Heterocyclic Compounds as Antiallergic Agents

This invention relates to novel heterocyclic compounds possessing lipoxygenase inhibitory and slow-reacting substance of anaphylaxis (SRS-A) antagonist activity which are useful as anti-inflammatory and antiallergic agents.

It is known that arachidonic acid (AA) is metabolized in mammals by two distinct pathways. The metabolism of arachidonic acid by cyclooxygenase enzymes results in the production of prostaglandins and thromboxanes. The physiological activity of these AA metabolites has been amply elucidated in recent years. The other pathway of AA metabolism involves lipoxygenase enzymes and results in the production of a number of oxidative products called leukotrienes. The latter are designated by the LT nomenclature system, and the most significant products of the lipoxygenase metabolic pathway are the leukotrienes C.sub.4 and D.sub.4. The substance denominated slow-reacting substance of anaphylaxis (SRS-A) has been shown to consist of a mixture of leukotrienes, with LTC.sub.4 and LTD.sub.4 as the primary products and having varying amounts of other leukotriene metabolites [see Bach et al., J. Immun. 215, 115-118 (1980); Biochem. Biophys. Res. Commun. 93, 1121-1126 (1980)].

The significance of these leukotrienes is that a great deal of evidence is accumulating showing that leukotrienes participate in inflammatory reactions, exhibit chemotactic activities, stimulate lysosomal enzyme release and act as important factors in the immediate hypersensitivity reaction. It has been shown that LTC.sub.4 and LTD.sub.4 are potent bronchoconstrictors of the human bronchi [see Dahlen et al., Nature 288, 484-486 (1980)], and another leukotriene, LTB.sub.4, is a powerful chemotactic factor for leukocytes [see A. W. Ford-Hutchinson, J. Roy. Soc. Med., 74, 831-833 (1981)]. The activity of leukotrienes and slow-reacting substances (SRS's) as mediators of inflammation and hypersensitivity is extensively reviewed in Bailey and Casey, Ann. Reports Med. Chem., 17, 203-217 (1982).

The biological activity of the leukotrienes and SRS's, and of lipoxygenase as the enzyme leading to the metabolism of AA to leukotrienes, indicates that a rational approach to drug therapy to prevent, remove or ameliorate the symptoms of allergies, anaphylaxis, asthma and inflammation must focus on either blocking the release of mediators of these conditions or to antagonize their effects. Thus, compounds which inhibit the biological effects of the leukotrienes and SRS's and/or which control the biosynthesis of these substances, as by inhibiting lipoxygenase, are considered to be of value in treating such conditions as allergic bronchial asthma, allergic rhinitis, as well as in other immediate hypersensitivity reactions.

The invention provides novel compounds of the formula ##STR2## n is 0-6;

A is O or S;

B is OR.sup.2, SR.sup.2 or N(R.sup.2).sub.2 ;

R.sup.1 is hydrogen, loweralkyl, loweralkoxy or halo;

R.sup.2 is hydrogen or loweralkyl;

and the pharmaceutically acceptable salts thereof.

The term "halo" refers to fluoro, chloro, and bromo. The terms "loweralkyl" and "loweralkoxy" refer to moieties having 1-6 carbon atoms in the carbon chain.

The compounds of the invention can be prepared in a number of ways. In those instances in which the side chain Z is ##STR3## the compounds can be prepared by the reaction of an appropriate aniline derivative with the halide of an appropriate derivative of a carboxy or thiocarboxyalkanoyl as follows: ##STR4## where X, W, Y, A, B, R.sup.1 and R.sup.2 and n are as defined hereinbefore and hal refers to a halo radical, for example, chloro or bromo. The reaction is carried out in an organic solvent, for instance tetrahydrofuran, and at reduced temperatures, with the reaction mixture being allowed to slowly warm to room temperature. Compounds in which the side chain Z is ##STR5## can be prepared in the exact same manner, except that the starting compound is an appropriate N-hydroxyaniline derivative instead of the aniline derivative.

