WO1993025528A1 - Novel aminopropanol derivatives, pharmaceutical compositions containing them and process for preparing same - Google Patents

Abstract

The invention relates to novel optically active and racemic aminopropanol derivatives of formula (I) wherein R means hydrogen or a C1-4alkyl group; Ar stands for a phenyl group optionally substituted by at most two halogens, C1-4alkyl, C1-4alkoxy or nitro group(s); or a naphthyl group; Y represents halogen, C1-4alkyl, C1-4alkoxy, phenyl, 2,3-(CH=CH)2- or 3,4-(CH=CH)2- group; and n is an integer of 0, 1, 2, 3, 4, or 5 as well as acid addition salt of these compounds. The invention further relates to pharmaceutical compositions containing these compounds as well as a process for the preparation of the compounds of formula (I). The compounds of formula (I) are useful for inhibiting the peroxidation of lipids and treating the sequels thereof as well as for protection from or treatment of the sequels of calcium-mediated injuries induced e.g. by ischemia, hypoxia or reperfusion and for treating various degenerative neurological diseases e.g. Alzheimer's disease or Parkinson's disease.

Description

NOVEL AMINOPROPANOL DERIVATIVES, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND PROCESS FOR PREPARING SAME

The invention relates to novel, therapeutically active aminopropanol derivatives of formula

wherein

R means hydrogen or a Ci-.^alkyl group;

Ar stands for a phenyl group optionally substituted by at most two halogens, C₁--.₄alkyl, Cι_4alkoxy or nitro group(s) ; or a naphthyl group; Y represents halogen,

phenyl, 2,3-(CH=CH)₂- or 3,4-(CH=CH)₂- group; and n is an integer of 0, 1, 2, 3, 4 or 5 in racemic or optically active form as well as their acid addition salts and pharmaceutical compositions containing these compounds. Furthermore, the invention relates to a process for the preparation of the above compounds and compositions.

The compounds of formula (I) according to the inven¬ tion are new and possess a valuable biological activity. Under in vitro conditions they show a significant anti- oxidant (lipid peroxidation inhibitory) and neuronal Ca -2 -

uptake inhibitory effect. By investigating under in vivo conditions, they exert a remarkable antihypoxic and anti- convulsive action.

Accordingly, the invention relates also to a method of treatment, which comprises administering a therapeutically effective amount of a compound of formula (I) or a pharma¬ ceutically acceptable acid addition salt thereof to a patent for inhibiting the lipid peroxidation and for protection from or treatment of Ca-mediated injuries and the sequels thereof.

In the formula (I) the C₁_₄alkyl group or

moiety of the C₁---,₄alkoxy group may be a straight or branched chain alkyl group. The term halogen includes e.g. fluorine, chlorine or bromine. There are compounds known from the literature, which are structurally related to the compounds of the formula

Antihypoxically active substances, which are similar to the compounds of the present invention but bear a heterocyclic (benzoxazole, benzothiazole) substituent on the a ino group of the 4-aminopiperidine moiety, are described in the European patent specification No. 184,257.

Analgetically active 4-arylaminopiperidine derivatives substituted by a 4-arylbutyl or a 3-aroylpropyl group and a 3-arylpropyl group, respectively on the nitrogen of the piperidine ring are described in US patent specifications Nos 3,686,187 and 3,691,171.

Analgetically active 4-(1-naphthylamino)piperidine derivatives substituted by a 2-arylethyl substituent in position 1 of the piperidine ring are disclosed in the Swiss patent specification Nos. 528,507 and 535,767. Due to hypoxia, ischemia (global or focal, per¬ manent or transient) or reperfusion, the cognitive functions are damaged [S. N. einachter et al.: Group Report 8: "Models of Hypoxia and Cerebral Ischemia" Phar acopsychiat. 21, pages 94 to 98 (1990)].

Depending on the severity and duration of hypoxia and ischemia, reversible or irreversible injuries occur: the structure and function of the membrane are damaged, which may lead to neuronal death.

As a consequence of hypoxia and ischemia, the ito- chondrial ATP production ceases and an aerobic glycolysis develops, which results in lactic acid acidosis. Due to the ATP definiciency, the function of ion-pumps is stopped [T. F. Hornbein: "Hypoxia and the Brain" in: R.G. Crystal and J. B. West eds. The Lung: Scientific Founda¬ tions, pages 1535 to 1541 (1991)]. The K⁺ concentration of the extracellular spaces significantly increases, which leads to membrane depolarization inducing the opening of potential-dependent Ca channels. The influx of Ca to the cell partly proceeds through these Ca channels.

The increase in the Na⁺ permeability of the membrane induces the release of a large amount of excitatory amino acids (glutamate, aspartate) . Glutamate activates the receptor-dependent Ca channels, through which Ca similarly may penetrate into the cell [B. K. Siesjδ et al.: "Calcium Fluxes, Calcium Antagonists and Calcium-Related Pathology in Brain Ischemia, Hypoglycemia, and Spreading De- pression: A Unifying Hypothesis. J. Cereb. Blood Flow Metab. 9, pages 127 to 140 (1989)].

The influx of Ca to the cell (pre- and postsynaptic Ca influx) may induce catabolic reactions. The increase in the intracellular Ca may initiate reactions significantly influencing the functions and integrity of the cells.

Ca-induced abnormal reactions include lipolysis, proteolysis, disintegration of the microtubules, high- grade phosphorylation of proteins, release of catechol- amines in remarkable amounts and formation of free radicals [B. K. Siesjδ et al.: "Brain Injury: Neuro- che ical Aspects" in: J. Povlishoch and C. Becker eds. Central Nervous System Trauma-Status Report, pages 513 to 532 (1984)].

Blocking of the Na and Ca channels may play an important role in the mode of action of cerebro- protective compounds.

Use of tetrodotoxin blocking the Na channel proved to be favourable in the protection from ischemic injuries [Y. Yamasaki et al.: "The Possible Involvement of Tetro- dotoxin-sensitive Ion Channels in Ischemic Neuronal

Damage in the Rat Hippocampus" Neurosci. Lett. 121. pages 251 to 254 (1991); as well as D. Ashton et al.: "Extra¬ cellular Ions During Veratridine-induced Neurotoxicity in Hippocampal Slices: Neuroprotective Effects of Flunarizine and Tetrodotoxin" Brain Res. 528. pages 212 to 222 (1990) ] .

