DE2845762A1 - MEDICINAL PRODUCTS WITH ANTIVIRAL EFFECT - Google Patents

Abstract

Improved antiviral medicines are those which contain as active component (S)- or (RS)-9-(2,3-dihydroxypropyl)adenine of the formula I <IMAGE> where appropriate combined with 9-( beta -D-arabinofuranosyl)adenine of the formula II <IMAGE> The compounds are effective medicines for viral diseases such as, for example, vaccinia, herpes simplex 1, herpes simplex 2, measles, vesicular stomatitis and the like. The advantages of these medicines include low toxicity of (S)- and (RS)-9-(2,3-dihydroxypropyl)adenine, their easy availability and high stability. Combination with 9-( beta -D-arabinofuranosyl)adenine reveals the synergistic effect of the two components. This results in a lowering of the dosage of the toxic and costly 9-( beta -D-arabinofuranosyl)adenine, with the same biological action being achieved, and its breakdown in the body is restricted.

Description

Remedies with antiviral effects

The invention relates to medicaments with an antiviral effect which, as active ingredient (S) - and / or (RS) -9- (2,3-dihydroxypropyl) adenine of the formula I contain.

Some derivatives of nucleosides, ie sugar derivatives of heterocyclic compounds, have antiviral effects, e.g. B. 9- (PD-arabinofuranosyl) adenine (hereinafter referred to as araA for short) of the formula II which has a pronounced effect against DNK viruses and ribavirin (1D-ribofuranosylI -1.2.4-triazole-3-carboxamide), which has broad antiviral activity against some RNA and DNA viruses (Science 177, 705 (1972); Chemotherapy 21, 505 (197.5). The cost of producing these remedies is, however, very high because of the complicated procedure, and their usability is also considerably restricted by the narrow concentration range in which they can be applied in view of their toxicity and degradation in the organism.

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

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

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 for short) is known. Both compounds can be prepared by reacting 1-0-p-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 product 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) are heated at 100 ° C. for 8 h, whereupon the mixture is evaporated in vacuo and the residue is removed from methanol is recrystallized. The product obtained is boiled with 80% acetic acid for 1 h and evaporated to dryness in vacuo, after which the dry residue is evaporated three times from 50 ml of ethanol each time and the remainder is crystallized from methanol.

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

(S) form: F. 202-203 OC, [] D25 = -35.4 ° (c = 1, water).

(RS) form: F. 207 - 208 ° C, UV spectrum (pH 7): Ämax = = 260 nm, E, ax - 14000 A min = 228 nm.

According to the CS copyright certificate No. ... (PV 1787-77), the (RS) form of the compound I (RS-DHPA) is obtained by heating adenine with a cyclic 1,2-carbomate of glycerol of the formula IV obtained with sodium or potassium hydroxide or carbonate in dimethylformamide or dioxane at 100 - 140 OG. 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) heated to boiling for 1 h and evaporated in vacuo; the residue is decolorized in 50 ml of boiling water with activated charcoal. After evaporation in vacuo, the residue is crystallized from methanol.

Yield: 60-70% (RS) form of compound I (RS-DHPA); F. 205 - 208 OC, UV spectrum (water): Amax = 260 nm, gmax = 14000.

Biological or pharmacological effectiveness indicates exclusively the D-glycero form, d. H. the (S) -form of the compound of the formula I, whereas the L-glycero form, d. H. the (R) -form, is completely ineffective. Since the racemic (RS) derivative contains 50% effective (S) form, it is accordingly about as effective as the (S) -enantiomer.

(S) -DHPA of the formula I exhibits high antiviral effectiveness RNA and DNA viruses with very low intrinsic toxicity. This effect is all the more surprising, as further, similar compounds up to one concentration of 100 / µg / ml have no relevant antiviral activity, e.g. B. derivatives of Adenine with another aliphatic component such as (RS) -2-hydroxypropyl-, 2-hydroxyethyl-, 2-aminoethyl-, ß -DL-alanyl-, (S) -3.4-dihydroxybutyl-, (RS) -3.4-dihydroxybutyl-, (RS) -threo-2.3.4-trihydroxybutyl-, (RS) -3. 5-dihydroxypentyl, 3-hydroxypropyl and 1,3-dihydroxy-2-propyl or 2,3-dihydroxypropyl derivatives of other heterocyclic ones Bases such as hypoxanthine, uracil, cytosine and thymine. Among other related compounds araA has an effect that is limited to DNA viruses, and ribavirin is also antiviral effective, but at the same time highly cytotoxic.

