阅读说明：本技术 C-7位卤代酰基头孢化合物、制备方法和应用 (C-7 halogenated acyl cephalosporin compound, preparation method and application ) 是由 范莉 李洋 杨大成 胡军华 吴玉珠 杨德蒙 占爽 韩海燕 于 2021-03-31 设计创作，主要内容包括：本发明公开了C-7位卤代酰基头孢化合物、制备方法和应用,属于药物合成技术领域。C-7位卤代酰基头孢化合物结构式如下。实验表明,合成的大多数C-7位卤代酰基头孢化合物具有抗人致病菌活性,其中7个化合物对于所测试的8株人致病菌活性强于或相当于测试的上市头孢药物,TM1f显示了最强最广的抑菌活性；此外,TM1s对柑橘褐斑病菌Al.6的抑菌活性与阳性农药咪鲜胺相当,且强于同类药物头孢噻吩。所得高活性分子对人致病菌和柑橘真菌病菌具有非常好的应用前景。(The invention discloses a C-7 halogenated acyl cephalosporin compound, a preparation method and application thereof, belonging to the technical field of drug synthesis. The C-7 halogenated acyl cephalosporin compound has the following structural formula. Experiments show that most of the synthesized C-7 halogenated acyl cephalosporin compounds have anti-human pathogenic bacteria activity, wherein the activity of 7 compounds on 8 tested strains of human pathogenic bacteria is stronger than or equal to that of tested cephalosporin drugs on the market, and TM1f shows the strongest and widest antibacterial activity; in addition, the bacteriostatic activity of TM1s on citrus brown spot pathogen Al.6 is equivalent to that of the positive pesticide prochloraz, and is stronger than that of the similar drug cephalothin. The obtained high-activity molecules have good application prospects on human pathogenic bacteria and citrus fungal pathogens.)

A C-7 haloacyl cephalosporin compound characterized by the following structural formula:

in the formula, Y is chlorine or bromine; r₁Is H, Cl, OAc orR₂Is H or OCH₃；R₃Is H or CHPh₂(ii) a n is 1, 2 or 3.

2. A compound as shown below, or a pharmaceutically acceptable salt thereof:

3. a process for the preparation of a C-7 haloacyl cepham compound as claimed in any of claims 1 and 2, comprising the steps of:

reacting 7-MAC of the cephalosporin parent nucleus with halogenated acyl chloride to prepare C-7 halogenated acylated target molecules TM1 a-TM 1 e;

removing benzhydryl Ph from TM1 a-TM 1e₂CH to prepare C-7 halogenated acylated target molecules TM1 f-TM 1 j;

reacting three cephalosporin mother nuclei which are collectively called 7-AXCA with halogenated acyl chloride to prepare C-7 halogenated acylated target molecules TM1 k-TM 1 y;

in the formula, Y, R₁And n is defined as Y, R in the structural formula of the C-7 halogenated acyl cephalosporin compound disclosed in any one of claims 1 and 2₁And n is as defined.

4. A process for the preparation of a C-7 haloacyl cepham compound as claimed in claim 3, comprising the steps of:

A. under the action of alkali, carrying out low-temperature reaction on the cephalosporin mother nucleus 7-MAC and halogenated acyl chloride in an organic solvent to prepare TM1 a-TM 1 e; the organic solvent is dichloromethane, chloroform, acetonitrile, tetrahydrofuran or N, N-dimethylformamide; the alkali is triethylamine, pyridine, 4-dimethylamino pyridine, sodium carbonate, potassium carbonate or sodium bicarbonate; the temperature is-40 ℃ to 4 ℃; the optimized reaction conditions are as follows: 7-MAC reacts with haloacyl chloride and pyridine in DCM solution at-25 ℃.

B. TM1 a-TM 1e reacts with a benzhydryl-removing acidic reagent in an organic solvent in the presence of a cation trapping agent, the temperature is controlled, stirring is carried out, and the solvent is separated out to prepare TM1 f-TM 1j. The organic solvent is dichloromethane, chloroform, acetone, ethyl acetate, tetrahydrofuran or diethyl ether; the benzhydryl-removing acidic reagent is aluminum trichloride, zinc chloride, copper bromide, concentrated hydrochloric acid, concentrated hydrobromic acid or trifluoroacetic acid; the cation trapping agent is phenol, resorcinol, anisole or 2-mercaptoethanol; the temperature is-20 ℃ to 40 ℃; the precipitation solvent is diethyl ether, methyl tert-butyl ether, petroleum ether, ethyl acetate, n-hexane or cyclohexane; the optimized reaction conditions are as follows: the solvent is dichloromethane, the acid reagent for removing the benzhydryl is trifluoroacetic acid, the cation trapping agent is anisole, the temperature is 4-19 ℃, and the precipitated solvent is diethyl ether and petroleum ether.

C. Under the action of alkali, reacting cephalosporin nucleus 7-AXCA with haloacyl chloride in an organic solvent and water to obtain TM1 k-TM 1 y; the organic solvent is dichloromethane, chloroform, acetone, acetonitrile, tetrahydrofuran or N, N-dimethylformamide; the alkali is sodium methoxide, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, triethylamine or pyridine. The optimized reaction conditions are as follows: 7-AXCA is reacted with a haloacyl chloride, sodium bicarbonate in acetone and water.

5. A pharmaceutical formulation comprising a compound according to claims 1 and 2 or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier and/or adjuvant.

6. A combination comprising a compound as claimed in claims 1 and 2 or a pharmaceutically acceptable salt thereof and other active ingredients.

7. The use of a compound as claimed in claims 1 and 2 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the control of pathogenic bacteria in humans.

8. The use according to claim 7, wherein the human pathogenic bacteria are Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

9. Use of a compound according to claims 1 and 2 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the control of citrus canker or citrus brown spot.

10. Use according to claim 9, wherein the citrus-related disease is citrus brown spot.

Technical Field

The invention relates to the technical field of drug synthesis, in particular to a C-7 halogenated acyl cephalosporin compound, a preparation method and application thereof.

Background

Infection refers to the process by which a microorganism interacts with a host and causes various degrees of pathological changes while living in the host. The infection is everywhere, causing many troubles, injuries and even death. At present, the medical treatment method for solving the infection mainly adopts chemotherapy. Chemotherapeutic drugs are of various types, such as antibiotic drugs, antibacterial drugs, antifungal drugs, antitubercular drugs, antiviral drugs, etc. The medicines are suitable for one or more infections, can solve the transmission of infectious diseases, and effectively protect human health.

