WO1988007044A1

WO1988007044A1 - Timolol derivatives

Info

Publication number: WO1988007044A1
Application number: PCT/US1988/000793
Authority: WO
Inventors: Hans Bundgaard
Original assignee: Insite Vision, Inc.
Priority date: 1987-03-17
Filing date: 1988-03-17
Publication date: 1988-09-22
Also published as: EP0305496A4; EP0305496A1

Abstract

Lipophilic, transient ester derivatives of timolol represented by general formula (I), wherein R1 represents a substituted or unsubstituted branched chain alkyl, straight or branched chain alkenyl, aryl, aralkyl or cycloalkyl group, a substituted or unsubstituted aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, or a substituted or unsubstituted group of the formula R2-O-, wherein R2 is an alkyl, aryl, aralkyl or cycloalkyl group or of the formula R'2-CONH, wherein R'2 is an alkyl, aryl, aralkyl or cycloalkyl group, and nontoxic pharmaceutically acceptable acid addition salts thereof, are disclosed as prodrug forms of the anti-glaucoma drug timolol which are also useful in the treatment of glaucoma and increased intraocular pressure and which exhibit either improved corneal absorption and/or impeded conjunctival absorption characteristics, thus permitting the use of smaller doses of such derivatives than of timolol, thereby reducing systemic timolol concentration while achieving the same therapeutic effect.

Description

DESCRIPTION

Timolol Derivatives

Field of the Invention

This application is a continuation-in-part of my pending application Serial No. 26,618, filed March 17, 1987, the disclosure of which is incorporated herein by reference. This invention relates to novel prodrug f o r m s o f t i m o l o l [ S - ( - ) -1-(tert-butylamino) -3-[ (4-morpholino-1,2,5-- thiadiazol-3- yl)oxy]-2-propanol] ["The Merck Index", Ninth Edition (Rahway, N.J.:Merck & Co., Inc. 1976), 9170]. More particularly, this invention relates to novel transient prodrug derivatives of timolol useful in the treatment of glaucoma. These timolol derivatives, when administered to the eye of a warm-blooded animal, e.g., to the human eye, are converted in the ocular tissue into the anti-glaucoma drug timolol. The conversion of the prodrug form to timolol in the ocular tissue proceeds in transient fashion, i.e., in a manner such that as timolol itself is released, the moieties which split off are nontoxic or are metabolized to nontoxic metabolic products.

This invention further relates to methods for preparing these novel transient (prodrug) timolol derivatives, to pharmaceutical compositions containing such derivatives, and to methods for using such derivatives.

Background of the Invention

Timolol, represented by the structural formula:

is a non-selective β-adrenergic receptor blocker widely used in the treatment of glaucoma and increased intraocular pressure. See Frosst U.S. Patents Nos. 3,619,370; 3,655,663 and 3,657,237; Wasson et al, J.Med.Chem. 15. 651 (1972); Franciosa et al, Clin. Pharmacol.Ther. 13. 128 (1972); Ulrych et al, Clin. Pharmacol.Ther. 13. 232 (1972).

Timolol's therapeutic usefulness is limited by a relatively high incidence (40%) of cardiovascular and respiratory side effects [Munroe et al.. Drug Intell. Clin. Pharm. 19. 85 (1985); Nelson et al. Am.J. Opthalmol. 102. 606 (1986)] which arise as a result of absorption of the topically applied drug into the systemic circulation [Alvan et al, Clin. Pharmacokin. 5. 95 (1980); Shell, Surv. Qphthalmol. 26. 207 (1982)].

It has now been discovered that the systemic absorption of topically applied timolol can be decreased, and its adverse effects thereby diminished, by administering certain transient ester derivatives (prodrugs) of timolol, and nontoxic pharmaceutically acceptable acid addition salts thereof, which are highly lipophilic in comparison to timolol itself and which, as a consequence, exhibit improved corneal absorption characteristics. The more efficient corneal absorption of the prodrug derivatives of this invention permits the use of smaller doses of such derivatives than of timolol, thereby reducing systemic timolol concentration while achieving the same therapeutic effect. Certain ester derivatives of timolol are disclosed in German Offenlegungsschrift No. 2,428,030. However, there is no suggestion in this published application that the compounds described, e.g., O-succinyl timolol, have any prodrug activity, and their topical administration for treatment of glaucoma is not explicitly or implicitly mentioned or claimed.

The synthesis of a "homologous series" of O-acyl prodrugs of timolol for ocular administration (the O-acetyl, O-valeryl and O-caprylyl esters are mentioned) is reported in an abstract by Chang and Buur et al entitled "Prodrug Approach to Minimize Systemic Absorption of Timolol", Proc. Acad. Pharm. Sci. (No. 140, p. 106), published in October, 1985.

Certain ester derivatives of 4-[3-(substituted amino)-2-hydroxypropoxy] -1,2,5-thiadiazole compounds are generically disclosed in U.S. Patent No. 3,655,663. The patent states that acetyl, propionyl, butyryl or benzoyl substituents may be substituted at the 2-hydroxy position of the generic compounds, but does not exemplify or specify any ester of timolol. There is no suggestion in this patent that any such esters would have any prodrug activity, and the topical or other administration of such compounds for treatment of glaucoma is not explicitly or implicitly mentioned or claimed.

