallo-β-lactamase, beta-lactam, aminoglycoside and tetracycline medication targeted therapy SAU infection can be cooperateed with: the curative effect of these antibiotic that the dosage of 1 μ g/mL makes improves 4-128 times, and the dosage of 64 or 8 μ g/mL makes Aneet to the drug-fast bacteria for producing NDM-1 or production CcrAE.coliCurative effect improve 32 times.

Description

What a kind of targeted therapy Methicillin-resistant Staphylococcus aureus, S. aureus L-forms and superbacteria infected The synthesis and application of antibacterials ASC

Technical field

The present invention relates to a kind of targeted therapy Methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus L-forms (SAU) infection and collaboration resists The synthetic method and application of the antibacterials ASC of raw extract for treating superbacteria infection, belongs to field of medicinal chemistry.

Background technique

" superbacteria " drug resistance that metallo-β-lactamase (Metallo- β-lactamases, M β Ls) mediates causes almost All antibiotic failures clinically used.Thus, it is the World Health Organization to the monitoring and inhibition that produce M β Ls drug-resistant bacteria (WHO), the hot spot that national governments and pharmaceuticals industry are paid close attention at present.Mistake of the antibiotic in terms of clinical, agricultural and animal husbandry Degree uses, and a large amount of drug-resistant bacteria is caused to generate, so that there are huge choosings when selection tackles the drug of these drug-resistant bacterias Select pressure.In recent years, strain such as pseudomonas aeruginosa, staphylococcus aureus, the Bao Man for the production M β Ls being clinically separated to are not Lever bacterium and Bacillus anthracis etc. obviously increase, and its drug resistance is more and more stronger, carry the " super of NDM-1 so that developing to Grade bacterium ", in global spread, there is no any drug to tackle so far from 2010.The drug resistance history of antibiotic is looked back, one new Antibiotic comes into operation, it is long then 4 years, it is short that then less than 1 year, there have been corresponding drug-resistant bacterias.As it can be seen that antibiotics are opened Hair speed does not catch up with the paces of bacterial resistance generation much.So tackling drug-resistant bacteria by continuous research and development antibiotics Road is passless.The ideal strategy for coping with antibiotics resistance is the inhibitor of research and development M β Ls, cooperates with to come with antibiotic with it Inhibit.

Since penicillin is found, beta-lactam antibiotic has been developed as main force's antibacterials clinically, The antibacterials about 60% clinically used at present are beta-lactam antibiotic.Beta-lactam antibiotic is broad spectrum antibiotic One of object, and the treatment most successful drug of bacterium infection.Beta-lactam antibiotic has hundreds of, and by structure, it is broadly divided into Four classes: penicillins, Carbapenems, cephalosporins and monoamides ring class.Beta-lactam antibiotic molecule contains one A beta-lactam nucleus, the structure change of side chain form different antibiotic.The mechanism of action of beta-lactam antibiotic is logical It is often the synthesis for inhibiting transpeptidase, to prevent the synthesis of cell wall.

Excessive use of the antibiotic in terms of clinical, agricultural and animal husbandry, causes a large amount of drug-resistant bacteria to generate.Bacterium pair Beta-lactam antibiotic generates there are many mechanism of drug resistance, and most important one mechanism is to generate beta-lactamase.Bacterium Beta-lactam nucleus by producing beta-lactam enzymatic hydrolysis beta-lactam antibiotic shows drug resistance.Currently, There are more than 1000 kinds through determining beta-lactamase, these enzymes are classified as tetra- groups of A, B, C and D.Wherein A, C and D group enzyme are referred to as For serine beta-lactamase (Serine- β-lactamases, S β Ls), it is interior that serine residue disguises as nucleophilic group attack β- The carbonyl carbon of amide ring is played a role with hydrolyzing antibiotic.And B group enzyme, i.e. metallo-β-lactamase (Metallo- β- Lactamases, M β Ls), active normal performance often relies on Zn (II) ion.According to gene order and with Zn (II) The difference of ions binding mode, M β Ls are further classified as tri- subgroups of B1, B2, B3.M β Ls hydrolysis is nearly all known Antibiotic still can clinically be used to handle the super drug-resistant bacteria for carrying M β Ls there are no any drug so far.

The bacterial resistance mediated in view of beta-lactamase gets worse the threat of human health, and people, which have done, largely to grind Study carefully in the inhibitor for finding beta-lactamase.1972, the inhibitor clavulanic acid of serine beta-lactamase was found.1984 Year, clavulanic acid is used for clinic by FDA approval as the inhibitor of first serine beta-lactamase.Clavulanic acid molecule contains There is a beta-lactam nucleus, an acyl group multienzyme complex can be formed in conjunction with target protein activity center, then resets and form amide The activity of intermediate and irreversible inhibition enzyme.Then, clavulanic acid and Amoxicillin combination are developed Amoxicillin-carat and are tieed up Sour potassium piece (also referred to as Amoxicillin/potassium Clavulanat), successfully realizes beta-lactamase inhibitor and cooperateing with for beta-Lactam antibiotic is antibacterial Efficiency.This is infected for the first time using collaboration theory treatment drug-resistant bacteria in human history.But regrettably, clavulanic acid and anti- The combination of raw element is but invalid to the drug-resistant bacteria for producing metallo-β-lactamase.

Staphylococcus aureus (Staphylococcus aureus) is gram-positive bacteria, belongs to firmicutes, is common in Nasal cavity, respiratory tract and the skin histology of human body.In recent years, S. aureus L-forms have become one of global main pathogenic bacteria, and partly cause is Because its wide-scale distribution in public places, increases the risk infected repeatedly.In addition, S. aureus L-forms get over the drug resistance of antibiotic To be more obvious.S. aureus L-forms can produce a variety of enzymes, such as protects the coagulase of bacterial cell, decomposes the hyaluronic acid of hyaluronic acid Enzyme, the lipase of fat of degrading, the staphylococcus kinases of solution fibrin, the deoxidation walnut nucleotidase for destroying DNA, Yi Jizao At S. aureus L-forms to drug resistant beta-lactamase of antibiotic etc..S. aureus L-forms can release penicillase, which can hydrolyze antibiosis The beta-lactam nucleus of element, so that such antibiotic loses the destruction to bacterium.

The staphylococcus aureus being positive to methicillin, oxacillin, Cefoxitin in drug resistance or mecA gene Be defined as methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus, MRSA).More than 40 years confessed one's crime since plant MRSA detection, MRSA infection are in rising trend always all over the world.In United States Hospital Monitoring of infection system (NNIS) report, 182 hospital MRSA in 1975 account for the 2.4% of S. aureus L-forms infection sum, rise within 1997 To 24.8%.After the nineties, the ground such as domestic capital, Shanghai large hospital MRSA separation rate be more than S. aureus L-forms infection sum 50% with On.The abuse of antibiotic results in MRSA multidrug resistant phenomenon and gets worse, such as to beta-lactam, macrolides, four The Multiple Classes of Antibiotics such as ring element class, aminoglycoside, fluoroquinolones and sulfamido produce different degrees of drug resistance.Glycopeptide class Antibiotic vancomycin is the last line of defense for treating MRSA infection, but vancomycin resistance staphylococcus aureus has occurred (Vancomycin intermediary-resistant Staphylococcus aureus, VISA) and vancomycin resistance gold Staphylococcus aureus (Vancomycin resistant Staphylococcus aureus, VRSA).In recent years, to benefit how azoles The anti-MRSA drug such as amine and Daptomycin also finds its drug-fast bacteria in succession.Therefore, the new drug that searching is effective against MRSA has been compeled In the eyebrows and eyelashes.

The resistance mechanism of MRSA specifically includes that (1) great expression is encoded by mecA gene and generates penicillin binding protein (PBP2a).Beta-lactam antibiotic can inhibit the activity of transpeptidase PBP2, but in the presence of antibacterials, PBP2a easily with PBP2 formed compound and supplement or compensatory antibacterials inhibit PBP2 function, to continue to the conjunction of bacteria cell wall At；(2) generate beta-lactamase, the beta-lactam nucleus of catalyzing hydrolysis beta-lactam antibiotic and show drug resistance.

To sum up, the bacterial resistance that beta-lactamase mediates gets worse the threat of human health.Although silks such as carat dimensions Propylhomoserin beta-lactamase inhibitor, which is combined antibiotic, has good inhibitory effect to Gram-negative drug-fast bacteria.However, its is right The drug-resistant bacteria for carrying metallo-β-lactamase is invalid.So far, can clinically be used to handle carrying gold without any drug Belong to the super drug-resistant bacteria of beta-lactamase.

Summary of the invention

The targeted therapy for having both antibacterial agent and bacterial resistance target enzyme inhibitor the object of the present invention is to provide one kind is golden yellow The novel antibacterial drug ASC and its synthetic method of color staphy lococcus infection.

To achieve the goals above, the present invention is realized by following technical proposals:

Compound shown in general structure (I):

R₁For

R is-NH₂,-CHO ,-COOH ,-OH ,-Cl or-Br；

R₂For-NH₂Or-H；

R₃For

X is N or S.

Further, any compound being as follows such as formula (I) compound represented:

The preparation method of above compound, comprising the following steps:

(1) in the presence of triethylamine, A1 uses R in acetonitrile₃- Cl acylation is prepared into A2；

(2) A2 sodium iodide iodide reaction is added in acetone, A3 is made；

(3) in the presence of N-methylmorpholine, A3, which reacts in chloroform with following sulfydryl azole compounds, is made A4；

(4) in the presence of methyl phenyl ethers anisole, A4 obtains ASC with trifluoroacetic acid deprotection group in methylene chloride；

It specifically describes, in above-mentioned steps (1), by A1 and 2,6- lutidines is dissolved in acetonitrile and is cooled to 0 DEG C, and acyl is added dropwise Then chlorine is added the triethylamine of equimolar ratio, is warmed to room temperature after being stirred overnight；Vacuum distillation removes solvent, and gained residual is dissolved in Ethyl acetate respectively washed once with saturated sodium bicarbonate solution and saturated salt solution, and organic phase is dry with anhydrous magnesium sulfate, takes out It filters, be evaporated under reduced pressure to A2.

