CN1124778A

CN1124778A - Antitumor antibiotic-Yunnan-mycin

Info

Publication number: CN1124778A
Application number: CN 95109425
Authority: CN
Inventors: 戚长菁; 甄永苏; 陈文君; 胡继兰; 薛玉川; 张春颖
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 1995-08-03
Filing date: 1995-08-03
Publication date: 1996-06-19
Anticipated expiration: 2015-08-03
Also published as: CN1050846C

Abstract

The cytosine nucleoside dipeptide, a new antibiotics named "Yunnanmycin" for inhibiting cancer, is prepared through such steps as ion exchange resin extracting from fermented liquid of Yunnan streptomycete strain "2321", chromatographic analysis for separation and purification, hydrolysis using concentrated hydrochloric acid, ion exchange resin extraction and laminar eduction, and features 87% inhibiting rate to cancer, slight poison to bone marrow, but slight inhibition to hemopoietic system.

Description

A kind of new antitumor antibiotics-Yunnan mycin

The invention belongs to new two peptide antibiotics of the cytidine(C with antitumor action.

Surplus the microbiotic of having found so far that derives from microorganism nearly ten thousand more than the kind, wherein many agricultural chemicals etc. have obtained widespread use as medicine, but good effect, antitumor antibiotics that toxicity is low are still very few for number.Therefore the new variety of further seeking the ideal antitumor action are still urgent day by day.

The present invention is from the process of Yunnan actinomycetes screening antitumor antibiotics, be separated to active substance in the nutrient solution by streptomycete bacterial strain 2321 with antitumor and anti-microbial effect, separation has obtained having the new antibiotic 2321C of antitumor action from the acid hydrolysis products of this material, names to be Yunnan mycin (Yunnanmycin).(the generation bacterium of this Yunnan mycin is deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", BeiJing ZhongGuanCun.100050, preservation date nineteen ninety-five, July 26, numbering 0234).

Through spectrum and MASS SPECTRAL DATA ANALYSIS, determine that Yunnan mycin has suc as formula structural formula shown in (1) R=H, this structural formula can be with all of Yunnan mycin and hitherto known cytidine(C dipeptides structure medical and agricultural antibiotic such as gougerotin (Gougerotin), qingfengmeisu (Qingfengmycin) and Ningnanmycin (Ningnanmycin) equiphase zone other, so the affirmation Yunnan mycin is a new microbiotic.

In the formula: R=H (Yunnan mycin),

R can be K, Na, NH ₄And CH ₃, C ₂H ₅As follows etc. content of the present invention and main points: one. the generation bacterium of Yunnan mycin, fermentation, anti-microbial activity and manufacture method 1. produce the bacterium source: Guan Ping wilderness area, Yunnan soil morphological characteristics: fibrillae of spores volution.Spore is polytypism: ellipse, garden shape, melon seeds shape, spore

The surface spinosity.

Cultural characteristic: see Table 1

Table 1

Culture medium growth produce the good white of mycelia substrate mycelium soluble pigment Isp.1 culture medium without light yellow complexion Isp.2 culture medium well white to brown ash colourless without Isp.3 culture medium appropriateness white to brown ash colourless to little ash without the Isp.4 culture medium well white to brown ash colourless to little ash without the Isp.5 culture medium well white to brown ash colourless to the good white of the little yellow Isp.6 culture medium of butterfat colourless without the good light gray moccasin of H2SIsp.7 culture medium look dirty yellow glucose asparagine agar appropriateness light gray colourless without the poor brown ash of sucrose Cha Shi agar colourless without the pale yellow glycerine Cha Shi of the good brown pale yellow greyness of glucose Cha Shi agar agar well in vain to the appropriate greyish white extremely grey colourless or butterfat of No. 1 agar of the yellow Gao Shi of the pale yellow brown dirt of light gray without or yellowish

Do not form melanochrome, do not produce H ₂S.

2. fermentation

The spore cultivation of going down to posterity: the Isp.5 substratum, cultivated in 10 days for 28 ℃.

Seed culture:

Medium component: glucose 1%, starch 1.5%, meat extract 0.5%, peptone (fish) 0.5%, NaCl0.3%, analysis for soybean powder 1%, no salt solution, PH7.0.28 ℃ of shaking culture 48 hours.

