CN112094279A

CN112094279A - P-aminosalicylic acid dihydroartemisinin derivative and preparation method and application thereof

Info

Publication number: CN112094279A
Application number: CN202011027532.6A
Authority: CN
Inventors: 范莉; 杨大成; 潘建芳; 唐雪梅; 任艳会; 周围; 徐兴然; 胡军华; 谢建平; 许峻旗; 吴玉珠; 韩海燕
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2020-12-18
Anticipated expiration: 2040-09-25
Also published as: CN112094279B

Abstract

The invention discloses a p-aminosalicylic acid dihydroartemisinin derivative, a preparation method and application thereof, belonging to the technical field of drug synthesis. The structural formula of the p-aminosalicylic acid dihydroartemisinin derivative is shown in the specification. The in vitro antibacterial activity test result shows that the overall antibacterial activity of the target compound is better; the in vitro antifungal activity test result shows that the target compound has good antibacterial activity on pichia pastoris; the determination result of the activity of resisting citrus germs shows that the compound has inhibitory activity on citrus colletotrichum gloeosporioides, citrus brown spot germs and citrus canker germs; the result of the activity test of the anti-mycobacterium smegmatis shows that the inhibitory activity of the intermediate IM1 'and IM2' is good. Thereby proving that the amino salicylic acid dihydroartemisinin derivatives and the intermediate thereof have potential application prospects in the fields of bacteria resistance, fungi resistance, citrus bacteria resistance and tuberculosis resistance.

Description

P-aminosalicylic acid dihydroartemisinin derivative and preparation method and application thereof

Technical Field

The invention relates to the technical field of drug synthesis, in particular to a p-aminosalicylic acid dihydroartemisinin derivative and a preparation method and application thereof.

Background

Para-aminosalicylic acid(PAS for short) is the earliest found tuberculosis treatment small-molecule drug and is also used for treating inflammatory bowel diseases. PAS amino derivatives can be classified into three major groups. The first is a PAS schiff base derivative. Research results show that the antibacterial concentration of the synthesized target compound to mycobacterium smegmatis and mycobacterium bovis is lower than PAS; schiff base derivatives of Pyrazinamide (PZA) and PAS, p-H₃₇The Rv inhibitory activity was stronger than PZA (MICs of 3.13 and 6. mu.g/mL, respectively). The second type is PAS hydrazone derivative, Isoniazid (INH) reacts with PAS to prepare a compound containing hydrazone group, and the experimental result shows that the compound is compared with a positive control (MIC)_INH＝1μg/mL、MIC_{Ciprofloxacin}1.5. mu.g/mL and MIC_{Norfloxacin hydrochloride}10. mu.g/mL) is preferred. In the third category, derivatives of amino groups with additional functional groups also exhibit good biological activity.

Dihydroartemisinin (DHA), the most important derivative of artemisinin, is not only better than artemisinin in solubility, bioavailability and bioactivity, but also has a hemiacetal hydroxyl group which provides a key reactive site for further modification while preserving the parent structure. Starting from DHA, a number of DHA derivatives have been successfully designed and synthesized that have excellent antimalarial efficacy or other potential activities, including antitubercular, anticancer, antiallergic, antibiotically enhancing, anthelmintic, antimycotic, antiviral and anti-HIV effects, among others.

Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis that can cause human death, and the mortality rate is second to acquired immune deficiency syndrome (HIV). Although more than 10 antituberculosis drugs exist, more than 1000 million tuberculosis patients are still newly added in 2018 all the world, and tuberculosis still represents a great infectious disease threatening human health.

The current tuberculosis treatment needs the combined medication of a plurality of medicines, the course of treatment of drug-sensitive tuberculosis (DS-TB) is 6 months, most of patients with widely drug-resistant tuberculosis (XDR-TB) may need to receive the treatment of 8 antibiotics, the course of treatment is generally 9-20 months, and some patients with widely drug-resistant tuberculosis (XDR-TB) are even longer, so that the patient compliance is poor, the probability of drug resistance is increased, or the clinical result at the end of the treatment is unsatisfactory. On a global scale, recent data show that DS-TB has a treatment success rate of 85%, multi-drug resistant tuberculosis (MDR-TB) of 56%, and XDR-TB of 39%. The main challenge in tuberculosis treatment is the duration and complexity of the drug course, both of which affect compliance; in addition, antituberculosis drugs have toxic and side effects, especially drugs for treating drug-resistant tuberculosis; further, there is a lack or limitation of pediatric drug formulations for second line therapy. The drug interactions between antitubercular drugs and antiretroviral therapy and drug-accumulated toxicity increase the risk of immune-reconstitution of inflammatory syndromes, further complicating the treatment of aids-tuberculosis comorbidities (HIV-TB), diabetes-tuberculosis comorbidities (DM-TB) and M/XDR-TB patients, with limited medication and great difficulty of treatment, leading to global panic, so that the WHO announces a tuberculosis global emergency in 1993 and 1998 for two degrees. There is a global urgent need for therapeutic agents that are more effective, affordable to patients, non-toxic, and capable of shortening the treatment time.

Fungi can cause various diseases of animals, plants and humans. Different fungi cause diseases in different ways and can be divided into the following types: (1) pathogenic fungal infections: caused by exogenous fungi, such as dermatophytosis; (2) conditionally pathogenic fungal infections: caused by endogenous fungi, such as candida albicans, etc.; (3) fungal hypersensitivity disorders: urticaria, asthma, etc. caused by inhalation or ingestion of hypha or spores; (4) fungal toxicosis: caused by eating mildewed grains containing mycotoxin; (5) mycotoxin: is associated with tumorigenesis. Antifungal agents commonly used for the treatment of mycoses, known are azole antifungal agents (luliconazole, bifonazole, ketoconazole, miconazole, itraconazole, clotrimazole, neticonazole, oxiconazole, tioconazole, miconazole, omoconazole, sulconazole and salts thereof and the like), benzylamine antifungal agents (butenafine and salts thereof and the like), allylamine antifungal agents (terbinafine and salts thereof and the like), morpholine antifungal agents (amorolfine and salts thereof and the like), thiocarbamic antifungal agents (liranaftate, tolnaftate, tolcyclamate and the like), and antibiotics (nystatin, gulcomycin, pseudopenicillin, dry helminthosporin, nitropyrrolidin, amphotericin and the like) and the like, however, these antibacterial drugs have strong accumulated toxicity, often cause liver and kidney injury, digestive tract irritation, dizziness, allergy and the like, therefore, the search for novel antibacterial drugs with unique action mechanism becomes one of the hotspots in the research and development of the current drugs.

The citrus canker is widely distributed, can harm dozens of rutaceae plants, and is a major epidemic disease affecting the worldwide citrus production. The damage to citrus leaves, branches and fruits typically causes canker spots, untimely treatment and aggravated diseases, and seriously damages citrus production and economic benefits. The citrus canker pathogenic bacteria line is complex in differentiation, high in incidence rate, rapid in propagation and wide in host range, so that the prevention and control of citrus diseases are always a worldwide problem, and no method can radically cure citrus diseases at present. When in production, the mixed liquid containing metal copper ions, such as Bordeaux mixture, is usually sterilized, and is required to be sprayed for a plurality of times, so that the generation of drug resistance can be accelerated, and the toxicity to soil and other probiotics can be generated. The development of novel anti-citrus drugs is urgent.

Disclosure of Invention

In view of the above, the present invention aims to provide a p-aminosalicylic acid dihydroartemisinin derivative, a preparation method and an application thereof.

Through research, the invention provides the following technical scheme:

1. a p-aminosalicylic acid dihydroartemisinin derivative shown as a formula I, a racemate, a stereoisomer, a tautomer, a nitrogen oxide compound or a pharmaceutically acceptable salt thereof:

in formula I, R is selected from:

R₁and R₂Independently selected from H or C1-C3 alkyl; r₃And R₄Independently selected from H or C1-C3 alkyl; r₅Is H orC1-C3 alkyl; r₆Is H or C1-C3 alkyl; r₇Is H, C1-C3 alkyl or amino; r₈H, C1-C3 alkyl, substituted or unsubstituted phenyl, wherein the substituents on the phenyl are one or more independently selected from halogen, hydroxy, amino or C1-C3 alkyl; r₉And R₁₀Independently selected from H or C1-C3 alkyl; r₁₁is-O-, -S-or-NH-; r₁₂And R₁₃Independently selected from the group consisting of H, halogen, or alkanoyl; l is₁Selected from: - (CH)₂)_n+1-、-(CH₂)_nCO-or-CO (CH)₂)_nCO-, n is 1,2, 3 or 4; l is₂Selected from: - (CH)₂)_m-m is 2, 3,4 or 5; x is selected from: C1-C6 alkyl.