In above-mentioned steps (2), A2 and sodium iodide are dissolved in acetone, are stirred at room temperature, remove gained residual after solvent be added water, Ethyl acetate extraction, merges organic phase, saturated sodium thiosulfate solution and saturated common salt water washing, organic phase anhydrous magnesium sulfate A3 is made in dry, vacuum distillation.

In above-mentioned steps (3), in the presence of N-methylmorpholine, TLC tracking rises in chloroform after reaction to A3 and B4 It warms to room temperature, removes solvent, column silica gel isolates and purifies to obtain A4.

In above-mentioned steps (4), in the presence of methyl phenyl ethers anisole, A4 uses trifluoroacetic acid deprotection group in methylene chloride, wait react After, vacuum distillation removes solvent, and gained residual washs to obtain ASC with ether.

Application of the above compound in preparation collaboration antibiotic magnetic target therapy infection of staphylococcus aureus drug.

The above compound is a kind of inhibitor of bacterial resistance target protein metallo-β-lactamase, can cooperate with as drug Beta-lactam antibiotic treats the super drug-fast bacteria that metallo-β-lactamase mediates, and treats MRSA infection.

The beta-lactam antibiotic is one of penicillins, cephalosporins and carbapenem antibiotic Or it is a variety of.The present invention provides at least the test of the drugs such as compound collaboration Cefazolin, Imipenem.

It the invention has the benefit that ASC provided by the invention is not only a kind of antibacterial agent in itself, but also is a kind of The broad spectrum type inhibitor of beta-lactam antibiotic drug resistance target protein metallo-β-lactamase.Beta-lactam of the present invention is anti- Raw element, refers in molecule and contains by the molecular beta-lactam nucleus class antibiotic of four originals, including common penicillins, cephalo One of bacteriums and carbapenem antibiotic are a variety of.ASC can cooperate with beta-lactam, aminoglycoside and tetracycline Class three classes 7-8 kind antibiotic (benzyl penicillin, ampicillin, Cefazolin sodium, Ceftriaxone Sodium, Cefotaxime Sodium, Imipenem, Kanamycins, tetracycline etc.) magnetic target therapy staphylococcus aureus infection.It is combined the ASC of 1 μ g/mL dosage, so that these The vigor of antibiotic improves 4-128 times.ASC and ampicillin show optimum synergistic Antifungal activity, that is, are combined 1 μ g/mL dosage ASC, using clinically the ampicillin of dosage 1/128 can inactivate staphylococcus aureus at present；One 2 μ g/mL dosage ASC-NA almost 100% ground treatment staphylococcus aureus infection, efficiency maintain at least 48 hours or more.

ASC can cooperate with Cefazolin targeted inhibition carry plasmid pET-26-L1, pET-26-NDM-1, pET-26-VIM-2, The super drug-resistant bacteria E.coli of pET-26-CcrA or pET-26-ImiS.ANB1H, ANB0H of combination 64 μ g/mL of dosage, ANB04N or ANA2C, so that Aneet treatment produces the vitality restoration of the super drug-resistant bacteria E.coli-DH10B of NDM-1 32 Times.It is combined ASC-NA, ANB04O or ANA2C of 8 μ g/mL, so that Aneet treatment produces the drug-fast bacteria E.coli-BL21's of CcrA Vigor improves 32 times.It is combined the ASC-NB or ABA2C of 16 μ g/mL, so that Aneet treats the vitality restoration of E.coli 64 Times.

In addition, ASC can effectively treat the infection of MRSA, wherein the therapeutic effect of ANB04N is best (MIC=2 μ g/mL), Effect than oxacillin inactivation MRSA is by force up to 4 times.ASC-NB shows dose dependent inactivation performance to MRSA, and a dosage is Almost 100% ground inactivates MRSA to the ASC-NB of 16 μ g/mL, and the efficiency that sterilizes maintenance 48 hours or more.Animal (mouse) tests table Bright, ASC-NB extremely effective treats the MRSA of mouse infection.The ASC-NB that one dosage is 35mg/kg, so that mouse heart It is killed with the MRSA almost all of pulmonary infection, after a week still without bacterial reproduction；With the ASC-NB of dosage, but also mouse The colonization amount of MRSA significantly reduces in liver, spleen and nephridial tissue.ASC-NB treats the efficiency of MRSA with administration concentration and administration time Difference and be in dependent change.Obviously, ASC-NB has good therapeutic effect to the MRSA of mouse infection, this indication: ASC tool There is a possibility that potential drug for becoming clinical treatment MRSA infection.This patent has filled up world's blank.

Detailed description of the invention

Fig. 1 be ASC-NA to staphylococcus aureus inactivation efficiency at any time and its variation of concentration；

Fig. 2 is the dosage and time dependent change that Aneet inactivates MRSA；

Fig. 3 is the dosage and time dependent change that ASC-NB inactivates MRSA；

Fig. 4 is mouse after blank, infection MRSA and respectively administration (ASC-NB or oxacillin) afterwards in organ-tissue The variation of MRSA colonization amount.

Specific embodiment

Embodiments of the present invention, but the implementation being not intended to restrict the invention are further illustrated below by specific example Range.

The synthetic route of intermediate product B4:

(1) it is warming up to 80 DEG C after mixing B1 by the molar ratio of 1:1 with hydrazine hydrate, is cooled to room after reaction reflux 8-12h Temperature has a large amount of white solid B2 to be formed in cooling procedure.

(2) B2 is dissolved in the ethyl alcohol of sufficient amount, is stirred at room temperature to clarification, be added ammonium thiocyanate that molar ratio is 1:1 it After add concentrated hydrochloric acid, be warming up to 80-100 DEG C of reflux 12-16h and obtain B3.

(3) it is warming up to 100-160 DEG C after B3 being dissolved in hydroxide sodium solution, is acidified to pH with hydrochloric acid after being stirred at reflux =5-6, white solid generated are washed with water to neutrality, obtain B4 after dry.

Embodiment 1

ASC-NB structural formula of the present invention is as follows:

Above-mentioned ASC-NB preparation sequentially includes the following steps:

1. the preparation step of intermediate product C4 is as follows:

(1) C1 is mixed with hydrazine hydrate by 1:1 molar ratio, is warming up to 80 DEG C of reflux 8-12h.To slowly cold after the reaction was completed But to room temperature, a large amount of white solid C2 generate in cooling procedure；

¹H NMR(400MHz,DMSO-d₆): δ 12.52 (s, 1H), 10.07 (s, 1H), 7.79 (d, 1H), 7.37 (t, 1H), 6.87(dd,2H),4.65(s,2H)。

(2) C2 is dissolved in the ethyl alcohol of sufficient amount, is stirred at room temperature to clarification, the ammonium thiocyanate of equimolar ratio is added, then Concentrated hydrochloric acid is added and is warming up to 80-100 DEG C of reflux 12-16h.To be cooled to room temperature after reaction, it is filtered to remove insoluble matter, gained Filtrate decompression distillation, dry acquisition C3.

(3) it C3 is dissolved in hydroxide sodium solution is warming up to 100-160 DEG C of reflux and be acidified to after completion of the reaction with hydrochloric acid PH=5-6, a large amount of white solids of gained are washed with water to neutrality, and vacuum drying obtains C4；

¹H NMR (400MHz, DMSO-d₆):δ13.70(s,1H),7.62(d,1H),7.79(d,1H),7.34(t,1H), 6.99(dd,2H),6.92(t,1H)。

2. the preparation step of intermediate product C5 is as follows:

(1) by A1 and 2,6- lutidines is dissolved in acetonitrile and is cooled to 0 DEG C, and the phenyllacetyl chloride 2h of 1.5 equivalents is added dropwise, The triethylamine of equivalent is added, is warmed to room temperature after being stirred overnight.To be evaporated under reduced pressure removing solvent after completion of the reaction, gained residual is molten In ethyl acetate, saturated sodium bicarbonate solution and saturated salt solution respectively be washed once, and organic phase is dry with anhydrous magnesium sulfate, takes out Solvent is removed in filter, decompression distillation, and A2 is made in silica gel column separating purification.

(2) A2 is dissolved in acetone, argon gas protection is lower to be added sodium iodide by 1:10 molar ratio, and 2.5h is stirred at room temperature.It has reacted It is extracted at rear ethyl acetate, merges organic phase, saturated sodium thiosulfate solution and saturated common salt water washing, the anhydrous sulphur of organic phase Sour magnesium is dry, vacuum distillation obtains A3.

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and is slowly added under nitrogen protection It is warmed to room temperature that the reaction was continued after C4,0 DEG C of stirring 2h.TLC tracking, to remove solvent after the reaction was completed, column silica gel isolates and purifies system Obtain C5；

¹H NMR (400MHz, CDCl₃): δ 7.32 (d, J=8.5Hz, 9H), 6.88 (d, J=8.7Hz, 3H), 6.11 (d, J =9.1Hz, 1H), 5.83-5.77 (m, 1H), 5.17 (s, 2H), 4.91 (d, J=4.8Hz, 1H), 4.29 (s, 2H), 3.80 (s, 3H), 3.64 (d, J=8.2Hz, 2H), 3.48 (s, 2H).

The preparation step of 3.ASC-NB is as follows:

C5 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h, TLC monitoring are added at 0 DEG C, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether, ASC-NB is made after drying；

¹H NMR (400MHz, MeOD): δ 7.32 (d, J=4.7Hz, 7H), 7.15 (d, J=8.5Hz, 2H), 6.87 (d, J =8.5Hz, 2H), 6.13 (s, 1H), 5.45 (d, J=3.8Hz, 1H), 5.29 (dd, J=8.6,3.7Hz, 1H), 4.67 (s, 1H),3.82(s,1H),3.79(s,1H),3.59(s,1H),3.56(s,1H).¹³C NMR(101MHz,MeOD):δ154.92, 154.57,154.16,151.85,145.58,134.59,133.98,130.32,126.38,125.51,123.65,122.83, 122.57,120.16,115.80,71.41,71.38,71.34,70.30,68.51,46.71,9.77.HRMS[M-H]^-(m/z) for C₂₄H₂₀N₅O₅S₂:calcd.522.0900,obsd.522.0930。

Embodiment 2

The structural formula of ASC-NA of the present invention is as follows:

1. the preparation step of intermediate product D3,

(1) D1 is mixed in acetonitrile and the 2h that flows back with thiosemicarbazides by 1:1 molar ratio, it is cooling after obtained solid ethyl alcohol Intermediate product D2 is washed to obtain repeatedly.

