CS199093B1 - 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

Certain nucleoside derivatives, i.e. sugar derivatives of heterocyclic compounds, have an antiviral effect. For example, 9- (β-D-arabinofuranosyl) adenine (in other araA) has significant activity against DNA viruses or ribavirin (1- (-D-ribofuranosyl) -1,2,4-triazole-3-carboxamide) has broad antiviral activity on some RNK- and DNKviruses (Science 177. 705 (1972); Chemotherapy 21 · 505 (1975) ·

The cost of manufacturing these drugs due to the complexity of the 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 disadvantages are overcome by the medicament according to the invention, which comprises (S) - or (RS) -9- (2,3-dlhydroxypropyl) adenine of the formula I as active ingredient.

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Effective ^- toxic concentrations of active ingredient are from 0.1 m ^g to 1000 mg / kg bodyweight per dose hmotnosti'při, 'the content of active ingredient to pharmaceutical carrier may be in the range of 0.1 to 100% by weight.

The process for the preparation of the (S) -enantiomer and the racemic (RS) form of the compound of formula I (hereinafter S-DHPA - or RS-DHPA) is known.

Both compounds can be prepared by reacting 1-O-p-toluenesulfonyl-2,3-O-isopropylidene-D- (or -DL -) glycerin of formula II p-ch ₃ c ₆ h ₄ s ₂ and ₂ ch-ch ₂ . Y «· ^{Ы зС} CH ₃ with the sodium with ^divided ateninu in dimethyl-formamide at ¹⁰⁰ ° C and boiling the resulting ^O - intermediate 8 (56 acetic acid (Collection Czech. Chem. Commun., £ 0, 187/1975 /) · thus e.g. ae, 11.4 g (40 'mmol) of the compound of formula II and 7,8 g of sodium salt of adenine (50 mmol) is heated in dimethyl ^formamide (1 ^{0 mL)} for ^{8 h d.} at 1 ° ⁰ C, evaporated in va ^to uu and ZB ^y tek kryetaluje from methanol. the product is boiled for 1 h with 8056 acetic acid - concentrated in vacuo. the residue was evaporated three times with 50 - ml- ethanol, and the residue was - crystallized from methanol. 50-60% of (S) or (RS) -9- (2,3-dlhydroxypropyl) adenine is obtained. (S) -Frma: - mp 202-203 ° C [α] D 25 -35.4 ° (c = X) ^{in a).} CCC) -form: mp 205-208 ° C. UV-Spectrum ^- (pH 7) J λωχ “260- mm, λ ^max ' ¹ 4 ⁰ ° 0, ^Amin = ^{- 228}

According to MS. 191-656, wherein the (RS) form of the compound I - (RS-DHPA) is obtained by heating adenine with a 1,2-cyclic glycerin carbonate of the formula -III ® 2 - CH - CH 2 OH

III

and hydroxide - or sodium or potassium carbonate in dimethylformamide or dioxan ^p s 80 to 140 ^- C. So ^EXAMPLE. Adentau mixture of ^{(1, 3,} 5 g, ^l mmol ^{W) -} glycerin - ^^ cykHckéfo carbonate (III) (2.0 .g), sodium carbonate (0.3 -g) with dimethylformamide ( 25 ml) was heated under boiling for 1 h, evaporated - under vacuum - the residue in 50 ml of boiling water was decolorized with activated carbon. After evaporation in vacuo, the residue of the crystal is from methanol: 60. to 70% (RS) -form ^- Substance ^Y I (RS-205 TT-DHPA - Oz 206 ° C, W-SPE microns (water) - = ^-260 nm max £! "4000- exhibits biological activity only The D-glycero- (S) -form of the compound of formula (I), whereas the L-glycero- (R) -form is completely ineffective, since the racemic (RS) derivative contains 56% The (S) -DHPA of formula (I) is characterized by a high anti-viral activity against RNK1 DNA viruses with very low intrinsic toxicity.

199 093 rather that other similar compounds, either ³ adenine derivatives having another aliphatic component, eg (R3) -2-hydroxypropyl, 2-hydroxyethyl, 2-aminoethyl,. -DL-alanyl-, (S) -3,4-dihydroxybutyl, (RS) -3,4-dihydroxybutyl, (RS) -threo-2,3,4-trifluoromethylbutyl. (RS) -3,5-dihydroxypentyl, 3-hydroxypropyl and 1,3-dihydroxy-2-propyl or -2,3-dihydroxypropyl derivatives of other heterocyclic bases, eg hypoxanthine, uracil,. cytosine and thymine show no significant antiviral activity up to a concentration of 100 µg / ml. Among other related compounds, said araA has an effect limited to DNA viruses and said ribavirin is highly cytotoxic.

