AU688316B2 - 1,5 benzodiazepine derivatives having CCK and/or gastrin antagonistic activity - Google Patents

Description

WO 94/24151 PCTIEP94/01130 BENZODIAZEPINE DERIVATIVES HAVING CCK AND/OR GASTRIN ANTAGONISTIC ACTIVITY This invention relates to novel 1,5-benzodiazepine derivatives, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medicine.

Cholecystokinins (CCK) and gastrin are structurally related peptides which exist in gastrointestinal tissue and in the central nervous system. Cholecystokinins include CCK-33, a neuropeptide of thirty-three amino acids in its originally isolated form, its carboxy terminal octapeptide, CCK-8 (also a naturallyoccurring neuropeptide), and 39- and 12-amino acid forms. Gastrin occurs in 34-, 17- and 14- amino acid forms, with the miniumum active sequence being the C-terminal tetrapeptide, Trp-Met-Asp-Phe-NH 2 (CCK-4), which is the common structual element shared by both CCK and gastrin.

CCK and gastrin are gastrointestinal hormones and neurotransmitters in the neural and peripheral systems and perform their respective biological roles by binding to particular receptors located at various sites throughout the body.

There are at least two subtypes of cholecystokinin receptors termed CCK-A and CCK-B and both are found in the periphery and in the central nervous system.

CCK and gastrin receptor antagonists have been disclosed for preventing and treating CCK-related and/or gastrin related disorders of the gastrointestinal and central nervous systems of animals, and more particularly humans.

US Patent No. 4,988,692 describes a group of 3-acylamino 1-alkyl-5-phenyl benzodiazepine derivatives as cholecystokinin antagonists. Further the specification teaches that the compounds have a significantly greater affinity for the CCK-A receptor over the CCK-B receptor.

We have now found a novel group of 3-ureido compounds which are potent and specific antagonists of gastrin and/or CCK and in particular antagonists of gastrin and /or CCK at the CCK-B receptor.

-s a I~ WO 94/24151 PCT/EP94/01130 2 The present invention thus provides compounds of the general formula (I)

CONRR

2

CH

2

N

(X)m NjNHCONHR 4 R, and physiologically acceptable salts thereof wherein the group NR 1

R

2 represents a 5-7 membered saturated heterocylic ring which may be substituted by one or two methyl groups; R3 is C 1 6 alkyl, C 3 6 cycloalkyl or phenyl optionally substituted by 1 or 2 halogen atoms; R4 is phenyl or phenyl substituted by one or two groups selected from halogen, Cl-4alkyl, trifluoromethyl, trifluoromethoxy or (CH 2 )nRs wherein n is zero or 1 and R5 represents C1-4alkoxy, hydroxy, nitro, cyano, C0 2

R

6 S(O)pCH 3

NR

7

R

8

CONR

7

R

8

SO

2

NR

7

CO(C

1 -4alkyl), tetrazolyl, carboxamidotetrazolyl, or a 3trifluoromethyl 1,2,4-triazolyl;

R

6 is hydrogen, C 1 4 alkyl or benzyl; R7 is hydrogen or C 1 -4alkyl, R, is hydrogen, C1.

4 alkyl, S0 2

CH

3 or S02CF 3 X represents hydrogen, C1-4alkyl or halogen; m is zero, 1 or 2, and p is zero, 1 or 2.

The compounds of the invention possess at least one asymmetric carbon atom and the compounds of the invention include both enantiomers and mixtures thereof including the racemate.

The term alkyl as used herein refers to both straight chain and branched chain alkyl groups. For example C 16 _alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl or hexyl.

a sldl C Ls L WO 94/24151 PCT/EP94/01130 3 The term halogen includes fluorine, chlorine, bromine or iodine.

When R 5 represents a tetrazolyl, carboxamidotetrazolyl or 3-trifluoromethyl 1,2,4-triazolyl group these are linked to the rest of the molecule via a carbon atom therein and the invention includes all tautomers thereof end the C 1 4 alkyl -N substituted derivatives thereof. Examples of such groups include (1H) carboxamido-1 H-tetrazol-5-yl, 2-methyltetrazol-5-yl and (3trifluoromethyl-1,2,4-triazol-5-yl.

The group NR 1

R

2 is linked to the rest of the molecule via the nitrogen thereof examples of such groups include pyrrolidino, piperidino, hexamethylenimino-, 3,3-dimethylpiperidino, 2,6-dimethylpiperidino or 4,4dimethylpiperidino.

When R 3 represents phenyl optionally substituted by halogen examples of such groups include phenyl optionally substituted by fluorine e.g. phenyl or 2fluorophenyl, or 4-fluorophenyl.

When R 3 represents C3-6cycloalkyl examples of such groups include cyclopropyl, cyclopentyl or cyclohexyl.

When R 3 represents C 1 6 alkyl examples of such groups include methyl, ethyl, propyl, butyl, 3-methylbutyl or 3,3-dimethylbutyl.

Examples of suitable groups R 4 include phenyl optionally substituted by halogen e.g. fluorine, alkyl e.g. methyl, alkoxy e.g. methoxy, nitro, cyano, thiomethyl, carboxamido, carboxyl, dimethylamino, cyanomethyl, 1 carboxymethyl, or N-methanesulphonylcarboxamido.

A preferred class of compound according to the invention are those wherein the group NRjR 2 represent pyrrolidino, piperidino, 3,3-dimethylpiperidino 4,4dimethylpiperidino, 2,6-dimethylpiperidino or 2,5-dimethylpyrrolidino. Within this class the group NR 1

R

2 is conveniently pyrrolidino, piperidino, or 3,3dimethylpiperidino.

WO 94/24151 WO 94/4 151CTIEP94/01 130 4 The group X is conveniently halogen e.g. bromine, fluorine or fluorine or more particularly hydrogen.

A further preferred class of compounds of formula are those wherein R 3 q is phenyl, 2-fluorophenyl or cyclohexyl and more particularly 2-fluorophenyl or cyclohexyl.

