GB2233974A - Dihydropyridine antiinflammatory agent - Google Patents

Abstract

The S(+) isomer of 4(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-2-[2- (2,4,5-trimethyl- 1-imidazolyl)ethoxymethyl]-1,4-dihydropyridine is a selective antagonist of platelet activating factor having clinical utility in the treatment of allergic, inflammatory and hypersecretory conditions in humans.

Description

PATENT SPECIFICATION "Therapeutic Agents" DESCRIPTION This invention relates to dihydropyridines and specifically to the S(+) isomer of 4-(2-chlorophenyl) 3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-2- [2- (2,4, - 5-trimethyl-1-imidazolyl)ethoxymethyl]-1,4-dihydro- pyridine and its use as a selective antagonist of platelet activating factor having clinical utility in the treatment of allergic inflammatory and hypersecretory conditions in humans and animals.

Platelet activating factor (PAF, l-0-alkyl-2acetyl-sn-glyceryl-3-phosphorylcholine) is an ether phospholipid whose structure was first elucidated in 1979. It is produced by, released from and interacts with many pro-inflammatory cells, platelets and the kidney. In addition to potent platelet aggregating activity, PAF exhibits a wide spectrum of biological activities elicited either directly or via the release of other powerful mediators such as thromboxane A2 or the leukotrienes which make PAF inhibitors of potential value in the treatment of a variety of conditions including allergic, infammatory and hypersecretory conditions such as asthma, arthritis, rhinitis, bronchitis and urticaria, the treatment of circulatory shock, gastic ulceration, psoriasis and cardiovascular conditions, including angina, thrombosis and stroke.

According to our European Patent no 0100189 we describe and claim a number of dihydropyridine calcium channel blockers as anti-isachaemic and antihypertensive agents. In particular we describe and claim the compound 4-(2-chlorophenyl) -3-ethoxycarbonyl- 5-methoxycarbonyl-6-methyl-2-t2-(2,4,5-trimethyl-1- imidazolyl) ethoxy-methyl) -1, 4-dihydropyridine.

We have discovered that this compound is also a selective antagonist of platelet activating factor (PAF). However its utility based on this finding is restricted because of the activity of the compound as a calcium channel blocker.

We have now unexpectedly discovered that resolution of this compound completely separates the two activities and the S(+) isomer is a potent and selective PAF antagonist which is devoid of calcium channel blocking activity.

Thus according to the present invention we claim the S(+) isomer of 4-(2-chlorophenyl)-3-ethoxycarbonyl 5-methoxycarbonyl-6-methyl-2- [2- (2,4, 5-trimethyl-l- imidazolyl) ethoxymethyl] -1, 4-dihydropyridine when substantially free of the R(-) isomer, and its pharmaceutically acceptable salts.

By substantially free of the R(-) isomer we mean that the compound contains less than 10% and preferably less than 5% of the R(-) isomer.

The compound of the invention is prepared by resolution of the corresponding 2-(2-azidoethoxymethyl) dihydropyridine (I) followed by esterification, reduction of the azide group and reaction with diacetyl and acetaldehyde ammonia to give the trimethylimidazole derivative as shown in the following reaction scheme:

Appropriate procedures for preparation of the compound of formula (I) are described in our European patent no 0089167; for example the Hantzsch synthesis may be employed, using as starting materials 4-(2azidoethoxy)-3-ketobutyric acid 2-cyanoethyl ester and 2-chlorobenzaldehyde. Resolution is achieved by fractional crystallisation of the salt formed with an appropriate optically active base. For example we have found cinchonidine to be suitable for this purpose.

The desired S(+) isomer is esterified to give the diester (II). The subsequent reduction and coupling steps are as described in our European Patent 0100189.

The product at each stage can be isolated and purified by conventional procedures. The final product may be isolated as the free base or converted to a pharmaceutically acceptable salt by conventional procedures.

The activity of the compound of the invention is shown by its ability to inhibit the platelet aggregating activity of PAF in vitro. Testing is performed as follows: Blood samples are taken from either rabbit or man into 0.1 vol disodium ethylenediamine tetraacetic acid buffer and the samples centrifuged for 15 minutes to obtain a platelet pellet which is washed with a buffer solution (4 mM KH2PO4, 6mM Na2HPO4, 100 mM NaCl, 0.1% glucose and 0.1% bovine serum albumin, pH 7.25) and finally resuspended in buffer solution to a concentration of 2 x 108 platelets/ml. A sample (0.5 ml) is pre-incubated with stirring for two minutes at 37 C in a Paton aggregometer, either with vehicle alone, or with vehicle containing the particular compound under test.PAF is added at a sufficient concentration to give a maximum aggregating response in the absence of test compound (10-8 to 10 9 molar), and the platelet aggregation is measured by following the increase in light transmission of the solution. The experiment is repeated in the presence of test compound at a range of concentrations and the concentration of compound required to reduce the response to 50% of its maximum value is recorded as the IC50 value.

