CN112094279B

CN112094279B - P-aminosalicylic acid dihydroartemisinin derivative and preparation method and application thereof

Info

Publication number: CN112094279B
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: 2022-11-18
Anticipated expiration: 2040-09-25
Also published as: CN112094279A

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 measurement of the anti-mycobacterium smegmatis shows that the inhibitory activity of the intermediates IM1 'and IM2' is good. Thereby proving that the derivatives and intermediates of the aminosalicylic acid dihydroartemisinin have potential application prospects in the fields of resisting bacteria, fungi, citrus germs and tuberculosis.

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

Aminosalicylic acid (PAS) is the earliest discovered small-molecule drug for treating tuberculosis 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-H ₃₇ The Rv inhibitory activity was stronger than PZA (MICs of 3.13 and 6. Mu.g/mL, respectively). The second type is PAS hydrazone derivative, the reaction of Isoniazid (INH) and PAS prepared compounds containing hydrazone groups, the experimental results show, the comparison of positive control (MIC) _INH ＝1μg/mL、MIC _{Ciprofloxacin} = 1.5. Mu.g/mL and MIC _{Norfloxacin hydrochloride} =10 μ g/mL) is good. In the third category, derivatives of amino groups with other functional groups also show excellent 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 combination of a plurality of drugs, the course of treatment of drug-sensitive tuberculosis (DS-TB) is 6 months, most of patients with widely drug-resistant tuberculosis (XDR-TB) can need to receive the treatment of up to 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 compliance of the patients 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 pharmaceutical 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 infection: caused by exogenous fungi, such as dermatophytosis; (2) conditionally pathogenic fungal infection: 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) mycotic toxicosis: caused by eating mildewed grains containing mycotoxin; (5) mycotoxins: is associated with tumorigenesis. Antifungal agents commonly used for treating mycoses, known are azole antifungal agents (luliconazole, bifonazole, ketoconazole, miconazole, itraconazole, clotrimazole, neticonazole, oxiconazole, tioconazole, miconazole, omoconazole, sulconazole, salts thereof and the like), benzylamine antifungal agents (butenafine, salts thereof and the like), allylamine antifungal agents (terbinafine, salts thereof and the like), morpholine antifungal agents (amorolfine, salts thereof and the like), thiocarbamic antifungal agents (liranaftate, tolnaftate, tolcyclamate and the like), antibiotics (nystatin, gulcomycin, pseudopenicillin, dry helminthosporin, nitropyrrolidin, amphotericin and the like), and the like, but these antibacterial agents have strong cumulative toxicity, often cause liver and kidney injury, digestive tract irritation, dizziness, allergy and the like, so that the search for novel drugs having unique action mechanism is one of the research and development of antibacterial 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:

R ₁ and R ₂ Independently selected from H or C1-C3 alkyl; r ₃ And R ₄ Independently selected from H or C1-C3 alkyl; r ₅ Is H or C1-C3 alkyl; r ₆ Is H or C1-C3 alkyl; r ₇ Is H, C-C3 alkyl or amino; r is ₈ Is H, C-C3 alkyl, substituted or unsubstituted phenyl, the substituents on said phenyl are one or more independently selected from halogen, hydroxy, amino or C1-C3 alkyl; r ₉ And R ₁₀ Independently selected from H or C1-C3 alkyl; r ₁₁ is-O-, -S-or-NH-; r ₁₂ And R ₁₃ Independently selected from the group consisting of H, halogen, or alkanoyl; l is ₁ Selected from: - (CH) ₂ ) _n+1 -、-(CH ₂ ) _n CO-or-CO (CH) ₂ ) _n CO-, n is 1,2,3 or 4; l is a radical of an alcohol ₂ Selected from: - (CH) ₂ ) _m -, m is 2,3,4 or 5; x is selected from: C1-C6 alkyl.

Preferably, in said formula I, R ₁ And R ₂ Independently selected from H or methyl; r ₃ And R ₄ Independently selected from H or methyl; r ₅ Is H or methyl; r ₆ Is H or methyl; r ₇ Is methyl or amino; r ₈ Is methyl, substituted or unsubstituted phenyl, and the substituent on the phenyl is one or more and is independently selected fromHalogen, hydroxy, amino or C1-C3 alkyl; r ₉ And R ₁₀ Are both methyl; r ₁₁ is-O-, -S-or-NH-; r ₁₂ And R ₁₃ Independently selected from H, halogen or acetyl; l is a radical of an alcohol ₁ Selected from the group consisting of: - (CH) ₂ ) _n+1 -or- (CH) ₂ ) _n CO-, n is 1,2,3 or 4; l is ₂ Selected from the group consisting of: - (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:

L ₁ selected from: - (CH) ₂ ) _n CO-, n is 1,2,3 or 4; l is ₂ Selected from: - (CH) ₂ ) _m -, m is 2,3 or 4; x is selected from: and (4) ethyl.

