CZ20013988A3 - 1,5-Benzodiazepine derivatives enriched with enantiomer - Google Patents

Abstract

An enantiomerically enriched compound of Formula (I) is disclosed, processes for its preparation, pharmaceutical compositions containing it and the use therefore, for the treatment of CCK-A mediated diseases or conditions, such as obesity.

Description

Enantiomer-enriched 1,5-benzodiazepine derivatives

Technical field

The present invention relates to enantiomer-enriched 1,5-benzodiazepine derivatives, a process for their preparation, pharmaceutical compositions containing them and their use in medicine.

BACKGROUND OF THE INVENTION

Cholecystokinin (CCK) is a gastrointestinal and central nervous system peptide (A.J.Prange et al., Ann. Reports Med. Chem. 17, 31, 33 (1982); J. A. Williams, Biomed Res, 3, 107 (1982);

V.Mutt, Gastrointestinal Hormones, G.B.J. Green, Ed., Raven Press, New York, 169).

CCK is, inter alia, the physiological satiety hormone entering the mechanism of appetite control (Della-Ferra et al., Science, 206, 471 (1979); Saito et al., Nature, 289, 599 (1981); GPSmith, Eating and Its Disorders, AJ Stunkard and E. Stellar, Eds., Raven Press, New York, 67 (1984)) by regulating gallbladder contractions and pancreatic enzyme secretion and inhibiting gastric emptying and further acting as a neurotransmitter (AJ Prange et al., Ann Reports Med Chem., 17, 31, 33 (1982), JA Williams, Biomed Res, 3, 107 (1982), JE Morley, Life Sci.30, 479, (1982)). Gastrin is a peptide affecting gastric acid and pepsin secretion in the stomach (L. Sandvik et al., American J. Psychology, 260, G925 (1991); CWLin et al., American J. Psychology, 262, G1113, (1992)) . CCK and gastrin have the same structure of the C-terminal tetrapeptide: Trp-Met-Asp-Phe.

Two subtypes of CCK receptors, referred to as CCK-A and CCK-B, have been found in the periphery and central nervous system. Recently it has been discovered that CCK-B receptors resemble gasterin receptors (J.R. Pisegna, A. de Weerth, K. Huppi, S.A. Wank: Biochem, Biophys. Res. Commun. 189, 296-303 (1992)). CCK-A receptors are predominantly located in peripheral tissues including pancreas, gallbladder, ileum, pyloric sphincter and afferent vagal nerve fibers. CCK-A receptors are to a lesser extent present in the brain (THMoran et al., Brain Res., 362, 175-179 (1986); DR Hill et al., Brain Res, 4545, 101, (1989); DRHill et al., Neursci Lett., 89, 33, (1988); RW Barret et al., Mol. Pharmacol., 36, 285 (1989); DR, Hill et al., J. Neurosci, 10, 1070 (1990) Dauge et al., Pharmacol Biochem Behac., 33, 637 (1989)). CCK-B receptors are more widely represented in the brain (VJLotti and RSL Chang, Proc. Natl. Acad. Sci., USA, 83, 4923 (1986); JN Crawley, Trends Pharm. Sci., 88, 232 (1991)) )).

The agonist effect on CCK receptors has been associated with inhibition of food intake in animals and thus with weight loss (Della-Fera et al., Science, 206, 471 (1979); KE Asin et al., Intl. Conference on Obesity, p. 40 (1990)). It is believed that inducing satiety as a CCK effect is not due to the direct influence of nerve centers, but is mediated via the vagal nerve fibers. (G. P. Smith and B. J. Cushin, Neuroscience Abstr., 4, 180 (1978); G. P. Smith, C. Jerome, B. J. Cushin, R. Etherno and K. J. Simansky, Science, 212, 687-689 (1981)).

U.S. Patent No. 5,646,140 to Sugg et al. Relates to certain 3-amino-1,5-benzodiazepine compounds having CCK-A receptor agonist activity, thereby affecting the action of gastrin and cholecystokinin (CCK) in mammals (see Example compound) 7). Some of these compounds have an antagonistic effect on CCK-B receptors.

SUMMARY OF THE INVENTION

The present invention relates to a compound of formula I enriched in an enantiomer or a pharmaceutically acceptable salt or solvate thereof.

O

/

NS)

O)

The compound of formula I is 3- {3- [1- (isopropylphenyl-carbamylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo [b] [1, 4] -diazepin-3-yl] -ureido} -benzoic acid. This compound has a chiral carbon located in the benzodiazepine ring. The inventors have found that the enantiomer turns slightly in the positive direction and has advantageous properties under the conditions described below. This entantiomer, referred to herein as the (+) enantiomer, has a (S) configuration according to the Cahn-Ingold-Prelog method of projection. The inventors have found that the isomer has better properties than the racemic mixture and is therefore more suitable for use than the racemic mixture for the treatment of obesity and other conditions or diseases susceptible to CCK-A receptors.

• · · · · · · · ·

I · · · ·

The term "enantiomer-enriched" means that more (+) enantiomers are present than (-) enantiomers based on the amount of that isomer in the racemic mixture containing the same amount of both isomers.

The term "compound of the invention" or "enantiomer-enriched compound of the invention" includes pharmaceutically acceptable salts and solvates thereof.

The term "(+) enantiomer" refers to the optical rotation of enantiomers, not to the optical rotation of salts and solvates thereof. Preferred salts and solvates are salts and solvates of the (+) enantiomer of a compound of Formula I, regardless of the optical rotation of the individual salt or solvate.

We prefer that the (+) enantiomer make up at least 90% of the total amount of the enantiomer-enriched compound. It is particularly preferred that the (+) enantiomer constitutes at least 96% of the total amount of the compound. Most preferably, the (+) enantiomer constitutes at least 99% of the total amount of the compound.