Another preferred class of compounds of formula are those wherein R 4 is phenyl or phenyl substituted by methyl e.g. 3-methylphenyl or dimethylphenyl, 3-dimethylaminophenyl, phenyl substituted by fluorine e.g. 4fluorophenyl, phenyl substituted by methoxy e.g. 3-methoxyphenyl or 4methoxyphenyl, 3-nitrophenyl, 3-cyanomethylphenyl, 3-carboxamidophenyt, 3carboxyphenyl, 3-carboxymethylphenyl, or 3-(1 A particularly preferred group of compounds according to the invention are those wherein NRIR 2 represents pyrrolidino, piperidine or 3,3-dimethyl-, piperidino, R 3 represents 2-fluorophenyl or cyclohexyl, and X represents a hydrogen atom. Within this group particularly preferred compounds include those wherein R 4 is phienyl, 4-fluorophenyl, 3-dimethylaminophenyl, 3-carboxyphenyl, 3-carboxymethylphenyl or 3-(1 A particularly preferred compound of the invention is:- 1 -Cyclohexyl-2,4-dioxo-5-(2-oxo-2-pyrrolidin-1 -yl-ethyl)-2,3 1 H-benzopb[1 ,4]diazepin-3-yl]-3-(4-fluoro-phenyl)urea and enantiomers thereof.

Further preferred compounds of the invention include -Cyclohexyl-2,4-dioxo-5-(2-oxo-2-pyrrolidin- 1 -yl-ethyl)-2,3, 1 H-benzotb][1 ,4]diazepin-3-yl]-ureido}-benzoic acid; -(2-Fluoro-phenyl)-2,4-dioxo-5-(2-oxo-2-pyrrolidin-1 -yl-ethyl)-2,3 tetrahydro-1 H-benzofbl[1I,4]diazepin-3-ylI-u reido}-phenyl)-acetic acid; methyl-piperidin- 1-yl)-2-oxo-ethylj-5-(2-fluoro-phenyl)-2,4dioxo-2,3,4,5-tetrahydro-1 H-benzo~b][1 ,4]diazepin-3-yI]-ureido}-benzoic acid and enantiomers thereof.

~IR~I WO 94/24151 PCT/EP94/01130 The physiologically acceptable salts of the compounds of formula include conventional salts formed for example from pharmaceutically acceptable inorganic or organic acids as well as quaternary ammonium acid addition salts.

Examples of suitable salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, pamoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulphonic, methanesulphonic, naphthalene-2-sulphonic, benzenesulphonic and the like. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts.

References hereinafter to a compound according to the invention includes both compounds of formula and their pharmaceutically acceptable salts and solvates.

Compounds of the invention modulate the effect of gastrin and/or CCK in mammals. In particular compounds of the invention are antagonists of gastrin and/or CCK.

Compounds of the invention have been shown to be antagonists of CCK, particularly at CCK-B receptors as demonstrated for example by the compound's ability to inhibit the contractile actions of CCK-4 in the presence of a CCK-A antagonist, in the guinea-pig isolated ileum longitudinal musclemyenteric plexus.

The preparation and use of guinea-pig isolated ileum longitudinal musclemyenteric plexus has been described by K-H Buchheit et al in Nauyn- Schmeideberg's Arch. Pharmacol., (1985), 329, p36-41 and by V.L. Lucaites et al in J. Pharmacol. Exp. Ther., (1991) 256, 695-703.

Compounds of the invention have also been shown to have a greater affinity for the CCK-B receptor than for the CCK-A receptor. This may be determined using the CCK receptor binding assays described by Fornos et al J. Pharmacol Exp.

Ther., 26 1056-1063, 1992.

I~

WO 94/24151 PCT/EP94/01130 6 Alternatively the binding affinity of compounds of the invention for CCK-.A and CCK-B receptors may be determined using HeLa cell membranes that are transfected with the human CCK-B receptor or, COS-M6 cell membranes that are transiently transfected with the human CCK-A receptor.

Compounds of the invention have also been shown to be antagonists of gastrin as demonstrated by their ability to inhibit pentagastrin-stimulated acid secretion from rat isolated gastric mucosa using the procedure described by J.J. Reeves and R. Stables in Br. J. Pharmac.. 1985 86, p.677-684.

Compounds of the invention have also been shown to inhibit pentagastrin stimulated acid secretion in conscious gastric fistula rats using the methods described by Hedges and Parsons Journal of Physiology 1977, 267, 181-194.

The compounds of the invention are therefore useful for the treatment and/or prevention of disorders in mammals, especially humans, where modification of the effects of gastrin or CCK is of therapeutic benefit. Thus the compounds of the invention are useful for the treatment of gastrointestinal disorders especially those where there is an advantage in lowering gastric acidity. Such disorders include peptic ulceration, reflux oesophagitis and Zollinger Ellison syndrome.

They may also be useful for the treatment of gastrointestinal disorders such as irritable bowel syndrome, excess pancreatic secretion, acute pancreatitis, motility disorders, antral G cell hyperplasia, fundic mucosal hyperplasia or gastrointestinal neoplasms. The compounds of the invention are also useful for the treatment of central nervous system disorders where CCK and/or gastrin are involved. For example anxiety disorders (including panic disorder, agoraphobia, social phobia, simple phobia, obsessive compulsive disorders, post traumatic stress disorder, and general anxiety disorder), depression, tardive dyskinesia, Parkinson's disease or psychosis. They may also be useful for the treatment of dependency on drugs or substances of abuse and withdrawal, Gilles de la Tourette syndrome, or dysfunction of appetite regulatory systems; as well as the treatment of certain tumours of the lower oesophagus, stomach, intestines and colon. Compounds of the invention are also useful for directly inducing analgesia, or enhancing opiate or non-opiate mediated analgesia, as well as anaesthesia or loss of the sensation of pain.

LWL ~qCI WO 94/24151 PCT/EP94/01130 7 The invention therefore provides a compound of formula or a pharmaceutically acceptable salt or solvate thereof for use in therapy, in particular in human medicine.

According to another aspect the invention provides the use of a compound of formula or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment of conditions where modification of the effects of gastrin and/or CCK is of therapeutic benefit.

According to a further aspect of the invention we provide a method for the treatment of a mammal, including man, in particular in the treatment of conditions where modification of the effects of gastrin and/or CCK is of therapeutic benefit which method comprises administering an effective amount of a compound of formula or a pharmaceutically acceptable salt or solvate thereof to the patient.

It will be appreciated by those skilled in the art that reference herein to treatment extends to prophylaxis as well as the treatment of established diseases or symptoms.

It will further be appreciated that the amount of a compound of the invention required for use in treatment will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. In general however doses employed for adult human treatment will typically be in the range of 1-2000mg per day e.g 10-500mg per day.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day.

Compounds of the invention which antagonise the function of CCK in animals, may also be used as feed additives to increase the food intake in animals in daily dosages of around 1 mg/kg to ~c -L le -1 WO 94/24151 PCT/EP94/01130 8 While it is possible that, for use in therapy, a compound of the invention may be administered as the raw chemical it is preferable to present the active ingredient as a pharmaceutical formulation.

