CH631995A5 - Process for the production of polyene macrolide N-glycosyl derivatives - Google Patents

Description

The invention relates to a process for the production of N-glycosyl derivatives of the polyene macrolides.

In a known process for the production of N-glycosyl derivatives of the polyene macrolides and their salts, the derivative and the unreacted sugar are isolated from a reaction medium by precipitation and washing out with ethyl ether, drying under reduced pressure, and then by secreting the derivative of sugar and its possible conversion into salt.

The disadvantage of the process described above is the indispensable use of expensive and easily flammable solvents and the implementation of a series of additional operations to remove the excess of unreacted sugar. In addition, the salts thus obtained, although soluble in water, do not give clear solutions, which considerably limits their use.

The object of the present invention is to provide a method which does not have the disadvantages mentioned above.

The process according to the invention for obtaining N-glycosyl derivatives of the polyene macrolides is characterized in that the polyene macrolide containing the amino group is reacted with a mono- or oligosaccharide from the aldose or ketose series in an organic solvent or in an organic solvent mixture and then the N produced Glycosyl derivative precipitates from the reaction medium by means of water or an aqueous solution of an inorganic salt, is purified by crystallization from a higher alkanol, and is optionally converted into a salt.

Higher alcohol is understood to mean alcohols with at least 4 carbon atoms.

An advantage of the process according to the invention is, inter alia, the possibility of obtaining previously unknown N-methylglucamine salts of the N-glycosyl derivatives of the polyene macrolides.

These salts, similar to the polyene macrolides, N-glycosyl derivatives of these macrolides and their known salts, have antibiotic activity against fungi. The spectrum of the new salts is basically identical to that of the starting antibiotics and their complexes, e.g. such as the water-soluble amphotericin B complex - with sodium deoxycholate - used as a fungizone, which has so far been used exclusively in healing.

However, unlike Fungizon, some of these salts have e.g. activity of the N-methylglucamine salt of the N-glycosyl derivative of the polyungine against Trichomonas vaginalis. It was found that polyfungin on Trichomonas vaginalis was effective at a concentration of 837.7 mcg / ml, whereas N-methylglucamine salt of the N-glycosyl derivative of the polyfungin was effective at a concentration of 268.4 mcg / ml.

In addition, such salts, such as. B. the N-methylglucamine salt of the N-glycosyl derivative of amphotericin B, against many microbial strains, such as Torula sp., Geotrichum candidum and Cryptococcus neoformans, by higher activity than fungizone. Table 1 below shows the activity in vitro of the N-methylglucamine salt of the N-glycosyl derivative of amphotericin B and of the fungi zone against some fungi.

Table 1

Lowest effective concentration CE10 in mcg / ml

Mushroom type

N-methylglucamine salt of the N-glycosyl derivative of the fungizon amphotericin B.

Torula sp. 1.66 2.39

Geotrichum candidum 1.07 1.48 Cryptococcus neoformans 1.25 1.48

In the area of the tribes z. B. the species Candida sp., There is differentiating sensitivity to N-methylglucamine salt of the N-glycosyl derivative of amphotericin B and fungizon, which is characterized in that the number of insensitive to fungizon is statistically greater than the number of those against the said Salt-insensitive strains. This preferably affects the strains Candida albicans ZM, HM and 1/75, the strains Candida tropicalis JD and 20/75, and the strains Candida krusei TS and 16/75, on which fungizone is ineffective, but N-methylglucamine salt of N -Glycosyl derivatives of amphotericin B successfully acts.

The coefficient of relative activity, which characterizes the antibiotic protective effect in the experimental therapy of a mouse infected with Candida albicana, is significantly more favorable for N-methylglucamine salts or N-glycosyl derivatives of the polyene macrolides than for fungizone (see Table 2).

Table 2

antibiotic

Coefficient of relative activity in the general infection of a mouse with Candida albicans

Fungizon

1

N-methylglucamine salt of the

N-glucosyl derivative of:

- Amphotericins B

2.35

- polyfungins

5.08

- Nystatins

2.41

In rats which were injected with itravenous N-methylglucamine salt of the N-glycosyl derivative of the polyene macrolides and comparable fungizone, a more favorable effect was obtained with salt

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found because its level in the blood serum is longer than that of the fungizone (see Table 3).

