KR20020012567A - 1,5-Benzodiazepine Derivatives - Google Patents

Abstract

Enantiomeric rich compounds of formula (I), methods for their preparation, pharmaceutical compositions containing such compounds, and uses for the treatment of CCK-A mediated diseases or disorders such as obesity:

<Formula I>

Description

1,5-benzodiazepine derivatives {1,5-Benzodiazepine Derivatives}

Cholecystokinin (CCK) is a peptide found in the gastrointestinal tract and central nervous system (AJ Prange et al., Ann. Reports Med. Chem. 17, 31, 33 (1982), Williams, JA Williams, Biomed Res. 3, 107 (1982) and V. Mutt (Gastrointestinal Hormones, GBJ Green, Ed., Raven Press, NY, 169). CCK is a physiological satiety hormone specifically related to appetite control (Della-Ferra et al., Science, 206, 471 (1979), Saito et al., Nature, 289, 599, (1981) ], See Smith and GP Smith, Eating and Its Disorders, AJ Stunkard and E. Stellar, Eds, Raven Press, New York, 67 (1984)), modulators of gallbladder contraction and pancreatic enzyme secretion, gastric emptying Inhibitors and neurotransmitters (AJ Prange, supra, JA Williams, Biomed Res., 3, 107 (1982), JE Morley, Life Sci. 30 , 479, (1982). Gastrins are peptides involved in gastric acid and pepsin secretion in the stomach (L. Sandvik et al., American J. Physiology, 260, G925 (1991), CW Lin et al., American J. Physiology. , 262, G1113, (1992). CCK and gastrin have structural homology in C-terminal tetrapeptide: Trp-Met-Asp-Phe.

Two subtypes of CCK receptors called CCK-A and CCK-B have been identified, both found in the peripheral and central nervous systems. Recently, it has been reported that the CCK-B receptor is similar to the gastrin receptor (Pisegna, JR, de Weerth, A, Huppy, K, and Wang, SA). See Biochem. Biophys. Res. Commun. 189, 296-303 (1992). CCK receptors are primarily located in peripheral tissues including the pancreas, gallbladder, ileum, pyloric sphincter and vagus nerve afferent nerve fibers; Fewer CCK-A receptors were found in the brain (TH Moran et al., Brain Res., 362, 175-179 (1986), DR Hill et al., Brain Res, 4545). , 101, (1988)], DR Hill et al., Neurosci Lett., 89, 133, (1988), RW Barret et al., Mol. Pharmacol., 36,285, (1989) , DR Hill et al., J. Neurosci, 10, 1070 (1990), and V. Dauge et al. (Pharmacol Biochem Behac., 33, 637, (1989)). In contrast, CCK-B receptors are found primarily in the brain (VJ Lotti and RS L Chang, Proc. Natl. Acad. Sci. USA, 83, 4923 (1986)), CJ Crawley See Trends Pharm. Sci., 88, 232, (1991).

CCK agonist activity is associated with suppressing animal food intake and consequently weight loss (Della-Fera et al., KE Asin et al., Intl. Conference on Obesity, abstract pp. 40 (1990)). CCK acts through the vagus nerve fibers in the periphery and has not been reported to act directly on the brain to produce a satiety state (Smith, GP) and Cushin (BJ), Neuroscience Abstr., 4, 180 (1978), Smith, GP, Jerome, C., Cushin, BJ, Eterno, R. and Shimansky, KJ (Science). , 212, 687-689, (1981).

U.S. Pat.No. 5,646,140 (Sugg et al.) Discloses a specific 3-amino 1,5-benzodiazepine compound that can modulate the hormones gastrin and cholecystokinin (CCK) in mammals by exhibiting agonist activity on CCK-A receptors (especially , See compound of Example 7). Some of these also exhibit antagonist activity at the CCK-B receptor.

The present invention relates to novel 1,5-benzodiazepines derivatives, methods for their preparation, pharmaceutical compositions containing them and medical uses. More specifically, it relates to compounds which exhibit agonist activity on the CCK-A receptor.

In short, in one aspect, the present invention provides an enantiomeric rich compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof:

Compound of formula I is 3- {3- [1- (isopropyl-phenyl-carbamoylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo [b] [1,4] diazepin-3-yl] -ureido} benzoic acid. This compound has chiral carbons in the benzodiazepine ring. Applicants have found that enantiomers under the following conditions, which rotate light in the positive direction, are preferred. This enantiomer is hereinafter referred to as (+) enantiomer and has (S) coordination according to the Cahn Ingold Prelog convention. Applicants have found that this isomer has improved properties over racemic mixtures and is therefore more suitable than racemic mixtures for the treatment of obesity and other CCK-A mediated diseases or disorders. As used herein, "enantiomer rich" means that (+) enantiomers are more than (-) enantiomers compared to racemic mixtures having the same amount of (+) and (-) enantiomer isomers. It means a lot. As used herein, expressions including "compounds of the invention" or "enantiomer rich compounds of the invention", and these or similar phrases, include pharmaceutically acceptable salts and solvates thereof. "(+) Enantiomer" refers to the optical rotation of an enantiomer, not to the optical rotation of its salts and solvates. Preferred salts and solvates will be salts and solvates of the (+) enantiomer of the compound of formula (I) regardless of the optical rotation of the salt or solvate.

Preferably, the (+) enantiomer is at least 90% of the total amount of the enantiomer rich compound. More preferably, the (+) enantiomer is at least 96% of the total amount of the compound. Most preferably, the (+) enantiomer is at least 99% of the total amount of the compound.