The invention thus further provides a pharmaceutical formulation comprising a compound of formula or a pharmaceutically acceptable salt thereof together with one or more pharmaceutically acceptable caRriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The compositions of the invention include those in a form especially formulated for oral, buccal, parenteral, implant, or rectal administration. Oral administration is preferred.

Tablets and capsules for oral administration may contain conventional excipients such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone; fillers, for example, lactose, sugar, microcrystalline cellulose, maize-starch, calcium phosphate or sorbitol; lubricants, for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica; disintegrants, for example, potato starch or sodium starch glycollate, or wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitoi syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan mono-oleate or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; and preservatives, for example, methyl or propyl hydroxybenzoates or ascorbic acid. The compositions may also be formulated

I

WO 94/24151 PCT/EP94/01130 9 as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.

For buccal administration the composition may take the form of tablets or lozenges formulated in conventional manner.

The composition according to the invention may be formulated for parenteral administration by injection or continuous infusion. Formulations for injection may be presented in unit dose form in ampoules, or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatorv agents such as suspending, stabilising and/or dispersing agents. Alternatively the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

The composition according to the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus for example, the compounds of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions according to the invention may contain between 0.1 99% of the active ingredient, conveniently from 30-95% for tablets and capsules and 3for liquid preparations.

Compounds of general formula and salts thereof may be prepared by the general methods outlined hereinafter. In the following description, the groups

R

1

-R

8 and X are as defined for the compounds of formula unless otherwise stated.

According to a first general process A, compounds of formula may be prepared by the reaction of an amine of formula (11) wherein R 1

R

2

R

3 X and m have the meanings defined in formula ps~ WO 94/24151 PCT/EP94/01130

NRR,

2 NH R3 (II) with a compound R 4 Y, wherein R 4 Y is an isocyanate of formula (111), carbamoyl chloride of formula imidazolide of formula or an optionally substituted phenyl carbamate of formula (VI) wherein Ra is an optionally phenxoy group.

0 oC-- =N--R 4

CICONHR

4 N N N-R 4

H

(III)

(IV)

(V)

RaCONHR 4

(VI)

The reaction conveniently takes place in the presence of a suitable solvent such as a halohydrocarbon dichloromethane), an ether tetrahydrofuran) or nitrile acetronitrile) or a mixture thereof at a temperature in the range of 0 0 -80 0

C.

Isocyanates of formula (1ll) may be purchased or prepared by the reaction of amines H 2

N-R

4 with phosgene or triphosgene in a suitable solvent such as methylene chloride. Carbamoyl chlorides of formula (IV) are also prepared by the reaction of amines H 2

NR

4 with phosgene or triphosgene in a suitable solvent such as methylene chloride. Imidazolides of formula (VI) are prepared by treatment of amines H 2

N-R

4 with carbonyl diimidazole in a suitable solvent (dichloromethane, ether, tetrahydrofuran) at a temperature ranging from 0-800 C (conveniently at room temperature). Optionally substituted phenyl carbamates of formula (VI) are prepared by the reaction of amines H 2

N-R

4 with the appropriate WO 94/24151 PCT/EP94/01130 11 chloroformate RaCOCI in the presence of a base (pyridine, triethylamine) in a suitable solvent (dichloromethane) and at a temperature of 0 500 C.

According to a further general process B, compounds of formula may be prepared by reaction of an intermediate of formula (VII).

NRR

rN R, (VII) wherein Y is the group -NCO, -NHCOCI or NHCORa wherein Ra is an optionally substituted phenoxy group or a 1-imidazole group with an amine (VIII)

H

2

N-R

4

(VIII)

and optionally in the the presence of a base such as a tertiary amine (e.g.

triethylamine).

The reaction conveniently takes place in a suitable solvent such as halogenated hydrocarbon dichloromethane) or an ether (e.g.

tetrahydrofuran) or an amide N,N-dimethyl formamide) optionally at a temperature ranging from room temperature to the reflux temperature of the solvent.

Conveniently the compound of formula (VII) are prepared insitu from the amine In a particular aspect of the process when Y is the group NHCORa and Ra is a 1-imidazole group, the imidazolide (VII) may be formed in situ in which case the amine of formula (VIII) will be.mixed with the compound of formula (II) WO 94/24151 PCT/EP94/01130 12

NRR,

V NH 2

R

3 0 (II) in the presence of carbonyldiimidazole under the aforementioned conditions.

For process B when Y is the group NHCORa and R a is optionally substituted phenoxy group the reaction with the primary amine (VIII) is preferably carried out in the presence of a base such as a tertiary amine e.g. triethylamine.

For process B when Y is the isocyanate group -N=C=O the reaction with the primary amine (VIII) is preferably carried out in an aprotic solvent such as a halohydrocarbon e.g. methylene chloride. Conveniently the isocyanate is generated in situprior to the addition of the primary amine (VIII).

The compounds of formula (VII) wherein Ra is an optionally substituted phenoxy group may be prepared from the primary amine (II) by reaction with the corresponding optionally substituted phenyl chloroformate in the presence of a base such as pyridine. The reaction may be carried out in a solvent such as a halohydrocabon e.g. dichloromethane and at a temperature from 0-500.

Compounds of formula (VII) wherein Ra is a 1-imidazole group may be prepared by reacting a compound of formula (II) with carbonyldiimidazole in the presence of a suitable solvent such as a halogenated hydrocarbon dichloromethane) or an ether tetrahydrofuran) at a temperature ranging from 00 to 800 (conveniently at room temperature).

Compounds of formula (VII) wherein Y is the isocyanate grouping -N=C=O or carbamoyl chloride -NHCOCI may be prepared from the primary amine (II) by reaction with phosgene (COC12) or triphosgene in a suitable solvent such as methylene chloride.

I I s WO 94/24151 PCT/EP94/01130 13 According to a further general process C compounds of formula may also be prepared by a reaction of the compound of formula (IX) H 0 N H H R4 N 0 R 0 (IX) with the halide R 2

R

1

NCOCH

2 Z wherein Z a leaving group chlorine or iodine.

e.g. bromine, The reaction is conveniently carried out by trea'ing the compound of formula (IX) with a strong base such as sodium hydride in a polar aprotic solvent such as N,N-dimethylformamide followed by reaction with the alkylating agent

R,R

2

NCOCH

2

Z.

Compounds of formula (II) may be prepared by formula (X) CONR R

I

reduction of compounds of

(X)

w wherein W is CH-N 3 or C=N-NHPh.

