CN108299534B - Pregnane steroid compound and preparation method and application thereof - Google Patents

Abstract

The invention relates to a pregnane compound, which has the structural formula shown as follows:

Description

Pregnane steroid compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to a novel pregnene steroid compound and a preparation method and application thereof.

Background

Multidrug Resistance (MDR) refers to the Resistance of tumor cells after contacting with a certain anti-tumor drug, and also generates cross Resistance to multiple anti-tumor drugs with different structures and mechanisms, which is the most common problem in clinical tumor chemotherapy and can not be solved so far. Since the discovery of this phenomenon, multidrug resistance reversal agents have gradually become the hot spots for the development of chemotherapeutic drugs. The reversal agent plays a role in inhibiting efflux protein P-gp and increasing the concentration of an antitumor drug in cells by being combined with the antitumor drug, and can effectively reverse multidrug resistance, but clinical tests of the reversal agent fail to complete. After the drugs are used together, the problems of high toxicity, low curative effect, complex drug interaction and the like greatly limit the use of the drugs.

Mielan blue（Aglaia) About 250-300 species of Meliaceae, Midland, are distributed in India, Malaysia, Australia and Borinula. The 7 varieties of 1 variety produced in China are distributed in the south, southwest and southeast. In the application of traditional Chinese medicine and folk medicine, various Mianella plants can be used as the medicine, and have the effects of promoting blood circulation, relieving swelling and pain, soothing liver, promoting qi circulation, expelling parasites and the like. Reduced aglaia odorata (A)Aglaia abbreviataC.y. Wu) is a plant of the genus meliaceae, the genus aglaia, shrub to small tree, which is produced in provinces such as Yunnan, Guangxi, etc., in China, and mainly grows in valley rain forests or evergreen broad-leaved forests.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a novel pregnane compound and a preparation method thereof. The test shows that: the pregnane compound can be used for preparing tumor cell inhibiting drugs or drug-resistant tumor cell drugs.

In order to achieve the purpose, the invention adopts the following technical scheme:

a pregnane compound having the formula:

。

the preparation method of the pregnane compound comprises the following steps:

1) taking the crushed contracted sequence aglaia odorata stem bark dry powder, leaching with 85-95% ethanol at room temperature, and concentrating the leaching liquor under reduced pressure to obtain ethanol extract;

2) suspending the ethanol extract in water, sequentially extracting with dichloromethane and ethyl acetate, and concentrating the extract to obtain dichloromethane extract;

3) dissolving the dichloromethane extract with water, passing through D101 macroporous resin, washing with water to remove sugar and other water-soluble impurities, sequentially eluting with 60-90% ethanol, detecting, and mixing to obtain 5 components, and sequentially recording by TLC (thin layer chromatography) according to the elution sequence: fr.a, fr.b, fr.c, fr.d, fr.e;

dissolving Fr.B with methanol, stirring with silica gel, passing through silica gel column, and mixing with 20: 1-1: 2, performing gradient elution by petroleum ether-ethyl acetate, detecting and combining to obtain 4 components, and recording TLC as follows according to the elution sequence: b1, b2, b3, b 4;

b2 was eluted through reverse phase C18 column with a 50-90% methanol-water gradient to give three fractions, in order of elution, recorded as: b2a, b2b, and b2 c;

and purifying Fr.B.2b with preparative HPLC (RP-C18) and 75% methanol-water as mobile phase at wavelength of 210nm and flow rate of 10 ml/min.

The invention provides application of the pregnane compound in preparing a medicament for inhibiting tumor cells.

The invention also provides application of the pregnane compound in preparation of drug-resistant tumor cell drugs.

More preferably, the tumor cell may be a leukemia cell, an oral epidermoid carcinoma cell, or the like.

Compared with the prior art, the invention has the beneficial effects that:

1) the compound has stable structure, belongs to pregnane steroid compounds, and the extraction raw material is from reduced-order aglaia odorata growing in low mountain shrubs or valley banks. The reduced-order aglaia odorata is widely distributed in southern areas of China, and resources are very rich. Compared with the preparation of the rape sterol plant compound by taking other steroid compounds as raw materials, the method has the advantages of mild production conditions, few experimental steps, small technical difficulty, low production cost and small environmental pollution; meanwhile, the raw materials are rich and belong to natural renewable resources; the extraction and separation technology has small difficulty, and the solvent can be recycled, thereby not causing the pollution of the ecological environment. 2) The test shows that: the compound of the invention shows better cytotoxic activity to various human tumor cell strains, and in addition, the cytotoxic activity of the compound to drug-resistant strains is better than that of sensitive strains.

Drawings

FIG. 1 is an HR-ESI-MS spectrum of Compound 1;

FIG. 2 is an IR spectrum of Compound 1;

FIG. 3 is a 1H NMR spectrum of Compound 1;

FIG. 4 is a 13C NMR spectrum of Compound 1;

FIG. 5 is an HSQC spectrum of Compound 1;

FIG. 6 is an HMBC spectrum of compound 1;

FIG. 7 is the ROESY spectrum of Compound 1.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto. In the following examples, unless otherwise specified, methanol and ethanol are referred to as volume percentage concentrations.