The positive enantiomers of the invention can be thought of as complete cholecystokinin agonists in that they exhibit CCK agonist activity, bind to the CCK-A receptor, sufficiently stimulate gallbladder contraction and reduce the feeding of typical animals. For example, the (+) enantiomers of the present invention can be used in obesity and related pathologies, such as hypertension, gallbladder congestion, and diabetes, indirectly through weight loss and directly through CCK-A mediated Useful for the treatment of delayed gastric emptying. In addition, the positive enantiomers disclosed herein induce a satiety state, provide appetite control, alter food intake, regulate appetite, treat obesity, and maintain weight loss in mammals, especially humans Provide a new way to do this.

Accordingly, the present invention provides a method of treating a CCK-A mediated disease or condition, comprising administering to a patient a therapeutically effective amount of a positive enantiomer of the invention in a mammal, including a human. to provide.

The present invention also provides the use of an enantiomer rich compound of the present invention or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment of a CCK-A mediated disease or condition.

As used herein, it will be understood by those skilled in the art that treatment extends to prevention as well as treatment of a disease or condition. It will also be appreciated that the amount of preferred enantiomer of the present invention required for use in therapy varies depending upon the nature of the disease to be treated and the age and condition of the patient and is entirely at the discretion of the treating physician or veterinarian. In general, however, the amount used in adult treatment will typically be in the range of 0.02-5000 mg per day, for example 1-1500 mg per day. This preferred dosage may conveniently be administered in one dose, or at appropriate intervals, for example, two, three, four or more times per day.

The enantiomeric rich compound of the present invention may be therapeutically administered in a crude state, but it is preferred to provide the active ingredient in a pharmaceutical composition. Accordingly, the present invention further comprises a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and / or excipients, and optionally, other therapeutic and / or prophylactic ingredients, together with the enantiomeric rich compounds of the present invention. to provide. Carriers and / or excipients used herein should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the doser.

The formulations of the present invention include those prepared especially for oral, oral, parenteral, transplantation, transdermal or rectal administration, but oral administration is preferred. For oral administration, the compositions may take the form of tablets or umbilical cords prepared by conventional methods. Tablets and capsules for oral administration may be prepared by conventional excipients such as binders (e.g. syrup, acacia, gelatin, sorbitol, tragacanth, starch or polyvinylpyrrolidone store), fillers (e.g. lactose). , Sugars, microcrystalline cellulose, corn starch, calcium phosphate or sorbitol), lubricants (eg magnesium stearate, stearic acid, talc, polyethylene glycol or silica), disintegrants (eg potato starch or sodium starch glycol Rate) or humectant (eg, sodium lauryl sulfate). Tablets may be coated according to methods well known in the art, including enteric coatings.

Alternatively, the preferred enantiomers of the invention can be incorporated into oral liquid formulations such as, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs. It is also possible to provide formulations containing this preferred enantiomer as a dry product for use in admixture with water or other suitable excipients. Such liquid preparations may be conventional additives such as suspending agents (eg sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats); Emulsifiers (eg lecithin, sorbitan monooleate or acacia); non-aqueous excipients (which may include edible oils) (eg almond oil, fractionated coconut oil, oily esters, propylene glycol or Ethyl alcohol) and preservatives (eg, methyl or propyl p-hydroxybenzoate or sorbic acid), such formulations containing conventional suppository bases such as, for example, cocoa butter or other glycerides. It may also be prepared as a suppository.

In addition, the compositions of the present invention may be prepared for parenteral administration by injection or continuous infusion. Injectable preparations may take the form of suspensions, solutions or emulsions in oily or aqueous excipients, and may contain formulations such as suspensions, stabilizers and / or dispersants. Alternatively, the active ingredient may be in powder form for use in admixture with a suitable excipient (eg, sterile, pyrogenic water).

The composition according to the invention can also be prepared as a depot preparation. Agents with such long activity can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, the preferred enantiomers of the present invention may be made of suitable polymers or hydrophobic materials (eg emulsions in acceptable oils), or of some soluble derivatives such as ion exchange resins or, for example, some soluble salts. .

The composition according to the invention may contain 0.1 to 99% by weight of active ingredient, which may contain 30 to 95% for convenience and 30 to 50% for liquid formulations.

The (+) enantiomer of the present invention can be prepared by first preparing a racemic mixture as disclosed in Example 7 of US Pat. No. 5,646,140 and then separating the enantiomers by chiral chromatography.

Alternatively, suitable enantiomers of the amines of formula II, ie, (S) -enantiomers, are isocyanates of formula III, imidazolides of formula IV or optionally substituted phenyl carbonates of formula V: After reaction with, the carboxyl protecting group R can be removed to prepare:

(Wherein R is a carboxyl protecting group such as t-butyl and R ₁ is hydrogen or a common phenyl substituent such as NO ₂ ).

The reaction is typically carried out at a temperature range of 0-80 ° C. in the presence of a suitable solvent such as ether (eg tetrahydrofuran) or halohydrocarbon (eg dichloromethane) or nitrile (eg acetonitrile). Happens.

Typically, the required enantiomers of the amines (II) can be used in the form of their salts, for example R-camphorsulfonic acid salts, in which the reaction is in the presence of a base, for example a tertiary amine such as triethylamine. Can be performed under the following conditions.