Table 3

antibiotic

Duration of retention of the antibiotic level in the blood serum after administration of the dose 1/5 DLa, (min)

Fungizon

5

N-methylglucamine salt of the

N-glycosyl derivative of:

- Amphotericins B

15

- polyfungins

60

- Nystatins

30th

The selective toxicity of the N-methylglucamine salts of the N-glycosyl derivatives of the polyene macrolides is likewise much more favorable for these salts than for fungizone. The table below shows the coefficients of selective toxicity

EK

■ 50

and

EH.

50

DK

■ 50

DKj,

summarized. They give the ratio of the lowest concentration of the antibiotic, which causes a loss of 50% potassium ions from the erythrocyte cells of human blood (EK50), as well as the lowest concentration of the antibiotic, which causes 50% hemolysis of the erythrocytes of human blood (EH50), to the lowest concentration of the antibiotic that causes the loss of 50% potassium ions from the cells of Saccharomyces cerevisiae (DK 50).

Table 4

British Patent No. 1,387,187 discloses water-soluble sodium, tris and imidazole salts of the N-glycosyl derivatives of the polyene macrolides. Tris and imidazole salts must not be used naturally in medicine, 5 however the use of sodium salts of the N-glycosyl derivatives of the polyene macrolides in the form of injection solutions is too difficult due to the pH value of the solutions being too high. In addition, the sodium salts of the N-glycosyl derivatives of the polyene macrolides are not permanent in aqueous solutions with such a high pH, which means that pharmaceutical preparations with lower biological activity compared to the activity of the starting substance are obtained. A very advanced degradation of the lactone ring can be observed in the IR spectrum. In contrast to 15 sodium salts of the N-glycosyl derivatives of the polyene macrolides, N-methylglucamine salts are more soluble in water, and their aqueous solutions are stable and have a lower pH (see Table 6).

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Table 6

Sodium salt of the N-glycosyl derivative of polyungine

N-methylglucamine salt of the N-glycosyl derivative of polyungine

25th

pH 1% of the aqueous solution 9.9

Spectra in the IR

30th

Biological performance in E / mg

35 Solubility in water changed in the range 1704 'cm, in Vergi, the absorption band is missing for the sample

1680

4-10%

8.9

unchangeable compared to the pattern

2365> 15%

Selective toxicity index

antibiotic

EK50

EH »

DK50

DK50

Fungizon

1.09

1.4-2.9

N-methylglucamine salt of the

N-glycosyl derivative of:

- Amphotericins B

1.37

24th

- polyfungins

11.4

31

- Nystatins

17.3

45

The strong toxicity DL50 of the N-methylglucamine salt of the N-glycosyl derivatives of the ployen macrolides when administered intravenously is lower for mice than that of the fungizone (see Table 5).

Table 5

40

45

50

N-methylglucamine salts of the N-glycosyl derivatives of the polyene macrolides can be used in fungal infections in both surfaces * and systemic diseases caused by Candida albicans, Cryptococcus neoformans, Aspergillus, Geotrichum, Torulopsis and others, in generally known forms, such as aerosols, Infusions, vaginal tablets, tampons, drops and solutions for parenteral use can be used.

Example I

antibiotic

DLm for mice in mg / kg of body weight when administered intravenously

Fungizon

7.1-9.3

N-methylglucamine salt of the

N-glycosyl derivative of:

- Amphotericins B

12.1-14.3

- polyfungins

13.8-16.4

- Nystatins

19.0

1 g of amphotericin B and 0.3 g of anhydrous glucose are suspended in 15 ml of dimethylformamide and left at a temperature of 37 ° C. for 16 hours. 200 ml of distilled water are added to this solution and the precipitate is spun off, washed twice with 50 ml of benzene and once with 50 ml of 55 hexane. The precipitate is then dried under reduced pressure and extracted at a temperature of 50 ° C. with 100 ml of methanol for 15 minutes. The undissolved precipitate is spun off and extracted again with the same volume of methanol. The combined 60 solutions are kept at a temperature of -5 ° C throughout the night. The precipitate which has separated out is centrifuged off, washed twice with 50 ml of benzene and once with 50 ml of hexane and dried under reduced pressure. In this way, 0.65 g of crystalline N-glycosyl-65 derivative of amphotericin B with Elfcm = 1450 at 381 nm, IC 50 = 0.04 mcg / ml is obtained.

Initial antibiotic: E1 Vcm = 1580 at 318 nm and IC5n = 0.03 mcg / ml.

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Example II

10 g of polyfungin and 2.4 g of glucose are dissolved in 50 ml of dimethylformamide and left at a temperature of 32 ° C. for 16 hours. The derivative is eliminated by adding 11 0.1N aqueous sodium chloride solution, centrifuged, washed with 100 ml of water and extracted, then washed eight times with 100 ml of methanol.

