GB2107708A - An isopropylamino-pyrimidine derivative - Google Patents

Abstract

2-Isopropylamino-pyrimidine-N- oxide and its therapeutically acceptable salts are of interest in the field of nervous regeneration and for the treatment of muscular dystrophy. This compound may be prepared by submitting, at from 15 to 45 DEG C, 2- isopropylamino-pyrimidine to a smooth oxidation by stoichiometric proportions or a slight excess of up to 10% of an appropriate oxidising agent. The compound, the process for preparing it and therapeutic compositions containing it are claimed.

Description

SPECIFICATION An isopropylamino-pyrimidine derivative The invention relates to an isopropylamino-pyrimidine derivative, to a process for its preparation and to therapeutic compositions containing it.

The invention provides 2-isopropylamino-pyrimidine-N-oxide, which has the formula:

and therapeutically acceptable salts thereof.

This compound is particularly interesting in the field of nervous regeneration and for the treatment of muscular dystrophy.

According to the invention, 2-isopropylamino-pyrimidine-N-oxide may be prepared by smooth oxidation of 2-isopropylamino-pyrimidine at from 15 to 45"C by appropriate oxidising agents such as hydrogen peroxide, m-chloro-peroxybenzoic acid, postassium peroxymonosulphate, chromic acid, perphosphoric acid, peracetic acid, sodium perborate or tertiobutyl hydroperoxide. The oxidising agent is used in stoichiemetric proportion or in a slight excess of up to 10% with respect to this proportion. The desired salts may be obtained by the usual techniques.

The invention is illustrated by the following example.

Example Into a 4 litre reactor fitted with stirring means and calcium chloride protection were poured 100 g (0.728 mol) of 2-isopropylamino-pyrimidine and 2 litres of acetone. After stirring, there was added 0.8 mol of m-chloroperoxybenzoic acid and the temperature was raised to about 35"C. Stirring was maintained for one hour after the addition and the mixture was then evaporated to dryness under reduced pressure. The residue was treated with 450 ml of water, which gives a precipitate, and with 245 ml (2.4 mol) of the soda wash saturated with sodium chloride.This solution was then treated with CHC13, which gives a new precipitate: after filtration, washing with diethyl ether and drying, there were obtained 99.2 g (yield 90%) of an oily product, elemental analysis of which showed it to correspond to the formula C7H110N3; the melting point of the base 74-76"C (Tottoli). This compound is highly soluble in water, methanol and has a good solubility in chloroform at room temperature. The pH of 5% water solution is 6.1-6.4.

The corresponding hydrochloride melts at 94"C. The succinate obtained by reaction of succinic acid on the base, in acetone at the boil, melts at 92 C; maleate, aspartate and orthophosphate were also prepared by usual routes. However, as the base itself has a good water solubility, a good stability and favourable organoleptic characteristics, it can be used as such. The base is hereafter designated by "BN 1041".

Toxicity The toxicity was determined on female wistar rats l.P. and P.O.. Values obtained by usual techniques were 1.2 g/kg for the first route and 1.9 g/kg for the second one.

Pharmacology The experimentations performed have shown the interest of the compound of the invention on the growth and the regeneration of nerves they have also evidenced a good analgesic activity, which is a highly interesting side effect.

10 Compared action on the growing of neuritis The action of the compound of the invention (BN 1041) was determined comparatively with that of 2-isopropylamino pyrimidine orthophosphate (IAPP) on the growing of neuritis of spinal cord cells and terminal ramifications (rats) according to the method described in "La Nouvelle Presse Medicale", 11, No 16 - 1238 - 1242. This in vitro experimentation was conducted on cultures of rats embryos (14 days) spinal cord cells, each 35 mm culture box containing half a spinal cord (5.106 cells).

Both compounds were tested at decreasing doses from 103 to 108 M in order to determine the toxicity limits, the concentrations leading to maximum and minimum action on neurone growth parameters and the results at three days for the optimum concentrations. The results are reported in the Table 1.

TABLE 1 IAPP BN 1041 Toxicity limit > 10-5 > lO2M Concentration at which growth appears despite 1O-3M 1O-3M the toxic action Best operating concentration 1O-5M 1O-5M It is to be noticed that a growth appears at the same concentration of 10-3M which is more (IAPP) or less (BN 1041) toxic although the best concentrations are different: 0M for IAPP or 1 0M for the compound of the invention. In these concentrations, the reference compound is toxic but the compound of the invention is deprived of any toxicity.

2 ) Reinnervation of Skeletal muscle (Rat) This experimentation was conducted comparatively with 2-isopropylamino pyrimidine orthophosphate as reference compound on adult male albinos rats. Three batches of each 5 rats were used : one for control, one for reference compound and one for the compound of the invention.

On all the animals, a lesion of the left sciatic nerve was provoked by 3 or 4 local applications of a liquid nitrogen cryode (at about-180 C on the same region of the nerve, which results in a frozen zone of about 2-3mm. This technique is more efficient, more reliable, more easily reproducible than the known technique of mechanical crushing of the nerve; moreover, the recovery is faster and more complete.

The day after the lesion, control batch animals were injected 1.P. 1 ml/100g of physiologic saline solution whereas the second batch animal received 1.P. 300 mg/Kg of reference compound and the third batch animals, 100 mg/kg of the compound of the invention.

The progress of nerve regeneration is checked on the following days by electric stimulation of the nerve.

The reinnervation is obtained at 16 days for batches 2 and 3 and at 18 days for control batch.

At 18 days the recovery is appreciated on the internal left gastrocnemic muscle by comparison with its right homologue, by the techniques of intracellular recording of motory plate potentials.