Preferably, the dihydroartemisinin p-aminosalicylate 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 IM1';

reacting the intermediate IM1 'with a linker reagent to obtain an intermediate IM2';

coupling the intermediate IM2' with oxazole to prepare TM4;

reacting dihydroartemisinin with a linker reagent to prepare an intermediate IM4;

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

in the formula, R, L ₁ X and L ₂ The definition of (1) and the structural formula of the p-aminosalicylic acid dihydroartemisinin derivative are shown in the specification ₁ X and L ₂ The definitions of (A) are the same; r is ₁₄ And R ₁₅ Independently selected from halogen.

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

A. reacting p-aminosalicylic acid with alcohol under the action of acid to obtain an intermediate IM1'; 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 IM2'; 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 TM4; 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 IM4; the organic solvent is diethyl ether;

E. coupling IM4 and TM4 under the action of an organic solvent and alkali to prepare a dihydroartemisinin p-aminosalicylate derivative TM7; 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. Intermediate IM2' obtained by the above preparation method, 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 a straight or branched chain saturated monovalent hydrocarbon radical having 1 to 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 reasonably modifies amino, carboxyl and phenolic hydroxyl of the p-aminosalicylic acid to construct a p-aminosalicylic acid dihydroartemisinin derivative with a novel structure, and the chemical structure of the product is shown in the specification ¹ H NMR, ¹³ C NMR and HR MS confirmation;

2) The in vitro antibacterial activity test result of the compound shows that the antibacterial activity of the TM7 series compound is better than that of the parent PAS, the intermediate IM1' -1 and the precursor TM4-1 series compound 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, with 6 molecules of MIC <64 μ g/mL, with 3 molecules of MIC =16 μ g/mL and 1 molecule (TM 7-10) of MIC =2 μ g/mL. In addition, the IM2' -1 intermediate has strong capability of inhibiting staphylococcus aureus, micrococcus luteus and salmonella, and is even stronger than some 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 pastoris is good as a whole and is stronger than that of the precursor TM4-1 series compound, the parent PAS, the intermediate IM1'-1 and IM2' -1; after 24h of culture, 12 molecules of 14 TM7 series compounds tested have MIC value =4 mug/mL, and the MIC value is 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. 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. For the citrus brown spot germ, the inhibition rate (64.29%) of the intermediate IM2' -1 is higher than that of the 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 citrus brown spot pathogen and the 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. The results prove that the aminosalicylic acid dihydroartemisinin derivative and the intermediate thereof have potential application prospects in the field of resistance of citrus germs;

5) The result of the activity determination of the anti-mycobacterium smegmatis shows 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 aminosalicylic 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.

1. Primary reagents and instruments

P-aminosalicylic acid, chloroacetyl chloride, methylene chloride, 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 ·Et ₂ O) (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.

2. Preparation of p-aminosalicylic acid dihydroartemisinin derivatives

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) ₂ CO ₃ ) Adjusting pH to 7-8, refrigerating, filtering, and washing filter cake with ice water. The filtrate was extracted with Dichloromethane (DCM) (3X 30 mL) and the organic phase was collected and washed with saturated NaCl solution. Drying with anhydrous sodium sulfate, and concentratingSteaming, combining with a filter cake, carrying out column chromatography, drying in vacuum and weighing to obtain intermediate IM1' -1.32g with the yield of 64%.

2. Synthesis of intermediate IM2' -1

Adding IM1' -1 5mmol, DCM 5mL and NaHCO into a reaction bottle ₃ 12.5mmol; 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 Na ₂ SO ₄ Drying, removing solvent by rotary evaporation, performing column chromatography to obtain pure product, drying, and weighing to obtain intermediate IM2' -1.16g with yield of 90%.

3. Preparation of Compound TM4-1

Adding oxazole, N-Dimethylformamide (DMF) and K into a reaction bottle in sequence ₂ CO ₃ Stirring 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 was performed, and the filter cake was washed with saturated brine (10 mL × 1), washed with ice water (5 mL × 1), and dried to obtain a crude product, which was purified by column chromatography (PE/EA = 10. 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 TM4-1. 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.C ₃ ·Et ₂ The reaction was continued at-15 ℃ with 1.6equiv of O solution, and 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. Adding 10mL of petroleum ether, refrigerating and filtering. And (3) carrying out spin-drying on the filtrate, carrying out column chromatography (PE/EA =50:1,v/v), adding 6mL of petroleum ether, refrigerating, crystallizing, carrying out suction filtration, combining filter cakes, and carrying out vacuum drying to obtain an intermediate IM4-1. The experimental conditions and results are shown in Table 2.