Preferably, in said formula I, R₁And R₂Independently selected from H or methyl; r₃And R₄Independently selected from H or methyl; r₅Is H or methyl; r₆Is H or methyl; r₇Is methyl or amino; r₈Is methyl, substituted or unsubstituted phenyl, and the substituent on the phenyl is one or more and is independently selected from halogen, hydroxyl, amino or C1-C3 alkyl; r₉And R₁₀Are both methyl; r₁₁is-O-, -S-or-NH-; r₁₂And R₁₃Independently selected from H, halogen or acetyl; l is₁Selected from: - (CH)₂)_n+1-or- (CH)₂)_nCO-, n is 1,2, 3 or 4; l is₂Selected from: - (CH)₂)_m-, m is 2, 3 or 4; x is selected from: C1-C3 alkyl.

Preferably, in the formula I, R is selected from:

L₁selected from: - (CH)₂)_nCO-, n is 1,2, 3 or 4; l is₂Selected from: - (CH)₂)_m-, m is 2, 3 or 4; x is selected from: and (4) ethyl.

Preferably, the para-aminosalicylic acid dihydroartemisinin derivative shown in the formula I is any one of the following compounds:

2. the preparation method of the p-aminosalicylic acid dihydroartemisinin derivative comprises the following steps:

esterifying carboxyl p-aminosalicylate to obtain an intermediate IM 1';

reacting the intermediate IM1 'with a linker reagent to obtain an intermediate IM 2';

coupling the intermediate IM2' with oxazole to obtain TM 4;

reacting dihydroartemisinin with a linker reagent to prepare an intermediate IM 4;

coupling the intermediate IM4 with TM4 to prepare a target molecule of the dihydroartemisinin p-aminosalicylate derivative;

in the formula, R, L₁X and L₂The definition of (1) and the structural formula of the p-aminosalicylic acid dihydroartemisinin derivative are shown in the specification₁X and L₂The definitions of (A) are the same; r₁₄And R₁₅Independently selected from halogen.

Preferably, the preparation method of the p-aminosalicylic acid dihydroartemisinin derivative comprises the following steps:

A. p-aminosalicylic acid reacts with alcohol under the action of acid to prepare an intermediate IM 1'; the alcohol is methanol or ethanol; the acid is sulfuric acid;

B. reacting the intermediate IM1 'with a linker reagent in an organic solvent under the action of alkali to obtain an intermediate IM 2'; the organic solvent is dichloromethane, chloroform, acetone, ethyl acetate, tetrahydrofuran or diethyl ether; the alkali is potassium carbonate, triethylamine or sodium bicarbonate;

C. coupling the intermediate IM2' with oxazole under the action of an organic solvent and alkali to prepare TM 4; the organic solvent is dichloromethane, chloroform, acetonitrile, tetrahydrofuran or N, N-dimethylformamide; the alkali is sodium bicarbonate, triethylamine, sodium hydroxide, sodium methoxide or potassium carbonate.

D. Under the action of an organic solvent and boron trifluoride, dihydroartemisinin and a linker reagent are prepared into an intermediate IM 4; the organic solvent is diethyl ether;

E. coupling IM4 and TM4 under the action of organic solvent and alkali to prepare a dihydroartemisinin p-aminosalicylate derivative TM 7; the organic solvent is dichloromethane, chloroform, acetonitrile, tetrahydrofuran or N, N-dimethylformamide; the alkali is sodium bicarbonate, triethylamine, sodium hydroxide, sodium methoxide or potassium carbonate.

More preferably, in the step B, the organic solvent is dichloromethane or chloroform; the base is sodium bicarbonate.

More preferably, in the step C, the organic solvent is N, N-dimethylformamide, and the base is potassium carbonate.

3. Prepared by the above preparation methodIntermediate IM2', i.e

4. Intermediate IM2' obtained by the above preparation method, i.e.

Application in antituberculosis drugs.

5. The application of the p-aminosalicylic acid dihydroartemisinin derivative in antibacterial drugs.

6. The application of the p-aminosalicylic acid dihydroartemisinin derivative in antifungal medicines is provided.

Preferably, the p-aminosalicylic acid dihydroartemisinin derivative is applied to the anti-pichia pastoris medicament.

7. The application of the p-aminosalicylic acid dihydroartemisinin derivative in the drug for resisting citrus bacteria is provided.

Preferably, the application of the p-aminosalicylic acid dihydroartemisinin derivative in the drug for resisting colletotrichum gloeosporioides is provided.

The term "racemate" as used herein means, unless otherwise specified, an optically inactive organic substance composed of equal amounts of enantiomers. "stereoisomers" refers to molecules that have the same atomic composition and bonding, but differ in the arrangement of the atoms in three-dimensional space. "Nitrogen oxide" means a tertiary nitrogen with an oxygen atom forming⁺N-O^-Organic matter of the structural unit. The "pharmaceutically acceptable salt" may be an acidic salt or a basic salt, such as an inorganic acid salt, an organic acid salt, an inorganic base salt or an organic base salt.

The term "C1-C3 alkyl" refers to straight or branched chain saturated monovalent hydrocarbon radicals having 1-3 carbon atoms such as methyl, ethyl, propyl and isopropyl.

The term "halogen" refers to F, Cl, Br and I.

The invention has the beneficial effects that:

1) the invention provides a p-aminosalicylic acid dihydroartemisinin derivative, which takes p-aminosalicylic acid as a parent nucleus and has amino groups, carboxyl groups and amino groupsThe phenolic hydroxyl group is reasonably modified to construct a para-aminosalicylic acid dihydroartemisinin derivative with a novel structure, and the chemical structure of the product is shown in the specification¹H NMR,¹³C NMR and HR MS confirmation;

2) the in vitro antibacterial activity test result of the compounds shows that the antibacterial activity of the TM7 series compounds is better than that of the parent PAS, the intermediate IM1' -1 and the precursor TM4-1 series compounds on the whole, and the MIC value is as low as 2 mu g/mL. In comparison, the TM7 series compounds have the best inhibitory activity against staphylococcus aureus, and have 6 MIC molecules <64 μ g/mL, wherein 3 MIC molecules are 16 μ g/mL, and 1MIC molecule (TM7-10) is 2 μ g/mL. In addition, the IM2' -1 intermediate has strong capability of inhibiting staphylococcus aureus, micrococcus luteus and salmonella, and even has stronger capability than that of certain fluoroquinolone medicaments. The results show that the p-aminosalicylic acid dihydroartemisinin derivative and the intermediate thereof have potential application prospects in the antibacterial field;

3) the in vitro antifungal activity test result of the compound shows that the antibacterial activity of the target compound TM7 series to pichia is overall good and is stronger than that of the precursor TM4-1 series compound, the parent PAS, the intermediate IM1'-1 and the intermediate IM2' -1; after 24h of culture, 12 molecules of 14 TM7 series compounds have MIC values of 4 mu g/mL, which are the same as the MIC of positive control fluconazole, and show very strong antibacterial activity; the inhibiting activity of the intermediate IM2' -1 on pichia pastoris reaches 64 mu g/mL, which is stronger than that of a mother nucleus PAS. These data prove that the derivatives and intermediates of the p-aminosalicylic acid dihydroartemisinin have potential application prospect in the antifungal field