It is an object of this invention to provide novel prodrug forms of timolol, useful for the treatment of glaucoma and increased intraocular pressure.

Another object of this invention is to provide prodrug forms of timolol, which prodrugs or transient derivatives, owing to their improved lipophilicity, exhibit superior bioavailability over timolol per sβ when administered to the eye.

A further object of the present invention is to provide prodrugs of timolol which cleave in such a manner as to enable the parent drug timolol to be released at its site of therapeutic activity in the eye while the remaining cleaved moiety is non-toxic or is metabolized in a nontoxic fashion.

A still further object of this invention is to provide prodrugs of timolol which, when administered topically to the eye of a warm-blooded animal, e.g., a human, give rise to decreased systemic concentrations of timolol relative to administration of timolol per se in equivalent amounts, while at the same time eliciting the pharmacodynamic and therapeutically useful response of the parent timolol molecule more efficiently and prolongedly.

These and other objects, as well as the nature, scope and utilization of this invention, will become readily apparent to those skilled in the art from the following description, the drawing and the appended claims.

Summary of the Invention The novel, lipophilic, transient ester derivatives (prodrugs) of timolol of this invention are represented by the general formula:

wherein R₁ represents a branched chain alkyl group, preferably one having from 3 to 8 carbon atoms, inclusive, such as isopropyl, tert-butyl, tert-amyl (neopentyl) or isooctyl, a straight or branched chain alkenyl group, preferably one having from 2 to 6 carbon atoms, inclusive, such as propenyl, butenyl or pentenyl, an aryl group, e.g., a phenyl or naphthyl group, an alkyl-substituted aryl group, such as methylphenyl or ethylphenyl, an alkylthio-, nitro-, carbalkoxy-, alkanoyl- or alkanoyloxy-substituted aryl group, an aralkyl group, preferably an alkylene-aryl group in which the aryl moiety is a phenyl or naphthyl group and the alkylene moiety is a straight or branched hydrocarbon chain of 1 to 6 carbon atoms, inclusive, such as methylene or 1,2-butylene, a cycloalkyl group, preferably one having from 3 to 7 carbon atoms, inclusive, such as cyclopropyl, cyclopentyl or cyclohexyl, an alkyl-, phenyl-, or aralkyl-substituted cycloalkyl group such as 1'-methylcyclopropyl or 2'- methylcyclopropyl, an aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoms elected from the group consisting of nitrogen, oxygen, and sulfur, or a group of the formula R₂-O- wherein R₂ is an alkyl, aryl, aralkyl or cycloalkyl group or a group of the formula R'₂-CONH wherein R'₂ is an alkyl, aryl, aralkyl or cycloalkyl group. Any of the above-mentioned groups represented by R₁ can be unsubstituted or substituted with one or more substituents such as a halogen atom, a hydroxy group, a carbonyl group, a straight or branched-chain alkoxy group having the formula R₃-O-, wherein R₃ represents an alkyl group or an aryl group, unsubstituted or substituted with one or more halogen atoms or hydroxy groups, an amino group having the formula -NR₄R₅, wherein R₄ and R₅ are the same or different and are hydrogen, an alkyl group or together with the adjacent nitrogen atom form a 5- or 6-membered heterocyclic ring, which in addition to the nitrogen may contain one or two further heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, a carbamoyl group having the formula

wherein R₄ and R₅ are as defined above, or an oxyacyl group having the formula R₂COO- wherein R₂ is as defined above.

When R₄ and R₅ in the formulas -NR₄R₅ and -CONR₄R₅ together with the adjacent nitrogen atom form a 5- or 6-membered heterocyclic ring which in addition to the nitrogen atom may contain 1 or 2 further heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, this ring may, for instance, be 1-piperidinyl, 1-pyrrolidinyl, 1-piperazinyl, 4-methyl-1-piperazinyl, morpholinyl, thiomorpholinyl and 1-pyrazolyl. The term "halogen" designates fluorine, chlorine, bromine or iodine, with chlorine being preferred. When R1 is an aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, this ring may, for instance, be 2-, 3- or 4-pyridinyl, 2- or 3-thienyl, 2-, 4- or 5-thiazolyl, 2-,4- or 5- oxazolyl, 2-imidazolyl, 5-isoxazolyl, or 2- or 3-furanyl.

Nontoxic pharmaceutically acceptable acid addition salts of the novel, lipophilic transient ester derivatives (prodrugs) oftimolol represented by formula II above also come within the scope of the invention. Such salts generally include those formed using nontoxic inorganic or organic acids. For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, nitric, phosphoric and the like; and the salts with organic acids such as acetic, propionic, succinic, fumaric, maleic, tartaric, citric, glycolic, lactic, stearic, malic, pamoic, ascorbic, phenylacetic, benzoic, p-acetamidobenzoic, glutamic, salicylic, sulfanilic, and the like.