The invention also relates to pharmaceutical agents which contain the active ingredient of the formula I in the (S) or (RS) form and 9- (-D-arabinofuranosyl) adenine of the formula II in combination in a ratio of 1: 1 to 1000: 1 of the compound of the formula I to the compound of the formula II.

(S) -DHPA acts as a strong inhibitor of the enzyme adenosine deaminase, which is very common in cells, tissues and body fluids. Put it there (S) -DHPA or (RS) -DHPA neither for adenosine deaminase from bacterial cells (Escherichia coli, Salmonella typhimurium) are substrates from rabbit kidneys. Adenosine deaminase deaminates, for example, said substance araA (II) to the inactive derivative of hypoxanthin (Ann.N.Y.

Acad.Sci. 284, 60 (1977)), whereby its use as a Virostaticum is limited. The connection S-DHPA resp.

RS-DHPA inhibits this process; together with araA occurs a synergistic one Effect on as the antiviral effectiveness compared with the effect of the two Single active ingredients as such is significantly higher, d. H.

similar to z. B. with CO-viridarabine ((R) -3- (2-deoxy-p-D-erythro-pentaSuranosyl) -6.6.7.8-tetrahydroimidazo- (4,5d) -1.3-diazepin-8-ol); (cf. Ann.N.Y.Acad.Sci. 284, 9 (1977) and 284, 60 (1977) and Table 2).

Further advantages of the medicaments according to the invention reside in their excellent stability, which allows a sterilization of neutral, weakly acidic or weakly basic solutions at elevated temperature allows in the practical unlimited stability in a solid state and finally in easy accessibility, especially with the racemic derivative (RS) -DHPA.

Other advantages are the relatively simple manufacturing processes and low production costs. at industrial production are the manufacturing costs compared to the production of the listed known ones antiviral drugs significantly lower.

The pharmaceutical preparations according to the invention with a content an (S) -DHPA and / or (RS) -DHPA> which can also be used in combination with araA as an active ingredient can be in the form of powders, suspensions, solutions, spray emulsions, Ointments and creams are available and parenterally (intravenous, intradermal, intramuscular, intrathecal, etc.) or orally, rectally, intravaginally, intranasally or locally (skin, Mucous membrane, eye lesions, etc.) can be administered. The preparations can go through Mixing or dissolving of the active ingredient (s) together with pharmaceutically customary Auxiliary and / or carrier materials are produced, e.g. B. with aqueous or non-aqueous Solvents, stabilizers, emulsifiers, dispersants, suspension stabilizers, Wetting agents and the like, suitable additives (e.g. polyethylene glycols) and if necessary also coloring agents or flavorings. The preparations can vary depending on the type of administration and the type of disease contain different concentrations of active ingredient, but at least 0.1 and at most 100% by weight.

The medicaments according to the invention are for the treatment of viral diseases such as B. vaccinia, herpes simplex-1, herpes simplex-2, measles, vesicular stomatitis etc.

advantageously usable.

The pharmacological effectiveness of (S) -DHPA, (RS) -DHPA and theirs Combination with araA is illustrated in the following by means of examples.

Example 1 Antiviral activity of (S) -DHPA on the tissue cultures The cells of each culture were exposed to 100 times the dose of the virus that caused the infection infected by 50% of the cells (ccID50); 1 h after infection was (S) -DHPA at a concentration of 0 to 40 / ug / ml (in some cases up to 200 / g / ml) admitted. The ID50 was determined for each combination of host cells and virus, i.e. the concentration of (S> DHPA that resulted in 50% inhibition of the control experiment certain cytopathological effect of the virus is required (after L.J. Rosenthal u. I.L. Schechmaister, Tissue Kultur, p. 510, Academic Press, New York 1973). The results of this test are given in Table 1, from which 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 cytopathogenic effect of the virus in the presence of (S) -DHPA is a result of the decrease in growth the amount of virus; continuous single layer cultures of human skin fibroblasts (HSF cells) in Petri dishes were infected with the vesicular stomatitis virus (VSV virus) (4.510gloCCID50 / 0.5 ml / dish) inoculated and after 1 h at 37 ° C. the substance (S) -DHPA (100 / µg / ml) exposed. The cultures were incubated at 37 ° C. in different Time intervals frozen to -70 ° C and the titer of the virus in cell homogenates determined by plaque formation on L-929 mouse fibroblasts. The values of Fig, prove the sharp drop in the virus titer at 100 ug / ml (S) -DHPA compared to the control experiment, which is 4 orders of magnitude 48 hours after virus infection.

Example 2 Antiviral Effect of (S) -DHPA in vivo.