Compounds decreasing the pre- and postsynaptic Ca up¬ take or altering the accumulation of Ca on intracellular sites may be of therapeutical importance. At present, the Ca antagonists are used for treating ischemic injuries mainly on the basis of their vascular effects; however, it seems more and more important that compounds possessing such a mechanism of action exert their antihypoxic and antiischemic effects through inhibition of the Ca influx to neurons [R. Hall et al.: "Brain Protection: Physio¬ logical and Pharmacological Considerations" Part II: The Pharmacology of Brain Protection" Can. J. Anaesth. 37, pages 762 to 777 (1990)].

During the reperfusion following ischemia, a large amount of free radicals are formed. Hydroxyl and hyper- oxide radicals may arise in the mitochondrial respiratory chain, in the arachidonic acid cascade in the course of functioning of cyclooxygenase and lipoxygenase, because of the activation of xanthine oxidase and as a result of the autooxidation of catecholamines [T. F. Hornbein: -5-

Hypoxia and the Brain" in: R. G. Crystal and J. B. West eds. The Lung: Scientific Foundations, pages 1535 to 1541

(1991)].

Due to their lipid peroxidation inhibitory effect, antioxidant compounds provide protection against injuries induced by free radicals under ischemic, hypoxic conditions. Thus, antioxidants as antiischemic and anti- hypoxic compounds can be used for the treatment of such clinical syndromes [R. J. Traystman et al.: "Oxygen Radical Mechanisms of Brain Injury Following Ischemia and Reper¬ fusion" J. Appl. Physiol. 11 , pages 1185 to 1195 (1991)].

Free radical reactions likely play a causal role in the pathogenesis of ischemia-induced injuries such as ischemic intestinal diseases, myocardial ischemia, haemorrhagic shock, cerebrovascular function disturbances accompanied by ischemia, ischemic liver injury and renal ischemia [R. J. Korthuis et al.: "Physiology of Oxygen Radicals" Chapter 17, pages 217 to 249 (1986)].

In vitro tests for investigation of the antioxidant effect

The antioxidant effect was studied by using two methods.

1. Effect on the NADPH-induced lipid peroxidation in brain microsomes This investigation was carried out on microsomes prepared from rat brain by following the method of T. J. Player and A. A. Horton [J. Neurochem. 3_7, (2) , pages 422 to 426 (1981) ] .

Male Hannover-Wistar rats weighing 150-250 g each were used for the preparation of microsomes. After decapitation the whole brain of the rat was removed and homogenized in a 10-fold volume of ice-cold 0.25 M sucrose solution. The homogenate was centrifuged in a Hitachi CR 26H equipment at 15,000 x g at 4 °C for 10 i- nutes, then the supernatant was collected and centrifuged -6-

in a Hitachi SCP85H equipment at 78000 x g at 4 °C for 60 minutes. The pellet was suspended in 0.15 M KC1 solution, the protein content was determined and then adjusted to 10 mg/ml. The microsome preparation thus 5 obtained was frozen in a dry ice-acetone mixture and stored at -70 °C until use.

The components of the incubation mixture were: 50 M TRIS.HC1 (pH 6.8), 0.2 mM FeCl₃, 1 mM KH₂P0₄, 0.5 mM ADP, 0.2 g of microsomes as well as the compound to be tested.

10 The incubation was carried out in a final volume of 1 ml with an incubation time of 20 minutes at a temperature of 37 °C. The lipid peroxidation was induced by adding 0.4 mM NADPH. (The blank samples did not contain NADPH.) The re¬ action was stopped by adding 0.375 ml of a stopping solu-

15 tion containing 40% trichloroacetic acid and 5 M HCl in a 2:1 ratio. The formation of malondialdehyde was determined by using thiobarbituric acid. After stopping the reaction 1 ml of 1% thiobarbituric acid was added to the samples, which were then placed in a water

20 bath of about 100 °C for 10 minutes. Subsequently, the samples were centrifuged at 2,000 x g in a Janetzki K70 equipment at 4 °C for 10 minutes. The absorbance values of the coloured supernatant were measured at 535 nm on a Hitachi 150-20 spectrophotometer by using malondialdehyde-

25 -bis(diethyl acetal) , as a reference compound.

2. Effect on the Fe²⁺-induced lipid peroxidation in brain ho ogenate This investigation was carried out on rat brain homogenate by following the method of J. M. Braughler et

30 al. [J. Biol. Chem. 262 (22), pages 10438 to 10440 (1987) ] .

After decapitating Hannover-Wistar rats weighing 150-220 g each, the whole brain was homogenized in 9 volumes of ice-cold Krebs-Ringer's buffer (containing

35 15 mM HEPES (pH 7.4), 140 mM NaCl, 3.6 mM KC1, 1.5 mM -7-

CaCl , 0.7 mM MgCl₂, 1.4 mM KH₂P0₄ and 10 mM glucose). Then the protein content of the solution was determined and adjusted to 10 mg/ l. After adding the inhibitory agent to be tested in a volume of 5 μl to 200 μl of the homogenate, the mixture was incubated at 37 °C for 20 minutes. The Fe²⁺-induced lipid peroxidation was accomplished by adding 5 μl of a 8 mM Fe(NH₄)₂(S0₄)₂ solution. After passing of the incubation time, the re¬ action was stopped by adding 1 ml of a stopping solution containing 0.8 M HCl and 12.5% of trichloroacetic acid, then the samples were centrifuged at 2,000 x g in a Janetzki K70 equipment at 4 °C for 10 minutes.

To a 0.5 ml portion of the supernatant 1 ml of an 1% thiobarbituric acid solution was added, then the samples were placed in a water bath of 100 °C for 20 minutes. The colour intensity developed was determined at 535 nm with the aid of a Hitachi 150-20 εpectrophoto eter by using malondialdehyde-bis(diethyl acetal) , as a reference compound. On the basis of the concentration/effect correlations of the tested compounds the IC5₀ values were determined; these results are indicated in Table I.