Cephalosporins are important drugs for the clinical treatment of infections. Cephalosporin drugs are roughly divided into five generations in clinic: the first generation of cephalosporins represented by cefradine, cefazolin and cephalexin have strong action on gram-positive bacteria and weaker action on gram-negative bacteria; the second generation of cefuroxime, cefaclor and ceftriaxone sodium represented by the cefuroxime, cefaclor and ceftriaxone sodium enhances the antibacterial activity to gram-negative bacteria on the basis of keeping strong action on gram-positive bacteria; the third generation cephalosporin drugs represented by ceftazidime, ceftriaxone and cefoperazone and the fourth generation cephalosporin drugs represented by cefepime, ceftizoxime and cefpirome have broad-spectrum antibacterial activity on gram-positive bacteria, gram-negative bacteria and anaerobic bacteria, and the fourth generation cephalosporin drugs have stronger anti-gram-positive bacteria activity than the third generation and have no toxicity on kidney; the fifth generation cephalosporin drugs comprise cefprozil, cefditoren and cefepime, and the antibacterial drugs have an ultra-broad spectrum and no nephrotoxicity. Analyzing the structure of the cephalosporin marketed drug molecules to discover that the cephalosporin drug molecules consist of three parts, namely a cephalosporin mother nucleus, a C-7 amino modifying group and a C-3 substituent (or no substituent); the cephalosporin nucleus is commonly 7-ACA, 7-ACCA,7-ANCA,7-MAC,7-MCA (7-MAC removes benzhydryl) (the structure is shown as the following formula); the amino-modifying group at the C-7 position of the cephalosporin may be a simple thiopheneacetyl group, L-phenylglycyl group, cysteinyl group, cyanomethylmercaptoacetyl group, aminothiazoleacetyl group, or a very complex structural fragment such as 2-furyl- (Z) -2-methoxyiminoacetyl group, 2- (4-amino) thiazole- (Z) -2-methoxyiminoacetyl group. The C-3 position modifying group is commonly acetoxymethyl, chlorine atom and methylmercaptotetrazolyl methyl. The main differences of the first generation cephalosporin to the fifth generation cephalosporin are the side chain of the amino at the C-7 position and the group at the C-3 position. Therefore, the derivation range of the cephalosporin molecules is wide, and the corresponding new molecules and the activity thereof are expected.

Humans are infected, many of which are the result of attack by human pathogenic microorganisms. The best simple measure for treating infectious diseases is symptomatic drug treatment. The search for drugs against different pathogenic strains is a long-term subject of drug research, and there is an urgent need for new drugs that are more effective, affordable, and nontoxic.

The citrus has long-term breeding and cultivation history worldwide, and origin and diffusion of citrus fungal diseases are promoted together with frequent communication of citrus germplasm and seedlings worldwide in the last hundred years. The citrus brown spot and the citrus anthracnose have serious harm to the growth of citrus, 4-8 times of pesticide prevention and control are needed every year, pathogenic bacteria can generate drug resistance after long-term use of chemical bactericides, and environmental pollution can be caused by a large amount of sprayed bactericides. Therefore, a new drug which is low in toxicity or harmless and can effectively inhibit citrus fungal diseases is urgently needed in production.

The citrus canker is widely distributed, can harm dozens of rutaceae plants, and is a major epidemic disease affecting the worldwide citrus production. The citrus canker germ line is complex in differentiation, high in morbidity, fast in propagation and wide in host range, and the prevention and control of the citrus canker is always a worldwide problem, and no method can radically cure the citrus canker germ line at present; in recent years, the use of streptomycin for agricultural use has been prohibited, and the control agents effective against ulcer germs have been seriously deficient, and the development of novel anti-citrus ulcer agents is urgently needed.

Disclosure of Invention

In view of the above, the present invention aims to provide C-7 halogenated acyl cephalosporin compounds, preparation methods and applications thereof.

Through research, the invention provides the following technical scheme:

1. a C-7 haloacyl cephalosporin compound characterized by the following structural formula:

in the formula, Y is chlorine or bromine; r₁Is H, Cl, OAc orR₂Is H or OCH₃；R₃Is H or CHPh₂(ii) a n is 1, 2 or 3.

2. The present invention also provides compounds of the structure:

3. a process for the preparation of a C-7 haloacyl cepham compound as claimed in any of claims 1 and 2, comprising the steps of:

reacting 7-MAC of the cephalosporin parent nucleus with halogenated acyl chloride to prepare C-7 halogenated acylated target molecules TM1 a-TM 1 e;

removing benzhydryl Ph from TM1 a-TM 1e₂CH to prepare C-7 halogenated acylated target molecules TM1 f-TM 1 j;

reacting three cephalosporin mother nuclei which are collectively called 7-AXCA with halogenated acyl chloride to prepare C-7 halogenated acylated target molecules TM1 k-TM 1 y;

in the formula, Y, R₁And n is defined as Y, R in the structural formula of the C-7 halogenated acyl cephalosporin compound disclosed in any one of claims 1 and 2₁And n is as defined.

Preferably, the preparation method of the C-7 halogenated acyl cephalosporin compound comprises the following steps:

A. under the action of alkali, carrying out low-temperature reaction on the cephalosporin mother nucleus 7-MAC and halogenated acyl chloride in an organic solvent to prepare TM1 a-TM 1 e; the organic solvent is dichloromethane, chloroform, acetonitrile, tetrahydrofuran or N, N-dimethylformamide; the alkali is triethylamine, pyridine, 4-dimethylamino pyridine, sodium carbonate, potassium carbonate or sodium bicarbonate; the temperature is-40 ℃ to 4 ℃.

B. TM1 a-TM 1e reacts with a benzhydryl-removing acidic reagent in an organic solvent in the presence of a cation trapping agent, the temperature is controlled, stirring is carried out, and the solvent is separated out to prepare TM1 f-TM 1j. The organic solvent is dichloromethane, chloroform, acetone, ethyl acetate, tetrahydrofuran or diethyl ether; the benzhydryl-removing acidic reagent is aluminum trichloride, zinc chloride, copper bromide, concentrated hydrochloric acid, concentrated hydrobromic acid or trifluoroacetic acid; the cation trapping agent is phenol, resorcinol, anisole or 2-mercaptoethanol; the temperature is-20 ℃ to 40 ℃; the precipitation solvent is diethyl ether, methyl tert-butyl ether, petroleum ether, ethyl acetate, n-hexane or cyclohexane.

C. Under the action of alkali, reacting cephalosporin nucleus 7-AXCA with haloacyl chloride in an organic solvent and water to obtain TM1 k-TM 1 y; the organic solvent is dichloromethane, chloroform, acetone, acetonitrile, tetrahydrofuran or N, N-dimethylformamide; the alkali is sodium methoxide, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, triethylamine or pyridine.

More preferably, in the step A, the organic solvent is dichloromethane; the base is pyridine; the temperature was-25 ℃.

More preferably, in the step B, the organic solvent is dichloromethane; the acid reagent for removing the benzhydryl is trifluoroacetic acid; the cation trapping agent is anisole; the temperature is-4 ℃ to 19 ℃; the precipitated solvent is diethyl ether and petroleum ether.

More preferably, in the step C, the organic solvent is acetone and water; the alkali is sodium bicarbonate.

4. The invention also provides a pharmaceutical formulation comprising a compound according to claims 1 and 2 or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier and/or adjuvant.

5. The invention also provides a compound medicament which comprises the compounds or the pharmaceutically acceptable salts thereof and other active ingredients in the claims 1 and 2.