The starting aniline derivatives employed in reaction sequence can be prepared as follows: ##STR6## Compounds in which the bridge Y is --CH.sub.2 S-- and --CH.sub.2 N-- can be prepared in a like manner, using the appropriate nitrothiophenol or nitroaniline in place of the nitrophenol. Compounds in which the bridge Y is ##STR7## can be prepared by using the appropriate acyl chloride or acyl N-imidazole and the appropriate N-substituted nitroaniline. Finally, the starting N-hydroxyaniline derivatives can be prepared as follows: ##STR8## The starting carboxy or thiocarboxy alkanoyl derivative halides are commercially available or can be prepared by conventional methods.

The starting compounds for the preparation of compounds in which the linking bridge Y is --O--, --S-- or ##STR9## can be prepared by the following reaction sequence: ##STR10##

Compounds of the invention which contain a basic nitrogen are capable of forming pharmacologically acceptable salts, including the salts of pharmacologically acceptable organic and inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, methanesulfonic, benzenesulfonic, acetic, citric, fumaric, maleic, succinic and the like. The compounds which are carboxylic acids or have a hydroxamic function are capable of forming alkali metal and alkaline earth carboxylates and carboxylates of pharmacologically acceptable cations derived from ammonia or a basic amine. Examples of the latter include but are not limited to cations such as ammonium, mono-, di, and trimethylammonium, mono-, di- and triethylammonium, mono-, di-, and tripropylammonium (iso and normal), ethyldimethylammonium, benzyldimethylammonium, cyclohexylammonium, benzylammonium, dibenzylammonium, piperidinium, morpholinium, pyrrolidinium, piperazinium, 1-methylpiperidinium, 4-ethylmorpholinium, 1-isopropylpyrrolidinium, 1,4-dimethylpiperazinium, 1-n-butyl-piperidinium, 2-methylpiperidinium, 1-ethyl-2-methylpiperidinium, mono-, di- and triethanolammonium, ethyl diethanolammonium, n-butylmonoethanolammonium, tris(hydroxymethyl)methylammonium, phenylmonoethanolammonium, and the like.

The compounds of the invention, by virtue of their ability to inhibit the activity of lipoxygenase enzyme and by their ability to antagonize the effects of LTD.sub.4 and LTC.sub.4, which are the major constituents of SRS-A, are useful for the inhibition of symptoms induced by these leukotrienes. Accordingly, the compounds are indicated in the prevention and treatment of those disease states in which LTD.sub.4 and LTC.sub.4 are causative factors, for example allergic rhinitis, allergic bronchial asthma and other leukotriene mediated naso-bronchial obstructive air-passageway conditions, as well as in other immediate hypersensitivity reaction, such as allergic conjunctivitis. The compounds are especially valuable in the prevention and treatment of allergic bronchial asthma.

When the compounds of the invention are employed in the treatment of allergic airways disorders, they can be formulated into oral dosage forms such as tablets, capsules and the like. The compounds can be administered alone or by combining them with conventional carriers, such as magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter and the like. Diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, tablet-disintegrating agents and the like may be employed. The compounds may be encapsulated with or without other carriers. In all cases, the proportion of active ingredients in said compositions both solid and liquid will be at least to impart the desired activity thereto on oral administration. The compounds may also be injected parenterally, in which case they are used in the form of a sterile solution containing other solutes, for example, enough saline or glucose to make the solution isotonic. For administration by inhalation or insufflation, the compounds may be formulated into an aqueous or partially aqueous solution, which can then be utilized in the form of an aerosol.

The dosage requirements vary with the particular compositions employed, the route of administration, the severity of the symptoms presented and the particular subject being treated. Treatment will generally be initiated with small dosages less than the optimum dose of the compound. Thereafter the dosage is increased until the optimum effect under the circumstances is reached. In general, the compounds of the invention are most desirably administered at a concentration that will generally afford effective results without causing any harmful or deleterious side effects, and can be administered either as a single unit dose, or if desired, the dosage may be divided into convenient subunits administered at suitable times throughout the day.