Table I

Compound Example Inhibition of the Inhibition of the

No. No. NADPH-induced lipid Fe²⁺-induced li- peroxidation pid peroxidation

IC₅₀ (μM) IC₅₀ (μM)

7. 7 9.5 1.2

57, 1 1, 6 1.6 1, 5 4, 7 7, 8

37, 9 5 7 2 5 2 5

12.5 10.5 51.0

33.2

N.I.: The reaction investigated is not inhibited by the compound

It can be seen from the data of Table I that each of the compounds prepared in the various Examples exerted an antioxidant (lipid peroxidation inhibitory) activity. The antioxidant effect was investigated both in an enzymatic (NADPH-induced) and a non-enzymatic (Fe ⁺-induced) lipid peroxidation test. ^■9-

The level of the antioxidant activity of the compounds was characterized by their IC5₀ values. The cerebroprotective idebenone, the native antioxidant vita¬ min E (DL-α-tocopherol) , the anticarcinogenic ellagic acid and the hepatoprotective silymarin were used, as reference compounds.

Based on the data of Table I, the tested compounds showed a much higher activity in the inhibition of the NADPH-induced (enzymatic) lipid peroxidation than the reference compounds (DL-α-tocopherol, ellagic acid, silymarin) . The antioxidant effect of the compounds Nos. 0107966, 0107968, 0108199 and 0109001 was comparable to that of idebenone; whereas the compounds Nos. 0108534, 0108535, 0108536, 0108858 and 0109223 exerted a stronger inhibitory effect on the NADPH-induced lipid peroxidation than that of idebenone.

Each of the compounds listed in Table I showed a much stronger inhibitory effect on the Fe²⁺-induced (non- -enzymatic) lipid peroxidation than the reference co - pounds. Compounds Nos. 0108199, 0108534, 0108535 and 0108536 proved to be particularly active since each of them was ten times as active as idebenone or DL-α-toco¬ pherol. Similarly, the compounds Nos. 0109222, 0109223, 0108651 and 0109001 inhibited the Fe²⁺-induced lipid peroxidation considerably. The antioxidant activity of the compounds Nos. 0107966, 0107968 and 0108858 also exceeded that of the reference compounds.

By comparing the data in the in vitro antioxidant tests it can be stated that the substances Nos. 0109223, 0108534, 0108535, 0108536 and 0108858 exerted a higher inhibitory effect on the lipid peroxidation induced in various ways, than idebenone, found to be the most active reference compound up to the present.

The compounds according to the invention possess a significant antioxidant effect namely, they are capable to inhibit the lipid peroxidation processes induced by free radicals arising from Fenton's reaction (catalyzed by Fe²⁺) or during the functioning of the NADPH-cytochrom C reductase enzyme. Abbreviations:

NADPH: β-Nicotinamide adenine dinucleotide phospate, reduced form TRIS : Tris (hydroxymethyl) aminomethane

ADP: Adenosine-5 ' -diphospate HEPES: 2-[4-(2-Hydroxyethyl)-l-piperazine]-ethane- sulfonic acid Idebenone: 6-(10-Hydroxydecyl) -2 , 3-dimethoxy-5-methyl-

-1 , 4-benzoquinone DL-α-Tocopherol: [2, 5,7, 8-Tetramethyl-2-(4 ' ,8' ,12'-tri- methyl-tridecyl) -chroman-6-ol

Ellagic acid: 2,3,7,8-Tetrahydroxy[l]benzopyrano[5,4, 3- cde] [,l]benzopyran-5, 10-dione Silymarin: Silybinin + silydianin + silychristin

In vitro tests used for investigating the neuronal ca uptake inhibitory effect

The Ca uptake was investigated in synaptosomes prepared from rat brain cortex by following the method of P. H. Wu et al. [J. Neurochem. .39., pages 700 to 708 (1982) ]. 1. Effect on the synaptosomal K⁺-induced ⁴⁵Ca uptake Male Hannover-Wistar rats weighing 180 to 200 g each were used for the preparation of synaptosomes. After decapitation of the rats, the whole brains were collected in ice-cold physiological saline, the brain cortex was removed and purified from the white matter. The tissue obtained was homogenized in a 10-fold volume of 0.32 M sucrose solution by using a glass-teflon homogenizer. After centrifuging the homogenate in a Janetzki K-70 device at 1,000 x g at 4 ^eC for 10 minutes, the super- natant was separated and further centrifuged in a Hitachi CR-26H device at 12,000 x g at 4 °C for 20 minutes. After suspending the pellets in a 0.32 M sucrose solution, the protein content was determined and adjusted to 20 mg/ml.

The composition of the incubation mixture used for measuring the K⁺-stimulated ⁴⁵Ca uptake was: 112 mM NaCl, 5 mM KC1, 1.3 mM MgCl₂, 1.2 mM NaH₂P0₄, 1.2 mM CaCl₂, 10 mM glucose and 20 mM TRIS. The incubation mixture was saturated by carbogen (containing 95% of 0₂ and 5% of C0 ) up to a pH value of 7.4. The compounds to be tested as well as the synaptosome preparation corresponding to 1 mg of protein were added to the medium. The final volume of the incubation mixture was 1 ml. The samples were pre-incubated at 37 °C for 20 minutes. The Ca uptake was initiated by adding a solution containing 2.8 kBq (75 nCi) of ⁴⁵CaCl₂. For investigating the'K⁺- -stimulated ⁴⁵Ca uptake KC1 was used in a concentration of 60 mM; NaCl of the same concentration was added to the control samples.

The incubation lasted for 20 minutes. The reaction was stopped by adding 5 ml of ice-cold stopping solution (120 mM NaCl, 5 mM KC1, 5 mM EGTA, 20 mM TRIS-HC1, pH 7.4). After filtering the samples on a Whatman GF/C filter, the protein remaining on the filter was washed twice with 5 ml of washing solution each (132 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 1.2 mM CaCl₂, 20 mM TRIS-HC1, pH 7.4).