On the killed animals (at 18 days) sciatic nerves of rats treated, by the product of the invention show more than 37% of multiple innervation, by reference compound, 30% ; control animals shown only 16% multiple innervation. It is to be noticed that the product of the invention leads to a more regular reinnervation, involving frequently 2 to 3 axones by motory plate (reference compound 1-2 axone only).

30) Analgesic activity This activity has been determined by using the dental pulp stimulation test in the rhesus monkey. For comparison purposes, glafenine (30 and 60 mg/kg p.o.),2-isopropylamino pyrimidine IAPP (30, 60 and 120 mg/kg p.o.) and the compound of the invention : BN 1041 (30, 60 and 120 mg/kg p.o.), have been tested for analgesic activity using inhibition of the pain response to stimulation of the dental pulp in the rhesus monkey (adult female rhesus monkeys Macaca mulatta).

The monkeys were trained to sit in individual restraining chairs. On the day of a test, prior to dosing, the electrode leads were connected to a Grass stimulator and the pain threshold for each animal was determined using a series of transient but increasing stimuli applied to the dental pulp. In each stimulation schedule the frequency, pulse width and duration of the stimulus remained constant at 10 Hz, 3 ms and lOs respectively; only voltage was varied. The pain threshold was determined as the voltage required to produce individual reactions such as yawning and licking of the tooth. The animals were dosed orally and threshold voltages were applied 15,30,60,90, 120, 150, 240, and 300 minutes post-dose the presence of absence of individual reactions to the threshold voltage were then noted.

An interval of at least seven days was allowed between each test.

Test compounds were administered in aqueous 0.5% carboxymethyl-cellulose by oral gavage using a constant dose-volume of 4 ml/kg. Control animals received vehicle only at a dose volume of a 4 ml/kg. The effects of oral administration of the various test compounds on the pain response to electrical stimulation of the dental pulp - i.e the analgesic acitivity - are summarised in Table 2. Approximate ED50 values derived from the results of Table 2 are given in Table 3.

All three test compounds showed activity in this test. The data in Table 2 shows that glafenine reached peak activity by 30 minutes post-dose and activity was stili present in one animal at 150 minutes after dosing.

Compound IAPP showed peak activity by 90 minutes post-dose, with some effect still present at 300 minutes post-dose. A more active compound than either glafenine or IAPP appeared to be BN 1041, whose peak activity was reached 120 minutes after dosing and with residual effects up to 300 minutes ; the ED50 value at time of peak activity was approximately 30 mg/kg.

TABLE 2 Dose No. of monkeys showing analgesia at Treatment (mg/kg) time (min) after dosing p.o.

15 30 60 90 120 150 180 240 300 Vehicle - 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 30 0/4 1/4 0/4 1/4 1/4 1/4 1/4 1/4 1/4 Glafenine 60 1/4 2/4 2/4 2/4 2/4 1/4 0/4 0/4 0/4 30 0/4 0/4 0/4 1/4 0/4 1/4 0/4 0/4 0/4 IAPP 60 0/4 0/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 120 1/4 1/4 1/4 2/4 2/4 2/4 2/4 1/4 1/4 30 1/4 1/4 1/4 1/4 2/4 2/4 1/4 1/4 1/4 BN 1041 60 1/4 2/4 2/4 2/4 3/4 3/4 3/4 3/4 2/4 120 0/4 2/4 2/4 4/4 4/4 4/4 3/4 2/4 1/4 TABLE 3 Approximate ED50 values (mg/kg) at time (min) post dost Treatment 15 30 60 90 120 150 180 240 300 Glafenine > 60 60 60 60 60 60 > 60 > 60 > 60 IAPP > 120 > 120 > 120 120 120 120 120 > 120 > 120 BN 1041 > 120 85 85 53 30 30 42 42 ? The approximate ED50 values were calculated using the method of moving averages (Thompson, W.R., Bacteriological Reviews, (1947), 11, 115-145).

Presentation - posology This compound can be presented in any therapeutically acceptable form and, for instance, in tablets or in gelatine capsules containing 50 mg per dosage unit together with an excipient such as lactose ; for injectable form the product may be dosed in phials containing at least 5 mg of active ingredient dissolved in water. As to the posology for human use, oral administration requires from 100 mg to 1 g per diem whereas injectable form may be administred at doses between 5 mg to 100 mg per diem.

An example of the tablet form is given hereunder: 2-isopropylamino-pyrimidine-N-oxide 50 mg Microcrystalline cellulose 20 mg Corn starch 15 mg Talc 7 mg Silicic acid 6 mg Magnesium stearate 7 mg 100 mg

Claims (4)

1. 2-lsopropylamino-pyrimidine-N-oxide, or a therapeutically acceptable salt thereof.

2. A process for the preparation of 2-isopropylamino-pyrimidine-N-oxide, the process comprising submitting at from 15 to 45"C, 2-isopropylaminopyrimidine to a smooth oxidation by stoichiometric proportions or a slight excess of up to 10% of an appropriate oxidising agent such as hydrogen peroxide, m-chloro-peroxybenzoic acid, potassium peroxymonosulphate, chromic acid, perphosporic acid, peracetic acid, sodium perborate ortertiobutyl hydroperoxide.

3. A process for the preparation of 2-isopropylamino-pyrimidine-N-oxide, the process being substantially as described herein with reference to the Example.

4. A therapeutic composition 2-isopropylamino-pyrimidine-N-oxide or a therapeutically acceptable salt thereof in admixture with a therapeutically acceptable diluent or carrier.