The antiviral effect was determined by the method of J. Gen. Virol. 5, 359 (1969) and J. Clin. Invest. 49> 1565 (1970) determined: 20-day-old female NMRI mice (average weight 15 g) were injected intranasally with VSV virus (2.5 loglOCCID50JO, Ol ml / mouse) infected; then repeated interperitoneal (S) -DHPA (2 mg / mouse, 133 mg / kg) at intervals of 1 hour, 1 day to 4 days after infection. The results are shown in Fig. 2 for the following 14 days. Repeated administrations of (S) -DHPA lead to a considerable increase in survival (67% versus 37.5 fJ in the control group); the difference between the two groups is beautiful Noticeable 9 days after infection. Since the 14th day after the infection, in no further deaths registered in either group. The experiments were carried out with different Dosages of (S) -DHPA performed; repeated doses of 0.08 mg / mouse (5.4 mg / kg) were ineffective, whereas repeated doses of 0.4 mg / mouse (27 mg / kg) resulted in a significant increase in survival compared to the control group (55% versus 37.5%) lead.

Toxicity of (S) -DHPA All test animals survived repeated administrations up to 1000 mg / kgd within 3 to 5 days without any symptoms. Accordingly, (S) -DHPA is very little toxic.

Example 3 Antiviral Effect of the Combination of (S) -DHPA and araA Contiguous single-layer primary cell cultures from rabbit kidneys (PRK cells) in Petri dishes were infected with vaccinia virus (4.5 loglOCCID50 / 0.5 ml / dish) and then the effect of (S) -DHPA, araA or exposed to the combination of both substances in concentrations of 1 to 100 / ug / ml. The cultures were incubated at 37 0C, after which the titer of the virus in different Time intervals by plaque technique on PRK cells in the same way as for the Vesicular stomatitis virus and L-929 mouse fibroblasts determined as in Example 1 became. The results shown in Table 2 show that the combination of (S) -DHPA (30 / ug / ml) and araA (3 / ug / ml) show a greater drop in virus titer than each of the two effective individual compounds causes itself; the combination is about 3 orders of magnitude more effective.

Example 4 Inhibitory Effect of (S) -DHPA on Deamination of araA by adenosine deaminase of the intestinal mucosa It was the method according to Biochem. J. 152, 681 (1975), based on the spectrophotometric measurement the decrease in absorption of araA at. = 265 nm is based.

For 50 nmol araA is -β265 = 0.11, for 100 nmol araA is 265 = 0.16. The results are shown in FIGS. 3 and 4, from them it can be seen that the delay in the cleavage of araA by (S) -DHPA increases the concentration of (S) -DHPA is proportional.

Example 5 Determination of the effective concentration of (RS) -DHPA im Blood serum after intraperitoneal and peroral application in mice. Two groups of 20 female NMRI mice each with an average weight of 12 g once 10 mg (RS) -DHPA in 0.2 ml physiological solution either intraperitoneally or administered orally. 0, 20, 40, LIo> 80, 160 and 320 min after application Blood samples were taken, which showed the antiviral effect of the blood serum on tissue cultures human skin fibroblasts infected with the vesicular stomatitis virus (cf. Example 1) were infected. The concentrations of (RS) -DHPA in the serum were made from the dilution of the serum necessary to achieve a 50% strength Inhibition was required, calculated according to the formula C = f ID50, where C is the concentration of (RS) -DHPA in the blood serum, f the dilution of the serum leading to 50% virus inhibition was required, and ID50 is the experimental value of the concentration of (RS) -DHPA, which leads to a 50% virus inhibition under the test conditions (20 g / ml), mean. All data are mean values of the samples from 3 mice. The test results are listed in Table 3.

Table 1 shows the antiviral effectiveness of (S) -9- (2,3-dihydroxypropyl) adenine in tissue cultures, from Table 2 the synergism of the antiviral effect of (S) -9- (2,3-dihydroxypropyl) adenine and 9 - (# - D-arabinofuranosyl) adenine on primary cell cultures of vaccinia virus infected Rabbit kidneys. Table 3 shows the concentration of (RS) -DHPA in blood serum indicated by mice after intraperitoneal and peroral application.

Fig. 1 illustrates the inhibitory effect of (S) -DIIPA the growth of vesicular stomatitis viruses in human skin fibroblasts; the ordinate indicates the virus titer (log10PFU / ml), where PFU is the unit of Plaque formation means, on the abscissa, the time after infection in hours; the circles correspond to the control experiment, the filled dots to the experiments with (S) -DHPA.

