ITMI972300A1 - AMINOCARBONIL GENESEROLINE DERIVATIVES WITH SELECTIVE ANTICOLINESTERASIC ACTIVITY AT BRAIN LEVEL - Google Patents

Description

The present invention relates to aminocarbonyl derivatives of geneseroline, in particular to derivatives which carry an optionally substituted aryl substituent on the side chain. The compounds according to the present invention are endowed with potent and selective anticholinesterase activity in the brain.

In the European patent n. 0599890 Aminocarbonyl derivatives of geneseroline of formula (la) are described

where R is defined as linear or branched C2-C20 alkyl group, C3-C7 cycloalkyl, phenyl and benzyl, optionally substituted with alkyl groups, halogen atoms, alkoxy. In the above application, the n-heptylaminocarbonylgeneseroline compound alone was actually prepared and its pharmacological activity was demonstrated. Neptylgeneseroline (hereafter CHF 2060) is a long-lasting brain acetylcholinesterase (AChE) inhibitor.

Symptomatic therapy of senile dementia associated with Alzheimer's disease can be carried out with substances with anticholinergic activity with the aim of raising cerebral levels of acetylcholine and restoring the functionality of cholinergic neurons. The first compound to enter clinical practice and endowed with these properties was tacrine (Cognex; Davis, K.L. et al., N. Eng. J. Med. 327: 1253-1259, 1993).

However, the side effects at the hepatic level and the poor selectivity at the central level of this product have stimulated pharmacological research to discover new compounds with greater activity and selectivity of action at the central level. In summary, the need would be to have a compound with high anticholinesterase activity, long duration of action, greater affinity "in vitro" on the enzyme acetylcholinesterase (AChE) compared to butyrylolinestarase (BuChE) and at the same time having an "in vivo" selectivity "in inhibiting cerebral AChE compared to that present in other peripheral organs, for example in the heart.

One of the anticholinesterases belonging to this class can be considered for example the compound SDZ-ENA 713 (trade name Exelon) described in Enz A. et al. Progress in Brain Res. 98, 431-438, 1993), which we used in some studies as a comparator.

It has now been found that the compounds of formula (I)

where R is a phenyl or benzyl group, optionally substituted with C1-C4 alkyl groups, halogen atoms, C ^ -C ^ alkoxy have better pharmacological properties than the derivatives of formula (la) where R is a C2- alkyl group Linear or branched C2O, C3-C7 cycloalkyl, in particular R is n-heptyl.

Summary of the invention

The present invention, which represents a selection with respect to EP 0599890, relates to compounds of formula (I), as defined above, and their pharmaceutically acceptable salts, a process for their preparation, pharmaceutical compositions containing them and their use as an active principle for the preparation of medicaments.

Detailed description of the invention

According to the present invention, examples of phenyl or benzyl group substituted with alkyl groups are 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl, 2-ethylphenyl, 3-ethylphenyl, 4- ethylphenyl, 2-ethylbenzyl, 3-ethylbenzyl, 4-ethylbenzyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-propylbenzyl, 3-propylbenzyl, 4-propylbenzyl, 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-butylbenzyl, 3-butylbenzyl, 4-butylbenzyl, it being understood that the terms propyl and butyl include both the linear terms and the branched isomers. Examples of phenyl or benzyl group substituted with halogen atoms are 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2- bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 2-iodophenyl, 3iodophenyl, 4-iodophenyl, 2-iodobenzyl, 3-iodobenzyl, 4-iodobenzyl. Examples of phenyl or benzyl group substituted with C1-C4 alkoxy group are 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyenyl, 2-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl, 2-ethoxyphenyl, 3-ethoxyphenyl, 4-ethoxyphenyl, 2-ethoxybenzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 2-propoxyphenyl, 3-propoxyphenyl, 4-propoxyphenyl, 2-propoxybenzyl, 3-propoxybenzyl, 4-propoxybenzyl, 2-butoxyphenyl, 3-butoxyphenyl, 4-butoxyphenyl, 2-butoxybenzyl butoxybenzyl, 3-butoxybenzyl, 4-butoxybenzyl, it being understood that the terms propoxy and butoxy include both the linear terms and the branched isomers.

Preferred compounds according to the present invention are those in which R is selected between 2-ethylphenyl (hereinafter CHF 2819), 3-methylphenyl (hereinafter CHF 2957) and 2-methylphenyl (hereinafter CHF 2822).

The compounds according to the present invention can be prepared according to the teachings contained in the aforementioned European patent no.

