Field of the Invention

The invention relates to novel, optically active cis and trans 14-(N-substituted aminomethyl)eburnane derivatives and their pharmaceutically acceptable salts, the preparation thereof and pharmaceutical com¬ positions containing them.

The novel compounds according to the invention are therapeutically useful, they possess mainly an antioxi¬ dative (lipid peroxidation-inhibiting) and cholinery (muscarine-1 agonist) effect. Thus, the invention relates to pharmaceutical compositions containing these novel compounds as active ingredients, too.

Background of the Invention

A number of pathological processes are known, where the extraordinarily reactive free oxygen radicals are accumulated. The formation of free radicals leads to the oxydation of unsaturated fatty acids (lipid peroxy- dation) , which are essential constituents of membranes. This is a cell-destroying process of low specifity, altering or deteriorating biomolecules. The functions of various levels of cells, organs or the whole organ¬ ism may also be injured. Reactions involving free radicals likely play a causal role in the pathogenesis of ischaemia-induced injuries such as ischaemic intes¬ tinal diseases, yocardial ischae ia, haemorrhagic shock, ischaemic cerebrovascular function disturbances

and real ischaemia [R. J. Konthuis et al.: Physiology of Oxygen Radicals, Chapter 17, pages .217 to 249 (1086) ].

Due to their lipid peroxidation-inhibiting effect, antioxidative compounds provide for a protective action against free radical-induced injuries under ischaemic hypoxic conditions; therefore, antioxidants as anti- ischaemic or antihypoxic substances are useful for the treatment of such clinical patterns.

The contributions of free radicals to the develop¬ ment of traumatic injuries of the central nervous sys¬ tem can be considered to be proven [J. M. Braughler et al. : Drugs of the Future, 124.(2), pages 143 to 152 (1989)].

Similarly, the partial contribution of free radical reactions to the development of disease symptoms of connective tissues and the primary aetiological role thereof in rheumatoid arthritis can be considered as a verified fact [J. Lunec et al.: Cellular Antioxidant Defense Mechanisms, Chapter 33, pages 143 to 159 (1988) ].

Several hepatotoxic compounds are known, the liver- damaging effect of which is presumably connected with pathologic free radical reactions. Antioxidative com¬ pounds protect to acute and chronic liver diseases [J. Feher and A. Vereckei: "Szabadgyδk-reakciόk jelentosege az orvostudomanyban (The importance of Free Radical Reactions in the Medicine), pages 99 to 104 (1985)]. The role of free radical reactions in the development of several haematological clinical patterns, e.g. sickle cell anaemia or beta-thalassaemia, has substan¬ tially been verified. Due to the decreased protective capacity, the danger of oxidative injuries may be strengthened by O ₂ therapy or phototherapy in the cases of newborn or premature infants. The use of some anti-

oxidants is advantageous in the treatment .of such clinical pictures.

Cholinergic receptors of muscarinic-1 (M-l) type are predominantly present in cerebral cortex and are involved in memory and learning processes. As for example in the case of Alzheimer's disease the loss of cortical cholinergic functions are highly associated with memory and learning disturbances, M-l receptors might be primary targets of therapeutical intervention aiming to restore of damaged cholinergic functions by compounds acting as selective agonists at M-l choli¬ nergic receptors.

It has been recognized that the eburnane derivati¬ ves of the invention exert a significant antioxidative (lipid peroxidation-inhibiting) and cholinerg ( usca- rine-1 agonistic) effects.

Summary of the invention

The invention relates to novel, optically active cis and trans l4-(N-substituted aminomethyl)eburnane derivatives of the formula (I) ,

wherein

Rl stands for hydrogen, a C^galkyl or C ₂ - ₆ alkenyl group;

R2 means a Cι_galkyl, C2- ₆ alkenyl or a hydroxy-Cx-galkyl group; or Rl and 2 represent together with the adjacent nitrogen atom

- -

to which they are attached, a 5 to 7-membered sat rated ring, optionally containing one oxygen at and/or one optionally substituted nitrogen atom heteroatom; and n is 2 or 3, as well as their pharmaceutically acceptable salts and pha maceutical compositions containing these compounds. Furthe more, the invention relates to a process for the preparati of these novel compounds.

