NO820394L - PROCEDURE FOR THE PREPARATION OF NEW APOVINCAMOLD DERIVATIVES - Google Patents

Description

The present invention relates to new apovincaminol derivatives, a method for their production and pharmaceutical preparations containing them.

According to one aspect of the invention, new apovincaminol derivatives of general formula I have been provided,

namely apovincaminol-3<1>,4',5<1->trimethoxy-benzoate, of formula Ia 9-nitro-apovincaminol-3451-trimethoxy-benzoate of formula Ib and 11-nitro-apovincaminol-3<1>,4 <1>,5'-trimethoxy-benzoate of formula Ic

and acid addition salts thereof.

1" 2

In general formula I, either both R and R denote hydrogen, or one of the symbols R 1 and R 9 denotes hydrogen and the other denotes nitro.

The acid addition salts of the compound of general formula I can be formed with inorganic or organic acids.

Of the salts formed with inorganic acids, the hydrochlorides, sulfates and phosphates are particularly useful, while among the salts formed with organic acids the hydrogen tartrates, succinates, citrates and ascorbates are particularly useful.

Apovincaminol and its acetate are known to exhibit an effect on the coronary artery (French Patent 2,035,784). It is also known that apovincaminol benzoate exhibits general, vasodilating properties (Hungarian patent document 166 476; CA 82, 129 279. v (1975)) and that the esters of apovincaminol formed with alkane carboxylic acids with 3-12 carbon atoms exhibit a cerebral vasodilating effect (Hungarian patent specification 171 662, the corresponding US patent specification 4 108 996 and BR patent specification 26 32 118).

Thus, all the known esters of apovincaminol exhibit a vasotropic effect. On the other hand, the new compounds inhibit the enzyme activity of phosphodiesterase and can be used primarily in the treatment of skin diseases associated with pathological cell division and in the prophylaxis of new cases of such diseases.

Diseases associated with pathological cell division of the epidermis are relatively frequent and involve a few % of the population. These diseases can be of both benign and malignant nature, such as psoriasis atopy dermatitis, primary contact dermatitis, baso- and spinocellular carcinoma, inchthyosi-s, premalignant hyperkeratosis, light-induced keratosis, acne and seborrhoea dermatitis. Some diseases occur only in humans, while others occur in both humans and animals.

As some of the skin diseases associated with pathological cell division (e.g. psoriasis) do not occur in animals, the activity of the compounds against psoriasis can only be confirmed indirectly in animal experiments.

Voorhees et al. (Arch. Perm. 104, 359-365, (1971)) have found that the pathological cell division is accompanied by the decrease in the level of cyclic adenosine monophosphate (c-AMP). It is known that c-AMP is formed under the action of adenyl cyclase and is cleaved by phosphodiesterase. Voorhees succeeded in affecting psoriasis with agents that stimulated the action of adenyl cyclase (such as nor-epinephrine) or inhibited the function of phosphodiesterase (eg, papaverine).

When planning the mock-up test, it was based on the assumption that the statement of Voorhees is relevant and also that the opposite case applies. Thus, if it can be shown that a certain compound inhibits the function of phosphodiesterase, this makes it likely in an indirect way that the specified compound is suitable for the treatment of skin diseases

associated with the pathological cell division.

This assumption turned out to be correct: compounds that showed phosphodiesterase inhibitory activity in in vitro tests were found to be active in the treatment of psoriasis in clinical trials.

Present model tests were carried out using phosphodiesterase isolated from animal body tissue (rat brain, ox brain, ox heart). The enzyme was isolated by the method of J. Schroder and H. V. Richenberg (Biochem. Biophys. Acta 302, 50 (1973)), the isolated phosphodiesterase was purified by the method described by J. G. Hardman and E. W. Sutherland (J. Biol. Chem. 240, 3704 (1965)) and finally the activity of the purified enzyme was measured by the radioisotope method according to G. Poch in the presence of an excess of tritiated c-AMP (10.1 millimoles of c-AMP substrate from which 3H-c-AMP is 2.59 K Bq) in an incubation system first without inhibitor and then in the presence of apovincaminol-3*,4',5'-trimethoxy-benzoate derivative as inhibitor after an incubation period of 20 minutes (N. S. Arch. Pharmacol. 268, 272 (1979)). A 1 millimolar solution was prepared from the test compound and varying amounts were added to the incubated enzyme preparation, using the specified solution so that the concentration of the test connection

-7

in the incubated sample should correspond respectively to 5x10,

1 x 10~<6>, 5 x 10~<6>, 1 x 10<-5>, 5 x 10~<5>and 10~<4>mol/liter.