(2) D2 is dissolved in sodium hydroxide solution, obtains D3 with dilute hydrochloric acid tune pH to acidity after being refluxed overnight；

¹H NMR(400MHz,CDCl₃): δ 3.26 (s, 1H), 2.53 (t, J=5.9Hz, 2H), 2.31 (t, J=5.5Hz, 2H), 1.89 (p, J=5.7Hz, 2H).

2. the preparation step of intermediate product D4,

(1) by A1 and 2,6- lutidines is dissolved in acetonitrile, and the acyl chlorides 2h of 1.5 equivalents is added dropwise at 0 DEG C, be then added etc. The triethylamine of equivalent is warmed to room temperature after being stirred overnight.TLC tracking, to be evaporated under reduced pressure removing solvent, gained residual after the reaction was completed It is dissolved in ethyl acetate, saturated sodium bicarbonate solution and saturated salt solution respectively washed once, and organic phase is dry with anhydrous magnesium sulfate, take out Solvent is removed in filter, decompression distillation, and silica gel column separating purification obtains A2.

(2) A2 is dissolved in acetone, argon gas protection is lower to be added sodium iodide by 1:10 molar ratio, and 2.5h is stirred at room temperature.Acetic acid second Ester extraction merges organic phase, saturated sodium thiosulfate solution and saturated common salt water washing, and organic phase is dry with anhydrous magnesium sulfate, Vacuum distillation obtains A3.

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and under nitrogen protection, slowly adds Enter D3, stirs and be warmed to room temperature that the reaction was continued after 2h.TLC tracking, to remove solvent after completion of the reaction, column silica gel isolates and purifies obtained D4；

¹H NMR(400MHz,CDCl₃):δ8.02(s,1H),7.35-7.16(m,9H),6.90-6.77(m,2H),5.73 (s, 1H), 5.15 (d, J=5.9Hz, 2H), 4.89-4.81 (m, 1H), 4.27 (d, J=13.7Hz, 1H), 3.94 (d, J= 13.6Hz, 1H), 3.74 (s, 3H), 3.60 (s, 2H), 3.45 (d, J=18.4Hz, 1H), 2.77 (t, 2H), 2.34 (t, 2H), 2.06-1.92(m,2H)。

The preparation step of 3.ASC-NA,

D4 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h is added at 0 DEG C.TLC monitoring, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether and obtains ASC-NA；

¹H NMR (400MHz, MeOD): δ 7.30 (s, 2H), 7.29 (d, J=1.4Hz, 2H), 7.26-7.20 (m, 1H), 5.67 (d, J=4.8Hz, 1H), 5.02 (d, J=4.7Hz, 1H), 4.38 (d, J=13.6Hz, 1H), 4.02 (d, J= 13.6Hz, 1H), 3.84-3.74 (m, 2H), 3.60-3.56 (m, 2H), 3.54 (d, J=7.0Hz, 1H), 3.48 (q, J= 7.0Hz, 1H), 2.83 (dd, J=14.7,7.1Hz, 2H), 2.37 (t, J=7.3Hz, 2H), 2.01 (p, J=7.4Hz, 2H), 1.17(m,2H).¹³C NMR(101MHz,MeOD):δ175.17,173.24,164.72,163.37,158.84,135.09, 129.36,128.87,128.22,126.66,125.45,113.43,59.23,57.68,41.80,34.83,32.44, 27.18,25.02,22.58.HRMS[M-H]^-(m/z)for C₂₂H₂₂N₅O₆S₂:calcd.516.1016,obsd.516.1051。

Embodiment 3

ASC-SB structural formula of the present invention is as follows:

1. the preparation step of intermediate product E4,

(1) E1 and hydrazine hydrate are mixed by 1:1 molar ratio, is warming up to 80-100 DEG C of reflux 8-10h.To cold after the reaction was completed But to room temperature, a large amount of white solid E2 is obtained after cooling.

(2) E2 is dissolved in the ethyl alcohol of sufficient amount, the ammonium thiocyanate of equivalent is added after being stirred at room temperature to clarification, is then added Concentrated hydrochloric acid is warming up to 80-100 DEG C of reflux 12-16h.To be cooled to room temperature after the reaction was completed, it is filtered to remove insoluble matter, filtrate decompression It is spin-dried for, vacuum drying obtains E3.

(3) E3 is dissolved in a small amount of concentrated hydrochloric acid, be stirred at reflux.To which reaction mixture is poured into ice water after the reaction was completed, have A large amount of yellow solids generate.Filter cake is washed with water to neutrality and is re-dissolved in sodium hydroxide, filters off insoluble matter, and filtrate is adjusted to pH with dilute hydrochloric acid =5-6 stands, filters, is dried to obtain E4；

¹H NMR(400MHz,CDCl₃): δ 7.30-7.21 (m, 5H), 7.15 (ddt, J=9.3,7.2,2.4Hz, 1H), 3.81(s,2H),1.65(s,1H)。

2. the preparation step of intermediate product E5

(1) by A1 and 2,6- lutidines is dissolved in acetonitrile, and 0 DEG C is added dropwise acyl chlorides 2h by 1:1.5 molar ratio, be then added etc. The triethylamine of equivalent is warmed to room temperature after being stirred overnight.TLC tracking, to after the reaction was completed, be evaporated under reduced pressure and remove solvent, gained is residual It stays and is dissolved in ethyl acetate, respectively washed once with saturated sodium bicarbonate solution and saturated salt solution, organic phase is dry with anhydrous magnesium sulfate It is dry, it filters, vacuum distillation removes solvent, and A2 is made in silica gel column separating purification.

(2) A2 is dissolved in acetone, sodium iodide is added by 1:10 molar ratio under argon gas protection, 2.5h is stirred at room temperature.Ethyl acetate Extraction merges organic phase, saturated sodium thiosulfate solution and saturated common salt water washing, and organic phase is dry with anhydrous magnesium sulfate, subtracts Pressure, which is distilled, is made A3.

(3) A3 is dissolved in chloroform, is added N-methylmorpholine by 1:1.5 molar ratio, 0 DEG C and slow in 2h under nitrogen protection E4 is added, is then warmed to room temperature and continues to stir.TLC tracking, to after completion of the reaction, remove solvent, column silica gel isolates and purifies to obtain E5；

¹H NMR(400MHz,CDCl₃): δ 7.26 (d, J=2.3Hz, 3H), 7.22-7.21 (m, 2H), 7.20-7.15 (m, 7H), 6.77 (d, J=8.6Hz, 2H), 6.71 (d, J=9.0Hz, 1H), 5.68 (dd, J=9.0,4.9Hz, 1H), 5.11 (s, 2H), 4.77 (d, J=4.9Hz, 1H), 4.52 (d, J=13.5Hz, 1H), 4.24 (s, 2H), 4.05-3.98 (m, 2H), 3.71 (s,1H),3.69(s,3H),3.53(s,2H)。

The preparation step of 3.ASC-SB

E5 is dissolved in dry methylene chloride, 0 DEG C of addition methyl phenyl ethers anisole and trifluoroacetic acid and stir about 2h, TLC monitoring, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether and obtains ASC-SB；

¹H NMR (400MHz, MeOD): δ 7.38-7.37 (m, 2H), 7.31 (s, 3H), 7.30 (s, 5H), 5.68 (d, J= 4.8Hz, 1H), 5.51 (d, J=4.3Hz, 1H), 5.44 (s, 1H), 5.19 (d, J=5.9Hz, 1H), 4.39 (s, 2H), 3.77 (s,2H),3.61(m,2H),3.59(s,2H).¹³C NMR(101MHz,MeOD)δ167.43,165.35,164.13,163.62, 158.25,135.73,135.46,128.87,128.84,128.35,127.12,126.25,124.17,60.01,57.80, 47.05,38.88,32.70,26.69.HRMS[M-H]⁺(m/z)for C₂₅H₂₁N₄O₄S₃:calcd.537.0719, obsd.537.0762。

Embodiment 4

ANB1H structural formula of the present invention is as follows:

Above-mentioned ANB1H preparation sequentially includes the following steps:

1. the preparation step of intermediate product F4 is as follows:

(1) F1 is mixed with hydrazine hydrate by 1:1 molar ratio, is warming up to 80 DEG C of reflux 8-12h.To slowly cold after the reaction was completed But to room temperature, a large amount of white solid F2 generate in cooling procedure；

¹H NMR(400MHz,DMSO-d₆):δ9.26(s,1H),7.28(m,5H),4.25(s,2H),3.37(s,2H)。

(2) F2 is dissolved in the ethyl alcohol of sufficient amount, is stirred at room temperature to clarification, the ammonium thiocyanate of equimolar ratio is added, then Concentrated hydrochloric acid is added and is warming up to 80-100 DEG C of reflux 12-16h.To be cooled to room temperature after reaction, it is filtered to remove insoluble matter, gained Filtrate decompression distillation, dry acquisition F3.

(3) F3 is dissolved in after sodium hydroxide solution and is warming up to 100-160 DEG C of reflux and is acidified to after completion of the reaction with hydrochloric acid PH=5-6, a large amount of white solids of gained are washed with water to neutrality, and vacuum drying obtains F4；

¹H NMR(400MHz,DMSO-d₆): δ 7.36 (dq, J=7.5,1.3Hz, 2H), 7.29 (t, J=7.5Hz, 2H), 7.15 (tt, J=7.3,2.0Hz, 1H), 3.91 (s, 2H).

2. the preparation step of intermediate product F5 is as follows:

(1) by A1 and 2,6- lutidines is dissolved in acetonitrile and is cooled to 0 DEG C, and the phenyllacetyl chloride 2h of 1.5 equivalents is added dropwise, The triethylamine of equivalent is added, is warmed to room temperature after being stirred overnight.To be evaporated under reduced pressure removing solvent after completion of the reaction, gained residual is molten In ethyl acetate, saturated sodium bicarbonate solution and saturated salt solution respectively be washed once, and organic phase is dry with anhydrous magnesium sulfate, takes out Filter, vacuum distillation remove solvent, and A2 is made in silica gel column separating purification.