4) The result of the activity determination of the compound against citrus pathogens shows that the tested compound has certain inhibitory activity against citrus colletotrichum gloeosporioides and citrus brown spot pathogen: for the citrus colletotrichum gloeosporioides, under the test concentration of 1 mu g/mL, the inhibition rate of 12 TM7 series compounds on the citrus colletotrichum gloeosporioides is more than 50% of that of a positive medicament, namely prochloraz, and the activity of 4 molecules is more than 80% of that of the prochloraz; under the test of 4 mu g/mL, the inhibition rate of 12 TM7 series compounds exceeds 50 percent of that of the positive drug prochloraz, wherein the activity of TM7-12 is close to 90 percent of that of the prochloraz. The inhibition rate (64.29%) of the intermediate IM2' -1 on the citrus brown spot pathogen is higher than that of a positive control prochloraz (50%), and the inhibition rate (50%) of TM7-2 is the same as that of the prochloraz. More importantly, the high-activity molecules TM7-5 and TM7-12 do not show drug resistance to the brown spot pathogen of citrus and colletotrichum gloeosporioides. For citrus canker pathogen, under the test concentration of 1.6 mu g/mL, the inhibition activity of TM7 series target molecules is 5 molecules with the inhibition rate higher than 35%, and under the test concentration of 0.64 mu g/mL, the inhibition rate is 4 molecules with the inhibition rate higher than 35%, wherein the activity of TM 7-1-TM 7-3 is stronger than that of norfloxacin. These results prove that the amino-salicylic acid dihydroartemisinin derivatives and the intermediates thereof have potential application prospects in the field of citrus germ resistance;

5) the results of the activity measurement of the anti-Mycobacterium smegmatis show that the MIC values of the intermediate IM1'-1 are both 0.19 mu g/mL, and the MIC value of the intermediate IM2' -1 is 0.38 mu g/mL. Thereby proving that the intermediate of the amino salicylic acid dihydroartemisinin derivative has potential application prospect in the anti-tuberculosis field.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the embodiments of the present invention, and it is obvious 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.

First, main reagent and instrument

P-aminosalicylic acid, chloroacetyl chloride, dichloromethane, N-dimethylformamide, imidazole, 2-methylimidazole, 4-methylimidazole, pyrazole, benzimidazole, 1,2, 4-triazole, 2, 4-dimethylpyrazole, 1H-tetrazole, 5-methyltetrazole, 4, 6-dimethyl-2-mercaptopyrimidine, 1-methyl-5-mercaptotetrazole, 1-phenyl-5-mercaptotetrazole, 2-mercapto-5-methyl-1, 3, 4-thiadiazole (AR); 2-amino-5-mercapto-1, 3, 4-thiadiazole (97.5%); concentrated sulfuric acid, ethanol, sodium bicarbonate and potassium carbonate; dihydroartemisinin (AR); 3-bromo-1-propanol (more than or equal to 97%); anhydrous diethyl ether, boron trifluoride-diethyl ether (BF)₃·Et₂O) (AR); the other reagents are all commercial chemical pure or analytical pure products.

Nuclear magnetic resonance apparatus (AV-600,600MHz, TMS as internal standard); high resolution mass spectrometer (Varian 7.0T); a melting point tester (X-6); an automatic polarimeter (WZZ-2S); an ultraviolet analyzer (ZF-1); a rotary evaporator (RE-2000); magnetic stirring low-temperature constant-temperature water tank.

Preparation of dihydroartemisinin derivatives of p-aminosalicylic acid

1. Synthesis of intermediate IM1' -1

Into the reaction flask were added PAS 20mmol and absolute ethanol 25mL, and the mixture was stirred at room temperature. And (4) performing ice bath, dropwise adding 50mmol of concentrated sulfuric acid, after dropwise adding, performing oil bath reflux reaction at the temperature of 80 ℃, and monitoring by Thin Layer Chromatography (TLC) until the reaction is finished. Cooling in ice bath, and saturating with sodium carbonate (Na)₂CO₃) Adjusting pH to 7-8, refrigerating, filtering, and washing filter cake with ice water. The filtrate was extracted with Dichloromethane (DCM) (3X 30mL) and the organic phase was collected and washed with saturated NaCl solution. Dried over anhydrous sodium sulfate, rotary evaporated, combined with filter cake, column chromatographed, vacuum dried and weighed to give intermediate IM1' -12.32 g in 64% yield.

2. Synthesis of intermediate IM2' -1

To the reaction flask were added IM1' -15 mmol, DCM 5mL, NaHCO₃12.5 mmol; cooled in ice bath, and 10mmol of chloroacetyl chloride is added dropwise. After the addition, the reaction was continued in an ice bath (3 ℃ C.) and monitored by TLC until the reaction was complete. Stopping stirring, adding 10mL of ice-cold saturated saline solution to dissolve the solid, adjusting pH to 4-5 with 2N HCl solution, stirring, transferring into separating funnel, extracting with Ethyl Acetate (EA) twice, mixing the organic phases, washing with saturated saline solution, and removing anhydrous Na₂SO₄Drying, removing solvent by rotary evaporation, performing column chromatography to obtain pure product, drying, and weighing to obtain intermediate IM2' -11.16 g with yield of 90%.

3. Preparation of Compound TM4-1

Adding oxazole, N-Dimethylformamide (DMF) and K into a reaction bottle in sequence₂CO₃Stirring at room temperature, adding the intermediate IM2' -1, transferring to a water bath at 45 ℃ for stirring reaction, and tracking and monitoring by TLC until the reaction is finished. Stopping stirring, adding ice-cold saturated NaCl solution, adjusting pH to 6-7 with 2N HCl solution, and refrigerating. Suction filtration, washing of the filter cake with saturated brine (10 mL. times.1), washing with ice water (5 mL. times.1), drying of the filter cake to obtain a crude product, and column chromatography (PE/EA is 10:1-1:3, v/v) to obtain a pure product. When oxazole with mercapto group is added, THF is added as organic solvent, Petroleum Ether (PE) is used for multiple times of dispersion before column chromatography, and other conditions and operations are the same as above. And (4) drying the pure product in vacuum, and weighing to obtain the target compound TM 4-1. The experimental conditions and results are shown in Table 1.

TABLE 1 Experimental conditions and results for the preparation of TM4-1

4. Preparation of intermediate IM4-1

Adding DHA 1equiv, proper amount of diethyl ether and 3-bromo-1-propanol 1.2equiv into a reaction bottle, and dropwise adding BF at-15 deg.C₃·Et₂The reaction was continued at-15 ℃ with 1.6equiv of O solution, and the progress of the reaction was monitored by TLC. And after the reaction is completed, adding saturated sodium bicarbonate aqueous solution and DCM, performing extraction separation, monitoring by TLC that no product exists in an aqueous phase, drying an organic phase by using anhydrous sodium sulfate, and performing rotary evaporation to obtain an oily crude product. After adding 10mL of petroleum ether, the mixture was refrigerated and filtered with suction. Spin-drying the filtrate, performing column chromatography (PE/EA is 50:1, v/v), and addingRefrigerating and crystallizing 6mL of petroleum ether, filtering, combining filter cakes, and drying in vacuum to obtain an intermediate IM 4-1. The experimental conditions and results are shown in Table 2.

TABLE 2 intermediate IM4-1 Experimental conditions and results

5. Preparation of p-aminosalicylic acid dihydroartemisinin derivative TM7

Adding TM4-1, IM4-1 and K into a reaction bottle respectively₂CO₃DMF, stirring at controlled temperature, and TLC to monitor the reaction progress. After the reaction, 15mL of water and 20mL of EA were added, stirred uniformly, and allowed to stand for liquid separation. EA was extracted several times until the aqueous phase was product free and combined with the previous organic phase. The organic phase was saturated NaHCO₃Washing the solution (10mL multiplied by 1), washing with saturated brine (10mL multiplied by 1), drying with anhydrous sodium sulfate, rotary steaming to obtain a crude product, performing column chromatography (PE/EA is 10:1-5:1, v/v), rotary steaming, vacuum drying the pure product, and weighing to obtain the target compound TM 7. The experimental conditions and results are shown in table 3.