When Ri and R2 represent, together with the adjacen nitrogen atom to which they are attached, a nitrogen-con taining 5 to 7-membered saturated ring, said nitrogen ato may be substituted by a C _galkyl, C ₂ - ₆ alkenyl, phenyl-C -g alkyl, diphenylmethyl or p_-hydroxyphenyl group.

Detailed Description of the Invention

The antioxidative effect of the compounds of formul

(I) was studied on rat brain homogenate by using the metho of J. N. Braughler et al. [J. Biol. Che . 262(22) , page

10438 to 10440 (1987)].

Effect on the Fe^-t-induced lipid peroxidation in ra brain homogenate

Wistar rats with about 200 to 250 g body-weight wer decapitated and their whole brain was homogenized in 9 volu mes of a Krebs-Ringer buffer solution containing 15 mM HEPE

(pH 7.4), 140 mM NaCl, 3.6 mM KC1, 1.5 mM CaCl ₂ , 0.7 m MgCl2, 1.4 mM KH ₂ O and 10 mM glucose. Thereafter, the pro tein content of the solution was determined and adjusted t 10 mg/ l.

The inhibitory agents were given in a volume of 5 μl t 200 μl of the above homogenizate under cooling by ice, the the incubation mixture obtained was incubated at 37°C for 2 minutes. The Fe ²⁺ -induced lipid peroxidation was elicited b adding 5 μl of a 8 mM Fe(NH4)2(SO4) _ solution. After th incubation period the reaction was stopped by adding 1 ml o

a stopping solution containing 0.8 M HC1 and 12.5% of tri- chloroacetic acid, then the samples were centrifuged with 2000 x g at 4°C in a Janetzky K-70 equipment for 10 minutes.

After adding 1 ml of a 1% thiobarbituric acid solution to a 0.5 ml portion of the supernatant, the samples were placed in a water bath of 100°C temperature for 20 minutes. The colour intensity development was determined at 535 nm in a HITACHI 150-20 spectrophotometer by using malondialdehyde- bis(diethylacetal) as standard.

Muscarinic-agonist receptor binding assay

Male Hannover-Wistar rats were decapitated and cerebral cortices were rapidly dissected. The tissues were homoge¬ nized in 100 vol of ice-cold buffer (K/Na phosphate, 10 mM, pH 7.4) using a motor driven teflon/glass Potter homoge- nizer, centrifuged at 1,000 g for 10 min at 0°C. The super¬ natant was ready for receptor assay.

The receptor binding studies were carried out using plastic test tubes in a final volume of 1 ml of the same buffer containing 970 μl membrane suspension (about 1.2 mg membrane protein) , 10 μl of DMSO with the appropriate con¬ centrations of the test compounds and 20 μl of radioligand. Nonspecific binding was defined in the presence of 1 μM of atropine. The reaction was initiated by the addition of 1.0 nM of 3H-Cis-methyl-Dioxolane (specific activity: 2.1 TBq/mmol; New England Nuclear) and continued at 25°C for 60 min. The incubation was terminated by rapidly filtering the mixture under vacuum through Whatman GF/B glass fiber filters presoaked in an aqueous polyethyleneimine solution (0.05%) for at least 30 min using a Brandel M 48R Cell Harvester. The filters were further washed with 20 ml of ice-cold washing buffer.

After drying of the filter sheets, radioactivity re¬ tained on the filters was determined by liquid scintillation spectroscopy using an LKB Rackbeta liquid scintillation counter.

IC ₅₀ values were expressed in nM.

Muscarinic-l receptor binding assay

Male Hannover-Wistar rats were decapitated and cerebral cortices were rapidly dissected. The tissues were homoge¬ nized in 10 vol of 0.32 M ice-cold sucrose using a motor driven teflon/glass Potter homogenizer, centrifuged at 1,000 g for 15 min at 0°C. The resulting supernatant was further centrifuged at 17,000 g for 20 min at 4°C. The final pellet was resuspended in 100 vol of Krebs HEPES buffer, pH 7.4 and used for receptor assay.