The lipoxygenase inhibitory and leukotriene antagonist effects of the compounds of the invention may be demonstrated by standard pharmacological procedures, which are described more fully in the examples given hereinafter.

These procedures illustrate the ability of the compounds of the invention to inhibit the polymorphonuclear leukocyte synthesis of the lipoxygenase products 5-HETE and 5,12-di-HETE; the ability of the compounds to antagonize LTC.sub.4 and LTD.sub.4 -induced bronchospasm mediated by exogenously administered leukotrienes; and measure the in vivo activity of the compounds as lipoxygenase inhibitors and leukotriene antagonists of endogenous mediators of bronchospasm.

The following examples show the preparation and pharmacological testing of compounds within the invention.

A. 2-chloromethylbenzthiazole

To a solution of aminothiophenol (8.3 g) in methylene chloride at 0.degree. C. is added methyl chloroacetimidate hydrochloride [prepared according to procedures described by R. Roger and D. G. Neilson, Chem. Rev., 61, 179 (1961)] (8.6 g). The reaction is allowed to warm to room temperature while stirring overnight. The mixture is washed with water (3X); dried over magnesium sulfate and concentrated to an oil. The oil is distilled (120.degree.-135.degree. C. at 0.5 mm Hg) to give 7.8 g (71% yield) of product.

B. 3-[(2-benzthiazolyl)methoxy]nitrobenzene

A mixture of 2-chloromethylbenzthiazole (17.6 g), 3-nitrophenol (13.2 g), cesium carbonate (30 g), sodium carbonate (10 g), potassium iodide (0.5 g) and acetone (600 ml) is heated at reflux overnight. The mixture is filtered and the resulting solution is partially concentrated. A crystalline solid forms which is filtered and dried giving 22.8 g (84% yield) m.p. 156.degree.-157.degree. C.

C. 3-[(2-benzthiazolyl)methoxy]aniline

To a suspension of 3-[(2-benzthiazolyl)methoxy]nitrobenzene (18.6 g) in ethanolic hydrochloric acid is added powdered iron. The reaction is stirred overnight at room temperature. After neutralization with saturated aqueous sodium bicarbonate, the mixture is extracted with methylene chloride (3X). The extract is dried over magnesium sulfate and concentrated to give 15.1 g (90% yield) of product. m.p. 117.degree.-120.degree. C.

D. 4-[[3-[(2-benzthiazolyl)methoxy]phenyl]amino]-4-oxobutanoic acid, methyl ester

To a solution of 3-[(2-benzthiazolyl)methoxy]aniline (2.0 g) and triethylamine (0.87 g) in tetrahydrofuran (50 ml) at room temperature is slowly added a solution of 3-carbomethoxypropionyl chloride (1.2 g) in tetrahydrofuran. The reaction is stirred for 1 hour. The mixture is filtered through a pad of celite and silica gel and the solvent is removed in vacuo giving a solid. The solid is recrystallized from ethanol giving 1.5 g (51% yield) of product. m.p. 143.degree.-144.degree. C.

Analysis for: C.sub.19 H.sub.18 N.sub.2 O.sub.4 S.1/4H.sub.2 O. Calculated: C, 60.86; H, 4.90; N, 7.47. Found: C, 60.88; H, 4.92; N, 7.35.

Following the procedure of Example 1 and using appropriate starting material and reagents, the following compounds are prepared:

(a) 4-[[3-[(2-benzoxazolyl)methoxy]phenyl]amino]-4-oxobutanoic acid, methyl ester, m.p. 104.degree.-105.degree. C.

(b) 4-[[3-[(1-methyl-2-benzimidazolyl)methoxy]phenyl]amino]-4-oxobutanoic acid, methyl ester, m.p. 136.degree.-137.degree. C.

(c) 3-[[3-[(2-benzthiazolyl)methoxy]phenyl]amino]-3-oxopropanoic acid, methyl ester, m.p. 109.degree.-112.degree. C.

(d) 3-[[3-[(1-methyl-2-benzimidazolyl)methoxy]phenyl]amino]-3-oxopropanoic acid, methyl ester, m.p. 167.degree.-169.degree. C.