Hydrolysis of carboxyl protecting groups can be carried out using conventional methods (Protecting groups in Organic Synthesis T. Greene, Ed, Wiley Interscience, New York, p168, 1981). Thus, for example, if R is a t-butyl group, it can be removed by net decomposition with a suitable acid, such as hydrochloric acid, trifluoroacetic acid or formic acid, using established methods. For example, it is reacted with hydrochloric acid in a solvent such as 1,4-dioxane or with formic acid while applying heat in a solvent such as acetone or aqueous acetone.

Formula II,

<Formula II>

The required (S) -enantiomers of the amines can be prepared by cleaving the corresponding racemic amines via chiral HPLC chromatography or by asymmetric cleavage by crystallization via the R-camphorsulfonic acid salt.

Racemic amine (II) can be prepared by the method described in Intermediate 11 of US Pat. No. 5,646,140.

Alternatively, racemic amine (II) can be prepared by simultaneously reducing and hydrocracking the oxime of formula VI:

(Wherein R ₂ is an optionally substituted benzyl group).

For convenience, a suitable palladium catalyst, for example palladium on carbon, for example in the presence of hydrogen or aqueous ammonium formate in a solvent such as an aqueous alkanol, for example ethanol, isopropanol or an industrial methylation spirit, or tetrahydrofuran The reaction is carried out with 5% Pd on charcoal. For convenience, the reaction is carried out, for example, while heating to a temperature of 40-80 ° C (eg 60 ° C).

Examples of suitable R ₂ groups for use in the reaction include benzyl, or substituted benzyl, such as p-methoxybenzyl, or benzhydryl.

Oxime (VI) may be prepared by reacting an o-phenylene diamine derivative of formula VII with an activated derivative of a diacid of formula VIII:

Wherein R ₂ is an optionally substituted benzyl group

For convenience, the activated derivative of diacid (VIII) is the corresponding diacyl halide, for example chloride, which is prepared in situ by reacting diacid (VIII) with oxalyl halide (eg oxalyl chloride). For convenience, the reaction is carried out in the presence of dimethylformamide in an aprotic solvent such as esters (eg ethyl acetate, toluene, dichloromethane, dimethoxyether or mixtures thereof).

For convenience, diacids (VIII) are dialkylketomalonates (eg diethyl) in the presence of a base (eg pyridine) in a solvent such as an alkanol (eg ethanol or industrial methylated spirit). Ketomalonate) and the corresponding hydroxylamine R ₂ ONH ₂ , followed by hydrolysis of the corresponding dialkyl-oxymino malonate with aqueous sodium hydroxide.

In a further aspect, the present invention relates to racemic amine (II), wherein the (S) enantiomer of the compound of formula (I) is substantially free of (R) enantiomer, wherein the amine (II) is an oxime (VI) ) And more specifically oxime (VI) is prepared from compounds (VII) and (VIII).

Isocyanates of formula III can be purchased or prepared by reacting the corresponding amines (VI) with phosgene or triphosgene in a suitable solvent such as methylene chloride. The imidazolides of formula IV are treated by treating the corresponding amines (VI) with carbonyl diimidazole at a temperature of 0 to 80 ° C. (conveniently at room temperature) in a suitable solvent (dichloromethane, ether, tetrahydrofuran) It can manufacture. Optionally substituted phenyl carbamate of formula (V) may be substituted with the corresponding amine (VI) and optionally substituted phenyl chloroformate at base (pyridine, triethyl) at a temperature of 0 to 50 ° C. in a suitable solvent (dichloromethane). Amine) in the presence of amine). Amines of formula VI are known compounds and may be prepared by methods analogous to the methods used to prepare these known compounds.

The following examples are intended to illustrate the present invention, and thus the present invention is not limited thereto.

Example abbreviations: EtOAc = ethyl acetate; MeOH = methanol, DMF = N, N-dimethylformamide; IPA = isopropyl alcohol; IMS = industrial methylated spirits.

Intermediate 1

(+)-2- (3-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo- [b] [1,4] diazepin-1-yl)- N-isopropyl-N-phenylacetamide camphorsulfonic acid salt

(+/-)-2- (3-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo- [b] [1,4] diazepin-1-yl ) -N-isopropyl-N-phenylacetamide (10 g) and R-camphorsulfonic acid (4.98 g) were stirred in tetrahydrofuran (35 ml) and toluene (65 ml) to give a solution. The solution was heated to 70 ° C. while forming a suspension. Water (0.4 ml) was added followed by 2-pyridinecarboxaldehyde (0.24 g) in toluene (5 ml). The mixture was heated at 70 ° C. for 3 hours and then cooled to 25 ° C. over 5 hours and then stirred at 25 ° C. for 16 hours. The suspension was frozen at 0-5 ° C for 1.5 h. The solids were collected by filtration washing with toluene / tetrahydrofuran (2: 1) (10 ml). Drying in vacuo at 50 ° C. gave the title compound as a white solid (12.6 g). Chromatographic analysis: eluent: 30% isopropyl alcohol, 70% heptane + 0.05% diethylamine; Column: 25 cm × 4.6 mm id, Chiralpak AD; Flow rate: 1 ml / min; Temperature: 40 ° C .; Detection: UV 230 nm; Injection volume: 10 μL; Sample solution: 0.1 mg / ml; Sample in 30% isopropyl alcohol, 70% heptane. Sample solution was injected immediately after preparation. Retention time: (+) enantiomer, 8.2 minutes. Unwanted (-) enantiomer (12.7 min) was below the detection limit.

