CN101538247B - Preparation method of alkaloid compound - Google Patents

Abstract

The invention belongs to the technical field of medicaments and discloses an alkaloid compound and a preparation method thereof. The compound is obtained by steps of the strain culture and fermentation of streptomycete YL05A-1070(CGMCC No.2362) screened from soil on the slope of Duyang Mountain in Taihua County, Guanxi province, China, the filtration, extraction, adsorption elution and freeze drying of the fermentation liquor, and the like. The purity of the compound reaches more than 95 percent. The compound and pharmaceutically acceptable salts thereof including sulfate, hydrochloride and organic acid salt have the capacity of suppressing A549 cells, HGC-2 cells, HGC-27 cells and MCF-7 cells obviously. This capacity gives an indication that the compound has antitumor activity and can be used for developing antitumor drugs.

Description

A kind of preparation method of alkaloid compound

Technical field

The invention belongs to medical technical field, relate to alkaloid compound and preparation method thereof, be specifically related to a kind of compound and its pharmacy acceptable salt with anti-tumor activity; The present invention also relates to the microbial fermentation processes and the purposes of this compound on the manufacturing antitumor drug of this compound.

Background technology

Tumour is the frequently-occurring disease and the common disease of one type of serious threat human health, and M & M is in rising trend always, has become first deadly disease at present.The cancer mortality front three is liver cancer, cancer of the stomach, lung cancer, and China is one of liver cancer country occurred frequently in the world, and sickness rate is about 1/0,000.Liver cancer is in the situation of the effective medicine of a kind of famine so far, owing to liver cancer is lacked effective control method, patient's prognosis extreme difference.Except that but 5 annual survival rates of early stage patient with operation reach 80～90%, can not patient with operation, the mean survival time to paresthesia epilepsy has only 3～4 months, and average 5 annual survival rates of the whole liver cancer cases of developing country are merely about 5%.In addition, in, a thorny problem of advanced liver cancer clinical treatment is pain caused by cancer, after the patient finally brings into use morphine class analgesic; Because its serious drug dependence; Have only the increase that continues rapidly through dosage, just can reach the lenitive effect, and continuously heavy dose of analgesic uses; Increased the weight of the burden of liver again, thereby the grade malignancy of cancer is further aggravated.Therefore, antitumor type of new medicine of exploitation just seems very urgent and necessary.

Summary of the invention

One object of the present invention is to provide a kind of antitumor type of new medicine A that is used to treat tumor disease, and its molecular formula is C ₇H ₉FN ₂O ₄, molecular weight is 216, chemical structural formula is suc as formula shown in (I).Fusing point 141-143 ℃, the outward appearance powder that is white in color, it is hydrochloride, vitriol or organic salt at pharmacy acceptable salt.

The structure of the compd A that the present invention relates to adopts the method for nucleus magnetic resonance, spectroscopy such as infrared to confirm.

Another object of the present invention provides the fermentation of this antitumor antibiotics with bacterial classification and preparation method.

Through preliminary test, compd A is at the external growth that can effectively suppress kinds of tumor cells, its IC ₅₀In 0.9-56.7 μ mol/L scope, has notable antitumor activity.

Formula 1

Indole ketone compound A of the present invention separates to extract in the fermenting culture that obtains false light gray streptomycete Y05A-1070 (CGMCC No.:2362) the soil on Yangshan slope from Guangxi province Tai Hua county to obtain, and preparing method's concrete steps are following:

(1) the slant culture method of streptomycete Y05A-1070 (CGMCC No.:2362): slant medium is formed (1L): Zulkovsky starch 20g, NaCl 0.5g, KNO ₃1g, K ₂HPO ₄0.5g, FeSO ₄0.01g, MgSO ₄.7H ₂O0.5g, agar part 20g, pH 7.0.28-30 ℃, cultivated 7 days.

(2) the seed liquor cultural method of streptomycete Y05A-1070 (CGMCC No.:2362): seed culture medium is formed (1L): analysis for soybean powder 5～15g, yeast extract paste 3～7g, glucose 10～30g, starch 10～30g, K ₂HPO ₄0.1-1.0g, (NH ₄) ₂SO ₄0.1-3.0g, MgSO ₄.7H ₂O 0.1-0.5g, CaCO ₃0.1-5.0g pH 7.2.Triangle shakes bottled amount 20-60%, rotating speed 100-250 rev/min, 28-32 ℃, cultivates 20-48 hour.