100 ml of n-butanol are added to the combined extracts and concentrated under reduced pressure to a volume of 50 ml and then left to crystallize at a temperature of -5 ° C. The precipitate which has separated out is spun off, washed twice with acetone and hexane and then dried. In this way, 7.2 g of N-glycosyl derivative of polyfine with E1 = 710 at 304 nm, IC5 |) = 0.25 mcg / ml is obtained.

Example III

5 g of candicide and 1.5 g of glucose are suspended in 25 ml of dimethylformamide and left at a temperature of 37 ° C. for 20 hours. The derivative is precipitated with 200 ml of 5% ammonium sulfate solution and washed twice with 20 ml of water. The N-glycosyl derivative of the candicide obtained in the reaction is extracted with 200 ml of methanol at a temperature of 36 ° C., then using a 200 ml methanol-n-butanol mixture. The water is evaporated and the water azeotropically with butanol, after which the derivative is crystallized from n-butanol. In this way, 1.14 g of N-glycosyl derivative of the candicide with E1 fem = 210 at 378 nm from the methanol fraction and 1.96 g of derivative with E'fcm = 270 at 378 nm from the methanol-butanol fraction are obtained. IC50 N-glycosyl derivative of candicide = 0.004 mcg / ml.

Initial antibiotic: Exi | m = 210 at 378 nm, IC50 = 0.0025 mcg / ml.

Example IV

According to the method described in Example III, the condensation reaction of 5 g aureofacin with E'f ^, = 410 at 378 nm and 1.5 g glucose is carried out. In this way, 2.93 g of N-glycosyl derivative of aureofacin with E'îtm = 290 at 378 nm from the methanol fraction and 1.82 g of derivative with E'fem = 320 at 378 nm from the mathanol-n-butanol fraction are obtained . IC50 of the N-glycosyl derivative of aureofacin = 0.008 mcg / ml. IC5fl of the starting antibiotic '= 0.006 mcg / ml.

Example V

3 g of aureofacin are condensed with 0.9 g of glucose in 35 ml of dimethylfromamide. The undissolved impurities are spun off and the derivative is precipitated with 200 ml of 10% aqueous ammonium sulfate solution. The precipitate is filtered off using a layer of celite, washed with 200 ml of water and 200 ml of acetone, and the derivative is extracted using a mixture of chloroform-methanol-water in a mixing ratio of 10: 10: 1. Chloroform and methanol are distilled off and the water is azeotropically distilled with n-butanol and left overnight at a temperature of -5 ° C. for crystallization from n-butanol. In this way, 1.85 g of the N-glycosyl derivative of aureofacin with E'fcm = 320 at 378 nm and IC3S = 0.008 mcg / ml are obtained. IC5 »of the original antibiotic = 0.006 mcg / ml.

Example VI

1 g of amphotericin B and 0.3 g of glucose are suspended in 15 ml of dimethylformamide and left for a period of 20

Hours at a temperature of 37 ° C. The N-glycosyl derivative is precipitated using 300 ml of 5% aqueous ammonium sulfate solution and washed with 50 ml of water. The precipitate is suspended in 50 ml of methanol and 40 ml of n-butanol and 0.1 mM sodium carbonate in 10 ml of water is added, methanol and water are distilled off azeotropically with n-butanol, and the product is left to crystallize. In this way, 1.2 g of sodium salt of the N-glycosyl derivative of amphotericin B with E'fcrn = 1200 at 380 nm and IC 50 = 0.05 mcg / ml are obtained.

Example VII

1 g of amphotericin B and 0.3 g of glucose are suspended in 15 ml of dimethylformamide and left for 20 hours at a temperature of 37 ° C. The N-glycosyl derivative is precipitated using 300 ml of 5% aqueous ammonium sulfate solution. The precipitate which has separated out is washed with 50 ml of water, dissolved in 50 ml of methanol and 1 mM of Tris and 40 ml of n-butanol are added, after which methanol and the water are distilled off azeotropically with butanol and the product is left to crystallize. In this way, Tris salt of the N-glycosyl derivative of amphotericin B is obtained with E'f ° m = 1040 at 380 nm and IC50 = 0.6 mcg / ml.