Compounds of formula wherein W is CH-N 3 may be reduced to a compound of formula (1I) by hydrogenation in the presence of a suitable catalyst such as palladium, on a support such as carbon or calcium carbonate, or platinum (IV) oxide. The reaction conveniently takes place in the presence of a solvent such as an alkanol ethanol) an ester ethyl acetate) or acetic acid.

II I WO 94/24151 PCT/EP94/01130 14 Compounds of formula wherein W is C=N-NHPh may be reduced to a compound of formula (II) by reaction with zinc and acetic acid. This reaction may be carried out a temperature with the range 0-500.

Compounds of formula wherein W is CHN 3 may be prepared from a compound of formula wherein W is CH 2 by treatment with a strong base such as sodium hydride or potassium tert-butoxide followed by tri-isopropyl benzenesulphonyl azide. The reaction conveniently takes place in a solvent such as an ether tetrahydrofuran) at a temperature in the range of -780 to 200.

Compounds of formula in which W is C=NNHPh may be prepared by reaction of the ortho-phenylenediamine (XI) with the diacid chloride (XII), in a suitable solvent such as an ether e.g. tetrahydrofuran

CONR

1

R

2 1 CH 2

NH

CIO

NNHPh

(XII)

(XI)

Compounds of formula wherein W is CH 2 may be prepared by reaction of the compound of formula (XI) with the diacid chloride (XIII)

CIO

CH

Clo 2

(XIII)

Compounds of formula (XI) are either known compounds or may be prepared by analogous methods. Thus for example a compound of formula (XI) may be prepared by alkylation of the amine (XIV).

s, WO 94/24151 PCT/EP94/01130

NH

2 -N J(XIV) rj"

R

3 Thus the amine (XIV) may be reacted with the compound R 1

R

2

NCOCH

2 Z in which Z is chlorine or bromine, optionally in the presence of sodium iodide in a solvent such as N,N-dimethylformamide.

In general, the compounds of formula (111), and (VI) are either known compounds or may be prepared according to methods simlar to those used for the preparation of known compounds, According to a further process a compound of formula may be prepared from the compound of formula (XV) wherein Rb is hydrogen CH2CO2R

NHCONHR

4

R

3 O (XV) by reaction of an activated derivative thereof with the amine R 1

R

2

NH.

Conveniently the reaction is carried out using the acid in the presence of a diimide such as dicyclohexyl carbodimide and hydroxybenzotriazole in a solvent such as dichloromethane or in the presence 2-ethoxy-1-ethoxycarbonyl-1,2dihydroquinoline in a solvent such as dimethoxyethane.

The compounds of formula (XV) wherein Rb is a hydrogen may be prepared by hydrolysis of the corresponding compound of formula (XV) wherein Rb is a tbutyl group, for example by reaction with trifluoroacetic acid. The compound of formula (XV) wherein Rb is t-butyl may be prepared by alkylation of the corresponding compound of formula (IX) with the halo ester Z-CH 2

CO

2 Rb wherein Z is halogen. Alternatively the compound (XV) wherein Rb is t-butyl may be prepared using the general processes A and B described above for I ,I WO 94/24151 PCT/EP94/01130 16 preparing the compounds of formula but starting with the appropriate Nsubstituted benzodiazepine derivative.

According to a further process a compound of formula may be converted into another compound of formula using conventional techniques.

Thus compounds of formula wherein R 4 is a phenyl group substituted by a carboxyl or carboxymethyl group may be prepared by hydrolysis of the corresponding compound of formula wherein R 4 is a phenyl group substituted by an alkoxycarbonyl or alkoxycarbonylmethyl group.

Compounds of formula (IX) may be prepared from the diamine (XIV), in which the primary amino group is protected as an p-methoxybenzyl derivative thereof, using the general procedures described above for preparing the compounds of formula from the corresponding orthophenylenediamine (XI) followed by removal of the N-protecting group p-methoxybenzyl using conventional procedures.

Compounds of formula contain at least one asymmetric carbon atom, namely the carbon atom of the diazepine ring to which the substituted urea grouping is attached. Specific enantiomers of the compounds of formula may be obtained by resolution of the racemic compound using conventional procedures such as chiral HPLC. Alternatively the required enantiomer may be prepared from the corresponding enantiomeric amine of formula (II) using any of the processes described above for preparing compounds of formula from the amine The enantiomers of the amine (II) may be prepared from the racemic amine (II) using conventional procedures such as salt formation with a suitably optically active acid such as R- camphorsulphonic acid or by preparative chiral HPLC.

In order that the invention may be more fully understood the following examples are given by way of illustration only.

In the Intermediates and Examples unless otherwise stated. Melting points (mp) are determined on a Gallenkamp mp apparatus and are uncorrected. All temperatures refers to OC. Column chromatography was carried out over silica gel. T.l.c. refers to thin layer chromatography on silica plates. Dried refers to I- ,1 I WO 94/24151 PCT/EP94/01130 17 solutions dried over anhydrous sodium sulphate. The following abbreviation are also used in the Examples. EA ethyl acetate, DMF N,N-dimethylformamide, THF tetrahydrofuran, DE diethyl ether, DCM dichloromethane, MeOH methanol, AcOH acetic acid, ee enantiomenc excess, RT retention time.

Intermediate 1 Cyclohexyl-(2-nitro-phenyl)-amine.

A mixture of 2-chloronitrobenzene (20g), potassium carbonate (35g) and copper iodide (1.21g) in cyclohexylamine (43.6ml) was heated at 1500 under nitrogen for 18h. The mixture was allowed to cool to room temperature and was adsorbed onto silica. This was chromatographed with hexane-EA (98:2) as eluent to give the title compound (22.94g) as an orange solid.

T.l.c. (98:2 hexane-EA) Rf 0.38 Intermediate 2 N-Cvclohexyl-benzene-1.2-diamine.

A solution of Intermediate 1 (10g) in ethyl acetate (400ml) was hydrogenated at 230 and 1 atm. pressure over 10% palladium on carbon (1g) for 4h. The catalyst was removed by filtration through hyflo and the filtrate evaporated to give the title compound (8.5g) as an orange oil.

hexane-EA) Rf 0.36 Intermediate 3 2-(2-Cyclohevylamino-phenvlamino)-1-pyrrolidin-1 -v-ethanone.