Intermediate 2

3-nitrobenzoic acid t-butyl ester

To a solution of 3 nitrobenzoyl chloride (5.00 g) in anhydrous tetrahydrofuran (70 ml) was added potassium t-butoxide (3.82 g) and stirred under nitrogen for 2 hours. The reaction mixture was concentrated in vacuo and partitioned between dichloromethane and water. After phase separation, the aqueous phase was back extracted with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The crude product was purified on flash grade silica gel using 0-5% gradient ethyl acetate in n-hexane. The portions containing the product were combined, concentrated in vacuo and then dried under high vacuum to afford the title compound as an oil (3.82 g). ¹ H NMR (300 MHz, CDCl ₃ ) δ = 1.63 (s, 9H); 7.62 (t, J = 7.9 Hz, 1 H); 8.29-8.41 (m, 2 H); 8.78-8.80 (m, 1H). MS (Cl): [M + H] ⁺ = 224.

Intermediate 3

3-Amino-benzoic acid t-butyl ester

A solution of 3-nitro-benzoic acid t-butyl ester (3.77 g) in anhydrous ethanol (50 ml) was combined with palladium on carbon (10 wt.%, 0.30 g) and stirred under atmospheric hydrogen for about 3 hours. The reaction mixture was filtered between pads of diatomaceous earth and then concentrated in vacuo to afford an oil. It dried under high vacuum to crystallize to yield the title compound as a tan solid (3.28 g). ¹ H NMR (300 MHz, CDC1 ₃ ) δ = 1.58 (s, 9H); 6.79-6.87 (m, 1 H), 7.19 (t, J = 8.5 Hz, 1 H); 7.24-7.34 (m, 1 H); 7.38 (d, J = 8.0 Hz, 1 H). MS (Cl): [M + H] ⁺ = 194.

Intermediate 4

3-isocyano-benzoic acid t-butyl ester

At 0-5 ° C. triphosphogen (13.428 g) was added to a solution of 3-amino-benzoic acid t-butyl ester (26.50 g) and triethylamine (38.23 ml) in anhydrous tetrahydrofuran (600 ml). 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 slurried in hexane (500 ml), filtered and the filtrate was concentrated in vacuo to afford the title compound as an oil (21.54 g, 71.6%). 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, 1 H); 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- (isopropyl-phenyl-carbamoylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo [b ] [1,4] diazepin-3-yl] -ureido} benzoic acid t-butyl ester

Method A

Intermediate (I; 66.30 g) was slowly added to a solution of 3-isocyano-benzoic acid t-butyl ester (21.54 g) in anhydrous tetrahydrofuran (750 ml). Triethylamine (13.70 ml) was added dropwise to the reaction mixture. The resulting reaction mixture was stirred at ambient temperature overnight. The reaction mixture was poured into water (3000 ml) to give a white solid. The solids were collected by filtration washing with water (3x 500 ml). Drying in vacuo gave the title compound as a white solid (65.01 g). The crude title compound was used without further purification. Chromatography Characterization: Eluent: 30% isopropyl alcohol, 70% heptane + 0.05% diethylamine; Column: 25 cm × 4.6 mm id, Chiralpak AD; Flow rate: 1 ml / min; Temperature: 40 ° C .; Detection: UV 230 nm; Injection volume: 10 μL; Sample solution: 0.1 mg / ml sample in 30% isopropyl alcohol, 70% heptane. Sample solution was injected immediately after preparation. Retention time: (+) enantiomer, 15.6 min. Unwanted (-) enantiomer (13.3 min) was below the detection limit.

Method B

With stirring at 20 ° C., a solution of 3-amino-benzoic acid t-butyl ester (15.8 g) in dichloromethane (40 ml) was added to a suspension of carbonyl diimidazole (13.2 g) in dichloromethane (55 ml) in 30 minutes. Dropwise over. The resulting solution was stirred at 20 ° C. for 1 hour. To this solution was added a solution of Intermediate 1 (50 g) in dichloromethane (130 ml) over 5 minutes. Water (200 ml) was added to quench the reaction and stir for 10 minutes. The phases were separated and the organic phase was washed with water. The organic phase was concentrated under atmospheric pressure and 100 ml of dichloromethane were removed by distillation. t-butyl methyl ether (700 ml) was added and the mixture was stirred at 20 ° C. overnight. The solid was collected by filtration, washed with t-butyl methyl ether (100 ml) and dried in vacuo at 45 ° C. to afford the title compound as a white solid (42 g, 63 mmol).

Intermediate 6

Diethyl 2-[(benzyloxy) imino] malonate

At 20 ° C., di-ethylketomalonate (60 g) was added to a stirred suspension of O-benzylhydroxylamine (57.8 g) in IMS (500 ml) containing pyridine (30 ml). The reaction was heated at 75 ° C. for 4 hours. The reaction was cooled and the solvent removed under reduced pressure. The residue was partitioned between EtOAc (500 ml) and water (300 ml) and the organic layer was separated, washed with water (250 ml) and dried over MgSO ₄ . The solvent was evaporated to give 95.3 g of the title compound as a colorless oil (99% th, residual EtOAc of about 3% w / w), 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 intermediate 6 (40 g) in MeOH (80 ml) was added 2M NaOH (200 ml) over 20 minutes. The reaction was stirred at rt for 2 h. MeOH was removed under reduced pressure and the remaining solution was cooled and acidified to pH 2 by dropwise addition of concentrated HCl (˜30 ml) while maintaining the temperature below 35 ° C. A thick white slurry was formed which was diluted with water (50 ml) to increase fluidity. The solids were collected by filtration, washed with cold water (25 ml) and dried under vacuum at 55 ° C. to afford the title compound as a white solid (17 g). It was found to contain about 10% w / w residual inorganic salts. Fertilization yield ~ 45% th. Use without further purification.