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and is slowly added under nitrogen protection It is warmed to room temperature that the reaction was continued after F4,0 DEG C of stirring 2h.TLC tracking, to remove solvent after the reaction was completed, column silica gel isolates and purifies system Obtain F5；

¹H NMR(400MHz,CDCl₃): δ 7.39-7.27 (m, 10H), 7.22 (d, J=7.4Hz, 2H), 6.87-6.83 (m, 2H), 6.27 (d, J=9.0Hz, 1H), 5.74 (s, 1H), 5.21 (d, J=11.8Hz, 1H), 5.12 (d, J=11.8Hz, 1H), 4.80 (d, J=4.8Hz, 1H), 4.34 (d, J=13.9Hz, 1H), 4.02 (m, 2H), 3.91 (d, J=13.8Hz, 1H), 3.78 (s, 3H), 3.61 (d, J=4.9Hz, 2H), 3.55 (d, J=17.6Hz, 1H), 3.43 (d, J=18.4Hz, 1H).

3.ANB1H preparation step it is as follows:

F5 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h, TLC monitoring are added at 0 DEG C, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether, ANB1H is made after drying；

¹H NMR (400MHz, MeOD): δ 7.34-7.29 (m, 7H), 7.27-7.25 (m, 3H), 5.64 (d, J=4.8Hz, 1H), 4.90 (m, 1H), 4.42 (d, J=13.6Hz, 1H), 4.07 (m, 2H), 3.98 (d, J=13.6Hz, 1H), 3.58 (d, J =8.8Hz, 2H), 3.50 (d, J=7.6Hz, 1H), 3.35 (m, 1H)¹³C NMR(101MHz,MeOD):δ168.03, 164.57,163.42,150.82,147.28,137.43,136.53,130.87,130.65,130.42,128.08,127.12, 124.44,123.17,59.01,57.80,48.05,30.11,29.45,26.12.HRMS[M-H]^-(m/z)for C₂₅H₂₂N₅O₄S₂:calcd.520.1107,obsd.520.1138。

Embodiment 5

The structural formula of ANA2C of the present invention is as follows:

1. the preparation step of intermediate product D3,

(1) G1 is mixed in acetonitrile and the 2h that flows back with thiosemicarbazides by 1:1 molar ratio, it is cooling after obtained solid ethyl alcohol Intermediate product G2 is washed to obtain repeatedly.

(2) G2 is dissolved in sodium hydroxide solution, obtains G3 with dilute hydrochloric acid tune pH to acidity after being refluxed overnight；

¹H NMR(400MHz,DMSO-d₆): δ 13.21 (s, 1H), 13.10 (s, 1H), 2.74 (t, J=6.1Hz, 2H), 2.60 (t, J=5.4Hz, 2H).

2. the preparation step of intermediate product G4,

(1) by A1 and 2,6- lutidines is dissolved in acetonitrile, and the acyl chlorides 2h of 1.5 equivalents is added dropwise at 0 DEG C, be then added etc. The triethylamine of equivalent is warmed to room temperature after being stirred overnight.TLC tracking, to be evaporated under reduced pressure removing solvent, gained residual after the reaction was completed It is dissolved in ethyl acetate, saturated sodium bicarbonate solution and saturated salt solution respectively washed once, and organic phase is dry with anhydrous magnesium sulfate, take out Filter, vacuum distillation remove solvent, and silica gel column separating purification obtains A2.

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and under nitrogen protection, slowly adds Enter G3, stirs and be warmed to room temperature that the reaction was continued after 2h.TLC tracking, to remove solvent after completion of the reaction, column silica gel isolates and purifies obtained G4；

¹H NMR(400MHz,CDCl₃): δ 8.02 (s, 1H), 7.31 (dd, J=7.6,5.4Hz, 4H), 7.27-7.24 (m, 3H), 6.98 (d, J=8.9Hz, 1H), 6.89-6.82 (m, 2H), 5.74 (dd, J=8.8,4.8Hz, 1H), 5.22-5.10 (m, 2H), 4.86 (d, J=4.8Hz, 1H), 4.24 (d, J=13.7Hz, 1H), 3.98 (d, J=13.6Hz, 1H), 3.78 (s, 3H), 3.62 (d, J=5.9Hz, 2H), 3.58 (s, 1H), 3.50-3.42 (m, 1H), 3.00 (t, J=6.7Hz, 2H), 2.75 (t, J= 6.8Hz,2H)。

The preparation step of 3.ANA2C,

G4 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h is added at 0 DEG C.TLC monitoring, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether and obtains ANA2C；

¹H NMR (400MHz, MeOD): δ 8.02 (s, 1H), 7.41-7.11 (m, 5H), 5.67 (d, J=5.1Hz, 1H), 5.01 (d, J=5.1Hz, 1H), 4.40 (d, J=13.1Hz, 1H), 4.01 (d, J=13.4Hz, 1H), 3.79 (d, J= 17.7Hz,1H),3.65-3.54(m,2H),3.56-3.42(m,1H),2.99(m,2H),2.79(m,2H).¹³C NMR (101MHz,MeOD):δ173.97,173.26,164.74,163.40,158.47,135.09,129.37,128.94, 128.87,128.23,126.67,125.42,59.21,57.66,48.11,35.66,30.34,27.23,21.29.HRMS[M- H]^-(m/z)for C₂₁H₂₀N₅O₆S₂:calcd.502.0849obsd.502.0821。

Embodiment 6

ANB0H structural formula of the present invention is as follows:

1. the preparation step of intermediate product H4,

(1) H1 is mixed with hydrazine hydrate by 1:1 molar ratio, is warming up to 80 DEG C of reflux 8-12h.To slowly cold after the reaction was completed But to room temperature, a large amount of white solid H2 generate in cooling procedure；

¹H NMR(400MHz,DMSO-d₆): δ 9.81 (s, 1H), 7.85 (dt, J=7.0,1.5Hz, 2H), 7.59-7.50 (m, 1H), 7.38 (t, J=7.4Hz, 2H), 4.52 (s, 2H).

(2) H2 is dissolved in the ethyl alcohol of sufficient amount, is stirred at room temperature to clarification, the ammonium thiocyanate of equimolar ratio is added, then Concentrated hydrochloric acid is added and is warming up to 80-100 DEG C of reflux 12-16h.To be cooled to room temperature after reaction, it is filtered to remove insoluble matter, gained Filtrate decompression distillation, dry acquisition H3.

(3) H3 is dissolved in sodium hydroxide solution and is warming up to 100-160 DEG C of reflux, after completion of the reaction, be acidified to pH with hydrochloric acid =5-6, a large amount of white solids of gained are washed with water to neutrality, and vacuum drying obtains H4；

¹H NMR(400MHz,DMSO-d₆): δ 13.5 (s, 1H), 8.01-7.86 (m, 2H), 7.62 (dd, J=8.6, 6.0Hz,1H),7.55-7.46(m,2H)。

2. the preparation step of intermediate product H5 is as follows:

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and is slowly added under nitrogen protection It is warmed to room temperature that the reaction was continued after H4,0 DEG C of stirring 2h.TLC tracking, to remove solvent after the reaction was completed, column silica gel isolates and purifies system Obtain H5；

¹H NMR(400MHz,CDCl₃): δ 7.78 (d, J=9.0Hz, 2H), 7.45-7.27 (m, 8H), 7.33-7.22 (m, 2H), 6.96-6.90 (m, 2H), 6.18 (d, J=9.0Hz, 1H), 5.86 (dd, J=9.1,4.9Hz, 1H), 5.24 (s, 2H), 4.97 (d, J=4.9Hz, 1H), 4.54 (d, J=11.8Hz, 1H), 4.44 (d, J=11.8Hz, 1H), 3.85 (s, 3H), 3.68 (d, J=8.1Hz, 2H), 3.63 (d, J=3.8Hz, 1H), 3.46 (m, 1H).

3.ANB0H preparation step it is as follows:

H5 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h, TLC monitoring are added at 0 DEG C, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether, ANB0H is made after drying；

¹H NMR (400MHz, MeOD): δ 7.95 (dt, J=5.8,3.6Hz, 2H), 7.55-7.43 (m, 2H), 7.32- 7.18 (m, 6H), 5.67 (d, J=4.7Hz, 1H), 5.04 (d, J=4.7Hz, 1H), 4.51 (d, J=13.8Hz, 1H), 4.11 (d, J=13.7Hz, 1H), 3.76 (d, J=12.1Hz, 1H), 3.64 (d, J=14.7Hz, 1H), 3.57 (d, J=8.4Hz, 2H),3.54-3.44(m,1H).¹³C NMR(101MHz,MeOD):δ173.26,164.79,164.01,152.48,135.28, 130.18,128.36,128.90,128.76,128.22,126.65,126.20,125.62,125.11,113.40,59.18, 54.27,41.26,34.59,27.50.HRMS[M-H]^-(m/z)for C₂₄H₂₀N₅O₄S₂:calcd.506.0951, obsd.506.0914。

Embodiment 7

ANB04O structural formula of the present invention is as follows:

Above-mentioned ANB04O preparation sequentially includes the following steps:

1. the preparation step of intermediate product O4 is as follows:

(1) O1 is mixed with hydrazine hydrate by 1:1 molar ratio, is warming up to 80 DEG C of reflux 8-12h.To slowly cold after the reaction was completed But to room temperature, a large amount of white solid O2 generate in cooling procedure；

¹H NMR(400MHz,DMSO-d₆):δ9.49(s,1H),7.97-7.42(m,2H),7.03-6.52(m,2H), 4.38(s,2H)。

(2) O2 is dissolved in the ethyl alcohol of sufficient amount, is stirred at room temperature to clarification, the ammonium thiocyanate of equimolar ratio is added, then Concentrated hydrochloric acid is added and is warming up to 80-100 DEG C of reflux 12-16h.To be cooled to room temperature after reaction, it is filtered to remove insoluble matter, gained Filtrate decompression distillation, dry acquisition O3.