Fermentation culture:

Medium component: glucose 2%, starch 1%, meat extract 0.5%, yeast powder 0.5%, peptone (fish) 0.5%, NaCl0.3%, analysis for soybean powder 1%, no salt solution, PH7.028 ℃ of shaking culture 96 hours.Active calibrating: test organism: intestinal bacteria B substratum: meat extract 0.3%, peptone 1%, agar 1.6%, no salt solution, PH7.0-7.2.Calibration method: paper dish method.3. the anti-microbial activity Yunnan mycin only has more weak activity to intestinal bacteria B.Paper dish method detected result sees Table 2

Table 2

Test organisms minimum inhibitory concentration (ug/ml) Bacillus subtilis6633＞500Staphylococcus aureus 229p＞500Sarcina lutea＞500Escherichia coia B 500Escherichia coli 0111＞500Proteus vulgaris ox19＞500Pseueomonas aeruginosa11＞500Klebsiella pneumoniae＞500Candida albicans＞500Penecillium avallaneum 5404＞500

4. manufacture method

The filtrate of the fermentation culture of streptomycete bacterial strain 2321, with the absorption of 2% (grams per milliliter) gac dress post, 50% acetone wash-out, concentrating under reduced pressure is removed the aqueous solution behind the acetone, transfers PH3, by strong acid cation resin (NH ₄ ⁺Type), exchanges, with 1N ammoniacal liquor wash-out, elutriant shows that to intestinal bacteria active part removes deammoniation, lyophilize, cold dry product neutral alumina post adsorption chromatography, 75% methanol-eluted fractions, go the lyophilize of methyl alcohol rear solution, use sephadex G-10 column chromatography after the drying again, the active part lyophilize, cold dry product adds an amount of concentrated hydrochloric acid, hydrolysis is 60 hours under room temperature, removes excessive hydrogen chloride gas, and the aqueous solution is by strong acid cation resin (NH ₄ ⁺Type). exchange, 0.1N ammoniacal liquor wash-out, lyophilize, dry product is expanded with sephadex G-10 column chromatography for separation, water, and active part is lyophilize again, and dry product (is made silica gel G F by oneself with the preparation of silica gel thin sheet chromatographic separation ₂₅₄Plate, 20 * 20cm), 60% methyl alcohol launches, and the cold dry product purity of isolating active part reaches more than 95%, claims Yunnan mycin.Two. the structure of Yunnan mycin and physicochemical data:

Yunnan mycin has structure as the formula (1), its UV, and IR, FAB-MS, molecular formula, ¹HNMR, ¹³CNMR reaches ¹³The C-DEPT data are as follows, and (1) formula is to reach according to these data ¹H- ¹Relevant spectrum of H (COSY) and anti-phase detection are long-range ¹H- ¹³The analysis of the relevant spectrum of C heteronuclear multikey (HMBC) is determined.

UV: 258nm λ _Mnax ^{0.1N NaOH}258nm λ _Max ^0.1NHCl275nm (seeing accompanying drawing 1) IR (KBr): 3200-3400,1640-1660,1485,1275,1060-1080,940,840,780cm ^-1(seeing accompanying drawing 2)

FAB-MS(m/z)：445(M+H) ^-

443 (M-H) ^-(seeing accompanying drawing 3)

Molecular formula: C ₁₆H ₂₄K ₅O ₉

¹H-NMR (400MHz in D ₂O, 30 ℃), see Table 3 and accompanying drawing 4

Table 3

¹H-NMR(400?MHz?in?D ₂O，30℃)

ppm multi assignment

7.83 1H，d，8Hz H-6

5.12 1H，d，8Hz H-5

5.70 1H，m H-1′

4.56 1H，t，5Hz Ser-α

4.05 1H，m H-4′

4.02 1H，d，11Hz H-5′

3.98 1H，d，16Hz Gly-α

3.93 1H，d，16Hz Gly-α

3.87 2H，m Ser-β

3.81 2H，m H-2′,3′

2.77 3H, s NCH ₃ ¹³C-NMR (100MHz in D ₂O, 30 ℃), see accompanying drawing 5. ¹³C-DEPT (100MHz in D ₂O, 30 ℃), see accompanying drawing 6. ¹H- ¹H COSY sees accompanying drawing 7. ¹H- ¹³C-HMBC sees Table 4 and accompanying drawing 8.