The activity of the compound of the invention is also demonstrated in vivo by its ability to protect mice from the lethal effect of an injection of PAF. A mixture of PAF (50 ug/kg) and DL-propranolol (5 mg/kg) in 0.9% w/v sodium chloride is injected (0.2 ml) via a tail vein into mice. The compound is either injected into the tail vein immediately prior to the PAF/propranolol injection or administered orally by gavage two hours earlier. The compound is tested at several doses in groups of 5 mice and the dose which reduces mortality to 50% is recorded as the PD50 value.

The compound is also tested for its ability to reduce PAF-induced bronchoconstriction in anaesthetised guinea pigs. In this test airways resistance and dynamic lung compliance are calculated from recordings of airflow and transpleural pressure and calculation of tidal volume. The bronchoconstriction induced by PAF (100 ng/kg) is determined. One hour after the initial dose of PAF the compound under test is administered and the PAF challenge repeated. The ability of the compound to reduce the bronchconstrictor effect of PAF is calculated as a ratio.

For therapeutic use the compound of the invention will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, it may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavouring or colouring agents. It may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, it is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.

For administration to man in the curative or prophylactic treatment of allergic bronchial conditions and arthritis, oral dosages of the compound will generally be in the range of from 2-1000 mg daily for an average adult patient (70 kg). Thus for a typical adult patient, individual tablets or capsules contain from 1 to 500 mg of active compound, in a suitable pharmaceutically acceptable vehicle or carrier.

Dosages for intravenous administration would typically be within the range 1 to 10 mg per single dose as required. For the treatment of allergic and bronchial hyper-reactive conditions, inhalation via a nebuliser or aerosol may be the preferred route of drug administration. Dose levels by this route would be within the range 0.1 to 50 mg per single dose as required. In practice the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case but there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Thus in a further aspect the invention provides a pharmaceutical composition comprising the compound of the invention, or a pharmaceutically acceptable salt thereof, for use in medicine, in particular in the treatment of allergic, inflammatory and hypersecretory conditions in a human being.

The invention also includes the use of the compound of the invention for the manufacture of a medicament for use as a PAF antagonist including the treatment of allergic, inflammatory and hypersecretory conditions in a human being.

The preparation of the compound of the invention is illustrated by the following Examples.

EXAMPLE 1. Preparation of 4-(2-azidoethoxy)-3-ketobutyric acid 2-cvanoethvl ester Azidoethoxy acetic acid (8.0 g, 55 mmole) in dry dichloromethane (100 ml) was stirred in an ice bath under nitrogen while N,N'-carbonyldiimidazole (10.0 g, 61 mmole) was added over 0.5 hours. The solution was allowed to stir at room temperature for 1 hour before adding a solution of Meldrum's acid (8.0 g, 55 mmole) and pyridine (4.45 ml) in dichloromethane (50 ml). The mixture was stirred overnight at room temperature. The solution was then washed with 2N hydrochloric acid (2 x 30 ml) dried (MgSO4) and the solvent evaporated to yield a red oil. This was dissolved in 3-hydroxy proprionitrile (15 ml) and stirred at 700C for 3 hours.

The mixture was cooled and diluted with dichloromethane (50 ml), washed with water (3 x 30 ml) dried (MgSO4) and the solvent evaporated to yield a red oil (4.3 g).

The crude product was chromatographed on silica eluting with diethyl ether to give the title cyanoethyl ester as a yellow oil (4.5 g).

2.PreParation of 2-r(2-azidoethoxv)methvll-4- (2-chloro henYl)-5-methoxvearbonyl-3-cyanoethOxy- carbonyl-6-methyl-1 4-dihvdropvridine The above cyanoethyl ester (4.0 g, 16.6 mmole), 2-chlorobenzaldehyde (2.35 g, 16.6 mmole) and the enamine derived by dissolving methylacetoacetate (1.92 g) in methanolic ammonia (20 ml), standing at room temperature for 5 hours and evaporating to dryness, were heated under reflux in methanol (50 ml) overnight. The solvent was evaporated and the residue chromatographed on silica eluting with ethyl acetate containing increasing diethylamine to give the title product (4.08 g, 54%).

3. 2-[(2-Azidoethoxy methyl]-4-(2-chlorohenvl - 5-methoxycarbonyl-6-methyl-1,4-dihydropyridine-3- carboxvlic acid The above cyanoethyl ester (4.0 g) was dissolved in dioxan (20 ml), treated with 2N sodium hydroxide (5 ml) and the solution stirred for for 3 hours at room temperature. The solution was evaporated to dryness and the residue partitioned between ethyl acetate and water. The aqueous phase was acidified to pH4 and extracted with ethyl acetate. The organic phase was washed, dried and the solvent evaporated to yield the title acid (3.4 g., 95%).