The aqueous solution of the compound used in the comparison (papaverine) was added to the enzyme prepared in a similar manner.

The activity of the control (the enzyme solution containing no inhibitors) was set to 100%, while the activity of the solutions containing the compound of general formula I and papaverine was expressed as the percentage of the control. The results measured on the enzyme isolated from rat brain are listed in the following table.

The results on enzyme isolated from ox brain and ox heart were measured in a similar way. Using the results obtained, the enzyme activity was plotted against the logarithm of the enzyme inhibitor concentration (expressed in μmol). The concentration of the enzyme inhibitor which reduces the enzyme activity by 50% (I5Q) was read from the curve. The results obtained are listed in the following table.

It appears from the above table that the new compounds were 33-50 and 4.5-9 times more active than papaverine used as reference compound on the enzyme isolated from ox heart and rat brain.

The first clinical tests were carried out with topical preparations containing the active ingredient (ointment, cream, solution, tincture, paste, aerosol). Creams containing 2%, 1%, 0.5%, 0.25% and 0.1% of (-)-9-nitro- or (-H11-nitro-apovincaminol-3',4',5',- trimethoxy benzoate was used.

Patients suffering from psoriasis were treated.

A fundamental feature of the selection was that the patients did not simultaneously receive a systemic treatment for their main disease (eg an immunosuppressive, cytostatic or glucocorticoidal treatment).

Groups consisting of five patients were each examined using the so-called plaque method. One side of symmetrical skin lesions was treated with the cream containing the active ingredient while the other side was treated with placebo. The other affected skin surfaces on the patient were treated with other topical methods - including an ointment that is generally used for the treatment of psoriasis, containing flumethasone pivalate and salicylic acid, which ointment was used as a reference substance.

The test was started with creams with the highest content of active ingredient and additional patients were treated with a cream with the lowest content of active ingredient, but which was still active. The treatment was carried out for two weeks,

twice per day, in an open bandage.

The effect was assessed by observing three different symptoms - inflammation, infiltration and desquamation. The intensity of the symptoms was expressed on the following scale between 0 and 3

The symptoms were assessed before treatment (I), after treatment for seven days (II) and after treatment for fourteen days (III). In the following table, the average number of points (total number of points divided by the number of patients) is indicated. A cream containing 2%. active ingredient was used.

Test connection

According to another aspect of the invention, a method is provided for the preparation of the new compounds of general formula I in which either both R 1 and R 2 denote hydrogen or where one of the symbols R<1> and R 2 is hydrogen and the other is nitro , and pharmaceutically acceptable acid addition salts thereof., which method is characterized in that apovincaminol or an acid addition salt thereof is reacted with 3,4,5-trimethoxy-benzoic acid or a reactive derivative thereof which is capable of carrying out acylation, whereupon the compound of formula Ia is isolated or that this is converted into a pharmaceutically acceptable acid addition salt, or if desired, that the compound of formula. Ia is nitrated and that the mixture of the compound of formula Ib and Ic is separated into the two components, and if desired, that a compound of general formula Ib or Ic is converted into a pharmaceutically acceptable acid addition salt.

The method according to the invention is preferably carried out in the presence of an organic solvent, in particular a chlorinated hydrocarbon or an aliphatic ketone or pyridine, in particular in methylene chloride, chloroform or acetone. If a 3,4,5-trimethoxy^benzoyl halide is used as acylating agent, the reaction is carried out in the presence of an equimolar amount or a small excess of a. antacid. For this purpose, e.g. an alkali carbonate, alkali potassium hydrogen carbonate or organic amine is used. If 3,4,5-trimethoxy-benzoic acid is used as acylating agent, the reaction is carried out in the presence of a catalytic amount of an acid.