The receptor binding studies were carried out in a final volume of 1 ml of Krebs HEPES buffer containing 970 μl of membrane suspension (about 0.8 mg membrane protein), 10 μl of DMSO with the appropriate concentrations of the test compounds and 20 μl of radioligand. Nonspecific binding was defined in the presence of 1 μM of atropine. The reaction was initiated by the addition of 1.0 nM of 3H-Pirenzepine (specific activity: 3.2 TBq/mmόl; New England Nuclear) and continued at 30°C for 60 min. The incubation was terminated by rapidly filtering the mixture under vacuum through What¬ man BF/B glass fiber filters using a Brandel M 48R Cell Harvester. The filters were further washed with 20 ml of ice-cold washing buffer.

After drying of the filter sheets, radioactivity re¬ tained on the filters was determined by liquid scintillation spectroscopy using an LKB Rackbeta liquid scintillation counter.

IC5 ₀ values were expressed in nM.

Muscarinic-2 receptor binding assay

Male Hannover-Wistar rats were decapitated and cerebel¬ lum was rapidly dissected. The tissues were homogenized in 10 vol of 0.32 M ice-cold sucrose using a motor driven tef¬ lon/glass Potter homogenizer, centrifuged at 1,000 g for 15 min at 0°C. The resulting supernatant was further centri¬ fuged at 17,000 g for 20 min at 4°C. The final pellet was

resuspended in 100 vol of Krebs HEPES buffer, pH 7.4 and used for receptor assay.

The receptor binding studies were carried out in a final volume of 1 ml of Krebs HEPES buffer containing 970 μl membrane suspension (about 0.8 mg membrane protein), 10 μl of DMSO with the appropriate concentrations of the test com¬ pounds and 20 μl of radioligand. Nonspecific binding was defined in the presence of 1 μM of atropine. The reaction was initiated by the addition of 1.0 nM of 3H-N-methyl- scopolamine (specific activity: 2.9 TBq/mmol; New England Nuclear) and continued at 30°C for 60 min. The incubation was terminated by rapidly filtering the mixture under vacuum through Whatman BF/B glass fiber filters using a Brandel M 48R Cell Harvester. The filters were further washed with 20 ml of ice-cold washing buffer.

After drying of the filter sheets, radioactivity re¬ tained on the filters was determined by liquid scintillation spectroscopy using an LKB Rackbeta liquid scintillation counter.

IC5 ₀ values were expressed in nM.

Table I

* McN-A-343: (4-Hydroxy-2-butynyl)-1-trimethyl- ammonium m-chlorocarbonilate chloride

It can be seen from the data of Table I that the anti¬ oxidative (lipid peroxidation-inhibiting) activity of the compounds of Examples is superior to that of idebenone, a known antioxidant used already in the therapy. In addition, the antioxidative activity of the compounds of the invention proved to be higher than that of vitamin E (DL-α-tocopherol) in the above test.

It is also evident that the compounds given in the table have stronger muscarinic affinity than McN-A-343. Moreover, M /M ₂ ratio is also favourable for the compounds of the examples than that of McN-A-343.

As for the cholinergic effects of the compoundsof the invention, they may provide therapeutical tools for the treatment of cognitive disorders. Alzheiner's disease (AD) , the most common type of dementia, where the cognitive dis¬ turbances such as memory and learning impairment are pre¬ dominant, has no effective treatment as yet. It is well established that the most consistent phatological feature of AD is the degeneration of cerebral cholinergic neurons.

According to another aspect of the invention, there is provided a process for the preparation of compounds of the formula (I) and their pharmaceutically acceptable salts, which comprises reacting a new, optically active cis or trans 14-(chloroalkylammomethyl)eburnane derivative of the formula (II) ,

_Cl -l

wherein n is as defined above, with a primary or secondary

amine of the formula (III) ,

wherein Ri and R are as defined above, in an inert organic solvent in the presence of an acid binding agent and, if desired, converting the compound of the formula (I) obtained to its pharmaceutically acceptable salt.

