NO301116B1 - Analogous Process for Preparing Therapeutically Active Tetrahydro-2-Saccharinylmethylaryl Carboxylates - Google Patents

Description

The present invention relates to the production of new tetrahydro-2-saccharinylmethylarylcarboxylates which inhibit the enzymatic activity of proteolytic enzymes, and which are used in the treatment of degenerative diseases.

The inhibition of proteolytic enzymes with non-toxic reagents is useful in the treatment of degenerative disorders, such as emphysema, rheumatoid arthritis and pancreatitis, where proteolysis is an essential element.

Protease inhibitors are widely used in biomedical research. Serine proteases are the most widespread class of proteolytic enzymes. Some serine proteases are characterized as chymotrypsin-like or elastase-like based on their substrate specificity.

Chymotrypsin and chymotrypsin-like enzymes normally cleave peptide bonds in proteins at a site at which the amino acid residue on the carboxyl side is typically Trp, Tyr, Phe, Met, Leu or another amino acid residue containing aromatic or large alkyl side chains.

Elastase and elastase-like enzymes normally cleave peptide bonds at a site where the amino acid residue on the carboxyl side of the bond is typically Ala, Val, Ser, Leu or other similar, smaller amino acids.

Both chymotrypsin-like and elastase-like enzymes are found in leukocytes, mast cells and pancreatic saliva in higher organisms, and are secreted by many types of bacteria, yeast and parasites.

JP patent 7200419 describes a number of 2-saccharinylmethyl benzoates including 2-saccharinylmethylbenzoate per se and 2-saccharinylmethyl-2,4-dichlorobenzoate and 4-nitrobenzoate. It is stated in the JP document that the compounds "have strong activity against rice blast, rice sheat blight, rice helminthosporium leaf spot and rice bacterial leaf blight disease".

Sunkel et al., J. Med. Chem., 31, 1886-1890 (1988) discloses a series of 2-saccharinyl-lower alkyl-1,4-dihydro-pyridine-3-carboxylates which have platelet aggregation inhibitory and anti-thrombotic activities.

US Patent 4,263,393 to Chen describes various 2-aroylmethylsaccharins useful as (in translation) "photographic elements and film units".

US Patent 4,195,023 to Mulvey et al. describes R1-2-R2C0-1,2-benzisothiazol-3-ones, where R^ is halogen, alkoxy, alkylamino, dialkylamino, alkoxycarbonyl, amino, nitro or hydrogen in the benzenoid ring and Rg is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl , halophenyl, heteroaryl or substituted heteroaryl, and R^—2-A-CO-saccharins where R^ has the same meanings as the benzenoid ring substituents in the 1,2-benzisothiazol-3-ones and A is alkyl, alkenyl, alkynyl, cycloalkyl, fluorophenyl , heteroaryl or substituted-heteroaryl. The compounds are reported to have elastase inhibitory activity and to be useful in the treatment of emphysema.

Zimmermann et al., J. Biol. Chem., 225(20), 9848-9851 (1980) describes N-acyl saccharins, where the acyl group is furoyl, thenoyl, benzoyl, cyclopropanoyl, ethylbutyryl and acryloyl, which have serine protease inhibitory activity.

JP patent 73/35457 describes 4-methylphenyl-2-saccharinyl carboxylate which is stated to have bactericidal and fungicidal activities.

Several classes of compounds are known to be serine protease inhibitors. E.g. US Patent 4,659,855 to Powers discloses arylsulfonyl fluoride derivatives useful as elastase inhibitors. US Patents 4,547,371 and 4,623,645 to Doherty et al. describes respectively cephalosporin sulfones and -sulfoxides which are stated to be potent elastase inhibitors which are useful in the treatment of inflammatory conditions, especially arthritis and emphysema.

Teshima et al., J. Biol. Chem., 257(9), 5085-5091 (1982) reports the results of studies of serine proteases (human leukocyte elastase, porcine pancreatic elastase, cathepsin G and bovine chymotrypsin Aa) with 4-nitrophenyl esters and thioesters of N-trifluoroacetylanthranilates, 2-substituted -4H-3,1-benz-oxazin-4-ones, 2-substituted-4-quinazolinones and 2-substituted-4-chloroquinazolines.

Cha, Biochem. Pharmacol., 24. 2177-2185 (1975) mentions kinetic methods in connection with the study of the binding of inhibitors to macromolecules, such as enzymes, and methods for determining such parameters as the inhibition constants, reaction rates and concentrations for bound and unbound enzyme.