(3) O3 is dissolved in sodium hydroxide solution and is warming up to 100-160 DEG C of reflux, after completion of the reaction, be acidified to pH with hydrochloric acid =5-6, a large amount of white solids of gained are washed with water to neutrality, and vacuum drying obtains O4；

¹H NMR(400MHz,DMSO-d₆):δ9.15(s,1H),7.76-7.69(m,2H),6.88-6.81(m,2H)。

2. the preparation step of intermediate product O5 is as follows:

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and is slowly added under nitrogen protection It is warmed to room temperature that the reaction was continued after O4,0 DEG C of stirring 2h.TLC tracking, to remove solvent after the reaction was completed, column silica gel isolates and purifies system Obtain O5；

¹H NMR (400MHz, MeOD): δ 7.76 (d, J=8.3Hz, 2H), 7.33-7.26 (m, 4H), 7.26-7.22 (m, 1H), 7.18 (d, J=8.3Hz, 2H), 6.91-6.86 (m, 2H), 6.86-6.78 (m, 2H), 5.66 (d, J=4.9Hz, 1H), 5.10 (d, J=11.9Hz, 1H), 5.02 (d, J=4.8Hz, 1H), 4.57 (d, J=13.6Hz, 1H), 3.95-3.88 (m, 1H), 3.87 (d, J=4.4Hz, 1H), 3.75 (s, 3H), 3.58 (d, J=1.4Hz, 1H), 3.55 (d, J=8.4Hz, 1H), 2.99(s,1H),2.86(s,1H)。

3.ANB04O preparation step it is as follows:

O5 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h, TLC monitoring are added at 0 DEG C, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether, ANB04O is made after drying.

¹H NMR(400MHz,MeOD):δ7.78(m,2H),7.30(m,4H),7.23(s,1H),6.92(m,2H),5.68 (d, J=6.9Hz, 1H), 5.05 (d, J=6.8Hz, 1H), 4.45 (d, J=4.6Hz, 1H), 4.15 (d, J=5.0Hz, 1H), 3.83 (d, J=3.9Hz, 1H), 3.67-3.61 (m, 1H), 3.61-3.52 (m, 2H)¹³C NMR(101MHz,MeOD):δ 173.27,163.49,160.08,158.86,158.42,151.32,135.08,129.04,128.86,128.46,128.22, 128.06,127.65,127.38,115.64,59.17,57.68,41.78,34.50,27.24.HRMS[M-H]^-(m/z)for C₂₄H₂₀N₅O₅S₂:calcd.522.0900,obsd.522.0923。

Embodiment 8

ANB04N structural formula of the present invention is as follows:

Above-mentioned ANB04N preparation sequentially includes the following steps:

1. the preparation step of intermediate product P4 is as follows,

(1) P1 is mixed with hydrazine hydrate by 1:1 molar ratio, is warming up to 80 DEG C of reflux 8-12h.To slowly cold after the reaction was completed But to room temperature, a large amount of white solid P2 generate in cooling procedure；

¹H NMR(400MHz,DMSO-d₆): δ 9.49 (s, 1H), 7.44 (dd, J=8.0,1.7Hz, 1H), 7.21-7.08 (m, 1H), 6.71 (d, J=8.2Hz, 1H), 6.51 (m, 1H), 6.36 (s, 2H), 4.40 (s, 1H).

(2) P2 is dissolved in the ethyl alcohol of sufficient amount, is stirred at room temperature to clarification, the ammonium thiocyanate of equimolar ratio is added, then Concentrated hydrochloric acid is added and is warming up to 80-100 DEG C of reflux 12-16h.To be cooled to room temperature after reaction, it is filtered to remove insoluble matter, gained Filtrate decompression distillation, dry acquisition P3.

(3) it P3 is dissolved in hydroxide sodium solution is warming up to 100-160 DEG C of reflux and be acidified to after completion of the reaction with hydrochloric acid PH=5-6, a large amount of white solids of gained are washed with water to neutrality, and vacuum drying obtains P4；

¹H NMR(400MHz,DMSO-d₆):δ7.60-7.54(m,2H),6.72-6.65(m,2H),4.65(s,2H)。

2. the preparation step of intermediate product P5 is as follows:

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and is slowly added under nitrogen protection It is warmed to room temperature that the reaction was continued after P4,0 DEG C of stirring 2h.TLC tracking, to remove solvent after the reaction was completed, column silica gel isolates and purifies system Obtain P5；

¹H NMR(400MHz,CDCl₃):δ7.18(m,12H),6.87-6.73(m,2H),6.04(s,1H),5.34(dd,J =8.5,3.9Hz, 1H), 5.21 (m, 1H), 5.04 (m, 2H), 4.64 (s, 2H), 4.04 (d, J=12.9Hz, 2H), 3.99 (m, 1H), 3.87 (d, J=13.9Hz, 1H), 3.72 (s, 3H), 3.52 (s, 2H).

3.ANB04N preparation step it is as follows:

P5 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h, TLC monitoring are added at 0 DEG C, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether, ANB04N is made after drying；

¹H NMR (400MHz, MeOD): δ 7.87-7.76 (m, 2H), 7.29 (d, J=4.4Hz, 3H), 7.23 (d, J= 4.5Hz, 2H), 7.01 (dt, J=8.3,3.9Hz, 2H), 5.67 (d, J=4.8Hz, 1H), 5.03 (d, J=4.7Hz, 1H), 4.64 (s, 2H), 4.46 (d, J=13.7Hz, 1H), 4.13 (d, J=13.7Hz, 1H), 3.86-3.75 (m, 1H), 3.66- 3.58 (m, 1H), 3.57 (d, J=8.2Hz, 1H), 3.53-3.45 (m, 1H)¹³C NMR(101MHz,MeOD):δ173.01, 168.95,164.37,156.70,152.74,150.25,135.09,128.95,128.27,127.83,127.20,126.70, 122.37,121.08,113.48,60.89,53.15,49.65,41.80,29.68.HRMS[M-H]^-(m/z)for C₂₄H₂₁N₆O₄S₂:calcd.521.1060,obsd.521.1051。

Embodiment 9

ANNB1H structural formula of the present invention is as follows:

Above-mentioned ANNB1H preparation sequentially includes the following steps:

1. the preparation step of intermediate product Q4 is as follows:

(1) Q1 is mixed with hydrazine hydrate by 1:1 molar ratio, is warming up to 80 DEG C of reflux 8-12h.To slowly cold after the reaction was completed But to room temperature, a large amount of white solid Q2 generate in cooling procedure.

(2) Q2 is dissolved in the ethyl alcohol of sufficient amount, is stirred at room temperature to clarification, Q2 and potassium hydroxide are pressed into 1:1.5 molar ratio Then carbon disulfide molar ratio (1:1.5) room temperature 3h is added in mixing.It is filtered to remove insoluble matter, the distillation of gained filtrate decompression, drying Obtain Q3.

(3) Q3 is mixed with hydrazine hydrate by 1:1 molar ratio, is heated to reflux 4h.After completion of the reaction, room temperature is cooled to add Water is acidified to pH=5-6 with hydrochloric acid, there is Precipitation, and washing vacuum drying obtains Q4.

¹H NMR(400MHz,DMSO-d₆):δ13.54(s,1H),7.36-7.17(m,5H),5.56(s,2H),4.03(s, 2H).

2. the preparation step of intermediate product Q5 is as follows:

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and is slowly added under nitrogen protection It is warmed to room temperature that the reaction was continued after Q4,0 DEG C of stirring 2h.TLC tracking, to remove solvent after the reaction was completed, column silica gel isolates and purifies system Obtain Q5；

¹H NMR(400MHz,CDCl₃):δ7.19(m,12H),6.81(m,2H),6.04(s,1H),5.35(m,1H), 5.21 (s, 1H), 5.04 (s, 2H), 4.64 (s, 2H), 4.04 (d, J=12.9Hz, 2H), 3.99 (t, J=5.3Hz, 1H), 3.87 (d, J=13.9Hz, 1H), 3.72 (s, 3H), 3.69-3.65 (m, 1H), 3.61 (d, J=5.3Hz, 2H), 3.52 (s, 2H)。

The preparation step of 3.ANNB1H is as follows:

Q5 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h, TLC monitoring are added at 0 DEG C, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether, ANNB1H is made after drying；

¹H NMR (400MHz, MeOD): δ 7.34-7.27 (m, 10H), 6.29 (s, 1H), 5.33 (d, J=6.9Hz, 1H), 5.30 (d, J=6.9Hz, 1H), 5.20 (d, J=5.3Hz, 1H), 4.78 (s, 2H), 4.25-4.13 (m, 2H), 4.11-4.01 (m, 2H), 3.76 (d, J=11.9Hz, 1H), 3.57 (m, 2H), 3.50-3.46 (m, 1H)¹³C NMR(101MHz,MeOD):δ 173.26,164.77,163.58,150.25,144.78,135.10,134.10,128.62,128.15,127.51,126.42, 125.56,122.15,118.13,59.18,57.66,41.78,34.59,30.51,27.20.HRMS[M-H]^-(m/z)for C₂₅H₂₃N₆O₄S₂:calcd.535.1217,obsd.535.1191。

Embodiment 10

The structural formula of ANB02C of the present invention is as follows:

Above-mentioned ANB02C preparation sequentially includes the following steps:

1. the preparation step of intermediate product R4 is as follows:

(1) R1 is mixed with hydrazine hydrate by 1:1 molar ratio, is warming up to 80 DEG C of reflux 8-12h.To slowly cold after the reaction was completed But to room temperature, a large amount of white solid R2 generate in cooling procedure.

(2) R2 is dissolved in the ethyl alcohol of sufficient amount, is stirred at room temperature to clarification, the ammonium thiocyanate of equimolar ratio is added, then Concentrated hydrochloric acid is added and is warming up to 80-100 DEG C of reflux 12-16h.To be cooled to room temperature after reaction, it is filtered to remove insoluble matter, gained Filtrate decompression distillation, dry acquisition R3.

(3) it R3 is dissolved in hydroxide sodium solution is warming up to 100-160 DEG C of reflux and be acidified to after completion of the reaction with hydrochloric acid PH=5-6, a large amount of white solids of gained are washed with water to neutrality, and vacuum drying obtains R4；

¹H NMR(400MHz,CDCl₃): δ 8.15 (dd, J=7.4,2.1Hz, 1H), 7.82 (dd, J=7.5,2.0Hz, 1H), 7.69 (td, J=7.4,2.0Hz, 1H), 7.62 (td, J=7.3,2.0Hz, 1H), 3.28 (s, 1H).