TABLE 2 Experimental conditions and results for intermediate IM4-1

5. Preparation of p-aminosalicylic acid dihydroartemisinin derivative TM7

Respectively adding TM4-1, IM4-1 and K into a reaction bottle ₂ CO ₃ DMF, temperature-controlled stirring reaction, and TLC for monitoring 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 NaHCO ₃ Washing the solution (10 mL × 1), washing with saturated brine (10 mL × 1), drying with anhydrous sodium sulfate, rotary-steaming to obtain a crude product, performing column chromatography (PE/EA = 10. 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 is 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；

¹ H NMR(600MHz,DMSO-d6)δ10.57(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). ¹³ C NMR(151MHz,DMSO-d6)δ172.40,166.81,165.39,159.53,144.13,138.78,132.97,128.42,121.12,114.61,110.74,103.51,100.81,87.15,80.83,64.95,62.95,60.60,55.34,52.38,49.74,44.24,36.61,34.39,30.98,28.83,26.06,24.51,21.49,20.63,14.59,13.19.LC MS calcd for C ₃₂ H ₄₃ N ₃ O ₉ ,[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；

¹ H NMR(600MHz,DMSO-d6)δ10.61(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). ¹³ C NMR(151MHz,DMSO-d6)δ172.41,169.12,166.87,165.38,161.82,159.54,145.41,131.35,126.30,121.49,110.93,106.77,103.49,87.12,80.82,61.57,60.60,55.35,52.36,49.18,44.22,36.59,34.37,33.60,30.98,26.06,24.53,21.50,20.64,19.36,14.54,13.19,12.94.LC MS calcd for C ₃₃ H ₄₅ N ₃ O ₉ ,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.55(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). ¹³ C NMR(151MHz,DMSO-d6)δ166.92,165.39,159.52,144.15,137.97,136.79,132.96,117.30,114.60,110.74,103.60,103.50,100.81,87.16,80.83,67.91,64.95,60.59,52.38,49.75,44.24,36.70,36.53,34.38,30.98,28.84,26.06,24.52,20.61,14.59,14.02,13.19,9.08.LC MS calcd for C ₃₃ H ₄₅ N ₃ O ₉ ,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.59(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). ¹³ C NMR(151MHz,DMSO-d6)δ166.57,165.39,159.53,144.12,139.55,132.97,132.06,114.62,110.77,105.78,103.61,103.50,100.79,87.14,80.84,64.93,62.90,60.59,55.35,54.98,52.40,44.24,36.69,36.54,34.38,30.98,28.82,26.07,24.51,20.63,14.59,13.20.LC MS calcd for C ₃₂ H ₄₃ N ₃ O ₉ ,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.76(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). ¹³ C NMR(151MHz,DMSO-d6)δ170.76,166.56,165.38,159.55,145.44,144.08,143.70,134.87,132.99,122.84,122.01,119.86,114.66,110.78,110.69,103.50,100.79,87.14,80.82,64.95,62.92,60.60,60.20,55.35,52.37,47.89,44.22,36.60,34.36,30.97,28.80,26.06,24.50,20.63,14.56,13.17.LC MS calcd for C ₃₆ H ₄₅ N ₃ O ₉ ,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.69(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). ¹³ C NMR(151MHz,DMSO-d6)δ165.59,165.37,159.52,151.88,146.07,143.91,133.00,114.80,110.81,103.67,103.50,100.80,87.14,80.83,64.97,62.92,60.62,52.39,52.33,44.24,36.69,36.53,34.38,30.98,28.81,26.06,24.52,24.48,20.62,14.58,13.19.HR MS calcd for C ₃₁ H ₄₂ N ₄ O ₉ ,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.83(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). ¹³ C NMR(151MHz,DMSO-d6)δ165.36,164.52,159.51,145.69,143.68,133.04,114.99,110.86,103.73,103.51,100.81,87.15,80.83,65.01,62.94,60.65,55.35,52.38,50.60,44.23,36.71,36.52,34.39,30.97,28.81,26.07,24.50,20.62,14.58,13.19.HR MS calcd for C ₃₀ H ₄₁ N ₅ O ₉ ,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.85(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). ¹³ C NMR(151MHz,DMSO-d6)δ165.35,164.44,159.51,153.89,143.62,133.04,115.03,110.92,103.78,103.50,100.79,87.13,80.82,65.02,62.91,60.65,52.37,49.76,44.22,36.68,36.52,34.37,30.98,28.80,26.06,24.53,24.48,20.62,14.58,13.18,8.75(s).HR MS calcd for C ₃₁ H ₄₃ N ₅ O ₉ ,[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；