0599890 and in particular with the method described in the Example below, starting from geneseroline. Geneseroline is a compound known from the literature (Yu Q.S. et al. Journal of Naturai Products 52 (2), 332-336, 1989).

In a Declaration filed in the United States during the granting procedure of US patent 5538968, corresponding to European patent no. 0599890, a particular selectivity towards cerebral acetylcholinesterase (AchE) with respect to piasmatic acetylcholinesterase (AchE) had previously been highlighted for the compounds of the invention, therefore a selectivity for the central cerebral district greater than that for the peripheral district.

We have now found that aminocarbonyl derivatives of geneseroline of formula (I) exhibit a higher inhibition power of cerebral acetylcholinesterase (AchE) in vivo than the derivatives substituted with an alkyl radical in the same position, as described in the European patent cited above and in particular for neptylgeneserine. The observed increase in potency occurs with the same efficacy of enzymatic inhibition. Furthermore, the compounds of formula (I) have shown, in addition to greater potency, a greater selectivity of enzymatic inhibition. In fact, they demonstrate both a greater selectivity at the tissue level (brain tissue compared to heart tissue) and a greater selectivity towards the enzyme acetylcholinesterase (AchE) than the enzyme butyrylcholinesterase (BuchE).

This selectivity is superior to that of n-heptylgeneserine and that of reference compounds such as physostigmine and SDZ-ENA 713.

EXAMPLE

Synthesis_ of_ N- (2-ethylphenylaminocarbonyleneseroline hydrochloride (CHF 2819)

75 g of geneseroline (0.3 moles) were dissolved in 750 ml of acetone, and 2 g of anhydrous potassium carbonate was added to the solution. After 10 minutes of stirring at room temperature, 47.8 g (0.324 moles) of 2-ethylphenylisocyanate dissolved in 100 ml of acetone were added in about 60 minutes, maintaining the temperature at 20'C. After 30 minutes of stirring it was filtered on celite, washing with a little acetone and the clear solution was concentrated up to about 500 ml. 73 ml of 4.75 M hydrochloric acid in ethyl acetate were added to the solution, maintaining the temperature below 25 ° C.

At the end of the addition the temperature was brought to 5 ° C, and after 120 minutes the suspension obtained was filtered by washing with 100 ml of acetone.

The solid obtained was dried at 50 ° C under vacuum to constant weight obtaining 106.04 g of crude product which, crystallized from absolute ethanol, yielded after drying 86.94 g (70.2%) of N- (2-ethylphenyl ) aminocarbonylgeneseroline hydrochloride as a white crystalline solid.

m.p .: 179-181'C (dee.).

With a similar procedure, by making geneseroline react with the appropriate isocyanate, the following can be prepared:

N- (3-methylphenyl) aminocarbonylgeneseroline hydrochloride (CHF 2957)

m.p .: 178.5-179.5 <e> C;

N- (2-methylphenyl) aminocarbonylgeneseroline hydrochloride (CHF 2822).

M.p .: 172-174-C (dee.).

In a first embodiment of the invention, the preferred compounds CHF 2819, CHF 2957 and CHF 2822 were evaluated for their enzyme inhibition potency and selectivity of action through a series of parameters compared to n-heptylaminocarbonylgeneseroline (hereinafter CHF 2060).

SD male rats weighing 150-250 g were divided into groups of 8 animals each according to the treatments with the different substances that were administered orally in distilled water, in a volume of 2 ml / kg. After 2 hours from the administration the animals were sacrificed in order to take the brain. The brain tissue was homogenized in an 11% solution of Triton 100 in 0.1 M phosphate buffer at pH 8. After centrifugation for 15 minutes at a temperature of 4 "C, the sumatant on which the determination of AChE was carried out with the method described by Ellman G.L. et al. (Biochem. Pharmacol.

7, 88-95, 1961). For each treatment the percentage of inhibition of the enzyme towards the controls (animals treated with distilled water) was determined.

In another series of experiments, the kinetics of inhibition of cerebral and cardiac AChE were evaluated at 1, 4 and 16 hours after single administration of the test substances. The dosage of the enzyme was performed on the tissue homogenates in the same way as described above. The dose administered for each product was that corresponding to 1/7 of their lethal dose (DLjg). From the obtained enzyme inhibition curve, the area under the curve (AUC) referred to the three evaluation times for brain and heart tissue was calculated.

The comparison between the different compounds in inhibiting brain AChE is shown in Table 1.

Table 1

Compound Dose Inhibition V EDS0 (mg / kg p.o) Potency order (*) (mg / kg p.o.) Brain AChE (95% L.C.)