The filters were placed in glass cuvets and then dried at 40 °C for 1 hour. Subsequently, 5 ml of a scintillation cocktail were added into each cuvet and the radio¬ activity of the samples was measured by using a liquid scintillation spectrophotometer (1219 Rackbeta, LKB Wallace) . 2. Effect on the synaptoso al veratrine-stimulated ⁴⁵Ca uptake

Male Hannover-Wistar rats weighing 180 to 200 g each were used for the preparation of synaptosomes. After de- capitation of the rats, the whole brains were collected in ice-cold physiological saline, the brain cortex was removed and purified from the white matter. The tissue obtained was homogenized in a 10-fold volume of ice-cold 0.32 M sucrose solution by using a glass-teflon homo- genizer. After centrifuging the homogenate in a

Janetzki K-70 device at 1000 x g at 4 °C for 10 minutes, the supernatant was further centrifuged in a Hitachi CR-26H device at 12000 x g at 4 °C for 20 minutes. After suspending the pellets in a 0.32 M sucrose solution, the protein content of the solution was determined and adjusted to 20 mg/ml.

The composition of the incubation mixture used for measuring the veratrine-stimulated ⁴⁵Ca uptake was: 132 mM NaCl, 5 mM KC1, 1.3 mM MgCl₂, 1.2 mM NaH₂P0₄, 1.2 mM CaCl , 10 mM glucose, 20 mM TRIS. The incubation mixture was saturated by carbogen (containing 95% of 0₂ and 5% of C0₂) up to a pH value of 7.4. The compounds to be tested as well as the synaptosome preparation corres¬ ponding to 1 mg of protein were added to the medium. The final volume of the incubation mixture was 1 ml. The samples were pre-incubated at 37 °C for 20 minutes. The Ca uptake was initiated by adding a solution containing 2.8 kBq (75 nCi) of ⁴⁵CaCl₂. For investigating the veratrine-stimulated ⁴⁵Ca uptake, veratrine was used in a concentration of 20 μM.

The incubation lasted for 20 minutes. The reaction was stopped by adding 5 ml of ice-cold stopping solution (120 mM NaCl, 5 mM KCl, 5 mM EGTA, 20 mM TRIS-HC1, pH 7.4). After filtering the samples on a Whatman GF/C filter, the protein remaining on the filter was washed -13-

twice with 5 ml of washing solution each (132 mM NaCl, 5 mM KC1, 1.3 mM MgCl₂, 1.2 mM CaCl₂ and 20 M TRIS-HC1, pH 7.4) .

The filters were placed into glass cuvets and then dried at 40 °C for 1 hour. Subsequently, 5 ml of a scintillation cocktail was added into the cuvet each and the radioactivity of the samples was measured by using a liquid scintillation spectrophotometer (1219 Rackbeta, LKB Wallace) .

The IC50 values were determined on the basis of con¬ centration/effect correlations of the tested compounds and are summarized in Table II.

Table II

Number of measurement are given in parentheses.

For the investigation of K⁺-induced ⁴⁵Ca uptake, the membrane depolatization is established by increasing the potassium ion concentration of the extracellular space. This leads to the opening of the potential-dependent Ca channels. The influx of Ca ions to the cell proceeds through these channels.

For the investigation of veratrine-induced ⁴⁵Ca uptake, the membrane depolarization is established by increasing the sodium ion concentration of the intra- cellular space since veratrine impedes the inactivation of Na channels. The thus induced membrane depolarization similarly results in the opening of the Ca channels. The Ca-antagonistic effect of the compounds was characterized by the IC5₀ values. The cerebroprotective nimopidine, flunarizine and εabeluzol were used as reference substances.

It can be seen from data of Table II that each of the compounds prepared (as described hereinafter in various Examples) possessed a Ca uptake inhibitory effect.

The compounds shown in Table II inhibited the K⁺- -induced ⁴⁵Ca uptake to a much higher degree than nimo¬ pidine. The.--compounds Nos. 0108536, 0108489 and 0108487 proved to be particularly effective. When investigating the K⁺-stimulated ⁴⁵Ca uptake, the Ca uptake inhibitory effect of these compounds surpasses even the Ca-antago¬ nistic effect of flunarizine, too. The compound No. 0108536 inhibited the K⁺-induced ⁴⁵Ca uptake twice as effectively as the cerebroprotective salubezol found to be most effective of the reference compounds.

Our studies carried out by using this test indicated that likely, the compounds of the invention inhibited the function of potential-dependent Ca channels. When investigating the veratrine-induced ⁴⁵Ca uptake, the activity of each compound according to the invention showed a higher activity in comparison to nimopidine. The veratrine-induced ⁴⁵Ca uptake inhibitory effect of compounds Nos. 0108487 and 0108534 approximated whereas that of the compound No. 0107966 exceeded the activity of -15-

flunarizine.

Based on study of the veratrine-induced ⁴⁵Ca uptake it can also be assumed that the compounds according to the invention might be capable to influence the function¬ ing of Na channels, too. Abbreviations:

EGTA: Ethylene glycol bis(2-aminoethyl) ether

N,N'-tetraacetic acid TRIS: Tris(hydroxy ethyl)aminomethane Nimodipine: l,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-

3,5-pyridinedicarboxylic acid 2-methoxy- ethyl l-methylethyl ester Flunarizine: 1-[bis(4-fluoropheny1)methyl]-4-(3-pheny1- -2-propenyl)piperazine Sabeluzol: N-Methyl-N-{l-[3-(4-fluorophenoxy)-2-hydroxy- propyl]piperidin-4-yl}-benzothiazole-2-amine The pharmacological effect of the compounds found to be effective in the in vitro biochemical test were supported by in vivo measurements. The effects of the compounds were tested on male

CFLP/LATI mice weighing 18 to 22 g each. The compounds to be tested were orally administered in a 5% Tween 80 suspension in a volume of 10 ml/kg of body weight. Nimopidine and flunarizine were used as reference substances. The control groups were treated with distilled water containing 5% of Tween 80.

The cytotoxic hypoxia (KCN) test

1 hour after the oral administration of the substances to be tested, the animals were intravenously (i.v.) treated with 5 mg/kg of potassium cyanide (KCN) . The time of survival was measured from the administration of potassium cyanide up to the last respiratory movement.

Animals having a survival time longer by 30% than the average survival time of the placebo-treated control group were considered to be protected. -16-

The ED5₀ values (i.e. the dose being effective in 50% of the animals) were calculated from the percentage of the surviving animals by using the probit analysis.

