CS199095B1 - Antiviral agent - 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

The present invention relates to a medicament having an antiviral effect.

Some nucleoside derivatives, i.e., sugar derivatives of heterocyclic compounds, exhibit an antiviral effect. For example, '9- ((1-D-arabinofuranosyl) adenine (araA) has a distinctive^atactivity^proti DNK-viruses or rlbavirin (1-⁽ (Z-D-ribofuaanosyl) -1,2,4 triazole-3-carboxamide) has broad antiviral activity on some RNK- and DNK-viruses (Science 177, 705 (1972); Chemotherapy 21, 505 (1975)). The cost of manufacturing these drugs due to the complexity of preparation is considerable and their use is greatly limited by the narrow range of concentrations at which they can be applied due to their toxicity and degradation in the body.

^These drawbacks it by ^h ^ ^dy ODS Tranum l ^ec e Nu prostfefck ^b 'according to WNA ^ .ezu, its ^{Z p t} Ata paragraph is that it contains as active ingredient a mixture of (S) - or (RS) -9- (2, 3-dihydroxypropyl) adenine of formula I

199 095

199 095 β 9- (β-D-erabinofuganoeyl) adenine of formula II

(II) in a 1: 1 ratio. to 1000: 1- of a compound of formula I to a compound of formula II.

^Teaches variables Netox ^even if CKE concentration ^ ve ^ with clippers are ^0.1 to ¹⁰⁰⁰ mg / kg žiy ^s weight at one dose, whereby "active ingredient content relative to farmaucetickému carrier may be in the range of 0.1 to 100% by weight.

The procedure for the preparation of both the (S) -enantiomer and the racemic (RS) - form of the compound of formula I (hereinafter referred to as S-DHPA or RS-DHPA) is known.

Both compounds can be prepared by the reaction of 1-O-p-tolueneulfonyl-2,3-O-isopropylidene-D- (or -DL-) glycine of formula III p-CH ₃ C ₆ H ₄ SO ₂ 0 CH in

/ \ HJC · _CH3 (III) sodium salt adentau in ^{dimethylaminosaccharin} l ^f ORMA ^id at ¹⁰⁰ ° C and the boiling VZN ^ikléh the margin ^ genus ^ US £ 056 acetic acid (Collection. Czech. Chem. Commun., 40, 187 (1975)). Thus e.g. 11.4 - g (40 mmol) of compound III, 7.8 sodium salt of adenine (50 mmol) was heated in ^formamide dimethy1 ⁽¹ °° - ^{ml) for 8} h at 100 ° C, evaporated in ^ va ^k uu. and ZBY ^ K: Cr ^y s ^t aluje from. meWanol. The product "boils LHA 8 (56 acetic acid, evaporated in vacuo, the residue was evaporated three times with 50 ml of ethanol s- and ee residue crystallized from methanol. There were obtained 50 to 60 56 (S) b n ^{e of} RS) -9- ( ^2, 3-dihydroxypropyl ^oxyp r ^a propyl) adenine ^ u ^. (S) -form: mp 202-203 -35 ⁴ DEG (c = 1, water). ⁽ RS) -Form: ^b .t. ^{- 2} ° 7 to ²⁰⁸ ° C. UV-JSP-to.um ^{(p H 7) AmnY} ^ ⁿⁿ = ^260>

' ¹⁴⁰⁰⁰ ' ^A nn ^{3,228 nm} ·

Podle.čs. 191 656, wherein the (RS) form of the compound I (RS-DHPA) is obtained by heating adenine with the glyceryl β-cyclic carbonate of formula IV

CH 2 -CH-OLOH · 1. J \ / (IV) c

II o

and sodium or potassium hydroxide or carbonate in dimethyl tertiarybide or dioxane at 80 to 140 ° C. For example, a mixture of adentau (1.35 ^g , 10 mmoU, glycerol-1'-cyclohexyl tartonate (IV) (2.0 g), sodium carbonate (0.3 g), and dimethylformamide (25 mL) is heated for 1 hour. h at boiling point, evaporate in vacuo and the residue in 50 ml of boiling water decolourised with activated carbon After evaporation in vacuo the residue was crystallized from methanol to give 60-70% of (RS) -format I (RS-DHPA), bt 205 206 ° C, UV-ektrum with ^p (water): «2 ⁶ ° NRN = 14000. the biological účinnost.vykazuje · exclusively D-glycero i.e. (s) -forma.látky formula I, whereas L-glycero Since the racemic (RS) derivative contains 50% of the active (S) -form, it is of the order of magnitude as effective as the (S) -teriomer.

