DE2845762C2 - - Google Patents

Description

The invention relates to medicaments with antiviral activity which are active ingredients (S) - and / or (RS) -9- (2,3-dihydroxypropyl) adenine in the formula I. contain

Some derivatives of nucleosides, ie sugar derivatives of heterocyclic compounds, had antiviral activity, e.g. B. 9- ( b -D-arabinofuranosyl) adenine (hereinafter referred to as AraA) of the formula II which has a pronounced effect on DNK viruses, and ribavirin (1- [ β- D-ribofuranosyl] -1,2,4-triazole-3-carboxamide), which has broad antiviral activity against some RNA and DNA viruses (Science 177 , 705 (1972); Chemotherapy 21, 505 (1975). However, the expenditure for the production of these remedies is very high due to the complicated procedure, and their usability is also due to the narrow concentration range in which they are given their toxicity and their degradation in the organism can be applied, significantly restricted.

These disadvantages are eliminated by the medicament according to the invention, which contains (S) - and / or (RS) -9- (2,3-dihydroxypropyl) adenine in the formula I as active ingredient contains, the active ingredient content, based on the total weight of the medicament, which may contain a pharmaceutical carrier, being 0.1 to 100% by weight.

The effective, non-toxic single dose is 0.1 to 1000 mg / kg.

The preparation of the (S) enantiomer and the racemic (RS) form of the compound of the formula I (hereinafter referred to as S-DHPA or RS-DHPA) is known. Both compounds can be prepared by reacting 1-op-toluenesulfonyl 2,3-I-isopropylidene-D- (or -DL -) - glycerol of the formula III with the sodium salt of adenine in dimethylformamide at 100 ° C and boiling the resulting intermediate with 80% acetic acid (Czech. Chem. Commun. 40, 187 [1975]). So z. B. 11.4 g (40 mmol) of the compound of formula III and 7.8 g of the sodium salt of adenine (50 mmol) in dimethylformamide (100 ml) heated at 100 ° C for 8 h, whereupon evaporated in vacuo and the residue from Methanol is recrystallized. The product obtained is boiled for 1 hour with 80% acetic acid and evaporated to dryness in vacuo, after which the dry residue is evaporated three times from 50 ml of ethanol each time and the rest is crystallized from methanol.

Yield: 50-60% (S) - or (RS) -9- (2,3-dihydroxypropyl) adenine.  

(S) shape: F. 202-203 ° C, = -35.4 ° ( c = 1, water). (RS) shape: F. 207-208 ° C, UV spectrum (pH 7): λ _max = 260 nm, ε _max = 14,000, λ _min = 228 nm.

According to CS patent application VP 1787-77, the (RS) form of the compound I (RS-DHPA) is obtained by heating adenine with a cyclic 1,2-carbonate of the glycerol of the formula IV obtained with sodium or potassium hydroxide or carbonate in dimethylformamide or dioxane at 100-140 ° C. So z. B. a mixture of adenine (1.35 g, 10 mmol), cyclic glycerol-1,2-carbonate (IV) 2.0 g), sodium carbonate (0.3 g) and dimethylformamide (25 ml) for 1 h at the boil heated and evaporated in vacuo; the residue is decolorized in 50 ml of boiling water with activated carbon. After evaporation in vacuo, the residue is crystallized from methanol.

Yield: 60-70% (RS) form of compound I (RS-DHPA); F. 205-208 ° C, UV spectrum (water): λ _max = 260 nm, ε _max = 14,000.

Has biological or pharmacological activity only the D-glycero form, d. H. the (S) shape of the Compound of formula I, whereas the L-glycero form, d. H. the (R) shape, is completely ineffective. Because the racemic (RS) derivative contains 50% effective (S) form, it is  accordingly approximately as effective as the (S) -enantiomer.