US Patent 4,276,298 to Jones et al. describes 2-R-1,2-benzisothiazolinone-1,1-dioxides, where R is phenyl substituted with fluorine, dinitro, trifluoromethyl, cyano, alkoxycarbonyl, alkylcarbonyl, carboxyl, carbamoyl, alkylacylamino, alkylsulfonyl, N,N-dialkylsulfamoyl, trifluoromethoxy, trifluoromethylthio , trifluoromethylsulfonyl and trifluoromethylsulfinyl, or pyridyl substituted with the same as R when R is phenyl with the exception that pyridyl can also be mono-nitro substituted. The compounds are stated to have protease enzyme inhibitory activity, particularly elastase inhibitory activity and to be useful in the treatment of emphysema, rheumatoid arthritis "and other inflammatory diseases".

Power, Biochem., 24» 2048-2058 (1985) describes studies of the inhibitions of four chymotrypsin-like enzymes, cathepsin G, rat mast cell proteases I and II, human skin chymase and chymotrypsin Aa, by N-furoylsaccharin and N-(2,4 -dicyano-phenyl1)sacchar in.

Svoboda et al., Coll. Czech. Chem. Commun., 51, 1133-1139

(1986) describe the preparation of 4-hydroxy-2H-1,2-benzothiazine-3-carboxylates by intramolecular Dieckmann condensation of 2H-1,2-benzisothiazol-3-one-2-acetate-1,1-dioxide esters.

US Patents 4,350,752 and 4,363,865 to Reczek et al. and US Patent 4,410,618 to Vanmeter et al. relates to photographic reagents (US Patent 4,350,752 to Reczek and US Patent 4,410,618 to Vanmeter et al.) and photographic dyes (US Patent 4,363,865 to Reczek) and describes various 2-substituted-saccharins useful for such applications, e.g. "photographic reagents" bonded through a heteroatom to an "imidomethyl-blocking" group (US Patent 4,350,751 to Reczek), "carrier-diffusible photographic dyes" bonded to the nitrogen atom of an imide through a 1,1-alkylene group (US- patent 4,363,865 to Reczek) and N-acylmethylimides which are described as "blocked photographic reagents" and which have (in translation) "a residue of an organic photographic reagent containing a heteroatom through which it is attached to the blocking group"

(Water meter).

US Patent 3,314,960 to Freed describes 2-(1,1,3-trioxo-1,2-benzisothiazol-2-yl)glutarimides which are said to be useful as sedatives.

2-Chloromethylsaccharin is described in FR Patent 1,451,417 as an intermediate for the preparation of N-methylsaccharin-d,1-trans-chrysanthemate, which is useful as an insecticide, and US Patent 3,002,884 to Lo describes 2-chloro -, 2-bromo- and 2-Iodomethyl-saccharins which are useful as fungicidal agents.

The present invention relates to the production of new 4,5,6,7-tetrahydro-2-saccharinylmethylarylcarboxylates which have protease enzyme-inhibiting activity and which are useful in the treatment of degenerative diseases. These therapeutically active compounds have the formula:

where:

R<4a> is hydrogen or lower alkyl;

R<6> is hydrogen or lower alkyl; or

R<4a> and r<6> together form a spirocyclopropyl;

R<7> is hydrogen or lower alkoxy;

Ar is phenyl substituted with from 1 to 3 groups selected from halogen and -SO 2 -N=B, where N=B is —NR-(alkylene)-N(alkyl) 21 where R is lower alkyl;

and therapeutically acceptable Je acid addition salts of basic compounds thereof.

The terms lower alkyl and lower alkoxy used in the present context mean monovalent aliphatic radicals, including branched radicals, with 1-10 carbon atoms. The lower alkyl part in such groups thus includes e.g. methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl, 2-methyl-3-butyl, 1-methylbutyl, 2-methylbutyl, neopentyl, n-hexyl, 1-methylpentyl, 3-methylpentyl, 1-ethylbutyl, 2-ethylbutyl, 2-hexyl, 3-hexyl, 1,1,3,3-tetramethylpentyl 1,1-dimethyloctyl and the like.

The term halogen used herein means fluorine, chlorine, bromine or iodine.

The term alkylene used herein means divalent, saturated radicals including branched radicals, with 2-10 carbon atoms and which have their free valences on different carbon atoms and thus include 1,2-ethylene, 1,3-propylene, 1,4-butylene, 1-methyl-1,2-ethylene, 1,8-octylene and the like.