(+) The enantiomer-enriched compound of the present invention agonists at CCK-A receptors. It can be considered as a full-length chloro-cystokinin agonist by binding to CCK-A receptors and stimulating gallbladder contractions and reducing the amount of food intake in animal models. (+) The enantiomer-enriched compound of the present invention can be used to treat obesity and obesity-related pathological symptoms (hypertension, gallbladder and diabetic stagnation) for the indirect effect on weight loss and direct effect on CCK-A receptors mediated slowing of gastric emptying. Administration of the (+) enantiomer-enriched compound of the present invention provides a new possibility to affect satiety, appetite and food intake in mammals, especially humans, to reduce appetite, treat obesity and result in weight loss.

The present invention further relates to a method of treating a mammal, including a human, of a condition or condition susceptible to the action of CCK receptors, comprising administering to a patient a therapeutically effective amount of the (+) enantiomer-enriched compound of the invention.

The present invention further relates to the use of an enantiomer-enriched compound of the present invention or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment of diseases or conditions susceptible to action by CCK-A receptors.

References to therapy include disease prevention as well as therapy for developed diseases or symptoms.

The amount of the preferred enantiomer of the present invention varies depending on the nature of the disease, the age and condition of the patient, and is entirely at the discretion of the physician or veterinarian. In general, doses administered to adults are in the range of 0.02-5000 mg / day and 1-1500 mg / day, respectively. The desired dose may be administered simultaneously or divided into two, three, four or more doses per day.

The enantiomer-enriched compound of the present invention can be administered in the raw chemical state. We prefer to administer the active ingredient as a component of a pharmaceutical composition.

The present invention relates to a pharmaceutical composition comprising an enantiomer-enriched compound of the invention with one or more pharmaceutical carriers and / or excipients and optionally other therapeutically and / or prophylactically active ingredients. Carriers and / or excipients must be "suitable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not injurious to the patient.

The pharmaceutical composition comprising the enantiomer-enriched compound of the present invention may be formulated for oral, buccal, parenteral, implant, topical or rectal administration. Oral administration is preferred. For buccal administration, the formulation may be formulated into tablets or lozenges by conventional means. Tablets and capsules for oral administration may contain conventional excipients such as binding agents (syrup, acacia syrup, gelatin, sorbitol, tragacanth, starch or polyvinylpyrrolidone), fillers (lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate or sorbitol), glidants substances (magnesium stearate, stearic acid, talc, polyethylene glycol or silica), disintegrants (potato starch or sodium starch glycolate) or wetting agents (sodium lauryl sulphate). Tablets may be coated, including an enteric coating, according to generally known methods.

The enantiomer-enriched compound of the present invention can be formulated for oral administration in the form of liquid pharmaceutical preparations (aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, etc.). The drug formulations containing the enantiomer-enriched compound of the present invention may be in the form of powders to prepare an aqueous or other solution prior to administration. Such liquid preparations contain commonly used ingredients - suspending agents (sorbitol syrup, methylcellulose, glucose / sucrose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated fats), emulsifying agents (lecithin, sorbitan monooleate, or acacia gum) vegetable oils - almond oil, fractionated coconut oil, oil esters, propylene glycol or ethyl alcohol) and preservatives (methyl or propyl p-hydroxybenzoates or sorbic acid).

The pharmaceutical composition comprising the compound of the present invention may be in the form of suppositories or contain corresponding suppository bases (cocoa butter or other glycerides), respectively.

The pharmaceutical composition comprising the enantiomer-enriched compound of the present invention may be prepared in a depot form.

These prolonged-release drugs can be administered by implantation (subcutaneously or intramuscularly) or by intramuscular injection.

The enantiomer-enriched compound of the present invention may be formulated using suitable polymeric or hydrophobic materials (emulsion in a suitable oil), an ion exchange resin, or as a partially soluble derivative (partially soluble salt).

The pharmaceutical composition contains the enantiomer-enriched compound of the present invention in an amount of 0.1-99% active ingredient, typically from 30-95% for tablets or capsules, and 3-50% for liquid preparations.

The (+) enantiomer-enriched compound of the present invention can be prepared from a racemic mixture, the preparation of which is described in Example 7 of US Patent No. 5,646,140, followed by separation of the enantiomers by chiral chromatography.

An alternative method of making the (+) enantiomer-enriched compound of the present invention is by reacting the corresponding enantiomers, namely the (S) -enantiomer of the amine of formula II:

with an isocyanate of formula III (where R is a carboxyl protecting group, i.e. t-butyl), an imidazole of formula IV (where R is a carboxyl protecting group, i.e. t-butyl), or an optionally substituted phenyl carbonate of formula V (where R is carboxyl protecting group, i.e. t-butyl and R 1 · a »δ

• «

9 · · * is a hydrogen atom or a suitable phenyl substituent (i.e., NCb), followed by removal of the protecting group R.

(IN)

The reaction is carried out in the presence of a suitable solvent (ether-tetrahydrofuran) or a halohydrocarbon (dichloromethane) or nitrile (acetonitrile) at a temperature ranging from 0 to 80 ° C.

The desired enantiomer of the amine of formula (II) may be a salt thereof, for example the salt of R-camphorsulfonic acid. The present invention relates to the preparation of the (+) enantiomer-enriched compound of the present invention by reacting the R-camphoric acid amine salt of the present invention in the presence of a tertiary amine (triethylamine) base.

9 · · ** ** ** «** ** ** ** ** ** ** ** **

Hydrolysis of the carboxyl protecting group is carried out using generally known procedures (Protec ng groups in Organic Synthesis, T. Green, Ed.Wiley Interscience, New York, p. 168, 1981). For the example of using t-butyl as a residue R, hydrolysis with the appropriate acid (hydrochloric acid, trifluoroacetic acid or formic acid) is carried out under suitable conditions - using a solvent (for example, for hydrochloric acid the solvent is 1,4-dioxane, for reaction with formic acid) the solvent is acetone or an aqueous acetone solution) and by heating.

