Withaferin A derivatives and preparation methods and applications thereof

Abstract

The present invention discloses a series of withaferin A derivatives and preparation methods and applications thereof, belonging to the field of medical technology. The applicant's test results show that the withaferin A derivatives of the present invention have good anti-cancer activity against a variety of cancer cell lines, are expected to be used in the treatment of cancer, and have good potential medicinal value.

Description

Technical Field

[0001] The invention relates to withaferin A derivatives and preparation methods and applications thereof, belonging to the technical field of medicines. Background Art

[0002] The treatment of cancer has always been a global problem. Its high mortality rate and expensive treatment costs seriously affect the quality of life of patients and their families. In 2020, there were 4.57 million new cancer cases and 3 million deaths in my country, and the number of new cancer cases ranked first in the world. The causes of cancer are diverse. Under different treatment strategies, chemotherapy has always been one of the most reliable options for treating tumors. Natural products have the characteristics of multiple pathways, multiple targets, multiple effects, pleiotropy and low toxicity and side effects, providing an important source for the screening and discovery of active ingredients with cancer chemoprevention effects. It is reported that more than 60% of anticancer drugs are currently derived from natural products, including paclitaxel, vinblastine and etoposide. As a typical secondary metabolite of W. Sonmifera in the Solanaceae family, withanolides were first reported for their anticancer activity in 1967. Withanolide is a natural steroid compound with 28 carbon atoms and has the skeleton structure of ergostane. According to the difference of C-17 side chain, withanolide can be divided into two types, Type A and Type B, as shown below:

[0003]

[0004] Withaferin A (WA) is one of the main biologically active components of lactone compounds, and its structure is shown in the following formula 1:

[0005]

[0006] Withaferin A has a wide range of biological properties, including anti-inflammatory, anti-tumor, immunomodulatory, and anti-angiogenesis. The most widely studied is the anti-cancer activity of Withaferin A. Studies have shown that Withaferin A can exert its anti-cancer effects by inducing apoptosis in a variety of human cancer cells, such as prostate, colon, breast, leukemia, pancreas, kidney, head and neck. The Developmental Therapy Program of the National Cancer Institute of the United States tested 60 human cancer cell lines and found that the average growth inhibitory concentration (IC) of Withaferin A on tumor cells was 50) is 620nM. Although a lot of research has been done on the potential anticancer properties of withaferin A, there are few reports on its structural modification. In fact, there are three positions on the steroidal skeleton of withaferin A that are suitable for nucleophilic attack and modification, including the α,β-unsaturated ketone group of the A ring, the 5b,6b-epoxy group of the B ring, and the α,β-unsaturated six-membered lactone on the E ring. After analysis, the inventor team of this application believes that based on the structural framework of withaferin A, structural modification of it will likely result in withaferin A derivatives with better anticancer activity. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a series of withaferin A derivatives with novel structures and significant anticancer activity, preparation methods thereof, and applications thereof.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The withaferin A derivative of the present invention is a compound having a structure shown in the following formula 4, 7, 8, 10, 12, 14 or 15 or a pharmaceutically acceptable salt thereof:

[0010]

[0011]

[0012] The preparation method of the withaferin A derivative of the present invention comprises:

[0013] The preparation method of the compound of the structure shown in Formula 4 is as follows: the compound of the structure shown in Formula 1 and the compound of the structure shown in Formula 2 are placed in a polar solvent, and reacted in the presence of an alkaline catalyst to obtain a compound of the structure shown in Formula 3; the compound of the structure shown in Formula 3 is placed in a polar solvent, a metal catalyst is added, and the reaction is carried out under a reducing atmosphere to obtain a compound of the structure shown in Formula 4;

[0014] The preparation method of the compound of the structure shown in Formula 7 is as follows: the compound of the structure shown in Formula 1 and the compound of the structure shown in Formula 5 are placed in a polar solvent for reaction to obtain a compound of the structure shown in Formula 6; the compound of the structure shown in Formula 6 is placed in a polar solvent, a metal catalyst is added, and the reaction is carried out under reducing atmosphere conditions to obtain a compound of the structure shown in Formula 7;

[0015] The preparation method of the compound of the structure shown in Formula 8 is as follows: taking the compound of the structure shown in the following Formula 1 and placing it in a polar solvent, adding a first oxidant to react, and obtaining the compound of the structure shown in Formula 8;

[0016] The preparation method of the compound of the structure shown in Formula 10 is as follows: the compound of the structure shown in Formula 1 and the compound of the structure shown in Formula 9 are placed in a polar solvent, and reacted in the presence of a basic catalyst to obtain the compound of the structure shown in Formula 10;

[0017] The preparation method of the compound of the structure shown in Formula 12 is as follows: the compound of the structure shown in Formula 1 is placed in a polar solvent, and a second oxidant is added to react to obtain the compound of the structure shown in Formula 12;

[0018] The preparation method of the compounds of formula 14 and formula 15 is as follows: the compound of formula 12 and the compound of formula 13 are placed in a polar solvent, and a condensing agent is added to react to obtain the compounds of formula 14 and formula 15;

[0019]

[0020] In the preparation method of the present invention, the polar solvent involved can be one or a combination of two or more selected from tetrahydrofuran (THF), dichloromethane (DCM), tert-butyl alcohol, methanol, water, acetone, pyridine and ethanol. The amount of the polar solvent can be determined as needed, preferably to fully dissolve the raw materials involved in the reaction.