Example 1

The compound of the invention is extracted from condensed aglaia odorata (Aglaia abbreviataC.y. Wu), the bark of the contracted order michelia was collected from sidsura longeparata in 10 months in 2014, identified by professor yuanwang jun, the university of south river, and the specimens were stored in the specimen museum of the college of pharmacy of south river university.

A pregnane compound having the formula:

。

the preparation method of the pregnane compound comprises the following steps:

1) taking 10 kg of pulverized stem bark dry powder of Sasa veitchii, extracting with 95% ethanol (3 × 35L) at room temperature for 3 times, mixing the 3 obtained extractive solutions, and concentrating under reduced pressure to obtain ethanol extract;

2) suspending the ethanol extract obtained in the step 1) in 1L of water, sequentially extracting with 5L of dichloromethane and 5L of ethyl acetate, and concentrating the extract to obtain 135g of dichloromethane extract;

3) dissolving dichloromethane extract (135 g) with 500mL of water, passing through D101 macroporous resin, washing with water to remove sugar and other water-soluble impurities, sequentially eluting with 60%, 70%, 80% and 90% ethanol, detecting and combining to obtain 5 components, and sequentially recording TLC as follows according to the elution sequence: fr.a, fr.b, fr.c, fr.d, fr.e;

Fr.B (15 g) was dissolved in 15mL of methanol, stirred on silica gel, gradient eluted through silica gel column with petroleum ether-ethyl acetate at volume ratios of 20:1, 15:1, 10:1, 5:1, 1:2, detected and combined to give 4 fractions, TLC in order of elution was assigned as: b1, b2, b3, b 4;

b2 was eluted through a reverse phase C18 column with a 50%, 70%, 90% methanol-water gradient to give three fractions, in order of elution, recorded sequentially as: b2a, b2b, and b2 c; b2b was purified by preparative HPLC (RP-C18) using 75% methanol-water as the mobile phase at a wavelength of 210nm and a flow rate of 10ml/min to give pregnane compound (56 mg, tR 10.5 min), which was designated as compound 1.

The relevant test data for compound 1 are given below (see in particular figures 1 to 7).

Colorless needle crystals (MeOH) having a melting point of 205 to 206 ℃ [ α ]]−38.9 (c= 0.075, CHCl₃); IR(KBr) ν_max: 3425, 2938, 1649, 1547, 1453, 1401, 611; ESI-MS: 401.2 [M+ Na]⁺;HR-ESI-MS: 401.2678 ([M+ Na]⁺, C₂₃H₃₈O₄Na; calcd 401.2662)。¹H NMR and¹³c NMR data are shown in table 1.

TABLE 1 preparation of Compound 1¹H (500 MHz) and¹³c (125 MHz) NMR data attribution

。

The structure of compound 1 was analyzed.

Colorless needle-like crystal (methanol) having a melting point of 205-206 ℃ and an optical rotation value of [ α ]]−38.9 (c0.075；CHCl₃). Is easily soluble in chloroform. After thin-layer chromatography, the color is developed with concentrated sulfuric acid-ethanol to be light red. Infrared spectrum shows hydroxyl (3425 cm)^-1) Ester group (1640 cm)^-1）。ESI-MS: 401.2 [M+ Na]⁺；HR-ESI-MS: 401.2678 ([M+ Na]⁺, C₂₃H₃₈O₄Na and calcd 401.2662) to obtain the molecular formula of C₂₃H₃₆O₄。

2 compounds can be found in the hydrogen spectrum of the compound 1Angular methyl unimodal signal (δ _H0.79 and 0.86), 3O-methine proton signals [ alpha ], [ alphaδ _H4.04 (t,J= 2.6Hz), 4.32 ( dt,J= 7.8, 4.1 Hz), 5.26 (dq,J=10.9, 6.0Hz)]And a methyl unimodal signal on acetyl group (δ _H2.00). According to the compound¹H and¹³c NMR data concluded that compound 1 is a trihydroxy substituted pregnane compound, and one of the hydroxyl groups was acetylated. From the HMBC correlation peaks, two hydroxyl substitutions were identified at positions 3 and 16, respectively, while the acetoxy substitution was identified at position 20, which was determined by an oxygen-containing proton at position 20 [5.26 (dq,J= 10.9, 6.0Hz)]determined in relation to HMBC at C-15, C-17 and acetyl.

The relative configuration of Compound 1 is determined by ROESY spectroscopy (see FIG. 7). The hydrogen proton at position 16 is correlated with the presence of ROESY in H-17, but not Me-18, indicating that the hydroxyl group at position 16 is β substitution.the configuration at position 20 in Compound 1 can be determined by Me-18 chemical shift asR*. Since Me-18 is not associated with H-20 by ROESY, the most stable configuration on the side chain is 20RHydroxy substitution, which is relatively close to Me-18 in this state, places the chemical shift of the methyl group in a low field region by a unshielding effect, while the configuration at position 20 of the compound isR*Thus far, compound 1 was structurally identified as (3 α,5 α,16 β, 20R)-3, 16-dihydroxypregn-20-acetyl, which is a novel compound searched by literature.