2. the preparation step of intermediate product R5 is as follows:

(3) A3 is dissolved in chloroform, N-methylmorpholine is added by 1:1.5 molar ratio, 0 DEG C and is slowly added under nitrogen protection It is warmed to room temperature that the reaction was continued after R4,0 DEG C of stirring 2h.TLC tracking, to remove solvent after the reaction was completed, column silica gel isolates and purifies system Obtain R5；

¹H NMR(400MHz,MeOD):δ7.61(m,3H),7.28(m,6H),7.23(m,2H),6.86(m,2H),5.67 (d, J=5.2Hz, 1H), 5.14 (d, J=11.9Hz, 1H), 5.08 (d, J=11.9Hz, 1H), 5.00 (d, J=4.8Hz, 1H), 4.44 (d, J=13.6Hz, 1H), 3.97 (d, J=13.6Hz, 1H), 3.80 (d, J=18.3Hz, 1H), 3.75 (s, 3H),3.60-3.55(m,2H),3.55-3.51(m,1H)。

3.ANB02C preparation step it is as follows:

R5 is dissolved in dry methylene chloride, methyl phenyl ethers anisole and trifluoroacetic acid stir about 2h, TLC monitoring are added at 0 DEG C, to anti- Answer object consumption completely, vacuum distillation removes solvent, and gained residual is washed repeatedly with ether, ANB02C is made after drying.

¹H NMR(400MHz,MeOD):δ8.00(s,1H),7.69-7.66(m,2H),7.61(m,1H),7.31-7.28 (m, 4H), 7.24 (m, 2H), 5.68 (d, J=4.7Hz, 1H), 5.05 (d, J=4.8Hz, 1H), 4.37 (d, J=13.6Hz, 1H), 4.17 (d, J=13.7Hz, 1H), 3.83-3.76 (m, 1H), 3.64 (d, J=5.6Hz, 1H), 3.59 (dd, J=7.7, 2.4Hz,2H).¹³C NMR(101MHz,MeOD)δ:173.28,168.52,164.80,161.99,158.04,151.68, 135.09,132.01,130.77,130.64,130.49,129.69,128.87,128.24,127.12,126.67,120.22, 113.40,59.12,53.93,42.16,34.77,27.34.HRMS[M-H]^-(m/z)for C₂₅H₂₀N₅O₆S₂: calcd.550.0849,obsd.550.0823。

Embodiment 11

Drug test mainly includes following part:

1) enzyme inhibitor, evaluation of the ASC to M β Ls inhibitory activity are used as.

2) compound ASC-NA, ASC-NB, ANB1H, ANB02C, ANB04O, ANB0H, ANB04N, ANNB1H, ANA2C and ASC-SB measures it and cooperates with beta-lactam antibiotic to carrying plasmid pET-26-L1, pET-26-NDM-1, pET-26-VIM- 2, the bacteriostatic activity (MIC) of the super drug-resistant bacteria E.coli of pET-26-CcrA or pET-26-ImiS.

3) with MRSA 43300, Bacillus coli communis DL219 (DE3) for mode bacterium, preliminary sieve is evaluated by Antifungal activity Select the compound with potential bacteriostatic activity or synergistic activity.

4) application of the ASC in treatment infection of staphylococcus aureus.

5) ASC-NB treats the effect of MRSA infection with the dependent change of its concentration and time.

6) preliminary assessment of MRSA infection is treated in the foundation of mouse systemic MRSA infection model with ASC in vivo.

(1) enzyme inhibitor, evaluation of the ASC to M β Ls inhibitory activity are used as；

This case expedition includes: as enzyme inhibitor, ASC evaluates the wide spectrum inhibitory activity of M β Ls；

ASC evaluates the inhibitory activity of M β Ls, by measurement ASC to the 503nhibiting concentration (IC of M β Ls₅₀) Lai Shixian.IC₅₀ Measurement use following steps:

(1) IC of the ASC to M β Ls₅₀The measurement of value carries out on 8453 spectrophotometer of Agilent UV, uses 50mM Tris (pH 7.0) is used as buffer.M β Ls is selected from three groups and representative NDM-1, VIM-2, CcrA, ImiS and L1 evaluate ASC, and ultimate density is respectively 13,50,58 and 100nM.Made using 50 μM of Cefazolin sodiums For the substrate of NDM-1, VIM-2, CcrA and L1, use 60 μM of Imipenems as the substrate of ImiS, monitoring wavelength is respectively 265 and 300nm.

(2) premixing in 50mM Tris (pH 7.0) by M β Ls and inhibitor at room temperature, then substrate is added, The initial rate of antibiotic hydrolysis is recorded under each inhibitor concentration.The measurement of all hydrolysis rates measures three times in parallel, takes it Average value is fitted to obtain IC₅₀Value.ASC is determined the inhibitory activity of four kinds of M β Ls by enzyme inhibition dynamics, measures institute Obtain IC₅₀Value is listed in table 1.

Inhibitory activity (IC of 1 ASC of table to M β Ls₅₀,μM)

As known from Table 1, ASC there is wide spectrum to inhibit efficiency, and inhibitory effect five kinds of M β Ls for adhering to three different groups separately Well (IC₅₀<45μM).Secondly, ASC has best inhibitory activity (IC to L1₅₀<3.5μM).Value, it is noted that this right The ASC that there is " superbacteria " drug resistance target protein M β Ls wide spectrum to inhibit efficiency, the problem for capturing bacterial resistance to the mankind are brought Wish.

(2) it is used as enzyme inhibitor, ASC cooperates with beta-lactam antibiotic to carrying plasmid pET-26-L1, pET-26- The antibacterial activity of the drug-resistant bacteria E.coli of NDM-1, pET-26-VIM-2, pET-26-CcrA or pET-26-ImiS are evaluated:

As enzyme inhibitor, ASC cooperate with beta-lactam antibiotic to carry plasmid pET-26-L1, pET-26-NDM-1, The antibacterial activity of the drug-resistant bacteria E.coli of pET-26-VIM-2, pET-26-CcrA or pET-26-ImiS are evaluated, and are to pass through survey Antibiotic realizes the change of bacterium minimum inhibitory concentration (MIC) in the presence of being scheduled on ASC.The measurement of MIC, using broth dilution Method carries out as follows:

(1) antibacterials storage liquid preparation: the antibacterials that preparation concentration is 256 μ g/mL store liquid, are stored in -80 DEG C Ultra low temperature freezer.The beta-Lactam antibiotic for selecting penicillins, cephalosporins and carbapenem series is antibacterials.

(2) preparation of culture medium: culture medium uses Mueller-Hinton (MH) broth bouillon.

(3) it the preparation of inoculum: selects E.coli, American tissue incubator ATCC MRSA 43300, carry plasmid pET- The drug-resistant bacteria E.coli of 26-L1, pET-26-NDM-1, pET-26-VIM-2, pET-26-CcrA or pET-26-ImiS are real Test bacterial strain.It is bacterium colony 3-5 similar with oese picking form, it is inoculated in the MH broth bouillon of 4-5mL, 37 DEG C of incubation 2- 6 hours.Logarithmic growth phase bacterium solution after increasing bacterium is with MH meat soup corrected concentrations to 0.5 Maxwell than turbid standard.

(4) it dilutes the preparation and bacterium solution inoculation of antibacterials: sterile tube 13 sequences is taken, except 1.6mL MH is added in the 1st pipe Outside meat soup, MH meat soup 1mL is added in remaining every pipe, and antibacterials stoste (128 μ g/mL) 0.4mL is added in the 1st pipe and mixes, then It draws 1mL to the 2nd to manage, draws the pipe of 1mL to the 3rd after mixing again, so continuous doubling dilution to the 11st pipe, and inhaled from the 11st pipe 1mL is taken to discard, the 12nd pipe is the blank control of not drug containing.At this time each pipe drug concentration be followed successively by 256,128,64,32,16, 8,4,2,1,0.5,0.25μg/mL.Above-mentioned each 1mL of the inoculum prepared is added in every pipe, makes every pipe bacterium solution final concentration about It is 5 × 10⁵CFU/mL.1st to the 11st pipe drug concentration is respectively 128,64,32,16,8,4,2,1,05,0.25,0.125 μ g/ mL.Measure inhibitor in the presence of MIC value when, take sterile test tube 13 sequences, except the 1st pipe addition 1.4mL MH meat soup in addition to, MH meat soup 0.9mL is added in remaining every pipe；Inhibitor 0.2mL solution is added in 1st pipe, and 2-13 pipe is separately added into 0.1mL, remaining step is same On.

(5) be incubated for: inoculated dilution tube is stoppered plug, sets in 37 DEG C of normal air incubators and is incubated for 16-20 hours.

(6) result: being observed, the drug concentration of the test tube of no bacterial growth with naked eyes or microplate reader, as tested bacterium MIC。

It is the good broad spectrum type inhibitor of antibiotics resistance target protein M β Ls in view of ASC, we determine ASC collaboration pioneer The MIC value of V couple of production NDM-1 drug-resistant bacteria E.coli-DH10B.The experimental concentration range of ASC is 1-64 μ g/mL.Measurement result It is listed in table 2.

2 ASC of table cooperates with Aneet to the MIC value (μ g/mL) for producing NDM-1 drug-resistant bacteria E.coli-DH10B

As can be seen from Table 2, ASC itself is a kind of fungicide, and MIC is 128 μ g/mL (in addition to ANNB1H), meanwhile, ASC It can effectively cooperate with antibiotic to improve its antibacterial activity, that is, reduce the MIC value of antibiotic；With the increase of ASC concentration, antibiotic pair The MIC value of drug-resistant bacteria reduces, i.e., its antibacterial activity enhances；When combination 64 μ g/mL of dosage ANB1H, ANB0H, ANB04N or When ANA2C, so that the vigor that Aneet treatment produces NDM-1 drug-fast bacteria E.coli improves 32 times.

It is the broad spectrum type inhibitor of antibiotics resistance target protein M β Ls in view of ASC, ASC cooperates with Aneet to production CcrA drug resistance The MIC value of bacterium E.coli-BL21 is measured.The experimental concentration range of ASC is 0.5-32 μ g/mL.Measurement result is shown in Table 3.

3 ASC of table cooperates with Aneet to the MIC value (μ g/mL) for producing CcrA drug-resistant bacteria E.coli-BL21

As can be seen from Table 3, ASC itself is a kind of fungicide, can also effectively cooperate with antibiotic to improve its antibacterial activity, that is, drop The MIC value of low antibiotic；With the increase of ASC concentration, antibiotic reduces the MIC value of drug-resistant bacteria, i.e., its antibacterial activity increases By force；As ASC-NA, ANB04O or ANA2C of combination 8 μ g/mL of dosage, so that Aneet treatment produces the drug-resistant bacteria of CcrA The vigor of E.coli-BL21 improves 32 times.

