WO2021016954A1

WO2021016954A1 - 3-acetyl-5-hydroxy-b-norcholesterol-6-(n-methyl)thiosemicarbazone, and preparation method therefor and use thereof

Info

Publication number: WO2021016954A1
Application number: PCT/CN2019/098727
Authority: WO
Inventors: 杨坤; 黄燕敏; 崔建国; 甘春芳; 潘玉秋
Original assignee: 广西万德药业有限公司
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2021-02-04

Abstract

Disclosed are a 3-acetyl-5-hydroxy-B-norcholesterol-6-(N-methyl)thiosemicarbazone, a preparation method therefor and the use thereof, and structural formula (I) of the 3-acetyl-5-hydroxy-B-norcholesterol-6-(N-methyl)thiosemicarbazone. The prepared 3-acetyl-5-hydroxy-B-norcholesterol-6-(N-methyl)thiosemicarbazone has obvious inhibitory effects on various tumor cell lines such as liver cancer, lung cancer, gastric cancer, cervical cancer, prostate cancer and colon cancer, has a low toxicity, and is not prone to causing drug resistance. Also provided in the present invention are a preparation method for the compound and the use of the compound. The preparation method is simple, is easy to implement, and facilitates market promotion and application.

Description

3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone, preparation method and use thereof

【Technical Field】

The invention belongs to the technical field of medicine preparation, and specifically relates to a 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone, a preparation method and uses thereof.

【Background technique】

Cancer is a major disease endangering human health. Overcoming malignant tumors is a difficult problem in modern medicine and the biggest challenge in today's pharmaceutical industry. At present, various different types of anti-tumor drugs continue to appear and are used clinically, but the anti-tumor drugs used in the clinic have a greater toxicity to human tissues, which greatly limits their scope of use. Therefore, looking for anticancer drugs with high efficiency, high selectivity and low side effects is the main direction of anticancer drug development.

It is reported in the literature that hydrazone compounds and their derivatives have good anti-tumor physiological activity, and have a good effect on Hey-1B (human ovarian cancer cells), SKBR3 (human breast cancer), etc. in inhibiting proliferation and growth in vitro ( Khan S A, Asiri A M, Yusuf M. Synthesis and biological evaluation of some thiazolidinone derivatives of steroid as antibacterial agents [J]. European Journal of Medicinal Chemistry, 2009, 40(36): 2597-2600). With the in-depth research on steroid hydrazone compounds, a variety of steroid hydrazone compounds with unique structures and excellent physiological activities have been synthesized. Cholesterol B-drop also has a good anti-tumor activity. Related personnel have carried out related research on the chemical modification of cholesterol, such as the Chinese invention patent "a type of B- with [6-5-6-6] steroid core structure The synthesis method of nor-D-homo-lactam steroids (Patent No.: ZL201310184259.1)" discloses a class of B-nor-D-homo-endo with [6-5-6-6] steroid core structure Amide steroid compound, the steroid nucleus structural formula of the compound is as follows:

among them:

X=O, NOH, NOCH3, NOCH2Ph, NNHCSNH2, NNHCSNHCH3, NNHCSNHCH2CH3, NNHCSNHHPh. The B-nor-D-homo-lactam steroid compound 5 with the [6-5-6-6] steroid nuclear structure of the present invention has an inhibitory effect on the growth and proliferation of liver cancer cell lines, and compounds 10 and 11 on colon cancer cell lines . It can be used as a drug intermediate or drug used in different drug manufacturing and uses. For example, the Chinese invention patent "6-substituted-4-aza-A-homo-3-oxosteroid and its application in the preparation of anti-tumor drugs (Patent No.: ZL201010107528.0)" discloses a 6- Substituted-4-aza-A-homo-3-oxo steroids have the chemical structural formula shown in formula I:

Where X = OH, =NOH, OSO3Na, =O, =NNH2, =NCSNH2, =NCONH2, =NCNHNH2, =NNHCSNH2, =NNHCONH2, OCH2CH2N(R')2, N(CH2CH2Cl)2, halogen, =S, SH , OR', wherein said R'is a common alkyl group. R is a straight chain with 1-14 C atoms or a carbon chain with different positions substituted branched or a carbon chain with different functional group substituents, or a functional group containing different heteroatoms such as OH, =NOH, OSO3Na, =O , =NNH2, =NCSNH2, =NCONH2, =NCNHNH2, =NNHCSNH2, =NNHCONH2, OCH2CH2N(R')2, N(CH2CH2Cl)2, halogen, =S, SH, OR'. Experiments prove that the 6-substituted-4-aza-A-homo-3-oxosteroid compound of the present invention has obvious inhibitory effect on various tumor cell lines, and can be used to prepare drugs for treating cancer. For example, the Chinese application for invention patent "3-substituted-B-Homo-steroid-B-cyclic lactam compound and its preparation method and application in the preparation of anti-tumor drugs (application number: 201110009124.2, application publication number: CN102146115A)" is published A class of 3-substituted-B-Homo-steroid-B-cyclic lactam compounds and their preparation methods and applications in the preparation of anti-tumor drugs. The 3-substituted-B-Homo-steroid-B-cyclic lactam compound has a chemical structural formula as in Formula I:

Experiments prove that the 3-substituted-B-Homo-steroid-B-cyclic lactam compound of the present invention has obvious inhibitory effect on various tumor cell lines, and can be used to prepare drugs for treating cancer.

[Content of the invention]

The invention provides a 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone, preparation method and application thereof, to solve the current clinical use of anti-tumor drugs Because of its greater toxicity to human tissues, it greatly limits the scope of their use.

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

The structural formula of 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone of the present invention is as follows:

In the present invention, cholesterol is used as a raw material, an acetyl group is introduced at the 3-position, and a 3-acetyl-B-cholesterol-lowering compound is obtained by condensation after breaking the ring and then introducing N-methylthiosemicarbazone at the 6-position 3-Acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone is obtained.

Starting from cholesterol, the target compound is synthesized according to the following chemical reaction formula:

Reagents and reaction conditions: (a) CH ₂ Cl ₂ /triethylamin; (b) O ₃ /(CH ₃ ) ₂ S; (c) Al ₂ O ₃ /Ph; (d) NH ₂ NHCSNHCH ₃

Further, the breaking and opening of the ring is carried out under the condition of passing ozone.

Further, the condensation is an aldol condensation reaction using neutral alumina at room temperature.

The invention also provides an application of 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone in the preparation of antitumor drugs.

Further, the tumor disease includes one of liver cancer, lung cancer, stomach cancer, cervical cancer, prostate cancer, and colon cancer.

The invention has the following beneficial effects:

The 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl) thiohydrazone prepared by the invention is effective for liver cancer, lung cancer, gastric cancer, cervical cancer, prostate cancer, colon cancer, etc. The tumor cell strain has obvious inhibitory effect, low toxicity, and resistance to drug resistance; the present invention also provides a preparation method and application of the compound, which is simple and easy to implement, and is beneficial to market promotion and application.

【Detailed ways】

In order to facilitate a better understanding of the present invention, the following examples are used to illustrate, these examples belong to the protection scope of the present invention but do not limit the protection scope of the present invention.

Starting from cholesterol, the target compound (compound 2, compound 3, compound 4) is synthesized according to the following chemical reaction formula:

In order to make the disclosure of the present invention more complete, more specific embodiments are used to illustrate the present invention.