¹ H NMR(600MHz,DMSO-d6)δ10.49(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-13and H-18),0.80(d,J＝7.3Hz,3H,H-12),0.61(d,J＝6.3Hz,3H,H-17). ¹³ C NMR(151MHz,DMSO-d6)δ169.64,167.59,167.43(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 C ₃₅ H ₄₇ N ₃ O ₉ S,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.65(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). ¹³ C NMR(151MHz,DMSO-d6)δ170.78,166.03,165.38,159.51,153.72,144.12,132.99,114.69,110.77,103.49,100.78,87.14,80.84,68.12,65.99,63.69,60.61,52.39,44.24,38.18,36.67,34.12,30.98,28.81,26.07,24.51,21.14,20.60,15.48,14.58,13.20.LC MS calcd for C ₃₁ H ₄₃ N ₅ O ₉ S,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.70(s,1H,H-5),7.76(d,J＝8.6Hz,1H,H-3),7.71～7.65(m,5H,H-24 and H-25and 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-14and 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). ¹³ C NMR(151MHz,DMSO-d6)δ165.80,165.38,159.52,154.32,144.12,133.51,132.98,131.15,130.56(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 C ₃₆ H ₄₅ N ₅ O ₉ S,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.63(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). ¹³ C NMR(151MHz,DMSO-d6)δ166.30,166.13,165.38,164.63,159.52,144.22,132.97,114.62,110.75,103.49(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 C ₃₂ H ₄₃ N ₃ O ₉ S ₂ ,[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℃；

¹ H NMR(600MHz,DMSO-d6)δ10.52(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-14 and H-15),1.31(t,J＝7.1Hz,3H,H-1),1.30～1.23(m,2H,H-14 and 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). ¹³ C NMR(151MHz,DMSO-d6)δ170.40,166.87,165.40,159.52,149.67,144.28,132.95,114.57,110.74,103.49(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 C ₃₁ H ₄₂ N ₄ O ₉ S ₂ ,[M+H] ⁺ 679.25,found 679.30.

3. biological activity detection of aminosalicylic acid dihydroartemisinin derivatives

1. In vitro antibacterial Activity assay

The activity (MIC value) of a compound against Staphylococcus aureus (Staphyloccocus 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 dilution 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 17h. After activation, the cells were diluted to 10 with brain heart infusion Broth (BHI) medium ⁵ CFU/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, and discarding the solution; at the moment, the concentration of the substance to be detected in each hole is 512,256,128,64,32,16,8,4 mug/mL from top to bottom in sequence. The last two columns of each plate are used as controls and contain no substance to be detected, one column is used as a bacterial growth control and added with bacterial liquid, and the other column is used as a negative control and added with no bacterial liquid. Finally, 50 mu L of diluted bacteria liquid is added into each row of 1-8 holes, a compound hole test is adopted, 5 compounds are tested on each plate, and the concentration of the substance to be tested in each hole, namely the final concentration, is 256,128,64,32,16,8,4,2 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, 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, with 6 molecules of MIC <64 μ g/mL, with 3 molecules of MIC =16 μ g/mL and 1 molecule (TM 7-10) of MIC =2 μ g/mL. In addition, the intermediate IM2' -1 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 the 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 DMSO into clear transparent liquid to prepare a solution of 3.2mg/mL, sealing by a sealing film, and storing in a refrigerator in a dark place. For partially poorly soluble compounds DMSO/tween-80 =200/1 (v/v) was used to increase solubility, tween-80 being a 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 shaking table 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 medium ⁵ CFU/mL of bacterial suspension for standby.