[0021] In the preparation method of the present invention, the compound with the structure shown in formula 1 is withaferin A, the compound with the structure shown in formula 2 is tert-butyldimethylchlorosilane (TBDMSCl), and its usage is usually 1 to 1.5 times the molar amount of the compound with the structure shown in formula 1; the compound with the structure shown in formula 5 is acetic anhydride, and its usage is usually 1 to 1.5 times the molar amount of the compound with the structure shown in formula 1; the compound with the structure shown in formula 9 is phenyl thiochloroformate, and its usage is usually 1 to 1.5 times the molar amount of the compound with the structure shown in formula 1; the compound with the structure shown in formula 13 is dimethyl sulfate, and its usage is usually 1 to 1.5 times the molar amount of the compound with the structure shown in formula 1.

[0022] In the preparation method of the present invention, the selection and dosage of the basic catalyst involved are conventional selections in the prior art. Specifically, the basic catalyst can be one selected from pyridine, 4-dimethylaminopyridine (DMAP), imidazole, sodium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, triethylamine and N,N-diisopropylethylamine, etc.; preferably, DMAP is used. The dosage of the basic catalyst is usually used in excess relative to the compound of the structure shown in Formula 1, preferably 1 to 2 times the molar amount of the compound of the structure shown in Formula 1.

[0023] In the preparation method of the present invention, the selection and dosage of the metal catalyst involved are conventional selections in the prior art. Specifically, the metal catalyst can be selected from palladium carbon catalyst (Pd / C), palladium hydroxide, palladium acetate, platinum, nickel and Ni / Al 2 O 3 The amount of the metal catalyst is usually 0.5 to 1 times the molar amount of the compound of the structure shown in Formula 3 or Formula 6. From the perspective of safe operation, it is preferred to add the metal catalyst under the protection of an inert atmosphere (such as nitrogen or helium).

[0024] In the preparation method of the present invention, the reducing atmosphere involved generally refers to a hydrogen atmosphere.

[0025] In the preparation method of the present invention, the first oxidant and the second oxidant are both oxidants, and the "first" and "second" are only used to distinguish the different oxidants used in the compounds of the structures shown in Formula 8 and Formula 12. In the preparation method of the compound of the structure shown in Formula 8 and Formula 12, the first oxidant and the second oxidant can be conventional oxidants in the prior art. In the preparation method of the compound of the structure shown in Formula 8, the purpose of adding the oxidant is to hydroxylate the C-2,3 carbon-carbon double bond. Therefore, the first oxidant is further preferably selected from potassium osmate dihydrate (K 2 O 4 ·2H 2 O), potassium permanganate, osmium tetroxide and sodium periodate, or a combination of two or more thereof. The amount of the first oxidant is generally 1 to 2 times the molar amount of the compound of the structure shown in Formula 1. When potassium osmate dihydrate is selected as the first oxidant, it needs to be used in an acidic system (acidic conditions of pH ≤ 6, preferably pH = 3 to 4; specifically, it can be adjusted with acids such as citric acid or acetic acid) and is used in combination with N-methylmorpholine-N-oxide (NMO), wherein the amount of NMO is generally 1 to 1.5 times the molar amount of the compound of the structure shown in Formula 1. In the preparation method of the compound of the structure shown in Formula 12, the purpose of adding the oxidant is to further oxidize the aldehyde group to a carboxyl group. Therefore, the second oxidant is further preferably Jones reagent or potassium dichromate, or a combination of Jones reagent and potassium dichromate, and more preferably Jones reagent. The amount of the second oxidant used should be more than 3 times the molar amount of the compound of the structure shown in Formula 1. In actual operation, the amount of the second oxidant used is usually set to 6 to 7 times the molar amount of the compound of the structure shown in Formula 1.

[0026] In the method for preparing the compound of the structure shown in Formula 12, the second oxidant first oxidizes the compound of the structure shown in Formula 1 to obtain the compound of the structure shown in the following Formula 11, and then further oxidizes it to obtain the compound of the structure shown in Formula 12.

[0027]

[0028] In the preparation method of the present invention, the condensing agent involved is a conventional choice in the prior art, such as a carbodiimide condensing agent, an organophosphorus condensing agent, etc., preferably potassium carbonate or a carbodiimide condensing agent (such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), etc.). The amount of the condensing agent is usually 2 to 5 times the molar amount of the structural compound shown in Formula 11. When the condensing agent is a carbodiimide condensing agent, it is preferred to also add a condensation activator, and the selection and amount of the condensation activator are the same as those in the prior art. Specifically, the condensation activator can be HOBt, DMAP, HOAt, 4-PPY, etc., preferably using DMAP / EDCI, or a combination of DMAP / DCC; the amount of the condensation activator is usually 0.8 to 1.2 times the molar amount of the condensing agent.

