Phthalidilidene derivatives of carnitine and alkanoylcarnitines and pharmaceutical compositions containing them

Abstract: (3-phtalidylidene) alkyl esters of carnitine and alkanoyl carnitines of general formula (I) wherein Y is a C₁-C₅ alkylene group, unsubstituted or substituted with one or more lower C₁-C₄ alkyl groups; R is hydrogen or C₂-C₆ alkanoyl; R₁, R₂,R₃, and R₄, identical or different, are hydrogen, halogen C₁-C₈ alkoxy, C₁-C₄ lower alkyl, halogen-substituted lower alkyl, amino, alkyl-substituted amino wherein the alkyl group has 1 to 4 carbon atoms, nitro, cyano, C₁-C₄ alkanoylamino, or R₁ and R₂ taken together, R₂ and R₃ taken together or R₃ and R₄ taken together form a C₁-C₄ alkylenedioxy group, and X⁻ is the anion of a pharmacologically acceptable salt are potent PAF (plateletactivating factor) and acetyl-cholinesterase antagonists. Pharmaceutical compositions containing the compounds of formula (I) are useful for the therapeutical treatment of inflammatory and allergic conditions, particularly asthma, anaphylactic shock, as contraceptives and for the treatment of pre-senile and senile dementias (typically, Alzheimer's disease).

Description: The present invention relates to (3-phthalidylidene) alkyl esters of carnitine and alkanoyl carnitines of general formula (I) wherein Y is a C₁-C₅ alkylene group, unsubstituted or substituted with one or more lower C₁-C₄ alkyl groups; R is hydrogen or C₂-C₆ alkanoyl; R₁, R₂,R₃ and R₄, identical or different, are hydrogen, halogen, C₁-C₈ alkoxy, lower C₁-C₄ alkyl, halogen-substituted lower alkyl, amino, alkyl-substituted amino wherein the alkyl group has 1 to 4 carbon atoms, nitro, cyano, C₁-C₄ alkanoylamino, or R₁ and R₂ taken together, R₂ and R₃ taken together or R3 and R4 taken together form a C₁-C₄ alkylenedioxy group, and X⁻ is the anion of a pharmacologically acceptable salt. The esters of general formula (I) are potent PAF (plateletactivating factor) and acetylcholinesterase antagonists. PAF is an endogenous phospholipid discovered in the early sixties, and was thus termed after its remarkable ability to aggregate platelets. PAF does not play exclusively a role in stimulating the platelets to aggregate, but also plays an important role in various pathological conditions, such as inflammatory and allergic conditions. The esters (I) can be used as active ingredients in pharmaceutical compositions for treating those pathological conditions wherein PAF is the etiological agent or one of the etiological agents. Moreover, because of their acetylcholinesterase antagonism, they are effective for the treatment of pre-senile and senile dementias (typically, Alzheimer's disease). In particular, in formula (I) the various substituents can have the following meanings: Y is C₁-C₂ alkylene; R is selected from hydrogen, acetyl, propionyl, butyryl, isobutyryl, valeryl and isovaleryl; R₁, R₂, R₃ and R₄, identical or different, are selected from hydrogen, fluorine, chlorine, methoxy, ethoxy, methyl, trifluoromethyl, trichloromethyl, amino, nitro, cyano and acetylamino; X⁻ is the anion of a pharmacologically acceptable acid selected from chloride; bromide; iodide; aspartate; particularly acid aspartate; citrate, particularly acid citrate; phosphate, particularly acid phosphate; fumarate; particularly acid fumarate; glycerophosphate; glucosephosphate; lactate; maleate; particularly acid maleate; orotate; oxalate; particularly acid oxalate, sulphate, particularly acid sulphate; trichloroacetate, trifluoroacetate and metansulphonate. PAF (1-0-alkyl-2-acetylglycero-3-phosphorylcholine) has formula PAF actually represents a family of phospholipids, because the alkyl group at position 1 can vary in lenght from C₁₆ to C₁₈. PAF is biosynthesized following a stimulus by platelets, neutrophils, monocytes, mast cells, eosinophils, renal mesangial cells, renal medullary cells and vascular endothelial cells. A recent thorough review of PAF biochemistry, pharmacology, pathobiology, receptors and antagonists has been published by P. Braquet et al. in Pharmacological reviews, vol. 39, n. 2, 97-145 (1987). It has been definetly ascertained that PAF is involved