(3) it is used as antibacterial agent, application of the ASC in treatment MRSA and Escherichia coli DL219 (DE3) infection:

This case expedition includes: ASC targeted therapy MRSA and collaboration antibiotic treatment Escherichia coli DL219 (DE3) infection Efficiency evaluation；ASC-NB evaluates the bactericidal effect and its concentration and time dependence of MRSA；Mouse systemic MRSA infects mould The foundation of type and the preliminary assessment of ASC interior therapeutic MRSA effect；

1. having by bacteriostatic experiment preliminary screening latent with MRSA 43300, Escherichia coli DL219 (DE3) for mode bacterium In bacteriostatic activity or the compound of synergistic activity.

It is the good broad spectrum type inhibitor of M β Ls in view of ASC, ASC cooperates with Aneet to the MIC of E.coli-DL219 (DE3) Value is measured.The experimental concentration range of ASC is 0.25-16 μ g/mL.Measurement result is shown in Table 4.

4 ASC of table cooperates with the MIC value (μ g/mL) of Aneet targeting inactivation E.coli-DL219 (DE3)

As shown in 4 column datas of table, 10 noval chemical compound ASC (in addition to ANNB1H) listed by the present invention, to E.coli- DL219 (DE3) has significant ground inactivation efficiency (μ of MIC≤64 g/mL)；ASC cooperates with Aneet, significantly improves the antibiotic Sterilizing activity.As the ASC-NB or ABA2C of combination 16 μ g/mL of dosage, so that the work of Aneet treatment E.coli-DL219 (DE3) Power is improved up to 64 times；When the amount ratio of Aneet and ASC-NB or ABA2C are 1:4, optimum synergistic sterilizing efficiency is shown.

It is the broad spectrum type inhibitor of antibiotics resistance target protein metallo-β-lactamase in view of ASC, ASC system listed by the present invention The MIC value of 10 compounds of column and oxacillin and methicillin (as control) targeted inhibition MRSA are measured, result It is listed in table 5.

The MIC (μ g/mL) of 5 ASC targeted inhibition MRSA of table

Compound	ASC-NA	ASC-NB	ANB1H	ANB02C	ANB04O	ANB0H
							MIC(μg/mL)	32	4	8	16	4	32
Compound	ANB04N	ANNB1H	ANA2C	ASC-SB	Oxacillin	Methicillin
							MIC(μg/mL)	2	128	64	8	8	8

As known from Table 5, ASC (in addition to ANNB1H) has good inhibitory effect (μ of MIC≤64 g/mL) to MRSA, The sterility of middle ASC-NB, ANB04O and ANB04N are 2-4 times stronger than oxacillin and methicillin, and the sterilizing of ANB04N is imitated Fruit is best (MIC=2 μ g/mL).These results indicate that ASC is entirely possible to become the potential antibacterial for tackling MRSA for clinic Drug.

(4) application of the ASC in treatment infection of staphylococcus aureus:

This case expedition includes: as antibacterial agent, bacteriostatic activity of the ASC to Gram-negative bacteria and gram-positive bacteria Evaluation；As enzyme inhibitor, ASC cooperates with the efficiency evaluation of antibiotic magnetic target therapy infection of staphylococcus aureus.

1.ASC evaluates the bacteriostatic activity of Gram-negative bacteria and gram-positive bacteria:

As antibacterial agent, the evaluation of the bacteriostatic activity of ASC be by measure its to the minimum inhibitory concentration (MIC) of bacterium come It realizes (measurement of MIC is detailed in down).Respectively using gram-positive bacteria and Gram-negative bacteria as experimental strain, using ASC as anti- The MIC value of bacterium reagent measurement is listed in table 6:

The MIC value (μ g/mL) of 6 ASC of table

Note: "-" indicates that in the presence of the ASC of 128 μ g/mL dosage, experimental strain is still active.

It as known from Table 6, is in experimental strain in 5 gram-positive bacterias and Gram-negative bacteria, ASC is only to golden yellow Color staphylococcus shows sterilizing activity, and sterilizing activity is very good (μ of MIC < 1 g/mL).This evaluation discloses, and ASC is to gold Staphylococcus aureus has specificity deactivation.

The efficiency evaluation of 2.ASC collaboration antibiotic magnetic target therapy infection of staphylococcus aureus:

As enzyme inhibitor, it is logical that ASC, which cooperates with the efficiency evaluation of antibiotic magnetic target therapy infection of staphylococcus aureus, Measurement antibiotic in the presence of ASC is crossed to realize the change of bacterium minimum inhibitory concentration (MIC).The measurement of MIC, using meat soup Dilution method carries out as follows:

(1) antibacterials storage liquid preparation: the antibacterials that preparation concentration is 128 μ g/mL store liquid, are stored in -80 DEG C Ultra low temperature freezer.Select the beta-Lactam antibiotic of penicillin, cephalosporin and carbapenem series as antibacterials；

(2) preparation of culture medium: culture medium uses Mueller-Hinton (MH) broth bouillon；

(3) E.coli, the ESBL-E.Coli being clinically separated to, pseudomonas aeruginosa, excrement the preparation of inoculum: are selected Enterobacteria, the ESBL- Klebsiella Pneumoniae (ATCC700603) of standard and staphylococcus aureus are experimental strain.Use oese Picking form similar bacterium colony 3-5, be inoculated in the MH broth bouillon of 4-5mL, 35 DEG C incubation 2-6 hours.After increasing bacterium Logarithmic growth phase bacterium solution is with MH meat soup corrected concentrations to 0.5 Maxwell than turbid standard；

(4) it dilutes the preparation and bacterium solution inoculation of antibacterials: sterile tube 13 sequences is taken, except 1.6mL MH is added in the 1st pipe Outside meat soup, MH meat soup 1mL is added in remaining every pipe, and antibacterials stoste (128 μ g/mL) 0.4mL is added in the 1st pipe and mixes, then It draws 1mL to the 2nd to manage, draws the pipe of 1mL to the 3rd after mixing again, so continuous doubling dilution to the 11st pipe, and inhaled from the 11st pipe 1mL is taken to discard, the 12nd pipe is the blank control of not drug containing.At this time each pipe drug concentration be followed successively by 256,128,64,32,16, 8,4,2,1,0.5,0.25μg/mL.Above-mentioned each 1mL of the inoculum prepared is added in every pipe, makes every pipe bacterium solution final concentration about It is 5 × 10⁵CFU/mL.1st to the 11st pipe drug concentration is respectively 128,64,32,16,8,4,2,1,05,0.25,0.125 μ g/ mL.Measure inhibitor in the presence of MIC value when, take sterile test tube 13 sequences, except the 1st pipe addition 1.4mL MH meat soup in addition to, MH meat soup 0.9mL is added in remaining every pipe；Inhibitor 0.2mL solution is added in 1st pipe, and 2-13 pipe is separately added into 0.1mL, remaining step is same On；

(5) be incubated for: inoculated dilution tube is stoppered plug, sets in 35 DEG C of normal air incubators and is incubated for 16-20 hours；

It is the good broad spectrum type inhibitor of antibiotics resistance target protein metallo-β-lactamase in view of ASC, we have rated The efficiency of ASC collaboration antibiotic magnetic target therapy infection of staphylococcus aureus.Evaluation experimental has chosen clinically routine use 8 kinds of antibiotic of three categories, the experimental concentration range of ASC is 0.06125-1 μ g/mL.1 chemical combination of various concentration embodiment of measurement The object ASC-NB and MIC that variety classes antibiotic targeted inhibition staphylococcus aureus is cooperateed with 3 compound ASC-SB of embodiment It is listed in table 7 and 8 respectively.

The sterilizing activity (MIC, μ g/mL) of 7 ASC-NB of table collaboration variety classes Antibiotics On Staphylococcus Aureus

As shown in table 7, in addition to kanamycins, ASC-NB can cooperate with beta-lactam and tetracycline antibiotics totally 7 kinds of realities Antibiotic (benzyl penicillin, ampicillin, Cefazolin sodium, Ceftriaxone Sodium, Cefotaxime Sodium, Imipenem, the Fourth Ring tested Element), the dose dependent sterilizing activity to staphylococcus aureus is shown, i.e., with the increase of ASC-NB dosage, antibiotic To the sterilizing activities of S. aureus L-forms in enhancing (MIC value reduction).As the ASC-NB for being combined 1 μ g/mL dosage, above-mentioned antibiotic is to golden Portugal The sterilization capability of bacterium has been respectively increased 64,128,4,32,16,2,8 times or more.ASC-NB and ampicillin show optimum synergistic Efficiency is combined the ASC-NB of 1 μ g/mL dosage, using clinically the ampicillin of 1/128 dosage of dosage can treat gold at present The infection of Portugal bacterium.

The sterilizing activity (MIC, μ g/mL) of 8 ASC-SB of table collaboration variety classes Antibiotics On Staphylococcus Aureus

As shown in Table 8, ASC-SB can cooperate with the totally 8 kinds of experiments of beta-lactam, aminoglycoside and tetracycline antibiotics Antibiotic (benzyl penicillin, ampicillin, Cefazolin sodium, Ceftriaxone Sodium, Cefotaxime Sodium, Imipenem, kanamycins, Tetracycline), the dose dependent sterilizing activity to staphylococcus aureus is shown, i.e., with the increase of ASC-SB dosage, is resisted Raw element is to the sterilizing activity of S. aureus L-forms in enhancing (MIC value reduction).When the ASC-SB for being combined 1 μ g/mL dosage, above-mentioned antibiotic pair The sterilization capability of S. aureus L-forms has been respectively increased 64,128,4,32,16,10,16,4 times or more.Equally, ASC-SB and ampicillin Optimum synergistic efficiency is shown, that is, is combined the ASC-NB of 1 μ g/mL dosage, uses the clinically ammonia benzyl of 1/128 dosage of dosage at present XiLin can both treat the infection of S. aureus L-forms.