Example VIII

3 g of polyfungin are suspended in 15 ml of dimethylformamide, 0.9 g of glucose are added and the mixture is left to stand at a temperature of 38 ° C. for 24 hours. The insoluble precipitate is spun off and the N-glycosyl derivative is precipitated from the solution with 115% aqueous ammonium sulfate solution. The precipitate is spun off and extracted three times with 40 ml of methanol at a temperature of 38 ° C. 3mM Tris is added to this solution, then 40 ml of n-butanol is added, the methanol is distilled off, the water is azeotropically distilled off with n-butanol, and the mixture is left to crystallize at a temperature of -5 ° C. In this way 2.85 g of trisalt of the N-glycosyl derivative of the polyfine are obtained with Blfcm = 570 at 304 nm and IC50 = 0.4 mcg / ml.

Initial antibiotic: E1 ^ = 660 at 304 nm, IC50 = 0.15 meg / ml.

Example IX

20 g of polyfungin and 6 g of glucose are dissolved in 100 ml of dimethylformamide and left to stand at a temperature of 36 ° C. for 18 hours. The solution obtained is filtered. 400 ml of 5% aqueous ammonium sulfate solution are added to the filtrate and left to stand for a period of 2 hours at a temperature of + 4 ° C. The precipitate which has separated out is filtered off, washed with water and then extracted three times, each time using 800 ml of methanol.

6 g of N-methylglucamine are dissolved in 60 ml of water and added to the methanol extracts. 1000 ml of n-butanol are added to the resulting solution and the whole is concentrated at a temperature of 45 ° C. under reduced pressure until the water and methanol have been completely eliminated. The rest is left to crystallize at a temperature of + 4 ° C. The precipitate is filtered, washed with n-butanol and then with petroleum ether and dried at room temperature. In this way, 16.5 g of N-methylglucamine salt of the N-glycosyl derivative of the polyfine are obtained with elfcm = 560 at 304 nm and IC5n = 0.42 mcg / ml.

Starting antibiotic: E '^ m = 660 at 304nmundIC5o = 0.16 mcg / ml.

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Example X

20 g of nystatin and 6 g of glucose are dissolved in 100 ml of dimethylformamide and the procedure is exactly as in Example IX. In this way, 18 g of N-methylglucamine salt of the N-glycosyl derivative of nystatin are obtained with E1 ïcm = 560 at 304 nm and IC5 (| = 0.45 mcg / ml.

Initial antibiotic: E '^ m = 790 at 304 nm, IC5n = 0.18 mcg / ml.

Example XI

20 g of amphotericin B and 6 g of glucose are dissolved in 200 ml of dimethylformamide and left to stand at a temperature of 35 ° C. for 18 hours. The solution obtained is filtered. 500 ml of 5% aqueous ammonium sulfate is added to the filtrate.

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Fat solution added and left for a period of 2 hours at a temperature of + 4 ° C. The precipitate is filtered off and washed with water. 1500 ml of methanol at a temperature of 50 ° C. and 6 g of 5 N-methylglucamine dissolved in 60 ml of water are added to the precipitate and mixed for 30 minutes, after which 11 n-butanol are added. The whole is concentrated at a temperature of 45 ° C under reduced pressure until the removal of the methanol and the water. The rest is left to crystallize at a temperature of + 4 ° C. The precipitate is filtered off, washed with n-butanol and then with petroleum ether, and dried at room temperature. In this way, 18 g of N-methylglucamine salt of the N-glycosyl derivative of amphotericin B are obtained with E '^ m = 1140 at 380 nm and IC50 = 0.1 mcg / 15 ml.

M

Claims (6)

661 995 PATENT CLAIMS 1. A process for the production of N-glycosyl derivatives of the polyene macrolides, characterized in that the polyene macrolide containing the amino group is reacted with a mono- or oligosaccharide of the aldose or ketose series in an organic solvent or in an organic solvent mixture and then the N produced Glycosyl derivative precipitates from the reaction medium by means of water or an aqueous solution of an inorganic salt, purified by crystallization from a higher alkanol, and optionally converted into a salt. 2. The method according to claim 1, characterized in that the purified N-glycosyl derivative is crystallized from a higher alcohol after conversion into salt. 3. The method according to claim 1, characterized in that the N-glycosyl derivative is precipitated by means of an ammonium sulfate solution. 4. The method according to claim 1, 2 or 3, characterized in that the N-glycosyl derivative is then purified by crystallization from n-butanol. 5. The method according to claim 1, characterized in that the purified, precipitated N-glycosyl derivative is converted into N-methyl-glucamine salt. 6. The method according to claim 1, characterized in that the purified N-glycosyl derivative is crystallized after conversion into salt from n-butanol.