The compounds of formula VI inhibit the activity of serine proteases, especially human leukocyte elastase and the chymotrypsin-like enzymes and are thus useful in the treatment of degenerative disease states such as emphysema, rheumatoid arthritis, pancreatitis, cystic fibrosis, chronic bronchitis, respiratory distress syndrome in adults, inflammatory bowel disease, psoriasis, bullous pemphigoid and α-l-antitrypsin deficiency.

The new compounds of formula VI are prepared according to the present invention by combining a 4,5,6,7-tetrahydro-2-halomethylsaccharin compound of the formula: where X is a halogen, with an arylcarboxylic acid of the formula:

in the presence of an acid acceptor,

and, if desired, converting a basic compound thus obtained into a therapeutically acceptable acid addition salt thereof.

The reaction is carried out as mentioned in the presence of an acid acceptor, such as an alkali metal carbonate, a trilower alkylamine or 1,8-diazabicyclo-[5.4.0]undec-7-ene, hereinafter referred to as DBU. The reaction is carried out in an organic solvent which is inert under the reaction conditions, e.g. acetone, methyl ethyl ketone (MEK), acetonitrile, tetrahydrofuran (THF), diethyl ether, dimethylformamide (DMF), N-methylpyrrolidinone, methylene dichloride (MDC), xylene, toluene or lower alkanols, at a temperature in the range from ambient temperature up to the boiling point of the solvent used .

For the production of intermediate products and starting material in connection with the present method for the production of the tetrahydrosaccharin compounds of formula VI, the reactions in the following reaction core can be used:

The 5-chloro-2-benzyl-2H-isothiazol-3-one-1-oxide can be oxidized with a suitable oxidizing agent, preferably hydrogen peroxide in acetic acid, to the 1,1-dioxide which is then reacted under typical Diels-Alder conditions with the appropriate the diene compound and is reduced to obtain the 2-benzyl-tetrahydrosaccharin compound which is hydrogenolysed as before to the tetrahydrosaccharin compound. This tetrahydrosaccharin compound gives, on reaction with formaldehyde in a lower-alkanol solvent, a hydroxymethylsaccharin compound which, on further reaction with a thionyl halide or a phosphorus halide, corresponds to the halomethylsaccharin derivative of formula VIII which is used as starting material in the present method. The 4,5,6,7-tetrahydrosaccharins which are starting materials for the final compounds of formula VI where R<7> is hydrogen can be synthesized using a method as follows:

A 3-alkyl-2-cyclohexenone compound is thus reacted with the appropriate alkyllithium cuprate 1 an ethereal solvent, preferably diethyl ether, at from -50 to +20°C, preferably approx. (<T>C, and the resulting adduct is treated in situ with methyl cyanoformate and hexamethylphosphoramide. The 6,6-dialkyl-2-oxocyclohexanecarboxylate thus prepared is reacted with benzyl mercaptan as described above and the mixture of 2-(benzylthio)cyclohexanecarboxylates is oxidatively chlorinated as described above to obtain a mixture of chlorosulfonyl esters which are treated with ammonia as before to obtain the desired 4,4-dialkyl-4,5,6,7-tetrahydrosaccharins The halomethyl derivative of formula VIII can be obtained as indicated above.

The aryl carboxylic acids, Ar—COOH, which are used to prepare the final products of formula VI are members of a known class and can be prepared using well-known, conventional synthetic methods.

Chloromethyl esters of the aryl carboxylic acid can be prepared by treating the carboxylic acid with formaldehyde or a formaldehyde equivalent, preferably paraformaldehyde, in the presence of (1) a chloric acid, preferably zinc chloride or hydrochloric acid, or (2) trimethylsilyl chloride plus stannous chloride.

Simple chemical transformations which are conventional and well known to those skilled in the art can be used to effect changes in functional groups in the compounds of the present invention. E.g. can catalytic reduction of nitro groups to prepare the corresponding amino-substituted compounds, acylation of amino-substituted compounds to prepare the corresponding amides, oxidation of sulfides or sulfoxides to prepare the corresponding respective sulfoxides or sulfones, saponification of esters to prepare the corresponding carboxylic acids, catalytic debenzylation of phenol ethers or of benzyl amines to prepare the corresponding phenols or debenzylated amines or reaction of phenols with an alkylating agent in the presence of base to prepare ethers as required are carried out.

In standard biological test methods, the compounds of formula VI have been found to possess human leukocyte elastase (HLE) and chymotrypsin inhibitory activities and are thus useful in the treatment of degenerative diseases such as emphysema, rheumatoid arthritis, pancreatitis, cystic fibrosis, chronic bronchitis, respiratory distress syndrome in adults, inflammatory bowel disease, psoriasis, bullous pemphigoid and α-l-antitrypsin deficiency.

