DD145271A5 - METHOD FOR THE PRODUCTION OF INORGANIC SALTS OF TRIMETHYLAMMONIUM DERIVATIVES OF POLYENEMACROLIDES - Google Patents

Abstract

The invention relates to a process for the preparation of inorganic salts of 'imethylammoniumderivaten of Polyenemakrolide, in particular of inorganic salts of ethyl esters of these derivatives. These compounds are used as antibiotics in the treatment M mycoses in humans and animals. The aim of the invention is the valuable antibiotics from the group to render the polyene macrolides water-soluble. According to the invention, derivatives of the general formulas or 2 wherein R is the residue of the methylene substituted polyene macrolide X is an anion of an inorganic salt such as methylsulfate, sulfate, chloride, phosphate and the remainder of the polymakrolide without the Karboxyl group. - Formulas 1 and 2 -

Description

Berlin, 19. 12. 1979

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55 985 12

Process for the preparation of trimethyl ammonium compounds of polyene macrolides

Anisendungsgebiet the invention

The invention relates to a process for the preparation of inorganic salts of trimethylammonium derivatives of polyene macrolides.

Antibiotics from the group of polyene macrolides find extensive use in the treatment of mycoses in humans and animals, as well as in combating or preventing the fungal infection.

Characteristic of the known technical solutions

Heretofore known is a series of polyenemacrolide derivatives having excellent physicochemical properties. "For example, a complex of amphotericin B with sodium desoxycholate, N-acyl derivatives - US Pat. No. 3,244,590, methyl ester hydrogen chlorides - Mechlinski W *, Schaffner CvP., J Antibiotics, 2 l, 256 (195 l) of condensation products with formaldehyde - FE-PS No. 41 272, or with oxidized polysaccharides - ΒΞ-Ρ8 Hr · 620 619, and ΪΓ-glycoside derivatives - BE-PS Ur. 787'531.

A disadvantage of the derivatives described above is that the improvement of the physicochemical

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Properties in most cases adversely affect the other properties of these compounds, in the case of N-acyl derivatives, a significant reduction of the antimycotic activity takes place, and the ester salts are very unstable

From DE-OS 2,706,156 information on the production of N, N, NO? Rimethylderivaten, such as amphoterizine B, nastatin and mykoheptin with tinsubstituierter carboxyl group is known, the structure of which has not been detected · At the same time, these compounds are water-insoluble, what their Immediate application in solutions impossible ·

Object of the invention

The aim of the invention is the provision of antibiotically active compounds from the group of polyene macrolides, which are water-soluble ₀

Explanation of the essence of the invention

The invention is based on the object "water-soluble compounds from the group of Pol ^ renemakrolide and to find methods for their preparation.

According to the invention, the inorganic salts of trimethylammonium derivatives of the polyene-ana-acrosides, in particular the inorganic salts of the methyl esters of these derivatives are prepared, which are characterized by the formula 1,

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Wherein E denotes the radical of the methylene-substituted carboxyl group polyene macrolide and X denotes the anion of an inorganic salt, such as methylsulfate, sulfate, chloride, phosphorate, or the general formula 2, wherein E denotes the radical of the polyene macrolide without the carboxyl group , and X has the meaning given above.

The process for the preparation of the inorganic salts of trimethylammonium derivatives of poly (e) macrolides, in particular of inorganic salts of the methyl esters of these derivatives of general formula 1, wherein R is the radical of the methylene-substituted polyene macrolide and X is an anion of an inorganic salt, such as methylsulfate, sulfate, Chloride, called phosphate, or with general formula 2, wherein R ^{f is} the radical of Polymakrolids without the carboxyl group Ib, and X has the meaning given above, is characterized in that the polyenemacolide in the organic solvent or in their mixture, at room temperature and with continuous stirring with dimethyl sulfate in the presence of a neutralizing agent, the whole is turned off to react, and then the product is isolated from the reaction medium with ethyl ether, dissolved in butanol, washed with water, reduced in pressure and thickened with ethyl acetate the end product of general formula 1, ie trimethylammonium methylsulfate of methyl ester of polyene macrolide, or the end product of general formula 2, ie trimethyl

  - 4- -

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excreted in polyene macrolide, which is then optionally converted into other inorganic salts by known methods. "

The polyene macrolides used are nystatin, polyfungin, amphotericin B, eandicidin, primarizine, trichomycin, levorizin, ehimozidine, perimycin, candidate and aureofazine. The organic solvents used are dimethylacetamide, dimethyl sulfoxide and aliphatic alcohols having a chain length of C1 to C5.

As neutralizing agent, sodium bicarbonate is used.