with a variety of physiophatological conditions, including artherial thrombosis, acute inflammation, endotoxic shock, acute allergic diseases and asthma. It is a potent bronchoconstrictor and promotes the accumulation of eosinophils in the lung, it causes tracheal and bronchial edema and it stimulates the secretion of mucus. All this has prompted remarkable interest in the development of PAF-antagonists. The compounds of the present invention are prepared by condensing carnitine or the appropriate alkanoylcarnitine with the suitable 3-(ω-bromo-alkylidene) phtalide, optionally substituted at positions 4,5,6 and 7 with R₄, R₃, R₂, R₁, respectively, the meanings of which have been previously defined. 3-(ω-bromoalkylidene) phthalides and their preparation are described in the prior art. For instance, in the Japanese patent application 85814/1978/publication 13221/1980) to Kanebo and in Chem. Pharm. Bull. 31 (8), 2698-2707 (1983), Fumio Sakamoto et al disclose the synthesis of (Z)- and (E)-3-(2-bromoethylidene) phthalides which these Authors use as intermediates for preparing penicillin (3-phtalidylidene) ethyl esters. These latter compounds are prodrugs of orally administrable penicillins, which are advantageously endowed with better bioavailability with respect to the antibiotics as such. (See also in this regard the Japanese patent application 106650/1978, publication n. 33444/1980 to Kanebo). The synthesis described by the foregoing Authors comprises (see the following reaction scheme) the bromination of known phthalides (I) (German Auslegeschrift 1.266.310; S.N. Chakravati and W.N. Perkin, J. Chem. Soc. 1927, 196) with N-bromo-succinimide (NBS) in carbontetrachloride to give the bromides (2) which with triphenylphosphine in benzene give the phosphonium bromides (3). These latter compounds, reacted with acetaldehyde in the presence of triethylamine, give the ethylidenephtalides (4) and (5), i.e. both (Z) and (E) isomers. By bromination of (4) and (5) with NBS, 3-(2-bromo-ethylidene) phthalides (6) in the (Z) isomer form only are obtained. The following non-limiting examples illustrate the preparation of some of the compounds of the present invention. Example 1 (Z)-(3-phthalidylidene)ethyl ester of L-carnitine bromide (ST 736). To a suspension of 3.98 g(0.0247 moles) L-carnitine (M.W. 161.2) in 30 mL dimethylformamide a solution of 5.9 g (0.0247 moles) (Z)-3-(2-bromoethylidene)phthalide (M.W. 239.07) (Chem. Pharm. Bull. 31 (8), 2698-2707, 1983) in 30 mL dimethylformamide was added, while keeping the temperature of the reaction mixture at +25°C. The reaction was monitored via TLC (eluant CHCl₃: MeOH:H₂O:iso-PrOH:AcOH 60:40:15:10:15). After stirring for 3 hours at 25°C, the very thin suspension was filtered and the solution slowly added dropwise to 800 mL ethyl ether. The suspension that formed was stirred for 2 hours and then allowed to stand at +5°C overnight. The precipitate was filtered and crystallized from isopropanol. 7.44 g (3-phthalidylidene)ethyl ester of L-carnitine bromide were obtained. Yield 75.2%. M.P. = 154-156°C (da isoPrOH) [α] 25 / D = -10.7°(c=1 in H₂O) TLC: single spot (eluant CHCl₃:MeOH:H₂O:isoPrOH:AcOH 60:40:15:10:15) silica gel plates 0.25 mm - 60 F₂₅₄ (E. Merck) Detectors: UV 0.254 nm and iodine vapors HPLC: Waters 990 Column: µBondapack C₁₈, inner diameter = 3.9 mm, length 300 mm; mobile phase: KH₂PO₄0.05 M/CH₃CN 70:30; t=25°C; flow rate: 1 ml/min; detector: UV λ = 205 nm; capacity factor (K′) calculated on the peak of Br⁻ anion: K′=1.36. ¹H-NMR: Varian 300 MHz(D₂0) δ (p.p.m.):2.95-2.70(2H,m,C H ₂COOR), 3.385(9H,s,+N(C H ₃)₃), 3.55(2H,d,C H ₂N),4.90-4.80(1H,m,C H OH), 4.953(2H,d,J AX =7.2Hz,C H ₂CH=), 5.806(1H,t,J AX =7.2Hz,CH₂C H =),7.40-7.70(4H,m,Arom) Attribution of configuration to the C=C double bound The (Z) configuration of the compound to the C=C double bound was attributed on the grounds of the accumulated (CD₃CN) ¹H-NMR N.O.E. spectra (Nuclear Overhauser Effect) and the proton-coupled carbon spectra. The experiment ¹H-NMR N.O.E. (Nuclear Overhauser Effect) was conducted by irradiating the triplet at δ 6.0 p.p.m.; a doublet at δ 7.932 and 7.907 