(preferably hydrochloric or sulfuric acid) or an activator of the carboxyl group and/or a dehydrating agent. The carboxyl group can be activated by a halogenated phenol, preferably pentachlorophenol. For example, N,N'-dicyclohexylcarbodiimide can be used as a dehydrating agent. The acylation can be carried out at a temperature between -20°C and the boiling point of the reaction mixture, preferably 20-60°C.

The compound of formula Ia can be isolated from the reaction mixture. by extraction and/or evaporation.

The product thus obtained can be converted into a pharmaceutically acceptable acid addition salt. The salt formation can be carried out using inorganic or organic acid (e.g. hydrochloric acid, sulfuric acid or phosphoric acid or tartaric acid, succinic acid, citric acid or ascorbic acid). The salt formation is carried out according to methods known in the field. One can proceed by adding a solution of the acid in ethyl ether or acetone to the solution of the base. Salt formation is carried out at a pH value of 3-6.

_D_en^^aål^d£^-eriiold±Æ fjarbJjxdeXs-e^^of -Xa can be -nitrated if desired. The nitration can preferably be carried out using concentrated nitric acid. The reaction is preferably carried out in

glacial acetic acid as a medium.' The nitration is preferably carried out during cooling, at a temperature of around 0°C. When the reaction is finished, the excess acid is neutralized and the mixture of the compound of formula Ib and Ic is isolated from the reaction mixture by extraction and/or evaporation.

The isomer mixture thus obtained can be separated

in the two components. The separation of the isomers can preferably be carried out by chromatographic methods.

The compounds of formula Ib and Ic can be converted into their pharmaceutically acceptable acid addition salts if desired by the methods described in connection with the preparation of the acid addition salts of the compound of formula Ia.

According to another feature of the invention, pharmaceutical preparations with a phosphodiesterase-inhibiting effect are provided which can be used in the treatment of skin diseases associated with pathological cell division and prophylaxis of new attacks of such diseases, which preparations comprise as active ingredient a compound of general formula I or a pharmaceutically acceptable acid addition salt thereof and optionally further therapeutically active compounds in admixture with -usual pharmaceutical carriers and/or diluents. The content of active ingredient in the pharmaceutical preparations is preferably 0.1 8.0%, in particular 0.2 - 2.0%. The preparations may optionally contain further pharmaceutically active compounds, such as antibiotics, cytostatic agents, prostaglandins, dithranol, salicylic acid, tar, anti-inflammatory agents, immunosuppressive agents, glucocorticoids and - in the case of preparations suitable for parenteral administration - local anesthetics. As glucocorticoid, theia^^olone~acetonide can preferably be used. The active ingredient can preferably be processed, in the form of preparations for topical use, such as creams, ointments, solutions, gels, aerosols, aerosol foams, adhesive plasters, etc.

The active ingredient can preferably be used in the form of the base, while acid addition salts are also used.

It is preferred to incorporate the active ingredient

in a cream that can be washed off.

The creams can be prepared by dissolving the active ingredient in a solvent .of. type of alcohol. preferably in propylene glycol or ethylene glycol or a mixture thereof formed with a little water, after which the solution thus obtained is mixed with an easily spreadable fat phase which is skin-compatible.

Specified fatty phase may comprise cetyl alcohol, stearyl alcohol, cetostearyl alcohol, paraffin oil, glycerol monostearate or the like.

The cream can also contain an emulsifier - preferably polyoxyethylene sorbitan monooleate or monostearate - and a preservative such as benzoic acid derivatives, preferably methyl p-hydroxybenzoate.

The creams may preferably contain 0.25 - 2.0% active ingredient, 45 - 50% glycol, 23 - 27% paraffin oil, 11 - 15% stearyl alcohol and possibly up to 100% of other auxiliary substances.

The active ingredient can also be formulated in form

of an ointment that cannot be washed off with water, by incorporating the active ingredient directly into the fatty phase.

The active ingredient can also be formulated in the form of a solution or tincture which can contain e.g. 20 - 40% propylene glycol or dipropylene glycol, 40 - 55% .96% ethanol and up to 100% distilled water.

The aerosol formulations can be prepared by adding to the solution active ingredient in propylene glycol a fatty substance - e.g. isopropyl myristate - and a propellant (e.g. freon).