[0029] In the preparation method of the present invention, the reaction when preparing each target compound can be carried out at room temperature or under ice bath or heating conditions, and whether the reaction is complete can be tracked and detected by thin layer chromatography. Preferably, when preparing the compounds of the structures shown in formula 4, 7, 8 and 10, the reaction is preferably carried out at room temperature; when preparing the compounds of the structure shown in formula 12, the reaction is preferably carried out under ice bath conditions; and when preparing the compounds of the structures shown in formula 14 or formula 15, the reaction is preferably carried out at room temperature or under heating (preferably 50 to 70°C).

[0030] The above methods all prepare crude products of the target compounds. Therefore, the method of the present invention also includes the step of purifying the crude target compounds. Specifically, the conventional purification method can be used to purify the target compounds to improve the purity of the target compounds. Specifically, the crude product can be purified by silica gel column chromatography. It is more preferred to extract the material obtained by the reaction and then perform silica gel column chromatography to reduce the burden on the silica gel column. Among them, the eluent used for elution during column chromatography is preferably a mixed solvent composed of petroleum ether (PE) and ethyl acetate (EtOAc), or dichloromethane and ethyl acetate. In the mixed solvent, the volume ratio of petroleum ether to ethyl acetate, or dichloromethane to ethyl acetate is preferably 10:1 to 1:2. If extraction is involved, the extractant is the same as the polar solvent used in the reaction, such as dichloromethane; it can also be a conventional extractant such as ethyl acetate.

[0031] The applicant has found through experiments that the withaferin A derivatives described in the present invention have good anti-cancer activity. Therefore, the present invention also includes the use of the compounds of the structures shown in the above formulas 4, 7, 8, 10, 12, 14 and 15 in the preparation of drugs for treating cancer.

[0032] Furthermore, the present invention also provides a drug for treating cancer, which comprises a therapeutically effective dose of the compound of the structure shown in the above formula 4, 7, 8, 10, 12, 14, 15. The dosage form of the above drug can be any pharmaceutically acceptable dosage form, specifically, it can be a conventional dosage form such as granules, tablets, pills, capsules or injections.

[0033] Compared with the prior art, the present invention provides a series of novel withaferin A derivatives and preparation methods thereof. The applicant's test results show that the withaferin A derivatives of the present invention have good anti-cancer activity against a variety of cancer cell lines, are expected to be used in the treatment of cancer, and have good potential medicinal value. DETAILED DESCRIPTION

[0034] In order to better explain the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0035] Some abbreviations appearing in the following embodiments are explained as follows:

[0036] Compound 1 represents a compound with a structure shown in Formula 1, Compound 2 represents a compound with a structure shown in Formula 2, Compound 3 represents a compound with a structure shown in Formula 3, Compound 4 represents a compound with a structure shown in Formula 4, Compound 5 represents a compound with a structure shown in Formula 5, Compound 6 represents a compound with a structure shown in Formula 6, Compound 7 represents a compound with a structure shown in Formula 7, Compound 8 represents a compound with a structure shown in Formula 8, Compound 9 represents a compound with a structure shown in Formula 9, Compound 10 represents a compound with a structure shown in Formula 10, Compound 11 represents a compound with a structure shown in Formula 11, Compound 12 represents a compound with a structure shown in Formula 12, Compound 13 represents a compound with a structure shown in Formula 13, Compound 14 represents a compound with a structure shown in Formula 14, and Compound 15 represents a compound with a structure shown in Formula 15.

[0037] Example 1: Preparation of Compound 4

[0038]

[0039] 30.0 mg of compound 1 (0.06 mmol, 1.0 equ.), 19.3 mg of compound 2 (tert-butyldimethylsilyl chloride, abbreviated as TBDMSCl, 0.1 mmol, 2.0 equ.), 6.5 mg of imidazole (0.1 mmol, 1.5 equ.), and 11.8 mg of DMAP (0.1 mmol, 1.5 equ.) were placed in a container, 2 mL of DCM was added to dissolve, and the reaction was stirred at room temperature for 6 to 7 hours, and the reaction was monitored by TLC thin layer plate. After the reaction was completed, 2 mL of ice water was added to quench, and the resulting mixture was extracted with DCM (1×10 mL), and the organic phase was washed with saturated brine (to better absorb the residual water in the organic phase and play a drying role), and then anhydrous Na 2 SO 4 Dry, filter and concentrate, and the residue is purified by TLC thin layer plate (PE / EtOAc=1 / 3) to obtain compound 3; then 25.0 mg of compound 3 (0.04 mmol, 1.0 equ.) is placed in a container, 2 mL of THF is added, and the mixture is stirred at room temperature under N 2 22.0 mg of Pd / C (0.2 mmol, 5.0 equ.) was added under the protection of 2 The reaction was stirred at room temperature for 12 to 13 hours and monitored by TLC. After the reaction was complete, Pd / C was removed by filtration, and the mixture was extracted with DCM (3 × 30 mL) and then with H 2 O (40 mL), the combined organic phase was washed with saturated brine (to better absorb the residual water in the organic phase and play a drying role), and then washed with anhydrous Na 2 SO 4 After drying, filtration and concentration, the residue was purified by silica gel flash chromatography (eluent: PE / EtOAc, 10 / 1 to 1 / 1, volume ratio) to obtain compound 4, which was characterized as follows:

[0040] Compound 4 (2,3-dihydro-24,25-dimethylwithaferin A): yield 52.0%, white solid; 1H NMR(600MHz,Chloroform-d)δ4.32(dt,J=12.0,3.9Hz,1H),3.51(t,J=3.7Hz,1H),3.13(s,1H),2.74–2.59( m,2H),2.23–2.59(m,1H),2.23–2.16(m,2H),2.14–2.09(m,1H),2.05–1.99(m,1H),1.91(dt,J=12.7,3.5Hz ,1H),1.79–1.73(m,1H),1.72–1.68(m,1H),1.65–1.61(m,1H),1.42–1.34(m,4H),1.30(s,3H),1.24(s,3H) ,1.14(d,J=7.1Hz,3H),1.12–1.05(m,4H),0.91(q,J=5.8Hz,6H),0.90–0.83(m,1H),0.64(d,J=3.0Hz,3H).

[0041] Example 2: Preparation of Compound 4

[0042] Repeat Example 1, except that:

[0043] 1) No imidazole was added, and only DMAP was used as the alkaline catalyst;

[0044] 2) Use methanol instead of DCM.

[0045] Finally, a white solid was obtained with a yield of 52%. The obtained white solid was determined to be compound 4 through characterization.

[0046] Example 3: Preparation of Compound 4

[0047] Repeat Example 1, except that:

[0048] 1) Using triethylamine instead of imidazole and DMAP as the basic catalyst;

[0049] 2) Replace DCM with water;

[0050] 3) Pd / C is replaced by palladium acetate.

[0051] Finally, a white solid was obtained with a yield of 34%. The obtained white solid was determined to be compound 4 through characterization.

[0052] Example 4: Preparation of Compound 7

[0053]

[0054] 20.0 mg of compound 1 (0.4 mmol, 1.0 equ.) was placed in a reaction flask, 1 mL of pyridine was added, and the mixture was stirred at room temperature. Subsequently, 30 μl of acetic anhydride was drawn with a syringe, diluted with a small amount of pyridine, and slowly added to the reaction flask. Stirring was continued at room temperature for 19 to 20 hours, and the reaction was monitored by TLC. After the reaction was completed, the mixture was extracted with EtOAc (3×10 mL), and then precipitated with H 2 O (40 mL), the combined organic phase was washed with saturated brine, and then washed with anhydrous Na 2 SO 4 Dry, filter and concentrate, and the residue is purified by TLC (PE / EtOAc=1 / 4) to obtain compound 6; then 23.0 mg of compound 6 (0.04 mmol, 1.0 equ.) is placed in a container, 1.5 mL of EtOH is added to dissolve, and the mixture is stirred at N 2 8.0 mg Pd / C (0.08 mmol, 0.2 equ.) was added under the protection of 2 The mixture was stirred at room temperature for 6 to 7 hours and monitored by TLC. After the reaction was complete, Pd / C was removed by filtration, and the mixture was extracted with DCM (3 × 30 mL) and then with H 2 O (40 mL), the combined organic phase was washed with saturated brine, and then washed with anhydrous Na 2 SO 4 After drying, filtration and concentration, the residue was purified by silica gel flash chromatography (eluent: PE / EtOAc, 10 / 1 to 1 / 1, volume ratio) to obtain compound 7, which is characterized as follows:

[0055] Compound 7 (2,3-dihydro-24,25-dimethyl-4-(1-carbonylethoxy)withaferin A): yield 63.0%, white solid, 1 H NMR(600MHz,Chloroform-d)δ4.56(t,J=3.2Hz,1H),4.32(dt,J=12.0,3.9Hz,1H),3.15(d,J=2 .0Hz,1H),2.72–2.67(m,2H),2.45–2.36(m,1H),2.27–2.23(m,1H),2.21–2.17(m,1H),2.16–2. 09(m,2H),2.05(s,3H),1.98–1.91(m,2H),1.78–1.69(m,1H),1.69–1.60(m,4H),1.45–1.31(m, 6H),1.27(s,3H),1.14(d,J=7.0Hz,3H),1.10–0.99(m,3H),0.92(t,J=6.5Hz,6H),0.65(s,3H).

[0056] Example 5: Preparation of Compound 7

[0057] Repeat Example 4, except that:

[0058] 1) Use tert-butyl alcohol instead of pyridine;

[0059] 2) Use Ni / Al 2 O 3 Replace Pd / C.