Table 4 ¹³C-NMR (100MHz in D ₂O；30℃)ppm muiti Correlations observed in HMBC (δH) assignment176.83 s 4.02 (H-5′) C-6′174.13 s 4.56 (Ser-α) 3.87 (Ser-β) Ser-CO169.88 s 4.56 (Ser-α) 3.98 (Gly-α) 3.93 (Ser-α) Gly-CO168.89 s 7.83 (H-6) 6.12 (H-s) C-4160.69 s 7.83 (H-6) 5.70 (H-1′) C-2144.78 d 6.12 (H-s) 5.70 (H-1′) C-699.92 d 7.83 (H-6) C-585.92 d 7.83 (H-6) 3.81 (H-2′or3′) C-1′80.83 d 4,05 (H-4′) C-5′76.65 d 5.70 (H-1′) 4.05 (H-4′) 3.81 (H-2′or3′) C-2′or3′74.66 d 5.70 (H-1′) 3.81 (H-2′or3′) C-2′or3′64.15 t 4.56 (Ser-α) Ser-β58.47 d 3.87 (Ser-β) Ser-α56.35 d 4.02 (H-s′) 3.81 (H-2′or3′) C-4′52.63 t 2.77 (NCH) Gly-α35.84 g 3.98 (Gly-α) 3.93 (Gly-α) NNH ₃

Three. the antitumor action of Yunnan mycin and toxicity

1. the antitumor action of Yunnan mycin

(1). anti tumor activity in vitro: KB clone (deriving from people's oral squamous cell carcinomas) is used in experiment, is containing 5%CO with the RPMI1640 nutrient solution that contains 10% calf serum ₂37 ℃ of incubators in cultivate to adopt the clone to generate to measure (Clonogenic assay) and judge the lethal effect of medicine tumour cell.The KB cell of taking the logarithm vegetative period adds 96 ^#In the culture plate, every ^#50 cell/0.2ml cultivate and add medicine after 24 hours, after 6 days under inverted microscope counting cells colony (containing 30 above persons of cell), calculate the colony inhibiting rate, the result proves that Yunnan mycin has lethal effect to the KB cell, and its action intensity is concentration dependent; Yunnan mycin is to the IC of KB cell ₅₀(half colony inhibition concentration) is 3 μ g/ml (seeing accompanying drawing 9).

(2). intravital test is to the curative effect of tumour: the Kunming mouse of 18-20g body weight is used in experiment, gets murine sarcoma 180 ascites and adds physiological saline (1: 3) dilution, and it is subcutaneous to be inoculated in the mouse armpit, every mouse 0.2ml.Knurl oral after 24 hours (po) administration in the inoculation, 2 times (respectively administration in 1,6 day) or 3 times (respectively administration in 1,4,7 day), first administration was put to death animal after 10 days, got tumour and weighed, and calculated tumor control rate.The result shows that Yunnan mycin has significant curative effect to murine sarcoma 180 (solid-type), and (135mg/kg, po x2), reach 87% (P＜0.01) to inhibition rate of tumor growth to use tolerable dose.See Table 5

Table 5. Yunnan mycin begins/finishes to change (g) mean ± SD (%) 1 contrast 10/10+10.1 3.80 ± 1.08 Yunnan mycin 60 3 10/10+5.7 2.03 ± 0.48 47 to heavy (g) tumour inhibiting rate lot number group (mg/kg) number of times of the restraining effect test dose administration number of animals body weight knurl of murine sarcoma 180 growths ^*

90 3 10/10 +4.5 1.74±0.31 54 ^**

120 3 9/10+4.3 0.84 ± 0.24 78 ^*II contrasts 10/10+7.3 2.94 ± 0.83 Yunnan mycin 105 2 10/10+2.0 0.71 ± 0.16 76 ^*

120 2 10/10 -0.3 0.55±0.24 81 ^**

135 2 10/10 +1.8 0.38±0.11 87 ^**

Mouse armpit subcutaneous vaccination tumour, oral (po) administration.