If the antibiotic molecule contains an aromatic amino group, it is subject to substitution with one or two alkyl groups. Solcaen antibiotics include levorin, candizidine, trichomycin ·

The investigations carried out have shown that the alkylation conditions proposed by us are sufficiently mild so as not to lead to the decomposition of the antibiotic. Under the alkylation conditions described in the literature, the enhancement of the dimethyl sulfate would cause the macrolide ring to open, cleave the sugar ester, and degrade the polyene chromophore, resulting in loss of biological activity.

The antifungal and hemolytic activity in vitro of the DMS derivatives of antibiotics compared with the

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The starting substance is shown in the attached table.

The structure of the derivatives obtained was detected by spectrometric methods. By way of example, we cite a method of identifying the candidate kandizidine derivative as a result of the alkylation according to the proposed method with dimethylsulfate, and hereinafter called DMS-candicidin.

Under the conditions of azido-lysis, the mycosamine residue is freed. The substance obtained as a result of cleavage of the O-glycoside bond by the action of the dilute acid solutions on the DMS candicidin was identified as HiN, 1T-trimethylnycosamine.

The structure of this compound was characterized by field ion spectroscopy and nuclear magnetic resonance spectroscopy (signal at <S = 3§68 indicative of nitrogen atom-linked methyl groups, with an intensity three times higher than that of the signal at (f = 1 | 68, indicative of three protons of the methyl group The presence of an absorption band in the infrared spectrum at V = 1730 cm with simultaneous lack of carboxylate band, is proof of the presence of the ester bond in the molecule of the antibiotic derivative.

-1

drawing for the starting antibiotic at V '= 1590cm. The absorption spectra, within the range of visible and ultraviolet radiation, of candicidin and its derivative differ only insignificantly

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at the intensity. The positions of the absorption maxima and the oscillation structure, however, are identical. This demonstrates an unchanged structure of the polyene chromophore in the DMS derivative.

In the spectrum of nuclear magnetic resonance of the peracidic DMS candicidin, bands were found at 0 = 3.65 and ο = 3.03, which are characteristic of hydrogens of the methoxyl and N-substituted methyl groups. In aqueous solutions of bases, p-aminoquinophenone is released from the candidate sugar, while N-dimethyl-, N-methyl-, and unsubstituted p-aminoacetophenone in molar ratios 3! 5 ί 2.

An advantage of the derivatives according to the invention consists in their complete water solubility, and thereby a high antifungal activity and excellent selective toxicity in vitro in relation to the starting antibiotics.

Execution ia game

The inorganic salts of the trimethylammonium derivatives of polyene macrolides, in particular the inorganic salts of the methyl esters of these derivatives and the process for their preparation illustrate the examples given below *

B e i s ρ i e 1 I

2.0 g of polyfungin with ^] J _m = 800 at 304 mn are dissolved in 20 ml of a mixture of dimethylformamide with methanol

· -

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712

dissolved in a ratio of 1: 1, after which 2 g of sodium bicarbonate are added and 2 ml of dimethyl sulfate are added dropwise. The whole yard is stirred at tree temperature for 10 hours. During this time, the entirety of the antibiotic is virtually completely reacted. It is then evaporated under reduced pressure with the addition of ethyl ether »methanol and the precipitate is precipitated, to which 50 ml of butanol are added. The whole is separated from the undissolved sodium bicarbonate and the solution in the butanol washed twice with 20 ml of water. The butanol fraction is azeotropically thickened with water under reduced pressure to a volume of about 15 ml. Thereafter, the methyl ester of the trimethylamine salt is precipitated by polyether with ethyl ether.

After washing twice, successively with ethyl ether and hexane, the derivative is dried in vacuo »obtained 1.6 g of methyl ester of 1T, N-Ö? Rimethylasmioniumsalzes of Polyf ungin with EJl = 700 at 304 nm, which is 80% of theoretical Yield corresponds.

Figure I II

20 g of nystatin with E _m = 860 at 304 nm are dissolved in 50 ml of methanol. Thereafter, 2 g of sodium bicarbonate are added and 2 ml of dimethyl sulfate dripped. The whole is stirred at a temperature of 25 ⁰ G over 4 × hours. The further procedure is as described in Example I.

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-8th -

Obtained 1.3 g of methyl ester of Ν, Ν, Ν-trimethylammonium of nystatin with E ^ = 760 at 304 nm,

1 cm

which corresponds to 75 % of the theoretical yield

Example III

1.0 g of amphotericin -B with E ^ _m = 1420 at 382 nm are dissolved in 20 ml of a mixture of dimethylformamide with methanol in a ratio of 10: 1, after which 1 g of sodium bicarbonate is added and 1 ml of dimethyl sulfate is added dropwise. The whole is stirred at a temperature of 15 ^° C. for 24 hours.

The further procedure is as described in Example I.