[0060] Finally, a white solid was obtained with a yield of 23%. The obtained white solid was determined to be compound 7 through characterization.

[0061] Example 6: Preparation of Compound 7

[0062] Repeat Example 4, except that:

[0063] 1) Use THF instead of EtOH;

[0064] 2) Use THF instead of pyridine;

[0065] 3) Replace Pd / C with platinum.

[0066] Finally, a white solid was obtained with a yield of 28%. The obtained white solid was determined to be compound 7 through characterization.

[0067] Example 7: Preparation of Compound 8

[0068]

[0069] 20.0 mg of compound 1 (0.04 mmol, 1.0 equ.) was placed in a flask, and 500 μl of tert-butyl alcohol, 250 μl of water, 7.4 mg of NMO (0.06 mmol, 1.5 equ.), and 16.1 mg of citric acid (0.08 mmol, 2.0 equ.) were added in sequence. After stirring at room temperature for 5 minutes, 2.8 mg of K 2 O 4 ·2H 2 O (0.01mmol, 0.2equ.), kept stirring at room temperature for 0.5-1 hour, and the reaction was monitored by TLC thin layer plate. After the reaction was completed, saturated sodium thiosulfate solution (3mL) was added to the reaction to quench the reaction, and then ethyl acetate (3×10mL) was added to the mixture and extracted, and the organic phases were combined and anhydrous Na 2 SO 4 After drying, filtration and concentration, the residue was purified by silica gel flash chromatography (eluent: PE / EtOAc, 10 / 1 to 1 / 1, volume ratio) to obtain compound 8, which is characterized as follows:

[0070] Compound 8 (2,3-dihydroxywithaferin A): yield 55.5%, white solid; 1 H NMR(600MHz,Chloroform-d)δ4.94(d,J=2.9Hz,1H),4.42(dt,J=13.4,3.5Hz,1H),4.35(d,J=4.8Hz,2H),3.76 (d,J=3.3Hz,1H),3.59(d,J=3.8Hz,1H),3.28(s,1H),2.54(dd,J=17.8,13.3Hz,1H),2.10(dt,J=14.7,3.3Hz,1 H),2.03(s,3H),1.95(dd,J=12.4,3.5Hz,1H),1.74(dd,J=13.3,3.5Hz,1H),1.71–1.59(m,6H),1.47(s,3H),1 .38–1.34(m,1H),1.25–1.24(m,3H),1.21–1.07(m,2H),1.02(d,J=6.6Hz,3H),0.93–0.78(m,1H),0.69(s,3H).

[0071] Example 8: Preparation of Compound 8

[0072] Example 7 was repeated, except that:

[0073] 1) Using acetone instead of tert-butyl alcohol and water as the polar solvent for the reaction;

[0074] 2) Use osmium tetroxide instead of K 2 O 4 ·2H 2 O, NMO and citric acid were used as the first oxidants.

[0075] Finally, a white solid was obtained with a yield of 78%. The obtained white solid was determined to be compound 8 through characterization.

[0076] Example 9: Preparation of Compound 8

[0077] Example 7 was repeated, except that:

[0078] 1) Using pyridine instead of tert-butyl alcohol and water as the polar solvent for the reaction;

[0079] 2) Use sodium periodate instead of K 2 O 4 ·2H 2 O, NMO and citric acid were used as the first oxidants.

[0080] Finally, a white solid was obtained with a yield of 54%. The obtained white solid was determined to be compound 8 through characterization.

[0081] Example 10: Preparation of Compound 10

[0082]

[0083] 10.0 mg of compound 1 (0.02 mmol, 1.0 equ.) and 5.2 mg of 4-dimethylaminopyridine (0.04 mmol, 2.0 equ.) were placed in dry N 2 In a flask under protection, 0.5 mL of DCM was added to dissolve, and then 6.0 μl of 1.25 g / mL compound 9 (0.04 mmol, 2.0 equ.) was added dropwise, and the reaction was stirred at room temperature for 0.5 to 1 hour, and the reaction was monitored by TLC thin layer plate. After the reaction was completed, saturated sodium bicarbonate solution (2 mL) was added to the reaction to quench the reaction, and the mixture was extracted with DCM (3×10 mL), and then the organic phases were combined and anhydrous Na 2 SO 4 After drying, filtration and concentration, the residue was purified by silica gel flash chromatography (eluent: PE / EtOAc, 10 / 1 to 1 / 1, volume ratio) to obtain compound 10, which is characterized as follows:

[0084] Compound 10 (27-(1-phenoxythiocarbonyl)withaferin A): yield 70.0%, white solid; 1 H NMR(600MHz,Chloroform-d)δ7.43–7.37(m,3H),7.16(dd,J=14.1,8.0Hz,1H),7.10–7.06(m ,2H),6.37(d,J=9.8Hz,1H),5.14(d,J=6.0Hz,1H),4.45–4.32(m,3H),3.31(s,1H),2.54–2.4 5(m,2H),2.34(dt,J=7.7,3.8Hz,2H),2.04(s,3H),2.01–1.99(d,J=2.2Hz,3H),1.97–1.94(m ,2H),1.33(s,3H),1.10–1.04(m,3H),1.00(d,J=6.9Hz,3H),0.85–0.76(m,5H),0.70(s,3H).