A. 2-bromomethyl benzofuran

A mixture of 2-methylbenzofuran (2.0 g), N-bromosuccinimide (2.7 g) and carbon tetrachloride is heated at reflux overnight. The reaction is filtered and the solvent is removed in vacuo giving 3.8 g (52% monobrominated) of product (62% yield).

B. 4-[[3-[(2-benzofuranyl)methoxy]phenyl]amino]-4-oxobutanoic acid, methyl ester

The title compound is prepared by the methods of Example 1, B-D, but using 2-bromomethylbenzofuran. A white solid is obtained, m.p. 112.degree.-113.degree. C.

Analysis for: C.sub.20 H.sub.19 NO.sub.5. Calculated: C, 67.97; H, 5.41; N, 3.96. Found: C, 67.60; H, 5.45; N, 3.94.

A. 3-[2-benzthiazolyl)oxy]nitrobenzene

A mixture of 2-chlorobenzthiazole (17.0 g) and 3-nitrophenol (13.9 g) is heated at 140.degree. C. for 6 hours. After cooling, the remaining oil is crystallized from diisopropyl ether giving 16.8 g (62% yield) of product, m.p. 86.degree.-88.degree. C.

B. 4-[[3-[(2-benzthiazolyl)oxy]phenyl]amino]-4-oxobutanoic acid, methyl ester

The title compound was prepared by the method of Example 1, C and D, but using 3-[(2-benzthiazolyl)oxy]nitrobenzene. A white solid is obtained, m.p. 78.degree.-82.degree. C.

Analysis for: C.sub.18 H.sub.16 N.sub.2 O.sub.4 S. Calculated: C, 60.66; H, 4.53; N, 7.86. Found: C, 60.30; H, 4.88; N, 7.48.

A. N-methyl-3-[(2-benzthiazolyl)methoxy]aniline

A mixture of 3-[(2-benzthiazolyl)methoxy]aniline (3.8 g), prepared as in Example 1C, triethyl orthoformate (25 ml) and 5-10 drops of trifluoroacetic acid is heated at reflux for six hours. The mixture is concentrated and the residual oil is dissolved in ethanol and treated with sodium borohydride (1.5 g) at 0.degree. C. The reaction is allowed to warm to room temperature while stirring overnight. The mixture is then refluxed for 3 hours and concentrated. The remaining oil is dissolved in methylene chloride and washed with water; dried over magnesium sulfate and concentrated. The remaining oil is crystallized from hexane giving 1.8 g (45% yield) of product, m.p. 93.degree.-95.degree. C.

B. 4-[N-methyl[3-[(2-benzthiazolyl)methoxy]phenyl]amino]-4-oxobutanoic acid, methyl ester

The title compound is prepared by the method of Example 1D, but using N-methyl-3-[(2-benzthiazolyl)methoxy]aniline. A white solid is obtained, m.p. 107.degree.-110.degree. C.

Analysis for: C.sub.20 H.sub.20 N.sub.2 O.sub.4 S.HCl.1/2H.sub.2 O. Calculated: C, 55.87; H, 5.16; N, 6.52. Found: C, 55.41; H, 4.71; N, 6.39.

A. 3-[(2-benzthiazolyl)methoxy]phenylhydroxylamine

To a solution of 3-[(2-benzthiazolyl)methoxy]nitrobenzene (1.6 g), prepared as in Example 1B, in tetrahydrofuran with 10% palladium on carbon is slowly added hydrazine hydrate (0.4 ml). Fresh catalyst is added twice over 4 hours. The mixture is filtered through celite and the solvent is removed in vacuo to give 1.3 g (81% yield) of product, m.p. 112.degree.-114.degree. C.

B. 4-[[3-[(2-benzthiazolyl)methoxy]phenyl]hydroxylamino]-4-oxobutanoic acid, methyl ester

The title compound is prepared by the method of Example 1D, but using 3-[(2-benzthiazolyl)methoxy]phenylhydroxylamine. A white solid is obtained, m.p. 123.degree.-125.degree. C.