In particular, the cyclopropanoyl esters and substituted cyclopropanoyl esters of timolol are preferred for use in this invention and, most particularly, the 1'-methylcyclopropanoyl ester is preferred.

Brief Description of the Drawing FIG. 1 is a graph comparing the total timolol concentration in the aqueous humor and plasma of pigmented rabbits at 5 and 30 minute post-instillation of 25 μl of 15 mM solutions of timolol and its prodrug O-pivalyl timolol.

Detailed Description of the Invention Preparation of the Prodrugs of Formula II The compounds of this invention can be prepared by a variety of synthetic routes. One method comprises reacting a timolol salt, e.g. timolol hydrochloride, with an acid chloride of the formula III

R₁-COCl

(III) wherein R₁ is as defined above. The reaction can conveniently be carried out in the absence of a solvent or in an inert solvent such as benzene, toluene, acetone, acetonitrile, dioxane, dichloromethane or the like, at a temperature of from room temperature (about- 25°C) to reflux, for from about 1 to about 100 hours. Instead of acid chlorides, acid bromides, acid anhydrides or mixed anhydrides may be used. Another method of preparing the compounds of formula II utilizes as a starting material timolol in which the amino group has been conveniently protected, e.g. by a benzyl, carbobenzoxy carbonyl or t-butyloxycarbonyl group. A compound of the formula III may then be reacted with N-protected timolol in a solvent such as acetone, acetonitrile, dioxane, water, pyridine or the like, at from about 0ºC to reflux, for from about 1 to about 24 hours, eventually in the presence of an acid scavenger such as an alkali metal carbonate or triethylamine. The N-protecting group is subsequently removed by, e.g., hydrogenation or acidic hydrolysis.

A third method of preparing the compounds of formula II comprises reacting N-protected timolol with an acid of the formula IV R₁-COOH

(IV) wherein R₁ is as defined above. The reaction is conducted in the presence of a suitable dehydrating agent, e.g., N,N-dicyclohexylcarbodiimide, and is conveniently carried out in an inert solvent such as dioxane, pyridine, dichloromethane or the like, at a temperature of from about 0ºC to about 60°C, for from about 1 to about 48 hours. The N-protecting group is subsequently removed from the reaction product to give compounds of formula II by, e.g., hydrogenation or acidic hydrolysis.

Preferred compounds of the invention are compounds in which R₁ represents one of the following groups: decyl, dodecyl, isopropyl, isobutyl, tert-butyl, isopentyl, neopentyl, isooctyl, 4-methylphenyl, 4-aminophenyl, 2-methylphenyl, 4-hydroxyphenyl, 2-hydroxyphenyl, 4-methoxγphenyl, 2,4-dimethylphenyl, 4-dimethylaminophenyl, benzyl, 2-thienyl, 2-furanyl, cyclopropyl, cyclohexyl, cyclopentyl, 1'-methylcyclopropyl, 2'methylcyclopropyl, methoxy, ethoxy, butoxy, cyclohexyloxy, 2-acetoxyphenyl, 4-acetoxyphenyl, 2-carbamoylethyl, nicotinyl, 2-N,N- dimethylcarbamoylethyl, and nontoxic pharmaceutically acceptable acid addition salts thereof.

Dose and Dosage Forms

The compounds of formula II may be administered per se, or in combination with any pharmaceutically appropriate inert ophthalmic vehicle or carrier system. The administered dose (either as a single dose, a daily dose, or other time-presented doses) depends on the requirements of the individual under treatment. The dosage administered is, therefore, not subject to specific limits. The dose of any compound of formula II will typically be an anti-glaucoma effective amount, or, expressed in another way, an amount of the compound of formula II which, inside the eye, produces an amount of timolol that achieves the desired pharmacological response. Generally, the single medical doses for warm-blooded animals, which include humans and primates, will be in the range of approximately 0.005 mg to 1 mg, with 0.05 mg to 0.5 mg being preferred. The number of doses per day ordinarily will be 1 or 2. The compounds of formula II may be administered in the form of a pharmaceutical composition, which may be a liquid application form, such as a solution, a suspension, or an emulsion, an ointment, a cream, an aerosol, a polymeric or solid controlled-release or monitoring drug delivery device (such as a membrane or capsule-type delivery system) or a polymeric solution that gels upon ophthalmic instillation, resulting in a controlled-release or monitoring drug delivery device or system.

Pharmaceutically appropriate inert vehicles for use in carrier systems for the ophthalmic administration of the compounds of this invention are well known to those skilled in the art of ophthalmic pharmaceutical formulations. Thus, pharmaceutically acceptable carriers for the preparation of eyedrops include conventional or common vehicle buffer systems, isotonic boric acid solutions, isotonic saline vehicles, and the like, with or without polymers or viscosity altering additives such as hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol or polyacrylamide. Suitable carriers for the preparation of ophthalmic oil solutions of the compounds of this invention include arachis oil, castor oil, mineral oil and the like. Further information concerning the formulation of ophthalmic pharmaceutical preparations is found in such texts as "Remington's Pharmaceutical Sciences", Sixteenth Edition (Easton, Pa.:Mack Publishing Company, 1980) and "Ophthalmic Drug Delivery Systems", J.R. Robinson, Ed. (Washington, D. C.:American Pharmaceutical Assn., 1980), as well as in Justin et al., Acta Pharm. Fenn. 90. 289 (1981).

