SU1075973A3 - Process for preparing meitensionoids - Google Patents

Abstract

PURPOSE:The acylation of maytansinol with a specific compound is effected in the presence of a carbodiimide to produce maytansine derivatives useful as carcinostatic agent or antiactynomyces agent with industrial advantage. CONSTITUTION:The acylation of maytansinol with a compond of formula I (R is lower alkyl) is conducted in the presence of a carbodiimide preferably dicyclohexylcarbodiimide to produce maytansine derivatives of formula II. Further the use of an acidic or basic catalyst in a molar ratio of 0.01-1 to 1 mol of the compound of forumula I increases the yield.

Description

This invention relates to the preparation of maytansinoid compounds, namely, maytansinoids of the formula of cn-cCh 1 g 13 CHjO where R is lower alkyl with C, possessing anti-microbial and pro-tumor properties that can be used in medicine Some of mattansin L are known -configurations, namely, the maite of Sivoids of the formula I, where K is CH3:. CH, CH3, obtained by a series of solvent extraction steps and isolated from plants such as Maytenus cerrata. In the course of the target products is, respectively, 0.00002%, 0.0000031%, 0.0000036% CI. The aim of the invention is to simplify the process, increase the yield and expand the range of target products. The goal is achieved by the method of producing maytansinoids of the formula I, namely, that metansinol is acted upon with an acid of the general formula CH3 CH, I 3, 3 RCW-CH-COOH where R - has the indicated values, in the presence of dicyclohexylcarbodiimide and, if necessary, in the presence of acid Lewis. The maytansinoids of the formulas I obtained include the L-form, the new D-form, or a mixture thereof. According to the proposed method, -metansinol is acylated with an acid of Form II in the presence of carbodiimide. The acid of formula II can be any of the L- and D-isomers, as well as they are mixed when the residue of an acid of formula I which is supposed to be introduced into the gyrxyl group in the 3-position, is an optically active group. you are formula II. However, even if an optically active acid of Formula II is directly produced, a mixture of D- and L-methancinoids of Formula I is prepared. With respect to maytansinol, an acid of Formula II can be used in an amount of about 1-500, preferably up to about 30 molar equivalents, and carbodiimide in an amount of about 1-700, preferably up to about 50 polar equivalents. If necessary, it is advisable to carry out the reaction in the presence of an acidic catalyst, such as Lewis acids; anhydrous zinc chloride, anhydrous aluminum chloride (AICI), anhydrous ferric chloride, titanium tetrachloride (.), tin tetrachloride (SnCl4), antimony tetrachloride, cobalt chloride, copper chloride, trifluoride etherate, and inJ, inorganic or organic strongly organic and inorganic strongly organic and inorganic organic chloride and copper chloride, tetrachloride (SnCl4), and tetrachloride. as sulfuric, perchloric, hydrochloric, hydrobromic, benzosulfonic, p-toluenesulfonic, trifluoroacetic acid, trichloroacetic acid, etc .; acidic ion exchange resins such as dowex-50 (), etc. The basic catalyst may be, for example, selected from organic amine compounds such as pyridine, alpha, beta and gamma picoline, 2,6-lutidine, 4-dimethyl-aminopyridine, dimethylaniline, diethylaniline, triethylamine, N-methylmorpholine, and others. ; alkali metal halides such as potassium fluoride, anhydrous lithium iodide, and others; salts of organic acids, such as sodium acetate, etc. Anhydrous zinc chloride is particularly desirable. The use of such a catalyst results in an improved yield of maytansinoids of the formula I; The catalyst is used in an amount sufficient to promote the acidification reaction. Such a suitable amount of catalyst in many cases can be selected from the range of about Or 001-10, preferably from about 0.01 to 1 molar equivalent with respect to the acid of the formula But When such a catalyst is used, the amount of acid of the formula IT with respect to maytansinol may be reduced, preferably to about 1-6 MOLAR EQUIVALENTS, This reaction may be carried out in a suitable solvent, such as ester, for example ethyl acetate, ethers, for example diethyl ether, dioxane, tetrahydrofur N, et al .; halogenated hydrocarbons, methane chloride ngschrimer, chloroform, and others. Sodium, for example

acetonitrile, aromatic hydrocarbons, such as benzene, nitromethane, pyridine, dimethylformamide, dimethyl sulfoxide, sulfolane, etc., as well as in a suitable solvent mixture.

The proposed method makes it possible to obtain the target products in a sufficiently high yield, in a single stage, in a sufficiently high yield, including in the new D-form.