Analysis for: C.sub.19 H.sub.18 N.sub.2 O.sub.5 S. Calculated: C, 59.05; H, 4.69; N, 7.25. Found: C, 58.81; H, 4.90; N, 6.93.

The compounds 5- and 12-hydroxyeicosatetraenoic acid (5-HETE and 12-HETE) and 5,12-dihydroxyeicosatetraenoic acid (5,12-diHETE) are early arachidonic acid oxidation products in the lipoxygenase cascade, which have been shown to mediate several aspects of inflammatory and allergic response. This is especially true with respect to 5,12-diHETE, which is also denoted as LTB.sub.4 [see Ford-Hitchinson, J. Roy. Soc. Med., 74, 831 (1981)]. The assay of this Example measures the ability of the compounds of the invention to inhibit the synthesis of 5-HETE and LTB.sub.4 by rat glycogen-elicited polymorphonuclear leukocytes.

The assay is carried out as follows:

Peritoneal PMN are obtained from female Wistar rats (150-250 g) that received an i.p. injection of 6% glycogen (10 ml). After 24 hours, rats are killed by CO.sub.2 asphyxiation and peritoneal cells are harvested by peritoneal lavage using Ca.sup.++ and Mg.sup.++ free Hanks' balanced salt solution (HBSS). The peritoneal exudate is centrifuged at 400 g for 10 minutes. After centrifugation, the lavaged fluid is removed and the cell pellet is resuspended in HBSS containing Ca.sup.++ and Mg.sup.++ and 10 mM L-cysteine at a concentration of 2.times.10.sup.7 cells/ml. To 1 ml portions of cell suspension, test drugs or vehicle are added and incubated at 37.degree. C. for 10 minutes. Following this preincubation, the calcium ionophore (10 .mu.M), A23187, is added together with 0.5 .mu.Ci [.sup.14 C] arachidonic acid and further incubated for 10 minutes. The reaction is stopped by the addition of ice cold water (3 ml) and acidifying to pH 3.5. Lipoxygenase products are then extracted twice into diethyl ether. The pooled ether extracts are evaporated to dryness under nitrogen and the residue is redissolved in a small volume of methanol and spotted on aluminum backed pre-coated thin layer chromatographic plates. The samples are then co-chromatographed with authentic reference 5-HETE, 12-HETE and 5,12-diHETE in the solvent system-hexane:ether:acetic acid (50:50:3). After chromatography, the areas associated with 5-HETE and 5,12-diHETE standards are identified by autoradiography, cut out and quantitated by liquid scintillation.

Results are expressed as % inhibition of [.sup.14 C]5-HETE and [.sup.14 C]LTB.sub.4 (5,12-HETE) synthesis. ##EQU1##

Testing compounds of the invention in this assay and using the antioxident 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW755C) as a standard, the following results are obtained.

Testing of several other compounds of the invention in this test, but at the level of 100 .mu.m gives the following percentages of inhibition:

The results show that compounds of this invention have significant activity in inhibiting the synthesis of the arachidonic acid lipoxygenase oxidation products 5-HETE and LTB.sub.4.

The assay of this Example measures the in vivo ability of the compounds of the invention to inhibit the bronchospasm induced in guinea pigs by the exogenously administered leukotrienes C.sub.4 and/or D.sub.4. This assay is essentially a measure of the SRS-A antagonist properties of the compounds tested.

This assay is carried out as follows:

Male Hartley strain guinea pigs (350-600 g) are anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The jugular vein is cannulated for injection of drugs and the carotid artery for monitoring blood prerssure. The trachea is cannulated for artificial ventilation by a miniature Starling pump and for indirect measurement of respiratory volume changes as described infra. Additional pentobarbital sodium (15 mg/kg, i.v.) is administered to arrest spontaneous respiration. Submaximal bronchoconstrictor responses are established in control animals by varying the dose-levels of leukotriene. Intravenous dose-levels of LTC.sub.4 range from 1 to 2 .mu.g/kg and for LTD.sub.4 the range is from 0.3 to 1 .mu.g/kg. The aerosol bronchoprovocation dose for LTC.sub.4 is generated from 1.6 .mu.M solution and for LTD.sub.4 from a 2.0 .mu.M solution.