Example 1

Synthesis of compound 2

Weigh 386 mg (1 mmol) of cholesterol into a 100 mL pear-shaped reaction flask, add 20 mL of dichloromethane and stir at room temperature until the cholesterol dissolves, add 1 mL of triethylamine, add 1 mL of acetyl chloride, and stir for 4 hours at 40°C. TLC tracking Reaction, stop the reaction when there is no obvious raw material. Pass the crude column with ethyl acetate, remove excess ethyl acetate and dichloromethane by distillation under reduced pressure, dissolve in dichloromethane, wash with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively, extract three times successively, and combine the organic Phase, the solvent was removed under reduced pressure to obtain a solid crude product, which was separated by column chromatography to obtain the target product compound 2. White solid: mp109～112℃. ¹ H NMR (CDCl ₃ , 300MHz) δ: 0.70 (3H, s, C18-H), 0.87 (3H, d, C26-H), 0.90 (3H, d, C27-H) ), 0.93 (3H, d, C19-H), 1.04 (3H, s, C21-H), 2.05 (3H, s, CH3), 4.64 (1H, m, C3-H), 5.39 (1H, m, CH-6). ¹³ C NMR (CDCl ₃ , 75MHz) δ: 170.52 (C=O), 139.66 (5-C), 122.64 (6-C), 73.99, 56.69, 56.15, 50.04, 42.32, 39.52, 38.13 ,36.59,35.79,31.90,31.87,28.01,23.83,22.82,22.56,21.43,19.31,18.72,11.86.IR(KBr)v/cm ^-1 :3451.24,3144.64,2955.19,2867.95,2820.59,2720.88,1723.79,1439.62 ,1464.55,1362.35,1245.19,1140.50,1035.80,975.98,953.54,903.69,794.01,736.68,659.40,614.53.HREIMS calcd for C ₂₉ H ₄₈ O ₂ K[M+K] ⁺ 467.3291,found 467.3274.

Example 2

Synthesis of compound 3

Weigh 2mmol of the corresponding precursor compound 2 in an eggplant-shaped reaction flask, add 30mL of dichloromethane to dissolve it, add 5mL of methanol dropwise, pass O ₃ , the solution appears blue, react for 2 hours, TLC tracking the reaction, no After the raw material point, stop feeding O ₃ , add 5 mL of dimethyl sulfide, stir and react for 4 hours, then spin dry under reduced pressure to obtain a white oil, continue to add 20 mL of benzene to dissolve, weigh 10 mg of neutral alumina into the reaction flask , Continue to stir for 5 hours, TLC follows the reaction, and stop the reaction when there is no raw material point. Pass through the crude column with ethyl acetate, and evaporate most of the solvent under reduced pressure. The crude product is separated by column chromatography to obtain the target product compound 3 with a yield of 60%. mp84～88℃. ¹ H NMR(CDCl _3, 300MHz)δ : 0.74 (3H, s, C18-H), 0.86 (3H, d, C26-H), 0.88 (3H, d, C27-H), 0.93 (3H, d, C19-H), 0.96 (3H, s , C21-H), 2.08 (3H, s, -CH3), 5.15 (1H, m, OH), 9.69 (1H, s, CHO). ¹³ C NMR (CDCl ₃ , 75MHz) δ: 203.91 (CHO), 169.74(C=O),83.65,70.36,64.53,56.00,55.65,51.51,45.59,44.64,42.20,39.46,39.34,36.17,35.60,27.99,24.98,24.46,23.81,22.79,22.54,21.55,21.38,18.74 ,18.20,12.48.IR(KBr)v/cm ^-1 :3590.83,3172.06, 2947.71,2867.95,1738.75,1693.88,1472.03,1387.27,1300.03,1252.67,1185.36,1150.47,1053.25,975.98,906.18,866.30,818.94,724.21 ,622.01,492.39.HREIMS calcd for C ₂₉ H ₄₈ O ₄ Na[M+Na] ⁺ 483.3450, found 483.3446.