Metrazole convulsion

After a l-hour pretreatment the animals were sub- cutaneously (s.c.) treated with a 125 mg/kg dose of pen- tylenetetrazole (metrazole) . The abolishment of tonic extensor convulsions and the survival were considered to be a protective effect.

The ED50 values were calculated from the percentage of protected animals by using probit analysis.

The results are summarized in Table III.

The antioxidant and Ca ⁺ uptake inhibitory effects of the substances measured under in vitro conditions were assayed by two pharmacological methods under in vivo conditions.

The cerebroprotective effect was supported by the KCN- -lethality inhibitory effect; whereas the Ca ⁺ uptake inhibitory effect was proven by the inhibitory effect on the metrazole convulsion.

By blocking the cytochrome C oxidase, KCN interferes with the metabolism of the cell and therefore results in a lactic acid acidosis and cytotoxic hypoxia; si ultaneous- ly, a large amount of Ca²⁺ ions flow into the cell.

In the cytotoxic hypoxia, the compounds Nos. 0108535 and 0108536 were 3 to 4.5 times as active, the compound No. 0108489 was 1.7 to 2.5 times as effective as the reference substances. The effects of other compounds according to the invention were also more favourable in this test.

Concerning the protection against convulsions, the anticonvulsive effect of the compounds Nos. 0108487, 0108489 and 0108536 was 1.5 to 2.4 times as pronounced as that of flunarizine, which latter is 4 times as active anticonvulsant as nimodipine. The anticonvulsive effect of other compounds was comparable to that of flunarizine and was approximately 2.0 to 2.5 times as strong as that of nimodipine. Compounds found to have a significant effect both in the antioxidant and Ca uptake inhibitory in vitro tests, were effective in the anticonvulsive test, too.

The compounds according to the invention may be useful for the protection from or treatment of the sequels of Ca- -mediated injuries induced e.g. by ischemia, hypoxia or reperfusion. In addition, the compounds tested may be utilized for the treatment of clinical syndromes, where the free radicals play the role of aetiological factors, e.g. cerebral and spinal trauma, apoplexy, stroke, ischemic injuries of cerebrovascular origin, hypoxia following atherosclerosis as well as in various degene¬ rative neurological diseases such as e.g. Alzheimer's disease or Parkinson's disease.

In the above clinical syndromes the expected therapeu- tical doses of the compounds of the invention are between 0.1 and 40 mg/kg of body weight, which are administered daily once or in several divided doses in oral or parenteral route.

According to the invention the novel aminopropanol derivatives of formula (I) as well as their acid addition salts can be prepared by reacting a 4-aminopiperidine derivative of formula

wherein R and Ar are as defined above, with a racemic or optically active epoxide derivative of formula

wherein Y and n are as defined above and, if desired, resolving the so-obtained compound of formula (I) and/or, if desired, converting it to an acid addition salt.

Hereinafter, the preparation of the novel compounds of formula (I) according to the invention will be described in detail. In the process of the invention an a inopiperidine -19-

derivative of formula (II) is reacted with up to 20% excess of a racemic or optically active epoxide derivative of formula (III) in a protic or aprotic solvent, e.g. alcohols or ether-type solvents, halogenated or aromatic hydrocarbons, such as toluene or xylene, at the boiling point of the solvent used. The reaction time is about 10 hours. In a preferable case the compounds of formula (I) obtained are precipitated from the reaction mixture by cooling or they can be separated after evaporation. Some derivatives may need a purification by column chromato- graphy. By using optically active epoxides of formula (III) , the compounds of formula (I) obtained will of course be optically active. By reacting racemic compounds of formula (III) , the compounds of formula (I) obtained will be in racemic form which, if desired, can be separat¬ ed to the pure enantio ers by using resolution methods known per se.

Of the 4-aminopiperidine derivatives of formula (II) used as starting substances, e.g. N-methyl-N-(3-methyl- phenyl)-4-aminopiperidine (see European patent specifica¬ tion No. 156,433) , N-phenyl-4-aminopiperidine [Chem. Pharm. Bull. 3_3, (5) pages 1826 to 1835 (1985)], N-(2,6- -di ethylphenyl)-4-aminopiperidine (United States patent specification No. 4,126,689), N-(4-methoxyphenyl)-4-amino- piperidine, N-(4-chlorophenyl)-4-aminopiperidine and N-(4- -fluorophenyl)-4-aninopiperidine (United States patent specification No. 3,686,187) are known. Other derivatives required can be prepared according to the methods described in the above literature references. Of the 4-naphthylaminopiperidine derivatives of formula (II) e.g. 4-(l-naphtyl)-aminopiperidine is described in Swiss patent specification No. 535,767. Other derivatives desired can be prepared by using the methods described therein. The epoxide derivatives of formula (III) are known compounds, a number of which are commercially available. Optically active substances of formula (III) can be prepared by following, e.g. the methods published in: J. Org. Che . 5A_» pages 1298 to 1304 (1989). If desired, the racemic or optically active compounds of formula (I) may be converted to their acid addition salts in a known manner.

The salt formation may be carried out in an inert organic solvent, e.g. by dissolving the compound of for- ula (I) in the selected solvent and then portionwiεe adding the appropriate acid to this solution until the pH value of the mixture becomes strongly acidic (pH value of about 1) . However, the salts may also be formed by adding a calculated amount of the desired acid dissolved in the solvent of choice to the above solution. Thereafter, the acid addition salt precipitated is separated from the re¬ action mixture in a suitable manner, e.g. by filtration.

The active agents of formula (I) can be formulated in pharmaceutical compositions by mixing them with non-toxic, inert, solid or liquid carriers and/or auxiliaries common¬ ly used in the therapy for parenteral or enteral administration. Useful carriers are e.g. water, gelatine, lactose, starch, pectin, magnesium stearate, stearic acid, talc, vegetable oils, such as peanut oil, olive oil and the like. The active agents can be formulated in any usual pharmaceutical composition, particularly solid composi¬ tion, e.g. rounded or edged tablet, dragee or capsule such as gelatine capsule, pill, suppository and the like. Optionally, these compositions may contain also other commonly used pharmaceutical auxiliaries, e.g. stabilizers, preservatives, wetting agents, surfactants, emulsifying agents and the like. The compositions can be prepared in a known manner, e.g. by sieving, mixing, granulating and compressing the components in the case of solid compositions. The compositions may be subjected to other usual operations of the pharmaceutical technology , e . g . sterilization.