The (S) -DHPA · of formula I is characterized by high antiviral activity against both RNK- and DNA-viruses and very low intrinsic toxicity. This effect is entirely unexpected, especially since the ^Star other similar ^compounds, either ³ derivatives edentate another and aUckou ^ s ^^ ou, e.g. ^(R) - ^{2-hydroxy p} ro ^p y ^{i 2} - ^{h h} hy ydroxye ^{i, 2-} amieoe ^{th i i,} fit - ^D L-α ⁱ anУ ⁱ -. ^{(S) -3,4-d} IHY ^d roxy ^b u ^h У ^{i, (RS)} -3,4-dihydroxybuhyl, (R ^^ - dihydrcxybutyl (RS) threo-2,3,4-trlhVdroxvbuh.yi. ( RS) -3,5 ihydroχyptetyi ^d, 3-hydroxypropyl, and 1,3-dihydroxy-2-propyl or 2,3-dihydraxypropylderlváty · other heterocyclic bases, eg. hypoxanthine, uracil, and thymine cytoainu no significant antivlrál ^ until concentrations of 100 µg / ml did not show.And other related compounds, araA has this effect limited to DNA viruses and highly cytotoxic ribavirin.

^(S) -D ^HPA is a Nu ^l inhibitor and ^{d and} e eeos ^{deaπú.eae-YR} in his enz ^y ^ i ^ o ^b rozštten unkách, tissues and body fluids. Neither (S) -DHPA nor (RS) -DHPA is a substrate of bacterial cell adenoin deaminase (Seche-eh-coli, Salmonella typhimurium) or rabbit kidney cells. Adteosiд-dtaeinaaa deamleujt, for example the araA to the inactive hypoxanthine derivative (Ann. N. Ϊ Acad · Sel. 284. 60/1977), limits its use as virotta. Substance S-DHPA and RS-DHPA this process is inhibited and · therefore acts in synergism with araA antiviral action strongly .zvýšeným · 8 in comparison with both active ingredients samotým ^ ^i. E. Similar as e.g. ⁰ 0 dаrа-virus ^{and b} in ( ^(R) - ^{3- (2-deoxy fi -} d-trУ ^{th ro-h} of pen urαeosyl ^f) -3,6,7, t-hehrahy ^d roim ^d-azo (4,5-d) - (l, ³ (diazpin-8-ol): see Ann, NY Acad Sci 284. 9 (1977) and 284. 60 (1977) - see below Table 1.

The advantage of the present medicament is to reduce the content of the active substance araA while attaining the same antiviral effect due to the synergistic action of (S) - or (RS) -DHPA and thus to the reduction of toxicity. araA-induced preparation.

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Another advantage is the relatively simple preparation and low production cost of the fabric. of formula I, which is used in excess in the combination of the two active ingredients.

Pharmaceutical preparations containing (S) -DHPA or (RS) -DHPA and araA as active ingredients may take the form of powders, suspensions, solutions, sprays, emulsions, ointments and creams and may be used for parenteral (i.e. administration, oral, rectal, oral, rectal, oral, inhalation or topical administration (skin, mucosal, ocular, etc.). These preparations may be prepared by mixing or dissolving the active compound (s) and pharmaceutically acceptable carriers for such purposes as, for example, aqueous or non-aqueous solvents, stabilizers, emulsifiers, dispersants, suspending agents, wetting agents and the like, with suitable additives (e.g. e.g., polyethylene glycols) and, if necessary, coloring or flavoring agents. The preparations may contain varying concentrations of the active ingredient depending on the nature of the administration and the nature of the disease, but at least 0.1 and at most 100% of the mono-mono-hormone.

In the following, the biological activity of (S) -DHPA or (KS) -DHPA with araA is illustrated in the examples without darkening. whatever he omme.

EXAMPLE 1 ACOCOvLtIcE Effect of Combination of (S) -DHPA a. araA.