(S) -DHPA of the formula I has high antiviral activity against RNA and DNA viruses and at the same time very low self-toxicity. This effect is all the more surprising since other, similar compounds up to a concentration of 100 µg / ml have no relevant antiviral activity, eg. B. derivatives of adenine with another aliphatic component such as (RS) -2-hydroxypropyl-, 2-hydroxyethyl, 2-aminoethyl-, b -DL-alanyl-, (S) -3,4-dihydroxybutyl-, (RS) -3,4-dihydroxybutyl-, (RS) -threo-2,3,4-thrihydroxybutyl-, (RS) -3,5-dihydroxypentyl, 3-hydroxypropyl- and 1,3-dihydroxy-2-propyl- or 2 , 3-Dihydroxyproylderivate from other heterocyclic bases such as hypoxanthine, uracil, cytosine and thymine. Among other related compounds, AraA has an activity restricted to DNA viruses, ribavirin is also antiviral, but at the same time highly cytotoxic.

In addition to the active ingredient of the formula I, the medicaments according to the invention can be in the (S) - or (RS) -form 9- ( β- D-arabinofuranosyl) adenine in the formula II in a ratio of 1: 1 to 1000: 1 of the compound of formula I to the compound of formula II.

(S) -DHPA acts as a strong inhibitor of the enzyme adenosine desaminase, which is very common in cells, tissues and body fluids. (S) -DHPA or (RS) -DHPA are neither substrates for adenosine desaminase from bacterial cells (Escherichia coli, Salmonella typhi murium) nor from rabbit kidneys. NY) Acad. Sci. 284, 60 [1977]), which limits its use as a virostatic. The compound S-DHPA or RS-DHPA inhibits this process; together with AraA there is a synergistic effect, since the antiviral activity is significantly higher compared to the effect of the two individual active substances as such, ie similar to e.g. B. CO-Viridarabin ((R) -3- (2-deoxy- β- D-erythro-pentafuranosyl) -6,6,7,8-tetrahydroimidazo- (4,5d) -1,3-diaz-epine -8-ol); (see Ann. NY Acad. Sci. 284, 9 [1977] and 284, 60 [1977] as well as Table 2).

Further advantages of the medicament according to the invention are in their excellent stability, the one Sterilization of neutral, weakly acidic or weakly enables basic solutions at elevated temperatures, in practically unlimited stability in a solid state and finally in easy accessibility, especially for the racemic derivative (RS) -DHPA.

Other advantages are the relatively simple manufacturing process and low production costs. At  Industrial production is the manufacturing cost compared to the production of the known known antiviral preparations significantly lower.

The pharmaceutical preparations according to the invention Containing (S) -DHPA and / or (RS) -DHPA, if necessary. in combination with AraA as an active ingredient, can be in the form of powders, suspensions, solutions, spray emulsions Ointments and creams are present and parenteral (intravenous, intradermal, intramuscular, intrahecal, etc.) or orally, rectally, intravaginally, intranasally or locally (Skin, mucous membrane, eye lesions, etc.) administered will. The preparations can be made by mixing or dissolving of the active ingredient (s) together with the usual pharmaceuticals Auxiliaries and / or carriers are produced, e.g. B. with aqueous or non-aqueous solvents, Stabilizers, emulsifiers, dispersants, suspension stabilizers, Wetting agent u. Like., suitable additives (e.g. polyethylene glycols) and if necessary also Colorants or flavorings. The preparations can depending on the type of administration and the type of disease contain different concentrations of active ingredient, at least but 0.1% by weight.

The medicaments according to the invention are for treatment of viral diseases such as B. vaccine, herpes simplex-1, Herpes simplex-2, measles of versicular stomatitis u. Ä., can be used advantageously.

The pharmacological efficacy of (S) -DHPA and (RS) -DHPA and their combination with AraA is discussed below Examples explained.  