The presently preferred administration form is an eyedrop solution.

A typical method for preparing aqueous eyedrops containing a compound of this invention is to dissolve the compound (e.g. as a water-soluble salt) in sterile water in a given concentration (e.g. 1-5 mg/ml), optionally adjust the pH to, e.g., 4-5 with a suitable buffer or with hydrochloric acid or sodium hydroxide, optionally add a preservative such as phenethanol or chlorobutanol, optionally add a viscosity altering additive such as methylcellulose, and sterilize the final solution by, e.g., membrane filtration.

An eyedrop preparation may also comprise the compound formulated as a sterile, solid preparation in any eyedrop container. Before dispensing, iostonic saline is added to dissolve the compound. In order that those skilled in the art can more fully understand this invention, the following examples are set forth. These examples are given solely for purposes of illustration, and should not be considered as expressing limitations unless so set forth in the appended claims. All parts and percentages are by weight, unless otherwise stated. The derivatives described all had spectroscopic properties (IR and 1 _H _NMR) and elemental analyses in agreement with their structures.

Example I

Timolol maleate (6 g) was dissolved in water (100ml). Sodium hydroxide (2 M) was added to give a pH of 10 and the mixture was extracted with ethyl acetate (2 x 75 ml). The extracts were dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was dissolved in ether (100 ml) and a slight excess of 3 M HCl in methanol was added. Petroleum ether was added and after standing overnight at 5ºC the white crystalline precipitate formed was filtered off and recrystallized from acetone-ether to give 4.1 g (84%) of timolol hydrochloride. Mp 130-131ºC.

Example II

Isobutyryl chloride (10 mmol) was added to a slurry of timolol hydrochloride (3 mmol, 1.05 g) in 12 ml of benzene. The mixture was refluxed with stirring for 3 hours and then evaporated in vacuo. Benzene (10 ml) was added and the mixture evaporated again. The solid residue was slurried in ether, filtered off, washed with ether and recrystallized from ethanol- ether to give O-isobutyryl timolol hydrochloride in an 86% yield. Mp 174-175ºC (from ethanol-ether). Analysis:

Calc. for C₁₇H₃₁Cl N₄O₄S: C 48.27 H 7.39 N 13.25

Found: C 48.20 H 7.31 N 13.21

Example III

O-pivalyl timolol hydrochloride was prepared from timolol hydrochloride and pivalyl chloride by the procedure described in Example II and ioslated in 65% yield. Mp 146-147ºC (from benzene-ethanol-petroleum ether). Analysis: Calc. for C₁₈H₃₃Cl N₄O₄S: C 49.47 H 7.61 N 12.82

Found: C 49.43 H 7.65 N 12.79

Example IV

Cyclohexanoyl chloride (10 mmol) was added to a slurry of timolol hydrochloride (3 mmol, 1.05 g) in 12 ml of benzene. The mixture was refluxed with stirring for 3 hours and then evaporated in vacuo. Benzene (910 ml) was added and the mixture evaporated again. The residue was taken up in water (40 ml) and ethyl acetate (50 ml), and 2 M sodium hydroxide was added with stirring to give a pH of about 9.5. The ethyl acetate layer was separated, washed with water (20 ml), dried over anhydrous sodium sulfate and evaporated in vacuo to give O-cyclohexanoyl timolol free base as an oil. This was dissolved in ether (50 ml) and a solution of fumaric acid (3 mmol, 348 mg) in 2-propanol (5 ml) was added. After standing for 1 hour at 5°C crystalline O-cyclohexanoyl timolol fumarate was isolated by filtration. The compound was washed with ether and recrystallized from ethanol- ether (79% yield). Mp 180-181°C. Analysis:

Calc. for C₂₄H₃₈N₄O₈S: C 53.12, H 7.06, N 10.32

Found: C 53.03, H 7.04, N 10.33

Example V A mixture of timolol hydrochloride (3 mmol, 1.05 g) and benzoyl chloride (2 ml) was stirred at 60ºC for 20 hours. After cooling, petroleum ether (20 ml) was added and a semi-solid residue precipitated. This residue was taken up in water (40 ml) and ethyl acetate (50 ml), and 2 M sodium hydroxide was added with stirring to give a pH of about 9.5. The organic layer was separated, washed with water (20 ml), dried over anhydrous sodium sulfate and evaporated in vacuo to give O-benzoyl timolol free base as an oil. This was dissolved in ether (50 ml) and a solution of fumaric acid (3 mmol, 348 mg) in 2-propanol (5 ml) was added. After standing for 1 hour at 5ºC crystalline O-benzoyl timolol fumarate was filtered off, washed with ether and recrystallized from ethanol-ether (83% yield). Mp 180-181°C. Analysis:

Calc. for C₂₄H₃₂N₄O₈S: C 53.72, H 6.01, N 10.44

Found: C 53.70, H 6.04, N 10.42

Example VI A mixture of timolol hydrochloride (3 mmol, 1.05 g) and benzoyl chloride (3 ml) in acetonitrile (25 ml) was kept at 80ºC for 45 hours. The solution was evaporated in vacuo and then treated as described in

Example V above. The O-benzoyl timolol fumarate isolated (80% yield) was identical to that described in Example V.