The compounds of formula VI which have basic functions can be converted into the acid addition salt form by interaction between the base and an acid. Similarly, the free base can be regenerated from the acid addition salt form in a conventional manner, ie by treating the salts with cold, weak aqueous bases, e.g. alkali metal carbonates and alkali metal bicarbonates. The bases thus regenerated can be reacted with the same or a different acid to recover the same or a different acid addition salt. The bases and all their acid addition salts can thus easily be interconverted.

Likewise, certain compounds of formula VI which have acid, i.e. carboxylic acid, functions can be converted into their salt forms by reacting the acid with a base, such as alkali metal or ammonium hydroxides or with organic bases such as alkyl, dialkyl or trialkylamines, and the acids can be regenerated from the salts by treating the salts with aqueous acids.

Formula VI not only represents the structural configuration of the bases and acids, but is also representative of the structural units that are common to all the compounds of formula VI either in the form of the free base, the free acids or in the form of the salts of the bases and the acids. By virtue of these common structural units, it has been found that the compounds of formula VI and their salts have inherent pharmacological activity of a type to be described more fully below. This inherent pharmacological activity can be obtained in useful form for pharmaceutical purposes by using the free bases or free acids in themselves or the salts formed from pharmaceutically acceptable acids and bases, i.e. acids or bases whose anions or cations are harmless to the animal organism in effective dosages of the salts such that useful properties associated with the common structural unit represented by the free bases and free acids are not impaired by side effects attributable to the anions or cations.

When utilizing this pharmacological activity of the salt, it is naturally preferred to use pharmaceutically acceptable salts. Although water insolubility, high toxicity or lack of crystalline character may render certain particular salt compounds unsuitable or less desirable for use as such in a given pharmaceutical application, the water insoluble or toxic salts may be converted to the corresponding pharmaceutically acceptable bases by decomposition of the salts with aqueous base or aqueous acid as explained above, or they may alternatively be converted to any desired pharmaceutically acceptable salt by double decomposition reactions involving the anion or cation, e.g. by ion exchange methods.

Besides their usefulness in pharmaceutical applications, the salts are also useful as characterizing or identifying derivatives of the free bases or free acids or in isolation or purification methods. Common to the salts, such characterization or purification salt derivatives can, if desired, be used to regenerate the pharmaceutically acceptable free bases or free acids by reacting the salts with aqueous base or aqueous acid, or they can alternatively be converted into a pharmaceutically acceptable salt by e.g. e.g. ion exchange methods.

The new feature of the compounds thus lies in the concept of the 2-saccharinylmethylarylcarboxylates of formula VI and not in any particular acid or base part or acid anion or base cation which is linked to the salt forms of the compounds.

The compounds of formula VI can be prepared for pharmaceutical use by including them in unit dosage form as

tablets or capsules for oral administration either alone or in combination with suitable aids such as calcium carbonate, starch, lactose, talc, magnesium stearate, gum acacia and the like. Furthermore, the compounds can be formulated for oral, parenteral or aerosol inhalation administration either in aqueous solutions of water-soluble salts of the compounds or in aqueous alcohol, glycol or oil solutions or oil-water emulsions in the same way as conventional medicinal substances are prepared.

The percentages of active component in such preparations can be varied so that a suitable dosage is achieved. The dose administered to a particular patient varies depending on the physician's judgment using as criteria: route of administration, duration of treatment, patient's size and physical condition, the effectiveness of the active component and the patient's response to this. An effective dosage amount of the active component can thus only be determined by the doctor after consideration of all criteria by applying the best judgment on behalf of the patient.

The molecular structures of the compounds of formula VI were assigned on the basis of study of their infrared and NMR spectra. The structures were confirmed through the agreement between calculated and found values for elemental analyzes by the elements or by analysis of high-resolution mass spectra.

The following examples illustrate the invention and include, in addition to the production of relevant final connections, also the production of output connections.

Example IA

To freshly distilled cyclopentadiene (25 ml) at 0°C was added 4-bromo-2-(tert-butyl)isothiazol-3(2H)-one-1,1-dioxide (Heiv. Chlm. Acta., 72, 1416 , 1989) (7.9 g, 0.03 mol). After stirring at 0°C under nitrogen for 16 hours, the reaction mixture was concentrated in vacuo. The residue was purified by filtration through silica gel, eluting with hexanes (500 mL), followed by 2056 ethyl acetate in hexanes (500 mL). The latter elutions were concentrated in vacuo to obtain 9.8 g (100% of the norbornene adduct) 3a-bromo-2-t-butyl-3a,4,7,7a-tetrahydro-4,7-methanol-1,2-benzisothiazole -3(2H)-one-1,1-dioxide as a white solid.

