WO2014056401A1

WO2014056401A1 - Double-bond substituted tripterygium wilfordii lactone derivative and preparation method and use thereof

Info

Publication number: WO2014056401A1
Application number: PCT/CN2013/084291
Authority: WO
Inventors: 樊兴; 秦继红
Original assignee: 上海汇伦生命科技有限公司
Priority date: 2012-10-09
Filing date: 2013-09-26
Publication date: 2014-04-17
Also published as: CN103709227B; CN103709227A

Abstract

Disclosed are a double-bond substituted tripterygium wilfordii lactone derivative as represented by formula (I), or pharmaceutically acceptable salt or hydrate thereof, or optical isomer thereof, wherein R₁=OH, R₂=H, or R₁=H, R₂=OH. Also disclosed are a preparation method and uses thereof in the preparation of drugs for treating cancers and cancer-related diseases.

Description

Double bond substituted triptolide derivative, preparation method and application thereof

The invention relates to structural modification and activity research of natural medicinal active ingredients, in particular to double bond substituted triptolide derivatives, preparation method and research for treating proliferative tumor diseases. Background technique

Tripterygium wilfordii, commonly known as the genus Echinochloa, is a woody vine of the genus Tripterygium (Cdastraceae), which is a plant with abundant resources in China. There are four species of Tripterygium, Tripterygium wilfordii Hook f., Tripterygium hypoglaucum Levi. Hutch, Tripterygium regelii Sprague et Takeda, and Cangshan Leigong. Tripterygium forretii Dicls, distributed in China. Tripterygium was first collected in Shennong Bencaojing. Its main chemical constituents are diterpenoids, triterpenoids, sesquiterpenes, alkaloids, etc. Up to now, nearly 200 compounds have been isolated from Tripterygium plants. Studies in the past two decades have shown that it has various activities such as anti-tumor, anti-inflammatory, immunosuppressive, anti-fertility, antibacterial and the like.

Tripterygium wilfordii has been used in the treatment of tumors for many years, and one of the active ingredients is triptolide. In addition to extensive anti-tumor effects, triptolide has also entered clinical trials for the treatment of leukemia, but the large side effects of triptolide, too narrow a therapeutic window, limits its clinical application. . In the past, the structural modification of triptolide was mostly limited to the introduction of water-soluble groups, and there was no fundamental change in the main structure. When such prodrugs enter the body, they are still converted to triptolide by hydrolysis or metabolism, which determines that they cannot fundamentally improve the side effects of triptolide. At the same time, when the C ₁₄ -position of triptolide was modified, it was found that the group configuration at this position had a significant effect on the activity of the compound, and the problem of the optical isomer added great difficulty to the synthesis and separation work.

The present invention focuses on the study of the structure-activity relationship of triptolide, and the structure is modified and modified differently from the prior art, and a batch of novel structure of triptolide derivatives is obtained, thereby completing the present invention. Summary of the invention

One of the technical problems to be solved by the present invention is in the case of triptolide, against the problems existing in the existing triptolide. A hydroxyl group is introduced at the ₅ -position or the c- ₁₅ -position, and an exocyclic double bond is introduced at the c ₁₄ -position to form a batch of double bonds to replace the triptolide derivative.

The second technical problem to be solved by the present invention is to provide a method for preparing a hydroxy-substituted triptolide derivative having a 14-double bond triptolide as a basic structure.

The third technical problem to be solved by the present invention is to study the pharmacological and toxicological experiments of the triptolide derivative by using a 14-double bond triptolide as a basic structure, and find that it has tumors for tumor diseases, especially reproductive systems. Good therapeutic effect, high efficiency, low toxicity, no cross-resistance with traditional cytotoxic anti-tumor drugs, and has a good development prospect for the treatment of proliferative diseases such as tumors.

The double bond-substituted triptolide derivative provided by the present invention is a compound of the formula (I) or a pharmaceutically acceptable salt or hydrate thereof or an optical isomer thereof:

(I)

Wherein R^OH, R ₂ =H, or RfH, R ₂ =OH. In a preferred embodiment of the invention, in the general formula (I), =011, R ₂ = H, that is, a compound represented by the following structural formula ( la):

( l a)

In a preferred embodiment of the invention, in the general formula (I), ί^=Η, R ₂ = OH,

( lb)

The preparation method of the present invention is as follows:

1. A method for preparing a compound of the formula (la) consisting of the following steps:

Step 1: Using triptolide (1) as a starting material, attacking the C14 carbonyl group of triptolide with an iodoformin reagent in an aprotic solvent to obtain (14S) -14β-methyl Ester alcohol (2); Step 2: (14S) -14β-methylepitolide (2) dehydrated with trifluoroacetic anhydride in an aprotic solvent to give the C14 position extra-double bond product 14-dehydroxy- 14-Methylene-tritolide (3); Step 3: 14-dehydroxy-14-methylene triptolide (3) is prepared by culturing in potato medium containing Neurospora crassa Hydroxy-14-methylene-5-hydroxy triptolide (I a)