A mixture of Intermediate 2 2-oxo-2-(pyrrolidin-1-yl)ethylbromide (9.4g) and potassium carbonate (18.5g) in dry DMF (250ml) was stirred at 230 under nitrogen for 18h. The mixture was poured into water (600ml) and extracted with ethyl acetate (3x300ml). The combined extracts were washed with water (3x300ml) and saturated brine (200ml), dried and evaporated to give a brown oil. This was chromatographed with hexane-EA as eluent to give the itlIe compound (9.8g) as a cream solid, m.p.108-1100.

T.l.c. (1:1 hexane-EA) Rf 0.42 I I _I WO 94/24151 PCT/E P94/01130 18 Intermediate 4 1 -Cyclobexyl-5-(2-oxo-2-pyrrolidin-1 -vl-ethyl)-3-(phenyl-hydrazono)-1 benzolb[1 .4]diazepine-24-dione.

Solutions of Intermediate 3 (9.8g) and 2-(phenyl-hydrazono)-propanedioyl dichloride (8.36g) in dry THF (75m) were added at equal rates to dry THF (75m1) cooled to -100 under nitrogen. When the addition was complete the mixture was allowed to warm to room temperature and stir for 3h. The solvent was removed by evaporation to give the crude title compound (19.5g) as a yellow crunchy foam.

T.l.c. (DE) Rf 0.23 Int~rmdiate 3-Amino-1 -cyclhexyl-5-(2-oxo-2-Dyrrolidin-1 benzo[bl[1 .4]diazepine-24-dione.

A solution of Intermediate 4 (19g) in glacial acetic acid (200ml) was added dropwise to a stirring suspension of zinc dust (15g) in glacial acetic acid. The mixture was stirred at 230 for 1.5h whereupon the zinc was removed by filtration through hyflo and the filtrate evaporated to give a red oil. This was partitioned between water (200ml) and ethyl acetate (75m). The aqueous portion was adjusted to pH9 with solid Na 2

CO

3 and extracted with ethyl acetate (4x100ml).

The combined organic extracts were dried and evaporated to give a brown oil which was chromatographed with DCM-MeOH (95:5) as eluent to give the tit! compound (7.4g) as a pink crunchy foam.

T.l.c. (95:5 DCM-MeOH) Rf 0.26 Intermediate 6 -Cyclohexyl-2.4-dioxo-5-(2-oxo-2-pyrrlidin-1 -yl-ethyl)-23. 1 H-benzob1 .4]diazepin-3-yll-ureido}-benzoic acid.benzyl ester.

Triethylamine (108l) and triphosgene (77mg) were added sequentially to a solution of 3-amino-benzoic acid benzyl ester (177mg) in dry THF (1Oml) at 00 under nitrogen. More triethylamine (108l) was added and stirring continued at 00 for 30min. A solution of Intermediate 5 (250mg) in dry THF (10mi) was added and stirring continued at 230 for 18h. The mixture was then partitioned between 2N hydrochloric acid (5Omi) and ethyl acetate (50ml). The dried organic extract i ~--F9eP I

I~

WO 94/24151 PCT/EP94/01130 19 was evaporated and the residue chromatographed with EA-hexane as eluent to give the title compound (320mg) as a pale yellow solid.

T.l.c. (2:1 EA-hexane) Rf 0.17 I.r. (Solution in CHCI 3 3384;2939;1690;1658;1423cm- 1 Intermediate 7 3-Nitro-benzoic acid tert-butyl ester.

A solution of 3-nitrobenzoic acid (1.4g) in dry toluene (10ml) was treated with N,N-dimethylformamide di-tertbutyl acetal (10ml). The mixture was heated under reflux for 18h then cooled, diluted with ethyl acetate and washed consecutively with water, 8% sodium bicarbonate solution and saturated brine. The dried organic phase was evaporated to give the tiLt compound (1.02g) as a yellow oil.

T.l.c. (97:3 DCM-MeOH) Rf 0.66 Intermediate 8 3-Amino-benzoic acid tert-butyl ester.

A solution of Intermediate 7 (1g) in ethanol (20ml) was hydrogenated at 230 and latm pressure in the presence of 10% palladium on carbon as catalyst (200mg).

After 2h the mixture was filtered through hyflo and the filtrate evaporated. The residue was chromatographed with DCM-MeOH (97:3) as eluent to give the title compound (727mg) as a colourless oil.

T.l.c. (97:3 DCM-MeOH) Rf 0.35 Intermediate 9 Nitro-phenyl)-acetic acid benzyl ester.

A mixture of 3-nitrophenylacetic acid benzyl alcohol (2.9ml) and 4,4dimethylaminopyridine (300mg) in dry dichloromethane (50ml) was treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide The resulting yellow mixture was stirred at 230 for 3days whereupon the solvent was removed in acu The residue was partitioned between water (100ml) and ethyl actate (100ml) and the organic phase washed with water (2x100ml) and saturated brine (100ml) then dried and evaporated. The residue was chromatographed with hexane-EA as eluent then again with isohexane-DCM as eluent to give the itle compound (2.05g) as a pale yellow oil.

14 WO 94/24151 PCT/EP94/01130 T.I.c. (4:1 hexane-EA) Rf 0.35 Intermediate Amino-phenyl)-acetic acid benzyl ester.

A solution of Intermediate 9 (2.05g) in ethyl acetate (100ml) was hydrogenated at 230 and latm pressure in the presence of 5% platinum on carbon (200mg) as catalyst. After 90min the mixture was filtered through hyflo and the filtrate evaporated to give the title compound (.77g) as a pale yellow oil T.l.c. (DCM) Rf 0.35 Intermediate 11 (2-Fluoro-phenvl)-(2-nitro-phenl)-amine.

A mixture of 2-fluoroaniline pota:,3ium carbonate (2.5g) and copper(l)iodide (414mg) in 2-fluoronitrobenzene (16.9ml) was heated to 1800 under nitrogen for 18h. The cooled mixture was poured into water (300ml) and extracted with ethyl acetate (2x250ml) then the combined extracts were washed with saturated brine and evaporated. The residual brown oil was azeotroped with ethanol/water then toluene. The residue was chromatographed with hexane-DE (100:0 to 95:5) to give the title compound (3.25g) as a bright orange solid, m.p.58-9°.

T.l.c.(95:5 hexane-DE) Rf 0.45 Intermediat 12 N-(2-Fluorohbenyl)-benzene-1.2-diamine.

A solution of Intermediate 11(15.6g) in ethyl acetate (400ml) was hydrogenated at 230 and latm pressure in the presence of 5% platinum on carbon (2g) as catalyst. After 1h the mixture was filtered through hyflo and the filtrate evaporated to give the title compound (13.45g) as a yellow solid.