The novel trans compounds of formula (II) used as start¬ ing substances can be prepared by using the process described in Examples 1 and 2. The novel cis compounds of formula (II) can similarly be prepared according to Examples 1 and 2, except that 14-hydroxymethyleburnane prepared according to Examples 6 and 7 of the Hungarian patent spec¬ ification No. 171,663 is used as starting substance.

In the course of preparing the starting substances of formula (II) , the possibility of epimer formation is given since the substituent bound to the C-14 atom can be in α- or β-position. In the case of the target compounds of formula (I) , the steric position of the group bound to the C-14 atom is the same as the steric position of the group bound to the C-14 atom of the starting compound of formula (II) ^" . ^" Off the compounds of formula (II) , both the 14-α and 14-β epimers are useful starting substances.

The primary and secondary amines of the formula (III) are known, commercially available products. Suitable primary amines are e.g. Cl-6alkylamines, C2-3 hydroxyalkylamines, benzylamine and phenethylamine. Useful secondary amines are e.g. symmetric or asymmetric dialkylamines or dialkenyl- amines or 5 to 7-membered saturated cyclic amines containing optionally oxygen and/or optionally substituted nitrogen in their ring.

The process according to the invention will hereinafter

be discussed in detail.

The starting 14-(chloroalkylammomethyl)eburnane deriva tive of the formula (II) is reacted with an amine of th formula (III) in an inert, high-boiling organic solvent at temperature of 100 to 160°C in the presence of an acid bind ing agent. Any inorganic or organic base can be employed a an acid binding agent, which is inert under the reactio conditions. Suitable inorganic acid binding agents are e.g. anhydrous potassium and sodium carbonate; useful organi acid binding agent is e.g. a dialkylaniline though even¬ tually, an excess of the a ine of formula (III) may prefer¬ ably be used as acid binding agent. The compound of formula (I) formed can be separated in such a way that after termi¬ nation of the reaction, the mixture is diluted with water, the organic phase is dried, filtered, the solvent is removed and the residue is recrystallized from a suitable solvent or purified through salt formation in a known manner.

If desired, the compounds of formula (I) can be con¬ verted to their acid addition salts by reacting them with an inorganic or organic acid. Suitable inorganic acids for salt formation are hydrogen bromide; sulfuric acid; perhalogenic acids such as perchloric acid; useful organic acids are e.g. formic, acetic and propionic acid as well as alkanesulfonic acids, e.g. methanesulfonic and ethanesulfonic acid; and a ino acids such as aspartic or glutamic acid.

The active agents of formula (I) or their pharmaceuti¬ cally acceptable salts can be formulated to pharmaceutical compositions by mixing them with non-toxic inert, solid or liquid carriers and/or auxiliaries commonly used in the therapy for parenteral or enteral administration. Suitable carriers are e.g. water, gelatin, lactose, starch, pectin, magnesium stearate, stearic acid, talc, vegetable oils such as peanut oil, olive oil and the like. The active ingredient may be formulated in the form of usual pharmaceutical compo¬ sitions, particularly in a solid form, e.g. in the form of

rounded or angled tablet, dragee, capsule such as gelatin capsule, pill, suppository and the like.

The amount of the solid carrier may be varied within a wide range; preferably, it is between about 25 mg and 1 g. Optionally, the pharmaceutical compositions may contain usual pharmaceutical additives, e.g. preservatives, stabili¬ zers, wetting or emulsifying agents and the like. The compo¬ sitions can be prepared by using well known methods, e.g. in the case of solid compositions by sieving, mixing, granulat¬ ing and compressing the components. The compositions may be subjected to other usual operation of the pharmaceutical technology, such as sterilization.

The invention also relates to a method of treating cognitive disorders and disorders connected with lipid per¬ oxidation. This method comprises administering a therapeu- tically effective amount of an active agent of the formula (I) or a pharmaceutically acceptable salt thereof to the patient. A daily dose of 0.1 to 50 mg/kg body weight is used generally for treating an adult human by administering this amount daily once or in divided doses.