Preferably ^p ředmětnáho ^medicinal preparation is His ^considerable N ^and SM-AC, which ^and amortized and Listeria-like! neu ^t parenterally, weakly acidic or weakly alkaline solutions at elevated temperature, a virtually unlimited stability in solid state and finally its accessibility, in particular by resolving a racemic derivative of (RS) -DHPA.

Another advantage is its relatively simple preparation and low production costs. For industrial production, ee is much lower. production costs compared to the preparation of the above-mentioned known antiviral preparations.

Pharmaceutical preparations containing (S) -DHPA or (RS) -DHPA as active ingredient may take the form of powders, suspensions, solutions, sprays, emulsions, ointments and creams and may be used for -parenteral (intravenous, intradermal, intramuscular, intrathecal and .) or for oral, rectal, intravaginal and intranasal administration or topical application (skin, mucosal, ocular, etc.). - These. The preparations may be prepared by mixing or dissolving the active compound (s) with pharmaceutically acceptable carriers for such purposes as, for example, aqueous or non-aqueous solvents, stabilizers, emulsifiers, suspending agents, wetting agents, suitable excipients (e.g. polyethylene glycols) and if desired 1 with coloring or flavoring. The preparations may contain different concentrations of active ingredient depending on the nature of the administration and the nature of the disease, but at least 0.1% and at most 100% by weight.

V. Others The biological activity of (S) -DHPA and (RS) -DHPA is illuminated. in the examples without being limited thereto.

Example 1

An ^AL n ^t Ivira ^{also learning} to structure the ^(S) - ^DHPA on rope culture ^and CH

Cells in ^{the DE} and to URE ^ ly in ^the feed is ^f ^ ston obkem virus dose infective for otřebné ^p i ^c% of cells for 1 h after infection were added (S) -DHPA at a concentration of 0-40 <.mu.g / ml (in in some cases up to 200 «^ / βΙ). It was determined for each combination of host cells and virus

ID 50 * concentration of (S) -DHPA required to 50% suppress the cytopathic effect of the virus as determined by control (according to L.J. Rosenthal, I.L. Schechmalster, Tissue

Culture, p. 510, Academic Prese, Nev. 1973). The results of these experiments are shown in Table I. It is clear that some viruses, such as vaccinia, herpes simplex type 1 and 2,

Measles, vesicular stomatitls are very sensitive to (S) -DHPA, while others such as poliomyelltis, Coxsackie, Sindbis are unaffected. The inhibitory effect of the cytopathogenic action of the virus in the presence of (S) -DHPA results from a reduction in virus growth: continuous monolayer cultures of human skin fibroblasts (HSP-cells) in Petri dishes were inoculated with vesicular stomatitis virus (VSV-virus) (4.5 log ^ Q) 0010? // 0.5 ml / ml) and after 1 h at 37 ° C exposed to (S) -DHPA (100 <µg / ml) (CCXD? Q is the dose needed to infect ⁵⁰ % cells ^{) )} . Kuitury ^{were i} n ^{k b} u ^y alkylated pm ³ 7 ° ^C in various SsovýS intarvataS ^- frozen at -7 ° ^C and ⁰ r ^tit virus in cell homogentoeS determined toorbou ^pl and ^{Ku -} L-929 myěích fibrobtastach. The data in FIG. ^{1 d} of ^the arts ^t uj ^s sdný podes ^{- tit} ru virus at. ^ / nú. ⁽ S ⁾ -D ^Hp A compared to control, four orders of magnitude 48 h after virus infection.

Example 2

The antiviral effect of (S) -DHPA in vivo was verified by the method of J. Gen. Virol. 359 (1969) and J. Clin. Invest. 42, 1565 (1970): 20 day old NMRI female mice - (mean weight 15 g) were infected intranasally with VSV virus (2.5 log ^ OCID ^^ / 0 ^ 0.1 ml / mouse) and then re-administered intraperitoneally (S) -DHPA (2 mg / kg, 133 mg / kg) at 1h intervals, 1 day to 4 days after infection. Results These are shown in Figure 2 for the following 14 days. Repeated doses of - (S) -DHPA produced a marked prolongation of survival (67% versus 37.5% in the control group); the difference in both groups is apparent already 9 days after infection. From the 14th day after infection no further deaths were recorded in both groups. These experiments were performed 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) induced a significant prolongation of survival. versus control group (55% vs 37.5%). Toxicity (S) -DHPA: All experimental animals survived repeated doses up to 100 mg / kg / day - for 3-5 days without any signs of disease.