T.i.c. (9:1 hexane-DE) Rf 0.25 Intermediate 13 2-[2-(2-Fluoro-phenylamino)-phenylanino]-1-pyrrolidin-1-yl-ethanone.

A solution of 2-oxo-2-(pyrrolidin-1-yl)ethylbromide (12.8g) in dry DMF was added dropwise to a mixture of Intermediate 12 (13.45g) and potassium carbonate (27.5g) in dry DMF (100ml) at 230 under nitrogen. The mixture was

-I

WO 94/24151 PCTfEP94/01130 21 stirred at 600 for 4h then poured into 2N sodium carbonate solution (500ml) and extracted with ethyl acetate (400ml). The organic extract was washed with water (2x250ml) and.saturated brine (250ml), dried and evaporated. The residue was chromatographed with hexane-DE (50:50 to 0:100) as eluent to give the title compound (14.12g) as a pale brown solid.

T.l.c. (DE) Rf 0.53 Intermediate 14 -(2-Fluoro-phenvl)-5-(2-oxo-2-pyrrolidin-1 -yl-ethvl)-3-(phenyl-hydrazono)-1.5dihydro-benzo[b][1.4]diazepine-2.4-dione.

Solutions of Intermediate 13 (14.12g) and 2-(phenyl-hydrazono)-propanedioyl dichloride (11.04g) in dry THF (100ml) were added simultaneously and dropwise at equal rates to dry THF (100ml) cooled to -100 under nitrogen.

When the addition was complete the mixture was allowed to warm to room temperature and stir for 3.5h. The solvent was removed by evaporation to give the crude title compound (23g) as a yellow crunchy foam.

T.l.c. (EA) Rf Intermediate 3-Amino-1 -(2-fluoro-phenyl)-5-(2-oxo-2-pyrrolidin-1 benzo[b][1.4]diazepine-2.4-dione.

A solution of Intermediate 14 (23g) in glacial acetic acid (200ml) was added dropwise to a stirred suspension of zinc dust (22.2g) in glacial acetic acid (100ml) in a cold water-bath. The mixture was stirred at 230 for 2.5h whereupon the zinc was removed by filtration through hyflo and the filtrate evaporated. The residue was partitioned between water (150ml) and ethyl acetate (100ml) and the aqueous portion basified with solid Na 2

CO

3 The layers were separated and the aqueous phase further extracted with ethyl acetate then the combined organic extracts were washed with saturated brine, dried and evaporated. The residue was chromatographed with EA-MeOH (100:0 to 95:5) as initial eluent followed by DCM-MeOH (80:20) to give the title compound (11.07g) as a fawn powder.

T.l.c. (95:5 DCM MeOH) Rf 0.23 st I WO 94/24 151PC/9/013 PCT/EPI)4/01130 22 Ineme iate 1 (3-{341l -(2-Fluoro-p2henvl)-2.4-dioxo-5-(2-oxo-2-pyrolidin-1 -yl-ethvl)-2 .3A45tetrahydro-1 H-benzo[b][1 .4ldiazepin-3-yl] ureido}-phenyl)-acetic acid benzyl eter, A solution of Intermediate 15 (3g) in dry dichloromethane (40m1) was added dropwise to a stirred solution of phosgene in toluene (60ml) under nitrogen. The resulting mixture was stirred at 230 for 4h then nitrogen was bubbled through the mixture into ammonia solution for 18h. The mixture was then evaporated and the residue dried at 600 invau for 3h to give the intermediate isocyanate (3.3g) as a buff solid which was used without further characterisation. A solution of this isocyanate (400mg) in dry dichloromethane (3ml) was added dropwise to a stirred solution of amino-phenyl)-acetic acid benzyl ester (241 mg) in dry dichloromethane (2ml) at 230 under nitrogen. After 60mmn the solvent was removed ijnya= and the residue triturated with diethyl ether. The solid was filtered off and dried to give the l~e cmpond (263mg) as an off-white solid, m.p.1 04-60.

T.l.c. (EA) Rf 0.54 Inemeiate 17 2-Bromno-1 -(3.3-dimethyl-piperidin-1 -yI)-ethanone.

A mixture of 3,3-dimethylpiperidine (l0g) and triethylamine (12.3m1) in dry dichloromethane (50mi) was added dropwise to an ice-cold solution of bromoacetyl bromide (7.7m1) in dry dichloromethane (lO0mI). The mixture was stirred at 230 for 1 8h then recooled to 00. Iced water (200ml) was added and the layers separated. The aqueous layer was further extracted with dichioromethane (2x200mI) then the combined extracts were washed with 2N HCl (200ml) and saturated brine (200m1), dried and evaporated to give the ftilQ cmpound (17.28g) as a brown oil.

T.l.c. (DE) Rf 0.49 Intemite 8 1 -(3.3-Dimethyl-piperidin-1 -vl)-2-[2-(2-fluoro-phenylaminio)-phenylaminojetanone.Q A mixture of Intermediate 12 and potassium carbonate (6.15g) in dry DMF (1 OmI) was treated with a solution of 2-bromo-1 -(3,3-dimethyl-piperidin-1 -yl)- WO 94/24151 PCT/EP94/01130 23 ethanone (3.5g) in dry DMF (10ml) and stirred at 230 under nitrogen for 3 days.

The mixture was poured into water and extracted with ethyl acetate. The combined extracts were washed with water and saturated brine, dried and evaporated to give a brown oil. This was chromatographed with DCM-MeOH (100:0 to 97:3) as eluent to give the title compound (3.17g) as a pale brown foam.

T.l.c. (DCM) Rf 0.23 Intermediate 19 1-[2-(3.3-Dimethyl-piperidin-1-yl)-2-oxo-ethy!]-5-(2-fluoro-phenyl)-3-(phenvlhydrazono)-1.5-dihydro-benzo[b][1.4]diazepine-2.4-dione.

Solutions of Intermediate 18 (3.08g) and 2-(phenyl-hydrazono)-propanedioyl dichloride (2.12g) in dry THF (100ml) were added at equal rates to dry THF cooled to -100 under nitrogen. When the addition was complete the mixture was allowed to warm to room temperature and stirred for 2h. The solvent was removed by evaporation to give the crude title compound (5.25g) as a yellow crunchy foam.

T.l.c. (95:5 DCM-MeOH) Rf 0.68 Intermediate 3-Amino-1-[2-(3.3-dimethyl-piperidin-1-vl)-2-oxo-ethyll-5-(2-fluoro-phenvl)-1.5dihydro-benzo[b][1.4]diazepine-2.4-dione.