6. The invention also provides application of the compounds or the pharmaceutically acceptable salts thereof in preparing medicaments for preventing and treating human pathogenic bacteria. The human pathogenic bacteria are preferably staphylococcus aureus, micrococcus luteus, bacillus subtilis, escherichia coli and pseudomonas aeruginosa.

7. The invention also provides application of the compounds or the pharmaceutically acceptable salts thereof in preparing medicines for preventing and treating citrus canker or citrus brown spot. The citrus-related disease is preferably citrus brown spot.

The invention has the beneficial effects that:

1) the C-7 halogenated acyl cephalosporin compound provided by the invention takes 4 cephalosporin mother nuclei 7-MAC, 7-ACA, 7-ACCA and 7-ANCA (the latter 3 are collectively called as 7-AXCA) as starting materials, and performs halogenated acylation modification on C-7 amino to obtain TM1 a-TM 1e and TM1 k-TM 1 y; removing benzhydryl from TM1 a-TM 1e under the action of an acidic reagent to further obtain TM1 f-TM 1j. The invention constructs C-7 halogenated acyl cephalosporin compounds with simple structure, and the chemical structure of the products is shown in the specification¹And (4) HNMR confirmation.

2) The C-7 halogenated acyl cephalosporin compound provided by the invention is subjected to in vitro anti-human pathogen activity determination, and the activity of all target compounds (namely halogenated acyl products) is found to be stronger than or equal to that of a parent molecule, stronger than or equal to or weaker than that of drugs on the cephalosporin market; TM1f showed the best overall inhibitory activity against the tested strain; the inhibitory activity of all target compounds on the garcinia microsphere strain is the strongest overall. Wherein, the bacteriostatic activity to staphylococcus aureus ATCC25129, ATCC14125 and TM1f is stronger than that of the tested 3 cephalosporin medicaments; for escherichia coli, the activity of 11 compounds in 25 cephalosporium halogenated acyl products is stronger than or equal to that of marketed drugs cephalothin, and TM1f and TM1g have the same inhibitory activity with marketed drugs cefoxitin sodium and ceftizoxime sodium and are 8 times that of cephalothin; for micrococcus luteus, 25 synthesized halogenated acyl products all show excellent inhibitory activity, wherein 16 compounds have equivalent activity with cephalothin, cefoxitin sodium and ceftizoxime sodium, and have good overall activity; the inhibitory activity of the TM1k/TM1n on salmonella is 2 times or equivalent to that of the marketed drug cephalothin; for pseudomonas aeruginosa, the TM1f inhibition activity is 4 times that of cefoxitin sodium, 8 times that of cephalothin and 16 times that of ceftizoxime sodium; the inhibition activities of the bacillus subtilis TM1a and TM1k are respectively 4 times and 8 times of that of cephalothin. Proves that the C-7 halogenated acyl cephalosporin compound has good application prospect in the antibacterial field.

3) The in vitro anti-citrus germ activity test result of the C-7 halogenated acyl cephalosporin compound provided by the invention shows that the inhibition rate of TM1s with the concentration of 4 mug/mL on Al.6 of the brown spot germ is as high as 90%, the inhibition rate is equivalent to that of positive control prochloraz, the inhibition rate is stronger than that of the similar drug cephalothin, and the compound is worthy of further development; under the concentration of 2 mu g/mL and 1 mu g/mL, the antibacterial activity of 7-MCA halogenated acyl products TM1 f-TM 1j is the best (the inhibition rates are 66% -71% and 54-70% respectively), is stronger than that of 7-MAC halogenated acyl products TM1 a-TM 1e (17-70% and-7-45%) under the same concentration, and is also stronger than that of cephalothin (12% and 7%). The research discovers for the first time that certain halogenated acyl derivatives of cephalosporin nucleus have the activity of inhibiting citrus canker bacteria and strong inhibition activity on citrus pathogenic fungi, and proves that C-7 halogenated acyl cephalosporin compounds have very good application prospects in the field of resistance to citrus bacteria.

Detailed Description

The technical solution of the present invention is described in detail and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The main reagents and instruments involved in the synthesis of the invention are: 7-MAC, 7-ACA, 7-ACCA and 7-ANCA (awarded by Chongqing Tiandi pharmaceutical Co., Ltd., AR); chloroacetyl chloride, 3-chloropropionyl chloride, 4-chlorobutyryl chloride and 4-bromobutyryl chloride (Shanghaineri Fine chemical Co., Ltd., AR); sodium bicarbonate, pyridine (AR, titanium new chemical limited, Chongqing); the other reagents are all commercial chemical pure or analytical pure products and are directly used without purification. A magnetic stirring low-temperature constant-temperature water tank (PSL-1810, Shanghai Ailang instruments Co., Ltd.); a melting point tester (X-6, Beijing Fukai Instrument Co., Ltd.); automatic polarimeters (WZZ-2S, Shanghai precision scientific instruments, Inc.); nuclear magnetic resonance apparatus (AV-600, Bruker, USA; TMS as internal standard).

EXAMPLE 1 Synthesis Condition exploration of TM1a

First, the selection of the base species is performed. 7-MAC 2mmol was taken in a 50mL round bottom flask, dissolved in 8mL Dichloromethane (DCM), divided into 8 portions and placed in 8 25mL round bottom flasks. Stirring in ice bath, adding different bases into each round-bottom flask, dropwise adding 2-chloroacetyl chloride after 10min, tracking and monitoring by TLC, and obtaining the experimental results shown in Table 1.

TABLE 1 Effect of base species on TM1a Synthesis

Wherein Et₃N is triethylamine, Py is pyridine, DMAP is 4-dimethylaminopyridine, DBU is 1, 8-diazabicyclo [5.4.0 ]]Undec-7-enes

TLC detection shows that the reaction is best when organic base pyridine is used. A search for the amount of pyridine was then performed.

TABLE 2 Effect of pyridine amount on TM1a Synthesis

The above process was carried out, and TLC detection showed that the reaction was best when 7-MAC/Py was 1:1.5 molar ratio.

TABLE 3 Effect of acyl chloride dosage on TM1a Synthesis

TLC monitoring parallel experiments show that the molar ratio of 7-MAC to 2-chloroacetyl chloride is 1:1.5 or 1:2, the reaction can be completed within 2h, and 1:1.5 is finally selected as the feed ratio of the synthesis.

TABLE 4 Effect of temperature on the synthesis of TM1a

Front of the selectionThe reaction conditions were explored and parallel experiments were performed with stirring in a low temperature reactor (-25 ℃) and ice bath (4 ℃), and TLC detection showed that chloroacetylation could complete the reaction in 2 h.¹HNMR characterization showed that some of the product double bonds of the reaction carried out at 4 ℃ were shifted to a mixture of isomers of the 2-position double bond (. DELTA.2) and the 3-position double bond (. DELTA.3) and were difficult to separate, while the double bonds of the reaction product at-25 ℃ were not shifted. Therefore, the reaction temperature should be controlled to-25 ℃.