p.p.m. (J=8.3 Hz) corresponding to C-H, was observed. Examples 2-31 The compounds of the examples 2-31 were prepared by following the procedures outlined in Example 1 and effecting the required substitutions which, however, are apparent to anyone having ordinary skill in organic syntheses. The formula of these compounds is obtained by substituting in the general formula (I) the meanings of R and R₂ given in the following table (R₁=R₃=R₄=H, unless otherwise indicated). In the same table, the main physico-chemical characterists of the obtained compounds are shown. In synthesizing the compounds of the invention, the selection of solvents and operating conditions can be broadened with respect to those set forth in Example 1, as hereinbelow disclosed. As reaction solvents, aprotic dipolar solvents are used such as e.g. dimethylformamide, acetonitrile, diethyleneglycol dimethyl ether, dimethyl sulfoxide, sulfolane, preferably dimethylformamide. The reaction temperature is comprised between +5°C and +80°C, preferably between +25°C and +35°C. The dropping time is comprised between 10 and 120 minutes and preferebly is 60 minutes. The reaction time is comprised between 1 and 48 hours, preferably 2-3 hours. When the reaction is completed, the stirring temperature is comprised between -20°C and +25°C, preferably +5°C, for times ranging from 2 to 48 hours, preferably 14 hours. The solvents for precipitating and isolating the final compounds are selected from symmetrical or asimmetrical, straight or branched lower alkyl ethers, such as diethyl ether, di -n-propyl ether, diisopropyl ether, di-n-butyl ether, preferably diethyl ether. Alternatively, the reaction mixture can be concentrated under vacuum at low temperature, and the residue recrystallized from suitable solvent(s). Some of the pharmacological tests conducted in order to assess the activity of the compounds of the present invention are reported hereinbelow. BINDING TO PAF RECEPTORS The binding of the compounds under examination to rabbit platelet receptors was studied following the procedure disclosed by D. Nunez et al, Eur J. Pharmacol. 123 , 197 (1986). Blood was drawn via intracardiac puncture from male rabbits (HY/CR strain-Charles River, Italy) weighing 2.5-3.0 Kg. Blood was diluted (1:9) in a ACD (0.8% citric acid, 2.2% trisodium citrate, 2.45% glucose, weight percentages) solution and centrifuged at 100 x g for 15 minutes. The precipitate was suspended in the incubation buffer at 2⁻⁴ x 10⁸ platelet/ml concentration, immediately before binding. The platelet number and possible erythrocyte contamination was controlled with a platelet counter DELCON CA480. Platelets were incubated in polyethylene tubes with ³H-PAF (0.5 nM) for 30 minutes at 25°C. The test compounds were added to a volume of 200 µl. The final volume was equal to 500 µl. Incubation was stopped by rapid filtration under vacuum through GF/C filters (WHATMAN) washed with (3x3 ml) cold incubation buffer using a Brondel Cell Harvester filtration system. The filters were counted in 8 mL OptiFluor (Packard) using a TRI-CARB 1900 CA-Packard liquid scintillation spectrometer (about 50% counting efficiency). RESULTS EVALUATION The percentage of inhibition of the specific binding receptor-radioligand was calculated for each concentration (mean of incubations in triplicate). The competition curve, the slope of the curve, and the IC₅₀ value (concentration which inhibits 50% of the specific radioligand-receptor binding) were calculated with the "All fit" program (A. De Lean et al., Am. J. Physiol. 