The invention is illustrated in detail by the aid of the following non-limiting Examples.

Example 1

Preparation of 14α-(chloroethylaminomethyl)-3α,16β- eburnane

4.25 g of 14α-(hydroxyethylaminomethyl)-3α,16β-eburnane hydrochloride were suspended in 50 ml of dichloroethane, 2.5 ml of dimethylformamide and 1.5 ml of thionyl chloride were added and the reaction mixture was boiled under reflux for 4 hours. Then, the mixture was evaporated to give a thick slurry and filtered at room temperature. The product fil¬ tered out was suspended in 50 ml of chloroform, 50 ml of water and then concentrated aqueous ammonia were added until reaching a pH value of 9. After thoroughly shaking the orga¬ nic phase was separated, dried over a drying agent, filtered

and evaporated to solvent-free. The residue was crystallized from a mixture containing 1 ml of methylene chloride and 6 ml of methanol to give 2.8 g (76%) of the title product, m.p.: 140-142°C, [α] _D ²⁰ = -121° (c=l, chloroform). Example 2

Preparation of 14 -(chloropropylaminomethyl)-3α ,16β- eburnane

After dissolving 4.5 g of 14α-(hydroxypropylamino- methyl)-3α.l6β-eburnane in 100 ml of dichloroethane, gaseous hydrogen • chloride was introduced into the solution until achieving a pH value of 1. Then, 5 ml of dimethylformamide and 1.5 ml of thionyl chloride were added to the crystalline suspension which was then boiled under reflux for 4 hours. After evaporating the mixture to a thick slurry and filter¬ ing at 0°C, the product obtained was suspended in 50 ml of chloroform, 50 ml of water and then concentrated aqueous ammonia were added until adjusting the pH value to 9 and the mixture was extracted. The organic phase was separated, dried over a drying agent, filtered and the solution was evaporated until solvent-free. The oily residue was crystal¬ lized from 8 ml of ethanol to obtain 3.0 g (64.8%) of the title product, m.p.: 79-81°C, [c_] _D ^2u = -120.7° (c=l, chloro¬ form) .

Example 3

Preparation of 14α-(mesyloxymethyl)-3α,16β-eburnane At 0°C 8 ml of methanesulfonyl chloride were added to a solution containing 15 g of (-)-14α-hydroxymethyl-3α,16β- eburnane in 90 ml of abs. pyridine. The mixture was stirred at 0°C for 30 minutes and then at 40°C for 1 hours. Sub¬ sequently, the solvent was distilled off under reduced pres¬ sure, the residue was dissolved in 200 ml of water and extracted 3 times with a total of 150 ml of methylene chlo¬ ride at a pH value of 9 adjusted by adding concentrated aqueous ammonia. The combined organic phase was dried over a drying agent, filtered and evaporated until solvent-free. In

this way 18 g (95.8%) of the oily title base were obtained, which was dissolved in 100 ml of ether, acidified to a pH value of 2 to 3 by adding isopropanolic hydrogen chloride, then the precipitated hydrochloride was filtered off to obtain 18.7 g (95%) of the title product, m.p.: 178-180°C, _ ^α _D ^{20 =} -25.4° (c=l, dimethylformamide) .

Example 4

Preparation of 14β-(mesyloxymethyl)-3α,16β-eburnane hyd¬ rochloride

The process described in Example 3 was followed, except that 15 g of (+)-14β-hydroxymethyl-3c_,16α-eburnane were used as starting substance to give 17.5 g (98.7%) of an oily residue. The salt formation was carried out according to the process described in Example 3 to obtain 17.6 g (92%) of the title hydrochloride, m.p.: 168-170°C, [α] _D ²⁰ = +90.5° (c=l, dimethylformamide) .