2. A method for preparing a compound of the formula (lb) consisting of the following steps:

Step 1: 14-dehydroxy-14-methylene triptolide (3) was prepared by incubation with rat liver microsomal protein in phosphate buffer solution to obtain 14-dehydroxy-14-methylene-15-hydroxyl Vinolactone (lb) The use of the fluorine-substituted triptolide derivative of the present invention for the preparation of a medicament for treating cancer and cancer-related diseases.

The cancer is kidney cancer, breast cancer, lung cancer, malignant melanoma, colon cancer, prostate cancer or ovarian cancer.

The terms used in this specification have the following meanings:

The "pharmaceutically acceptable salt" specifically includes organic acids and aspartic acid such as propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, and the like. a salt formed by an acidic amino acid such as glutamic acid and then formed with an inorganic base, such as a sodium, potassium, calcium, aluminum salt and an ammonium salt, or a salt formed with an organic base such as methylamine salt, ethylamine salt or ethanolamine salt. Or a salt of a mineral acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid which is esterified with a basic amino acid such as lysine, arginine or ornithine, or with formic acid or acetic acid. a salt of an organic acid such as picric acid, methanesulfonic acid or ethanesulfonic acid.

The meaning of "hydrate" includes monohydrate, dihydrate, and trihydrate.

The meaning of "optical isomer" includes enantiomers, diastereomers, mixtures of optical isomers, and pure optical isomers.

001-99. 9% by weight and an appropriate amount of a pharmaceutically acceptable carrier, the double bond-substituted triptolide derivative, a pharmaceutically acceptable salt or an optical isomer thereof. Various formulations, such as those suitable for oral, injectable or enteral administration.

The therapeutically effective amount can be administered to the subject according to the age (months or weeks of age) of the subject, the general health condition, the severity and duration of the disease, the route of administration, the sensitivity of the human body to the drug, and the like. A pharmaceutical preparation of a compound of the invention. DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the amount-effect curve of 14-dehydroxy-14-methylene-5-hydroxylogenide alcohol prepared in Example 1 of the present invention for inhibiting culture of human ovarian cancer SK-OV-3 cells in vitro.

Fig. 2 is a graph showing the amount-effectiveness curve of 14-dehydroxy-14-methylene-15-hydroxytriptolide prepared in Example 2 of the present invention for inhibiting human ovarian cancer SK-OV-3 cells in vitro.

detailed description

The invention is further illustrated by the following examples, but these examples are in no way intended to limit the scope of the invention, and the scope of the invention is determined by the claims.

First, the preparation example

Example 1

Structure (l a) Preparation of 14-dehydroxy-14-methylene-5-hydroxy triptolide Example 1

Preparation of (2) (14S)-14P-methylepitolide

Under a argon atmosphere, 10 mL of anhydrous diethyl ether was added to a reaction flask containing magnesium chips (264 mg, llmmol), and Mel (1.42 g, 10 mmol) dissolved in 5 mL of anhydrous diethyl ether was added dropwise to the above reaction system through a constant pressure dropping funnel. in. The dark grey reaction system was added and stirred at room temperature for half an hour. 1.37 mL of freshly prepared MeMgl format reagent solution was added dropwise to triptolide 1 (90 mg, 0.25 mmmol) which had been dissolved in 8 mL of dry tetrahydrofuran. After reacting for 2 hours at room temperature The reaction was quenched and the reaction was quenched with EtOAc EtOAc (EtOAc)EtOAc. =1 : 5 ) A white solid (14S)-14P-methylepitolide 2 (70 mg, yield 73%) was obtained. MS (ESI): m/z 375 [M+l]+. _3⁄4 NMR (CDC1 ₃ , 300MHz): δ 4.66 (s, 2H), 3.75 (d, IH, J=3.2Hz), 3.74 (d, IH, J=3.2Hz), 2.75-2.63 (m, IH), 2.47 (sept, IH, J=6.9Hz), 2.34-2.22 (m, IH), 2.21-2.03 (m, 2H), 1.85 (t, IH, J= 14.1 Hz), 1.58-1.48 (m, 4H) , 1.24-1.10 (m, IH), 1.03 (s, 3H), 0.94 (d, 3H, J = 6.9 Hz), 0.79 (d, 3H, J = 6.9 Hz).