**P＜0.01

(3). the antitumor action of Yunnan mycin and 5-fluor-uracil and toxicity compare: toxicity dose (1/3LD such as employing ₅₀) compare.With two .1. (2) In vivo assay Cells, at mouse armpit subcutaneous vaccination sarcoma 180 ascites 0.2ml, begin oral administration (po) after 24 hours, totally 2 times (respectively administration in 1,6 day).First administration was put to death animal after 10 days, got tumour and weighed, and calculated tumor control rate; Take out a side femur simultaneously, check the bone marrow nucleated cell number, calculate the medullary cell inhibiting rate.Experimental result, the tumor-inhibiting action of Yunnan mycin is stronger than 5-fluor-uracil, and bone marrow toxicity is lighter, uses to be equivalent to 1/3LD ₅₀Dosage, the inhibiting rate of Yunnan mycin (120mg/kg) and 5-fluor-uracil (80mg/kg) is respectively 81% (P＜0.01) and 66% (P＜0.01), both relatively, P＜0.01; Yunnan mycin and 5-fluor-uracil are respectively 22% (P＜0.05) and 83% (P＜0.01) to the inhibiting rate of bone marrow nucleated cell, and both compare, and P＜0.01 sees Table 6

Table 6 Yunnan mycin and 5-fluor-uracil are to the restraining effect of murine sarcoma 180 and to the toxicity of marrow relatively

Dosage number of animals tumor weight (g) bone marrow nucleated cell number (10 ⁷/ femur)

(mg/kg) beginning/end X ± SD inhibiting rate (%) X ± SD inhibiting rate (%) contrast 10,/10 2.94 ± 0.83 1.41 ± 0.27 Yunnan mycin 120 10,/10 0.55 ± 0.24 81 ^*1.10 ± 0.29 22 ^*5-fluor-uracil 80 10,/10 0.99 ± 0.33 66 ^*0.24 ± 0.18 83 ^*Mouse armpit subcutaneous vaccination tumour is irritated stomach (po) administration; Use is equivalent to 1/3LD ₅₀Dosage, totally 2 times.

* P＜0.05, * * P＜0.01, with control group relatively.

The above results shows that use waits toxicity dose (1/3LD ₅₀, po) comparing, Yunnan mycin is stronger than 5-fluor-uracil to the restraining effect of murine sarcoma 180 (solid-type), and lighter to bone marrow toxicity.

2. the toxicity of Yunnan mycin

(1) chmice acute toxicity: the LD of oral administration (po) ₅₀Be 360mg/kg.

The LD of intraperitoneal administration (1p) ₅₀Be 65mg/kg.

(2) use therapeutic dose (120mg, po, * 2, inhibiting rate 81%), hemopoietic system is had slight restraining effect, (medullary cell inhibiting rate 22%); Histopathologic examination, the various organs of treatment animal comprise that the heart, lung, tracheae, liver, food official, stomach, small intestine, spleen, kidney, suprarenal gland etc. there is no the toxicity pathology.

Four. manufacture method embodiment:

Get fermented liquid 10 liters, add diatomite filtration, discard mycelia, (water wets and adorns post filtrate, and post 5.5 * 55cm) adsorbs by 200 gram activated carbon columns, 50% acetone wash-out is collected portion for per 250 milliliters, and 1-4 part active part merges, remove acetone, about 500 milliliters of the aqueous solution is transferred PH3.0, by 50 milliliter of 001 * 7 ammonium type cation exchange resin column is housed, after the less water flushing, use 1N ammoniacal liquor wash-out, collect a for per 100 milliliters, 1-5 part active part merges, and removes ammonia, aqueous solution lyophilize, whole cold dry products are by 100 milliliters of post decolourings of neutral alumina (the wet dress of methyl alcohol), 75% methyl alcohol exhibition layer is received portion for per 100 milliliters, and 1-10 part active part merges, after removing methyl alcohol, lyophilize gets 2.3 gram dry products, gets a gram dry product through sephadex G-10 column chromatography for separation, (post: 1.2 * 100cm), washing, every 5ml milliliter is collected a, and 7-15 part active part merges lyophilize and gets cold dry product 0.56 gram, (HPLC detects purity 95%, C ₁₈Waters post 4.6 * 250mm, moving phase is 75% methyl alcohol, flow velocity 1 ml/min detects UV268nm, retention time 8.3 minutes).Get cold dry product 1 gram of this 95% purity, add 40 milliliters of concentrated hydrochloric acids, the room temperature hydrolysis is after 60 hours, removes the residue behind the hydrogen chloride gas, adds 200 milliliters in water, by strong acid cation resin Dowex * 50w * 4 (NH ₄ ⁺Type) 10 milliliter, exchange, after the washing,, collect a for per 3 milliliters with 0.1N ammoniacal liquor wash-out, 11-20 part active part merges, deammoniation gas postlyophilization obtains 550 milligrams of cold dry products, get 150 milligrams of these cold dry products by sephadex G-10 post (1.2 * 100cm), the washing, the lyophilize of drawing together property part, cold dry product (is made silica gel G F by oneself with the silica gel thin sheet chromatographic separation ₂₅₄Plate, 20 * 20cm), 60% methyl alcohol exhibition layer separates 60 milligrams of cold dry products that obtain active part, and (HPLC detects purity 96%, C ₁₆Waters post 4.6 * 250mm, moving phase is 75% methyl alcohol, flow velocity 1 ml/min detects UV268nm, retention time 2.9 minutes), claim Yunnan mycin.