TABLE 3 Experimental conditions and results for the Synthesis of Compound TM7

6. The TM7 product structure was characterized as follows:

TM7-1:Ethyl 4-(2-(1H-imidazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate Colorless oil；

(CH₂Cl₂,c＝2.0mg/mL)；¹H NMR(600MHz,DMSO-d6)10.57(s,1H,H-5),7.75(d,J＝8.6Hz,1H,H-3),7.63(s,1H,H-24),7.54(s,1H,H-26),7.15(s,1H,H-6),7.12(dd,J＝8.6Hz,1.3Hz,1H,H-4),6.90(s,1H,H-25),5.07(s,1H,H-22),4.93(s,2H,H-23),4.69(d,J＝3.2Hz,1H,H-10),4.23(q,2H,H-2),4.06～4.00(m,2H,H-7),3.43～3.36(m,2H,H-9),2.10(td,J＝14.1,3.7Hz,1H,H-11),2.00(dt,J＝14.1,5.6Hz,2H,H-8),1.93～1.87(m,3H,H-16and H-20),1.67～1.61(m,1H,H-19),1.52(dt,J＝13.2,6.1Hz,2H,H-14 and H-15),1.31(t,J＝7.1Hz,3H,H-1),1.25(s,3H,H-21),1.21～1.16(m,2H,H-14 and H-15),1.13～1.09(m,1H,H-13),1.06(d,J＝7.0Hz,1H,H-18),0.97(dd,J＝11.2,4.8Hz,1H,H-19),0.80(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)172.40,166.81,165.39,159.53,144.13,138.78,132.97,128.42,121.12,114.61,110.74,103.51,100.81,87.15,80.83,64.95,62.95,60.60,55.34,52.38,49.74,44.24,36.61,34.39,30.98,28.83,26.06,24.51,21.49,20.63,14.59,13.19.LC MS calcd for C₃₂H₄₃N₃O₉,[M+H]⁺614.3,found 614.3.

TM7-2:Ethyl 4-(2-(5-methyl-1H-imidazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate Colorless oil；

(CH₂Cl₂,c＝2.5mg/mL)；¹H NMR(600MHz,DMSO-d6)10.61(s,1H,H-5),7.76(d,J＝8.7Hz,1H,H-3),7.55(d,J＝1.3Hz,1H,H-6),7.36(d,J＝1.9Hz,1H,H-24),7.06(d,J＝3.4Hz,1H,H-4),6.74(s,1H,H-25),5.04(s,1H,H-22),4.69(d,J＝3.3Hz,1H,H-10),4.34(q,J＝7.1Hz,2H,H-23),4.25～4.20(m,2H,H-2),4.15～4.06(m,2H,H-7),3.41～3.35(m,2H,H-9),2.37～2.30(m,1H,H-11),2.23(s,3H,H-26),2.10～1.94(m,4H,H-8 and H-14 and H-15),1.64～1.47(m,4H,H-14 and H-15 and H-16 and H-20),1.32(d,J＝3.5Hz,3H,H-1),1.24(s,3H,H-21),1.20～1.08(m,3H,H-19 and H-20),1.02～0.93(m,2H,H-13 and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.64(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)172.41,169.12,166.87,165.38,161.82,159.54,145.41,131.35,126.30,121.49,110.93,106.77,103.49,87.12,80.82,61.57,60.60,55.35,52.36,49.18,44.22,36.59,34.37,33.60,30.98,26.06,24.53,21.50,20.64,19.36,14.54,13.19,12.94.LC MS calcd for C₃₃H₄₅N₃O₉,[M+H]⁺628.3,found 628.3.

TM7-3:Ethyl 4-(2-(4-methyl-1H-imidazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.101.3～102.7℃；

(CH₂Cl₂,c＝2.1mg/mL)；¹H NMR(600MHz,DMSO-d6)10.55(s,1H,H-5),7.75(d,J＝8.4Hz,1H,H-3),7.54(d,J＝1.5Hz,1H,H-6),7.48(s,1H,H-24),7.11(d,J＝8.6Hz,1H,H-4),6.83(d,J＝5.4Hz,1H,H-26),5.07(s,1H,H-22),4.69(d,J＝3.1Hz,1H,H-10),4.23(q,J＝13.6,6.5Hz,2H,H-2),4.16～4.06(m,2H,H-23),4.06～3.99(m,2H,H-7),3.44～3.34(m,2H,H-9),2.40～2.29(m,1H,H-11),2.08(s,3H,H-25),2.04～1.86(m,4H,H-8 and H-14 and H-15),1.67～1.46(m,4H,H-14 and H-15 and H-16 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.28～1.26(m,1H,H-19),1.25(s,3H,H-21),1.21～0.91(m,4H,H-13 and H-18 and H-19 and H-20),0.80(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.4Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)166.92,165.39,159.52,144.15,137.97,136.79,132.96,117.30,114.60,110.74,103.60,103.50,100.81,87.16,80.83,67.91,64.95,60.59,52.38,49.75,44.24,36.70,36.53,34.38,30.98,28.84,26.06,24.52,20.61,14.59,14.02,13.19,9.08.LC MS calcd for C₃₃H₄₅N₃O₉,[M+H]⁺628.3,found 628.3.

TM7-4:Ethyl 4-(2-(1H-pyrazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.93.8～95.2℃；

(CH₂Cl₂,c＝2.0mg/mL)；¹H NMR(600MHz,DMSO-d6)10.59(s,1H,H-5),7.77(d,J＝2.1Hz,1H,H-26),7.77(d,J＝8.5Hz,1H,H-3),7.55(d,J＝1.5Hz,1H,H-6),7.47(d,J＝1.4Hz,1H,H-24),7.12(dd,J＝8.6Hz,1.6Hz,1H,H-4),6.29(t,J＝2.0Hz,1H,H-25),5.76(s,2H,H-23),5.05(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.23(q,J＝7.1Hz,2H,H-2),4.14～4.07(m,1H,H-9),4.06～3.97(m,2H,H-7),3.44～3.35(m,1H,H-9),2.39～2.31(m,1H,H-11),2.14～1.58(m,6H,H-8 and H-14 and H-15 and H-20),1.56～1.44(m,2H,H-14 and H-15),1.32(t,J＝7.1Hz,3H,H-1),1.27(d,J＝7.0Hz,1H,H-16),1.25(s,3H,H-21),1.21～0.92(m,4H,H-19 and H-13 and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)166.57,165.39,159.53,144.12,139.55,132.97,132.06,114.62,110.77,105.78,103.61,103.50,100.79,87.14,80.84,64.93,62.90,60.59,55.35,54.98,52.40,44.24,36.69,36.54,34.38,30.98,28.82,26.07,24.51,20.63,14.59,13.20.LC MS calcd for C₃₂H₄₃N₃O₉,[M+H]⁺614.3,found 614.3.

TM7-5:Ethyl 4-(2-(1H-benzo[d]imidazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.112.4～114.3℃；

(CH₂Cl₂,c＝2.2mg/mL)；¹H NMR(600MHz,DMSO-d6)10.76(s,1H,H-5),8.24(s,1H,H-28),7.77(d,J＝8.6Hz,1H,H-3),7.68(d,J＝7.3Hz,1H,H-24),7.56(d,J＝1.3Hz,1H,H-6),7.50(d,J＝7.4Hz,1H,H-27),7.26～7.20(m,2H,H-25 and H-26),7.13(dd,J＝8.6Hz,1.5Hz,1H,H-4),5.05(s,1H,H-22),4.68(d,J＝3.2Hz,1H,H-10),4.23(q,J＝7.1Hz,2H,H-2),4.13～4.05(m,1H,H-9),4.06～3.96(m,3H,H-7 and H-9),2.39～2.30(m,1H,H-11),2.16～1.93(m,5H,H-8 and H-14 and H-15 and H-20),1.67～1.43(m,3H,H-14 and H-15 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.28～1.25(m,1H,H-16),1.24(s,3H,H-21),1.18(t,J＝7.1Hz,2H,H-19),1.12～1.05(m,1H,H-13),0.98～0.92(m,1H,H-18),0.78(d,J＝7.3Hz,3H,H-12),0.63(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)170.76,166.56,165.38,159.55,145.44,144.08,143.70,134.87,132.99,122.84,122.01,119.86,114.66,110.78,110.69,103.50,100.79,87.14,80.82,64.95,62.92,60.60,60.20,55.35,52.37,47.89,44.22,36.60,34.36,30.97,28.80,26.06,24.50,20.63,14.56,13.17.LC MS calcd for C₃₆H₄₅N₃O₉,[M+H]⁺664.3,found 664.3.