Example 1 Antiviral efficacy of (S) -DHPA in tissue cultures

The cells of each culture were infected with the hundredfold dose of the virus which is necessary to infect 50% of the cells (CCID₅₀); 1 h after infection, (S) -DHPA was added in a concentration of 0 to 40 µg / ml (in some cases up to 200 µg / ml). For each combination of host cells and virus, the ID₅₀ was determined, ie the concentration of (S) -DHPA, which is necessary for a 50% inhibition of the cypathological effect of the virus determined by the control experiment (according to LJ Rosenthal and IL Schechmaister, "Tissue Culture ", p. 510, Academic Press, New York 1973). The results of these tests are shown in Table 1. From this it can be seen that some viruses, e.g. B. vaccinia, herpes simplex type 1 and 2, measles and vesicular stomatitis, are very sensitive to (S) -DHPA, whereas other viruses such as poliomyelitis, Coxsackia and Sindbis remain unaffected. The inhibition of the cytophatogenic effect of the virus in the presence of (S) -DHPA is a consequence of the reduction in the increase in the amount of virus; Continuous monolayer cultures of human skin fibroblasts (HSF cells) in Petri dishes were vaccinated with the vesicular stomatitis virus (VSV virus) (4.5 log ₁ CCID₅₀ / 0.5 ml dish) and after 1 h at 37 ° C. of the substance ( S) -DHPA (100 µg / ml) exposed. The cultures were incubated at 37 ° C. and frozen at -70 ° C. at various time intervals, after which the titer of the virus in cell homogenates was determined on the basis of the plaque formation on L-929 mouse fibroblasts. The values of FIG. 1 show the sharp drop in the virus titer at 100 μg / ml (S) -DHPA compared to the control test, which is 4 orders of magnitude 48 hours after the virus infection.

Example 2 Antiviral effects of (S) -DHPA in vivo

The antiviral effect was determined using the method of J. Gen. Virol. 5, 359 (1969) and J. Clin. Invest. 49, 1565 (1970): 20-day-old female NMRI mice (average weight 15 g) were infected intranasally with VSV virus (2.5 log₁₀CCID₅₀ / 0.01 ml / mouse); then intraperitoneal (S) -DHPA (2 mg / mouse, 133 mg / kg) was applied repeatedly at intervals of 1 hour, 1 day to 4 days after the infection. The results are shown in Fig. 2 for 14 consecutive days. Repeated doses of (S) -DHPA lead to a significant increase in survival (67% vs. 37.5% in the control group); the difference between the two groups is noticeable as early as 9 days after infection. No further death was recorded in either group from the 14th day after infection. The experiments were carried out with different doses of (S) -DHPA; repeated doses of 0.08 mg / mouse (5.4 mg / kg) were ineffective, whereas repeated doses of 0.4 mg / mouse (27 mg / kg) significantly increased survival compared to the control group (55% versus 37 , 5%) lead.

Toxicity of (S) -DHPA

All animals survived repeated administrations up to 1000 mg / kg · d within 3 to 5 days without any signs of illness. (S) -DHPA is accordingly very little toxic.  

Example 3 Antiviral effect of the combination of (S) -DHPA and AraA

Contiguous primary monolayer cell cultures of Rabbit kidney cells (PRK cells) were placed in Petri dishes within one hour at 37 ° C with vaccinia virus (4.5 log₁₀CCID₅₀ / 0.5 ml / bowl) and then vaccinated Effect of (S) -DHPA, AraA or the combination of both Exposed to substances in concentrations of 1 to 100 µg / ml. The cultures were incubated at 37 ° C, after which the titer of the virus at different time intervals of plaque formation on PRK cells in the same way as for the vesicular stomatitis virus and L-929 mouse fibroblasts as determined in Example 1. In the Results shown in Table 2 demonstrate that the combination of (S) -DHPA (30 µg / ml) and AraA (3 µg / ml) one greater decrease in virus titer than either of the two effective ones Individual connections caused for themselves; the combination is about 3 orders of magnitude more effective.

Example 4 Inhibitory effect of (S) -DHPA on deamination of AraA by adenosine desaminase of the intestinal mucosa

It became the Biochem method. J. 152, 681 (1975), which is based on the spectrophotometric measurement of the decrease in absorption of AraA at λ = 265 nm. For 50 mol AraA, Δ ₂₆₅ = 0.11 for 100 nmol AraA, Δ ₂₆₅ = 0.16. The results are shown in Figures 3 and 4; from them it can be seen that the delay in cleavage of AraA by (S) -DHPA is proportional to the concentration of (S) -DHPA.