[0085] Example 11: Preparation of Compound 10

[0086] Example 10 was repeated, except that:

[0087] 1) Use pyridine instead of DCM as the polar solvent for the reaction;

[0088] 2) Pyridine was used instead of 4-dimethylaminopyridine as the basic catalyst.

[0089] Finally, a white solid was obtained with a yield of 78%. The obtained white solid was determined to be compound 10 through characterization.

[0090] Example 12: Preparation of Compound 10

[0091] Example 10 was repeated, except that:

[0092] 1) Use methanol instead of DCM as the polar solvent for the reaction;

[0093] 2) Potassium carbonate was used instead of 4-dimethylaminopyridine as the alkaline catalyst.

[0094] Finally, a white solid was obtained with a yield of 70%. The obtained white solid was determined to be compound 10 through characterization.

[0095] Example 13: Preparation of Compound 12

[0096]

[0097] Under argon, a solution of 500 mg of compound 1 (1.0 mol, 1.0 equ.) was added to acetone (2 mL) at 0°C, and 2.65 mL of 2.5 mol / L Jones reagent (6.6 mol, 6.25 equ.) was slowly added dropwise (the preparation method of Jones reagent is as follows: 25 g of chromium trioxide was dissolved in 75 mL of water in a 500 mL beaker, and 25 mL of concentrated sulfuric acid was slowly added dropwise under ice bath conditions while stirring. The temperature of the solution was maintained at 0-5°C.), and the reaction mixture was stirred at 0°C for 0.5-1 hour, and the reaction was monitored by TLC thin layer plate. After the reaction was completed, 2-propanol (3 mL) was added to the reaction to quench the reaction, and 10 w / w% NaHCO 3 The solution was neutralized and extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with saturated brine and then with anhydrous Na 2 SO 4 After drying, filtration and evaporation, an oil was obtained, which was purified by flash chromatography on silica gel (dichloromethane / ethyl acetate, 10 / 1 to 1 / 2, volume ratio) to obtain compound 12, which was characterized as follows:

[0098] Compound 12 (4-oxowithaferin A-25-carboxylic acid): yield 50.0%, white solid; 1H NMR(600MHz,Chloroform-d)δ6.89–6.84(m,2H),4.48(dt,J=13.5,3.4Hz,1H),3.43 (d,J=2.6Hz,1H),2.74(dd,J=19.0,13.4Hz,1H),2.56(s,3H),2.34(dd,J=18.9,3.0 Hz,1H),2.16(dt,J=15.0,3.3Hz,1H),2.11–2.06(m,1H),1.66(d,J=7.9Hz,1H),1.4 8–1.44(m,2H),1.38(s,4H),1.03(d,J=6.6Hz,3H),0.89–0.80(m,3H),0.73(s,3H).

[0099] Example 14: Preparation of Compound 12

[0100] Example 13 was repeated, except that:

[0101] 1) Potassium dichromate was used instead of Jones reagent.

[0102] Finally, a white solid was obtained with a yield of 31%. The obtained white solid was determined to be compound 12 through characterization.

[0103] Example 15: Preparation of Compound 12

[0104] Example 13 was repeated, except that:

[0105] 1) Use DCM instead of acetone.

[0106] Finally, a white solid was obtained with a yield of 28%. The obtained white solid was determined to be compound 12 through characterization.

[0107] Example 16: Preparation of Compounds 14 and 15

[0108]

[0109] 24.0 mg of compound 12 (0.05 mmol, 1.0 equ.) was placed in a container, 1.0 mL of acetone was added, and the mixture was stirred at room temperature. Then 20.7 mg of ground potassium carbonate (0.15 mmol, 3.0 equ.) was added, and 5.2 μl of compound 13 (0.05 mmol, 1.1 equ.) was slowly added dropwise at 50° C., and stirred at 50° C. for 15 minutes. After the reaction was completed, a saturated sodium bicarbonate solution (2 mL) was added to the reaction to quench the reaction, and the mixture was extracted with EtOAc (3×10 mL), and the organic phases were combined and anhydrous Na 2 SO 4After drying, filtration and concentration, the residue was purified by silica gel flash chromatography (eluent: PE / EtOAc, 10 / 1 to 1 / 1, volume ratio) to obtain Compound 14 and Compound 15, which were specifically characterized as follows:

[0110] Compound 14 (4-oxo-25-methoxycarbonyl-withaferin A): yield 20.0%, white solid; 1 HNMR(600MHz,Chloroform-d)δ6.89–6.84(m,2H),4.46(dt,J=13.1,3.4Hz,1H),3.86(s,3H),3.43(d,J=2.5Hz,1H),2.52(dd,J=18.0,13.1Hz,1H),2.16 (dt,J=14.9,3.2Hz,1H),2.09(s,3H),1.70–1.57(m,5H),1.45(d,J=10.7Hz, 1H),1.38(s,3H),1.01(d,J=6.7Hz,4H),0.87(t,J=6.9Hz,1H),0.72(s,3H).