The adduct (0.4 g, 1.2 mmol) in 25 mL of ethyl acetate containing 556 Pd on CaCO 3 (0.2 g) was stirred under a hydrogen atmosphere for 4 h and the reaction mixture was filtered through a pad of silica gel, eluting with ethyl acetate ( 100 ml). The elutions were concentrated in vacuo and the residue crystallized from hexanes to give 0.4 g (10056) of the bromonorbornane compound as a white crystalline solid.

To a solution of the bromonorbonane compound (3.7 g, 0.011 mol) in toluene (25 mL) at 0°C was added diazabicyclonone (1.37 g, 0.011 mol) in toluene (10 mL). After stirring at 0°C for 20 min. silica gel (25 g) was added to the reaction mixture. The resulting slurry was placed on top of a 15 cm pad of silica gel and eluted with 205 g of ethyl acetate in hexanes (800 mL). The eluates were concentrated in vacuo to give 2.8 g (10056 ) of the dehydrobrominated compound as a white solid.

2-t-butyl -4,5,6,7-tetrahydro-4,7-methanol-1,2-benzisothiazol-3(2H)one-1,1-dioxide (2.8 g, 0.011 mol) in trifluoroacetic acid (30 mL) was heated at reflux for 48 hours and allowed to stand at room temperature for 4 days. The resulting mixture was concentrated in vacuo, treated with methanol (20 mL) and evaporated to dryness. The residue was taken up in ether (100 ml) and

washed with saturated NaHCO 3 (1 x 50 mL). The layers were separated, the aqueous phase acidified to pH 1 with 2N HCl and extracted with MDC (2 x 100 mL). The combined organic extracts were dried and concentrated in vacuo to afford 0.9 g (4256) of the bicyclo(2.2.1)-saccharin derivative as a white solid.

A mixture of the bicyclo(2.2.1)saccharin derivative (0.9 g, 5 mmol), chloromethylphenyl sulfide (0.07 g, 7 mmol) and tetrabutylammonium bromide (0.36 g, 0.16 mmol) in toluene (50 mL) was refluxed under nitrogen for 16 hours, cooled to room temperature and evaporated to dryness under vacuum. The residue was purified by flash chromatography on silica gel (100 g) using 10056 MDC as eluent to afford 1.05 g (7256) of the sulfide as a viscous oil.

The sulfide (1.05 g, 3 mmol) in dichloromethane (100 mL) was treated with sulfuryl chloride (0.66 g, 5 mmol) and stirred for 2 h. The resulting yellow solution was diluted with MDC (100 mL), washed with saturated NaHCO3 solution, dried and concentrated in vacuo. The residue was purified by flame chromatography on silica gel (3356 MDC in hexanes) to give 0.66 g (8156) of 2-chloromethyl-4,5,6,7-tetrahydro-4,7-methano-1,2- benzisothiazol-3(2H)-one-1,1-dioxide.

The 2-chloromethyl compound (0.66 g, 2.7 mmol) was treated with 2,6-dichlorobenzoic acid (0.56 g, 2.9 mmol), anhydrous potassium carbonate (0.55 g, 4.0 mmol) and tetrabutylammonium bromide (0.2 g, 0.6 mmol) in DMF (2.5 mL) at 70 °C for one hour. The resulting mixture was concentrated in vacuo, diluted with ethyl acetate (100 mL) and filtered. The filtrate was washed with water, saturated NaHCO3, water and brine. The organic phase was concentrated in vacuo and the residue purified by flash chromatography on silica gel (3:6:1, MDC:hexanes:ether) to give 0.5 g (4756) 2-(2,6-dichlorobenzoyloxymethyl)-4,5 ,6,7-tetrahydro-4,7-methanol-1,2-benzisothiazole-3(2H)-one-1,1-dioxide as a colorless foam.

Examples IB and 1C

By a method analogous to that in example IA, cyclohexadiene and 1,1-dimethylcyclopentadiene can be converted respectively into 4,5,6,7-tetrahydro-4,7-ethane-1,2-benzisothiazol-3(2H)-one -1,1-dioxide and 8,8-dimethyl-4,5,6,7-tetrahydro-4,7-methanol-1,2-benzisothiazol-3(2H)-one-1,1-dioxide.