The desired (S) -enantiomer of the amine of formula II

is prepared by resolution of the corresponding racemic amine by chiral HPLC or by asymmetric resolution induced by crystallization using an R-camphorsulfonic acid salt.

The racemic amine of formula (II) can be prepared as described for the preparation of intermediate 11 of U.S. Patent No. 5,646,140.

·······················································

The racemic amine of the formula II may be concomitant reduction and hydrogenolysis of the oxime of the formula IV wherein R ₂ is suitably a substituted benzyl group.

The reaction is carried out in the presence of a suitable catalyst (palladium on charcoal and 5% palladium on charcoal) respectively in the presence of hydrogen or an aqueous solution of ammonium formate and in a solvent or aqueous solution of alkanol (ethanol, isopropanol or technical methylated ethanol) or tetrahydrofuran. The reaction is carried out by heating to 40-80 ° C and 60 ° C, respectively.

Examples of R ₂ groups suitable for use in the reaction are benzyl or substituted benzyl (p-methoxybenzyl or benzhydryl).

• · · · ·

An oxime of formula VI is prepared by reacting an orthophenylenediamine derivative of formula VII:

with an activated diacid derivative of the formula IIIa, wherein R 2 is an optionally substituted benzyl group.

(Vlil)

A suitably activated diacid derivative of the formula VIIIa is the corresponding diacyl halide (chloride), which is prepared in situ by reacting the diacid of the formula VIIIa with oxalyl halide (oxalyl chloride). The reaction is carried out in a suitable aprotic solvent ester (ethyl acetate, toluene, dichloromethane, dimethoxyether or mixtures thereof) and in the presence of dimethylformamide.

»· · Ti · ·

The acid of formula VIIIa is prepared by reacting a dialkyl ketone malonate (diethyl ketomalonate) with the corresponding hydroxylamine R2ONH2 in a solvent (alkanol-ethanol or technical methylated ethanol) and in the presence of a base (pyridine) followed by hydrolysis of the corresponding di-alkyloximinomalonate using aqueous sodium hydroxide solution.

The present invention relates to a process for preparing the (S) enantiomer of a compound of formula I substantially free of the (R) enantiomer from a racemic mixture of amine II described above, wherein the racemic amine of formula II is prepared from an oxime of formula VI produced from compounds VII and VIII.

Isocyanates of formula VII are commercially available or can be prepared by reacting the corresponding amine of formula VI with phosgene or triphosgene in a suitable solvent (methylene chloride). The imidazoles of formula IV are prepared by treating the corresponding amine of formula VI with carbonyldiimidazole in a suitable solvent (dichloromethane, ether, tetrahydrofuran) at a temperature in the range of 0 to 80 ° C (preferably at room temperature). The optionally substituted phenylcarbamates of formula V can be prepared by reacting the corresponding amine of formula VI with an optionally substituted phenyl chloroformate in the presence of a base (pyridine, triethylamine) in a suitable solvent (dichloromethane) and at a temperature in the range of 0 to 50 ° C. The amines of formula (VI) are generally known compounds or can be prepared in a manner known and used for known compounds.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are illustrative and not limiting of the scope of the present invention.

Used shortcuts:

EtOAc = ethyl acetate,

MeOH = methanol,

DMF = Ν, Ν-dimethylformamide

IPA = isopropyl alcohol,

IMS = industrial methylated spirits.

Intermediate 1 (+) - 2- (3-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-benzo [b] - [1,4] diazepin-1-yl) - Camphorsulfonic acid N-isopropyl-N-phenylacetamide

Mixture of 10 g (+/-) - 2- (3-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo [b] - [1,4] diazepine-1- yl) -N-isopropyl-N-phenylacetamide and 4.98 g of R-camphorsulfonic acid are stirred in a mixture of 35 ml of tetrahydrofuran and 65 ml of toluene to form a reaction solution which is heated to 70 ° C to form a suspension. 0.4 ml of water is added, followed by a solution of 0.24 g of 2-pyridinecarboxaldehyde in 5 ml of toluene. The mixture was heated at 70 ° C for 3 hours and then cooled to 25 ° C over 5 hours and stirred at 25 ° C for 16 hours. The suspension was cooled to 0-5 ° C for 1.5 hours. The solid product was collected by filtration, washing with 10 mL of a 2: 1 mixture of toluene and tetrahydrofuran. Drying in vacuo at 50 ° C afforded 12.6 g of the title compound as a white solid.

Chromatography: eluent: 30% isopropyl alcohol, 70% heptane + 0.05% diethylamine, column: 25 cm x 4.6 mm ID, Chiralpak AD, flow rate: 1 mL / minute, temperature: 40 ° C, detection at: UV 230nm, applied volume: 10 μΙ, test substance solution: 0.1 mg / ml, test substance in 30% isopropanol, 70% heptane. The test compound solution was applied immediately after preparation.

• ·

Retention time: (+) enantiomer = 8.2 minutes. The undesired (-) enantiomer (12.7 minutes) was not detectable.

Intermediate 2 3-Nitrobenzoic acid t-butyl ester

To a solution of 3.00 g of 3-nitrobenzyl chloride in 70 ml of anhydrous tetrahydrofuran was added 3.82 g of potassium t-butoxide, and the reaction mixture was stirred under a nitrogen atmosphere for 2 hours. The reaction mixture was concentrated in vacuo and partitioned between dichloromethane and water. After separation of the portions, the aqueous portion was back-extracted with ethyl acetate. The organics were combined, dried over anhydrous magnesium sulfate, filtered, and then concentrated in vacuo. The crude product was purified by flash chromatography on silica gel eluting with a gradient of 0 to 5% ethyl acetate in n-hexane. The product containing fractions were combined, concentrated in vacuo, and then dried under high vacuum to give 3.82 g of the title compound as an oil.

¹ H NMR (300 MHz, CDCl 3): d = 1.63 (s, 9H), 7.62 (t, J = 7.9 Hz, 1H), 8.29-8.41 (m, 2H), 8.78-8.80 (m, IH).