The activity experiment of the compound 1 for inhibiting tumor cells and drug-resistant tumor cells.

2.1 laboratory instruments, materials and animals.

24. A96-well cell culture plate (CoStar. Inc.), Biofuge centrifuges high speed low temperature centrifuge (ThermoInc.) CKX 41 microscope (OLYMPUS Inc.), HEPA Class 100 carbon dioxide incubator (Thermo Inc.) YXQ-LS-50 II model full automatic digital display steam sterilizer (Shanghai Boxun Co., Ltd.). Human leukemia cells (K562) and adriamycin-resistant human leukemia cells (K562/A02), human breast cancer cells (MCF-7) and adriamycin-resistant human breast cancer cells (MCF-7/ADM), human oral epidermoid carcinoma (KB) and vincristine-resistant human oral epidermoid carcinoma (KB/VCR), and human liver cancer cells (SMMC-7721) were purchased from the cell resource center of Shanghai Life sciences institute, Chinese academy of sciences. Dimethyl sulfoxide (DMSO) and thiazole blue (MTT) were purchased from Sigma inc., fetal bovine serum was purchased from PAA Laboratories GmbH, RPMI 1640 medium was purchased from Invitrogen inc., and trypsin was purchased from Amresco inc.

2.2 Experimental methods: MTT method for detecting cell proliferation inhibition experiment

1) Taking cells in exponential growth phase with good state, adding 0.25% trypsin digestive juice for digestion to make adherent cells shed, counting by 1 × 10⁴Preparing cell suspension per ml;

2) inoculating the cell suspension on a 96-well plate at 150 mu l/well, and placing the plate at the constant temperature of 37 ℃ and 5% CO₂Culturing in an incubator for 24 hours;

3) changing the liquid, adding test drugs with different concentrations, 100 mu l/hole (final concentration: 0.01,0.1,1, 10, 100μM), culturing for 72 hours by a conventional method;

4) discarding the supernatant, adding 100 mu l of MTT (methyl thiazolyl tetrazolium) with the concentration of 0.5 mg/ml into each hole, and incubating for 4 hours in an incubator;

5) discarding the supernatant, adding 100 mul of DMSO into each well, and oscillating the upper surface of the oscillating table for 15 minutes;

6) measuring by using a microplate reader, wherein the measuring wavelength is 570nm, and calculating the inhibition rate:

inhibition rate = [ A ]_{(negative control)}-A_(sample)]/ [A_{(negative control)}-A_(blank)]×100 。

2.3 results of the experiment.

The compound 1 is subjected to primary cytotoxicity experiments of seven human tumor cell lines, namely human leukemia cells (K562) and adriamycin-resistant human leukemia cells (K562/A02), human breast cancer cells (MCF-7) and adriamycin-resistant human breast cancer cells (MCF-7/ADM), human oral epidermoid carcinoma (KB) and vincristine-resistant human oral epidermoid carcinoma (KB/VCR) and human liver cancer cells (SMMC-7721). The results of the experiment are shown in table 2.

As can be seen from table 2: the compound 1 shows better cytotoxic activity to human tumor cell lines such as KB, KB/VCR, K562/A02 and the like, and the value is noted that the cytotoxic activity of the compound 1 to drug-resistant strains KB/VCR and K562/A02 is better than that of sensitive strains.

TABLE 2 cytotoxic Activity data for various compounds ^* (IC₅₀,μM)

^*Results are expressed as mean ± s.d. of each experiment (n = 3).

Claims (1)

1. The preparation method of the pregnane compound is characterized in that the structural formula of the compound is as follows:

the method comprises the following steps:

1) taking the crushed contracted sequence aglaia odorata stem bark dry powder, leaching with 85-95% ethanol at room temperature, and concentrating the leaching liquor under reduced pressure to obtain ethanol extract;

2) suspending the ethanol extract in water, sequentially extracting with dichloromethane and ethyl acetate, and concentrating the extract to obtain dichloromethane extract;

3) dissolving the dichloromethane extract with water, passing through D101 macroporous resin, washing with water, sequentially eluting with 60-90% ethanol, detecting, and mixing to obtain 5 components, and sequentially recording by TLC (thin layer chromatography) according to the elution order: fr.a, fr.b, fr.c, fr.d, fr.e;

dissolving Fr.B with methanol, stirring with silica gel, passing through silica gel column, and mixing with 20: 1-1: 2, performing gradient elution by petroleum ether-ethyl acetate, detecting and combining to obtain 4 components, and recording TLC as follows according to the elution sequence: b1, b2, b3, b 4;

b2 was eluted through reverse phase C18 column with a 50-90% methanol-water gradient to give three fractions, in order of elution, recorded as: b2a, b2b, and b2 c;

and purifying Fr.B2b by preparative HPLC with 75% methanol-water as mobile phase at wavelength of 210nm and flow rate of 10 ml/min.

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