Example 5

Preparation of 14α-(hydroxyethylaminomethyl)-3α,16α- eburnane

A mixture containing 18 g of 14α-hydroxymethyl-3α,16α- eburnane base (obtained according to Example 3) , 18 ml of ethanolamine and 18 ml of chlorobenzene was boiled under reflux for 2 hours. Thereafter, chlorobenzene was removed from the reaction mixture under reduced pressure, the resi¬ due was treated with 100 ml of water, the product separated was filtered off, washed with water until neutral and dried to obtain 14.5 g (88%) of a product, which was recrystal- lized from 100 ml of acetonitrile to give 11.6 g (80%) of the title compound, m.p.: 150-153°C, [α] _D ²⁰ = -73.1° (c=l, chloroform) .

Example 6

Preparation of 14β-(hydroxyethylaminomethyl)-3α,16α- eburnane dihydrochloride

A mixture containing 17.5 g of 14β-(mesyloxymethyl)- 3α,16α-eburnane base (prepared as described in Example 4),

17.5 ml of ethanolamine and 17.5 ml of chlorobenzene was boiled under reflux for 2 hours, then the mixture was distilled under reduced pressure until solvent-free and the residue was treated with 100 ml of water. Water was decanted from the oily precipitate and the treatment by water described above was twice repeated. The thus-obtained oil was dissolved in 80 ml of methylene chloride, dried over a drying agent and after filtering the methylene chloride solution was evaporated to dryness. The water-free oil obtained was dissolved in 100 ml of acetone and isopropa- nolic hydrogen chloride was added until reaching a pH value of 2. The precipitate was filtered off to yield 15 g (78%) of the title dihydrochloride, m.p.: 280-285°C, [α]D ²⁰ = 76.7° (c=l, dimethylformamide) . Example 7

Preparation of 14α-(chloroethylaminomethyl)-3α,16α-ebur- nane dihydrochloride

Gaseous hydrogen chloride was introduced into the solu¬ tion of 10.6 g of 14α-(hydroxyethylammomethyl)-3α,16α-ebur- nane in 100 ml of dichloroethane until a pH of 2 was reached, then 5 ml of dimethylformamide and 2.5 ml of thio- nyl chloride were added and the mixture was boiled under reflux for 4 hours. Subsequently, the mixture was evaporated to one third of its original volume and the crystalline pre¬ cipitate was filtered off at room temperature to give 13 g (97%) of the title dihydrochloride, m.p.: 290-295°C. Example 8

Preparation of 14β-(chloroethylaminomethyl)-3α,16α-ebur- nane dihydrochloride

5 ml of dimethylformamide and 2.5 ml of thionyl chloride were added to a solution containing 12.8 g of 14β-(hydroxy- ethylaminomethyl)-3α,16α-eburnane dihydrochloride in 100 ml of dichloroethane and the reaction mixture was boiled under reflux for 4 hours. Then, the mixture was evaporated to the third of its original volume and the crystalline precipitate

was filtered off at room temperature to obtain 12.4 g (92.5%) of the title dihydrochloride, m.p.: 299-301°C.

Example 9

Preparation of 14α-[4-(p_-hydroxyphenylpiperazine-l-yl)- ethylaminomethyl]-3α,lδα-eburnane

8.9 g of 14o!-(chloroethylaminomethyl)-3α,16o:-eburnane dihydrochloride were suspended in 100 ml of chlorobenzene, 20 ml of water and then concentrated aqueous ammonia were added to adjust a pH value of 9. The mixture was thoroughly shaken, the organic phase was separated, dried over a drying agent, filtered off and 8.9 g of 4-(p_-hydroxyphenyl)pipera- zine were added. The mixture was boiled under reflux for 8 hours and then filtered off at 10°C. After evaporating the filtrate to dryness, the residue was crystallized from 15 ml of acetonitrile to obtain 6.2 g (60.5%) of the title pro¬ duct, m.p.: 188-190°C, [ ] _D ²⁰ = -48.6° (c=l, chloroform).