The invention also relates to a method for inhibiting lipid peroxidation as well as treating the sequels thereof furthermore , for the protection from or treatment of the sequels of Ca-mediated injuries . This method comprises administering a therapeutically effective amount of an active agent of the formula (I ) or a pharmaceutical¬ ly acceptable acid addition salt thereof to the patient . The invention is illustrated in detail by the aid of the following non-limiting Examples . Example 1

Preparation of (±) -l-phenoxy-3-{4-[ ( 1 -naphthyl ) amino] - piperidin-l-yl}-2-propanol [ (I) , Ar = l-naphthyl, R = H, Y = H; compound No. 0107966]

After suspending 11.7 g ( 0.030 mol) of 4- [ ( l-naph¬ thyl) amino] piperidine dihydrochloride [ (II) , Ar = l- naphthyl , R = H] in 240 ml of chloroform, 120 ml of 4 N aqueous sodium hydroxide solution are added to the above suspension and the mixture is then stirred until dissolution of the solid material . After separating the two phases the aqueous layer is extracted with 30 ml of chloroform and the combined organic phase is washed with 30 ml of water. The chloroform solution is dried and evaporated under reduced pressure. The oily residue is dissolved in 120 ml of xylene, 6. 1 ml ( 0.045 mol) of (±) -l-phenoxy-2 , 3-epoxypropane [ (III) , Y = H] are added to the solution, then the reaction mixture is boiled under reflux for 10 hours while stirring. After cooling the product precipitated from the solution is filtered and recrystallized from 200 ml of acetonitrile to give the title compound in a yield of 9.0 g (85. 5%) , m.p. : 160-162 ^βC.

By following the above process, the (±) derivatives of formula (I) listed hereinafter were prepared by reacting -22-

the corresponding piperidine derivatives of formula (II) with the suitably substituted epoxy compounds of for¬ mula (III) .

Similarly, by using the above method, (±)-l- (4-fluoro- phenoxy)-3-{4-[ (2-naphthyl) amino]piperidin-l-yl}-2-pro- panol [(I), Ar = 2-naphthyl, R = H, Y •= 4-F] [m.p.: 166-168 ^βC (after recrystallization from ethyl acetate) , compound No. 0108650] was prepared from 4- [ (2-naphthyl) - a ino] piperidine dihydrochloride [(II), Ar = 2-naphthyl, R =■ H] and (±) -l-(4-f luorophenoxy) -2,3-epoxypropane [(III), Y = 4-F]. 0033

-23-

Example 2

Preparation of (±)-l-(4-chlorophenoxy)-3-{4-[(1- naphthyl)amino]piperidin-l-yl}-2-propanol [ (I) , Ar = i-naphthyl, R = H, Y = 4-C1; compound No. 0108536]

After adding 200 ml of 4 N aqueous sodium hydroxide solution to a suspension containing 15.0 g (0.050 mol) of 4-[ (l-naphthyl)amino]piperidine dihydrochloride [(II), Ar = 1-naphthyl, R = H] in 400 ml of chloroform, the mixture is stirred until dissolution of the solid material. After separating the two phases the aqueous layer is extracted with 50 ml of chloroform, then the combined organic phase is washed with 50 ml of water. The chloroform solution is dried and evaporated under reduced pressure. After dissolving the oily residue in 200 ml of xylene, 10.2 g (0.055 mol) of (±)-l-(4-chlorophenoxy)- -2,3-epoxypropane [(III), Y = 4-C1] are added, then the reaction mixture is boiled under reflux for 10 hours whil stirring. After cooling down, the solution is evaporated under reduced pressure and the residue is recrystallized from 100 ml of ethanol to obtain the title compound in a yield of 19.2 g (93.5%), m.p.: 165-167 °C.

Similarly, by using the above process, the (±) deri¬ vatives of formula (I) listed hereinafter were prepared b reacting the corresponding piperidine derivatives of formula (II) with the suitably substituted epoxy compound of formula (III) . -24-

-25-

Similarly, by using the above method, (±)-1-(3-phen- oxy)-3-{4-[ (2-naphthyl)amino]piperidin-l-yl}-2-propanol [(I), Ar = 2-naphthyl, R « H, Y = H; compound No. 0508649] was prepared from 4-[ (2-naphthyl)amino]piperidine dihydro- chloride [(II), Ar = 2-naphthyl, R = H] and (±)-1-phenoxy- -2,3-epoxypropane. [(Ill), Y = H]. The melting point of the compound obtained is 156-157 °C (after recrystalliza- tion from ethanol) . Example 3 Preparation of (±)-l-(l-naphthyloxy)-3-{4-[ (1- naphthyl)amino]piperidin-l-yl}-2-propanol [ (I) , Ar = l-naphthyl, R = H, Y = 2,3-(CH=CH)₂-) ; compound Mo. 0108535]

After adding 40 ml of 4 N aqueous sodium hydroxide solution to a suspension containing 3.9 g (0.010 mol) of 4-[ (1-naphthyl)amino]piperidine dihydrobro ide [(II), Ar = 1-naphthyl, R = H] in 80 ml of chloroform, the mixture is stirred until dissolution of the solid material. After separating the two phases, the aqueous layer is extracted with 10 ml of chloroform, then the combined organic phase is washed twice with 10 ml of water each. The chloroform solution is dried and evaporated under reduced pressure. The oily residue is dissolved in 40 ml of toluene, and after adding 2.2 g (0.011 mol) of (±)-l-(l-naphtyloxy)-2,3-epoxypropane [(III), Y = 2,3-

(CH=CH)2~] to the above solution, the reaction mixture is boiled under reflux for 10 hours while stirring. After evaporating the solution under reduced pressure, the residue is purified by chro atography on a silica gel column (particle size 0.063-0.200 mm) by using a 9:1 mixture of chloroform and methanol as eluent. The fractions containing the pure product are evaporated and the residue is recrystallized from 30 ml of ethanol to give the title product in a yield of 1.8 g (42%), m.p.: 111-113 ^βC. -26-

By following the above process the (±) derivatives of formula (I) listed hereinafter were prepared by reacting the corresponding 4-aminopiperidine derivatives of formula (II) with the suitably substituted epoxy compounds of for- ula (III).