The optimized reaction conditions for the synthesis of TM1a are, thus far: the feeding ratio of 7-MAC to chloracetyl chloride and pyridine is 1:1.5:1.5, and the mixture is stirred and reacted in DCM solution at the temperature of minus 25 ℃ rapidly. By adopting the optimized conditions, the effective synthesis of target molecules TM1 a-TM 1e is realized (see Table 5), and the yield is higher than 85%.

Example 2 Synthesis of TM1 a-TM 1e

Adding 7-MAC 5mmol and DCM 4mL into a 100mL round-bottom flask, stirring, placing in a low-temperature reactor after complete dissolution, controlling the temperature (-25 ℃) to stir, adding pyridine (Py)7.5mmol after uniform dissolution, stirring for 20min, and dropwise adding 1mL DCM solution of acyl chloride 7.5mmol at the dropping speed of about 1d/4 s. After the addition was completed, the reaction was stirred continuously at-25 ℃ and the progress of the reaction was monitored by Thin Layer Chromatography (TLC). After completion of the reaction, ice-cooled saturated brine (20 mL) and DCM (20 mL) were added thereto, and the mixture was rapidly stirred, and the pH was adjusted to 2-3 with 0.5N HCl solution. Transferring to a separating funnel, separating, extracting (20mL multiplied by 1) the water phase by DCM, combining the organic phase, washing with saturated brine (20mL multiplied by 2), collecting the organic phase, and anhydrous Na₂SO₄Drying, rotary steaming and vacuum drying to obtain target molecules TM1 a-TM 1 e.

TABLE 5 results of synthetic experiments for TM1 a-TM 1e

Characterization data for the synthesized molecules were as follows:

TM1a light yellow solid, m.p. 96-97℃, ¹H NMR(600 MHz,DMSO-d6)δ_H 9.68(s,1H),7.54(d,J＝7.5Hz,2H),7.46(d,J＝7.5Hz,2H),7.36(t,J＝7.5 Hz,2H),7.33(t,J＝7.5Hz,2H),7.29(dd,J＝13.5,7.3Hz,2H),6.89(s,1H),5.22(s,1H),4.39(d, J＝13.3Hz,1H),4.22(d,J＝3.8Hz,2H),4.18(d,J＝13.4Hz,1H),3.88(s,3H),3.76(d,J＝17.9 Hz,1H),3.57(d,J＝17.8Hz,1H),3.44(s,3H).

TM1b light yellow solid, m.p. 96-97 ℃, ¹H NMR(600MHz, DMSO-d6)δ_H 9.42(s,1H),7.54(d,J＝7.5Hz,2H),7.46(d,J＝7.6Hz,2H),7.36(t,J＝7.5Hz, 2H),7.33(t,J＝7.5Hz,2H),7.29(dd,J＝14.1,7.2Hz,2H),6.88(s,1H),5.18(s,1H),4.37(d,J＝ 13.3Hz,1H),4.16(d,J＝13.3Hz,1H),3.87(s,3H),3.79(t,J＝6.2Hz,2H),3.75(d,J＝18.1Hz, 1H),3.55(d,J＝17.9Hz,1H),3.42(s,3H),2.76(tt,J＝12.3,8.0Hz,2H).

TM1c (r) yellow oil, ¹H NMR(600MHz,DMSO-d6)δ_H 9.33(s,1H),7.54(d,J＝7.4Hz,2H),7.45(d,J＝7.5Hz,2H),7.36(t,J＝7.6Hz,2H),7.32(t,J＝ 7.5Hz,2H),7.29(dd,J＝14.2,7.3Hz,2H),6.87(s,1H),5.16(s,1H),4.37(d,J＝13.3Hz,1H), 4.16(d,J＝13.3Hz,1H),3.88(s,3H),3.74(d,J＝17.9Hz,1H),3.65(t,J＝6.5Hz,2H),3.55(d,J ＝17.9Hz,1H),3.41(s,3H),2.44–2.40(m,2H),1.98–1.93(m,2H).

TM1d (r) yellow oil, ¹H NMR(600MHz,DMSO-d6)δ_H 9.41(s,1H),7.54(d,J＝7.5Hz,2H),7.46(d,J＝7.6Hz,2H),7.36(t,J＝7.5Hz,2H),7.33(t,J＝ 7.5Hz,2H),7.29(dd,J＝14.1,7.2Hz,2H),6.88(s,1H),,5.17(s,1H),4.37(d,J＝13.3Hz,1H), 4.16(d,J＝13.3Hz,1H),3.87(s,3H),3.75(d,J＝18.0Hz,1H),3.64(t,J＝6.3Hz,2H),3.55(d,J ＝17.9Hz,1H),3.43(d,J＝5.0Hz,3H),2.90(m,J＝16.4,7.8Hz,2H).

TM1e (r) yellow oil, ¹H NMR(600MHz,DMSO-d6)δ_H 9.35(s,1H),7.55(d,J＝7.4Hz,2H),7.47(d,J＝7.6Hz,2H),7.37(t,J＝7.5Hz,2H),7.33(t,J＝7.5Hz,2H),7.31–7.27(m,2H),6.89(s,1H),5.17(s,1H),4.39(d,J＝13.3Hz,1H),4.17(d,J＝ 13.4Hz,1H),3.87(d,J＝14.8Hz,4H),3.76(d,J＝17.9Hz,1H),3.56(dd,J＝12.3,5.3Hz,2H), 3.46(s,3H),2.45–2.40(m,2H),2.05(td,J＝13.5,6.9Hz,2H).

example 3 example of operation of TM1 f-TM 1j Synthesis

TM1 a-TM 1e 2.0.0 mmol and DCM 1.0mL are added into a 100mL round bottom flask, stirred and dissolved, 2.0mmol of anisole and 1.0mL of trifluoroacetic acid (TFA) are added into a glacial salt bath and stirred, and then added dropwise. After the addition, the mixture was stirred at room temperature and the progress of the reaction was monitored by TLC. And after the reaction is finished, slowly adding 5mL of diethyl ether and 20mL of petroleum ether, stirring, freezing, separating out a solid, performing suction filtration, washing with petroleum ether to obtain solid powder, and performing vacuum drying to obtain the target molecules TM1 f-TM 1j.

TABLE 6 results of synthetic experiments for TM1 f-TM 1j

The TM1 f-TM 1j characterization data are as follows:

TM1f light yellow solid, m.p. 131-, ¹H NMR(600MHz, DMSO-d6)δ_H 9.61(s,1H),5.14(s,1H),4.36(d,J＝13.4Hz,1H),4.23(d,J＝8.8Hz,1H),4.19(t, J＝10.7Hz,2H),3.94(s,3H),3.73(d,J＝17.5Hz,1H),3.49(d,J＝17.9Hz,1H),3.40(s,3H). TM1g light yellow solid, m.p. 100-, ¹H NMR(600MHz, DMSO-d6)δ_H 9.36(s,1H),5.10(s,1H),4.35(d,J＝13.4Hz,1H),4.20(d,J＝13.4Hz,1H),3.94 (s,3H),3.78(t,J＝6.2Hz,2H),3.73(d,J＝18.2Hz,2H),3.48(d,J＝17.9Hz,1H),3.39(s,3H), 2.82–2.70(m,2H).