Description of drawings:

Fig. 1 is the UV of Yunnan mycin;

Fig. 2 is the IR of Yunnan mycin;

Fig. 3 is the FAB-MS of Yunnan mycin;

Fig. 4 is a Yunnan mycin ¹H-NMR;

Fig. 5 is a Yunnan mycin ¹³C-NMR;

Fig. 6 is a Yunnan mycin ¹³C-DEPT;

Fig. 7 is a Yunnan mycin ¹H- ¹HCOSY;

Fig. 8 is a Yunnan mycin ¹H- ¹³CHMBC

Fig. 9 is the lethal effect of Yunnan mycin to KB born of the same parents, and the clone generates and measures drug treating time 6 days.

Claims

1. the antitumor antibiotics of a following structural formula

In the structural formula: R=H (Yunnan mycin)

R can be K, Na, NH ₄And CH ₃, C ₂H ₅Deng

2. preparation method of the antitumor antibiotics of structural formula according to claim 1, it is characterized in that streptomycete bacterial strain 2321 on substratum compositions such as glucose, starch, shaking culture fermentation under the neutrallty condition, strongly acidic cation-exchange extracts, sephadex chromatography separates, concentrated hydrochloric acid hydrolysis, purifying obtains finished product.

3. antitumor action of the compound of structural formula according to claim 1 is characterized in that tumour cell (KB cell) adopting the clone to generate and measuring in vitro culture, judges the lethal effect of medicine to tumour cell; Get murine sarcoma 180 cell inoculations and grown, form solid tumor, use the Yunnan mycin treatment,, calculate tumor control rate by taking by weighing the knurl body weight to trying the subcutaneous breeding of mouse armpit.

4. according to the preparation method of the described antitumor antibiotics of claim 2, it is characterized in that streptomycete bacterial strain 2321 fibrillaes of spores twist, its surperficial spinosity does not form melanochrome, does not produce hydrogen sulfide, and it is on the Isp5 substratum that spore goes down to posterity, and cultivates 10 days for 28 ℃; The seed culture medium composition is a glucose 1%, meat extract 0.5%, fish peptone 0.5%, NaCl0.3%, analysis for soybean powder 1%, no salt solution, PH7.0,28 ℃ of shaking culture 48 hours; Fermention medium composition glucose 2%, starch 1%, meat extract 0.5%, yeast 0.5%, peptone 0.5%, NaCl0.3%, analysis for soybean powder 1%, no salt solution, PH7.0,28 ℃ of shaking culture 96 hours.

5. according to the preparation method of the described antitumor antibiotics of claim 2, it is characterized in that streptomycete 2321 fermented liquids are adsorbed through charcoal, the aqueous acetone wash-out, the strong acid type cationic resin exchange, the aluminum oxide decolouring, sephadex chromatography separates, concentrated hydrochloric acid hydrolysis, by the strongly acidic cation-exchange exchange, gel column separates, silica gel G F again ₂₅₄Thin-layer chromatography, separation and purification, lyophilize gets product.

6. according to the antitumor action of the described compound of claim 3, it is characterized in that Yunnan mycin is concentration dependent to the lethal effect of KB cell, to the IC of KB cell ₅₀Be 3 μ g/ml; In vivo test has significant curative effect to murine sarcoma 180 (solid-type), uses tolerable dose (135mg/kg, po, * 2) that inhibition rate of tumor growth is reached 87% (P＜0.01).Toxicity doses such as employing compare, and the tumor-inhibiting action of Yunnan mycin is strong than 5-fluor-uracil, and bone marrow toxicity is lighter.