NL8204175A - LINK WITH ANTIVIRUS ACTIVITY AND METHOD FOR THE PREPARATION THEREOF. - Google Patents

Description

i * VO 3813

Compound with antivirus activity and method for its preparation.

The invention relates to a new compound with an antivirus activity, namely 1-N- [2- (2-dimethylaminoethoxy) acetyl-amlno] adamantane phosphone acetate of the formula 1 of the formula sheet. Phosphonacetic acid has anti-virus activity against vaccine 5 herpes, type 1 herpes and type 2 herpes which has been known for a long time.

Reference is made in this regard to Appl. Microb. 26, 264-7 (1973), Invest. Ophthalmol. 12, 532-4 (1973), as well as Antimicrob. Agents and Chemoth. 7, 285-8 (1975), as well as 9, 308-11 (1976).

The activity of 1-N- [2- (2-dimethylaminoethoxy) acetylamino] -10 adamantane, also known as tromantadine, against the same viruses has also been described previously in Arzneimittelforschung 23, 577 (1973) and 23, 718 (1973).

It has now been found that the compound of formula 1, or tromantadine phosphonacetate, has a surprisingly strong activity against herpes viruses compared to the above two compounds and an activity of the same magnitude against the yaccin virus. The invention therefore also relates to pharmaceuticals with an antivirus activity containing the compound of the formula 1 as the active ingredient.

The invention furthermore relates to a process for the preparation of tromantadine phosphonacetate, namely by converting phosphonacetic acid and tromantadine in a molar ratio of substantially 1: 2 into polar solvents such as water or, more preferably, lower alcohols and isolating the salt thus formed by the solution. to evaporate or to add a non-solvent therefor. This reaction is preferably carried out at room temperature. The following example further illustrates this facet of the invention.

EXAMPLE

a) 1-N- (2-chloroacetylamino) adamantane.

50 g of 1-aminoadamantane hydrochloride was placed in a 10% aqueous solution of 21.3 g of sodium hydroxide, after which a solution was added under magnetic stirring and cooling with ice below 5 ° C. 3 3 of 21.5 cm chloroacetyl chloride in Π) cm. CH ^ Cl ^. This reaction mixture was allowed to stir for about 2 hours, after which the organic phase was separated, 8204175-2- washed with water, dried with magnesium sulfate and the solvent was distilled off under reduced pressure. The resulting residue was recrystallized with diisopropyl ether: isopropanol 80:20, after which the product obtained in a yield of 80% had a melting point of 122-123 ° C.

b) 1-N- [2- (2-dimethylaminoethoxy) acetylamino] adamantane (tromantadine).

To a solution of 31.3 g (0.35 mol) dimethylaminoethano1 in 150 ml anhydrous tetrahydrofuran was added 8.4 g (0.35 mol) NaB while stirring and maintaining the temperature at 15-20 ° C.

After no more hydrogen developed, 40 g (0.17 mol) of 1-N- (2-chloroacetyl-amino) adamantane was then added and the whole was boiled for 15 hours, after which it was poured into ice. The organic phase was extracted with ethyl ether, washed with water and dried with 10 CO 2. The solvent was distilled off under reduced pressure and the residue purified by chromatography on silica, eluting first with diethyl ether and then with ethanol.

The pure product, a dense yellowish oil, was isolated in 53% yield.

c) Tromantadine phosphonacetate (1).

20 g (0.142 mol) of phosphonacetic acid and 80 g (0.285 mol) of tromantadine whezdai dissolved in 120 cm of absolute ethanol. The resulting solution was evaporated to dryness under vacuum. This yielded a low-melting crystalline material (melting point ca. 33-40 ° C), which was very hygroscopic and very soluble in water, methanol and ethanol.

25 Elemental analysis

For C34H5N4P09 (mol. Wt. = 700.83),% C = 58.26; H = 8.77; N = 7.99 found% C = 57.78; H = 9.04; N = 7.82 IR spectrum (nujol; the values for the absorption bands are shown in cm *): bond N - Hi wide, bands 3550-2500 o - h; binding C = 0 (acid) wide, band 1700-1670 C = 0 (amide) i -8-2 0 4 1 7 5 · «··· * -3- NMR H ^ spectrum (recorded in CDCl ^, internal standard 3MS, the chemical shift values are shown in i): 2.4 (s, 121, 2 (CH3) 2); 2.6-3 (m, 41.2 (C12-N)); 3.3-3.9 (m, 101.2 (C0-C12-0), 2 (^ -0); P-C12-C001); 6.8 (s, 11, mobile C01) 8.3 (s, 41, mobile NI).

10 The toxicological pharmacological properties of tromantadine phosphonacetate (referred to below as AFP-723) are as follows: Cell toxicity

The neutral red incorporation method was used, which indicates the vitality of the cells in contact with the drug.