(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 1024,512,256,128,64,32,16,8 mug/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 512,256,128,64,32,16,8,4 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 results were normalized by determining that the bacteria grew normally in the blank drug-free control (negative control) wells and no bacteria grew 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 IM2' -1; after 24h of culture, 14 tested TM7 series compounds have MIC values between 4 and 64 mu g/mL, wherein 12 molecules have MIC values =4 mu g/mL, and the MIC values are the same as that 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. 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 drug stock solution was diluted to the desired concentration with an appropriate solvent and diluent (sample mass 1.0mg, drug stock solution 1.0mg/1ml =1.0mg/mL was prepared first, 2 dilution concentrations were 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 48h. Measurement: the colony diameter was measured using a cross method. And (3) calculating: inhibition% = (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 gloeosporides strain Co.3 of the citrus Colletotrichum to obtain a virulence equation. TM7-5 and IM2' -1 carry on the rescreening to the citrus brown spot germ Alternaria alternata bacterial strain Al.6, obtain the 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 diluting by 100, 250, 500, 1000, 2000 and 4000 times. Preparation of a medicament culture medium: 50, 20, 10, 5, 2.5 and 1.25. Mu.L of the drug stock solution and 5mL of 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 2 ℃ for 48h in a 16h incubator under light. And (3) 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 KD ₅₀ 、KD ₉₀ R, 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. For the citrus brown spot germ, the inhibition rate (64.29%) of the intermediate IM2' -1 is higher than that of the positive control prochloraz (50%), and the inhibition rate (50%) of TM7-2 is the same as that of the prochloraz.

Re-screening the high-activity molecules TM4-1-10, TM7-4, TM7-5, TM7-12 and TM7-14 obtained by primary screening to obtain 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, TM7-5 and TM7-12 did not exhibit drug resistance, and the potential application prospect of the amino-salicylic acid bis-artemisinin derivative in the field of anti-citrus germs is proved.

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 antitubercular Activity

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

Determining the culture medium used by the mycobacterium smegmatis as a 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 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 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 detected into the first hole of the row 1, fully blowing and beating the liquid to be detected and the bacterial liquid for at least 3 times by using a pipette gun (fully mixing the object to be detected and the bacterial liquid uniformly), sucking 100 mu L of the liquid to be detected and adding the liquid to the first hole of the row 2, fully blowing and beating the liquid to be detected and the bacterial liquid uniformly, sucking 100 mu L of the liquid from the first hole of the row 2 and 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 mug/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 jumping holes occur, 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 can be seen from the analysis in Table 10, the MIC values of the intermediate IM1' -1 are 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 aminosalicylic 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

1. A dihydroartemisinin p-aminosalicylate derivative as shown in formula I, or a pharmaceutically acceptable salt thereof:

in formula I, R is selected from:

R ₁ and R ₂ Independently selected from H or C1-C3 alkyl;

R ₃ and R ₄ Independently selected from H or C1-C3 alkyl;

R ₅ is H or C1-C3 alkyl;

R ₆ is H or C1-C3 alkyl;

R ₇ is H, C-C3 alkyl or amino;

R ₈ is H, C-C3 alkyl, substituted or unsubstituted phenyl, the substituents on said phenyl are one or more independently selected from halogen, hydroxy, amino or C1-C3 alkyl;

R ₉ and R ₁₀ Independently selected from H or C1-C3 alkyl;

L ₁ selected from: - (CH) ₂ ) _n+1 -、-(CH ₂ ) _n CO-or-CO (CH) ₂ ) _n CO-, n is 1,2,3 or 4;

L ₂ selected 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,

R ₁ and R ₂ Independently selected from H or methyl;

R ₃ and R ₄ Independently selected from H or methyl;

R ₅ is H or methyl;

R ₆ is H or methyl;

R ₇ is methyl or amino;

R ₈ is 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;

R ₉ and R ₁₀ Are both methyl;

L ₁ selected from: - (CH) ₂ ) _n+1 -or- (CH) ₂ ) _n CO-, n is 1,2,3 or 4;

L ₂ selected 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:

L ₁ selected from: - (CH) ₂ ) _n CO-, n is 1,2,3 or 4;

L ₂ selected from the group consisting of: - (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 IM1';

coupling the intermediate IM2' with oxazole to prepare TM4;

coupling the intermediate IM4 and TM4 to prepare a target molecule p-aminosalicylic acid dihydroartemisinin derivative;

in the formula, R, L ₁ X and L ₂ The 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 are ₁ X and L ₂ The definitions of (A) are the same; r ₁₄ And R ₁₅ Independently selected from halogen.

6. Use of a dihydroartemisinin derivative of para-aminosalicylic acid as claimed in any one of claims 1 to 4 in the preparation of an antibacterial medicament.

7. Use of dihydroartemisinin derivatives of aminosalicylic acid as claimed in any one of claims 1 to 4 for the preparation of antifungal agents.

8. Use of dihydroartemisinin derivatives of aminosalicylic acid as claimed in any one of claims 1 to 4 for the preparation of a drug against citrus pathogens.