Example VII

A mixture of timolol maleate (2 mmol, 864 mg) and 4-methoxybenzoyl chloride (2.og) in acetonitrile (15 ml) was stirred at 85º C for 20 hours. The acetonitrile was removed in vacuo and the residue treated as described in Example V. O-4-methoxybenzoyl timolol fumarate was recrystallized from ethanol-ether, yield: 75%. Mp 201-203°C. Analysis:

Calc. for C₂5H₃₄N₄O₉S: C 52.99, H 6.05, N 9.89

Found: C 53.08, H 6.00, N 9.86

Example VIII A mixture of timolol maleate (2 mmol, 864 mg) and

4-methylbenzoyl chloride (2 ml) in acetonitrile (15 ml) was stirred at 85°C for 18 hours. The acetonitrile was removed by evaporation under reduced pressure and the residue obtained treated as described in Example V. O-4-methylbenzoyl timolol fumarate was recrystallized from ethanol-ether, yield: 82%. Mp 204-206ºC. Analysis:

Calc. for C₂₅H₃₄N₄O₈S: C 54.53, H 6.22, N 10.18

Found: C 54.43, H 6.29, N 10.17

Example IX

A mixture of timolol maleate (2 mmol, 864 mg) and 2-furoyl chloride (1.5 ml) in acetonitrile (15 ml) was stirred at 80°C for 40 hours. The acetonitrile was removed by evaporation under reduced pressure and the residue obtained treated as described in Example. V. O-2-furoyl timolol fumarate was recrystallized from ethanol-ether, yield: 76%. Further examples are listed below. The compounds were prepared by stirring a mixture of timolol maleate (2 mmol, 864 mg) and the appropriate acid chloride (2 ml) in acetonitrile (15 ml) at 75ºC for 18 hours. The acetonitrile was removed by evaporation under reduced pressure and the residue obtained treated as described in Example V. The esters obtained as fumarate salts were recrystallized from acetone or ethanol-ether.

Example Compound Yield(%) m.p. (°C) X O-cyclopropanoyl timolol 79 172-173

XI O-1'-methylcyclopropanoyl timolol 74 174-175

XII O-2'-methylcyclopropanoyl timolol 77 161-175

XIII O-cyclopentanoyl timolol 78 172-173 xrv O-2-ethylbutyryl timolol 75 137-138 XV O-3,3-dimethylbutyryI timolol 81 173-174

XVI O-2-methylbenzoyl timolol 80 162-164

XVII O-2-methoxybenzoyl timolol 75 126-127 XVII I O-2-benzoyloxymethylbenzoyl timolol 55 150-152 XIX O-3-thienyl timolol 79 149-150 XX O-2-acetoxybenzoyl timolol 60 135-136

Example XXI

A mixture of timolol maleate (0.7 mmol, 302 mg), isatoic anhydride (0.9 mmol, 147 mg) and N,N-dimethylaminopyradine (0.07 mmol, 8.5 mg) in 2 ml of N,N-dimethylformamide was stirred at 60ºC for 5 h. After cooling, the solution was poured into a mixture of water (30 ml) and ethyl acetate (40 ml). Sodium hydroxide (2 M) was added while stirring to give a pH of 9-9.5 and the compound isolated as a fumarate salt following the procedure described in Example V. O-2-Aminobenzoyl timolol fumarate was recrystallized from acetone. Yield: 67%. Mp 169-170ºC.

Example XXII

O-2-Methylaminobenzoyl timolol fumarate was prepared as described in Example XXI using N- methylisatoic anhydride instead of isatoic anhydride. Yield: 70%. Mp. 165-167ºC.

Example XXIII

A mixture of timolol maleate (450 mg) and benzoylisocyanate (1.0 ml) in acetonitrile (8 ml) was stirred at 60ºC for 1 h and then evaporated under reduced pressure. The residue obtained was treated as described in Example V. O-Benzoylcarbamate ester of timolol (fumarate salt) was isolated in a yield of 65%. Mp. 144-145°C.