Mass spectrum (CI): [M + H] + = 224.

Intermediate 3 3-Amino-benzoic acid t-butyl ester

A solution of 3.77 g of 3-nitrobenzoic acid t-butyl ester in 50 ml of absolute ethanol is combined with 0.30 g of palladium on charcoal (10% by weight) and the reaction solution is stirred under a hydrogen atmosphere for about 3 hours. The reaction mixture was filtered through a pad of diatomaceous earth, and then concentrated in vacuo to give an oil which crystallized on drying under high vacuum to give 3.28 g of the title compound as a tan solid.

• • · · · · ¹ H NMR (300 MHz, CDCl _3): δ = 1.58 (s, 9H), 6.79 to 6.87 (m, 1H), 7.19 (t, J = 8 1.5 Hz, 1H), 7.24-7.34 (m, 1H), 7.38 (d, J = 8.0 Hz, 1H).

Mass spectrum (CI): [M + H] + = 194.

Intermediate 4 3-Isocyanobenzoic acid t-butyl ester

To a solution of 26.50 g of 3-amino-benzoic acid t-butyl ester and 38.23 ml of triethylamine in 600 ml of anhydrous tetrahydrofuran at 0-5 ° C is added 13.428 g of triphosgene. The reaction mixture was stirred at 0-5 ° C for 2 hours and then concentrated in vacuo to give a white solid. The crude product was suspended in 500 mL of hexane, filtered and the filtrate was concentrated in vacuo to give 21.54 g of the title compound as an oil in 71.6% yield. The crude isocyanate was used without further purification. ¹ H NMR (300 MHz, CDCl ₃ ): δ = 1.59 (s, 9H), 7.23 (bd, J = 7.8 Hz, 1H), 7.36 (t, J = 7.8 Hz) 1 H, 7.69 (bs, 1 H), 7.81 (d, J = 7.8 Hz, 1 H).

Intermediate 5 (+) - 3- [3- [1- (Isopropylphenylcarbamylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1 H -benzo [b] - tert -butyl ester [1,4'-Diazepin-3-yl] -ureido} -benzoic acid

Procedure A:

To a solution of 21.54 g of 3-isocyanobenzoic acid t-butyl ester in 750 ml of anhydrous tetrahydrofuran was slowly added 66.30 g of intermediate 1. To the reaction mixture was added dropwise 13.70 ml of triethylamine. The resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into 3000 mL of water to give a white solid. The solid product was collected by filtration, washing three times with 500 ml of water. After drying under vacuum filtration, the title compound is obtained as a white solid (65.01 g).

9 ·· 9 9 · «·· · · · solids. The crude resulting compound was used without further purification. Chromatography characteristics: eluent: 30% isopropyl alcohol, 70% heptane + 0,05% diethylamine, column: 25cm x internal diameter

4.6 mm, Chiralpak AD, flow rate: 1 ml / minute, temperature: 40 ° C, detection at: UV 230 nm, applied volume: 10 μΙ, solution of test substance:

0.1 mg / ml test substance in 30% isopropyl alcohol and 70% heptane. Apply the test substance solution immediately after preparation.

Retention time: (+) - enantiomer: 15.6 minutes.

The undesired (-) enantiomer (13.3 minutes) was not detectable.

Procedure B:

To a suspension of 13.2 g of carbonyl-diimidazole in 55 ml of dichloromethane stirred at 20 ° C was added dropwise a solution of 15.8 g of 3-aminobenzoate in tert-butyl ester in 40 ml of dichloromethane over 30 minutes. The resulting solution was stirred at 20 ° C for 1 hour. To this solution was added a solution of 50 g of intermediate 1 in 130 ml of dichloromethane over 5 minutes. The reaction is quenched by the addition of 200 ml of water and stirred for 10 minutes. The aliquots were separated and the organic portion was washed with water. The organic portion was concentrated at atmospheric pressure and 100 mL of dichloromethane was removed by distillation. After addition of 700 ml of t-butyl methyl ether, the mixture was stirred overnight at 20 ° C. The solid product was collected by filtration, washed with t-butyl methyl ether (100 mL) and dried in vacuo at 45 ° C to give the title compound as a white solid (42 g, 63 mmol).

Intermediate 6 diethyl 2 - [(benzyloxy) imino] malonate

To a stirred suspension of 57.8 g of O-benzylhydroxylamine in 500 ml of IMS containing 30 ml of pyridine was added 60 g of diethyl ketomalonate at 20 ° C. The reaction mixture was heated at 75 ° C for 4 hours. The reaction mixture was cooled and the solvents were removed under reduced pressure.

·

The residue was partitioned between 500 mL of ethyl acetate and 300 mL of water, and the organic portion was separated, washed with 250 mL of water, and dried over magnesium sulfate. The solvent was removed by evaporation to give 95.3 g of the title compound as a 99% colorless oil (about 3% residual ethyl acetate) which was used without further purification.

¹ H NMR (300 MHz, CDCl ₃ ): 7.4 (m, 5H), 5.35 (s, 2H), 4.35 (m, 4H), 1.3 (m, 6H).

Intermediate 7

2 - [(benzyloxy) -imino] -malonic acid

To a solution of 40 g of intermediate 6 in 80 ml of methanol was added 200 ml of 2M sodium hydroxide solution over 20 minutes. The reaction mixture was stirred at room temperature for 2 hours. The methanol was removed under reduced pressure and the resulting solution was acidified to pH 2 with cooling by dropwise addition of concentrated hydrochloric acid (approximately 30 mL) such that the temperature did not exceed 35 ° C. The thick white suspension formed was diluted with 50 ml of water, the solid collected by filtration, washed with 25 ml of cold water and dried under vacuum at 55 ° C to give 17 g of the title compound as a white solid, containing approximately 10% residual weight inorganic salts. The corrected yield is approximately 45%. The product was used without further purification.

¹ H NMR (300 MHz, D ₂ O): 7.4 (m, 5H), 5.2 (s, 2H).