A solution of Intermediate 19 (5.15g) in glacial acetic acid (75ml) was added dropwise to a stirring suspension of zinc dust (4.47g) in glacial acetic acid (50ml) in a cold water-bath. The mixture was stirred at 230 for 6h whereupon the zinc was removed by filtration through hyflo and the filtrate evaporated. The residue was partitioned between water and ethyl actate and the aqueous portion basified with solid Na 2

CO

3 The organic extract was washed with saturated brine, dried and evaporated. The residue was chromatographed with DCM-MeOH (95:5) as eluent to give the titleQ c.nound (2.38g) as a pale brown solid.

T.l.c. (95:5 DCM-MeOH) Rf 0.18 ~LL~s-- WO 94/24151 WO 9424151PCTJE P94/01 130 24 Inemdiate 21 .3-Di methyl-piperidin-1 -yl)-2-oxo-ethyl]-5-(2-fli'oro-phenyl)-2.4dioxo-2.3 .4.5-tetrahydro-1 H-benzofb[1 .41diazepin-3-yl]-ureidol-benzoic acid tet-utl ester Triethylamine (0.31 t-l) and triphosgene (217mg) were added sequentially to a solution of 3-amino-benzoic acid tert-butyl ester (423mg) in dry THF (l0mi) at.

00 under nitrogen. More triethylamine (0.31 ml) was added and stirring continued at 00 for 30mmn. A solution of !ntermediate 20 (800mg) in dry THF (l0mi) was added and stirring continued at 230 for 18h. The mixture was then partitioned between phosphate buffer solution (pH6.5) and dichloromethane.

The combined organic extracts were washed with saturated brine, dried and evaporated and the residue chromatographed with DCM-MeOH (97:3) as eluent to give a pale green solid which was triturated with DE-hexane to give the tile cm'o (1 .07g) as a white solid, m.p.1 T.l.c. (97:3 DCM-MeOH) Rf 0.15 l-il-Qyclohx.-4-dioxo-5-(2-xo-2-pyrrolidin-1 -yl-ethyl)-2.3 .4.5-te rahydro- IU-bnzQ=]t 4]iazepin-3-yC-3-(4-fluoro -phenyl)urea.

4-FLIWOcPhanyl isocyanate (650i) was added to a solution o'f Intermediate (2g) in dry dichioromethane (50ml) at 230 under nitrogen. TVhe resulting mixture was stirred for 1 h then adsorbed onto silica and chromatographed with EA- MeOH (100:0 to 95:5) as eluent to give the lijW-Q=p n (1.92g) as a white T.I. c. (EA) Rf 0.42 The compound of Example 1 (1 .7g) was separated into its 2 enantiomars (Isomer 1 and Isomer 2) by chir& HPL.C.

Column: Chiralcel OD 25cmi x 20mm id Eluent: Hexane-EtOH (70:30) Flow rate: 20 ml min- 1 Detection: uv 254nm Isomer 1, (452mg) as a white solid RT 8.6 min H.p.l.c. >99.5% ee WO 94/24151 WO 9414151PEP94O 1130 T.I.c. (EA) Rf 0.42 l.r. (Solution in CHCI 3 3622;3091 ;2938;2895;2403;1 657;1 51 5;1 425;1 189; 1 047;929cm- 1 Isomer 2, (488mg) as a white solid RT 15.1 min. H.p.l.c. 98.8%ee T.l.c. (EA) Rf 0.42 I.r. (KBr disc) 3366;2935;1 695;1 657;1 558;1 51 0;1 422;1 206;833;763cm- 1 1 -Cyclohexyl-2.4-dioxo-5-(2-oxo-2-pyrrolidin-1 -yl-ethyl)-2.3.4.5-tetrahydro- 1 H-benzoQfblt iZepin-a-yI]-ureidol-benzoic acid.

A solution of Intermediate 6 (248mg) in ethyl acetate (1lOmI) and THIF (5mI) was hydrogenated at 230 and 1 atm pressure overnight in the presence of palladium on carbon (25mg) as catalyst. More catalyst (25mg) was added and hydrogenation continued for 2h whereupon the mixture was filtered through hy-flo and the filtrate evaporated to give the Iitlii cQMpund (216mg) as a white solid, m.p.275-6 0 T.l.c. (100:2 EA-AcOH) Rf 0.25 l.r. (KBr disc) 3369;2935;1 700;1 659;1 557;1 499;1 431 ;1 233;760cm- 1 (3-{3-rl -(2-Fluoro-p2henyl)-2.4-dio2xo-5-(2-oxo2-2-pyrrolidi n-i -yl-ethyl)-2 .3.4.5tetrahydro-1 H-bpnzo[bl .4ldiazepin-3-vll-ureid ol Phenyl)-acetic acid.

A solution of Intermediate 16 (1 77mg) in THF (l0mI) was hydrogenated at 230 and latin pressure in the presence of 10% palladium on carbon (10mg) as catalyst. After 90min the mixture was fitered through hyflo and the filtrate evaporated to give the title cmpound..(67mg) as a white solid, m.p. 197.90.

T.l.c. (EA) Rf 0.17 1. r. (KBr disc) 3331 ;1 708;1 651 ;1 562;1 499;1 454;1403;1 238;761 cm- 1 Exmple 4 3-{3-f1 .3-Di methyl-piperidin-1 -yl)-2-oxo-ethyll-5-(2-f Luor-phenvl)-2 .4di~x-a4.5A.trahydro-1 H-benzo~b][1, 14diazepin-3-yll-ureido}-benzoic acid.

A solution of Intermediate 21 (400mg) indichloro methane (20ml) was treated with trifluoroacetic acid (2m1) and stirred at 230 under nitrogen for 1 h. The WO 94/24151 PCTIEP94IOI 130 26 mixture was concentrated hnyL= and the residue chromatographed with 0CM- MeOH (95:5) as eluent to give the ftile cmpound (344mg) as a white solid, m.p.225 0 dec.

T.l.c. (95:5 DCM-MeCH) Rf 0.26 Using the general processes A, B, C and D described above the following compounds of the invention have also been prepared.

