SU1165680A1 - D neplanocyn of its derivatives as intermediate products in synthesis of a neplanocyn - Google Patents

Abstract

NEW MATERIAL:Neplanocin D of formula I (R1 is OH, SH, SCH3, or Cl; R2 is H or protecting group), and its derivative. EXAMPLE:Neplanocin D of formula II. USE:An intermediate for the synthesis of anti-cancer drugs, capable of synthesizing anti-cancer drug Neplanocin A of formula III via the derivative. PROCESS:Ampullariella sp.-A11079 strain (FERM-P No.4494) is incubated in a liquid medium aerobically for 2-4 days, and Neplanocin D is adsorbed on active carbon from the culture filtrate, and eluted by hydrous methanol. The eluate is then separated by and adsorbent and ion exchange resin, and purified by recrystallization to give Neplanocin D. A protecting group and mercapt group are introduced, and the reaction product is alkylated to afford a novel derivative.

Description

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o The invention relates to new non-planinoc D and its derivatives in general formulas rt., x CongC RO OR where, SH, ZSNz; or which are used as intermediate products in the synthesis of neplanocine A. Neplanocine A has a suppressive effect on pathogenic microorganisms of plants, and also has anti tumor activity. It is obtained by inoculating a strain of the Ampullariella A.11079 FERM-P No. 4494 strain in a nutrient medium. The isolation of the antibiotic is carried out by absorption by adsorbents, followed by elution with aqueous organic solutions lj. The disadvantages of the method of obtaining n planocin A are its low yield, the laboriousness of the process, the cumbersomeness of the instrumentation of production. The aim of the invention is the chemical synthesis of neplanocine A from neplancine D and its derivatives, which is obtained as a by-product in the microbiological production of neplanocine A. Neplanocin D is obtained by incubating the strain of the species Ampullariel la A11079 FERM-P No. 4494 in a nutrient medium. Microorganisms can be grown in a variety of ways in an artificial or natural environment, liquid or solid culture. In industrial production, a liquid medium is preferred. Digestible carbon and nitrogen sources, inorganic salts and other substances can be used to make up the medium. This medium is used for microorganisms producing antibiotics-nonplanocine. Glucose, sucrose, glycerin, soluble starch and the like can be used as the carbon source. Pentone, soy bean powder, wheat extract, meat extract, rice bran, casein hydrolyzate, nitrate, ammonium salt, etc. can be used as sources of assimilable nitrogen. Inorganic salts, such as sodium chloride, calcium phosphates, 1 agni, iron III or manganese, may also be used. A defoaming agent such as silicone or soybean oil may be added. In the case of a liquid culture, it is preferable to use a submerged aerated culture. In this case, the cultivation is carried out at the optimum temperature for the microorganisms, preferably at 25-30 C. The duration of the cultivation depends on the applied conditions and is usually 2-4 days. The pH of the medium during cultivation is regulated from neutral to slightly acidic. This cultural environment contains antibiotics-nonplanocines. The isolation of antibiotics-nonplanocins can be accomplished by standard allocation or separation (by methods used to isolate the products of microbial metabolism). Since nonplanocins are weak bases, they can be absorbed by absorption by suitable adsorbents, followed by elution. By using a suitable solvent, activated carbon, cation exchange resins, activated alumina or silica gel can be used as adsorbents. Eluting solvents are selected depending on. from the used adsorbent, for example. it can be water-miscible organic solvents, such as aqueous methanol, aqueous acetone or aqueous dioxane. or solutions of salts, or acids, alkaline solutions. In addition, non-planocins can be isolated and purified as substances of a slightly alkaline nature. For example, antibiotics are absorbed by a cation-exchange resin, such as Amberlite 1PC-50 (t. Trademark product of 3 Rum and Haas, USA) or Douvec 50 (Dow Chemical trademark, USA) and eluted with acceptable acid, alkaline or saline . Preferably, a combination of an adsorbent and an ion exchange resin is used for the separation, elution and purification of antibiotics. For example, the filtrate of the loading culture in Amberlite IR-120 (trade mark) cation-exchange resin to absorb the antibiotic is eluted with an alkaline solution, for example, 3.7 K with aqueous ammonia, to obtain an active fraction, and after adjusting the pH to neutral or weakly alkaline medium antibiotics adsorb with activated charcoal, followed by elution with 70% methanol. The eluent is adsorbed with Amberlite IRA-410 anion-exchange resin (trademark) and again eluted with water to collect the active fractions. The combined fractions are concentrated to give a crude product, which is then purified on silica gel. Further purification is carried out by recrystallization. The purity of the compound can be determined by melting point or one spot on paper chromatography, 804 thin-layer chromatography and paper electrophoresis at 2630 A of UV light. For this, the three hydroxyl groups in the cyclopentene ring of neplancin D are protected by -interaction with benzoyl chloride. The hydroxyl group in the adenine structure of nonplanocin D is replaced by a marcapto group, which is reacted with pentahydric phosphorus, then the suction group for the hydroxyl group j is removed. resulting in a derivative of neplanocine D (GI). Methylation with methyl iodide gives a derivative of neplanocin D (IV), which is treated with methanolic ammonia, as a result of which neplanocine A is obtained. In addition, three hydroxyl groups on the cyclopentene ring of neplanocin D are protected by acetylation. This is achieved by interaction with acetic anhydride. Then, the said compound is converted to a derivative of neplancin D (VI) by reaction with thionyl chloride. The derivative of neplanocin D (VI) can be converted to neplanocin A via the derivative of neplanocine D by treatment with methanolic ammonia. The described reactions can be illustrated by the following scheme:

benufpi (lorid

HE

th

BzOCH,

But he

Neplanocine J /

5H

rt:

BzOCHoxv

OVG OBz

P

Obzobz

J

I

ys

N

BEARING

I

OH he

sh

ZSNz

 Methanolic SNO I H 7 Amnian

NOSE,

he he

Proprivodnoe neplanotsina V (III)

Neplanocin A

Ieplanocin} if

acetic anhydride

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Neplanocin A

Example 1. A 500 ml flask was charged with 1.00 ml of an aqueous medium (pH 6.5) containing 2% glucose, 2% starch, 1% yeast extract, 1% casein hydrolyzate and 0.2% calcium carbonate. The contents of the flask were sterilized for 15 minutes at 120 ° G. One loop with a crop of Ampullariel-la A 11079 FERM-P No. 4494 was sown in two flasks of this medium and shaken at. After four days, the culture medium is transferred to a 20 liter of the same as indicated sterilized medium, which is in a 30 liter fermentation tank, and grown with 300 l / min with stirring and aeration at 20 l / min.

For 48 hours

The medium grown in this way is transferred to 200 liters of aqueous medium (pH 6.5 containing 4% glucose, 1% soy bean powder, 0.4% meat ex, tract, 0.4% peptone, 0.1% yeast extract, 0.25% sodium chloride and 0.1% calcium carbonate, and grown at 30 ° C with stirring at 180 rpm and aeration

AEPA

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VI

Production6; 0doe neplanocina}

rJO l / min for 40 hours. The resulting broth (200 l) is filtered, and the mycelium is washed with water. The filtrate and wash water are combined to give about 140 liters of clear filtrate.

To the filtrate is added 3.6 kg of activated carbon powder. After stirring for 40 minutes, the activated carbon is filtered and thoroughly washed with water. Elution is carried out with a 70% methanol solution. 40 l of the eluate is dried under vacuum to obtain 80 g of crude powder containing neplanocine D.