Obtained 0.9 g of methyl ester of 2T, N, N-trimethylammonium salt of amphotericin B with E ^ = 900 at 382 nm, which corresponds to 90 % of the theoretical yield.

The resulting precipitate is distributed in countercurrent distribution in the system: chloroform: methanol: water (2: 2: 1). 0.3 g of methyl ester of the N, N, N-trimethyl ammonium salts of amphotericin B = 1400 at 282 are obtained nm.

Cm _ΛΤΒ ι cm

In s ρ ie II?

1 g of candidate with S ^ _m = 820 at 383 nm is dissolved in 20 ml of a mixture of dimethylacetamide with methane! in the ratio 10: 1 resolved. Thereafter, 1 g of sodium bicarbonate

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Ii 712 -9-

added and added dropwise 1 ml of dimethyl sulfate.

The whole is stirred at a temperature of 20 ⁰ O for 15 hours.

The further process route is as described in Example I.

Obtained 0.5 g of methyl ester of N, I \ f, N-trimethyl

155 esmonium salt of candidate with Ey = 550 at 383 ¹ ^ *

which corresponds to 80 % of the theoretical yield.

Bas product obtained is purified by column chromatography on water-saturated silica gel, in the system; Chloroform: methanol: water (20: 10: 1)

Obtained 2.0 g of candicidin derivative with ~ & V ° = at 378 nm.

Seispiel V

1 g. Trichomycin with B ^ _m = 4-50 at 378 nm is dissolved in 20 ml of a 10: 1 mixture of dimethylacetamide and methanol. Thereafter, 1 g of sodium bicarbonate are added and added dropwise 1 ml of dimethyl sulfate. The whole is stirred at a temperature of 20 ⁰ C for 15 hours. The further procedure is as described in Example I.

Obtained are 0.6 g of MS-trichomycin with Ά ^ _Μ = 560 at 378 nm.

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- 10 Be i β ρ i θ 1 VI

1 g pimarycine with E ^ = 960 at 304 mn is dissolved in 2 ml of methanol. Thereafter, 1 g of sodium bicarbonate are added and 1 ml of dimethyl sulfate is added dropwise. The whole is stirred at a temperature of 25 ^° C. for 15 hours. The further procedure is as described in Example I.

0.85 g of methyl ester of the Ν, Ν, Κ-trimethylammonium salt of pimarycine is obtained with E '= 700 at 304 · mn, which corresponds to 80 % of the thexetic yield.

Example VII

1 g of levorin with E ^ j ^ = 800 at 378 nm is dissolved in 20 ml of a mixture of dimethylformamide with methanol in the ratio 10: 1. Thereafter, 1 g of sodium bicarbonate are added and added dropwise 1 ml of dimethyl sulfate.

The whole is stirred at a temperature of 20 ⁰ G over 15 hours »The further procedure is as described in Example I.

Obtained are 0.7 g of DMS-levorin with S ^ j ^ = 600 at 304 mn, which corresponds to 70 % of the theoretical yield.

B e i s ρ ie I VIII

1 g of Ehimozy & in with £ ^ _m = 600 at 304 nm ¥ i / ird in 20 ml of a mixture of dimethylformamide with methanol in. .. ''''' ^: . '..' · · '''''- - 11

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2 14 71

- 11 -

Ratio 10 ϊ 1 dissolved · Thereafter, 1 g of sodium bicarbonate are added and added dropwise 1 ml of dimethyl sulfate. The whole is stirred at a temperature of 20 ⁰ C for 15 hours · The further procedure is as described in Example I.

Obtained are 0.8 g of methyl ester of the N, N, N-trimethylammonium salt of rhimozydin with sj ^ = 560 at 304 nm, which corresponds to 80 % of the theoretical yield.

Example IX

0.1 g perimycin with ^} / L · = 65Ο ^The 380 nm are dissolved in 2 ml of a mixture of thylaz et amide with methanol in a ratio of 10: 1. Thereafter, 0.1 g of sodium bicarbonate are added and added dropwise 0.1 ml of dimethyl sulfate. The mixture is stirred at a temperature of 20 ⁰ C for 10 hours and further as described in Example I beseh £ i.eben treated ·

Obtained are 0.05 g of Ν, Ν, Ν-trimethylammonium salt of perimycin with E Ύ * = 600 at 38Ο nm, which corresponds to • 50 % of the theoretical yield.

Example X

1 g of aureofazine with S ^ = 800 at 378 nm is dissolved in 20 ml of a mixture of dimethylformamide and methanol in a ratio of 10: 1. Thereafter, 1 g of sodium bicarbonate are added and added dropwise 1 ml of dimethyl sulfate. The whole thing is at a temperature of 20 ⁰ C.

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stirred for 20 hours and further treated as described in Example I.