Intermediate 8

2- [3 - [(benzyloxy) imino] -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-1,5-benzodiazepinyl) -N-isopropyl-N-phenylacetamide

To a stirred suspension of 40 g (corrected for salt content to 31.4 g) of intermediate 7 in 200 ml of ethyl acetate containing 0.5 (5 mol%) DMF was added dropwise 38.3 g of oxalyl chloride over 1 hour. The mixture was stirred at 25 ° C for 0.5 h, filtered through a Dicalite pad, washed with 40 mL of ethyl acetate to give a clear yellow solution. The solution was added to a stirred suspension of 50 g of N-isopropyl-N-phenyl-2- (2-phenylamino) -acetamide in 120 ml of ethyl acetate at 25 ° C over 5 minutes. The reaction mixture was heated to 60 ° C to form a dark red solution. After 1 hour, 200 mL of ethyl acetate was removed by atmospheric distillation. 120 ml of isopropanol and 40 ml of water are added and the mixture is further distilled to remove 80 ml of solvent. After addition of 40 ml of isopropyl alcohol and 40 ml of water, an additional 80 ml of solvent is removed by distillation. The reaction mixture was cooled to 25 ° C over 1.5 hours and the solid was collected by filtration. The solid product is washed twice with 120 ml of isopropanol, once with 120 ml of water and finally once again with 40 ml of isopropyl alcohol, and then dried under vacuum at 55 ° C to give 56.6 g of the title compound as a salmon pink powder.

¹ H NMR (300 MHz, CDCl 3): 2: 1 isomer mixture at oxime 7.6-6.95 (m, 18H), 6.9 (t, 1H), 5.3 (m, 2H), 4, 95 (m, H), 4.65 (d, 0.33H), 4.4 (d, 0.67H), 4.1 (d, 0.67H), 4.0 (d, 0.33H), 0, 95 (m, 6 H).

Intermediate 9 (±) -2- (3-Amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-benzo [b] - [1,4] diazepin-1-yl) -N-isopropyl -N-phenylacetamide

To a stirred suspension of 3 g of intermediate 8 and 2.08 g of ammonium formate in 30 ml of technical methylated ethanol (IMS) and 3 ml of water was added 0.25 g of 5% palladium on charcoal (50% by weight water) as a catalyst. The mixture was heated under nitrogen at 60 ° C overnight. The hot reaction mixture was filtered through Dicalite to remove the catalyst. The catalyst was washed with 60 ml of hot IMS and filtered. The filtrate was concentrated under reduced pressure to give 2.34 g of the title compound as a white solid.

• ·

Example 1

Chromatographic resolution of the enantiomers of (+) and (-) - 3- {3- [1- (isopropyl-phenyl-carbamylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo - [b] - [1,4] diazepin-3-yl] -ureido} -benzoic acid

Racemic acid 3- {3- [1- (isopropylphenyl-carbamylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo [b] - [1,4] - diazepin-3-yl-ureido-benzoic acid was prepared according to the procedure of Example 7 of US Patent No. 5,646,140 and the enantiomers were resolved by chiral HPLC under the following conditions: 5 µm Diacel Chiracel OD-R 250 x 4.0 µm internal diameter column, eluent mixture of acetonitrile, water, triethylamine and acetic acid in a ratio of 80: 20: 0.1: 1, UV detection at 230 nm, room temperature, flow rate of 1 ml / min and an applied volume of 20 µl.

Under these conditions, the retention time of the (+) enantiomers is 6.50 minutes and the (-) isomers 3.89 minutes.

Example 2 (+) - 3- {3- [1- (Isopropyl-phenyl-carbamylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1 H -benzo [b] - [1,4] -diazepin-3-yl] -ureido} -benzoic acid

Procedure

65.01 g of (+) - 3- {3- [1- (isopropylphenyl-carbamyl-methyl) -2,4-dioxo-5-phenyl- </RTI> t-butyl ester is stirred for 6 hours in 280 ml of 4N hydrochloric acid in dioxane. 2,3,4,5-tetrahydro-1H-benzo [b] [1,4] diazepin-3-yl] -ureido} -benzoic acid (interm. 5) at room temperature. The reaction mixture was concentrated in vacuo and the solid / oil formed was triturated with water to give a white solid. The white solid formed was collected by filtration and washed twice with 500 mL of water. The crude product was dissolved in 250 ml of hot acetone and 275 ml of water was added to the solution until it became cloudy. After an additional 40 ml of acetone was added, the solution was heated to a clear solution. After cooling, the white solid formed was collected by filtration, washed three times with 100 ml of water and dried under vacuum from a vacuum manifold at 40-50 ° C to give 40.496 g of the title compound as a white solid. Purity analysis was performed by chiral chromatography (see chromatography conditions).

Optical rotation (0.712 g in 100 mL acetone) [α] D = +84.3.

1 H NMR (300 MHz, DMSO): δ = 0.95 (d, J = 7.5 Hz, 3H), 0.97 (d, J = 7.2 Hz, 3H), 4.18 (d, J = 16.7 Hz, 1H), 4.48 (d, J = 16.7 Hz, 1H), 4.78 (m, 1H), 5.02 (d, J = 7.8 Hz, 1H), 6.91 (d, J = 7.8Hz, 1H), 6.95 (bd, J = 8.1Hz, 1H), 7.22-7.57 (m, 19H), 8.00 (s, 1H), 9.34 (s, 1H), 12.78 (s, 1H). Mass spectrum (ES): [M + 1] = 606.1, [M + Na] = 628.1, [M-1] = 604.1.

Procedure B

To a mixture of 15 ml of acetone and 25 ml of formic acid, 5 g of intermediate 5 was added at room temperature, and the resulting suspension was heated to 55 ° C and stirred for 5 hours. 30 ml of water are added dropwise to the solution at a temperature above 50 ° C. The resulting suspension was stirred at 55 ° C for 1 hour, cooled to room temperature and stirred overnight. The suspension is filtered and washed three times with 25 ml of water. The resulting residue was added to 50 ml of technical methylated ethanol, the suspension was heated to 45 ° C and stirred overnight. The suspension was cooled to room temperature, filtered, and the wet cake was dried under vacuum at 55 ° C to give 3.40 g of the title compound as a white solid.