Example 5 Determination of the effective concentration of (RS-) DHPA in the blood serum after intraperitoneal and oral application Mice

Two groups of 20 female NMRI mice each with an average weight of 12 g was once 10 mg each of (RS) -DHPA in 0.2 ml of physiological solution either administered intraperitoneally or orally. 0, 20, 40, Blood samples were taken 80, 160 and 320 min after the application decreased, whereupon the antiviral effect of the blood serum on tissue cultures of human skin fibroblasts with the vesicular stomatitis virus (see Example 1) had been infected. The concentrations of (RS) -DHPA in the serum were determined from the dilution of the serum, required to achieve 50% inhibition was, according to the formula

C = fID₅₀

where C is the concentration of (RS) -DHPA in blood serum, f the dilution of the serum, which is 50% Virus inhibition was required, and ID₅₀ the experimental Value of the concentration of (RS) -DHPA leading to a 50% virus inhibition under the test conditions leads (20 µg / ml) mean. All data are mean values samples from 3 mice. The test results are in Table 3 listed.

Table 1 shows the antiviral activity of (S) -9- (2,3-dihydroxypropyl) adenine in tissue cultures, and Table 2 shows the synergism of the antiviral activity of (S) -9- (2,3-dihydroxypropyl) adenine and 9- ( β- D-arabinofuranosyl) adenine on primary cell cultures of rabbit kidney cells infected with vaccinia virus. Table 3 shows the concentration of (RS) -DHPA in the blood serum of mice after intraperitoneal and oral administration.

Figure 1 illustrates the inhibitory effect of (S) -DHPA on the growth of vesicular stomatitis viruses in human skin fibroblasts; on the ordinate the virus titer (log₁₀PFU / ml) is given, where PFU means the unit of plaque formation, on the abscissa the time after infection in hours; the circles correspond to the control test, the filled in points to the tests with (S) -DHPA.

FIG. 2 shows the antiviral activity of (S) -DHPA illustrated in the vesicular stomatitis virus intranasally infected mice; the abscissa shows the number of days after infection, the ordinate the total mortality in%, the circles correspond to the control test again, the filled points of the application of (S) -DHPA.

The dependence of the cleavage of AraA by adenosine desaminase in the presence of (S) -DHPA is graphically illustrated in FIG. 3; the abscissa shows the concentration of (S) -DHPA in µg / ml, the ordinate shows the decrease in absorption at λ = 265 nm (0 = 50 nmol AraA, 0 = 100 nmol AraA).

Fig. 4 illustrates a Dixon plot of the dependence of the cleavage of araA by adenosine deaminase in the presence of (S) -DHPA group; the abscissa shows the concentration of (S) -DHPA in µg / ml, the ordinate the reciprocal of the initial cleavage rate (l / V) ( K _i = inhibition constant; 0 = 50 nmol AraA, 0 = 100 nmol AraA).

Table 1

Antiviral activity of (S) -9- (2,3-dihydroxypropyl) adenine in tissue cultures.

Table 2

Synergism of the antiviral activity of (S) -9- (2,3-dihydroxypropyl) adenine and 9- ( β- D-arabinofuranosyl) adenein in primary cell cultures from rabbit kidney cells infected with vaccinia virus

Table 3

Content of (RS) -DHPA in the blood serum of mice after peritoneal and oral application

Claims (2)

1. Medicament with antiviral activity, characterized in that it is (S) - and / or (RS) -9- (2,3-dihydroxypropyl) adenine in the formula I as active ingredient in an amount of 0.1 to 100 wt .-%, based on the total weight. 2. Medicament according to claim 1, characterized in that it is 9- ( β- D-arabinofuranosyl) adenine of the formula II as a further active ingredient in a ratio of 1: 1 to 1000: 1 of the compound of formula I to the compound of formula II.