* EDS0 CHF 2060 n-10 L.C. * ED ^ or Compound Confidence Limits

On the basis of the calculated ED ^ g values it is evident that the aryl derivatives are more potent than the reference compound CHF 2060 in inhibiting AChE after oral administration in the rat. In particular, the compound CHF 2819 was found to be 8 times more potent than the

product CHF 2060, while compounds CHF 2822 and CHF 2957 were 7 and 3 times more active, respectively. The analysis of the enzyme inhibition kinetics on brain and heart tissue homogenates is reported in Table 2.

Table 2

Compound Dose Inhibition% Maximum ACJC AUC Index of <(> mg / kg ACh £ ACh £ Cerebral Cardiac selectivity p. O. <)> Cerebral Cardiac (% inhib. X h) (t inhib. X hi (*)

* Cerebral AUC / Cardiac AUC n = 10

The results show that the aryl derivatives of geneseroline, unlike the alkyl ones, have a greater selectivity for brain tissue than for cardiac tissue.

In a further series of experiments, compounds CHF 2819, CHF 2957 and CHF 2822 were evaluated to inhibit erythrocyte AChE and human piasmatic BuChE "in vitro" compared to neptylaminocarbonylgeneseroline (CHF 2060) and standard f isostigmine and SDZ. -ENA 713. The method used is similar to that mentioned on page 8, line 3, Ellman G.L. et al. :

Table 3

I.S .: selectivity index IC5Q BuChE / ICso AChE n = 4-8.

All the products show an affinity (evaluated as IC ^ Q or the concentration of compound inhibiting the enzyme 50%) at the micromolar level for AChE. Among these, the compound SDZ-ENA 713 was less active than the other products. Relevant differences emerged in the inhibition of the BuChE enzyme. In fact CHF 2060, physostigmine and SDZ-ENA 713 are much more potent in inhibiting Buche than AChE. On the basis of the selectivity indices calculated, the CHF 2819 contribution was 115 times more selective on the AChE. CHF 2957 also has good selectivity while CHF 2822 was a little less selective.

It is important to underline that, of the two known forms of cholinesterase, BuChe is mainly distributed in the brain at the level of the glia and in the peripheral tissues. The affinity shown by some of the compounds examined for this enzyme form could help explain their activity in inhibiting the enzyme "in vivo" at the peripheral level.

From what has been described above, the compounds of formula (I) are useful for the preparation of a medicament having an inhibitory activity of acetylcholinesterase. In particular, CHF 2819 is characterized, compared to the other compounds considered, by a favorable "in vitro" selectivity on AChE compared to BuChE, a good duration of action and an "in vivo" selectivity in inhibiting cerebral AChE.

In a preferred aspect of the invention, said medicament is useful for the treatment of Alzheimer's disease and other pathologies on a neurodegenerative basis. Advantageously, said medicament has no peripheral side effects.

The medicament will be formulated in forms and dosages that can be determined by the expert clinician in the field, depending on the type of pathology, its severity and the patient's condition.

As regards the aspects pertaining to industrial application, the present invention also provides pharmaceutical compositions comprising an effective amount of active principle in admixture with conventional carriers and excipients in pharmaceutical use.

Said compositions can be prepared with techniques known in the art, as described for example in "Remington's Pharmaceutical Sciences Handbook", Mack Publishing Company, New York, U.S.A ..

Examples of compositions are oral, solid or liquid forms, such as tablets, capsules, solutions, suspensions, syrups; injectable forms, such as solutions, suspensions, emulsions; controlled release forms.

The daily dose of active principle to be administered with these compositions can vary from 1 to 50 mg and preferably can be comprised between 5 and 20 mg.

Claims (7)

1. Compounds of formula (I) where R is a phenyl or benzyl group, optionally substituted with C1-C4 alkyl groups, halogen atoms, C ^ -C4 alkoxy and their pharmaceutically acceptable salts. 2. Compounds according to claim 1, where R is selected from the group consisting of 2-ethylphenyl, 3-methylphenyl and 2-methylphenyl. 3. Pharmaceutical compositions containing an effective amount of a compound of claim 1 in admixture with pharmaceutically acceptable carriers and excipients. 4. Use of the compounds of claim 1, as active ingredients for the preparation of a medicament having inhibitory activity of acetylcholinesterase. Use according to claim 4, wherein said medicament is selective towards cerebral acetylcholinesterase. Use according to claim 5, wherein said medicament is useful for the treatment of Alzheimer's disease and other pathologies on a neurodegenerative basis. 7. Use according to claim 6, wherein said medicament is devoid of peripheral side effects.