TM7-6:Ethyl 4-(2-(1H-1,2,4-triazol-1-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.104.7～106.1℃；

(CH₂Cl₂,c＝2.3mg/mL)；¹H NMR(600MHz,DMSO-d6)10.69(s,1H,H-5),8.55(s,1H,H-25),8.00(s,1H,H-24),7.77(d,J＝8.6Hz,1H,H-3),7.53(d,J＝1.4Hz,1H,H-6),7.12(dd,J＝8.6Hz,1.6Hz,1H,H-4),5.17(s,2H,H-23),5.05(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.23(q,J＝7.1Hz,2H,H-2),4.13～4.08(m,1H,H-9),4.05～4.00(m,2H,H-7),3.42～3.36(m,1H,H-9),2.39～2.30(m,1H,H-11),2.14～1.87(m,5H,H-8and H-14 and H-15 and H-20),1.67～1.44(m,3H,H-14 and H-15 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.28–1.26(m,1H,H-16),1.25(s,3H,H-21),1.20–0.91(m,4H,H-13 and H-18 and H-19),0.80(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)165.59,165.37,159.52,151.88,146.07,143.91,133.00,114.80,110.81,103.67,103.50,100.80,87.14,80.83,64.97,62.92,60.62,52.39,52.33,44.24,36.69,36.53,34.38,30.98,28.81,26.06,24.52,24.48,20.62,14.58,13.19.HR MS calcd for C₃₁H₄₂N₄O₉,[M+H]⁺615.3025,found 615.3040.

TM7-7:Ethyl 4-(2-(2H-tetrazol-2-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.112.3～114.7℃；

(CH₂Cl₂,c＝2.4mg/mL)；¹H NMR(600MHz,DMSO-d6)10.83(s,1H,H-5),9.42(s,1H,H-24),7.77(d,J＝8.5Hz,1H,H-3),7.51(d,J＝1.4Hz,1H,H-6),7.11(dd,J＝8.6Hz,1.6Hz,1H,H-4),5.06(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.23(q,J＝7.1Hz,2H,H-2),4.12～4.06(m,2H,H-23),4.05～4.00(m,2H,H-7),3.41～3.36(m,2H,H-9),2.39～2.28(m,1H,H-11),2.14～1.84(m,5H,H-8 and H-14 and H-15 and H-20),1.68～1.42(m,3H,H-16 and H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.29～1.26(m,1H,H-19),1.25(s,1H,H-21),1.20～0.92(m,4H,H-13 and H-14 and H-15 and H-18),0.79(d,J＝7.3Hz,3H,H-12),0.65(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)165.36,164.52,159.51,145.69,143.68,133.04,114.99,110.86,103.73,103.51,100.81,87.15,80.83,65.01,62.94,60.65,55.35,52.38,50.60,44.23,36.71,36.52,34.39,30.97,28.81,26.07,24.50,20.62,14.58,13.19.HR MS calcd for C₃₀H₄₁N₅O₉,[M+Na]⁺638.2796,found 638.2788.

TM7-8:Ethyl 4-(2-(5-methyl-2H-tetrazol-2-yl)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.108.6～109.9℃；

(CH₂Cl₂,c＝2.3mg/mL)；¹H NMR(600MHz,DMSO-d6)10.85(s,1H,H-5),7.78(d,J＝8.5Hz,1H,H-3),7.51(d,J＝1.4Hz,1H,H-6),7.11(dd,J＝8.6Hz,1.7Hz,1H,H-4),5.04(s,1H,H-22),4.68(d,J＝3.2Hz,1H,H-10),4.23(q,J＝6.8Hz,2H,H-2),4.13～4.08(m,2H,H-23),4.05～3.99(m,2H,H-7),3.42～3.35(m,2H,H-9),2.50(s,3H,H-24),2.37～2.32(m,1H,H-11),2.13～1.86(m,5H,H-8 and H-14 and H-15 and H-20),1.66～1.45(m,3H,H-16 and H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.28～1.25(m,1H,H-19),1.24(s,3H,H-21),1.20～0.92(m,4H,H-13 and H-14 and H-15 and H-18),0.79(d,J＝7.3Hz,3H,H-12),0.64(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)165.35,164.44,159.51,153.89,143.62,133.04,115.03,110.92,103.78,103.50,100.79,87.13,80.82,65.02,62.91,60.65,52.37,49.76,44.22,36.68,36.52,34.37,30.98,28.80,26.06,24.53,24.48,20.62,14.58,13.18,8.75(s).HR MS calcd for C₃₁H₄₃N₅O₉,[M+Na]⁺652.2953,found 652.2942.

TM7-9:Ethyl 4-(2-((4,6-dimethylpyrimidin-2-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate Colorless oil；

(CH₂Cl₂,c＝2.0mg/mL)；¹H NMR(600MHz,DMSO-d6)10.49(s,1H,H-5),7.74(d,J_H4-H3＝8.6Hz,1H,H-3),7.53(d,J＝1.5Hz,1H,H-6),7.14(dd,J＝8.6Hz,1.7Hz,1H,H-4),6.97(s,1H,H-25),5.05(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.22(q,J＝7.1Hz,2H,H-2),4.06(s,2H,H-23),4.04～4.00(m,2H,H-7),3.58～3.33(m,2H,H-9),2.32(s,6H,H-24 and H-26),2.23～2.16(m,1H,H-11),2.14～2.05(m,2H,H-8),2.04～1.96(m,2H,H-14 and H-15),1.92～1.87(m,1H,H-20),1.65～1.44(m,3H,H-16 and H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.24(s,3H,H-21),1.17～1.06(m,3H,H-14 and H-15 and H-19),0.97～0.83(m,2H,H-13 and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.61(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)169.64,167.59,167.43(3C),165.41,159.55,144.57(s),132.92,116.55,114.27,110.65,103.46(d,J＝8.4Hz),100.76,87.13,80.82,64.88,62.86,60.55,52.37,44.23,36.63,36.53,36.05,34.36,30.98,28.80,26.06,24.50,23.77(3C),20.60,14.59,13.19(s).HR MS calcd for C₃₅H₄₇N₃O₉S,[M+H]⁺686.3106,found 686.3096.

TM7-10:Ethyl 4-(2-((1-methyl-1H-tetrazol-5-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.109.4～110.7℃；

(CH₂Cl₂,c＝2.2mg/mL)；¹H NMR(600MHz,DMSO-d6)10.65(s,1H,H-5),7.75(d,J＝8.6Hz,1H,H-3),7.48(d,J＝1.5Hz,1H,H-6),7.12(dd,J＝8.6Hz,1.7Hz,1H,H-4),5.04(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.32(s,2H,H-23),4.22(q,J＝7.1Hz,2H,H-2),4.12～4.08(m,2H,H-7),3.98(s,3H,H-24),3.56～3.41(m,2H,H-9),2.38～2.32(m,1H,H-11),2.14～2.04(m,2H,H-8),2.02(s,2H,H-14 and H-15),1.92～1.88(m,1H,H-20),1.65～1.43(m,3H,H-16 and H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.24(s,3H,H-21),1.20～1.14(m,1H,H-19),1.12～1.07(m,2H,H-14 and H-15),0.98～0.84(m,2H,H-13 and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.62(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)170.78,166.03,165.38,159.51,153.72,144.12,132.99,114.69,110.77,103.49,100.78,87.14,80.84,68.12,65.99,63.69,60.61,52.39,44.24,38.18,36.67,34.12,30.98,28.81,26.07,24.51,21.14,20.60,15.48,14.58,13.20.LC MS calcd for C₃₁H₄₃N₅O₉S,[M+H]⁺662.3,found 662.3.