4. S(+) isomer of 2-r(2-azidoethoxvmethvll-4-(2- chlorophenvl -5-methoxvcarbonvl-6-methvl-l .4 dihvdrovridine-3 -carbocylic acid The above acid (3.25 g. 8 mmole) and cinchonidine (2.36 g. 8 mmole) were dissolved in hot methanol, the solution evaporated to dryness and the salt taken up in hot methanol (100 ml). The solution was diluted with water (20 ml) and stored in a refrigerator overnight.

The resulting precipitate was filtered off and recrystallised twice from 10% aqueous methanol to give the cinchonidine salt (1.53 g. Mp 165-1680C. 25 -72.30 (c=1.05, MeOH).

The cinchonidine salt was suspended in ethyl acetate (50 ml) and washed with 2M hydrochloric acid (50 ml) and then water. The organic phase was dried and the solvent evaporated to give the title isomer 25 (800 mg). m.p. 134-145 C [α]D25 +34.7 (c=0.98 NeOH).

5. S(+) 2-r (2-Azidoethoxv)methv1 1-4-(2-chloro- phenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6- methvl-1 4-dihvdrovridine A solution of the above acid (750 mg, 1.8 mmole) in methylene chloride (10 ml) at 0 0C was treated with N, N' carbonyldiimdazole (330 mg, 2.03 mmole) in 3 portions. After 15 minutes the ice bath was removed but stirring was continued at room temperature for 30 minutes. The solvent was evaporated and the residue taken up in ethanolic sodium ethoxide (sodium (45 mg) in ethanol (15 ml)). The orange solution was stirred for 45 minutes and diluted with ethyl acetate (30 ml), washed with 2N hydrochloric acid (15 ml), water, dried (MgSO4) and evaporated to an oil which crystallised on standing (745 mg).A portion was recrystallised from ethanol. m.p. 84-86 C. [α]D25 +34.2 (c=1.05 MeOH).

6. S(+) 2- (2-Aminoethoxy) methyl-4-(2-chloro- phenyl-3-ethoxycarbonyl-5-methoxycarbonyl-6 - methvl-1,4-dihvdropvridine The above azide (750 mg) was dissolved in ethanol (20 ml) and hydrogenated over Pd/CaCO3 at 20 p.s.i.

(1.38 bar), for 3 hours. The mixture was filtered and the filtrate evaporated to dryness. The residue was taken up in ethyl acetate and a saturated solution of maleic acid in ethyl acetate (10 ml) added. The maleate salt was collected by filtration (840 mg,93%) 0 172-174 C 25 0 m.p. 172-174 C (] =+26.2 (c=1.05 MeOH) 7. S(+) 4-(2-Chlorophenyl)-3-ethoxycarbonyl-5- methoxvcarbonyl-6-methyl-2-[2-(2,4,5-trimethyl- 1-imidazolyl)ethoxymethvl]-1,4-dihydropyridine The above product (as the free base) (200 mg,0.49 mmole) was dissolved in methanol (5 ml) and cooled to -10 C. Diacetyl (170 mg, 2.0 mmole) was added in 4 portions and stirring continued for 1 hour.

Acetaldehyde ammonia (230 mg,1.25 mmole) was added below -5 C,and when homogenous the solution was stored in a refrigerator overnight. Excess ammonia (SG 0.880) added and the product extracted into ethyl acetate. The organic phase was washed with water, dried (NgSO4) and evaporated to an oil which was chromatographed on silica eluting with ethyl acetate containing increasing acetonitrile to yield the title product (60 mg). as a glass [α]D25 = 28 (c=1% MeOH) no m.p.; Found: C,61.88; H,6.67; N,8.38. C26H32C1N305 requires C,62.21; H,6.38; N,8.37%.

Claims (5)

1. S(+) 4- (2-Chlorophenyl) -3-ethoxycarbonyl-5- methoxycarbonyl-6-methyl- (2- (2,4, 5-trimethyl-l- imidazolyl) ethoxymethyl) -1, 4-dihydropyridine substantially free of the R(-) isomer and pharmaceutically acceptable salts thereof.

2. A pharmaceutical composition comprising a compound as claimed in claim 1 together with a pharmaceutically acceptable diluent or carrier.

3. A compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof, for use in medicine, particularly for the treatment of allergic, inflammatory and hypersecretory conditions.

4. The use of a compound as claimed in claim 1, for the manufacture of a medicament for use as a PAF antagonist including use in the treatment of allergic, inflammatory and hypersecretory conditions in a human being.

5. A process for preparing the compound claimed in claim 1 which comprises reacting S(+) 2-(2-amino ethOxy)methyl-4-(2-chlorophenyl)-3-ethoxyzarbonyl-5- methoxycarbonyl - 6 -methyl-l ,4 -dihydropyridine with diacetyl and acetaldehyde ammonia.