TM1h light yellow solid, m.p. 100-102 deg.C, ¹H NMR(600MHz, DMSO-d6)δ_H 9.28(s,1H),5.09(s,1H),4.36(d,J＝13.4Hz,1H),4.19(d,J＝13.4Hz,1H),3.94 (s,3H),3.72(d,J＝17.8Hz,1H),3.65(t,J＝6.0Hz,2H),3.48(d,J＝17.9Hz,1H),3.37(s,3H), 2.39(dd,J＝16.0,8.8Hz,2H),1.95(dt,J＝13.6,6.7Hz,2H).

TM1i light yellow solid, m.p.:110-, ¹H NMR(600MHz, DMSO-d6)δ_H 9.36(s,1H),5.10(s,1H),4.35(d,J＝13.5Hz,1H),4.20(d,J＝13.4Hz,1H),3.94 (s,3H),3.75–3.69(m,2H),3.63(t,J＝6.2Hz,2H),3.48(d,J＝17.9Hz,2H),3.39(s,3H),2.95– 2.84(m,2H).

TM1j light yellow solid, m.p. 106-, ¹H NMR(600MHz, DMSO-d6)δ_H 9.29(s,1H),5.09(s,1H),4.36(d,J＝13.4Hz,1H),4.19(d,J＝13.4Hz,1H),3.94 (s,3H),3.73(dd,J＝14.5,7.5Hz,1H),3.54(t,J＝6.1Hz,2H),3.48(d,J＝17.9Hz,1H),3.37(s, 3H),2.41(dd,J＝16.6,7.4Hz,2H),2.04(td,J＝13.6,6.9Hz,2H).

example 4 example of operation of TM1 k-TM 1y Synthesis

7-AXCA (5mmol), acetone (5 mL) and water (1 mL) were added to a 100mL reaction flask in this order, and stirred at room temperature, followed by addition of sodium bicarbonate (1.26g,1.5mmol) and a salt-ice bath, dropwise addition of acid chloride (1.0mmol), and stirring. TLC monitored the progress of the reaction. After completion of the reaction, ice-cold 2NHCl was added to adjust pH to 7, the solvent acetone was removed by rotation, and a solid was precipitated by freezing, and ice-cold 2NHCl was added to adjust pH to 3, and a large amount of solid was precipitated. And (4) carrying out suction filtration and drying to obtain solid TM1 k-TM 1 y. The results are shown in Table 7.

TABLE 7 results of synthetic experiments of TM1 k-TM 1y

The TM1 k-TM 1y characterization data are as follows:

TM1k white solid, m.p. 182 ℃ 185 ℃, ¹H NMR(600MHz, DMSO-d6)δ_H 8.93(d,J＝8.0Hz,1H),5.68(dd,J＝8.0,5.0Hz,1H),5.10(d,J＝5.0Hz,1H), 5.00(d,J＝12.8Hz,1H),4.69(d,J＝12.8Hz,2H),4.17(s,2H),3.55–3.47(m,2H),2.03(s,3H).

TM1l white solid, m.p. 164-, ¹H NMR(600MHz, DMSO-d6)δ_H 13.53(s,1H),9.01(d,J＝8.2Hz,1H),5.77–5.71(m,1H),5.11(d,J＝4.4Hz,1H), 5.01(d,J＝12.8Hz,1H),4.69(d,J＝12.8Hz,1H),3.79(t,J＝6.0Hz,2H),3.63(d,J＝18.1Hz, 1H),3.49(d,J＝18.1Hz,1H),2.70(s,2H),2.03(s,3H).

TM1m white solid, m.p. 189-, ¹H NMR(600MHz, DMSO-d6)δ_H 8.93(d,J＝8.0Hz,1H),5.69–5.66(m,1H),5.09(d,J＝4.7Hz,1H),5.00(d,J＝ 12.6Hz,1H),4.69(d,J＝12.6Hz,1H),3.64(s,2H),3.62(d,J＝8.6Hz,1H),3.49(d,J＝18.2Hz, 1H),2.35(t,J＝7.3Hz,2H),2.03(s,3H),1.95(dd,J＝8.3,5.2Hz,2H).

TM1n white solid, m.p. 153-, ¹H NMR(600MHz, DMSO-d6)δ_H13.56(s,1H),9.00(d, J ═ 8.3Hz,1H),5.73(dd, J ═ 8.2,4.8Hz,1H),5.11(d, J ═ 4.8Hz,1H),5.01(d, J ═ 12.8Hz,1H),4.69(d, J ═ 12.8Hz,1H),3.65(dd, J ═ 8.4,3.6Hz,2H), 3.62(d, J ═ 10.3Hz,1H),3.49(d, J ═ 18.1Hz,1H),2.84(dd, J ═ 12.8,6.4Hz,2H),2.03(s,3H), TM1o: white solid, m.178 p.:176 c, ¹H NMR(600MHz, DMSO-d6)δ_H 8.93(d,J＝8.0Hz,1H),5.68(dd,J＝8.0,4.8Hz,1H),5.10(d,J＝4.8Hz,1H), 5.00(d,J＝12.8Hz,1H),4.69(d,J＝12.8Hz,1H),3.66–3.61(m,2H),3.55–3.47(m,2H),2.36 (t,J＝7.0Hz,2H),2.25–2.13(m,1H),2.04(d,J＝7.3Hz,3H),1.98–1.91(m,1H).

TM1p light yellow solid, m.p. 235 ℃ and 238 ℃, ¹H NMR(600MHz, DMSO-d6)δ_H 13.95(s,1H),9.10(s,1H),5.75(dd,J＝8.1,4.8Hz,1H),5.19(d,J＝4.8Hz,1H), 4.17(s,2H),3.97(d,J＝18.0Hz,1H),3.63(d,J＝18.0Hz,1H).

TM1q light yellow solid, m.p. 236 ℃ 237 ℃, ¹H NMR(600MHz, DMSO-d6)δ_H 8.95(d,J＝8.3Hz,1H),5.72(dd,J＝8.3,4.9Hz,1H),5.19(d,J＝4.9Hz,1H), 3.97(d,J＝18.0Hz,1H),3.82(ddd,J＝57.5,16.8,6.0Hz,2H),3.68(d,J＝18.0Hz,1H),2.72(t, J＝6.0Hz,2H).

TM1r light yellow solid, m.p.:237-, ¹H NMR(600MHz, DMSO-d6)δ_H 8.90(d,J＝8.0Hz,1H),5.68(dd,J＝8.0,4.9Hz,1H),5.18(d,J＝4.9Hz,1H), 3.99–3.95(m,1H),3.69–3.64(m,1H),3.09–3.05(m,2H),2.63(t,J＝7.2Hz,7H),1.67–1.61 (m,2H).