0.7 B of EMS-aureofazine with E ^ _m = 760 is obtained at 378 nm, "which corresponds to 6 p % of the theoretical yield.

Example XI

0.1 g of Kandizidin with E ^ = 400 at 378 nm are dissolved in 5 ml of a mixture of dimethylformamide and methanol in the ratio 10: 1. Thereafter, 0.1 g of sodium bicarbonate are added and added dropwise 0.1 ml of dimethyl sulfate. The whole is stirred at a temperature of 20 ° C for 15 hours and further, as described in Example I, treated.

Obtained are O ₁ OAg of DMS-Kandizidin with E ^ _m = 560 at 378 nm, which corresponds to 45 % of the theoretical yield.

Example XII

20 g of a mixture of dimethyl acetamide and methanol in a ratio of 10: 1 and 1 ml of dimethyl sulfate are added dropwise to 1 g of mykoheptin with E _m =? 00 at 378 nm. The whole is stirred at a temperature of 20 ^?? C. for 15 hours and further, as described in Example I, treated »

Obtained werdän 0.8 g of methyl ester of Ν, Ν, ΙΓ-trimethylammonium of mykoheptin with E ^ = 680 at 378 nm,

1 cm

which corresponds to 85 % of the theoretical yield. . ' , · '. ^: . ". ''- ^: - 13 -

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! 4 7!

- 13 -

Table

Inflammatory and hemolytic activity of polyene macrolides and their derivatives

antibiotic Primarizin ^IC 50 (Mcg / ml) Primarizin (Mch / ml) 100 Polyfungin 1 400 EMS Polyfungin 1.7 20 nystatin 0.08 120 HiS nystatin 0.15 50 Ehimozidin 0.1 100 IMS Ehimozidin 0.25 30 Amphotericin B 1.5 50 IMS Amphotericin B 2.5 5 Mykoheptin -0.03 5 Mykoheptin 0.08 15 Kandidin 0.05 65 IMS Kandidin 0.2 20 Kandizidin 0.054 70 LMS Kandizidin 0.15 2.5 Aureofazin 0.005 15 IMS Aureofazin 0.005 0.35 Levorin 0.005 IMS; Levorin 0,003 2.5 iriciiomyzin 0,007 20 ems; Trichomyzin 0.009 3 Perimyzin 0.005 10 ras Perimyzin 0.01 3 0,002 12 MS. 0,003

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-14 -

Wherein:

IMS .- denotes an inorganic salt of

Methyl ester of trimethylammonium derivative of polyene macrolide or an inorganic salt of trimethylamonium derivative of polyene macrolide

  - indicates the concentration of the substance which causes in 50% the inhibition of the growth of the cells of Saccharomyces cerevisiae on liquid medium, determined spectrophotometrically at X = 660 nm after 24 hours incubation at a temperature of 28 ° 0

- refers to the concentration of the substance which under normal conditions causes the detachment of hemoglobin in 50 % , determined spectrophotometrically at X = 550 nm.

- 15 -

14 712 - ¹ > -

X COOCH

formula 1

R-N (CH

₃ )

formula 2

Claims (4)

19. 12. 1979 AP C 07 D / 214 712 2 t 4 712 Invention claim. Process for the preparation of inorganic salts of trimethylammonium derivatives of polyenemakrolides, in particular of inorganic salts of methyl esters of these derivatives having the general formula 1, viorin R the radical of the methylene-substituted polyene macrolide and X an anion of an inorganic salt such as methylsulfate, sulfate, Chloride or phosphate, or with the general formula 2, wherein R ¹ denotes the radical of the polymacrolide without the carboxyl group and X has the meaning indicated above, characterized in that the polyene macrolide in an organic solvent or in its mixture, at room temperature and with continuous stirring with dimethylsulfate in the presence of a neutralizing agent, the whole is turned off to react, and then the product is isolated from the reaction medium with ethyl ether, thickened under reduced pressure, and with ethyl ether the final product of the general form el 1, that is to say methyl sulfate of methyl ester of the trimethylammonium derivative of polyene macrolide, or the end product of general formula 2, that is to say methyl sulfate of the trimethylammonium derivative of polyenemacolide, which is then converted into other organic salts, optionally by known methods - 16 - 19. 12. 1979 AP C 07 D / 214- 2e method of item 1 'characterized in that as Polyenemakrolide nystatin, Polyfungin, amphotericin B, K _a ndizidin, Primarizin, Trochomyzinj. Levicin, ehimozidine, perimycin, candidate and aureogazine. 3 «method according to item 1, characterized in that are used as organic solvents Dirnethylazetamid, dimethyl sulfoxide and aliphatisehe alcohols having a chain length of 01 ... C5. 4 * Method according to item 1, characterized in that sodium hydrogen carbonate is used as a neutralizing agent. feiL / LSeiten formulas