Fig. 2 illustrates the antiviral effect of (S) -DHPA with the Vesicular stomatitis virus intranasally infected mice; on the abscissa is the number of days after infection, the ordinate is the total mortality indicated; eDen in fi the circles again correspond to the control experiment, the filled ones Points of application of (S) -DHPA.

In Fig. 3 is the dependence of the cleavage of araA by adenosine deaminase graphically illustrated in the presence of (S) -DHPA; on the abscissa is the concentration of (S) -DHPA in / ug / ml, the ordinate shows the decrease in absorbance at A = 265 nm indicated (0 = 50 nmol araA, = 100 nmol araA).

Fig. 4 shows a Dixon diagram of the dependence of the cleavage on araA by adenosine deaminase in the presence of (S) -DHPA; is on the abscissa is the concentration of (SY-DHPA in / µg / ml, on the ordinate the reciprocal of the initial Cleavage rate (l / V) indicated (Ki = inhibition constant; O = 50 nmol araA, = 100 nmol araA).

Table 1 Antiviral efficacy of (S) -9- (2,3-dihydroxypropyl) adenine in tissue cultures virus cells ID50 (/ ug / ml) I DNA viruses: vaccinia PRK 10-20 vaccinia HSF 10-20 Herpes simplex-1 strain KOS PRK 10 Herpes simplex-1 strain KOS HSF 20 Herpes simplex-2 strain 333 PRK 4-10 Herpes simplex-2 strain 333 HSF 7-20 RNA viruses: vesicular Stomatitis PRK 7-10 Vesicular stomatitis HSF 2-7 Vesicular stomatitis HeLa> 200 Poliomyelitis-l HSF> 200 Poliomyelitis-l HeLa> 200 Coxsackia B-4 HeLa> 200 Coxsackia B-4 Vero> 200 Measles Vero 4-40 Sindbis BHK> 200 Abbreviations: PRK primary culture of rabbit kidneys; HSF human skin fibroblasts; Vero continuous Monkey kidney cell line; BHK continuous cell line from newborn hamster kidneys.

Table 2 Synergism of the antiviral efficacy of (S) -9- (2,3-dihydroxypropyl) adenine and 9- (-D-arabinofuranosyl) adenine on vaccinia virus infected primary Cell cultures from rabbit kidney virus titre remedies (logl0PFU / ml) after the time Infection (d) 1 2 3 1. Control (not incubated with the medicinal product) 3.6 5.0 5.3 2. (S) -DHPA (30 1g / ml) 3.6 4.9 5.4 3. araA (3 / ug / ml) 2.1 4.0 5.0 4. (S) -DHPA (30th / ug / ml) + araA (3 / ug / ml) 2.0 1.8 2.1 Table 3 Content of (RS) -DHPA in blood serum of mice after peritoneal and oral administration time concentration of (RS) -DHPA after application (min) (1 g / ml serum) intraperitoneally orally O 100 100 20 1200 1800 40 800 600 80 400 400 160 200 400 320 200 400 The invention In summary, concerns remedies with antiviral effects that are used as active ingredient (s) (S) - and / or (RS) -9- (2,3-dihydroxypropyl) adenine or a mixture of these compounds with 9- (-D-arabinofuranosyl) adenine as a further active ingredient in a ratio of 1: 1 to 1000: 1 included.

The medicaments according to the invention are for the treatment of viral diseases such as vaccinia, herpes simplex-1, herpes simplex-2, measles, vesicular Stomatitis and the like are excellently suited. The benefits of these remedies include the low toxicity of (S) - and (RS) -9- (2,3-dihydroxypropyl) adenine, their slight Accessibility as well as their high stability. In combination with 9- (la -D-arabinofuranosyl) adenine a synergistic effect occurs. This can reduce the dosage of the toxic and expensive 9 - (* D-arabinofuranosyl) adenine with the same pharmacological effect be lowered, as its breakdown in the organism is reduced.

Claims (4)

Claims 1. Medicinal product with antiviral effect, characterized in that it is (S) - and / or (RS) -9- (2,3-dihydroxypropyl) adenine of the formula I as the active ingredient in an amount of 0.1 to 100% by weight, based on the total weight. 2. Remedy according to claim 1, characterized in that there is 9- (ß-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 the formula I to the compound of the formula II. 3. Remedy according to claim 1 or 2, characterized in that it is present in dosage units for a dosage of 0.1 to 1000 mg / kg live weight per single dose are suitable. 4. A method for producing the medicament according to any one of the claims 1 to 3, characterized in that the or the active ingredients, £ sat. with the addition of pharmacologically more suitable, more common Auxiliary and / or carrier materials, are brought into a form suitable for administration.