(3) fermentation process of streptomycete Y05A-1070 (CGMCC No.:2362): fermention medium is formed (1L): peanut powder 10-25g, yeast extract paste 5-30g, glucose 0.5-30g, peptone 0.1-10g, (NH ₄) ₂SO ₄0.1-10g, KCl 0.1-3g, pH 7.0.Fermentor tank loading amount 20-60%, inoculum size 3-12%, 28-32 ℃, cultivated 5-7 days by rotating speed 100-250 rev/min.After the fermentation ends, tunning obtains mycelium and ferment filtrate two portions after filtering through plate-and-frame filter press.

(4) the mycelium part that obtains of step (3),

(a) with clear water wash 2-3 all over after, squeeze out most of moisture, airing under the normal temperature;

(b) mycelium is with the methanol extraction several, and the extract concentrating under reduced pressure obtains medicinal extract;

(c) medicinal extract disperses with zero(ppm) water, with ethyl acetate extraction for several times, separates and recrystallization purifying through 100-200 order silicagel column (moving phase is chloroform-ETHYLE ACETATE gradient elution), obtains compd A.

The external growth that can effectively suppress kinds of tumor cells of compound through the present invention's preparation has notable antitumor activity.

Used false light gray streptomycete (Streptomyces pseudogriseolus) Y05A-1070 of the present invention has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica); Preserving number is CGMCC No.:2362), preservation day is on January 25th, 2008.

Embodiment

The preparation of embodiment 1 compd A

(1) the slant culture method of streptomycete Y05A-1070 (CGMCC No.:2362): slant medium is formed (1L): Zulkovsky starch 20g, NaCl 0.5g, KNO ₃1g, K ₂HPO ₄0.5g, FeSO ₄0.01g, MgSO ₄.7H ₂O 0.5g, agar part 20g, pH 7.0.28-30 ℃, cultivated 7 days.

(2) the seed liquor cultural method of streptomycete Y05A-1070 (CGMCC No.:2362): seed culture medium is formed (1L): analysis for soybean powder 5～15g, yeast extract paste 3～7g, glucose 10～30g, starch 10～30g, K ₂HPO ₄0.1-1.0g, (NH ₄) ₂SO ₄0.1-3.0g, MgSO ₄.7H ₂O 0.1-0.5g, CaCO ₃0.1-5.0g pH 7.2.Triangle shakes bottled amount 20-60%, rotating speed 100-250 rev/min, 28-32 ℃, cultivates 20-48 hour.

(3) fermentation process of streptomycete Y05A-1070 (CGMCC No.:2362): fermention medium is formed (1L): peanut powder 10-25g, yeast extract paste 5-30g, glucose 0.5-30g, peptone 0.1-10g, (NH ₄) ₂SO ₄0.1-10g, KCl 0.1-3g, pH 7.0.Fermentor tank loading amount 20-60%, inoculum size 3-12%, 28-32 ℃, cultivated 5-7 days by rotating speed 100-250 rev/min.After the fermentation ends, tunning obtains mycelium and ferment filtrate two portions after filtering through plate-and-frame filter press

(4) the mycelium part that obtains of step (3),

(a) with clear water wash 2-3 all over after, squeeze out most of moisture, airing under the normal temperature;

(b) mycelium is with the methanol extraction several, and the extract concentrating under reduced pressure obtains medicinal extract;

(c) medicinal extract disperses with zero(ppm) water, with ethyl acetate extraction for several times, separates and recrystallization purifying through 100-200 order silicagel column (moving phase is chloroform one ETHYLE ACETATE gradient elution), obtains compd A.

The experimental data of compd A: outward appearance is a white powder, m.p.141-143.Be slightly soluble in benzene, chloroform, ether equal solvent, water-soluble, ethanol, methyl alcohol.MS(m/z)：216。IRυ/cm ^-1(KBr)：1761(υ _C＝O)，1724(υ _C＝O)，1657(υ _C＝O)。 ¹H-NMR(CDCl ₃)δ：1.31(3H，t，J＝7.2Hz，-CH ₃)，4.26(2H，q，J＝7.2HZ，-COOCH ₂-)，4.68(2H，s，-CH ₂N＜C-9)，7.32(1H，d，C-H，J＝3.0Hz)，9.93(1H，br?s，H-N＜)。

The anti-tumor activity of embodiment 2 MTT reduction method detection compound A

1. material:

1.1 four Cuo salt (MTT): with the phosphate buffered saline buffer (PBS) of 0.01M dissolving MTT (3-(4,5-dimethythiaZol z-yl) 2,5-diphenyltetrazolium bromide, SIGMA) final concentration 5mg/mL, filtration sterilization, 4 ℃ keep in Dark Place after the packing.

