NO158254B - ANALOGUE PROCEDURE FOR THE PREPARATION OF BICYCLO (3,2,1) OCTAN CARBOXYLIC ACID DERIVATIVES. - Google Patents

Description

Process for the production of antiviral-active 5-nitro-2'-deoxyuridine.

The present invention relates to a method for the production of antiviral-active β-hitro-2'-deoxyuridine with the formula:

The antiviral active compound 5-nitro-2'-doxyuridine, which has a very high activity, has in many respects a much higher activity than previously known antiviral agents of similar structure, e.g. 5_iodo-2'-deoxyuridine.

According to the present invention, an aqueous solution containing 5-nitrouracil and thymidine in a ratio of 10:1 to 1:10 is incubated at 30° - 4-0°C for 2-5 hours at a pH of between approx. 6 and 7 in the presence of the enzymatic activity produced by fermentation of Lactobacillus leichmannii.

For this purpose, the enzyme is first prepared using a microbiological method, by fermenting a microorganism in a culture medium containing an assimilable carbon source, an assimilable nitrogen source and mineral salts in such quantities as are usual for fermentation processes, and this microorganism forms said en-

zyme during the fermentation. While it is intended that any such microorganism can be used, provided it gives a reasonable yield of the enzyme, it has been found that a particularly suitable organism is Lactobacillus leichmannii ATCC 7830- The fermentation can be carried out as usual, e.g. by incubating the microorganism at a temperature between 30° and 4-0°C for a period varying from 6 to 24 hours. At the end of the fermentation, the cells of the micro-organism are isolated, e.g. by centrifugation and then crushed to release the enzyme in the cells. An ultrasonic disintegrator is convenient for this purpose and acts on a buffer suspension of the cells. The enzyme dissolves and can be stored at low temperatures after excess cell material has been removed.

For the enzymatic reaction, 5-nitrouracil is incubated with the enzyme in the presence of thymidine. In the course of this incubation, half of the thymidine's deoxyribose is transferred to 5-nitrouracil, so that 5-nitro-2'-deoxyuridine is formed. Incubation takes place at temperatures between approx. 30° and 40°C, preferably 37°C, for 2-5 hours in a buffer solution, with pH between approx. 6 and 7, which solution contains thymidine and 5-nitrouracil in varying mutual ratios,

which may be between 10 - 1 and 1 - 10 and contain an appropriate volume of the impure enzyme, prepared as described above. At the end of the incubation, the product 5-nitro-2'-deoxyuridine can be isolated using a number of processes, but preferably by chromatography. Referring to the examples, which give a better explanation of the procedures used in the isolation, it can be mentioned that water is a good solvent, especially in this case if chromatography is

is turned. The properties of the manufactured product are given in example 1.

divided into 100 ml portions divided into 500 ml Erlenmeyer flasks are inoculated with the culture L..Leichmannii grown on a microinoculum medium and incubated at 37°C for 10 hours with alternating shaking. The cells from . the centrifugation is washed with M/15 phosphate buffer (pH 6.5) and after another centrifugation at moderate speed (5000 g), they are resuspended in 20 ml of said phosphate buffer. This suspension is subjected to ultrasonic disintegration, and the resulting finely divided cell material is centrifuged at 10,000 g for 10 minutes. The upper layer represents the unpurified enzyme and is stored in a frozen state at

-40°C

For the enzymatic reaction, a solution consisting of

of thymidine (0.5 g), 0.1 g 5-nitrouracil and 50 ml of the purified enzyme, and this is diluted to 500 ml with M/15 phosphate buffer (pH 6.5). The solution is incubated at yj°Q for 3 hours, heated on a water bath for