Step 2

Structure (3) Preparation of 14-dehydroxy- 14-methylene triptolide

MsCl (0.18 mL, 2.2 mmol), dry TEA (2.17 mL, 15.6 mmol) and dried pyridine (2.4 mL, 29.6 mmol) (14S)-14P-methylepitolide 2 (130 mg, 0.35 mmol) was dissolved in 5 mL of dry DCM at room temperature, added to the above reaction system, and trifluoroacetic anhydride was added dropwise to the reaction system with stirring. (0.24 mL, 1.7 mmol), reacted at room temperature for 2 h and then quenched with water. The solvent was evaporated under reduced pressure. The concentrated crude product was purified by column chromatography (ethyl acetate:ield:ield:::::::::::::::::::::::::::::::::::::::::: MS (ESI): m/z 355 [M+l]+. ^ NMR (CDC1 ₃ , 300MHz): δ 5.53 (s, IH), 5.39 (s, IH), 4.83-4.80 (m, 2H), 3.97 (d, IH, J=3.0Hz), 3.64 (d, IH , J=3.0Hz), 3.54-3.52 (m, IH), 3.07-3.05 (m, IH), 2.87-2.83 (m, IH), 2.53-2.42 (m, IH), 2.29-2.14 (m, 2H ), 1.99-1.89 (m IH), 1.53-1.46 (m, IH), 1.42-1.33 (m, IH), 1.09 (s, 3H), 0.97 (d, 3H, J=6.9Hz), 0.87 (d , 3H, J=6.9Hz).

Step 3

Preparation of 14-dehydroxy-14-methylene-5-hydroxy triptolide

From the test tube bevel, the Phytophthora aeruginosa was picked into a 250 mL flask (with 60 mL of potato medium:), shaken at 200 rpm, 25 ° C for 1 day, and then added with 200 μl of Omg/mL of 14-dehydroxy-14-- An ethanol solution of piracetin 3 (containing 10% DMSO) was further cultured for 4 days under the same conditions. The fermentation broth was filtered, and the filtrate was extracted three times with an equal volume of ethyl acetate, and the genus Streptomyces was ultrasonically extracted with an appropriate amount of ethyl acetate for 30 minutes. The two portions of ethyl acetate were combined and the solvent was evaporated to give a residue. The residue was separated by semi-preparative high performance liquid chromatography, and the fractions were collected. The solvent was concentrated under reduced pressure at 40 ° C to obtain 14-dehydroxy-14-methylene-5-hydroxy triptolide 1 a (0.4 mg, yield 20%). MS (ESI): m / z 373 [M + lfo ^ NMR (CD 3 OD, 300MHz): δ 5.55 (s, IH), 5.42 (s, IH), 4.84-4.80 (m, 2H), 3.90 (d , IH, J=2.7Hz), 3.65 (d, IH, J=2.7Hz), 3.07-3.06 (m, IH), 2.52-2.43 (m, IH), 2.28-2.06 (m, 3H), 1.91- 1.81 (m, IH), 1.32-1.21 (m, 2H), 1.13 (s, 3H), 0.99 (d, 3H, J=6.3Hz), 0.87

(d, 3H, J = 6.3 Hz). Example 2

Structure (l b) Preparation of 14-dehydroxy- 14-methylene-15-hydroxy triptolide Example 1

Preparation of (l b) 14-dehydroxy- 14-methylene-15-hydroxy triptolide

A stock solution of 14-dehydroxy-14-methylene triptolide 3 (50 mM) was prepared in methanol, and the final incubation system had a methanol content of 0.1%. The volume of the single incubation system was 10 mL, and the medium was 100 mM phosphate buffer (PBS, pH 7.4), including rat liver microsomal protein at a final concentration of 0.5 mg/mL, and 14 μdehydroxy-14-Asia at 50 μM. Methyl triptolide 3 and 1 mM NADPH were incubated for 2 h at 37 ° C in a water bath. The 14-dehydroxy-14-methylene triptolide 3 rat liver microsome incubation solution was extracted with ethyl acetate, and concentrated under reduced pressure at 40 ° C to dissolve the solvent in 5 ml of methanol: water 65:35. After separation by semi-preparative high performance liquid chromatography, the fractions were collected, and concentrated under reduced pressure at 40 ° C to obtain 14-dehydroxy-14-methylene-15-hydroxy triptolide lb (10 mg, yield 10%) ). MS (ESI): m/z 373 [M+l]+. _3⁄4 NMR (CDC1 ₃ , 400MHz): δ 5.82 (s, IH), 5.65 (s, IH), 4.84-4.80 (m, 2H), 3.97 (d, IH, J=3.3Hz), 3.87 (d, IH , J=3.3Hz), 3.07-3.06 (m, IH), 2.87-2.83 (m, IH), 2.28-2.08 (m, 3H), 1.97-1.92 (m, 1H), 1.52-1.38 (m, 2H), 1.35 (s, 3H), 1.34 (s, 3H), 1.09 (s, 3H). ¹³ C NMR (CDC1 ₃ , 100MHz): δ 176.68, 164.65 , 140.77, 125.90, 119.11, 72.52, 72.08, 67.01, 66.68, 64.93, 62.58, 57.48, 42.13, 37.30, 31.90, 27.28, 27.05, 25.22, 18.51, 14.63.