1.2 the preparation of target cell (is example with the A549 cell): from liquid nitrogen container, take out the frozen pipe of human lung carcinoma cell line A549 cell, insert rapidly in 37 ℃ of water-baths, do not stop to shake and make it to dissolve rapidly, aseptic technique moves in the centrifuge tube; To 10mL, the centrifugal 5s of 1000rpm abandons supernatant to add full nutrient solution (RPMI1640 (Gibcol BRL company) that contains 10% calf serum); Repeat above operation once; Move in the culturing bottle 5%CO after making the cell mixing with the piping and druming of full nutrient solution ₂, 37 ℃ of cultivations; The observation of cell growing state is in time changed nutrient solution, divides bottle.The cell in vegetative period of taking the logarithm, trysinization, adjustment cell count to 1 * 10 ⁵/ mL; Cell suspension is joined in 96 orifice plates, and each hole adds A549 cell 100 μ L (1 * 10 ⁵/ mL), 5%CO ₂, cultivate 8hr for 37 ℃.Add different concns B6-9 (using full nutrient solution preparation) solution 100 μ L, contrast adds full nutrient solution 100 μ L, continues to cultivate 48hr.Every hole adds each 10 μ L of MTT solution, continues to cultivate 4hr.Remove nutrient solution, every hole adds DMSO100 μ L, and the 5-10min that vibrates gently makes the particle dissolving.Use the enzyme linked immunological appearance to measure every hole OD value under the 570nm.Calculate tumor cell destruction and inhibiting rate,, try to achieve IC with the logarithm mapping of inhibiting rate to drug level ₅₀Lung cancer cell line A549, hepatoma cell strain HepG-2, stomach cancer cell line HGC-27, the TP of breast cancer cell strain MCF-7 is the same.

Tumor cell destruction %=[the average OD value that (the average OD value of average OD value-dosing group mensuration that control group is measured)/control group is measured] * 100%

1.3 experimental result

Experimental result shows that compd A all can effectively suppress lung cancer cell line A549, hepatoma cell strain HepG-2, stomach cancer cell line HGC-27, the growth of breast cancer cell strain MCF-7.Compd A is to the IC of these cancer cells ₅₀Value (μ mol/L) is seen table 1.

The cell in vitro poison test-results (IC of table 1 B6-9 ₅₀μ mol/L)

Cell strain	Compd A
		Lung cancer cell line A549	?0.9
Hepatoma cell strain HepG-2	?15.3
		Stomach cancer cell line HGC-27	?31.0
Breast cancer cell strain MCF-7	?56.7

Claims (1)

1. the preparation method of an alkaloid compound A; Described compd A structural formula is following; It is characterized in that: this method comprises that cultivation can produce the culture condition of false light gray streptomycete (Streptomyces pseudogriseolus) the Y05A-1070CGMCC No.2362 of said compd A; And from fermented liquid, reclaim resulting compd A, its concrete steps are following:

(1) the slant culture method of false light gray streptomycete Y05A-1070CGMCC No.2362: the 1L slant medium is formed: Zulkovsky starch 20g, NaCl 0.5g, KNO ₃1g, K ₂HPO ₄0.5g, FeSO ₄0.01g, MgSO ₄.7H ₂0 0.5g, agar powder 20g, pH7.0, cultivated 7 days by 28-30 ℃;

(2) the seed liquor cultural method of false light gray streptomycete Y05A-1070 CGMCC No.2362: the 1L seed culture medium is formed: analysis for soybean powder 5～15g, yeast extract paste 3～7g, glucose 10～30g, starch 10～30g, K ₂HPO ₄0.1-1.0g, (NH ₄) ₂SO ₄0.1-3.0g, MgSO ₄.7 H ₂O 0.1-0.5g, CaCO ₃0.1-5.0g pH7.2, triangle shake bottled amount 20-60%, rotating speed 100-250 rev/min, 28-32 ℃, cultivate 20-48 hour;

(3) fermentation process of false light gray streptomycete Y05A-1070 CGMCC No.2362: the 1L fermention medium is formed: peanut powder 10-25g, yeast extract paste 5-30g, glucose 0.5-30g, peptone 0.1-10g, (NH ₄) ₂SO ₄0.1-10g, KCl 0.1-3g, pH7.0, fermentor tank loading amount 20-60%; Inoculum size 3-12%, 28-32 ℃, cultivated 5-7 days by rotating speed 100-250 rev/min; After the fermentation ends, tunning obtains mycelium and ferment filtrate two portions after filtering through plate-and-frame filter press;

(4) the mycelium part that obtains of step (3),

(a) with clear water wash 2-3 all over after, squeeze out most of moisture, dry under the normal temperature;

(b) mycelium is with the methanol extraction several, and the extract concentrating under reduced pressure obtains medicinal extract;

(c) medicinal extract disperses with zero(ppm) water, with ethyl acetate extraction for several times, through 100-200 order silicagel column, is the eluent gradient wash-out with chloroform one ETHYLE ACETATE, separates and recrystallization purifying, obtains compd A