Conversion of Timolol Esters to Timolol

The timolol esters of this invention were shown to be hydrolyzable to the parent timolol. Aqueous solutions of the esters were kept at 37ºC and at various times analyzed by HPLC assays for intact esters as well as for timolol. A typical HPLC assay used a reversed-phase C-8 column eluted with methanol-0.03 M K₂PO₄ solution pH 4.5 (1:1 v/v) and with the column effluent being monitored at 294 nm. Analysis of the solutions of pH 2-10 showed a complete conversion of the esters to timolol. Some rate data for the conversion are shown in Table 1. At pH 3-4 the hydrolysis of timolol esters is quite slow, especially at lower temperatures. Thus, less than 10% degradation of O-4-methylbenzoyl timolol fumarate occurs for an aqueous solution (0.5 mg/ml) of pH 4 when stored at 5°C for two years. For an aqueous solution of O-4-methoxybenzoyl timolol fumarate of pH 5, less than 10% degradation occurs during storage at 5°C for four years. For an aqueous solution of O-1'-methylcyclopropanoyl timolol fumarate of pH 4, less than 10% degradation occurs during storage at 5°C for five years. It is readily possible to select esters of this invention with acceptable shelf-lives in aqueous solutions. This is in sharp contrast for simple alkyl esters of timolol. Thus, whereas the O-butyryl timolol ester was shown to possess a shelf-life of only 12 days at pH 4 and 25ºC, the O-1'-methylcyclopropanoyl and O-2'-methylcyclopropanoyl esters had a shelf-life greater than 10 months at similar conditions. For the O-2-aminobenzoate ester a shelf-life of one year at 25°C and pH 4 is obtainable.

It was surprisingly found that even very small amounts of tromethamine (Tris) (about 10^-4 - 10^-3 M) inhibited the rate of hydrolysis of timolol esters at weakly acidic pH values. Thus, by adding tromethamine to eyedrop formulations of the timolol esters it is possible to increase their utilization times significantly. However, many of the esters of the present invention are sufficiently stable to hydrolysis in formulated solution that addition of tromethamine or other stabilizers is not necessary to achieve satisfactory formulations, e.g. ocular solution formulations. Among such particularly hydrolysis-stable esters are the O-cycloalkanoyl timolol esters and substituted cycloalkanoyl esters, including particularly substituted cylopropanoyl timolol esters.

The hydrolysis of timolol esters to timolol was found to be catalyzed markedly in vitro by various ocular tissues. Some results are given in Table 1 below. These experiments show that the timolol esters of this invention are in fact prodrug derivatives of timolol in that the parent active compound is released at conditions similar to those prevailing in vivo. As described below the ready conversion of timolol esters to timolol was also proved to occur in vivo following topical administration of the esters.

Lipophilicity of Timolol Ester Prodrugs The apparent partition coefficients (P) for some timolol esters and timolol between octanol and 0.05 M phosphate buffer (pH 7.4) were measured. The lipophilicity of the compounds was also evaluated by reversed-phase HPLC and expressed in terms of capacity factors (k'). Values for log P and k' are listed in Table 2 below. The results show that the derivatives listed are all more lipophilic than the parent timolol.

TABLE 2

Ester log P³ k'⁴ t_1/2 (min)⁵

O-Pivalyl 2.68 4.17 215

O-Isobutyryl 2.00 2.81 68 O-Cyclohexyl 3.25 7.72 94

O-Benzoyl 2.41 3.57 120

O-(4-Methoxybenzoyl) 2.42 - 540

O-(4-Methylbenzoyl) 2.90 - 295

Unesterified Timolol -0.04 0.93 - O-Cyclopropanoyl 1.74 2.14 245

O-1'-Methylcyclopropanoyl 2.22 3.02 594

O-2-Aminobenzoyl 2.51 3.09 2200

³ Partition coefficients (P) between octanol and phosphate buffer solution pH 7.40.

⁴ HPLC conditions: RP-8 column (100 x 3 mm), eluent: acetonitrile-methanol, 0.01 M acetate buffer pH 4.0 (45:10:45 v/v).

⁵ At pH 7.40 (0.02 M phosphate buffer) and 37°C.

It should be recognized that the in vivo enzymatic hydrolysis of the present timolol prodrugs is thought to be a desired mechanism whereby timolol is released into a free form particularly suited to achieving therapeutic in vivo effects, e.g. the treatment of increased intraocular pressure associated with glaucoma. Thus, although stability of the present timolol prodrugs against hydrolysis while in formulated solution form is desirable in order to increase the shelf-life of the formulations, it is also preferred that in vivo enzymatic hydrolysis of the timolol prodrugs be relatively labile. It has been surprisingly discovered that many of the timolol esters of the present invention, including particularly the O-cycloalkanoyl esters, and more particularly the alkyl-substituted cyclopropanoyl timolol esters, combine the desirable characteristics of high stability against hydrolysis in formulation solution and relatively labile enzymatic hydrolysis in, for example, in vivo environments associated with, for example, the intraocular environment.

Ratio of Corneal to Conjunctival Permeability Coefficients of Timolol Prodrugs

A simple procedure to estimate the potential usefulness of a prodrug candidate in improving the therapeutic index of timolol is the ratio of corneal to conjunctival permeability coefficients. These coefficients can be obtained by monitoring the rate of appearance of prodrug and timolol across the isolated cornea or conjunctiva sandwiched between two compartments in the Ussing chamber. The corneal and conjunctival permeability coefficients as well as their ratios for selected alkyl, cycloalkyl, and aryl timolol ester prodrugs are listed in Table 3. It can be seen that all the prodrugs afford improved ratios of corneal to conjunctival absorption, and hence ocular to systemic absorption, when compared to timolol. Specifically, the respective ratios for the chemically stable prodrugs O-pivalyl, cyclopropanoyl, 1'-methylcyclopropanoyl, and 2'-methylcyclopropanoyl timolol are 0.57, 0.64, 0.77, and 0.44, all better than the ratio of 0.34 for timolol.