Injectable solutions for parenteral administration, preferably for subcutaneous or intracutaneous administration,

can be prepared by dissolving a salt of the active ingredient in a 0.72% aqueous sodium chloride solution, after which

The pH of the solution is adjusted to 5.

The pharmaceutical preparations can be prepared according to known methods within the pharmaceutical industry. One can "proceed" by""a"mixing the aXtTve-Ee~sCahddéI"""""and optionally' further therapeutically active compounds with suitable inert, non-toxic, known pharmaceutical carriers and/or additives and processing the mixture thus obtained into a form suitable for medical use.

The following examples illustrate the invention.

Example 1

Preparation of • (-,)-apovincaminol-3',4'5'-trimethoxy-benzoate 3.10 g (10.1 millimoles) (-)-apovincaminol was dissolved

in 60 ml of anhydrous dichloromethane, whereupon 3.10 g of anhydrous sodium carbonate and 2.50 g (10.9 mmol) of 3,4,5-trimethoxy-benzoyl chloride were added, and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with 100 ml. water, the organic phase was separated and the aqueous layer extracted twice with 20 ml of dichloromethane each time.

The combined dichloromethane phases were dried over magnesium sulfate, filtered, whereupon the filtrate was evaporated in vacuo. 4.50 g of the title compound were thus obtained, yield 89.1%.

Gross formula C3o<H>34<N>2°5<*>Molecular weight 502.61.

-1 -1

IR spectrum (film): u max 1725 cm ( -C = 0) ; 1620 cm ( =C=C=)

NMR spectrum (deuterochloroform): δ: 1.01 (t, 3H, CH 3 CH 2 ~ );

6: 3.72 (s, 6H, 2 x -OCH 3 ); 6: 3.85 (s, 3H, -OCH-j); 6: 4.25

(s, 1H, annealing) ; 6: 5.29 (s, 1H, -CH = ) ; 6: 5.4 (m, 2H, -OCH 2 )

6: 7.0 - 7.8 (m, 6H, aromatic).

MS (m/e): 502(53), 432(100), 290(17), 261(41), 220(19),

216 (23) , 212 (18), 195(35).

(a)^<5>= -22.0°C (c = 0.7;, dichloromethane).

Example 2

Preparation of (-)- apovincaminol- 3', 4', 5'- trimethoxy- benzoate hydrogen tartarate

The compound prepared according to Example 1 was resolved

in diethyl ether. A saturated solution of D-tartaric acid in diethyl ether was added to the solution until the precipitation of the hydrogen tartrate salt is complete. The salt was filtered off and dried. M.p.: 120-121°C. IR spectrum: (KBr): u max 1730 cm (-C=0); 1640-1665 cm"<1>(=C=C=).

(ct)p<5>-8.5° (c = 1; pyridine). Molecular weight: 652.7.

Example 3

Preparation of '(-)-9-nitrd-apoviricaminol-3',4',5'-trimethoxy-benzoate and (-)-1T-nitro-apovincaminol-3',4',5'-trimethoxy-benzoate 5 g (-)-apovincaminol-3',4',5<1->trimethoxy-benzoate was dissolved in 50 ml of glacial acetic acid. A mixture of 20 ml of glacial acetic acid and 10 ml of concentrated nitric acid (d = 1.52) was added to the solution obtained at 0°C and with stirring. The reaction mixture was poured into 350 mg of ice-cold water and the pH was adjusted to 9 with the addition of a 25% aqueous ammonium hydroxide solution. The alkaline solution was extracted first with 250 ml and then twice with 200 ml of dichloromethane each time. The combined organic solution was dried over anhydrous sodium sulfate, filtered and then the filtrate was evaporated to dryness in vacuo.

The isomer mixture thus obtained was separated into the two components by chromatographic methods using silica gel 60 as adsorbent. and a. 10:2 mixture of. benzene and acetone as developer. Under these conditions, the R 2 value of the 9-nitro compound is greater than that of the 11-nitro derivative. Thus 1.7 g of the 9-nitro compound and 1.8 g of the 11-nitro derivative were obtained.

The physical constants for (-)-9-nitro-apovincaminol-3',4',5'-trimethoxy-benzoate are as follows:

M.p.: 77-78°C. (a) D = -78.5° (c = 1; chloroform).