TM7-11:Ethyl 4-(2-((1-phenyl-1H-tetrazol-5-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propox-y)benzoate White solid；m.p.117.4～119.0℃；

(CH₂Cl₂,c＝2.3mg/mL)；¹H NMR(600MHz,DMSO-d6)10.70(s,1H,H-5),7.76(d,J＝8.6Hz,1H,H-3),7.71～7.65(m,5H,H-24 and H-25 and H-26 and H-27 and H-28),7.51(d,J＝1.5Hz,1H,H-6),7.12(dd,J＝8.6Hz,1.7Hz,1H,H-4),5.04(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.43(s,2H,H-23),4.23(q,J＝7.1Hz,2H,H-2),4.07～3.99(m,2H,H-7),3.47～3.36(m,2H,H-9),2.39～2.29(m,1H,H-11),2.15～1.95(m,4H,H-8 and H-14 and H-15),1.93～1.85(m,1H,H-20),1.66～1.57(m,1H,H-16),1.57～1.44(m,2H,H-14 and H-15),1.21～1.14(m,1H,H-19),1.12～1.04(m,2H,H-19 and H-20),1.31(t,J＝7.1Hz,3H,H-1),1.24(s,3H,H-21),0.99～0.88(m,2H,H-13 and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.62(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)165.80,165.38,159.52,154.32,144.12,133.51,132.98,131.15,130.56(3C),124.84(2C),114.69,110.77,103.49,100.79,87.14,80.83,68.12,64.97,63.69,62.92,60.60,52.39,44.24,38.26,36.60,34.33,30.98,28.82,26.07,24.51,20.60,14.59,13.19.LC MS calcd for C₃₆H₄₅N₅O₉S,[M+H]⁺724.3,found 724.3.

TM7-12:Ethyl 4-(2-((5-methyl-1,3,4-thiadiazol-2-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.108.7～109.9℃；

(CH₂Cl₂,c＝2.5mg/mL)；¹H NMR(600MHz,DMSO-d6)10.63(s,1H,H-5),7.75(d,J＝8.6Hz,1H,H-3),7.51(d,J＝1.5Hz,1H,H-6),7.13(dd,J＝8.6Hz,J＝1.7Hz,1H,H-4),5.04(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.29(s,2H,H-23),4.22(q,J＝7.1Hz,2H,H-2),4.06～4.01(m,2H,H-7),3.40～3.36(m,2H,H-9),2.67(s,3H,H-24),2.39～2.30(m,1H,H-11),2.14～2.05(m,2H,H-19 and H-20),2.01～1.97(m,2H,H-8),1.93～1.87(m,1H,H-20),1.67～1.57(m,1H,H-16),1.56～1.45(m,2H,H-14 and H-15),1.31(t,J＝7.1Hz,3H,H-1),1.24(s,3H,H-21),1.20～1.15(m,1H,H-19),1.13～1.02(m,2H,H-14 and H-15),0.99～0.84(m,2H,H-18 and H-13),0.80(d,J＝7.3Hz,3H,H-12),0.62(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)166.30,166.13,165.38,164.63,159.52,144.22,132.97,114.62,110.75,103.49(2C),100.78,87.13,80.83,68.12,64.95,62.90,60.60,55.35,52.39,44.24,38.65,36.60,34.35,30.98,28.81,26.07,24.52,20.61,15.64,14.59,13.21.HR MS calcd for C₃₂H₄₃N₃O₉S₂,[M+Na]⁺700.2333,found 700.2334.

TM7-13:Ethyl 4-(2-((5-amino-1,3,4-thiadiazol-2-yl)thio)acetamido)-2-(3-(((3R,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)propoxy)benzoate White solid；m.p.113.4～114.7℃；

(CH₂Cl₂,c＝2.3mg/mL)；¹H NMR(600MHz,DMSO-d6)10.52(s,1H,H-5),7.75(d,J＝8.5Hz,1H,H-3),7.51(d,J＝1.4Hz,1H,H-6),7.30(s,2H,H-24),7.12(dd,J＝8.6Hz,1.7Hz,1H,H-4),5.04(s,1H,H-22),4.69(d,J＝3.2Hz,1H,H-10),4.22(q,J＝7.1Hz,2H,H-2),4.14～4.04(m,2H,H-7)，4.03(s,2H,H-23),3.41～3.31(m,2H,H-9),2.39～2.31(m,1H,H-20),2.15～2.05(m,1H,H-19),2.10(td,J＝14.1,3.7Hz,1H,H-11),2.05～1.97(m,2H,H-8),1.93～1.87(m,1H,H-16),1.66～1.59(m,1H,H-20),1.57～1.47(m,2H,H-14and H-15),1.31(t,J＝7.1Hz,3H,H-1),1.30～1.23(m,2H,H-14and H-15),1.24(s,3H,H-21),1.22～1.16(m,1H,H-19),1.13～1.02(m,1H,H-13),0.98～0.91(m,1H,H-18),0.81(d,J＝7.3Hz,3H,H-12),0.63(d,J＝6.3Hz,3H,H-17).¹³C NMR(151MHz,DMSO-d6)170.40,166.87,165.40,159.52,149.67,144.28,132.95,114.57,110.74,103.49(2C),100.78,87.14,80.85,64.94,62.91,60.59,52.41,44.25,39.25,36.67,36.54,34.34,30.99,28.82,26.07,24.55,24.49,20.63,14.59,13.22.LC MS calcd for C₃₁H₄₂N₄O₉S₂,[M+H]⁺679.25,found 679.30.

and thirdly, detecting the biological activity of the derivatives of the p-aminosalicylic acid dihydroartemisinin

1. In vitro antibacterial Activity assay

The activity (MIC value) of the compounds against staphylococcus aureus (staphylococcus aureus ATCC 25129), Micrococcus luteus (Micrococcus luteus), Escherichia coli (Escherichia coli ATCC 25922), Acinetobacter baumannii (Acinetobacter baumannii ATCC 19606), Salmonella (Salmonella Enteritidis ATCC 13076) and Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 27853) was determined by the broth microdilution method.

A sample of 3.2mg was taken, and a DMSO solution containing 5% Tween 80 was first used to prepare a stock solution at a concentration of 3.2mg/mL, and 200. mu.L of the stock solution was then aspirated and diluted with a broth to 500. mu.L to obtain a test solution at a concentration of 1.28. mu.g/. mu.L.

Inoculating the preserved strain into a common liquid culture medium, and placing the strain in a constant temperature shaking table at 37 ℃ for activation culture for 17 h. Respectively diluting with brain heart infusion Broth (BHI) culture medium to 10⁵CFU/mL of bacterial suspension was ready for use (i.e., 5mL broth/5. mu.L of bacteria).

The operation is as follows: add 60. mu.L of blank broth to the first well of each column of the 96-well plate, and 50. mu.L of blank broth to the remaining wells; adding 40 mu L of the solution to be tested into the first hole of each row, and then diluting the substance to be tested twice, namely: adding the solution to be detected into the first hole, fully blowing and beating the solution to be detected by using a liquid transfer gun (at least three times, so that the substance to be detected and broth are fully and uniformly mixed), sucking 50 mu L of the solution to be detected, adding the solution to the second hole, fully blowing and beating the solution to be detected, uniformly mixing the solution to be detected, sucking 50 mu L of the solution to be detected, adding the solution to the third hole, repeating the steps until the eighth hole is reached, sucking 50 mu L of the solution to the eighth hole; at the moment, the concentration of the analyte in each hole is 512,256,128,64,32,16,8 and 4 mu g/mL from top to bottom. 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. Finally, 50 mu L of diluted bacteria liquid is added into each row of 1-8 holes, and a multi-hole test is adopted, wherein each plate tests 5 compounds, and the concentration of the substance to be tested in each hole, namely the final concentration, is 256,128,64,32,16,8,4 and 2 mu g/mL from top to bottom.

And (3) placing the inoculated 96-well plate into a constant-temperature incubator at 37 ℃ for culturing for 20-24h, and observing the growth condition of bacteria in the hole. And determining that the bacteria in the growth control hole grow normally and no bacteria grow in the negative control hole. The concentration of the drug in wells with no bacterial growth was observed as the MIC value of the drug against the bacteria.

The results of the in vitro bacteriostatic activity test on the aminosalicylic acid dihydroartemisinin derivatives and the intermediates are shown in table 4.