Example 10

Preparation of 14β-[ (4-diphenylmethylpiperazine-l-yl)- ethylamino ethyl]-3α,16α-eburnane

To the suspension of 8.9 g of 14β-(chloroethylamino- methyl)-3α,I6β-eburnane dihydrochloride in 100 ml of xylene 20 ml of water and concentrated aqueous ammonia were added to adjust a pH value of 9. After thoroughly shaking the organic phase was separated, dried over a drying agent, fil¬ tered off and 12.6 g of diphenylmethylpiperazine were added. After refluxing for 2 hours the mixture was filtered off at 0°C. The filtrate was washed with a total of 100 ml of water in two portions, dried over a drying agent, filtered off and the filtrate was acidified until a pH value of 1 by adding isopropanolic hydrogen chloride. The crystalline precipitate was filtered off, boiled with 50 ml of methanol and filtered at 0°C. The hydrochloride obtained of the title product was dissolved in 100 ml of distilled water, clarified by activ¬ ated carbon, filtered and alkalinized to pH 9 by adding con¬ centrated aqueous ammonia. The precipitated title product

was filtered off and washed with distilled water until neutral. In this way a yield of 6.1 g (51.8%) was obtained, m.p.: 78-80°C, [α] _D ²⁰ = +51.8° (c=l, chloroform). Example 11

Preparation of 14α-[(4-diphenylmethylpiperazine-l-yl)- ethylaminomethyl]-3α,16β-eburnane dihydrochloride

6 g of I4α-(chloroethylaminomethyl)-3α,16β-eburnane were refluxed with 6 g of benzhydrylpiperazine in 250 ml of xylene in the presence of 6 g of finely powdered potassium carbonate by using a Marcusson device for 10 to 12 hours. After the termination of the reaction 200 ml of water were added to the mixture at 20°C, the xylene phase was sepa¬ rated, dried over a drying agent, filtered off and stirred with 0.5 g of activated carbon and 0.5 g of Brockmann II aluminum oxide for 2 hours. After filtration the xylene solution was acidified to a pH value of 1 by adding isopro- panolic hydrogen chloride. The precipitate was filtered off and washed in two portions with a total of 20 ml of methanol at 40 to 45°C to give 7.1 g (67%) of the title hydrochlo¬ ride, m.p.: 278-280°C, [α]π ²⁰ = -58° (c=l, dimethylform- amide/NH ₃ ) .

Example 12

Preparation of 14α-[(4-p_-hydroxyphenylpiperazine-l-yl)- ethylaminomethyl]-3α,16β-eburnane

A solution containing 10 g of 14 -(chloroethylamino- methyl)-3α,16β-eburnane and 15 g of 4-(p_-hydroxyphenyl)pipe- razine in 100 ml of chlorobenzene was boiled under reflux for 10 to 12 hours. After the termination of the reaction the mixture was cooled to 10°C, filtered off and the fil¬ trate was evaporated until solvent-free under reduced pres¬ sure. The residue was boiled with 50 ml of methanol and fil¬ tered at 20°C to yield 8.5 g (61.6%) of the title product, m.p.: 218-220°C, [α]o ²⁰ = -72.4° (c=l, dimethylformamide). Example 13 Preparation of 14α-(piperidinylethyla inomethyl)-3α,16β-

eburnane dihydrochloride

A solution containing 3.7 g of l4ct-(chloroethylamino- methyl)-3c.,I6β-eburnane and 8 ml of piperidine in 20 ml of chlorobenzene was boiled under reflux for 2 hours. After termination of the reaction the mixture was distilled until solvent-free, the residue was treated with 100 ml of water and the undissolved part was dissolved in 50 ml of methylene chloride, dried over a drying agent, filtered off and evapo¬ rated to dryness. The residue was dissolved in 30 ml of ace¬ tone and acidified to pH 2 by adding isopropanolic hydrogen chloride. The precipitate was filtered off and washed with a little amount of acetone to obtain 3.5 g (70.9%) of the title dihydrochloride, m.p.: 270-275°C, [α] _D ²⁰ = -85.8° (c=l, dimethylformamide/NH ₃ ) .

Example 14

Preparation of 14α-(pyrrolidinylethylaminomethyl-3α,16β- eburnane

The process described in Example 13 was followed, except that 8 ml of pyrrolidine were used instead of 8 ml piperi¬ dine to give the title substance in a yield of 3.6 g (76%), m.p.: 258-262°C, [C_]D ²⁰ = -46.4° (c=l, dimethylformami- de/NH ₃ ).