Examples 2A - 2D

(i) General procedure for the preparation of methyl 2-alkylcyclohexan-6-one carboxylate: To a suspension of anhydrous Cul (10 mmol) in anhydrous THF (100 mL) was added Me 2 S (100 mmol) and the resulting solution was cooled to -78°C. The Grignard reagent (20 mmol) was added over a period of 15 min. After stirring at -78°C for one hour, a solution of cyclohexenone in THF was added and stirring continued for a further 15 min. To the resulting mixture was added HMPA (5 mL) and, after 15 min, methyl cyanoformiate (30 mmol ) in THF (20 mL) and the reaction mixture warmed to room temperature and stirred overnight. The reaction mixture was quenched with 2N HCl (50 mL). The layers were separated and the aqueous phase extracted with EtgO (1 x 100 mL). The combined organic extracts were washed with saturated N^Cl solution (3 x 50 mL), water (2 x 50 mL), brine (1 x 50 mL) and dried (Na 2 SO 4 ). Solvent removal in vacuo and purification by either Kugelrohr distillation or flame chromatography gave the desired methyl 2-alkylcyclohexan-6-one carboxylate (Table A). (ii) General procedure for the preparation of methyl 2-benzylthio-6-alkylcyclohex-2-ene carboxylate and 2-benzylthio-6-alkylcyclohex -1-ene carboxylate: A mixture of methyl 2-alkylcyclohexan-6-one carboxylate (1 eq.), benzyl mercaptan (1.1 eq. ) and the acid bed montmorillonite, KSF (1.5 times the weight of methyl-2-alkylcyclo-hexan-6-one carboxylate) in anhydrous toluene ( (50-100 mL) was refluxed under nitrogen with azeotropic removal of water for 12-14 hours and cooled to room temperature. The solids were filtered off and washed with ether. The combined filtrate was washed with 1056 Na 2 CO 3 , water, brine and dried. Removal of the solvent in vacuo and purification of the residue by flame chromatography in on silica gel (1056 ether in hexanes) gave a mixture of methyl 2-benzylthio-6-alkyl-cyclohex-2-ene carboxylate and 2-benzylthio-6-alkylcyclohex-1-ene carboxylate (Table B) which was used in the next step as a mixture.(iii) General procedure for the preparation of 4-alkyl-tetrahydro-saccharins: A solution of methyl 2-benzylthio-6-alkylcyclohex-2-ene carboxylate and 2-benzylthio-6-alkylcyclohex -1-ene carboxylate (1-10 mmol of the mixture) in 10 ml of MDC was diluted with 20-50 ml of glacial acetic acid and 1-5 ml of water, mixts. n was cooled to -10°C, and chlorine gas was bubbled through the mixture until the exothermic reaction ceased. The mixture was then stirred

for 10 minutes and brought to dryness to obtain a mixture of methyl 2-chlorosulfonyl-6-alkylcyclohex-2-ene carboxylate and 2-chlorosulfonyl-6-alkylcyclohex-1-ene carboxylate, which was dissolved in 10 mL of THF and added to 25 ml of a solution of concentrated ammonium hydroxide while cooling in an ice/acetone bath. After stirring for 2 hours, the reaction mixture was concentrated in vacuo, the residue taken up in water, acidified to pH 1 with 2N HCl and extracted with MDC. The organic phase was dried and concentrated in vacuo to obtain a mixture of methyl 2-aminosulfonyl-6-alkyl cyclohex-2-ene carboxylate and 2-aminosulfonyl-6-alkyl cyclohex-1-ene carboxylate. The mixture was dissolved in methanol and added to a freshly prepared solution of sodium methoxide (10-50 mmol) and stirred at ambient temperature for 12 hours. The reaction mixture was concentrated in vacuo, diluted with water and extracted with ether. The organic phase was discarded and the aqueous phase acidified to pH 1 with concentrated HCl and extracted with MDC. Washing with brine, drying and evaporating to dryness gave the organic extracts 4-alkyl-4,5,6,7-tetrahydrobenzoisothiazol-3-one-1,1-dioxide or 4-alkyltetrahydrosaccharin (Table C).