Preparation of (±)-l-(4-fluorophenoxy)-3-{4-[ (2,6- dimethylphenyl)amino]piperidin-l-yl}-2-propanol dihydrochloride [(I), Ar = 2,6-di-CH₃-phenyl, R = H, Y = 4-F; compound No. 0108945]

By following the procedure described in Example 1, 3.70 g (0.010 mol) of 4-[ (2 ,6-dimethylphenyl)amino]- piperidine dihydrobro ide [(II), Ar = 2,6-dimethylphenyl, R = H] are converted to the free base form. After dissolv¬ ing the evaporation residue in 40 ml of xylene and adding 1.85 g (0.011 mol) of (±)-1-(4-fluorophenoxy)-2,3-epoxy- propane [(III), Y = 4-F], the reaction mixture is boiled under reflux for 10 hours while stirring. After cooling down, the solution is evaporated under reduced pressure and the residue is purified by chromatography on a silica gel column (particle size 0.063-0.200 mm). A 95:5 mixture of chloroform and methanol is used as eluent. After eva- -27-

porating the fractions containing the pure product, the oily residue is dissolved in 20 ml of ether and an ethyl acetate solution containing hydrogen chloride is dropwise added to the solution until achieving a pH value of 1. The precipitate is filtered and dried on air to obtain the title porduct in a yield of 3.3 g (74%), m.p.: 241-242 °C.

By following the above process the hydrochlorides of the (±) derivatives of formula (I) listed hereinafter were prepared by reacting the corresponding starting substances of formula (II) and formula (III) .

Preparation of (+)-l-phenoxy-3-{4-[ (l-naphthyl)a ino]- piperidin-l-yl}-2-propanol [(I), Ar = l-naphthyl, R = H, Y = H;]

After adding 40 ml of 4 N aqueous sodium hydroxide solution to a suspension containing 3.0 g (0.010 mol) of 4-[ (1-naphthyl)a ino]piperidine dihydrochloride [(II), Ar = 1-naphthyl, R •= H] in 80 ml of chloroform, the mixture is stirred until the dissolution of the solid material. After separating the two phases, the aqueous layer is extracted with 10 ml of chloroform, then the combined organic phase is washed twice with 10 ml of water each. The chloroform solution is dried then evaporated under reduced pressure. After dissolving the oily residue -28-

in 40 ml of xylene and adding 1.65 g (0.011 mol) of (+)-i-phenoxy-2,3-epoxypropane [(III), Y = H], the re¬ action mixture is boiled under reflux for 10 hours while stirring. After cooling the solution, the precipitate is filtered and recrystallized from 20 ml of ethyl acetate to obtain the title product in a yield of 1.63 g (43%), m.p.: 153-155 °C, [α]_D ²⁵ = +5.8° (c = 1, dimethylforma ide) .

(-)-l-Phenoxy-3-{4-[ (1-naphthyl)amino]piperidin-l-yl}- 2-propanol [(I), Ar •= 1-naphthyl, R = H, Y = H] , [m.p.: 162-164 °C (after recrystallization from ethyl acetate) , [°.]D²⁵ = -4.8° (c = 1, dimethylformamide)] was prepared by following the above method and using 4-[(1-naphthyl)- amino]piperidine dihydrochloride [(II), Ar = 1-naphthyl, R = H] and (-)-l-phenoxy-2,3-epoxypropane [(III), Y = H] as starting substances. Example 6

Preparation of (-)-l-phenoxy-3-[4-{[N-(l-naphthyl)- -N-methyl]amino}piperidin-l-yl]-2-propanol [ (l), Ar = l-naphthyl, R = CH₃, Y = H] After liberating the base from 3.2 g (0.010 mol) of 4-{[N-methyl-N-(1-naphthyl) ]amino}piperidine dihydro¬ chloride 20 [(II), Ar = 1-naphthyl, R = CH₃] as des¬ cribed in Example 5, the oily residue is dissolved in 40 ml of xylene and, after adding 1.65 g (0.011 mol) of (-)-l-phenoxy-2,3-epoxypropane [(III), Y = H] , the reac¬ tion mixture is boiled under reflux for 10 hours while stirring. After evaporation of the solution under reduced pressure, the residue is purified by chromatography on a silica gel column (particle size 0.063-0.200 mm). Acetone is used as eluent. After evaporating the fractions containing the pure product, the residue is recrystallized from 20 ml of ethanol to give the title product in a yield of 0.82 g (21%), m.p.: 115-117 °C, [ct]_D25 = -10.8^β (c = 1, dimethylformamide) . By using the above process (+)-l-phenoxy-3-[4-{[N-(l- -naphthyl) -N-methyl]amino}piperidin-l-yl]-2-propanol was prepared from 4-{ [N-methyl-N-( 1-naphthyl) ]amino}piperidine dihydrochloride [(II), Ar = 1-naphthyl, R = CH₃] and

(+) -l-phenoxy-2, 3-epoxypropane [(III), Y = H] . The melting point of the compound obtained is 120-122 °C,

OID^{25 =} +10.9° (c * 1, dimethylformamide) .

Claims

C l a i m s

1. Aminopropanol derivatives of the formula

OH (Y

Ar-N—/ N-CH₂-CH-CH₂-0- f (I)

wherein

R means hydrogen or a Ci-^alkyl group;

Ar stands for a phenyl group optionally substituted by at most two halogens, C₁_₄alkyl,

or nitro group (s) ; or a naphthyl group;

Y represents halogen, C₁_₄alkyl, C₁_₄alkoxy, phenyl,

2,3-(CH=CH)₂- or 3,4-(CH=CH)₂- group; and n is an integer of 0, 1, 2, 3, 4 or 5 in racemic or optically active form, as well as acid addi- tion salts thereof.