Example 1 In a 50 ml round bottom flask, 47.3 MF (0.0837 mmol) of maytansinol and 145 mg (1.0 mmol) of N-methyl-acetyl-b-alanine were placed. After adding 10 ml of anhydrous methylene chloride, a homogeneous solution. To this solution was added 10 ml of solution 309, mg (1.5 mmol) of dicyclohexylcarbodiimide in anhydrous dichloromethane, after which the colorless crystals were immediately separated. -When refining, this suspension was heated under reflux in an oil bath at a bath temperature for 10 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, and the insoluble substances were separated by filtration. The insoluble substances were washed with a small amount of methylene chloride, and the washings; You combined with the filtrate. The combined filtrate was concentrated under reduced pressure, and the residue was chromatographed on a column filled with 40 g of silica gel using 45: 1 v / v chloroform-methanol. The first fractions containing a byproduct — dicyclohexylurea as the predominant paqTBopeHHoro substance — were removed, and the bio-autographically positive fractions merged. From the subsequent fraction, 27 mg of maytansinol was recovered from the column.

The bioautographically active fractions were combined, quantified and chromatographed on a column filled with 27 g of silica gel, and the elution was carried out with an ethyl acetate mixture; ethyl acetate saturated with water D2: 1 /). Using this procedure, 7.0 mg of a colorless, bioavtographically active conjugate was isolated. The fact that this product represents one compound was confirmed by thin layer chromatography on a TLC plate (Kieselgel 60 P 5715, Merck, HGF) in two solvent systems of rloroform: methanol (9: 1 by volume) and ethyl acetate, saturated with vodbA.

It has been confirmed that the structure of this compound corresponds to the compound of formula I, where R is methyl.

Elemental analysis.

Calculated,%: C 59.00; H 6.70; 5 N 6.07.

SE4.H4bS1MzO O

Found,%: C 58.68; H 7.02; N 6.54.

Mass spectrum (in CHCI a) m / e: 0 691 (M, traces), 630 (HjO-HNCO), 485, 470, 450.

UV spectrum (in ethanol) max .: 233, 243, 254, 282, 290 nm.

EXAMPLE 2-9. The following base solutions were prepared:

Solution A. Solution of maytansinol i in anhydrous methylene chloride, containing 1 mg of maytansinol in 50 μl of solution,

Solution B. A solution of dicyclohexylcarbodiimide in anhydrous methylene chloride containing 6 mg of dicyclohexylcarbodiimide in 50 μl of solution. Solution C. A solution of N-acetyl-N-methyl-N-ala in anhydrous methylene chloride containing N-acetyl-N-methyl-b-alanine at a concentration of 3 mg / 50 ml of solution.

50 µl of solution C, 50 µl of solution B and 50 µl of solution A were added in a specified order to a glass tube of size 4 about 4 mm in diameter and about 14 cm in length. After stirring, the catalyst was added as it is in in the case of a solid catalyst, or previously diluted with methylene chloride to a suitable concentration, when it is liquid (the amount of catalyst is about 0.3 mg). Finally, the reaction mixture was adjusted to 200 µl with methylene chloride. Glass tube: sealed at a distance of about 12 cm: along its length. With the intermittent shaking of the tube, the tube was left to stand at the indicated temperature for about 4 hours. Then the tube was opened and the doses of the contents, corresponding to 2 μl, were applied to a TLC plate (CQ; Kgel P 5715, 20 cm x 20 cm. Merck , Germany). After occurrence with Chloroform-methanol (9: 1 by volume) a mixture of methanoxin was detected with 1 xRS scan scan Shimazzu device at a wavelength of

detection of 250 nm and wavelength; control of 350 nm, and, according to a previously prepared calibration curve, absolute and percent outputs of maytansine were reported. At the same time, the percentage yield of mathansinol was determined in a similar way when it was vibrated. The results are shown in Table. one.

Table 1

examples 10-12. To the mixtures of 50 µl each of solutions C, B, and A obtained in glass tubes and the tubes, in the same way as in example 2–9, about 2 mg of catalysts of the torus were added, the reaction was carried out as in

Example 13. In 20 ml of anhydrous methylene chloride, 159 mg of n-methyl-n-propionyl-b-alanyl- were dissolved per q followed by the addition of 309 mg of dicyclohexylcarbodiimide and 48 mg of mayitol. The mixture was heated at the temperature of deflenmation, and to exclude moisture, for 1 h. The reaction mixture was concentrated and the insoluble substances were filtered off. The filtrate was concentrated under reduced pressure, and the yost of impregnators 2-9. Then the amount of matane obtained was determined. The percentage of mathansinon is also determined simultaneously. The data are given in table. 2

table 2

the current was passed through a column filled with 40 g of silica gel. The manifestation was carried out with a mixture of chloroform, methanol (45si by volume) and bioavtographically positive fractions merged. The solvent was distilled off, and the residue was rechromatographed on a silica gel column (27 g silica gel; the developing solvent was ethyl acetate-ethyl acetate,

saturated with water, 2: 1 by volume).