TM1s light yellow solid, m.p. 144-148 ℃, ¹H NMR(600MHz, DMSO-d6)δ_H 9.15(d,J＝8.2Hz,1H),6.35(dd,J＝17.1,8.2Hz,1H),5.79(dd,J＝8.2,4.9Hz, 1H),5.23(d,J＝4.8Hz,1H),5.20(d,J＝4.8Hz,1H),4.00(d,J＝9.3Hz,1H),3.72–3.70(m,1H), 2.84(dt,J＝13.0,6.6Hz,2H).

TM1t light yellow solid, m.p. 172 ℃ and 176 ℃, ¹H NMR(600MHz, DMSO-d6)δ_H 13.45(s,1H),8.82(d,J＝8.2Hz,1H),5.68(dd,J＝8.2,4.8Hz,1H),5.09(d,J＝ 4.8Hz,1H),3.62(d,J＝18.1Hz,1H),3.46(d,J＝18.1Hz,1H),3.08(dd,J＝13.2,7.0Hz,2H), 2.63(t,J＝7.0Hz,3H),1.65(dt,J＝13.7,7.0Hz,2H).

TM1u white solid, m.p. 170 ℃ and 173 ℃, ¹H NMR(600MHz, DMSO-d6)δ_H 13.17(s,1H),9.24(d,J＝8.2Hz,1H),6.50(dd,J＝6.3,2.3Hz,1H),5.74(dd,J＝ 8.2,5.0Hz,1H),5.08(d,J＝5.0Hz,1H),4.17(s,2H),3.65(dd,J＝19.0,2.3Hz,1H),3.56(dd,J ＝19.0,6.4Hz,1H).

TM1v white solid, m.p. 170 ℃ and 173 ℃, ¹H NMR(600MHz, DMSO-d6)δ_H 13.19(s,1H),9.00(d,J＝8.2Hz,1H),6.49(dd,J＝6.2,2.1Hz,1H),5.75(dd,J＝ 8.2,5.0Hz,1H),5.05(d,J＝5.0Hz,1H),3.79(t,J＝5.5Hz,2H),3.63(dd,J＝19.0,2.1Hz,1H), 3.54(dd,J＝19.0,6.2Hz,1H),2.71(td,J＝6.0,3.0Hz,2H).

TM1w white solid, m.p. 149-, ¹H NMR(600MHz, DMSO-d6)δ_H 8.89(d,J＝8.2Hz,1H),6.35(dd,J＝6.1,2.0Hz,1H),5.66(dd,J＝8.2,4.9Hz, 1H),5.00(d,J＝4.9Hz,1H),3.65–3.62(m,2H),3.59(dd,J＝18.8,2.2Hz,1H),3.49(dd,J＝ 18.8,6.3Hz,1H),2.36(dd,J＝10.8,3.9Hz,2H),1.97–1.92(m,2H).

TM1x white solid, m.p. 151-, ¹H NMR(600MHz, DMSO-d6)δ_H 13.04(s,1H),8.98(d,J＝8.2Hz,1H),6.49(dd,J＝6.3,2.3Hz,1H),5.75(dd,J＝ 8.2,5.0Hz,1H),5.05(d,J＝5.0Hz,1H),3.65(dd,J＝6.0,2.2Hz,2H),3.62(dd,J＝12.6,2.5Hz, 1H),3.54(dd,J＝19.0,6.3Hz,1H),2.89–2.78(m,2H).

TM1y white solid, m.p. 152-, ¹H NMR(600MHz, DMSO-d6)δ_H 12.84(s,1H),8.81(d,J＝8.2Hz,1H),6.48(dd,J＝6.2,2.2Hz,1H),5.71(dd,J＝ 8.2,4.9Hz,1H),5.04(d,J＝4.9Hz,1H),3.62(dd,J＝18.9,2.1Hz,1H),3.52(dd,J＝19.0,6.4Hz, 1H),3.07(m,2H),2.63(m,2H),1.68–1.61(m,2H).

example 5 Activity test against human pathogenic bacteria

5.1 test strains

Gram-positive bacteria: staphylococcus aureus (Staphylococcus aureus ATCC 25129), Staphylococcus aureus (Staphylococcus aureus ATCC 14125), Micrococcus luteus, and Bacillus subtilis. Gram-negative bacteria: escherichia coli (Escherichia coli ATCC 25922), Acinetobacter baumannii (Acinetobacter baumannii ATCC 19606), Salmonella (Salmonella Enteritidis ATCC 13076), Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 27853).

5.2 media and reagents

Ordinary liquid medium: 0.3% of beef extract, 1% of peptone, 0.5% of sodium chloride and deionized water. Adjusting the pH value to 7.0-7.2, subpackaging in 500mL conical flasks, and sterilizing with high-pressure steam at 121 ℃ for 30 minutes for later use;

reagent: distilled water and 0.9% physiological saline are prepared, and then high-pressure steam sterilization is carried out for standby at the temperature of 121 ℃ for 30 minutes. Dimethyl sulfoxide (DMSO, AR), and tween-80 (AR, New titanium chemical Co., Ltd., Chongqing).

5.3 instruments

An electronic balance (FA1104 model, Shunhui science instruments, Inc., Shanghai), an ultra-clean bench (SW-CJ-1FD model single-person single-side clean bench, Suzhou clean equipment, Inc.), a constant temperature oscillator (ZD-85 model, Changzhou Australian instruments, Inc.), a digital display constant temperature water bath (Beijing Heifeng instruments, Inc.), an automatic steam sterilization pot (product of APL, Tokyo, Japan), a liquid transfer gun (Finnpipette), an ultrasonic instrument (KQ3200, ultrasonic instruments, Inc., Kunshan).

5.4 Minimum Inhibitory Concentration (MIC) assay

(1) Preparation of sample solution: adding 3.2mg of TM1 a-TM 1y samples accurately weighed in a drying chamber into each 2mLPE tube, adding 1mL of DMSO (dimethyl sulfoxide) by using a pipette gun to prepare a clear transparent solution of 3.2mg/mL, sealing by using a sealing film, and storing in a freezer in a dark place. Some poorly soluble compounds were dissolved using DMSO/tween-80 ═ 200/1 (v/v).

(2) Preparing a solution to be detected: and sucking 320 mu L of sample solution, and diluting the sample solution to 1mL by using the culture medium to obtain a solution to be detected with the concentration of 1024 mu g/mL.

(3) Preparation of bacterial suspension: inoculating the preserved strain into common liquid culture medium, activating and culturing in a constant temperature shaker at 37 deg.C for 24 hr, and diluting to 10% with culture medium⁵CFU/mL of bacterial suspension is ready for use.

(4) Sample adding operation: operating under aseptic conditions. Adding 50 mu L of culture medium into each well of a 96-well plate; adding 50 mu L of the solution to be detected into the first hole and the second hole of the first row, diluting the solution twice, changing the concentration to 512 mu g/mL, fully blowing and beating the solution for at least more than 4 times by using a liquid-moving gun to fully and uniformly mix the object to be detected and the culture medium, sucking 50 mu L of the solution, adding the solution into the corresponding holes of the second row, and fully blowing and beating the solution to be detected and the culture medium to be fully and uniformly mixed; repeating the above steps until the eighth row, sucking 50 μ L per hole of the eighth row, and discarding; at the moment, the concentration of each row of substances to be detected is 512,256,128,64,32,16,8 and 4 mu g/mL from high to low in sequence; and then adding 50 mu L of diluted bacterial liquid into each hole, wherein the concentration of the substance to be detected in each hole is the final substance to be detected, and the concentration is 256,128,64,32,16,8,4 and 2 mu g/mL from high to low. The other columns of the 96-well plate were also operated in this manner. The last two columns of each plate were used as controls and contained no test substance, one column was used as control for bacterial growth with added bacteria solution, the other column was used as negative control without added bacteria solution. Each plate was tested for 5 compounds.