to coagulate proteins and centrifuged. The upper layer is evaporated to dryness under reduced pressure. The residue is extracted with 10 x 10 ml of hot ethanol and the combined extracts are evaporated to dryness under reduced pressure. The rest is redissolved in a minimum amount of water. This solution is chromatographed on a 4 x 30 cm column of Dowex format, which is equilibrated with 0.1 M ammonium formate adjusted to pH 6.0. The buffer first elutes thymidine closely followed by thymine. The pH is then changed to 3*5 and under these conditions 5-nitro-2'-deoxyuridine is eluted. The solution is evaporated to dryness at 40°C under reduced pressure and then evaporated twice more with absolute ethanol. After this, the precipitate is extracted with 5 x 30 ml of ethanol, the combined extracts are evaporated at 40°C under reduced pressure and give a colorless oil which solidifies after a while. The solidified product is dissolved in 20 ml of ethanol and evaporated to a small volume,

which on standing gives 5-nitro-2'-deoxyuridine.

Ultraviolet spectra.

The Rf value in ascending thin-layer chromatography in ethanol-ammonium acetate 7' 3 (pH 7-5) is 0.55. The Rf value in descending paper chromatography in NaHPO^ solution saturated with isoamyl alcohol is 0.82.

Example 2

Two liters of a medium with the following composition

divided into 100 ml portions divided into $ 00 ml Erlenmeyer flasks in-

inoculated with the culture L. Leichmannii grown on microinoculum medium (Difco) and incubated at 37°C for 12 hours. The cells obtained by centrifugation at a speed of $000 g are washed with M/30 phosphate buffer (pH 6.5), and after another centrifugation at the same speed, they are resuspended in a sufficient amount of said phosphate buffer, so that a suspension of a density of 6.5% light transmission

(Clap). This suspension is subjected to ultrasonic disintegration and the resulting finely divided cell material is centrifuged at 10,000 g for 10 minutes. The upper layer represents the unpurified enzyme and is stored frozen at -40°C.

For the enzymatic reaction, a solution of 5-nitrouracil (1 g) is used to which 0.2 g of thymidine and 80 ml of impure enzyme are added, and the whole is diluted to 160 ml with 0.25 M acetate buffer pH $. 8. The solution is incubated at 37°C for 150 minutes. After this incubation, the proteins are precipitated by adding three volumes of ethanol and centrifuged off at moderate speed. The upper layer is concentrated to 25 ml under reduced pressure, filtered and transferred onto a 4 x 30 cm column of Dowex 1 formate, which is equilibrated with 0.1 M ammonium formate adjusted to pH 6.0. Chromatographic analysis gives first thymidine followed by thymine. The pH is then changed to 3-5 °g under these conditions 5-nitro-2'-deoxyuridine is eluted. The fractions are collected and concentrated to approx. 50 ml at 40° C under reduced pressure. This solution is extracted 16 times with n-butanol saturated with water. The butanol extract is evaporated to dryness under reduced pressure at 45°C. The residue is dissolved in 25 ml of absolute ethanol.

This solution is slowly concentrated under reduced pressure to a small volume. Upon cooling in ice, '-deoxyuridine crystallizes out, and this gave a yield of 110 mg of pure product.

A comparison was made between the in vitro antiviral activity of 5-nitr,o-2'-deoxyuridine and other deoxynitrosides. The results are summarized in the following table.

The in vitro activity of 5-nitro-2'-deoxyuridine and other deoxynitrosides.

5-nitro-2'-deoxyuridine is also active against sheeppox virus at the same concentration as vaccinia.

The concentrations mentioned above indicate the levels at which complete inhibition is achieved; of cytopathic effect in the relevant virus.

With regard to therapy used in humans, 5-nitro-2'-deoxyuridine can be administered topically in the form of ointments or solutions. Alternatively, the drug can be given orally, or parenterally.

Claims (1)

Process for the production of antiviral-active 5_nitro-2'-deoxyuridine with the formula: n characterized, in that an aqueous solution containing 5-nitrouracil and thymidine in a ratio of 10:1 to 1:10 is incubated at 30° - 4-0°C for 2-5 hours at a pH of between approx. 6 and 7 in the presence of the enzymatic activity produced by fermentation of Lactobacillus leichmannii.