Ocular and Systemic Absorption of Topically Applied Timolol Esters in Pigmented Rabbits

When compared with timolol, the timolol esters of the present invention were shown to be absorbed two to four times more readily across the cornea into the aqueous humor of the pigmented rabbit eye at 5 and 30 minutes postinstillation of 15 mM solutions. Surprisingly, the plasma timolol concentration at the same time points was slightly reduced. The improved ocular absorption of O-pivalyl timolol suggests that the duration of intraocular pressure lowering by the timolol derived from O-pivalyl timolol may be extended by one to two half-lives in the aqueous humor, thereby leading to reduced dosing frequency and hence systemic drug load. At the same time, a two- to fourfold reduction in the instilled dose of O-pivalyl timolol is expected to result in more than this extent of reduction of plasma timolol concentration without compromising ocular absorption. Experimentally, 25 μl of a 15 mM O-pivalyl timolol prodrug solution in isotonic Tris buffer (pH 7.4) was instilled in the conjunctiva sac of each eye of six pigmented rabbits. At 5 or 30 minutes post dosing, the rabbits were killed by an overdose of pentobarbital solution administered via a marginal ear vein. The eyes were immediately rinsed with saline and blotted dry, and about 100-150 μl of aqueous humor was aspirated from the anterior chamber. One minute prior to killing each animal, 5 ml of blood was collected from a precannulated ear artery and was immediately centrifuged at 4°C to yield plasma. Both aqueous humor and plasma samples were frozen immediately and stored at -20ºC until assayed.

At the time of assay, an aqueous humor sample was mixed with an equal volume of methanol containing 6% perchloric acid and 0.5 μg/ml propanolol. Following centrifugation, 10 to 20 μl of the supernatant was injected into the HPLC. The plasma sample (2 ml) was mixed with 0.1 ml of propranolol solution (2 μg/ml) and 0.5 ml OF 1 M ammonium acetate buffer (pH 9), extracted with 5 times its volume of diethyl ether by vortexing for 1 minute, and then centrifuged at 1,500 x g for 10 minutes. The upper organic layer was transferred to a 15 ml screw-capped conical centrifuge tube containing 100 μl of 0.1 N HCl, vortexed for 1 minute, and centrifuged at 1,500x g for 10 minutes. The organic phase was discarded, while 10-50 μl of the aqueous phase, containing timolol, its prodrug, and propanolol, was injected into the HPLC. The extraction efficiency was better than 75%, and less than 1% of prodrug was decomposed during the entire extraction procedure when conducted in the cold. The HPLC procedure utilized a reversed phase ODS- C-18 column (4.6 mm x 250 mm, 5 μm) was used. The mobile phase was a mixture of acetonitrile and water containing 1% triethylamine HCl at pH 3.0. The proportion of acetonitrile in the mobile phase was increased linearly from 20 to 60% for the first 3 minutes and was kept at 60% for the next 15 minutes at a flow rate of 1.0 ml/minute. Timolol and its prodrug were monitored at 294 nm. The retention time was 7 minutes for timolol, 11.3 minutes for O-pivalyl timolol and 9.7 minutes for propranolol, the internal standard. The assay sensitivity was 5 nmoles with respect to timolol and its prodrug. The intra- and inter-run variations were less than 5 and 7.55, respectively.

The results indicate that, in general, the prodrug caused a 2- to 4-fold improvement in the corneal absorption of timolol coupled with a 30% and 50% reduction in plasma timolol concentration (p < 0.01) at 5 and 30 minutes, respectively, post-instillation of 15 mM timolol prodrug solution. This is shown in FIG. 1, which illustrates that at 5 minutes, the percent of

O-pivalyl timolol in hydrolyzed form in the aqueous humor was 33.8%, while at 30 minutes this increased to 82.8%. In the plasma, no intact prodrug was detected at either 5 or 30 minutes. Error bars represent standard error of the mean. Asterisks denote that in each case results which were significantly different from timolol administration (p < 0.01) were obtained. Key: crosshatched columns represent timolol; barred columns represent O-pivalyl timolol.

These findings suggest that at least a twofold reduction in the topical dose of timolol would be possible by using the prodrug, perhaps achieving the same intraocular pressure lowering effect as a higher dose of timolol while significantly reducing the systemic drug load.

The above discussion of this invention is directed primarily to preferred embodiments and practices thereof. It will be readily apparent to those skilled in the art that further changes and modifications in the actual implementation of the concepts described herein can readily be made without departing from the spirit and scope of the invention as defined by the following claims.