Corresponding monolayable direct cultures of Ikaline kidneys. (PPK-buukk) in Petri dishes were seeded with lvenmlVcinit (1 log ₁₀ ^cc 0D ₅ q / 0.5 dQ / mc) for 1 h at 37 ° C and then developed (S) -DHPH, aarA 1сПь Ikominacc (both 11 ° (, i lo at concentrations of 1 to 000 µg / m. Kultuyi УуУ (tnkbOvvtyy at. 37 ^° C) and boiling titer determined at various time intervals by plaque technique on PKK-b fats as reported for vesicular virus 80 µO / ml. And L-929 / V fibrobl8080 as shown in Example 1. The results shown in Table 1 showed that the combination of (S) -DHPA (ЗО ^ д / Ш) and araA (3 mg / ml) was obtained. ) causes a greater decrease in tHru virus than any of the active substances alone (is about 3 orders of magnitude more accountable).

Example 2

Inhibic. effect of (S) hDHPA. to dominate the araA cdeno8ic-dna / itcsot intestinal mucosa.

The method according to. Biochem. J. 152. 681 (1975), based on an apectrophotometric measurement of the decrease in araA ace at ²⁶⁵ . nm. Pro 50 nmol araA ≥ 265 = pro. 100 nmol. araA ~ °> 1 ⁶ · ^In ββ ^day are listed on. FIGS. 3 and ^{4, and} with ^d Toer шmn ^{C UTI} that. inhibition of araA cleavage by (S-hDHPA) is proportional to β-πD ηθοί (S) DDHPA.

Still in Table 1 is. listed .synergism. the antiviral effect of (S) -9- (2,3-dilyldimoxypropyl). adenif and 9- (β) - D-arcbitsfurctoayl) on the primoculture of rabbit kidney cells and vaccinia virus.

Figure 1 is a graph depicting the effect (S-DDhPA) on the growth of the yesiktillum atomaitis virus in human skin fibroblasts, the y-axis shows the virus titer (log10. PFU / 10Q), where PFU is the unit plaque formation, the x-axis shows the time after infection in hours, - control, - after (S) -DHPA application, Figure 2 is a graph showing the antiviral effect. (S) -DHPA on In the case of the infected cells of the vestitis virus, the x-axis shows the number of days after infection, the y-axis shows the total potency in%; - · - -o- (S) -DHPA; is a graphical representation of the araA cleavage of cdetosit-deamincsot in the .alpha. (S) -DHPA; x is the concentration of (S) dDHPA in .mu.g / ml on the y-axis; with absorbance at λ = 265 nm, - · - 50 nmol.

199,095 araA, -100 nmol araA; Fig. 4 Dixon plot of araA cleavage by adenosine deaminase in the presence of (S) -DHPA; the x-axis shows the concentration of (S) -DHPA in µg / ml, the y-axis inverse of the initial cleavage rate (1 / V); = inhibition constant;

- · - 50 nmol araA, -o- nmol araA.

Table 1

Synergism of the antiviral effect of (S) -9- ( ^2,3- dihydroxypyryl) -adenine and 9- (β-D-arabinofuranotyl) adenine on the primoculture Vaccine virus infected rabbit kidney cells

Medicinal product Virus titer (log-Lo m / m) time after infection (days) 1 2 3 1. Co-control (not incubated with active substance) 3.6 5.0 5.3 2. (S) -DHPA (10 µg / ml) 3.6 4.9 5.4 3. araA (3 µg / ml) 2.1 4.0 5.0 4. (S) -DHPA (30 µg / ml) + + araA (3 µg / ml) 2,0 1,8 2.1

б

188085

Claims (1)

Р 8 Е D Μ Т ТPL Medicinal product having an anti-viral action, characterized in that it contains, as active ingredient, a mixture of (S) - or (RS) - (M 2, 3-dihydroxypropyl) adenine of formula I (I) and 9- (D-arabinofuranoeyl) adenine of formula II (II) in a ratio of 1 to 1000: 1 of the compound of formula I to the compound of formula II; 4 drawings 199 095 ---- 1 ---- Г — I ---- 1 ---- 1 ---- 1 ----! ----- 1 ----- 1 Giant. 1