TABLE 4 inhibitory Activity of the Compounds against 6 pathogenic bacteria (MIC, μ g/mL)

Analysis table 4 shows that: the parent PAS and the intermediate IM1' -1 have almost no antibacterial ability; the TM4-1 series compounds have poor activity on six strains as a whole, and the MIC value of the molecule with the best activity is 16 mu g/mL; the antibacterial activity of the TM7 series compounds is better than that of the TM4-1 series compounds on the whole, and the MIC value is as low as 2 mu g/mL. In comparison, the TM7 series compounds have the best inhibitory activity against staphylococcus aureus, and have 6 MIC molecules <64 μ g/mL, wherein 3 MIC molecules are 16 μ g/mL, and 1MIC molecule (TM7-10) is 2 μ g/mL. In addition, the IM2' -1 intermediate has strong capability of inhibiting staphylococcus aureus, micrococcus luteus and salmonella, and even has stronger capability than that of certain fluoroquinolone medicaments. The results show that the p-aminosalicylic acid dihydroartemisinin derivative and the intermediate thereof have potential application prospects in the antibacterial field.

2. In vitro antifungal Activity assay

The activity of the compounds against pichia pastoris (MIC values) was determined using the NCCLS recommended broth microdilution method with fluconazole as positive control drug. The specific operation is as follows:

(1) preparation of sample solution: accurately weighing 3.2mg of a sample in a 2mL PE tube by a ten-thousandth balance in a drying chamber, adding 1mL DMSO into the PE tube by a pipette gun to dissolve the sample into clear transparent liquid to prepare a solution of 3.2mg/mL, sealing by a sealing film, and storing in a freezer in a dark place. For some poorly soluble compounds DMSO/tween-80 ═ 200/1(v/v) was used to increase solubility, tween-80 was the co-solvent.

(2) Preparing a solution to be detected: preparing stock solution with concentration of 3.2mg/mL with appropriate solvent and diluent, sucking 320 μ L of stock solution, adding Sabouraud's medium to total volume of 0.5mL, with concentration of 2048 μ g/mL, to obtain solution B.

(3) Preparation of bacterial suspension: inoculating the preserved strain into a Sabouraud's agar liquid culture medium, and placing the strain in a constant temperature shaker at 30 ℃ for activation culture for 24 hours. After activation, the surface colonies of agar were washed with distilled water and diluted to 10 with Sabouraud's medium⁵CFU/mL of bacterial suspension is ready for use.

(4) Sample adding operation: under the aseptic condition, 50 mu L of the Sabouraud's medium is added into each hole of a 96-hole plate; adding 50 mu L of solution to be tested into the first hole and the second hole of the first row, and diluting by twice to obtain solution with the concentration of 1024 mu g/mL; fully blowing and beating the first hole and the second hole by using a pipette gun to fully and uniformly mix the object to be detected with the culture medium, sucking 50 mu L of the liquid to be detected, adding the liquid to the first hole and the second hole of the second row, blowing and beating the liquid to be detected to fully and uniformly mix the liquid and the culture medium, repeating the steps until the eighth row is reached, sucking 50 mu L of the liquid in each hole of the eighth row, and discarding; at the moment, the concentration of the substance to be detected in each hole is 1024,512,256,128,64,32,16 and 8 mu g/mL from high to low (from top to bottom); then 50 mu L of diluted bacterium liquid is added into each hole of a 96-hole plate, and the concentration of the substance to be detected in each hole, namely the final concentration, is 512,256,128,64,32,16,8 and 4 mu g/mL from high to low (from top to bottom).

(5) Culturing and judging results: and putting the inoculated 96-well plate into a constant-temperature incubator at 30 ℃ for 24 hours and 30 hours. And taking out after the culture is finished, and observing the growth condition of bacteria in the holes. The result was normalized by determining the normal growth of bacteria in the blank drug-free control (negative control) wells and the no growth of bacteria in the positive control (medium + strain + positive drug) wells. The concentration of the drug in the wells with no bacterial growth was visually observed as the MIC of the drug against the bacteria.

The results of the in vitro antifungal activity test on the aminosalicylic acid dihydroartemisinin derivatives and the intermediates are shown in Table 5.

TABLE 5 inhibitory Activity of Compounds on Pichia pastoris (MIC, μ g/mL)

The data in the table 5 are analyzed, and the antibacterial activity of the target compound TM7 series on pichia pastoris is better than that of the precursor TM4-1 series compound, the parent PAS, the intermediate IM1'-1 and the intermediate IM2' -1; after 24h of culture, 14 tested TM7 series compounds have MIC values of 4-64 mu g/mL, wherein 12 molecules have MIC values of 4 mu g/mL, and the MIC values are the same as that of positive control fluconazole, and the compounds show very strong antibacterial activity; the inhibiting activity of the intermediate IM2' -1 on pichia pastoris reaches 64 mu g/mL, which is stronger than that of a mother nucleus PAS. The data prove that the amino salicylic acid dihydroartemisinin derivative and the intermediate thereof have potential application prospect in the antifungal field.

3. Determination of biological Activity against Citrus bacteria (preliminary screening)

(1) Preparing a medicament mother solution: the stock solution of the drug is diluted to the desired concentration with a suitable solvent and diluent (sample mass 1.0mg, stock solution of drug 1.0mg/1mL to 1.0mg/mL is prepared first, 2 dilution concentrations are set for each drug, 0.001mg/mL (i.e. diluted 1000 times) and 0.004mg/mL (i.e. diluted 250 times)).

(2) The operation is as follows: preparation of a medicament culture medium: preparing a medicament culture medium diluted by 1000 times: fully and uniformly mixing 5 mu L of medicament and 5mL of hot PDA culture medium in a 10mL centrifuge tube; preparation of a medicament culture medium diluted by 250 times: mix well 20 μ L of drug with 4980 μ L of hot PDA medium in a 10mL centrifuge tube. Control group: PDA medium without drug and drug medium with prochloraz added (1000-fold dilution and 250-fold dilution) were used as controls. Inoculating bacteria: the prepared medicament culture medium is poured into a 24-well plate, one well is poured for each concentration of each medicament of each strain, and numbering marks are made. Mycelia of the strain cultured at 28 ℃ for 7 days were picked and inoculated into the center of each well. Culturing: the 24-well plate is placed in an incubator with 28 ℃ and 16h of illumination for 48 h. Measurement: the colony diameter was measured using a cross method. And (3) calculating: inhibition [% ] is (CK colony diameter value-measured colony diameter value) × 100%/CK colony diameter value. Screening: the inhibition rates of different agents were compared with the inhibition rate of prochloraz agent.

Double sieve

And (3) carrying out secondary screening on the high-activity molecules TM7-4, TM7-5, TM7-12 and TM7-14 obtained by primary screening to inhibit Colletotrichum gloeosporioides strain Co.3 of the citrus Colletotrichum gloeosporioides, and obtaining a virulence equation. TM7-5 and IM2' -1 carry out secondary screening on the Alternaria alternata strain Al.6 of the citrus limosa to obtain a virulence equation.

The operation is as follows: and (3) diluting the medicament in a gradient manner: set up 6 dilution gradients: 0.01, 0.004, 0.002, 0.001, 0.0005 and 0.00025mg/mL, namely 100, 250, 500, 1000, 2000 and 4000 times of dilution. Preparation of a medicament culture medium: 50, 20, 10, 5, 2.5 and 1.25. mu.L of the mother solution of the drug and 5mL of the hot PDA medium were mixed well in a 10mL centrifuge tube. The agent medium was poured into the wells and each agent was repeated 4 times per gradient. PDA medium and prochloraz were used as control groups. Inoculating bacteria: mycelia of the strain cultured at 28 ℃ for 7 days were picked and inoculated into the center of each well. Culturing: the 24-well plate was placed at 28 ℃ in a 16h incubator under light for 48 h. Measurement: the cross method measures the colony diameter. And (3) calculating: processing the data by using a pesticide indoor bioassay data processing system (PBT data processing system) to obtain a regression equation and KD₅₀、KD₉₀R, standard error, chi-squared value, and 95% confidence value.

The results of the measurement of the resistance of the aminosalicylic acid dihydroartemisinin derivatives and the intermediates to citrus germs are shown in tables 6 to 9.