1H NMR (300MHz, 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

Oxalyl chloride (38.3 g) was added dropwise (˜1 hour) to a stirred suspension of intermediate 7 (40 g, corrected to 31.4 g salt content) in EtOAc (200 ml) containing DMF (0.5 ml, 5 mol%). The mixture was stirred at 25 ° C. for 0.5 h and then filtered through a pad of Dicalite and washed with EtOAc (40 ml) to give a clear yellow solution. At 25 ° C., this solution was added to a stirred slurry of N-isopropyl-N-phenyl-2- (2-phenylaminophenylamino) -acetamide (50 g) in EtOAc (120 ml) (˜5 min). The mixture was warmed to 60 ° C. and a dark purple solution formed. After 1 h, EtOAc (200 ml) was removed by atmospheric distillation. IPA (120 ml) and water (40 ml) were added and the mixture was further distilled to remove more solvent (80 ml). IPA (40 ml) and water (40 ml) were added and additional amount of solvent was distilled off (80 ml). The reaction mixture was cooled to 25 ° C. over 1.5 hours and the solids collected by filtration. The solid was washed again with IPA (2 × 120 ml), water (1 × 120 ml) and finally IPA (1 × 40 ml) and dried under vacuum at 55 ° C. to give the title compound as a salmon-colored powder (56.6 g).

2: 1 mixture of isomers for 1 H NMR (300 MHz, CDCl ₃ ) 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, 6H)

Intermediate 9

(±) -2- (3-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo- [b] [1,4] diazepen-1-yl)- N-isopropyl-N-phenylacetamide

To a stirred suspension of intermediate 8 (3 g) and ammonium formate (2.08 g) in IMS (30 ml) and water (3 ml) was added 5% Pd / C (50% w / w water) (0.25 g). The mixture was heated at 60 ° C. under nitrogen overnight. The hot reaction mixture was filtered through decalite to remove the catalyst. The catalyst was washed with hot IMS (60 ml) and filtered. The filtrate was concentrated under reduced pressure to afford the title compound as a white solid (2.34 g).

Example 1

Chromatographic Fractionation of Enantiomers

(+) And (-)-3- {3- [1- (isopropyl-phenyl-carbamoylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro- 1H-benzo [b] [1,4] diazepin-3-yl] -ureido} benzoic acid.

Racemic 3- {3- [1- (isopropyl-phenyl-carbamoylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo [b] [1,4] diazepin-3-yl] -ureido} Benzoic acid was prepared as described in Example 7 of US Pat. No. 5,646,140 and partitioned by chiral HPLC under the following conditions: 250 × 4.0 μm (id) column , 5um diacel chilacell OD-R; Eluents are 80: 20: 0.1: 1, 80 parts acetonitrile, 20 parts water, 0.1 parts triethylamine, and 1 part acetic acid; UV detection wavelength 230 nm; Ambient temperature; Flow rate 1 ml / min; And 20 μl injection volume. Under these conditions, the residence time of the (+) enantiomer was 6.50 minutes and the residence time of the (-) isomer was 3.89 minutes.

Example 2

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

Method A

(+)-3- {3- [1- (isopropyl-phenyl-carbamoylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo [ b] [1,4] diazepin-3-yl] -ureido} Benzoic acid t-butyl ester (Intermediate 5: 65.01 g) was stirred in 4N hydrochloric acid in dioxane (280 ml) at ambient temperature for 6 hours. The reaction mixture was concentrated in vacuo and the resulting solid / oil was triturated in water to give a white solid. The white solid was collected by filtration and washed with water (2 x 500 ml). The crude product was dissolved in hot acetone (250 ml) and water (275 ml) was added until the solution was cloudy. Additional acetone (40 ml) was added and the solution was heated until a clear solution was obtained. The solution was left to cool. The resulting white solid was collected by filtration, washed with water (3 × 100 ml) and dried at 40-50 ° C. under house vacuum (20-25 in Hg) to afford the title compound as a white solid (40.496 g). Purity analysis was performed by chiral chromatography (see chromatographic splitting protocol). 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, 3 H); 4.18 (d, J = 16.7 Hz, 1 H); 4.48 (d, J = 16.7 Hz, 1 H); 4.78 (m, 1 H); 5.02 (d, J = 7.8 Hz, 1H); 6.91 (d, J = 7.8 Hz, 1 H); 6.95 (bd, J = 8.1 Hz, 1H); 7.22-7.57 (m, 19 H); 8.00 (s, 1 H); 9.34 (s, 1 H); 12.78 (s, 1 H). MS (ES): [M + 1] = 606.1; [M + Na] = 628.1; [M-1] = 604.1.

Method B

At room temperature, intermediate 5 (5 g) was added to a mixture of acetone (15 ml) and formic acid (25 ml) and the resulting suspension was heated to 55 ° C. and stirred for 5 hours. Water (30 ml) was added dropwise to the solution while maintaining the temperature of the contents above 50 ° C. The resulting slurry was stirred at 55 ° C. for 1 hour, cooled to room temperature and stirred overnight. The slurry was filtered and washed with water (3 x 25 ml). The residue was added to IMS (50 ml) and the slurry was heated to 45 ° C. and stirred overnight. The slurry was cooled to room temperature, filtered and the wet mass was dried in vacuo at 55 ° C. to afford the title compound as a white solid (3.40 g).

Chiral chromatography analysis showed that the required reaction product contained 0.7% of the unwanted (-) enantiomer.