Example 3

Synthesis of compound 4

Weigh 1mmol of the corresponding precursor compound 3 in a 100mL pear-shaped reaction flask, add 20mL of absolute ethanol, stir to dissolve at room temperature, add 110mg of 4-methylthiosemicarbazide, and react for 5 hours at 60°C. The reaction was followed by TLC, and the reaction was stopped when there was no raw material point, and absolute ethanol was distilled off under reduced pressure. The crude product was separated by column chromatography to obtain the target compound 4 (3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone). White solid, yield 60%. mp93～94℃. ¹ H NMR(CDCl _3, 300MHz)δ:0.68(3H,s,C18-H), 0.86(3H,d,C26-H), 0.88(3H, d,C27-H),0.92(3H,d,C19-H),0.95(3H,s,C21-H),2.07(3H,s,CH ₃ ), 4.62(1H,m,C3-H), 5.39(1H,d,CH-6), 7.08(1H,m,NH-CH3), 7.40(1H,m,CH=N), 9.09(1H,S,NH). ¹³ C NMR(CDCl ₃ ,75MHz )δ: 187.37(C=S), 170.23(3-C=O), 148.32(6-C=N), 83.00(5-C), 70.00(3-C), 56.36(17-C), 55.84 (14-C), 53.13(7-C), 45.08(9-C), 44.55(13-C), 42.73(8-C), 40.47(10-C), 39.58(12-C), 39.45( 24-C), 36.19(4-C), 35.58(22-C), 30.50(NH--CH3), 28.54(20-C), 28.00(16-C), (24.89(25-C), 23.83 (23-C), 22.79(26-C), 22.54(27-C), 21.73(3ɑ-C), 21.46(11-C), 18.75(21-C), 18.26(19-C), 12.38( 18-C).IR(KBr)v/cm ^-1 :3144.64,2950.21,2867.95,1723.79,1544.31,1464.55,1397.24,1250.17,1073.19,1023.34,958.53,903.69,734.18; HREIMS calcd. for C ₃₁ H ₅₂ N _{_{^{3 O 3 S [MH] -}}} 546.3729, found 546.3734.

Example 4

Evaluation of anti-tumor activity of 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone (compound 4)

In order to evaluate the in vitro inhibitory activity of the synthesized 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl) thiohydrazone on the growth and proliferation of tumor cells, we used MCF-7 (human breast cancer) Cells), SKOV3 (ovarian cancer cells), T47D (human breast ductal carcinoma cells), and human renal epithelial cells (HEK-293T) as the research objects, using the MTT method to control 3-acetyl-5-hydroxy-B-cholesterol Sterol-6-(N-methyl) thiohydrazone (Compound 4) was tested for anti-tumor activity in vitro. The test data are shown in Table 1.

Table 1 Inhibitory activity of compound 4 in vitro (MTT method) (IC ₅₀ , μmol/L)

It can be seen from the test data in Table 1 that the 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl) thiohydrazone of the present invention has an effect on human breast cancer cells, human breast duct cancer cells, Human ovarian cancer cells have a significant effect of inhibiting the growth and proliferation of tumor cells.

The 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl) thiohydrazone of the present invention has low toxicity and is not easy to produce drug resistance; in addition, the preparation method of the compound is simple and easy to implement. Conducive to market promotion and application.

The above content cannot be considered that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be It is deemed to belong to the patent protection scope of the present invention determined by the submitted claims.

Claims

A 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl) thiohydrazone, which is characterized in that it has the following chemical structural formula:
A method for preparing 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone according to claim 1, characterized in that it comprises the following steps: Sterol is used as the raw material, the acetyl group is introduced at the 3-position, the 3-acetyl-B-cholesterol is obtained by condensation after breaking the ring, and then the N-methylthiosemicarbazone is introduced at the 6-position to obtain 3-acetyl 5-hydroxy-B-cholesterol-6-(N-methyl)thizone.
The preparation method of 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl) thiohydrazone according to claim 2, characterized in that the breaking and opening of the ring is carried out by passing ozone Under the conditions.
The method for preparing 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl) thiohydrazone according to claim 2, characterized in that the condensation is performed at room temperature using neutral Aldol condensation reaction of alumina.
An application of the 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone according to claim 1 in the preparation of antitumor drugs.
The use of 3-acetyl-5-hydroxy-B-cholesterol-6-(N-methyl)thizone in the preparation of anti-tumor drugs according to claim 1, wherein the tumor diseases include One of liver cancer, lung cancer, stomach cancer, cervical cancer, prostate cancer, and colon cancer.