(5) ASC inactivates dosage and timeliness the dependence evaluation of MRSA and staphylococcus aureus:

As a kind of novel antibacterial drug, we select ASC-NA, ASC-NB (using Aneet as control) to have rated it Inactivate the dosage and time dependence of MRSA or staphylococcus aureus.By ASC-NA, ASC-NB (Aneet) of various concentration Solution is added to isometric isodensity (OD₆₀₀=0.5) in MRSA or staphylococcus aureus bacterium solution, in different time, difference The OD of measurement experiment bacterium when dosage₆₀₀Value.Measurement result is as shown in Fig. 1~3.

Fig. 1 clearly illustrates, ASC-NA to staphylococcus aureus show dose dependent inactivation efficiency (this with it is aforementioned MIC measurement result is consistent), but the ASC-NA for a use of dosage being 2 μ g/mL, the infection of staphylococcus aureus is almost 100% ground is suppressed, and sterilizing efficiency maintains at least 48 hours or more.

Fig. 2 clearly shows that Aneet shows dose dependent inactivation performance (with aforementioned MIC measurement result one to MRSA It causes).But when the Aneet for a use of dosage being 16 μ g/mL, when the time reaching 48h, the infection of MRSA is not 100% ground is inactivated, and produces certain drug resistance again.

Comparative diagram 2 sees Fig. 3, and it is found that ASC-NB shows dose dependent inactivation efficiency to MRSA, (this is measured with aforementioned MIC As a result consistent), i.e., being gradually increased with dosage, the density of drug-resistant bacteria gradually decrease；When a use of dosage being only 16 μ The ASC-NB of g/mL can maintain MRSA almost 100% ground inactivation, sterilizing efficiency at least 48 hours or more.Obviously, ASC It is a kind of good anti-MRSA drug.

(6) foundation of mouse systemic infection MRSA model and the preliminary assessment of ASC interior therapeutic MRSA effect:

Establish stablize, reliable MRSA systemic infection model, with to the systematically pathogenesis of research MRSA infection and Experiment basis is established in treatment.The bacterial strain MRSA 43300 that adopts international standards establishes OD₆₀₀- CFU standard curve, through intraperitoneal injection way Diameter infects kunming mice, fixed from the selection of infective dose, the survival rate of mouse, mouse weight variation, blood and multi viscera bacterium The levels such as the amount of growing carry out changes with time monitoring, carry out system evaluation to the mouse model of foundation.The mouse model established through this approach, Lethal dose is every 5.0 × 10⁹CFU, sublethal dose are every 1.0 × 10⁹CFU (survival rate 60%-70%).Material Method is as follows:

(1) experimental animal: kunming mice, male female fifty-fifty, 6-8 week old, weight 18-22g are purchased from Xi'an Communications University's medicine Institute's animal experimental center.

(2) bacterial strain: international standard bacterial strain MRSA 43300 is purchased from American tissue incubator (American Tissue Culture Collection,ATCC)。

(3) the infection culture and preparation of bacterial strain: recovery international standard bacterial strain MRSA 43300, streak inoculation are flat in MH Plate, 37 DEG C, appropriate revolving speed cultivates 6-8h, supernatant is abandoned after centrifugation, gained bacterium colony is washed twice again with sterile saline, finally Thallus is resuspended with sterile saline, measures its absorbance (A at 600nm₆₀₀).According to previous experiment gained A₆₀₀- CFU mark Directrix curve, adjustment bacterial concentration is about 5.0 × 10⁹CFU/mL。

(4) mouse the foundation of mouse model: is randomly divided into 4 groups, every group 10.Wherein 3 groups of experimental group, 1 group of control group. All mouse provide enough feeds and drinking-water.Experimental mice (the I, the II, III group) is resuspended in through 0.2mL is injected intraperitoneally respectively Thallus in physiological saline, control group mice is through being injected intraperitoneally equivalent sterile saline.It is 35mg/kg's by dosage after 4h ASC-NB or oxacillin sodium are injected intraperitoneally respectively into the II, the III experimental mice body, and control group and experimental group (I) are not given Medicine.After treatment, mouse is dissected, takes its five internal organs to be ground into and is homogenized and be coated on culture dish, count bacterium after 37 DEG C of breedings overnight Fall number.As a result as shown in Figure 4.

From fig. 4, it can be seen that the heart of MRSA infected group mouse, liver, spleen, lung and kidney bacterial colonization amount reach maximum, make With the mouse for the oxacillin treatment infection MRSA that dosage is 35mg/kg, bacterial colonization amount of each organ-tissue of mouse in addition to spleen It is substantially reduced, i.e., oxacillin has certain curative effect to MRSA；However, the bacterium using the ASC-NB of same dosage, in mouse the five internal organs The more significant reduction of colonization amount ratio oxacillin, the MRSA almost all of especially heart and pulmonary infection are killed, and are tieed up It holds and MRSA is not observed within one week and grows.It is clear that ASC-NB is effective in cure to MRSA, curative effect is more preferable than oxacillin, special It is not the treatment to the MRSA of heart and pulmonary infection, there is the potential using value for clinical treatment MRSA infection.

To sum up, it is also most intractable pathogenic bacteria that staphylococcus aureus and MRSA, which are clinically most important at present,.Base In our researchs to bacterial drug resistance over 20 years, discovery ASC is a kind of antibiotics resistance target protein metallo-β-lactamase (M β Ls) good broad spectrum type inhibitor.As a kind of novel antibacterial drug, ASC important clinical value is: ASC can cooperate with 7-8 Kind of antibiotic (benzyl penicillin, ampicillin, Cefazolin sodium, Ceftriaxone Sodium, Cefotaxime Sodium, Imipenem, kanamycins, Tetracycline etc.) magnetic target therapy staphylococcus aureus infection.It is combined the ASC of 1 μ g/mL dosage, so that these antibiotic Vigor improves 4-128 times or more.It is combined the ASC of 1 μ g/mL dosage, using clinically the ampicillin of dosage 1/128 is just at present Staphylococcus aureus can be inactivated；The ASC-NA of one 2 μ g/mL dosage almost 100% ground treatment staphylococcus aureus sense Dye, efficiency maintain at least 48 hours or more.In addition, ASC can effectively treat the infection of MRSA, the wherein therapeutic effect of ANB04N Most preferably (MIC=2 μ g/mL), the effect than oxacillin inactivation MRSA is by force up to 4 times.ASC-NB shows dose-dependant to MRSA Property inactivation performance, almost 100% ground inactivates MRSA to the ASC-NB that dosage is 16 μ g/mL, and sterilizing efficiency maintains 48 hours More than.Animal (mouse) experiment shows that ASC-NB extremely effective treats the MRSA of mouse infection.One dosage is 35mg/kg ASC-NB so that the MRSA almost all of mouse heart and pulmonary infection is killed, after a week still without bacterial reproduction；Same dosage ASC-NB, but also in the liver of mouse, spleen and nephridial tissue MRSA colonization amount significantly reduce.The efficiency of ASC-NB treatment MRSA It is in dependent change with administration concentration and the difference of administration time.Obviously, ASC-NB has well the MRSA of mouse infection Therapeutic effect, this indication: ASC has a possibility that potential drug for becoming clinical treatment MRSA infection.

The above content is the further descriptions for combining specific preferred embodiment to make the present invention, cannot recognize It surely is unique embodiment.Those of ordinary skill in the art adopt technical solution of the present invention by reading description of the invention Any equivalent transformation taken, is regarded as claims of the present invention and is covered.

Claims

1. compound shown in general structure (I):

R₁ForR is-NH₂、- CHO ,-COOH ,-OH ,-Cl or-Br；

R₂For-NH₂Or-H；

R₃For

X is N or S.

2. a kind of compound according to claim 1, it is characterised in that: structural formula are as follows:

3. the preparation method of compound described in claim 1, it is characterised in that the following steps are included:

(2) A2 sodium iodide iodide reaction is added in acetone, A3 is made；

4. the preparation method of compound according to claim 3, it is characterised in that: in step (1), by A1 and 2,6- dimethyl Pyridinium dissolution is cooled to 0 DEG C in acetonitrile, and acyl chlorides is added dropwise, and the triethylamine of equimolar ratio is then added, is warmed to room temperature after being stirred overnight； Vacuum distillation removes solvent, and gained residual is dissolved in ethyl acetate, respectively washs one with saturated sodium bicarbonate solution and saturated salt solution Secondary, organic phase anhydrous magnesium sulfate is dry, filters, is evaporated under reduced pressure to A2.

5. the preparation method of compound according to claim 3, it is characterised in that: in step (2), A2 and sodium iodide are dissolved in Acetone is stirred at room temperature, and water, ethyl acetate extraction is added in gained residual after removing solvent, merges organic phase, saturated sodium thiosulfate Solution and saturated common salt water washing, organic phase is dry with anhydrous magnesium sulfate, is evaporated under reduced pressure obtained A3.

6. the preparation method of compound according to claim 3, it is characterised in that: in step (3), exist in N-methylmorpholine Under, TLC tracking is warming up to room temperature to A3 and B4 after reaction in chloroform, removes solvent, and column silica gel isolates and purifies to obtain A4。

7. the preparation method of compound according to claim 3, it is characterised in that: in step (4), in the presence of methyl phenyl ethers anisole, A4 uses trifluoroacetic acid deprotection group in methylene chloride, to which after reaction, vacuum distillation removing solvent, gained residual uses ether Wash to obtain ASC.

8. compound described in claim 1 is in preparation collaboration antibiotic magnetic target therapy infection of staphylococcus aureus drug Using.

9. a kind of for treating the drug of infection of staphylococcus aureus, it is characterised in that: contain claims 1 or 2 any one The described compound, and containing one of beta-lactam antibiotic, aminoglycoside antibiotics, tetracycline antibiotics or It is a variety of.

10. compound as claimed in claim 1 or 2 is in preparation and for inhibiting drug resistance target protein metallo-β-lactamase or for controlling Treat the application in the drug for the super drug-resistant bacteria infection that metallo-β-lactamase mediates.

11. a kind of for inhibiting drug resistance target protein metallo-β-lactamase or for treating the super of metallo-β-lactamase mediation The drug of drug-resistant bacteria infection, it is characterised in that: contain any compound of claims 1 or 2 and beta-lactam Antibiotic.

12. compound as claimed in claim 1 or 2 is in preparation and for treating methicillin-resistant staphylococcus aureus MRSA infection Drug in application.

13. a kind of for treating the drug of methicillin-resistant staphylococcus aureus MRSA infection, it is characterised in that: containing having the right It is required that 1 or 2 any compounds.