Example 15

Preparation of 14α-[ (α-phenethylamino)ethylaminomethyl]- 3α,16β-eburnane dihydrochloride

The process described in Example 13 was followed, except that 4.8 g of α-phenethylamine were used instead of 8 ml of piperidine. The title dihydrochloride was obtained in a yield of 3.3 g (62%), m.p.: 255-260°C, [α] _D ²⁰ = -42.9° (c=l, dimethylformamide/NH ₃ ) .

Example 16

Preparation of 14c.-[ (benzylamino)ethylaminomethyl]- 3α,16β-eburnane dihydrochloride

The process described in Example 13 was followed, except that 4.2 g of benzylamine were used instead of 8 ml of pipe-

ridine to give 3.5 g (68%) of title dihydrochloride, m.p.: 250-255°C, [c-] _D 2 ⁰ _ -89.2° (c=l, dimethylformamide/NH ₃ ) . Example 17

Preparation of 14α-[(diallylamino)ethylaminomethyl]- 3c,16β-eburnane

A solution containing 4.5 g of 14α-(chloroethylammo- methyl)-3α,16β-eburnane and 10 ml of diallylamine in 10 ml of xylene was boiled under reflux for 2 hours. After the termination of the reaction the mixture was evaporated to dryness and the residue was treated with 100 ml of water. The undissolved material was dissolved in 50 ml of benzene, dried over a drying agent, filtered off and the filtrate was clarified by activated carbon and Brockmann II aluminum oxide under stirring for 2 hours. After filtration the solu¬ tion was evaporated to dryness. The residue was crystallized from acetonitrile to give 2.2 g (42%) of the title compound, m.p.: 60-63°C, [α]D ²⁰ = -98.1° (c=l, chloroform). Example 18

Preparation of 14a-[(hydroxyethylamino)ethylaminome- thy1]-3 ,16β-eburnane dihydrochloride

The process described in Example 13 was followed, except that 10 ml of ethanolamine were used ins ^' tead of 8 ml of piperidine. The title dihydrochloride was obtained in a yield of 3.5 g (71.6%), m.p.: 288-290°C, [α] _D ²⁰ = -90 ^ό (c=l, dimethylformamide) . Example 19

Preparation of 14α-(morpholinylethylaminomethyl)-3α,16β- eburnane dihydrochloride

The process described in Example 13 was followed, except that 8 ml of morpholine were used instead of 8 ml of piperidine to obtain the title dihydrochloride in a yield of 3.4 g (68%), m.p.: 278-280°C (after recrystallization from ethanol) , [ct] _, ²⁰ = -48.6° (c=l, dimethylformamide). Example 20 Preparation of 14α-[(diethylamino)ethylaminomethyl]-

3α,16β-eburnane dihydrochloride

The process described in Example 13 was followed, except that 8 ml of diethylamine were used instead of 8 ml of pipe¬ ridine to give the title dihydrochloride in a yield of 3.6 g (74.8%), m.p.: 260-265°C, [α] _D ²⁰ = -44.4° (c=l, dimethyl¬ formamide) .

Example 21 Preparation of 14α-[ (4-diphenylmethylpiperazine-l-yl)- propylaminomethyl]-3α,16β-eburnane dihydrochloride

After refluxing 3.85 g of 14α-(chloropropylaminomethyl)- 3α,16β-eburnane with 9 g of benzhydrylpiperazine in 80 ml of xylene, 80 ml of water were added and after separation the xylene phase was dried, filtered off and clarified by acti¬ vated carbon and Brockmann II aluminum oxide under stirring for several hours. The solution was filtered off and the solution was evaporated to dryness. After dissolving the residue in 25 ml of acetone, the solution was acidified to pH 2 by adding isopropanolic hydrogen chloride. The precipi¬ tate was filtered off to obtain 3.7 g (54.8%) of the title dihydrochloride, m.p.: 260-265°C, [α]D ⁰ = -57° (c=l, di¬ methylformamide) .