Preparation

Oral solution

0.5-800 mg active ingredient

Polyethylene glycol 400 NF as much as 50 ml

The active ingredient was suspended in polyethylene glycol 400 and then dissolved by sonication to prepare an oral solution.

Oral suspension

0.5-80 mg of active ingredient

Polysorbate 80 NF (Tween 80) 0.02 ml

As much as 20ml of cleaning sterilizing water

The active ingredient was added to 0.1% (v / v) Tween 80 solution (20 ml) and the mixture was then sonicated or shaken to prepare an oral suspension.

refine

a.

6 mg active ingredient

Anhydrous Lactose USP 136.2mg

Sodium Starch Glycolate USP / NF 6mg

Stearic Acid USP / NF 1.5mg

Colloidal Silicon Dioxide USP / NF 0.3mg

Compressed Weight 150mg

The active ingredient, lactose and sodium starch glycolate were sieved through a 590 micron sieve and mixed in a suitable mixer. Stearic acid (beaten with 250 micron sieve) and colloidal silicon dioxide were added to the active blend and mixed. The blend was compressed into tablets using a suitable punch.

b.

6 mg active ingredient

Microcrystalline Cellulose USP / NF 136.5mg

Crospovidone USP / NF 6mg

Magnesium Stearate USP / NF 1.5mg

Compressed Weight 150mg

The active ingredient, microcrystalline cellulose and crosvidone were sieved 590 microns and mixed in a suitable blender. Magnesium stearate was sieved through a 250 micron sieve and mixed with the active blend. The resulting blend was compressed into tablets using a suitable tablet punch.

capsule

a.

6 mg active ingredient

Microcrystalline Cellulose USP / NF 128.25mg

Sodium Starch Glycolate USP / NF 15mg

Magnesium Stearate USP 0.75mg

Filling weight 150 mg

The active ingredient, microcrystalline cellulose, and sodium starch glycolate were sieved through a 590 micron mesh sieve, mixed together and lubricated with magnesium stearate (beaten with 250 microns sieve). The blend was filled into capsules of suitable size.

b.

6 mg active ingredient

Lactose Monohydrate USP 130.5mg

Povidone USP 6mg

Crospovidone NF 6mg

1.5 mg magnesium stearate

Filling weight 150 mg

The active ingredient and lactose were mixed together and granulated with povidone solution. The wet mass was dried and ground. Magnesium stearate and crospovidone were sieved through a 250 micron sieve and mixed with the granules. The resulting blend was filled into hard gelatin capsules of suitable size.

Biological assay

(+) And (-) enantiomers and racemic mixtures were characterized in the following assays. The results of this assay are summarized in the table below.

Guinea Pig Gallbladder Tissue Specimen. Gallbladder was removed from male Hartley guinea pig killed in CO ₂ atmosphere. Adhesive connective tissue was removed from the isolated gallbladder and cut into two rings from each animal (2-4 mm long). The ring was suspended in a tissue chamber with physiological saline (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO ₄ , 2.5 mM CaCl ₂ , 1.2 mM KH ₂ PO ₃ , 25 mM NaHCO ₃ , 11.1 mM dextrose). The bathing solution was maintained at 37 ° C. and aerated with 95% O ₂ /5% CO ₂ to maintain pH = 7.4. The tissue was connected to an isometric force displacement transducer (Grass, Model FT03 D) via a gold chain and stainless steel mounting wire. The reaction was then recorded on polygraph (Grass, Model 7E). Tissue from each animal served as time / solvent control and did not receive test compounds. The ring was held throughout the experiment by gradually pulling to a base stop tension of 1 gm (over 120 minutes). During the base tension adjustment period, the ring was exposed to acetylcholine ( ^10-6 M) four times to confirm tissue contractility. The tissue was then exposed to a submaximal amount of sulfated CCK-8 (Sigma, 3 × 10 ⁻⁹ M). After a stable reaction was obtained, tissues were washed three times quickly and every 5 to 10 minutes for 1 hour to reestablish a stable baseline.

Agonist EC50. The compound was dissolved in dimethylsulfoxide (DMSO) and then diluted with water and assayed through a progressive concentration-response curve for the test compound (10 ^-11 to 3 x 10 ^-5 M), then in the presence of the highest concentration of test compound Assay, by concentration-response curve for sulfated CCK-8 ( ^10-10 to ^10-6 M). As a final test, acetylcholine (1 mM) was added to cause maximal contraction. For each test compound, the minimum of three activity measurements was defined.

Establishment of stable CCK receptor containing cell lines. The cDNA clones for human CCK-A ¹⁸ or CCK-B ¹⁹ receptors were linked to pcDNA-1-neo vector of Invitrogen Corp. (San Diego, Calif.) For direct transfection. DNA was prepared by alkaline lysis and transfected into CHO-K1 cells (ATCC, Rockville, MD) using Lipofectin Reagent ²⁴ (Gibco BRL, Gaithersberg, MD). Stable transfectants were first selected using Geneticin (Gibco BRL) and fluorescent-activated cell sorting based on the binding of Fluorescein-Gly-[(Nle28,31] -CCk-8 Receptor bearing resistant cells were enriched, followed by substantial dilution of clone cell lines.