Analysis by chiral chromatography showed that the product contained 0.7% of the undesired (-) enantiomer.

Pharmacological examples:

Oral solutions

Active substance

Polyethylene glycol 400 NF

0.5-800 mg q.s. to 50 ml

The active ingredient is suspended in polyethylene glycol 400, and then dissolved by sonication to form a solution for oral administration.

Oral suspension

Active Ingredient 0.5 - 80 mg

Polysorbate 80 NF (Tween 80) 0.02 ml

Sterile water q.s. to 20 ml

The active ingredient is added to 20 ml of a 0.1% Tween 80 solution by volume and the mixture is then sonicated or shaken to form an oral suspension.

Tablets

Procedure A:

Active ingredient 6 mg Anhydrous lactose USP 136.2 mg Sodium starch glycolate USP / NF 6 mg Stearic acid USP / NF 1.5 mg Colloidal silica USP / NF 0.3 mg Weight of compressed tablet 150 mg

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The active ingredient, lactose and sodium starch glycolate are sieved through a 590 micron sieve and mixed in a suitable mixer. Stearic acid (sieved through a 250 micron sieve) and colloidal silica were added. The blend is intimately mixed with the blend containing the active ingredient and compressed into tablets using suitable tablet punches.

Procedure B:

Active ingredient 6 mg Microcrystalline cellulose USP / NF 136.5 mg Cross-linked povidone USP / NF 6 mg Magnesium stearate USP / NF 1.5 mg Weight of compressed tablet 150 mg

The active ingredient, microcrystalline cellulose and cross-linked povidone are sieved through a 590 micron sieve and mixed in a suitable mixer. After passing the magnesium stearate through a 250 micron sieve, it is mixed with the active ingredient mixture. The resulting mixture is compressed into tablets using a suitable tablet punch.

Capsules

Procedure A:

Active ingredient 6 mg

Microcrystalline cellulose USP / NF 128.25 mg

Sodium starch glycolate USP / NF 15 mg Magnesium stearate USP 0.75 mg

Cartridge weight 150 mg

The active ingredient, microcrystalline cellulose and sodium starch glycolate are sieved through a sieve with a mesh size of 590 microns, mixed and magnesium stearate is added as a glidant which is sieved through a sieve with a mesh size of 250 microns. The mixture is filled into capsules of appropriate size.

Procedure Β:

Active ingredient 6 mg Lactose monohydrate USP 130.5 mg Povidone USP 6 mg Cross-linked povidone NF 6 mg Magnesium stearate 1.5 mg Filling weight 150 mg

The active ingredient and lactose are mixed and granulated with a povidone solution. The wet granulate is dried and ground. The magnesium stearate and cross-linked povidone are passed through a 250 micron sieve and mixed with the granulate. The resulting mixture is filled into hard gelatin capsules of appropriate size.

Biological tests:

The (+) and (-) - enantiomers and the racemic compound of the present invention are characterized by the following assays. The test results are summarized in the following table.

Preparation of guinea pig gall bladder tissue preparation:

Gallbladders are collected from male Hartley guinea pigs after carbon dioxide sacrifice. The prepared gall bladders are cleaned of adhering connective tissue and the specimens from each animal are cut into two rings of 2-4 mm thickness. The sections are placed in a culture chamber containing saline consisting of 118.4 mmol sodium chloride, 4.7 mmol potassium chloride, 1.2 mmol magnesium sulfate, 2.5 mmol calcium chloride, 1.2 mmol potassium hydrogen phosphate, 25 mmol bicarbonate sodium, 11.1 mmol of dextrose. The bath solution is maintained at 37 ° C and bubbled with a mixture of 95% oxygen and 5% carbon dioxide at pH = 7.4. Tissues are attached with gold chains and stainless steel clamping wires to an isometric force transducer (Grass, Model FT03 D). Responses are written to a printing device (Grass, Model 7E). One tissue section from each animal serves as a time and solvent control without application of the test substance. The sections were gradually stretched (over 120 minutes) to a basal resting tension of 1 g, which was maintained throughout the experiment. During the time period during which the basal resting tension was changed, the sections were exposed four times to acetylcholine (10 ^-6 mol) to verify contractility. Tissues were exposed to a submaximal dose of sulfated CCK-8 (Sigma, 3 X 10 ^-9 mol). After obtaining a stable response, sections were washed quickly three times, and then washed every five to ten minutes to induce a stable basal value.

Agonist EC50 values:

Compounds are dissolved in dimethylsulfoxide (DMSO), diluted with water and checked cumulative response curve the concentration of the test compound (10 ¹¹ to 3 x 10 ^-5 mol), followed by cumulative response curve the concentration of sulfated CCK-8 (10 ^-10 up to 10 ⁶ mol). The final assay is the addition of 1 mmol acetylcholine to induce maximal contraction. Three activity measurements are performed for each test compound.

Introduction of stable CCK receptor-bearing cell lines:

0 1Q

CDNA clones for human CCK-A or CCK-B receptors were inserted into the pcDNA1-Neo vector (Invitrogen Corp, San Diego, CA) for direct transfection. DNA was prepared by alkaline lysis and transfected into CHO-K1 cells (ATCC, Rockville, MD) using Lipofectin reagent24 (Gibco BRL, Gaithersberg, MD). Stable transfectants were selected using Geneticin (Gibco BRL). Suspensions of resistant receptor-bearing cells are enriched with sorted cells using fluorescence binding by Fluorescein-Gly- [(Nle28,31) -CCK-8. Clone cell lines are then introduced by the limiting dilution method.