235 , E97, 1978) runned in a 55-SX IBM computer. The results are shown in Table 2. PLATELET AGGREGATION ANTAGONISM INDUCED BY PAF (Platelet activating factor) and ADP (Adenosinediphosphate) PAF-INDUCED AGGREGATION Blood was collected from male rabbits (HY/Charles River, Italy) by heart puncture using a plastic syringe and transferred immediately into plastic tubes containing 3.8% (by weight) solution of sodium citrate (0.1 ml/ml blood). PRP (Platelet Rich Plasma) was obtained by plasma centrifugation at 145 x g for 5 minutes at 22°C. Platelet number was determined with a CA 580A (Delcon) "platelet counter". The platelet number was adjusted to the fixed value of 350,000 platelet/ml by adding PPP (Platelet Poor Plasma). PPP was obtained by centrifuging blood at 1600 x g for 10 minutes at 22°C. Platelet aggregation was determined photometrically (G.V.R. Born, Nature 194 , 927; 1962) with an ELVI 840 aggregometer in plastic tubes under continuous stirring at 1000 rpm. Maximal light transmission (100%) was recorded by reading PPP, while 0% transmission was recorded by reading PRP. The PRP aggregation degree induced by the aggregation factor (PAF) was calculated as percentage of maximal light transmittance obtained with PPP using 250 µL samples of PRP. For each test, the concentration of aggregating agent needed to obtain 50-60% aggregation was determined and was shown to range between 10⁻⁷ and 9 x 10⁻⁸ M. The compounds were dissolved in bidistilled water at the concentration of 10⁻⁵ M and tested after 2 minutes of incubation. Aggregation inhibition was calculated as percentage of the value obtained in the presence of the aggregating agent only. ADP-INDUCED AGGREGATION Blood was drawn from the abdominal aorta of male rats (Crl:(WI)BR, Charles River, Italy) under barbiturate anaesthesia and quickly transferred into plastic tubes containing 0.1 mL/mL blood of 3.8% (by weight) solution of sodium citrate. PRP was obtained by plasma centrifugation at 115 x g for 5 minutes at 22°C and PPP by centrifugation at 1600 x g for 10 minutes. The final PRP platelet number was adjusted to the fixed value of 350,000/mL of PRP by adding PPP. Platelet count was made with a CA580 A(Delcan) platelet counter. The same procedure outlined in the paragraph "PAF aggregation" was followed for the ADP-induced aggregation. The concentration of the aggregating factor (ADP) ranged from 5 x 10⁻⁶ to 1 x 10⁻⁵ M. The results are shown in Table 4. OBSERVATION OF THE BEHAVIOUR AND DEATH RATE IN MICE The observation of normal behaviour in mice was conducted following the method by S. Irwin, Psychopharmacologia 13 , 222 ( 1968). This method allows alterations of some behavioural, neurophysiological and neurovegetative parameters directly detectable by the experimenter to be visualized. The test was conducted in male mice [Crl;(CD-I)(ICR)BR, Charles River - Italy] weighing 22-25 g, following oral administration of the compounds suspended in carboxymethylcellulosa (0.5% by weight in H₂O) to groups of 4 animals/dose. The animals were kept under continuous observation for five hours following treatment and twice a day for the subsequent five days. Death rate was also observed during the whole test period. The initial administered dose was 1,000 mg/kg per os; lower doses were administered following death of some animals or after marked effects at the initial dose were dected. The results are shown in Table 3. CONCLUSION The compounds were shown to be active in antagonizing PAF binding to its receptors at pharmacologically meaningful concentrations. The most active ones among the compounds were subsequently tested in the PAF-induced platelet aggregation test in order to assess whether the compounds behave as agonists or antagonists towards PAF receptors. The results obtained showed that the compounds are antagonists. Moreover, it was shown that their activity is specific because the same compounds were inactive in antagonizing the aggregation triggered by an aggregating agent (such as ADP) endowed with a different mode of action. Observation of behaviour and death rate in mice COMPOUND DOSE mg/kg per os SYMPTOMS DEATH RATE ST 736 600 n.e. 0/4 1000 convulsion, salivation 3/4 ST 739 600 n.e. 0/4 1000 jerks 2/4 ST 768 600 n.e. 0/4 1000 convulsion, salivation diarrhea, lacrimation 1/4 ST 775 1000 n.e. 0/4 ST 776 600 n.e. 0/4 1000 convulsion, salivation 2/4 ST 801 1000 salivation 0/4 ST 804 600 salivation 0/4 1000 salivation 1/4 ST 805 600 salivation 0/4 1000 salivation 1/4 ST 806 600 salivation 0/4 1000 salivation 1/4 ST 803 1000 diarrhea 0/4 n.e. = no effect Inhibition to PAF - and ADP -induced platelet aggregation COMPOUND CONCENTRATION % INHIBITION PAF ADP ST 776 10⁻⁵M 100% 5% ST 801 " 97% 7% ST 804 " 96% 13% ST 805 " 40% 10% ST 806 " 95% 18% ST 803 " 72% 8%