(iv) A mixture of 4-alkyl-4,5,6,7-tetrahydrobenzisothiazol-3-one-1,1-dioxide (4-alkyltetrahydrosaccharin) (1.0 eq.), chloromethylphenyl sulfide (1.5 eq. .) and tetrabutylammonium bromide (0.2 eq.) in toluene (25 ml/g saccharin) were refluxed under nitrogen for 16-24 hours and then cooled to room temperature. The resulting mixture was evaporated to dryness and the residue chromatographed on silica gel eluting with hexanes/MDC (1:1 to 1:3) to afford the corresponding 2-phenylthiomethyl-4-alkyl-4,5,6,7-tetrahydrobenzisothiazole- 3-one-1,1-dioxide or 2-phenylthiomethyl-4-alkyltetrahydro saccharin (Table D). (v) A solution of 2-phenylthiomethyl-4-alkyltetrahydrosaccharin (1.0 eq.) was treated with sulfuryl chloride (1.5 eq.) and stirred for 2 h. The resulting yellow solution was brought to dryness to obtain 2-chloromethyl-4-alkyltetrahydrosaccharin which was treated with 2,6-dichlorobenzoic acid (1.1 equiv), anhydrous potassium carbonate (1.5 equiv) and tetrabutylammonium bromide (0.2 equiv) in DMF (25 mL) at 70°C for one hour. The resulting mixture was concentrated in vacuo, diluted with ethyl acetate (100 mL) and filtered. The filtrate was washed with water, saturated NaECOs, water and brine. The organic phase was concentrated in vacuo, and the residue purified by flash chromatography on silica gel (2:1 MDC/hexanes) to give 4-alkyl-4,5,6,7-tetrahydro-2-saccharinylmethyl-2,6-dichlorobenzoate ( Table E).

Example 2E

By following a procedure similar to that described in Example 1AA 1 NO patent 300373, 2-chloromethyl-4-isopropyl-4,5,6,7-tetrahydrobenzisothiazol-3-one-1,1-dioxide was treated with 2,6-dichloro -3 - [ [2- (N ,N-dimethylamino )ethyl]-N-methylamino-sulfonyl]benzoic acid to obtain 4-isopropyl-4,5,6,7-tetrahydro-2-saccharinylmethyl-2,6-dichloro -3-[[2-(N,N-dimethylamino)ethyl]-N-methylaminosulfonyl]benzoate hydrochloride, m.p. 121°C (decomp.).

Example 3

Methyl- 2. 2- dimeth, cyclohexane- 6-one- carboxylate:

To a suspension of anhydrous Cul (70.0 g, 0.37 mol) in anhydrous ether (500 mL) at 0°C was added halogen-free methyllithium (520 mL of 1.4M solution in ether, 0.73 mol). After stirring at 0°C for 15 minutes, a solution of 3-methyl-2-cyclohexenone (20.0 g, 0.18 mol) in ether (50 mL) was added and stirring continued for a further hour. To the resulting mixture was added THF (50 mL) and HMPA (25 mL) and after 15 min. methyl cyanoformate (45.0 g, 0.53 mol) in THF (20 mL) and the reaction mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched with 2N HCl (50 mL). The layers were separated and the aqueous phase extracted with Et 2 O (1 x 500 mL). The combined organic extracts were washed with saturated NH 2 Cl solution (3 x 50 mL), water (2 x 50 mL), brine (1 x 50 mL) and dried (Na 2 SO 4 ). Removal of the solvent in vacuo and purification by Kugelrohr distillation gave 34.0 g (9956) of methyl 2,2-dimethylcyclohexane-6-one carboxylate, bp. 80-84°C/0.6 mm.

The cyclohexanone compound was converted to 4,4-dimethyl-4,5,6,7-tetrahydro-2-saccharinylmethyl-2,6-dichlorobenzoate, m.p. 121-123°C, following the procedure described above for Example 2D.

Example 4

(i) Reaction of isothiazole-5-carboxaldehyde with lithium 3-(triphenylphosphoranylidene)propanoate under standard Wittig conditions gives 4-(5-isothiazolyl)-3-butenoic acid which is reduced and cyclized with aluminum chloride to give 4-oxo-4 ,5,6,7-tetrahydrobenzisothiazole. The 4-oxo compound is reacted with methylenetriphenylphosphorane under standard Wittig conditions and a methylene is introduced into the resulting 4-methylene compound via a Simmons Smith reaction to obtain 6,7-dihydrospiro[benzisothiazole-4(5H),1'cyclopropane ] which is oxidized with hydrogen peroxide in acetic acid to obtain 6,7-dihydrospiro [benzisothiazole-4 ( 5 ), 1'-cyclopropane-1,1-dioxide-(4-spiro-cyclopropyl-tetrahydrosaccharin). This compound is chloromethylated according to Preparation IA in NO patent 300373 to obtain 2-chloromethyl-4-spirocyclopropyl-4,5,6,7-tetrahydrosaccharin. (ii) According to the procedure in Example 4 in NO patent 300373, 2-chloromethyl-4-spirocyclopropyl-4,5,6,7-tetrahydrosaccharin, as obtained in (i) above, is coupled with 2,6-dimethyl-benzoic acid to obtain 4-spirocyclopropyl-4,5,6,7-tetrahydro-2-saccharinylmethyl-2,6-dimethylbenzoate.