Test drugs are administered either intravenously, by aerosol or orally at 1 or 10 minutes before induction of bronchospasm by administration of either LTC.sub.4 or LTD.sub.4 at the predetermined dose-levels. Aerosols of soluble drugs or leukotrienes are produced in-line for 10 seconds only by actuation of an ultrasonic nebulizer (Monaghan). Aerosolized drug dosage is expressed in terms of solution concentration and by a fixed aerosol exposure time (approximately 10 seconds). Control animals receive saline in place of drug.

Respiratory volume changes are determined by a calibrated piston whose travel is recorded, via a linear transducer, on a Beckman Dynograph recorder. Maximal bronchoconstrictor volume is determined by clamping off the trachea at the end of the experiment. Overflow volumes at 3 and 5 minutes are obtained from the recorded charts.

The overflow volume at 3 and 5 minutes is expressed as a percentage of maximal bronchoconstriction. Combined group values are used from each of these time intervals to determine the inhibitory effect of drugs. ##EQU2## Students t-test for unpaired data is used to determine statistical significance. Dose response curves are generated and ED.sub.50 doses are interpolated from the regression lines.

Results for compounds of the invention in this assay, using LTD.sub.4 for induction of bronchospasm, are given below:

The results show that compounds of the invention have significant in vivo activity against LTD.sub.4 induced bronchoconstriction.

The assay of this Example measures the in vivo ability of the compounds of the invention to inhibit the bronchospasm induced in guinea pigs by endogenous mediators of the bronchoconstriction.

The assay is carried out as follows:

Male Hartley strain guinea pigs weighing 350-600 g are sensitized to ovalbumin (OA) (5 mg i.p. and 5 mg s.c.) on day 0. Three to five weeks later, the animals are anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The jugular vein is cannulated for injection of drugs and the carotid artery for monitoring blood pressure. The trachea is cannulated for artificial ventilation by miniature Starling pump and for indirect measurement of respiratory volume changes as described, infra. Additional pentobarbital sodium (15 mg/kg, i.v.) is administered to arrest spontaneous respiration. A cyclooxygenase inhibitor, indomethacin (10 mg/kg in tris buffer, i.v. at 9 min.) is administered to shunt arachidonic metabolism to lipoxygenase pathways. One minute later, chlorpheniramine (1.0 mg/kg in saline, i.v.) is given to attenuate the histaminic component of anaphylactic bronchoconstriction. Test drugs are administered either intravenously or by aerosol at 2 or 10 minutes before antigen challenge. Anaphylactic bronchoconstriction is induced by administration of aerosolized OA (1%). Aerosols of OA or soluble drug are produced in-line for 10 seconds only by actuation of an ultrasonic nebulizer (Monaghan). Aerosolized drug dosage is expressed in terms of solution concentration and by a fixed aerosol exposure time (approximately 10 seconds). Control animals receive saline in place of drug.

Respiratory volume changes are determined by a calibrated piston whose travel is recorded, via a linear transducer, on a Beckman Dynograph recorder. Maximal bronchoconstrictor volume is determined by clamping off the trachea at the end of the experiment. Overflow volumes at minutes 3 and 5 are obtained from the recorded charts.

The overflow volume at 3 and 5 minutes is expressed as a percentage of maximal bronchoconstriction. Combined group values are used from each of these time intervals to determine the inhibitory effect of drugs. ##EQU3## Students t-test for unpaired data is used to determine statistical significance. Dose response curves are generated and ED.sub.50 doses are interpolated from the regression lines.

The results for three compounds of the invention, with administration of the compounds at 10 and 20 minutes before ovalbumin challenge, and at varying doses, are presented below:

The results show that compounds of the invention have significant in vivo activity in inhibiting ovalbumin induced bronchospasm mediated by endogenous products of the lipoxygenase oxidation of arachidonic acid.