[0111] Compound 15 (25-methyl-23-double bond-4-oxowithaferin A-25-carboxyl): yield 44.2%, white solid; 1 H NMR(600MHz,Chloroform-d)δ6.95–6.81(m,2H),5.51–5.38(m,1H),5.03–4.93(m,1H),3.42(d,J=2.6Hz,1H),3.01–2.90(m,2H),2.16(dt, J=14.9,3.3Hz,1H),2.05–1.91(m,2H),1.78(s,3H),1.67–1.55(m,1H),1.46–1.41(m,3H),1.36(s,3H),0.82(d,J=6.6Hz,3H),0.70(s,3H).

[0112] Example 17: Preparation of Compounds 14 and 15

[0113] Example 16 was repeated, except that:

[0114] 1) Using water instead of acetone as the polar solvent for the reaction;

[0115] 2) DMAP / EDCI was used as the condensation agent instead of potassium carbonate, wherein the amount of DMAP was 3 times the molar amount of compound 12, and the amount of EDCI was 1.5 times the molar amount of DMAP.

[0116] Finally, two white solid products were obtained, one of which had a yield of 30% and was identified as compound 14 after characterization; the other had a yield of 45% and was identified as compound 15 after characterization.

[0117] Example 18: Preparation of Compounds 14 and 15

[0118] Example 16 was repeated, except that:

[0119] 1) Using ethanol instead of acetone as the polar solvent for the reaction;

[0120] 2) The reaction is carried out at room temperature and the reaction time is controlled to be 15 to 20 minutes.

[0121] Finally, two white solid products were obtained, one of which had a yield of 27% and was identified as compound 14 after characterization; the other had a yield of 36% and was identified as compound 15 after characterization.

[0122] Example 1: Anti-cancer activity test of target compounds 4, 7, 8, 10, 12, 14, and 15.

[0123] The anti-tumor activity of compound 1 and the target compound of the present invention was tested by MTT colorimetric method, and cisplatin (DDP) was selected as a positive control drug.

[0124] Instruments: clean bench (SW-CJ-1FD, AIRTECH, Sujing Antai), constant temperature CO2 incubator (3111, Thermo, USA), inverted biological microscope (IX71, OLYMPUS, Japan), enzyme-linked immunosorbent assay (Mode1680, BIO-RAD, USA), flat shaker (Kylin-bell lab Instruments), high pressure sterilizer (YX0.SG41.280, Shanghai Huaxian), centrifuge (SIGMA).

[0125] Reagents: DMEM (GIBCO), fetal bovine serum (GIBCO), trypsin (SIGMA), DMSO (SIGMA).

[0126] Cell lines: Human colon cancer HCT-116 cells, human large cell lung cancer H460 cells, human osteosarcoma HOS cells, nasopharyngeal carcinoma 5-8F cells, breast cancer MCF-7 cells, and triple-negative breast cancer M231 cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences.

[0127] Cell culture: The frozen cell lines were revived and placed in a constant temperature of 37°C CO 2Culture in an incubator, change the medium once a day, and plate when it is in the exponential growth phase and in good condition. Add 1 ml of 0.25% trypsin digestion solution, digest for 1 to 2 minutes, observe the cell state under a microscope, and when the adherent cells become round and shrink, remove the digestion solution, add 1 to 2 ml of DMEM medium containing 10% fetal bovine serum to make a cell suspension, count the cells, and count 5×10 cells per well. 4 The amount of cell suspension required was calculated based on the number of cells and the total number of wells. The cell suspension was inoculated on a 96-well plate at 100 μl / well, sealed with PBS, and placed in a constant temperature 37°C CO 2 Culture in an incubator for 24 h.

[0128] The anti-tumor activity was analyzed using the MTT method: the test drug, positive control DDP, and blank control DMSO were prepared with DMEM culture medium to a final concentration of 5 μM / well, with 3 replicates for each drug and cultured for 48 hours. The MTT reagent was added to a 96-well plate at 10 μl / well and incubated for another 4 hours. The culture medium in the plate was aspirated, 100 μl DMSO was added to each well, and the plate was shaken on a shaker for 10 minutes to dissolve the crystals. The absorbance of each well was detected at a wavelength of 570 nm using an enzyme-linked immunosorbent assay, and the cell inhibition rate was calculated according to the following formula. The average of the three initial screening results was the final inhibition rate, and compounds with an initial screening inhibition rate greater than 60% were subjected to concentration gradient screening (5-fold dilution) to prepare for the calculation of the IC of the test drug. 50 The results of three repeated experiments are the final IC values ​​of the tested compounds. 50 value.

[0129] Statistical analysis: IC50 was analyzed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA). Data are expressed as mean ± standard deviation (x±SD), and P<0.05 was considered to indicate a significant difference.