; Bioavtographic positive fractions were collected and 5 mg of a colorless compound was isolated. This product was identified as a compound of the formula I, where I is -ethyl, in the same manner as in the example Mass spectrum, m / e: 644), 485, 470. Example 14, using a procedure similar to that described in example HZ , 173 mg of H-isobutyryl-H-methyl-b-alanine, 309 mg of dicyclohexylcarbamide diimide and 48 mg of maytansinol were reacted in 20 ml of anhydrous methylene chloride. The reaction mixture was further processed as in Example 13 and 3.2 mg of a colorless compound was isolated. On the basis of its mass spectrum, this product was identified as a compound of formula I where CH 3 11 CH Mass spectrum, m / e: 658 (),, 485, 570. P 15, C ps 4, similar to that described in example 13, 187 mg H -methyl-H-isovaleryl-b-alanine, 309 mg of dicyclohexylcarbodiimide and 48 mg of maytansinol were reacted in 20 ml of anhydrous methylene chloride. The reaction mixture was treated as in Example 13 and 2.5 mg of a colorless compound were isolated. Based on its mass spectrum data, the product was identified as a compound of formula I, where H is CH2CH (CH3) 2. Mass spectrum, m / e: 672 (), 485, 470, for example, m 16, Maitansinol (300 mg, 0.5316 mmol) and s-acetyl-methyl-b-alanine (1.585 g, 10 , 62 mmol) was dissolved in 80 ml of dichloromethane, to the solution was added 3.285 g of dicyclohexylcarbodime and 72.5 mg (0.582 mmol) of anhydrous zinc chloride. The mixture was stirred at a temperature of about 20 ° C for 6 hours and kept at that temperature for 11 hours. Then more H-acetyl-H-methyl-La-aiii (530 mg), dicyclohexylcarb diimide (1095 mg) and anhydrous chlorine zinc ( 150 mg). After 2 hours, the reaction mixture was filtered and the filter was washed with approximately iSOSO tm and dried over anhydrous sodium sulfate. The insoluble products were filtered off and the filtrate was separated by chromatography on a silica gel pad (60 g) and chloroform: methanol 40: 1 (by volume) as the selection after several previous fractions were removed, fractions of 25 g each. Fractions 14- 2 were combined, concentrated and re-separated by chromatography using silica gel (65 g) and a mixture of ethyl acetate: ethyl acetate saturated with 2: 1 water (by volume), selected after several preparative fractions of 16 g each, to obtain 149.3 mg of a compound of formula I (Compound A) from fractions 25-60, Fractions 23, 24 and 61- 100 after the second chromatography was combined and 20 ,, 5 mg of the substance was evaporated, from which 6.3 mg of compound A was additionally separated, subjected to preparative thin-layer chromatography on silica gel with 10% isopropanol in chloroform. Fractions 101-153 from the second chromatography were combined and concentrated to obtain 320 mg of product from which 95.7 mg of isomer of compound of formula I was obtained (compound c) after repeated chromatography on a column of silica gel (75 g) with chloroform and methanol in a 40: 1 ratio (by volume). The total yield of Compound A was 155.6 mg, and Compound B 95.7 mg, Substance A was dissolved in a mixture of ethyl acetate and diethyl ether, and this solution was left in the refrigerator to obtain crystalline compound A, which was then recrystallized once from a mixture ethyl acetate-diethyl ether and twice from a mixture of methylene chloride and diethyl ether, to give crystals of compound A: colorless plates, mp, pl. 191195c (with decomposition). Compound B crystallized when chloroform solution of veterinary B was treated with diethyl ether. The crystals thus obtained were subsequently recrystallized twice from the same solvent system, obtaining the crystals of compound B, melting at 155-178 (with gradual decomposition). Example 17, Maitansinol (100 mg, 0.177 mmol) and N-aietil-Nmethyl-b-alanide (522 mg, 3.59 mmol) were dissolved in 25 ml of dichloromethane. To this solution, while cooling in an ice bath Tei iepaTyre below 5 ° C was added 1100 mg (5.38 mmol) of dicyclohexylcarbodiimide and 24 mg of anhydrous zinc chloride. The mixture was stirred at a temperature below 5c for 20 hours, then filtered. The filtrate was concentrated and the residue was subjected to chromatographic separation on a column of silica gel (55 g) with a mixture of chloroform: methanol 40: 1 (by volume), selecting 25-Grg1Mm fractions. Fractions 15-30 were combined and evaporated to give 147 mg of the mixture of substances. From fractions 31-220, 43 mg of maite sinol was administered. 