TABLE 6 inhibitory Activity of Compounds on Citrus fungal pathogens (preliminary screening results)

As can be seen from the data in Table 6, the compounds tested all have certain inhibitory activity against Colletotrichum citrinum gloeosporioides strain Co.3 and Alternaria citrina alternata strain Al.6: for the citrus colletotrichum gloeosporioides, under the test concentration of 1 mu g/mL, the inhibition rate of 12 TM7 series compounds on the citrus colletotrichum gloeosporioides is more than 50% of that of a positive medicament, namely prochloraz, and the activity of 4 molecules is more than 80% of that of the prochloraz; under the test of 4 mu g/mL, the inhibition rate of 12 TM7 series compounds exceeds 50 percent of that of the positive drug prochloraz, wherein the activity of TM7-12 is close to 90 percent of that of the prochloraz. The inhibition rate (64.29%) of the intermediate IM2' -1 on the citrus brown spot pathogen is higher than that of a positive control prochloraz (50%), and the inhibition rate (50%) of TM7-2 is the same as that of the prochloraz.

Carrying out secondary screening on the high-activity molecules TM4-1-10, TM7-4, TM7-5, TM7-12 and TM7-14 obtained by primary screening on the citrus colletotrichum gloeosporioides; and (3) re-screening the citrus brown spot germs by using high-activity molecules IM2' -1 and TM 7-5.

TABLE 7 inhibitory Activity of highly active molecules against Colletotrichum citricola (rescreening results)

TABLE 8 inhibitory Activity of highly active molecules against Phoma citrifolia (rescreened results)

As can be seen from the analysis in tables 6 and 7, the TM7-5 and TM7-12 do not show drug resistance, and prove that the derivatives of the amino-salicylic acid artemisinin have potential application prospect in the field of anti-citrus germs.

TABLE 9 inhibitory Activity of Compounds on Sclerotinia citrea

As can be seen from the data in table 9, the parent PAS was very weak at the concentrations tested above. Under the test concentration of 1.6 mu g/mL, the inhibition activity of TM7 series target molecules is 5 molecules with the inhibition rate higher than 35%, under the test concentration of 0.64 mu g/mL, the inhibition rate is 4 molecules with the inhibition rate higher than 35%, and the activities of TM 7-1-TM 7-3 are stronger than that of norfloxacin.

4. Determination of antituberculotic Activity

Test strains: mycobacterium smegmatis (strain ATCC 700084/mc (2)155)

The culture medium for determining Mycobacterium smegmatis is 7H9 culture medium; all reagents and tools need to be sterilized in advance; all operations should be performed under ultra clean bench aseptic conditions.

Preparing a solution to be detected: 10.0mg of sample to be detected is accurately weighed, and proper solvent and diluent are used for preparing the solution to be detected with the concentration of 1.0 mu g/mu L. Taking 1.0 mu g/mu L of the solution to be detected, and filtering the solution to be detected by using a disposable filter (the filtering diameter is 13-30 mm) to obtain a solution C to be detected.

The operation is as follows: adding 200 mu L of cultured wild type mycobacterium smegmatis bacterial liquid into the 1 st column of a 96-well plate, and respectively adding 100 mu L of bacterial liquid into the 2 nd to 12 th columns; adding 10 mu L of liquid to be tested into the first hole of the row 1, fully blowing and beating the liquid to be tested and the bacterial liquid for at least 3 times by using a pipette gun, sucking 100 mu L of the liquid to be tested, adding the liquid to the first hole of the row 2, fully blowing and beating the liquid to be tested and the bacterial liquid to be tested to be uniformly mixed, sucking 100 mu L of the liquid to be tested from the first hole of the row 2, adding the liquid to the first hole of the row 3, and repeating the steps until the row 11; column 12 is a negative control of 100. mu.L of the bacterial suspension. At this time, the concentration of the analyte in each well is 50,25,12.5,6.25,3.12,1.56,0.78,0.39,0.19,0.09, 0.05. mu.g/mL from left to right, and the last column of each plate is a negative control. And (3) placing the inoculated 96-well plate into a constant-temperature incubator at 37 ℃ for culturing for 3d, and observing the growth condition of bacteria in the hole. And determining the normal growth of the mycobacterium smegmatis in the blank drug-free control hole and the aseptic growth of the negative control hole. The concentration of the drug in the wells where no growth of M.smegmatis was observed visually was taken as the MIC of the drug against the bacteria. Each strain is repeated for 3 times, if a plurality of jump holes appear, the result is not reported, and the test needs to be repeated. And (3) determining MIC of the p-aminosalicylic acid dihydroartemisinin derivative and the intermediate to the mycobacterium smegmatis. Blank control, negative control and positive control are set in the determination process, and the results are shown in Table 10.

TABLE 10 inhibitory Activity of Compounds against Mycobacterium smegmatis (MIC values)

As analyzed in Table 10, the MIC values of the intermediate IM1' -1 were all 0.19. mu.g/mL; the MIC value of IM2' -1 was 0.38. mu.g/mL. Thereby proving that the intermediate of the amino salicylic acid dihydroartemisinin derivative has potential application prospect in the anti-tuberculosis field.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

in formula I, R is selected from:

R₁and R₂Independently selected from H or C1-C3 alkyl;

R₃and R₄Independently selected from H or C1-C3 alkyl;

R₅is H or C1-C3 alkyl;

R₆is H or C1-C3 alkyl;

R₇is H, C1-C3 alkyl or amino;

R₈h, C1-C3 alkyl, substituted or unsubstituted phenyl, wherein the substituents on the phenyl are one or more independently selected from halogen, hydroxy, amino or C1-C3 alkyl;

R₉and R₁₀Independently selected from H or C1-C3 alkyl;

R₁₁is-O-, -S-or-NH-; r₁₂And R₁₃Independently selected from the group consisting of H, halogen, or alkanoyl;

L₁selected from: - (CH)₂)_n+1-、-(CH₂)_nCO-or-CO (CH)₂)_nCO-, n is 1,2, 3 or 4;

L₂selected from: - (CH)₂)_m-m is 2, 3,4 or 5; x is selected from: C1-C6 alkyl.

2. The dihydroartemisinin derivative of para-aminosalicylic acid as claimed in claim 1, wherein in the formula I,

R₁and R₂Independently selected from H or methyl;

R₃and R₄Independently selected from H or methyl;

R₅is H or methyl;

R₆is H or methyl;

R₇is methyl or amino;

R₈is methyl, substituted or unsubstituted phenyl, and the substituent on the phenyl is one or more and is independently selected from halogen, hydroxyl, amino or C1-C3 alkyl;

R₉and R₁₀Are both methyl;

R₁₁is-O-, -S-or-NH-; r₁₂And R₁₃Independently selected from H, halogen or acetyl;

L₁selected from: - (CH)₂)_n+1-or- (CH)₂)_nCO-, n is 1,2, 3 or 4;

L₂selected from: - (CH)₂)_m-, m is 2, 3 or 4;

x is selected from: C1-C3 alkyl.

3. The dihydroartemisinin derivative of para-aminosalicylic acid as claimed in claim 2, wherein R in formula I is selected from:

L₁selected from: - (CH)₂)_nCO-, n is 1,2, 3 or 4;

L₂selected from: - (CH)₂)_m-, m is 2, 3 or 4;

x is selected from: and (4) ethyl.

4. The derivatives of dihydroartemisinin p-aminosalicylate as claimed in claim 3, wherein the derivatives of dihydroartemisinin p-aminosalicylate represented by formula I are any one of the following compounds:

5. a process for the preparation of dihydroartemisinin p-aminosalicylate derivatives as claimed in any one of claims 1 to 4, comprising the following steps:

esterifying carboxyl p-aminosalicylate to obtain an intermediate IM 1';

coupling the intermediate IM2' with oxazole to obtain TM 4;

in the formula, R, L₁X and L₂The definition of (a) and the structural formula of the dihydroartemisinin p-aminosalicylate derivative as described in any one of claims 1 to 3, wherein R and L are₁X and L₂The definitions of (A) are the same; r₁₄And R₁₅Independently selected from halogen.

6. Intermediate IM2' prepared by the process of claim 5, i.e.

7. Intermediate IM2' prepared by the process of claim 5, i.e.

Application in antituberculosis drugs.

8. The use of dihydroartemisinin derivatives of aminosalicylic acid as claimed in any one of claims 1 to 4 in antibacterial drugs.

9. The use of dihydroartemisinin p-aminosalicylate derivatives as claimed in any one of claims 1 to 4 in antifungal medicaments.

10. Use of dihydroartemisinin derivatives as claimed in any one of claims 1 to 4 in the preparation of anti-citrus bacteria medicament.