Second, pharmacodynamic evaluation test case

14-dehydroxy-14-methylene-5-hydroxy triptolide prepared in Example 1 of the present invention and 14-dehydroxy-14-methylene-15-hydroxy triptolide 2 prepared in Example 2 Growth inhibition of human ovarian cancer SK-OV-3 cells cultured in vitro.

Human ovarian cancer SK-OV-3 cells were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum at 37 ° C, 5% C0 ₂ , and tumor cells 0.7 x lO ⁴ /well inoculated into 96- plate, 24 hours later, dimethyl sulfoxide stock solution was added configuration (10_ ² M), each compound diluted with physiological saline, culture medium in a final concentration of 10- ^5, 10 ^"6 M, 72 hours. Discard the culture solution, fix the cells with 10% cold trichloroacetic acid, stain with Sulforhodamine B (SRB) solution, wash away the unbound SRB, Tris dissolves the protein-bound SRB, and the microplate reader is at 560 nM. The OD value was measured at the wavelength, and the cell growth inhibition rate was calculated according to the following formula: Inhibition rate = (00 value " _3⁄4 - OD value administration hole" / OD value to the kitchen _L x l00%. The 14-dehydroxy group prepared in Example 1 was obtained - 14-Methylene-5-hydroxy triptolide (see Figure 1) and 14-dehydroxy-14-methylene-15-hydroxy triptolide prepared in Example 2 for in vitro culture of human ovarian cancer SK- A dose-response curve for OV-3 cell inhibition (see Figure 2).

14-dehydroxy-14-methylene-5-hydroxy triptolide prepared in Example 1 and 14-dehydroxy-14-methylene-15-hydroxy triptolide prepared in Example 2 for human ovarian cancer The results of growth inhibition of SK-OV-3 cells are shown in Table 1.

Table 1. Growth inhibition of compounds on human ovarian cancer SK-OV-3 cells

The role of cancer SK-OV-3 cell growth. The above compound preparation examples and pharmacodynamic evaluation test examples are merely illustrative of the structure of the compound provided by the present invention, the preparation method thereof and the pharmacological test results, but various modifications and changes can be made to those skilled in the art. The appended claims cover all such modifications as are within the scope of the invention.

Claims

Claim

1. A double bond of the formula (I) in place of a triptolide derivative or a pharmaceutically acceptable salt or hydrate thereof or an optical isomer thereof:

(I)

Wherein RfOH, R ₂ =H, or Ri=H, R ₂ =OH.

2. The double bond-substituted triptolide derivative according to claim 1, wherein RfOH, R ₂ =H, is a compound represented by the following structural formula (I a):

( l a)

3. The double bond-substituted triptolide derivative according to claim 1, wherein Ι^=Η, R ₂ =OH, that is, a compound represented by the following structural formula ( lb):

( l b)

A method for producing a double bond-substituted triptolide derivative according to claim 2, wherein the compound of the formula (la) is prepared by the following steps:

Step 1: Using triptolide (1) as a starting material, attacking the C14 carbonyl group of triptolide with an iodoformin reagent in an aprotic solvent to obtain (14S) -14β-methyl Ester alcohol (2);

Step 2: (14S) -14β-methyl-tallinolide (2) dehydrated with trifluoroacetic anhydride in an aprotic solvent to give the C14-position extra-double bond product 14-dehydroxy-14-methylene tert Vine lactone (3);

Step 3: 14-dehydroxy-14-methylene triptolide (3) is prepared by culturing in a potato medium containing Neurospora crassa, 14-dehydroxy-14-methylene-5-hydroxy Tripterygium Ester alcohol (I a).

A method for producing a double bond-substituted triptolide derivative according to claim 3, wherein the compound of the formula (lb) is prepared by the following steps:

Step 1: 14-dehydroxy-14-methylene triptolide (3) was prepared by incubation with rat liver microsome protein in phosphate buffer solution to obtain 14-dehydroxy-14-methylene-15-hydroxyl Vine lactone (Ib).

The use of the double bond-substituted triptolide derivative according to claim 1 for the preparation of a medicament for treating cancer and a disease associated with cancer.

The use according to claim 6, wherein the cancer is kidney cancer, breast cancer, lung cancer, malignant melanoma, colon cancer, prostate cancer or ovarian cancer.