Claims: Claims for the following Contracting States : AT, BE, CH, LI, DE, DK, FR, GB, IT, LU, NL, SE (3-phthalidylidene) alkyl esters of carnitine and alkanoyl carnitines of general formula (I) wherein: Y is a C₁-C₅ alkylene group, unsubstituted or substituted with one or more C₁-C₄ lower alkyl groups; R is hydrogen or C₂-C₆ alkanoyl; R₁, R₂, R₃ and R₄, identical or different, are hydrogen, halogen, C₁-C₈ alkoxy, lower C₁-C₄ alkyl, halogen-substituted lower alkyl amino, alkyl-substituted amino wherein the alkyl group has 1 to 4 carbon atoms, nitro, cyano, C₁-C₄ alkanoylamino, or R₁ and R₂ taken together, R₂ and R₃ taken together or R₃ and R₄ taken together form a C₁-C₄ alkylenedioxy group, and X⁻ is the anion of a pharmacologically acceptable salt. The esters of claim 1, wherein Y is methylene or ethylene. The esters of claim 1 or 2, wherein R is selected from acetyl, propionyl, butyryl, isobutyryl, valeryl and isovaleryl. The esters of anyone of the preceding claims, wherein the halogen is selected from fluorine and chlorine. The esters of anyone of the preceding claims, wherein the alkoxy group is selected from methoxy and ethoxy. The esters of anyone of the preceding claims, wherein the halogen-substituted lower alkyl is selected from trifluoromethyl and trichloromethyl. The esters of anyone of the preceding claims, wherein the alkanoylamino is acetylamino. The esters of claim 1, wherein Y is methylene, R₁ = R₂= R₃= R₄= hydrogen and R is selected from hydrogen, acetyl, propionyl, butyryl, isobutyryl, valeryl and isovaleryl. The esters of claim 1, wherein Y is methylene, any three groups from R₁, R₂, R₃ and R₄ are hydrogen and the remaining group is fluorine or chlorine, and R is selected from hydrogen, acetyl, propionyl, butyryl, isobutyryl, valeryl and isovaleryl. The esters of claim 9, wherein R₁=R₃=R₄= hydrogen and R₂ is fluorine or bromine. A pharmaceutical composition comprising as active ingredient a compound according to anyone of the preceding claims, and a pharmacologically acceptable excipient therefor. The pharmaceutical composition of claim 11 for the treatment of diseases whereof PAF is the etiological agent or is one of the etiological agents. The pharmaceutical composition of claim 12 for the treatment of inflammatory and allergic conditions. The pharmaceutical composition of claim 13 for the treatment of asthma. The pharmaceutical composition of claim 12 for the treatment of shock. The pharmaceutical composition of claim 15 for the treatment of anaphylactic shock. The pharmaceutical composition of claim 11 as contraceptive. The pharmaceutical composition of claim 11 for the treatment of pre-senile and senile dementias. The pharmaceutical composition of claim 18 for the treatment of Alzheimer's disease. Claim for the following Contracting States : ES, GR A process for producing (3-phthalidylidene) alkyl esters of carnitine and alkanoyl carnitines of general formula (I) wherein: Y is a C₁-C₅ alkylene group, unsubstituted or substituted with one or more lower C₁-C₄ alkyl groups; R is hydrogen or C₂-C₆ alkanoyl; R₁, R₂,R₃ and R₄, identical or different, are hydrogen, halogen, C₁-C₈ alkoxy, C₁-C₄ lower alkyl, halogen-substituted lower alkyl, amino, alkyl-substituted amino wherein the alkyl group has 1 to 4 carbon atoms, nitro, cyano, C₁-C₄ alkanoylamino, or R₁ and R₂ taken together, R₂ and R₃ taken together or R₃ and R₄ taken together form a C₁-C₄ alkylenedioxy group, and X⁻ is the anion of a pharmacologically acceptable salt, which comprises condensing carnitine or an alkanoylcarnitine wherein the alkanoyl group has 2 to 6 carbon atoms with 3-(ω-bromoalkylidene) phtalide which is optionally substituted at 4,5,6 and 7 positions thereof with, respectively, the previously defined R₄,R₃,R₂ and R₁ groups, in the presence of aprotic, dipolar solvents, at a temperature from +5 to +80°C, for reaction times from 1 to 48 hours.

Inventors: PICCIOLA GIAMPAOLO, PICCIOLA, GIAMPAOLO
Applicants: SIGMA TAU IND FARMACEUTI, SIGMA-TAU INDUSTRIE FARMACEUTICHE RIUNITE S.P.A.
Application year: 1991
Main classification: C07D307
IPC codes: C07D307, C07D493