Claims

Claims: 1. A lipophilic, transient ester derivative of timolol represented by the general formula:

wherein R₁ represents a substituted or unsubstituted branched chain alkyl, straight or branched chain alkenyl, aryl, aralkyl, cycloalkyl or alkyl-, phenyl- or aralkyl- substituted cycloalkyl group, a substituted or unsubstituted aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, or a substituted or unsubstituted group of the formula R₂-O-wherein R₂ is an alkyl, aryl, aralkyl or cycloalkyl group or of the formula R'₂-CONH wherein R'₂ is alkyl, aryl, aralkyl or cycloalkyl group, said ester derivative, when substituted in its R₁ moiety, containing one or more of a halogen atom, a hydroxy group, a carbonyl group, a straight or branched-chain alkoxy group having the formula R₃-O-, wherein R₃ represents an alkyl group or an aryl group, unsubstituted or substituted with one or more halogen atoms or hydroxy groups, an amino group having the formula -NR₄R₅, wherein R₄ and R₅ are the same or different and are hydrogen, an alkyl group or together with the adjacent nitrogen atom form a 5- or 6-membered heterocyclic ring, which in addition to the nitrogen may contain one or two further heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, a carbamoyl group having the formula wherein R₄ and R₅ are as defined above, or an oxyacyl group having the formula R₂ COO-, wherein R₂ is as defined above.

2. A timolol ester as recited in claim 1 wherein R₁ represents a substituted or unsubstituted branched chain alkyl group having from 3 to 8 carbon atoms, inclusive.

3. A timolol ester as recited in claim 2 wherein the branched chain alkyl group is an isopropyl group.

4. A timolol ester as recited in claim 2 wherein the branched chain alkyl group is a tert-butyl group.

5. A timolol ester as recited in claim 2 wherein the branched chain alkyl group is an isooctyl group.

6. A timolol ester as recited in claim 1 wherein R₁ is a tert-amyl group.

7. A timolol ester as recited in claim 1 wherein R₁ represents a substituted or unsubstituted aryl group.

8. A timolol ester as recited in claim 1 wherein R₁ represents a substituted aryl group.

9. A timolol ester as recited in claim 8 wherein the aryl group is a phenyl group.

10. A timolol ester as recited in claim 8 wherein the aryl group is a 4-methoxy phenyl group.

11. A timolol ester as recited in claim 1 wherein R₁ represents a substituted or unsubstituted cycloalkyl group having from 3 to 7 carbon atoms, inclusive.

12. A timolol ester as recited in claim 11 wherein the cycloalkyl group is a cyclohexyl group.

13. A timolol ester as recited in claim 11 wherein the cycloalkyl group is a 1'-methylcyclopropyl group.

14. A timolol ester as recited in claim 11 wherein the cycloalkyl group is a cyclopropyl group.

15. A timolol ester as recited in claim 1 wherein R₁ represents a substituted or unsubstituted aromatic 5- or 6- membered heterocyclic ring containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.

16. A timolol ester as recited in claim 1 wherein R₁ represents a substituted or unsubstituted group of the formula R₂-O- wherein R₂ is an alkyl, aryl, aralkyl or cycloalkyl group.

17. A nontoxic pharmaceutically acceptable salt of a timolol ester of any one of claims 1-16, inclusive.

18. O-isobutyryl timolol hydrochloride.

19. O-pivalyl timolol hydrochloride.

20. O-cyclohexanoyl timolol fumarate.

21. O-benzoyl timolol fumarate.

22. O-4-methoxybenzoyl timolol.

23. O-4-methoxybenzoyl timolol fumarate.

24. O-4-methylbenzoyl timolol.

25. O-4-methylbenzoyl timolol fumarate.

26. O-2-furoyl timolol.

27. O-2-furoyl timolol fumarate.

28. O-1'-methycyclopropanoyl timolol.

29. O-1'-methylcyclopropanoyl timolol fumarate.

30. O-cyclopropanoyl timolol.

31. O-cyclopropanoyl timolol fumarate.

32. A pharmaceutical composition for treating glaucoma and increased intraocular pressure comprising an effective amount of a lipophilic transient ester derivative of timolol as recited in claim 1, or a nontoxic pharmaceutically acceptable salt of said ester derivative, in a pharmaceutically appropriate inert ophthalmic vehicle or carrier system.

33. A pharmaceutical composition as recited in claim 32 wherein said ester derivative is O-isobutyryl timolol.

34. A pharmaceutical composition as recited in claim 32 wherein said ester derivative is O-isobutyryl timolol hydrochloride.

35. A pharmaceutical composition as recited in claim 32 wherein said ester derivative is O-cyclopropanoyl timolol.

36. A pharmaceutical composition as recited in claim 32 wherein said ester derivative is O- cyclopropanoyl timolol fumarate.

37. A pharmaceutical composition as recited in claim 32 wherein said ester derivative is O-pivalyl timolol.

38. A pharmaceutical composition as recited in claim 32 wherein said ester derivative is O-pivalyl timolol hydrochloride.

39. A pharmaceutical composition as recited in claim 32 wherein said ester derivative is 0-1'-methylcyclopropanoyl timolol.

40. A pharmaceutical composition as recited in claim 32 wherein said ester derivative is 0-1'-methylcyclopropanoyl timolol fumarate.

41. A pharmaceutical composition as recited in any one of claims 32-40, inclusive, wherein said vehicle is an eyedrop solution.

42. A pharmaceutical composition as recited in claim 41 wherein said eyedrop solution contains a hydrolysis inhibiting amount of tromethamine.