[0130] Cell inhibition rate % = [(blank control OD value - drug group OD value) / blank control group OD value] × 100%

[0131] Results: The inhibition rates of the tested compounds were all greater than 50%, so IC 50 The test results are shown in Table 1 below.

[0132] Table 1. Inhibitory effects of the tested compounds on various cancer cell lines

[0133]

[0134] Note: a Each experiment was repeated three times, and the results were expressed as mean ± SD.

[0135] b Data are expressed as IC 50 , that is, the concentration of compound that inhibits 50% of free radicals (mean ± SD).

Claims

1. A withaferin A derivative having a structure represented by the following formula 8, 10, 12, 14 or 15 or a pharmaceutically acceptable salt thereof:

2. A method for preparing a withaferin A derivative having a structure shown in the following formula 4, 7, 8, 10, 12, 14 or 15, characterized in that: The preparation method of the compound of the structure shown in Formula 4 is as follows: the compound of the structure shown in Formula 1 and the compound of the structure shown in Formula 2 are placed in a polar solvent, and reacted in the presence of an alkaline catalyst to obtain a compound of the structure shown in Formula 3; the compound of the structure shown in Formula 3 is placed in a polar solvent, a metal catalyst is added, and the reaction is carried out under a reducing atmosphere to obtain a compound of the structure shown in Formula 4; The preparation method of the compound of the structure shown in Formula 7 is as follows: the compound of the structure shown in Formula 1 and the compound of the structure shown in Formula 5 are placed in a polar solvent for reaction to obtain a compound of the structure shown in Formula 6; the compound of the structure shown in Formula 6 is placed in a polar solvent, a metal catalyst is added, and the reaction is carried out under reducing atmosphere conditions to obtain a compound of the structure shown in Formula 7; The preparation method of the compound of the structure shown in Formula 8 is as follows: taking the compound of the structure shown in the following Formula 1 and placing it in a polar solvent, adding a first oxidant to react, and obtaining the compound of the structure shown in Formula 8; The preparation method of the compound of the structure shown in Formula 10 is as follows: the compound of the structure shown in Formula 1 and the compound of the structure shown in Formula 9 are placed in a polar solvent, and reacted in the presence of a basic catalyst to obtain the compound of the structure shown in Formula 10; The preparation method of the compound of the structure shown in Formula 12 is as follows: the compound of the structure shown in Formula 1 is placed in a polar solvent, and a second oxidant is added to react to obtain the compound of the structure shown in Formula 12; The preparation method of the compounds of formula 14 and formula 15 is as follows: the compound of formula 12 and the compound of formula 13 are placed in a polar solvent, and a condensing agent is added to react to obtain the compounds of formula 14 and formula 15; In the above preparation method, In the preparation method of the compound of the structure shown in Formula 4, the alkaline catalyst is imidazole, 4-dimethylaminopyridine or triethylamine, or a combination of imidazole and 4-dimethylaminopyridine; the metal catalyst is a palladium carbon catalyst or palladium acetate; the polar solvent is dichloromethane, methanol or water; In the preparation method of the compound of the structure shown in Formula 7, the metal catalyst is a palladium carbon catalyst, platinum or Ni / Al2O3; the polar solvent is pyridine, tert-butanol or tetrahydrofuran; In the preparation method of the compound of the structure shown in Formula 8, the first oxidant is one or a combination of two or more selected from potassium osmate dihydrate, osmium tetroxide and sodium periodate; when the first oxidant is potassium osmate dihydrate, it needs to be used under acidic conditions of a system pH ≤ 6 and is used in combination with N-methylmorpholine-N-oxide, wherein citric acid or acetic acid is used to adjust the system to acidity; the polar solvent is pyridine or acetone, or a combination of tert-butanol and water; In the method for preparing the compound of the structure shown in Formula 10, the alkaline catalyst is 4-dimethylaminopyridine, pyridine or potassium carbonate; the polar solvent is dichloromethane, pyridine or methanol; In the method for preparing the compound of the structure shown in Formula 12, the second oxidant is Jones reagent or potassium dichromate, or a combination of Jones reagent and potassium dichromate; the polar solvent is dichloromethane or acetone; In the preparation method of the compound of the structure shown in Formula 14 and Formula 15, the condensation agent is potassium carbonate or DMAP / EDCI; the polar solvent is acetone, water or ethanol; 3. The preparation method according to claim 2, characterized in that: The method also includes a step of purifying the obtained target compound.

4. Use of the withaferin A derivative of the structure represented by formula 10 in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating colon cancer, lung cancer, osteosarcoma, nasopharyngeal carcinoma or breast cancer.

5. Use of the withaferin A derivative having a structure shown in formula 8, 12, 14 or 15 of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating lung cancer, osteosarcoma, nasopharyngeal carcinoma or breast cancer.

6. A pharmaceutical composition comprising the withaferin A derivative according to claim 1 or a pharmaceutically acceptable salt thereof.

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