In a column filled with 6 liters of silica gel with a mixture of solvents C-propanol: a concentrated aqueous ammonia: water solution (10: 1: 1), 80 g of a crude powder is introduced and then eluted with the same mixture of solvents. Fractionate every 600 g of eluate. Neplanocine D is found in Fractions No., No. 20-37. These active fractions are collected, concentrated in vacuo to 30 ml and maintained at cooling. The precipitate is separated by filtration, resulting in 7 A60 mg of crude crystalline neplanocin D, 460 g of crude crystals are dissolved in 3 liters of hot water and injected to Sephadex G-15 (1850 ml., Filled with water), then eluted with water. Each 100 ml of eluate is fractionated, fractions of eluate No., No.-15-16 (200 ml) are collected and concentrated under reduced pressure to 20 ml. The resulting 11 concentrate is held at room temperature. 314 mg of leached non-planocin D in the form of white crystals are obtained. Neplanocin D has the following physico-chemical characteristics: elemental analysis,%: C 49.70; H 4.64; N 21.20; a pier mass (determined from mass spectral analysis): 264; m.p. 236-237 ° C; specific rotation 145 ° (, 6% HjO). Ultraviolet absorption spectrum. In water: L, 2510 A E ,,. . ,, In acidic water: L 2510 A E, 53. In alkaline water: 2360 A. Infrared absorption spectrum (KBr): absorption bands at 34203120, 2940-2880, 1680, 1580. 1545. 1510.1440.13390,1305.12210.1110.1075 1040, 1000, 990, 975, .900, 845, 780 cm. Soluble in water, dimethyl sulfide, side, dimethylformamide, acetic acid and pyridine. Insoluble in ethyl acetate, chloroform, benzene and hexane. Color reaction. Positive: decolorizes potassium permanganate and oxidizes periodate; negative: ninhydrin, faling and ferric chloride (III). Color: white needles RI magnitude (silica gel, Tokyo Kasei company, silica gel (f): H-tanol: acetic acid: water (6: 1: 1) R, 0.27; And -butanol: concentrated alcohol: water (10: 0.5: 2), 07; H-propanol: concentrated natural alcohol: water (10: 1: 1) R.0.18; acetone (10: 1), 33. Example 2. 376 mg The plantation of D, dissolved in 10 ml of anhydrous pyridine, is thoroughly stirred at 55 C. 0.8 ml of benzoyl chloride is added dropwise to the solution and stirred for 2 hours at 60-65 ° C. Then the solution is maintained at 40- 45 ° C for 8 hours at room temperature. With stirring, add 10 ml of water are dried and 25 minutes later dried under reduced pressure in an evaporator. The dried material is dissolved in 30 ml of chloroform, washed three times with 10 ml of one-normal hydrochloric acid and twice with one-normal sodium bicarbonate solution and then twice washed with 20 ml of water. The chloroform layer is dried anhydrous magnesium sulfate and concentrated to obtain 747 mg of a compound of formula (1) with a pale pink color (yield 91%). Compound formulas; (I) has the following physicochemical characteristics:) (, 6, methanol); The molecular formula is -3H1 7. mass 576 (mass spectrum); m.p. 116-177 C. UV Absorption Spectrum: A / yage 232 A .704. K.-magnitude (chloroform: methanol 10: 1), 53. 735 mg of the compound of formula (I) prepared above and 1.0736 g of phosphorus pentasulfide dissolved in 19 ml of pyridine are thoroughly mixed. To the mixture is added dropwise 0.19 ml of water. After boiling under reflux for 4 v with stirring, the reaction mixture is cooled and concentrated in vacuo. A brown oil is obtained as a result. The oily product is poured into boiling water with stirring and further stirred for 30 minutes to obtain a yellowish material. It is filtered to separate the precipitate, washed with a mixture of ethyl alcohol and sulfuric ether (1: 1), further washed with ethyl ether and dried, resulting in 561 mg of the compound of formula (II). The yield is 74.3%. The compound of formula II has the following physico-chemical characteristics: And -286, 9 (, 6, CHCl,); molecular formula C 2 H 2f S OA S; a pier weight 592; m.p. 235-239 C (foam decomposition). UV spectrum (in methanol): 2320 D E, 858.5; 3230 A 327.5. Rj-value (chloroform: 10: 1), 82. Example 3. In 31 ml of anhydrous methyl alcohol, 525 mg of the compound of formula (II) obtained in Example 2 is dissolved. 