Thus, (S) -DHPA is very little toxic.

Example 3

Determination of effective (RS) -DHPA concentration in blood serum after intraperitoneal and oral administration to mice. Two groups of 20 mice of NMRI female with an average weight ^of 12 g was administered Disposa ^ZOV of ^about 0 * After ¹ mg of (RS) -DHPA in ^0, 2 ml of solution ^B fyztotogickSo mu intoaperitoneálně or * orally. Blood samples were taken at times 0, 20, 40, 80, 160 and 320 ^m in ^u * t after the ^AP and publications for antivirus ^{AL nus Prior} INEK bevního ^e r ^{ь у.} fixed to ^the U ^L tatóových tours kožních- human fibroblasts infected with vesicular stomatitis (see Example 1). Serum (RS) -DHPA concentrations were calculated from the serum dilutions required to achieve 50% virus inhibition according to formula c = .f. . id ₅₀ , where - - C Is the concentration of (RS) -DHPA in blood serum, f Is the serum dilution required to achieve

50% inhibition of the virus and is an experimental value - the concentration (RS) -DHPA inducing

50% virus inhibition under experimental conditions (20% u / m) · All determinations are the average of the samples from three masses. The results of the experiment are shown in Table 3. Table 1 shows the anti-viral activity of (S) -9- (2,3- dihydroxypropyl) adenine in tissue cultures ·

Table 2 shows the level of (RS) -DHPA in the serum measured after intraperitoneal and peroral administration.

Figure 1 is a graph showing inhibition of the effect of (S) -DHPA on the increase in vesicular stomititis virus in human skin fibrous areas. The y-axis shows the tltr virus (log 10 PFJ / m), where PFU is the plaque formation unit; the x-axis shows the time after infection in hours; - control, -O- after application of (S) -DHPA; Figure 2 is a graph expressing the arthiral effect of (S) -DHPA in vesicles infected with intranasal vein-steroid virus; the x-axis shows the number of days after infection; on the y-axis, the total intensity in% is plotted; - control, -O- (S) -DHPA; Figure 3 is a graphical representation of the araA cleavage of adenosine deaminase in the presence of (S) -SHPA; the x-axis shows the concentration of (S) -DHPA in% g / ml, the y-axis shows the decrease in absorbance at λ = 265 nm; - 50 nmol araA, -0- 100 nmol araA; Figure 4 is a Dixon diagram of the araA cleavage of adenosine deaminase in the present. (S) -DHPA; the x-axis shows the concentration of (S) -DHPA in% iu / mL _t on the y-axis the inverse of the initial cleavage rate (1 / V); K i = inhibitory constant; - · - 50 nmol ara A, -0- 100 nmol ara A ·

Table - 1

(S) -9- (2,3-dihydroxyprspyl) adenine activity in tissue cultures

Virus DNKviry: Cells i ^ ^d 50 Q ^ m.) Vaccine PRK 10 to 20 Vaccine HSF 10 to 20 Herpes simplex- 1 strain KOS. PRK 10 Herpes simplex- 1 strain KOS HSF 20 May Herpes simplex- 2 strain 333 PRK 4 to 10 Herpes simplex- 2 strain 333 HSF 7 to 20 RNKvvry: Veeicular stomaaitis PRK 7 to 10 Veeicular Atomarnis HSF 2 to 7 Total hundred hundred ths HeLa > 200 Polissnyeltie-1 • HSF > 200 Poliomeeit is-1 HeLa > 200 Coxsackie B-4 HeLa > 200

б

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Coxsackie B-4 Věro > 200 Measles Věro 4 to 40 Sindbis BHK > 200 Abbreviations i PRK ..... rabbit kidney direct culture; HSF. ···· human skin fibroblasts:

Verily ... a continuous line of monkey kidney cells; BHK ..... continuous hamster kidney cell line.

Table 2

The (RS) -DHPA level in the blood aeroid is now following intraperitoneal and oral administration

бае (min) Concentration (RS) -DHPA (jpg / ml serum) after application intraperitoneal oral 0 100 ALIGN! 100 ALIGN! 20 May 1200 1800 40 800 600 80 400 400 160 200 400 320 200 400

P ft E D Μ £ T FACTS

Claims (4)

P ft E D Μ £ T FACTS Medicinal product and antiviral action, characterized as being active (S) - or (RS) -9- ( 2, 3-dihydroxypropyl) adenine of component I 4 drawings (I) 199 093 1--1--1 --- 1--1--1 — I — i — г г / О 5 6 7 8 9 1011 121314 Giant. 3 Giant. 2