Preparation of cell membranes:

CHO-K1 cells stably transfected with human CCK-A or CCK-B receptor cDNA are grown at 37 ° C in a humidified atmosphere of 95% oxygen and 5% carbon dioxide on Ham's F12 nutrient medium supplemented with 5% heat inactivated fetal bovine serum. Cells are passaged twice a week and cultured to a density of 2-4 million cells / ml. Cells were harvested by centrifugation at 600g at 4 ° C for 15 minutes and suspended in 20 ml of buffer at pH 7.4 containing 25 mmol TrisHCl, 5 mmol EDTA, 5 mmol EGTA, 0.1 mmol phenylsulfonyl fluoride and 100 µg. / ml trypsin inhibitor from soybeans. Cells are broken using a glass homogenizer with a Teflon plunger and driven by an electric motor (25 plunger plunges) and the homogenate is centrifuged for 10 minutes at a slow rotation speed (600 X g) at 4 ° C. The supernatant was collected and centrifuged at 4,000C for 15 minutes at high speed (500,000 x g) to form particle sediment. The sediment obtained by low speed centrifugation is again homogenized three times and centrifuged. The high-speed centrifuged aliquots were pooled and resuspended in buffer (1-5 mg protein per ml buffer) and frozen at -80 ° C. Protein concentrations are determined by the manufacturer (BioRad reagent and bovine serum albumin as standard).

Receptor Binding Assay:

¹²⁵ l Bolton Hunter labeled receptors, CCK-8 (Amersham, 74x10 ^'8

Bq / mmol) is dissolved in binding buffer (pH 7.4) at 100,000 counts per minute / 25 µl (containing 20 mmol HEPES, 118 mmol *).

sodium chloride, 5 mmol of potassium chloride, 5 mmol of magnesium chloride and 1 mmol of EGTA. Non-specific binding was determined using 10 pmol MK-329 ²⁰ (CCK-A) or 10 pmol L365 260 ²¹ (CCK-B). Test compound is dissolved in DMSO at a stock concentration that is 100-fold higher than the final assay concentration and diluted to the desired concentration with binding buffer. The binding assay is performed in triplicate using 96-well plates, in which: 25 µL of test compound, 25 µΙ ¹²⁵ L-Bolton Hunter CCK-8, 150 µΙ buffer (pH 7.4) and 50 µΙ receptor suspension are added sequentially. . The final DMSO concentration is 1% in all wells. After 3 hours at 30 ° C, incubation is terminated by rapid filtration of the mixture on glass filters (Whatman GF / B) followed by washing to remove unbound ligands. Binding radioactivity is quantified by a gamma counter.

Measurement of intracellular calcium:

CHO-K1 cells stably transfected with CCK-A or hCCK-B receptors are cultured on glass coverslips to 75-90% coverage. Cells were placed in serum-free culture medium containing 50 mmol FURA2-AM and 2.5 mmol probenecid for 50 minutes. Changes in intracellular calcium concentration are measured using a JASCO CAF-102 calcium analyzer using standard ratio techniques at 340 nm and 380 nm excitation wavelengths. Cells are washed with increasing concentrations of CCK8 (in duplicate) or compound (in duplicate) until a constant 340/380 ratio is formed. Allow at least 10 minutes between each stimulation after each wash. The maximal response was related to the maximum response induced by CCK-8. EC 50 values were calculated. To determine concentration-response curves, CHO-K1 cells that expressed CCK-B receptors were washed for 1 hour with three concentrations (10 ⁸ , 10 ⁷ , 10 ⁶ M, in duplicate) of the compounds. Consequently, concentration-response curves for CCK-8 are determined over a range of ^10-12 to 10 ^-6 M.

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Anorexia test:

Long-Evans rats weighing 225-300 g are given a tasty liquid diet (Bio-Serve F1657, Frenchtown, NJ) for two weeks without food. The day before the test, the rats were not fasted for 100 minutes, followed by intraperitoneal administration of drug vehicle (1 ml / kg propylene glycol, PG) and orally 0.9% sodium chloride solution at 8 ml / kg. After 20 minutes a liquid diet is given and its consumption is read after 30, 90 and 180 minutes.

To justify the study of the effect of the test compound, rats must consume a minimum of 8 ml of liquid food after the first 30 minutes the day before the test. The following day, after 100 minutes without food, rats are given vehicle (8-10 animals per dose) intraperitoneally or orally (1 ml / kg propylene glycol) or various doses of test compound (0.01 to 10 pmol / kg) dissolved in 1 ml / kg of propylene glycol followed by immediate administration of saline orally. The animals are re-administered 20 minutes later and food intake is read after 30, 90 and 180 minutes. The food intake values from each animal are related to the respective values from the previous day. The EC50 at 30 min and the IC50 at 30 min at 1 pmol / kg are calculated.

Gall bladder emptying test:

Literature: F. Makovec; M. Bani; R. Cereda; R. Chiste, M. A. Pacini, L. Rev., L. C. Rovati: Antispasmodic Activity on the Gallbladder of the Mouse of CR1409 (Lorgiumide), and Potent Antagonist of Peripheral Chlolecystokinin, Pharmacol. Res. Commun. 1987,19,41-51.

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PApp values:

Intestinal permeability measurement using an in vitro assay according to P. Artursson and J. Karison, 1991, Biochem. Biophys Res. Common. 175,880-885 (Correlation between oral drug absorption in humans and apparent drug permeability co-efficients in human intestinal epithelical (CACO-2) cells).