(5) Culturing and judging results: and putting the inoculated 96-well plate into a constant-temperature incubator at 37 ℃ for culturing for 16-20 h. After incubation, the 96-well plate was removed from the incubator and bacterial growth in the wells was observed and recorded. Before the results are judged, the results are meaningful only when the bacteria in the blank drug-free control hole grow normally and the bacteria in the negative control hole grow normally. The concentration of the test substance in the wells where no bacteria grew was visually observed was taken as the MIC of the test substance for the bacteria. If the hole jumping phenomenon occurs or the two hole results are different, repeated tests are needed for verification.

The test results are shown in Table 8.

TABLE 8 test results of inhibitory Activity of target Compound TM1 against human pathogenic bacteria (MIC value, μ g/mL)

The activity of all target compounds (namely halogenated acyl products) is stronger or equivalent to that of a parent molecule, stronger or equivalent to that of a drug on the cephalosporin market or weaker than that of the drug on the cephalosporin market; TM1f showed the best overall inhibitory activity against the tested strain; the inhibitory activity of all target compounds on the garcinia microsphere strain is the strongest overall.

For Escherichia coli, the overall activity of the halogenated acyl product is TM1 f-TM 1j, TM1 k-TM 1o, TM1 a-TM 1e, TM1 u-TM 1y, TM1 p-TM 1 t; the activity intensity is 7-MCA >7-ACA >7-MAC >7-ANCA >7-ACCA considered from mother nucleus of cephalosporium. The activity of 7-MAC halogenated acyl products TM1 a-TM 1e is weaker than that of products TM1 f-TM 1fj (7-MCA type) after the benzhydryl is removed, which shows that the antibacterial activity of C-4 carboxyl type molecules is obviously stronger than that of corresponding ester type molecules. 11 compounds of 25 cephalosporanic halogenated acyl products have MIC of 2-16 mug/mL, and the activity is stronger than or equal to that of a marketed drug cephalothin (16 mug/mL, MIC value, the same later); TM1f and TM1g have the same inhibitory activity (2 mu g/mL) as that of cefoxitin sodium and ceftizoxime sodium which are medicines on the market, and are 8 times of that of cephalothin.

The inhibitor has no obvious inhibiting activity (more than or equal to 256 mu g/mL) on salmonella, marketed drugs of cefoxitin sodium and ceftizoxime sodium, 4 cephalosporin nuclei and most haloacyl products, but the inhibiting activity (32/64 mu g/mL) of the 7-ACA haloacyl product TM1k/TM1n is 2 times or equal to that of marketed drug of cephalothin (64 mu g/mL).

The compound has no obvious inhibition activity (more than or equal to 256 mu g/mL) on Acinetobacter baumannii, cephalosporin nucleus, cefoxitin sodium and synthesized halogenated acyl products, however, TM1k is equivalent to cephalothin and ceftizoxime sodium (128 mu g/mL).

For pseudomonas aeruginosa, although the acyl derivatives of the cephalosporin nucleus 7-ANCA, 7-ACCA and 7-MAC have poor activity (MIC is more than or equal to 256 mug/mL), the halogenated acyl products of 7-MCA and 7-ACA show obvious inhibition activity (4-128 mug/mL), and the inhibition activity of the halogenated acyl products TM1 f-TM 1j of 7-MCA is 4-8 times that of the halogenated acyl products TM1 a-TM 1e of 7-MAC; in particular, TM1f inhibitory activity (4. mu.g/mL) was 4 times that of cefoxitin sodium (16. mu.g/mL), 8 times that of cefotaxime (32. mu.g/mL), and 16 times that of cefotaxime sodium (64. mu.g/mL), which is worthy of further investigation.

For Micrococcus luteus, 25 synthesized halogenated acyl products all show excellent inhibitory activity, wherein the MIC values of 16 compounds are 2 mu g/mL, and are equivalent to those of cephalothin, cefoxitin sodium and ceftizoxime sodium (2 mu g/mL), especially 7-MCA derived TM1 f-TM 1j and 7-ACA derived TM1 k-TM 1o, the MIC values are 2 mu g/mL, and the overall activity is very good.

The halogenated acyl products of staphylococcus aureus ATCC25129, ATCC14125 and 7-MCA generally have antibacterial activity (8-128 mu g/mL), wherein the antibacterial activity of TM1f is stronger than that of the tested 3 cephalosporin drugs; TM1a and TM1k in 7-MAC and 7-ACA halogenated acyl products also have good antibacterial activity (16-64 mu g/mL).

The antibacterial activity (8-32 mu g/mL) on bacillus subtilis and 7-MCA haloacyl products TM1 f-TM 1j is better, the inhibitory activity (16,8 mu g/mL) of a person (TM1a, TM1k) with chloroacetyl in 7-MAC and 7-ACA haloacyl products is 4 times and 8 times of that of cephalothin (64 mu g/mL), the inhibitory activity of TM1a is equivalent to that of cefoxitin sodium (16 mu g/mL), and the inhibitory activity of TM1k is 2 times of that of cefoxitin sodium.

Example 6 anti-Pichia Activity assay

MIC values for inhibition of Pichia pastoris by TM1 a-TM 1y were determined using the broth microdilution method recommended by NCCLS.

6.1 test strains and materials

(1) Test strains: pichia pastoris

(2) Test compounds: TM1 a-TM 1y, and a positive control drug fluconazole.

(3) Culture medium: a Sha's medium is selected and composed of peptone 1%, glucose 4% and deionized water 95%. The components are prepared according to the proportion, heated and dissolved into homogeneous phase, subpackaged in 500mL conical bottles, and sterilized by high-pressure steam at 121 ℃ for 20 minutes for later use. Reagent: 0.9% normal saline, DMSO, and Tween-80, which are commercially available analytical pure drugs.

(4) The instrument comprises the following steps: the same as 5.3.

6.2 method for determining minimum inhibitory concentration MIC

(1) And 5.4, preparing a sample solution and preparing a solution to be detected.

(2) Preparation of bacterial suspension: inoculating the preserved strain into a Sabouraud's agar liquid culture medium, and placing the strain in a constant temperature shaking table at 30 ℃ for activation culture for 24 hours; washing the surface colony of agar with distilled water, and diluting to 10 with Sabouraud's medium⁵CFU/mL of bacterial suspension is ready for use.

(3) Sample adding operation: the same procedure as for sample application in 5.4 was carried out.