0.62 ml of freshly prepared two-normal solution of sodium methylate in methanol is added and boiled under stirring for 2 hours with reverse cold . the addition of benzoyl groups. After distilling off the methyl alcohol, about 50 ml is added and the pH is adjusted to 7.5 by addition of acetic acid. The solution is extracted twice with 20 ml of sTttnoBoro acetic ester and the aqueous layer is concentrated to obtain the neplanocine D derivative of formula (III) in the form of needle crystals. Re-crystallization from hot water is carried out, a half MO of mg of the derivative of neplancin D of formula (III). Yield 4.3%. The non-planocin D derivative of formula (III) has the following physicochemical properties: the specific rotation cannot be measured due to the change in magnitude in the measurement. Molecular formula: C massa 280 (by mass spectrum); m.p. 26 262 С (with decomposition). UV spectrum at 2260 L (388.6), 3240 A (968.0). The value of R (propanol: conc. Water ammonia: water 10: 1: 1), 21. And p and me R 4. To a solution of 37 mg of the derivative of neplancin D of formula (III) prepared according to example 3 in 0.3 ml 0.4 n. Sodium hydroxide is added with 0.009 ml of methyl iodide and shaken at room temperature for 10 minutes. Then add 0.05 ml 0.4 n. sodium hydroxide and additionally add 0.009 ml of methyl iodide, shake again and hold at room temperature. To this solution, add 10 ml of water, concentrate in vacuo to 2 ml, and add to the 1.4x49 column filled with Sephadex G-15. The elution is carried out with water and fractionated with 3 g of eluate. Collect fractions Nos. 17-19, concentrate and cool to obtain 35 mg of the crystalline derivative of neplanocin D of formula (IV). Yield 90%. The derivative of planocin D of formula (IV) has the following physicochemical characteristics: “g) 141.7 (, 7, water); molecular formula mol. mass 294 (mass spectrum); m.p. 177-1J8 ° C. UV spectrum in water: 2220 A (E} J ;, 415.8) 5 2880 I (,); 2950 X (, 8): 2880 A (676.3); 2950 i (Ej ;; 670, 7). The value of R (propanol: conc. Aqueous solution of ammonia: water 1: 1: 1),, 42. Example 5. Synthesis of neplanocin A from a derivative of neplancin D of formula (IV). 50 mg of the derivative neplanocin P of formula (IV), prepared according to the same procedure as opDHa in example 4, dissolved in 1.5 ml of methanolic ammonia solution (, by expression), are placed in a sealed tube and kept at 146-148s in after 7 hours. After removing the solvent under reduced pressure, the residue is dissolved in 3 ml of hot water, then cooled, to give 33 mg of nonplanocin A in the form of needle crystals. Yield 74%. Example 6. To 1.14 g of neplanocin D in 12 ml of anhydrous pyridine was added 1 ml of acetic anhydride and held at room temperature for 8 hours. 30 ml of N-hexane are added and cooled in ice water to obtain 1 g of a crystalline compound of formula (V). 1 g of the compound of the formula V is added to 25 ml of a solution of 0.8 ml of thionyl chloride and 0.4 ml of dimethylformamide in chloroform and heated under reflux for 3.h at room temperature. Chloroform was distilled off under reduced pressure. The residue is poured into 100 ml of ice water with stirring. Extraction is carried out with 100 ml of chloroform, washed with sodium bicarbonate and water, and dried with sodium sulfate. After drying in vacuo, the nonplanocin D derivative of formula (VI) is obtained. Example 7. Synthesis of Noplanocin A from a derivative of neplancin D of formula (VI). To a derivative of neplancin D of formula (VI), prepared in prin116568012

measure 6 and placed in steel for- Physico-chemical characteristics

a covered tube, add 35 ml of nas-identical to those that respond to the leaked solution of ammonia n to methyl-known neplanocin A. alcohol and are reacted in a SO-c. Presented chemical sync for 5 h. After cooling, the recombination of non-planocin A from nonplanocin D

the stock mass is dried in a vacuum and takes place at higher

crystalline poro-concentration is obtained (chemical process),

shock, which is subjected to chromium-than during fermentation, which allows

tographic cleaning on sealy-use small compact

kagele. 286 mg of non-reactive vessels and simple conditiononocin A are obtained. The reaction is carried out.

Claims (2)

Neplanocin Ώ or its derivatives of the general formula Ko ok where K = H; X = OH, 5H, '8CH ₃ ; or K = Ac; X = C1, as intermediates in the synthesis of neplanocin A. 511,1165680 one 1 165680 2