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4 * 44 4 4 4 4 4 4 4 4 44 444 44 44

Pharmacological comparison of enantiomer and racemate

Exam (+) enantiomer (-) enantiomer racemate In vitro GPGB EC50 (nmol) 9.3 63 40 HCCK-A _IC50 (nM) 148 191 123 HCCK-A EC50 (nmol) 150 298 252 HCCK-B Ki (nmol) 3.2 22.3 10 HCCK-B EC50 (nmol) antagonist antagonist antagonist Selectivity ratio ¹ 46 8.6 12.3 PApp (10 x ⁷ cm / sec) 1.6 0.7 In vivo anorexia rats ED ₅₀ ip (pmol / kg) 0,034 0.48 0.06 ED ₅₀ po (pmol / kg) 1.1 inactive ³ 2,0 gallbladder mice emptying test ED ₅₀ ip (pmol / kg) 0,002 0.012 0.007 ED ₅₀ po (pmol / kg) 0.007 inactive ² 0,055

* ¹ selectivity ratio = IC 50 (hCCK-A) / Ki (hCCK-B) inactive up to 10 pmol / kg without significant feed reduction at doses up to 10 pmol / kg.

Three unexpected properties distinguish the (+) enantiomer from the (-) enantiomer and the racemate. Two of these effects are related to increased CCK-A activity (IC 50), which improves the desired effect of this enantiomer. A third property relates to the antagonist effect on CCK-B (+) enantiomer receptors, which is beneficial for reducing toxicity.

(+) The enantiomer was four times more potent than the racemate in the guinea pig gallbladder test (GPGB) in vitro. (+) The enantiomer was eight times more potent than the racemate in the mouse gall bladder emptying assay (oral dosing). Increasing this effect is beneficial in the treatment of gallbladder stasis and obesity therapy, since gallbladder stasis and rapid weight loss are serious problems.

Anorectic agents should be administered over a long period of time and therefore minimal toxicity is important. The primary toxicity associated with the use of cholecystokinin is a concomitant factor of the CCK-B receptor agonist effect. Activation of CCK-B receptors is primarily associated with increased anxiety and increased secretion of acid gastric juice. The ability of antagonists to CCK-B receptors was investigated for anxiolytic and antiulcerative effects. References: J. Lowe, Cholecystokinin-B Receptor Antagonists, Exp. Opin. Ther. Patents, 5 (3): 231-237 (1995).

The pre-amination of the rodent pancreatic CCK receptor subtype is CCK-A, and the irritation of this receptor subtype induces pancreatic hyperstimulation and hypertrophy in rodents. Both effects are considered undesirable. Recently, the tissue distribution of CCK receptors in humans has been described. The predominant subtype of CCK receptors in humans is the CCK-B subtype. Literature: S.A. Wank, Cholecystokinin Receptors, American Journal of Physiology-Gastrointestinal & Liver Physiology, 32 · »··

I * * «» »· ·

(5), pp. G628-G646, (1995). It follows that receptor irritation

CCK-B (agonist effect on CCK-B receptors) would result in anxiety, acidic gastric secretion and, in long-term therapy, pancreatic hyperstimulation and hypertrophy.

In order to reduce the adverse effects of agonist action on CCK-B receptors in vivo, the test substance should demonstrate affinity for CCK-B receptors and measurable CCK-B receptor antagonist activity in vitro assays. Both the enantiomers and the racemate have antagonistic activity at CCK-B receptors. Although all three formulations have similar IC 50 and EC 50 for human CCK-A receptors, the (+) enantiomer has the highest IC 50 and selectivity (46-fold) for CCK-B receptors. For this reason, we prefer the (+) enantiomer for efficacy given by IC 50 and EC 50 values to CCK-A receptors, and furthermore to very little possibility of CCK-B receptor irritation-induced side effects.

The (+) enantiomer-enriched compound of the present invention is nontoxic at therapeutically effective doses. Single dose oral toxicity studies indicate that the maximum non-lethal dose in rats is greater than 2000 mg / kg, 1000 mg / kg in male mice and 500 mg / kg in female mice.

Claims (12)

PATENT CLAIMS 1. Enantiomer-enriched 3- {3- [1- (isopropyl-phenyl-carbamylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo [b] - [1] 4] -diazepin-3-yl] -ureido} -benzoic acid or a pharmaceutically acceptable salt or solvate thereof. The enantiomer-enriched compound of claim 1, wherein the (+) enantiomer of the compound or a pharmaceutically acceptable salt or solvate thereof is at least 90% present. The enantiomer-enriched compound of claim 1 or claim 2, wherein the (+) enantiomer of the compound or a pharmaceutically acceptable salt or solvate thereof is at least 99% present. A pharmaceutical composition comprising an enantiomer-enriched compound according to claims 1 to 3 in addition mixture with one or more pharmaceutically acceptable carriers or excipients. A method of treating diseases treatable by CCK-A receptors, comprising administering an effective amount of a compound according to any one of claims 1 to 3. 6. A method of treating diseases treatable by CCK-A receptors comprising administering the pharmaceutical composition of claim 4. The method of claim 5 or claim 6, wherein said disease or condition is obesity, gall bladder stasis or diabetes The method of claim 5 or claim 6, wherein said disease or condition is obesity. ···· · · · tt ·· · · · · · · · · · · · · · · · · · Use of a compound according to any one of claims 1 to 3 in the manufacture of a pharmaceutical composition for the treatment of conditions and diseases susceptible to CCK-A receptors. A process for the preparation of a compound according to claim 1, comprising the steps of: a) Resolution of racemic 3- [3- [1- (isopropyl-phenyl-carbamylmethyl) -4,5-tetrahydro-1H-benzo [b] - [1,4] diazepin-3-yl] -benzoic acid chiral HPLC, b) reacting the corresponding enantiomer of an amine of formula II (II) with an isocyanate of formula III, an imidazole of formula IV or an optionally substituted phenylcarbamate of formula V, followed by removal of the carboxyl protecting group R. 34 .... · · ··· · ············· A process according to claim 11, characterized in that the compound according to claim 1 is prepared from a racemic amine of formula (II) which is produced by simultaneous reduction and hydrogenolysis of oxime (VI) and wherein R 2 is an optionally substituted benzyl group. VI A process according to claim 11, wherein the oxime of formula VI is prepared from an ortho-phenylenediamine of formula VII and an activated diacid derivative of formula VIII, wherein R 2 is an optionally substituted benzyl group.