For a more accurate determination of the type and extent of inhibition, the following parameters were also examined: a) growth time and cell division capacity; B) incorporation of 1-thimidine into the DNA; C) incorporation of H-uridine into the RNA; D) incorporation of C-leucine into the proteins.

The results obtained demonstrated an absence of toxicity up to amounts of 250 µg drug / cm medium.

Above this concentration, a slow reduction of the incorporation of the radioactive labels occurs accompanied by an approx. 50% feremination of the protein synthesis above 500 µg / cm.

The inhibition of the DNA appears to be considerably less strong; these 3 are only reduced by 50% at concentrations of about 100 µg / cm.

Human diploid cells j (Wi-38) were used in primary or secondary cultures while monkey kidney cells were used in continuous culture.

. The toxicity values shown above give a very favorable therapeutic coefficient in combination with the very high antivirus activity shown below.

-8-20 4 1 75 -4-

Local tolerance

This was determined using both an aqueous solution containing 5% of the compound and a 5% ointment in white petrolatum.

Both the aqueous solution and the ointment were applied to the backs of New Zealand rabbits shaved with an electric razor twice a day for four weeks. Possible erythemas and edemas were checked daily; only around the 20th day of treatment, a low percentage (10%) of the rabbits treated with the aqueous solution showed mild transient erythema, which did not increase further in the following days until the end of treatment.

Absence of systemic effects

The same rabbits were used as in the previous experiment. After four weeks of treatment, possible effects on circulation and respiration were assessed. The most important hematochemical and biochemical parameters were also determined. The main organs were removed and a histological examination was performed. No effect on circulation or respiration was observed. The hemato-20 chemical and biochemical parameters were found to be within normal limits, and the histological examination also revealed no pathological change.

Antivirus activity

Human diploloid cells (WI 38) and monkey kidney cells grown in Petri dishes were used for this. As the infectious virus, three DNA-containing viruses (vaccine, herpes 1 and herpes 2) as well as three RNA-containing viruses (influenza virus A, polio virus and synovia virus) were used.

The AFP-723 connection inhibited it. DNA-containing vaccine virus.

And herpes viruses already at concentrations of 1-5 µg / cm and 100 µg / cm ^ respectively with a TCID ^ q of about 50 µg / cm ^.

When the cells with 5 jig / ca? AFP-723 had been pretreated, the infection rate decreased by 99.9% (from 10 ^ 3 to less than 10).

The antivirus activity of the present compound takes place intracellularly at an intermediate stage of virus morphogenesis, -8-2 0-4 1 Z5 ______________________ -5- s * as could be demonstrated by electron microscopic means.

3

At concentrations of 50-100 µg / cm, AFP-723 blocks the vaccine virus when it is in an immature form, while herpes 1 and herpes 2 can be blocked in the intranuclear capsid stage. A concentration of 10-50 µg / cm 2 completely inhibits the production of plaque-forming viruses.

The following table presents the data presented above in a diagram comparing equimolar doses of phosphonic acetic acid (PPA) and tromantadine.

10 Infectious virus Vaccine Herpes 1 Herpes 2 AFP 723 +++ +++++ +++++ PPA +++ ++ ++

Tromantadine +++ +++ +++

The activity of AFP-723 against herpes viruses is surprisingly high compared to that of the comparative drugs, while its activity against vaccine virus is of the same order of magnitude.

These properties suggest that AFP-723 can be used selectively in fighting herpes virus infections and especially Herpes labialis, Herpes genitalis and Herpes cornealis.

The invention also relates to all industrially applicable aspects related to the use of AFP-723 for the treatment of herpes virus infections. To this end, an essential aspect of the invention is the pharmaceutical compositions containing predetermined amounts of AFP-723. The compound can be administered topically, for example in the form of a cream, gel, stick or solution, in a concentration of between 0.5 and 5% active substance.

_8204 1 75 ____-________________

Claims (3)

1. The compound 1-N- [2- (2-dimethylaminoethoxy) acetylamino] -adamantane phosphonic acetate of the formula 1 of the formula sheet. 2. Process for preparing the compound according to claim 1 / characterized in that phosphonacetic acid and 1-N- [2- (2-dimethylaminoethoxy) acetyl-amino] adamantane (tromantadine) in a molar ratio of substantially 1: 2 into polar solvents, such as water or, more preferably, lower alcohols, and isolates the salt thus formed by evaporation or by adding a non-solvent therefor. 10 Pharmaceutical preparations for treating Herpes infections, more in particular Herpes labialis, Herpes genitalis and Herpes cornealis, containing the compound according to claim 1, as the active ingredient. 8204 1 75 ............_........ ..........