Cell membrane specimen. CHO-K1 cells stably transfected with human CCK-A or CCK-B receptor cDNA, in a humidified atmosphere (95% O ₂ /5% CO ₂₎ in Ham's F12 medium supplemented with 5% heat inactivated fetal bovine serum ) At 37 ° C. Cells were passaged twice weekly and grown to a density of 2-4 million cells per mL. Cells were harvested by centrifugation (600 × g, 15 min, 4 ° C.), trisHCl (25 mM), EDTA (5 mM), EGTA (5 mM), phenyl sulfonyl fluoride (0.1 mM) and soybean trypsin inhibitor ( Resuspend in buffer (20 mL, pH 7.4) containing 100 μg / mL). Cells were disrupted with a vibrating glass Teflon homogenizer (25 strokes) and the homogenate was centrifuged at low speed (600 × g, 10 min, 4 ° C.). The supernatant was collected and centrifuged at high speed (500,000 × g, 15 min, 4 ° C.) to pellet the particle portions. The low speed pellets were processed three more times. The fast particle portions were combined and resuspended in buffer (1-5 mg protein / mL) and frozen at -80 ° C. Protein concentration was measured using BioRad reagent and bovine serum albumin as standard according to the manufacturer's instructions.

Receptor Binding Assay. Binding 125I-Bolton Hunter CCK-8 (Amersham, 2000 Ci / mmol) containing HEPES (20 mM), NaCl (118 mM), KCl (5 mM), MgCl 2 (5 mM) and EGTA (1 mM) Lysis in buffer (pH 7.4, 100,000 cpm / 25 μL). Nonspecific binding was measured with MK-329 ²⁰ (10 μΜ, CCK-A) or L-365,260 ²¹ (10 μΜ, CCK-B). Test compounds were dissolved in DMSO at stock concentrations 100 times the final assay concentration and diluted to appropriate concentrations with binding buffer. Binding assays were performed three times using a 96-well plate, followed by test compound (25 μL), 125 I-Bolton Hunter CCK-8 (25 μL), buffer (pH 7.4, 150 μL) and receptor preparation (50). μL) was added. The final concentration of DMSO was 1% in all assay wells. After 3 hours at 30 ° C., the mixture was quickly filtered on a glass filter (Whatman GF / B) to complete the incubation and then washed to remove unbound ligand. Binding radioactivity was quantified by gamma counting.

Intracellular Calcium Measurements: CHO-K1 cells stably transfected with hCCK-A or hCCK-B receptors were grown to 75-90% confluency on glass coverslips. Cells were loaded for 50 minutes in serum-free cultures containing 5 mM FURA2-AM and 2.5 mM probenecid. Changes in intracellular calcium concentration were measured using a JASCO CAF-102 calcium analyzer by standard ratioometer techniques using excitation wavelengths of 340 nm and 380 nm. Cells were perfused with increasing concentrations of CCK-8 (n = 2) or compound (n = 2) until a plateau of 340/380 ratio was achieved. More than 10 minutes of wash / recovery cycles were allowed between successive simulations. Maximum response was normalized to the maximum response induced by CCK-8. EC ₅₀ was calculated at the concentration necessary to elicit half of the maximal response. In addition to the agonist concentration-response curves, CHO-K1 cells expressing human CCK-B receptors were perfused with three concentrations of compound (10 ⁻⁸ , 10 ⁻⁷ , 10 ⁻⁶ M, n = 2) for 1 hour, then obtain a concentration-response curve for CCK-8 (10 ^-12 to 10 ^-6 M).

Anorexia Assay: Male Long-Evans rats (225-300 g) were adjusted for 2 weeks to eat delicious liquid food (Bio-Serve F1657, Frenchtown, NJ) after 2 hours fasting. On the day of pretreatment, rats were fasted (100 min) and injected with oral preload of saline (0.9% NaCl, 8 mL / kg) with IP with drug excipient (propylene glycol, PG, 1 mL / kg). . After 20 minutes liquid food was available and measurements were taken at 30, 90 and 180 minutes. To be suitable for drug treatment studies, rats had to eat at least 8 mL of liquid food within the first 30 minutes of the pretreatment day. The next day, after starving for 100 minutes, treatment with test compound at various doses (0.01 to 10 μmol / kg) dissolved in IP or PO with excipient (PG, 1 mL / kg), or PG (1 mL / kg) Was immediately followed by saline oral preload treatment. Food was made available again after 20 minutes and food intake was measured at 30, 90 and 180 minutes. All food intake data for each rat was normalized to each value obtained on the day of pretreatment. Efficacy was measured at 30 minutes and efficacy at 30 minutes, 1 μmol / kg dose.

Mouse gallbladder excretion assay: Makovec, F .; Bani, M .; Cereda, R .; Chiste, R .; Pacini (MA); Level (Revel, L.); Rovati, LC, Antitispasmodic Activity on the Gallbladder of the Mouse of CR 1409 (Lorglumide), a Potent Antagonist of Peripheral Chlolecystokinin. Pharmacol. Res. Commun. 1987, 19, 41-51.

PApp values: Artursson P. and Carison J., 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).

Pharmacological comparison of enantiomers and racemates

black (+) Enantiomer (-) Enantiomer Racemate In vitro GPGB EC ₅₀ (nM) 9.3 63 40 HCCK-A IC ₅₀ (nM) 148 191 123 HCCK-A EC ₅₀ (nM) 150 298 252 HCCK-B Ki (nM) 3.2 22.3 10 HCCK-B EC ₅₀ (nM) Antagonist Antagonist Antagonist Selectivity rain ¹ 46 8.6 12.3 PApp (x 10 ^-7 cm / sec) 1.6 0.7 In vivo Rat loss of appetite ED ₅₀ IP (μmol / kg) 0.034 0.48 0.06 ED ₅₀ PO (μmol / kg) 1.1 Inactive ³ 2.0 Mouse gallbladder Emission black ED ₅₀ IP (μmol / kg) 0.002 0.012 0.007 ED ₅₀ PO (μmol / kg) 0.007 Inactive ² 0.055 Selectivity ratio = IC50 (hCCK-A) / Ki (hCCK-B) 2. Inert at less than 10 μmol / kg 3. At the doses below 10 μmol / kg, the amount of food observed is not significantly reduced.