<1>H-NMR (CDCl 3 ): δ: 3.8 (s, 6H, 2 x CH 3 O-); 6: 3.91 (s, 3H, 1 x CH 3 O - ); 6: 7.3 (d, 1H, H12); 6: 7.96 (d, 1H, H₂) 6: 8.1 (d, 1H, H₂).

The physical constants for (-)-11-nitro-apovincaminol-3',4*,5'-trimethoxybenzoate are as follows:

M.p.: 72-73°C. (a) D = -134.3° (c = 1; chloroform).

<1>H-NMR (CDCl 3 ): δ: 3.8 (s, 6H, 2 x CH 3 O-); <5 : 3.9 (s, 3H,

1 x CH30-) ; 6: 7.56 (d, 1H, Hg); 6: 8.1 (d, 1H, H 2 ); 6: 8,9

(d, 1H, H12).

Example 4

Forward<s>tillin<g>off. 9- nitro- a<p>Ovincamindl-3',4'5'- trimethoxy-benzoate- hydrochloridedg 1 T- nitrorrapovincaminol- 3''-, 4 ' , 5'- trimethoxybenzoate- hydrdchloride

9-nitro- or 11-nitro-apovincaminol-3',4',5'-trimethoxybenzoate prepared according to example 3 was dissolved in methanol and the pH of the solution was adjusted by adding a solution of hydrogen chloride and methanol. The hydrochloride salt formed was precipitated by the addition of diethyl ether. The salt was filtered off, washed and dried. The hydrochloride of the 9-nitro derivative melts at 144-150°C, while. the melting point of the hydrochloride of the 11-nitro derivative is 141-144°C.

Example 5

A cream with the following composition was prepared:

The active ingredient was dissolved in propylene glycol in a water bath (bath temperature not exceeding 50°C). The other components were heated until they melted and were then cooled to 40-45°C with constant stirring. To the mixture, the solution of the active ingredient was added with stirring and the cream thus obtained was cooled with stirring.

In an analogous way, creams containing 0.25%,

0.5%, 1.0% and 1.5% active ingredient prepared.

The active ingredient 9-nitro- or 11-nitro-apovincaminol-3',4',5'-trimethoxybenzoate can also be used.

Example 6

A cream with the following composition was prepared:

The procedure was analogous to that described in example 5, with the exception that the two active ingredients were dissolved in propylene glycol. The 11-nitro derivative of apovincaminol-3',4<1>,5<1->tri-methoxybenzoate can also be used as an active ingredient.

Example 7

A tincture solution with the following composition was prepared:

The above formulation can also be prepared using the 11-nitro derivative as active ingredient.

Example 8

A tincture solution with the following composition was prepared:

Example 9

An aerosol with the following composition was produced:

The .9-nitro or 11-nitro derivative can also be used as an active ingredient.

Example 10

An aerosol foam with the following composition was prepared:

Claims (5)

1. Process for the preparation of therapeutically active compounds of general formula I in 2 wherein either R and R both denote hydrogen or one of 1 2 the symbols R and R are hydrogen and the other is nitro, and pharmaceutically acceptable acid addition salts thereof, characterized in that apovincaminol or an acid addition salt thereof is reacted with 3,4,5-trimethoxy-benzoic acid or a reactive derivative thereof, whereupon the compound of formula Ia , is isolated, or that this is converted into a pharmaceutically acceptable acid addition salt thereof, or if desired, that one is held compound, of formula Ia is nitrated, and that the obtained mixture of the compounds of formulas Ib and Ic ...■. _.. _ _ v is separated into the two components, and if desired, that a compound of general formula Ib or Ic is converted into a pharmaceutically acceptable acid addition salt. 2. Process according to claim 1, characterized in that 3,4,5-trimethoxy-benzoyl chloride is used as acylating agent. 3. Method according to claim 1 or 2, characterized in that the acylation is carried out in the presence of an organic solvent. 4. Method according to claim 1, characterized in that the nitration is carried out in the presence of glacial acetic acid. 5. Method according to claim 1 or 4, characterized by ., that the nitration is carried out at a temperature around 0°C.