Example 5

(i) 2-benzyl-4-isopropyl-6-oxo-tetrahydrosaccharin is reduced with sodium borohydride and methylated with methyl iodide in the presence of sodium hydride to give 2-benzyl-4-isopropyl-6-methoxytetrahydrosaccharin. This compound is debenzylated and chloromethylated as in Preparation 23 in NO patent 300373 to obtain 2-chloromethyl-4-isopropyl-6-methoxy-4,5,6,7-tetrahydrosaccharin. (ii) According to the procedure in Example 4 in NO patent 300373, 2-chloromethyl-4-isopropyl-6-methoxy-4,5,6,7-tetrahydro-saccharin, as obtained in (i) above, is coupled with 2,6 -dimethyl-benzoic acid to obtain 4-isopropyl-6-methoxy-4,5,6,7-tetrahydro-2-saccharinylmethyl-2,6-dimethylbenzoate.

BIOLOGICAL TEST RESULTS.

Measurement of the inhibition constant, , of an HLE-inhibitor complex has been described for "truly reversible inhibition constants" which usually concern competitive inhibitors. [Cha, Biochem. Pharmacol., 24» 2177-2185,

(1975)]. However, the compounds in the present invention do not form truly reversible inhibitor complexes, but are consumed to a certain extent by the enzyme. Thus, instead of measuring a K^-value, a K-^^-value was calculated and this is defined as the ratio k0ff/con» the reactivation rate for the enzyme to the inactivation rate for the enzyme. The values for kDff and con are measured and Kj<*> is then calculated.

The inactivation rate, con, of enzymatic activity was determined for the test compounds by measuring the enzyme activity of an aliquot of the respective enzyme as a function of time after addition of the test compound. When plotting the log value for the enzyme activity against time, an observed inactivation rate, k0bs» is obtained which can be represented as k0^s = ln2/t^ where t^ is the time required for the enzyme activity to fall by 50#. The inactivation rate will then be equal

where [I] is the concentration of the inhibiting compound. The reactivation constant, k0ff, is determined in the same way, and the inhibition constant, Kj<*>, is then calculated as

The values obtained for con and K^<*> for specific substituted saccharin derivatives are shown in Table 1, the compounds being identified by the above example numbers in which their preparations are described.

Claims (3)

1. Analogous process for the preparation of therapeutically active 2-saccharinylmethylarylcarboxylates of the general formula: where: R<4a> is hydrogen or lower alkyl; r<6> is hydrogen or lower alkyl; or R<4a> and R<*> together form a spirocyclopropyl; R<7> is hydrogen or lower alkoxy; Ar is phenyl substituted with from 1 to 3 groups selected from halogen and -SO 2 -N=B, where N=B is -NR-(alkylene)-N(alkyl) 2 , where R is lower alkyl; and therapeutically acceptable acid addition salts of basic compounds thereof, characterized in that a 4,5,6,7-tetrahydro-2-halomethylsaccharin compound of the formula: where X is a halogen, with an arylcarboxylic acid of the formula: ArCOOH in the presence of an acid acceptor, and, if desired, converting a basic compound thus obtained into a therapeutically acceptable acid addition salt thereof.; 2. Analogous process according to claim 1, for the preparation of compounds where R<4a> is hydrogen, methyl, ethyl or isopropyl, R*> is hydrogen or methyl, R<7> is hydrogen or methoxy and Ar is phenyl substituted with from 1 to 3 halogen -atoms, characterized by the use of correspondingly substituted starting materials. 3. Analogous process according to claim 2, for the preparation of the compounds: 4,5,6,7-tetrahydro-2-saccharinylmethyl-2,6-dichlorobenzoate, 4-methyl-4,5,6,7-tetrahydro-2-saccharinylmethyl-2, 6-dichloro-benzoate, 4-ethyl-4,5,6,7-tetrahydro-2-saccharinylmethyl-2,6-dichloro-benzoate, 4-isopropyl-4,5,6,7-tetrahydro-2-saccharinylmethyl-1- 2,6-dichlorobenzoate or 4,4-dimethyl-4,5,6,7-tetrahydro-2-saccharinylmethyl-2,6-dichlorobenzoate, characterized by using correspondingly substituted starting materials.