147 mg of the product was subjected to the 2nd purification on a chromatographic column with a mixture of ethyl acetate: ethyl acetate and water, 2.1 (by volume). Several of the previous fractions were removed and then the eluate was collected in 15 g fractions, obtaining 34 metansine, 17 mg of a mixture of metanesin and some other substances, and 47 m of the substance containing the isite maite sin. At preparative thin layer chromatography (PZC plates, 60 Art. 5717 silica gel, Merck HGF) with eluant methylene chloride: isopropyl alcohol 9: 1 (or 17 mg of the product), another 5.0 mg of meytansin was obtained. 47 mg of the product was subsequently chromatographed on a column of silica gel (60 g), eluent chlorofo ethanol 40: 1 (by volume), to give 20.0 mg of the isomer of metanesin. The total yield of metanesin and its isomer was 39 and 20 mg, respectively. Example 18. Methansinol (100 mg, 0.177 mmol), H-methyl-K-propionyl-b-alanine (84.5 mg, 0.531 mmol) and dicyclohexylcarbodiimide (164 mg, 0.796 mmol) were dissolved in 15 ml of dry dichloromethane. Immediately formed suspension with a white precipitate. About 24 mg (0.176 mmol) of anhydrous zinc chloride was added to this suspension with stirring at room temperature. After stirring at the same temperature for 1 h, another 84.5 mg of y-methyl-N41 propionyl-b-alanine, 164 mg of dicyclohexylcarbodiimide, about 24 mg of anhydrous zinc chloride and 10 m of dichloromethane were added, and the mixture was further stirred for another 1 h and 50 min. The reaction mixture was then filtered to remove a small amount of insoluble material, and the filtrate was diluted with dichloromethane to a volume of approximately 100 ml, taken in water, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure, and the separated precipitate was filtered and washed with a small amount of ethyl acetate. The combined filtrate and washing of the liquid was subjected to chromatography on a column of silica gel (65 g), eluent ethyl acetate-ethyl acetate, saturated with water 2: 1 (by volume); Selection of 15-gram fractions after several preceding fractions have been removed. Fractions -... C were combined, and by evaporation, 66 mg of the compound of formula I (substance C), and from fractions 40-97 58 mg of the isomer of the compound of formula G was obtained (in C2He7 compound D) Crystals of this substance when standing at room temperature. The crystals were collected and recrystallized from a mixture of dichloromethane-diethyl ether to obtain the crystals of compound C: colorless needles, which were observed at 185-189 ° C (with poor decomposition). . A solution of substance D in a small amount of ethyl acetate gave crystals of compound D, which was recrystallized from a mixture of dichloromethane and diethyl ether to give crystals of compound D: colorless needles, melting at 192 197 s (with decomposition). 1 Example 19. Methansinol (100 mg, 0.177 microl), N-isobuty. Aryl-N-methyl-b-alanine (92 mg, 0.532 mmol) and dicyclohexylcarbardiimide (164 mg, 0.796 mmol) were dissolved in 15 ml of dry dichloromethane. To this solution, while cooling in an ice-water bath with stirring, 24 mg of anhydrous zinc chloride was added, and the mixture was stirred for 0.5 h while cooling with ice. The ice-water bath was then removed, and the reaction mixture was heated to room temperature (), 92 mg of H-isobutyryl-M-methylb-alanine, 164 mg of dicyclohexylcarbodiimide and mg of anhydrous zinc chloride were added after 1 h, together with 10 ml dry dichloromethane. After another 45 minutes, the next portion of N-isobutyryl-methyl-b-alanine (31 mg), dicyclohexylcarbodiimide (55 mg) and anhydrous zinc chloride (mg) was added, and the reaction mixture was further stirred for 45 minutes. The total reaction time was 3h. Then, the reaction mixture thus obtained was treated as in Example 17, and the resulting material was chromatographed on silica gel (65 g) with eluent ethyl acetate: ethyl acetate saturated with 2: 1 water (by volume), selecting 15-gram fractions as in Example 17. Fractions 8 -22 were pooled, the solvent was evaporated, and 70.5 mg of the compound of formula I (((SN)) .2, compound E) was collected. Fractions 27-55 gave, after mixing and evaporating the solvent, 57.5 mg ichmer of the compound of formula I ((CH3) 2, compound F), Crystals of the compound E in a small amount of ethyl acetate precipitated out, which recrystallized from a mixture of dichloromethane-diethyl ether, obtaining crystals: substance B: colorless needles, melting at 185-187 ie (with decomposition).

Claims (1)

METHOD FOR PRODUCING MATHANESINOIDS of the general formula where R is lower alkyl with С ^ _ *, characterized in that, in order to simplify the process, increase the yield and expand the assortment of target products, methane synol is acted by an acid of the general formula sn ₃ sn ₃ rcn-sn-soyon where R has the indicated meanings, in the presence of dicyclohexylcarbodiimide and, if necessary, in the presence of a Lewis acid. SU ,. 1075973 A