2. A compound as claimed in claims 1 which is selected from the group consisting of l-(4-chlorophenoxy)-3-{4-[ (1-naphthyl)amino]piperidin-1- yl}-2-propanol, l-(l-naphthyloxy)-3-{4-[ (1-naphthyl)amino]piperidin-l-yl}-

2-propanol, l-(4-f luorophenoxy)-3-{4-[ (1-naphthyl) amino] piper idin-1- y}-2-propanol, l-(4-f luorophenoxy)-3-{4-[ (4-chlorophenyl)amino]piperidin-l- -yl}-2-propanol, l-(4-chlorophenoxy)-3-{4-[ ( 4 -chlorophenyl) amino] piper idin-l-

-yl } -2-propanol , in racemic or optically active form as well as acid addi¬ tion salts of these compounds. 3. A pharmaceutical composition, w h i c h c o m p r i s e s as active ingredient a racemic or optically active aminopropanol derivative of formula

wherein

R means hydrogen or a C₁_₄alkyl group;

Ar stands for a phenyl group optionally substituted by at most two halogens, C₁_4alkyl, C₁_₄alkoxy or nitro group(s); or a naphthyl group; Y represents halogen, Cι_₄alkyl, Cι_ alkoxy, phenyl, 2,3-(CH=CH)₂- or 3,4-(CH=CH)₂- group; and n is an integer of 0, 1, 2,

3, 4 or 5 or a pharmaceutically acceptable acid addition salt thereof in admixture* with a carrier and/or other additive commonly used in the pharmaceutical industry.

4. A process for the preparation of the novel amino¬ propanol derivatives of formula

wherein

R means hydrogen or a C₁--.₄alkyl group;

Ar stands for a phenyl group optionally substituted by at most two halogens, C-_j^alkyl, C₁_₄alkoxy or nitro group(s); or a naphthyl group; Y represents halogen, C₁_₄alkyl, Cι_ alkoxy, phenyl, -32-

2,3-(CH=CH)₂- or 3,4-(CH=CH)₂- group; and n is an integer 0, 1, 2, 3, 4 or 5 in racemic or optically active form and acid addition salts of these compounds, • w h i c h c o m p r i s e s reacting a 4-aminopiperidine derivative of formula

5 wherein R and Ar are as defined above, with a racemic or optically active epoxide derivative of formula

wherein Y and n are as defined above and, if desired, resolving the so-obtained compound of formula (I) and/or, if desired, converting it to an acid addition salt.

5. A process as claimed in claim 4, w h i c h c o m p r i s e s reacting the 4-aminopiperidine derivative of formula (II) with an excess of the epoxide derivative of formula (III) .

6. A process as claimed in claim 4, w h i c c o m p r i s e s carrying out the reaction of the compound of formula (II) with the compound of formula (III) in an organic protic or aprotic solvent, at the boiling point of the solvent used.

7. A process for the preparation of a pharmaceutical composition, w h i c h c o m p r i s e s mixing as active ingredient a novel racemic or optically active aminopropanol derivative of formul

wherein

R means hydrogen or a C₁-_₄alkyl group;

Ar stands for a phenyl group optionally substituted by at most two halogens, Cι_₄alkyl, Cι_₄alkoxy or nitro group(s) ; or a naphthyl group; . Y represents halogen, C₁_₄alkyl, Cι_₄alkoxy, phenyl,

2,3-(CH=CH)₂- or ^'3,4-(CH=CH)₂- group; and n is an integer of 0, 1, 2, 3, 4 or 5 or a pharmaceutically acceptable acid addition salt thereof with a carrier and/or other additive commonly used in the pharmaceutical industry and converting the mixture to a pharmaceutical composition.

8. Method for inhibiting lipid peroxidation and treat- ing the sequels thereof as well as for protection from or treatment of the sequels of calcium-medicated injuries induced e.g. by ischemia, hypoxia or reperfusion and for treating various degenerative neurological diseases such as e.g. Alzheimer's disease or Parkinson's disease w h i c h c o m p r i s e s administering to a patient to be treated a therapeutically effective amount of an optically active or racemic aminopropanol derivative of formula Ar

wherein

R means hydrogen or a Cι_₄alkyl group; Ar stands for a phenyl group optionally substituted by at most two halogens, C₁_₄alkyl, C₁_₄alkoxy or nitro group(s) ; or a naphthyl group; Y represents halogen,

phenyl,

2,3-(CH=CH)₂- or 3,4-(CH=CH)₂- group; and n is an integer of 0, 1, 2, 3, 4 or 5 or a pharmaceutically acceptable acid addition salt thereof alone or in the form of a pharmaceutical composi¬ tion.

9. The use of a compound of formula

wherein

R means hydrogen or a C₁-_₄alkyl group;

Ar stands for a phenyl group optionally substituted by at most two halogens, C₁_ alkyl,

or nitro group (s) ; or a naphthyl group; Y represents halogen, C₁_ alkyl, C₁_ alkoxy, phenyl,

2,3-(CH=CH)₂- or 3,4-(CH=CH)₂- group; and n is an integer of 0, 1, 2, 3, 4 or 5 or a pharmaceutically acceptable acid addition salt thereof for the preparation of a pharmaceutical composi¬ tion for inhibiting lipid peroxidation and treat¬ ing the sequels thereof as well as for protection from or treatment of the sequels of calcium-medicated injuries induced e.g. by ischemia, hypoxia or reperfusion and for treating various degenerative neurological diseases such as e.g. Alzheimer's disease or Parkinson's disease.

10. The use of a compound of formula

wherein

R means hydrogen or a C₁_₄alkyl group;

Ar stands for a phenyl group optionally substituted by at most two halogens, Cι_₄alkyl, Cι_₄alkoxy or nitro group (s); or a naphthyl group;

Y represents halogen, Cι_₄alkyl, C₁_₄alkoxy, phenyl,

2,3-(CH=CH)₂- or 3,4-(CH=CH)₂- group; and n is an integer of 0 , 1 , 2 , 3 , 4 or 5 or a pharmaceutically acceptable acid addition salt thereof for inhibiting lipid peroxidation and treat¬ ing the sequels thereof as well as for protection from or treatment of the sequels of calcium-medicated injuries induced e.g. by ischemia, hypoxia or reperfusion and for treating various degenerative neurological diseases such as e.g. Alzheimer's disease or Parkinson's disease.