(4) Culturing: the inoculated 96-well plate is put into a 30 ℃ constant temperature incubator to be cultured for 48h, and then the results of 24h and 48h are observed and recorded.

(5) And (4) judging a result: and judging the result in the same way as 5.4. The test results are shown in Table 9.

TABLE 9 test results of Pichia pastoris inhibitory Activity of the target Compound TM1 (MIC value, μ g/mL)

Table 9 shows that 4 cephalosporin nuclei have no inhibitory activity and that cephalothin is also poor, but TM1a (MIC ═ 32 μ g/mL) and TM1f (MIC ═ 64 μ g/mL) show significant inhibitory activity, 8-fold and 4-fold respectively that of cephalothin.

Example 7 Activity test against Citrus pathogenic fungi

7.1 materials and test strains

(1) Test compounds TM1 a-TM 1y (1mg/mL), positive control prochloraz (1mg/mL), 24-well plates, PDA culture medium, 10mL centrifuge tube, 5mL micropipette and suction head.

(2) The strain is as follows: colletotrichum citricola Co.3 strain (Colletotrichum gloeosporioides, Co.3) and Curcubeba citricola Al.6 strain (Alternaria alternate, Al.6).

7.2 assay methods

(1) Preparing a mother solution of a compound to be tested: 1.0mg of each desired test compound was accurately weighed and dissolved in 1mL of an appropriate solvent to give a 1.0mg/mL compound mother liquor.

(2) Compound culture medium preparation: preparation of a culture medium containing 2 mu g/mL of a compound: taking 10 mu L of compound mother liquor and 4990 mu L of hot PDA culture medium, and fully and uniformly mixing in a 10mL centrifuge tube; preparation of a culture medium containing 4 mu g/mL of a compound: mu.L of the compound stock solution was mixed well with 4980. mu.L of hot PDA medium in a 10mL centrifuge tube. Control group: PDA medium without compound and PDA medium with prochloraz added (2. mu.g/mL and 4. mu.g/mL) were used as negative and positive controls.

(3) Inoculating and culturing: the prepared compound medium was poured into 24-well plates, 4 wells per concentration of each compound per strain and numbered. Mycelia of the strain cultured at 28 ℃ for 7 days were picked and inoculated into the center of each well. The 24-well plate is placed in an incubator with 28 ℃ and 16h of illumination for 48 h.

(4) Measurement and calculation: the diameter of the colony was measured by the cross method and the inhibition rate was calculated according to the following formula

Inhibition [% ] is (CK colony diameter value-measured colony diameter value) × 100%/CK colony diameter value.

The results are shown in Table 10.

TABLE 10 test results of inhibitory activity of target compound TM1 on citrus pathogenic fungi

TABLE 11 result of Al.6 rescreening of M1s on Physalospora citricola

When the concentration is 2 mug/mL and 4 mug/mL, most of the synthesized cephalosporin halogenated acyl products have no inhibitory activity to citrus colletotrichum gloeosporioides Co.3 and citrus brown spot germ Al.6, but the inhibition rate of TM1s with the concentration of 4 mug/mL to brown spot germ Al.6 is up to 90%, and the inhibition rate is equivalent to that of positive control prochloraz and is stronger than that of the similar medicine cephalothin, so that the cephalosporin halogenated acyl products are worthy of further development. The halogenated acyl cephalosporin products have strong inhibitory activity on citrus pathogenic fungi, and are not reported in the literature at present.

Example 8 resistance to Citrus canker pathogen test

8.1 materials and test strains

(1) Preparation of a culture medium: LB liquid culture medium was selected for the experiment.

Preparation of bacterial suspension: activating RL strain of Citrus canker pathogen on PDA culture medium, culturing at 28 deg.C for 3d, scraping a small amount of colony on ultraclean bench with blue gun head, placing in sterile water, and shake culturing at 28 deg.C and 200r for 2h on shaking table to obtain bacterial suspension.

Preparing compound TM1 a-TM 1y and positive control benziothiazolinone mother liquor: accurately weighing the compound, and dissolving the compound in sterile water or an organic solvent to prepare a mother solution of 1mg/mL for later use.

(2) Method of producing a composite material

The experiment was performed in 96-well plates, each compound was set with 4 concentration gradients, each concentration was repeated 5 times, and different concentrations corresponded to control CK1 without addition of bacterial suspension (thereby excluding the effect of the compound's own color), and at the same time, to Control (CK) with addition of bacteria only without addition of compound. Each compound was set at 4 dilutions, 2. mu.g/mL (i.e., 500-fold dilution), 1. mu.g/mL (i.e., 1000-fold dilution), 0.5. mu.g/mL (i.e., 2000-fold dilution), and 0.2. mu.g/mL (i.e., 5000-fold dilution). Namely: taking 10 mu L of compound mother liquor as a sample solution (10 mu g/mL, namely 100 times dilution) in 990 mu L of sterile water, then sequentially diluting the sample solution by adopting a multiple dilution method, sequentially adding 40 mu L, 20 mu L, 10 mu L and 4 mu L of the sample solution into 4 concentration gradients of an experimental group, then adding 140 mu L of LB liquid culture medium into each hole of a row gun, and supplementing sterile water into each hole to ensure that the volume of the total solution in each hole reaches 180 mu L. Finally, 20. mu.L of the prepared bacterial suspension is added into each hole of the calandria gun to prepare mixed bacterial liquid with the total volume of 200. mu.L, so that the final concentrations of the samples are A (2. mu.g/mL), B (1. mu.g/mL), C (0.5. mu.g/mL) and D (0.2. mu.g/mL). The volume of the bacterial suspension in CK1 was replaced with 20. mu.L of LB medium, and the volume of the compound in CK was made up with sterile water. Mixing well, and culturing in 28 deg.C incubator for 48 h.

Determination of OD Using Spectrophotometer₆₀₀And (4) calculating the light absorption value and the bacteriostasis rate.

Bacteria inhibition rate%₆₀₀- (Compound OD)₆₀₀The compound CK1 OD₆₀₀)/CKOD₆₀₀X100

The results are shown in Table 12.

TABLE 12 test results of inhibitory Activity of target Compounds TM1 a-TM 1y against Sclerotinia citrulli

The results in table 12 show that the activity of the synthesized compound for inhibiting citrus canker pathogen is weakened along with the reduction of the concentration of the compound, and the compound has a better activity-dosage relation; under the concentration of 2 mu g/mL and 1 mu g/mL, the antibacterial activity of 7-MCA halogenated acyl products TM1 f-TM 1j is the best (the inhibition rates are 66% -71% and 54-70% respectively), is stronger than that of 7-MAC halogenated acyl products TM1 a-TM 1e (17-70% and-7-45%) under the same concentration, and is also stronger than that of cephalothin (12% and 7%); the activity strength and weakness sequence of the corresponding halogenated acyl products of the cephalosporin nucleus is 7-MCA >7-MAC >7-ACA ≈ 7-ACCA > 7-ANCA. The research reports for the first time that certain acyl derivatives of cephalosporin nucleus have the activity of inhibiting citrus canker pathogen.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.