In three unexpected biological activities. (+) Enantiomers are distinguished from (-) enantiomers and racemates. Two of these activities relate to enhanced CCK-A efficacy, which should demonstrate the beneficial activity of this enantiomer. The third relates to CCK-B antagonist activity of (+) enantiomers, which should prove beneficial through reduced toxicity.

Positive enantiomers were four times more potent than racemates in an in vitro test of isolated guinea pig gallbladder (“GPGB”). Positive enantiomers were eight times more potent than racemates in mouse gallbladder excretion assays (oral administration). Increased efficacy is expected to be beneficial for the treatment of gallbladder congestion and for the treatment of obesity. Gallbladder congestion is a serious problem in rapid weight loss.

As edible depressants are intended for long-term use, it is important to minimize the risk of toxicity. The main toxicity associated with the use of cholecystokinin is incidental CCK-B receptor agonist activity. Activation of the CCK-B receptor is mainly associated with increased anxiety and increased gastric acid secretion. The usefulness of CCK-B antagonists has been studied for the development of anxiolytic agents and antiulcers. See, eg, Lowe, J. "Cholecystokinin-B Receptor Antagonists" in Exp. Opin. Ther. Patents, 5 (3), pp 231-237 (1995).

The dominant CCK receptor subtype in the rodent pancreas is the CCK-A subtype, and activation of this subtype causes aberrant stimulation and aberrant hypertrophy of the pancreas in rodents. Both of these activities are considered undesirable. Recently, the tissue distribution of CCK receptors in human tissues has been reported. Surprisingly, the dominant receptor subtype in the human pancreas is the CCK-B receptor subtype. See, eg, Wang, S. A., "Cholecystokinin Receptors" in American Journal of Physiology-Gastrointestinal & Liver Physiology, 32 (5) :, pp G628-G646, (1995). Thus, in humans, the CCK-B receptor (CCK-B agonist activity) not only increases anxiety and gastric acid secretion, but can also cause pancreatic abnormal irritation and abnormal hypertrophy with prolonged use.

In order to reduce the risk of undesired CCK-B agonist activity in vivo, preferred compounds must have affinity for the CCK-B receptor and have measurable CCK-B antagonist activity in an in vitro assay. Both enantiomers and racemates are CCK-B antagonists. All three compositions have similar human CCK-A receptor affinity (IC50) and efficacy (EC50), but the (+) enantiomer has the highest CCK-B receptor affinity (IC50) and selectivity (46-fold). Thus, positive enantiomers are preferred not only in terms of CCK-A efficacy and efficacy, but also in minimizing the potential for toxic side effects caused by CCK-B.

The positive enantiomers of the present invention are substantially nontoxic in therapeutically effective amounts. For example, single dose oral studies have shown that the maximum nonfatal dose was at least 2000 mg / kg in rats, at least 1000 mg / kg in male mice and at least 500 mg / kg in female mice.

Claims (12)

Enantiomer rich 3- {3- [1- (isopropyl-phenyl-carbamoylmethyl) -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 rich compound of claim 1, wherein the (+) enantiomer, or a pharmaceutically acceptable salt or solvate thereof, is at least 90% of the compound. The enantiomer rich compound according to claim 1 or 2, wherein the (+) enantiomer, or a pharmaceutically acceptable salt or solvate thereof, is at least 99% of the compound. A pharmaceutical composition comprising an enantiomer rich compound as defined in any one of claims 1 to 3, mixed with one or more pharmaceutically acceptable carriers and / or excipients. A method of treating a CCK-A mediated disease or condition comprising administering an effective amount of a compound as defined in any one of claims 1 to 3. A method of treating a CCK-A mediated disease or condition comprising administering a pharmaceutical composition as defined in claim 4. 7. The method of claim 5 or 6, wherein the disease or condition is obesity, gallbladder congestion, or diabetes. 7. The method of claim 5 or 6, wherein said disease or disorder is obesity. Use of a compound as defined in any one of claims 1 to 3 in the manufacture of a medicament for the treatment of a CCK-A mediated disease or condition. (a) racemic 3- {3- [1- (isopropyl-phenyl-carbamoylmethyl) -2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo [b] [1,4] diazepin-3-yl] -ureido} benzoic acid partitioning by chiral HPLC; (b) reacting a suitable enantiomer of an amine of formula II with an isocyanate of formula III, an imidazolide of formula IV or an optionally substituted phenyl carbamate of formula V, and then the carboxy protecting group R Steps to remove Method for producing a compound as defined in claim 1 comprising: <Formula II> <Formula III> <Formula IV> <Formula V> The process according to claim 10, wherein the required compound of claim 1 is prepared via racemic amine (II) prepared by simultaneously reducing and hydrocracking the oxime of formula VI: <Formula VI> Wherein R ₂ is an optionally substituted benzyl group. 12. The process of claim 11, wherein the oxime (VI) is prepared from o-phenylene diamine of formula (VII), and activated derivatives of diacids of formula (VIII): <Formula VII> <Formula VIII> Wherein R ₂ is an optionally substituted benzyl group.