Table 1

CONQI

CH 2 N H CO N H

H

3 4-00H 3 3-F 3-NO 2 3-C H 2

CN

H

3-CON H 2 3-N(CH 3 2 4-F 254-50 180-20 1 88-900 173-50 210-20 216-80 275-70 251-30 *Rf 0.3 T.l.c. cyclohexane E A:AcOH 1:1:0.0.4 PCT/E P94/01 130 WO 94/24151 Table 2 co

N-

Ex. Q 14 16 17 18 19 Table 3 Ph Ph Ph 2-F-Ph 2 F-Ph 2F-Ph 3-tetrazolyl 3-CH 3 3-CONHSO 2

CH

3 4-F 3-C H 3 3-CO 2

H

2430 234-60 1 99-2000 1 55-70 1 95-70 -225-70

NR

1

R

2 NHCONH

-F

WO 94/24151 PCT/EP94/01130 Ex. No.

Proces

N

N

N

No 2400 1700 2600 220-5 0 dec 255-80 Pharmacy Examples Tablets a. Active ingredient Lactose anhydrous USP Microcrystalline Cellulose NF Pregelatinised starch Ph.Eur.

Magnesium stearate USP Compression weight 163mg 69mg 300mg The active ingredient, microcrystalline cellulose, lactose and pregelatinised starch are sieved through a 500 micron sieve and blended in a suitable mixer.

The magnesium stearate is sieved through a 250 micron sieve and blended with the active blend. The blend is compressed into tablets using suitable punches.

I

WO 94/24151 PCT/EP94/01130 29 b. Active ingredient Lactose monohydrate USP 120mg Pregelatinised starch Ph.Eur. Crospovidone NF 8mg Magnesium stearate USP 2.m Compression weight 200mg The active ingredient, lactose and pregelatinised starch are blended together and granulated with water. The wet mass is dried and milled. The magnesium stearate and Crospovidone are screened through a 250 micron sieve and blended with the granule. The resultant blend is compressed using suitable tablet punches.

Capsules a. Active ingredient Pregelatinised Starch Ph.Eur. 148mg Magnesium stearate USP 2mg Fill weight 200mg The active ingredient and pregelatinised starch are screened through a 500 micron mesh sieve, blended together and lubricated with magnesium stearate (meshed through a 250 micron sieve). The blend is filled into hard gelatin capsules of a suitable size.

b. Active ingredient Lactose monohydrate USP 223mg Povidone USP 12mg Crospovidone NF 12mg Magnesium stearate .3mg Fill weight 300mg The active ingredient and lactose are blended together and granulated with a solution of Povidone. The wet mass is dried and milled. The magnesium WO 94/24151 PCT/E94/01130 stearate and Crospovidone are screened through a 250 micron sieve and blended with the granule. The resultant blend is filled into hard gelatin capsules of a suitable size.

CCK-B. Receptor Binding Affinity CCK-B receptor binding studies were carried out using HeLa cell membranes which had been stabily transfected with the human CCK-B receptor cloned from temporal cortex cDNA library.

Measurement of CCK-B binding affinity.

The transfected HeLa cell membranes were incubated with 30pM [12 5 1]BHsCCK-8 in the presence of various concentrations of the test compound (1pM to 1 JM). All experiments were performed in an assay volume of 2 50 pl.

Membranes were harvested onto GF/C glass fibre filter paper (Whatman, UK) by rapid filtration using a Cell Harvester (Brandel model M-24R) and washed three times with HEPES Wash buffer chilled to 4°C. The radioactivity trapped on individual filter discs was counted over 60 seconds using a gamma counter (Mini Gamma counter, LKB, Wallac, Finland) at a counting efficiency of 77%. A control experiment was also carried out in the absence of a test compound.

Analysis of the data was performed by computer-assisted non-linear regression (ALLFIT programmes DeLean et al., 1978;). IC50 values for the test compounds were converted to K i values using the Cheng Prussof equation (K i ICo/(1 (Cheng and Prussof, 1973). Results obtrained with representative compounds of the invention in this test are given below and expressed as pKi values.

L I WO 94/24151 PCT/EP94/01130 31 CCK-A Receptor Binding Affinity CCK-A receptor binding studies were carried out using COS-M6 cell membranes which have been transiently transfectJd with human gall bladder CCK-A receptor cDNA.

The clone was transiently transfected into COS-M6 cells grown to confluency using the dimethylaminoethyl dextran method based on the method of Seed and Araffo. Proc. Natl, Acad, Sci. USA 84, 3365 1987. The required transfected cell membranes were then isolated in a conventional manner.

The affinity of compounds of the invention for the CCK-A receptor was determined using the procedures described above for the CCK-B receptor however, in these experiments the test compound was tested at concentrations in the range 10pM to CCK-B Binding Studies Example No pKi 1 1(isomer 1) 1 (isomer 2) 8.1 2 8.7 3 8.4 4 8.8 7 8.3 8 8.6 8.4 12 14 8.8 8.7 as st Ib I I':\OPE\MIC\~5675.94006- 6/1/98 -32- The compounds of the invention are essentially non-toxic at therapeutically active doses.

Thus no untoward effects were observed when the compound of Example 1 (isomer 1) was administered to the gastrin fistula rat at doses of 0.3 and 1 mg/kg iv. At these doses gastric acid secretion was significantly inhibited.

Throughout this specification and claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

o 0 o 0 0

Claims (9)

1. Compounds of the general formula (1) CON RR CH 2 1 0* NIHCONHR 4 0 (1) R M and physiologically acceptable salts thereof wherein the group NRIR, S:represents a 5-7 membered saturated heterocylic ring which may be substituted by one or two methyl groups; R, is C 1 -6alkyi, C 3 6 cycloalkyl or phenyl optionally substituted by 1 or 2 halogen atoms; R 4 is phenyl or phenyl substituted by one or two groups selected from halogen, C 1 4 alkyl, trifluoromethyl, trifluoromethoxy or R 5 wherein n is zero or 1 and R 5 represents C 1 4 alkoxy, hydroxy, nitro, cyano, C0 2 R 6 S(O)pCH 3 NR 7 R 8 CONH 7 R 8 SO 2 NR 7 CO(Cl. 4 alkyl), tetrazolyl, carboxamidotetrazolyl, or a 3- trifluoromethyl 1,2,4-triazolyl; R 6 is hydrogen, C 1 4 alkyl or benzyl; R 7 is hydrogen or C 1 4 alkyl, R8 is hydrogen, C 1 4 alkyl, SO 2 CH 3 or SO 2 CF 3 X represents hydrogen, C 1 4 alkyl or halogen; m is zero, 1 or 2, and p is zero, 1 or 2.

2. Compounds as claimed in Claim 1 wherein NR 1 R, represents pyrrolidino, 2,5-dimethylpyrrolidino, piperidino, 3,3-dimethylpiperidino, 4,4- dimethylpiperidino or 2,6-dimethylpiperidino.

3. Compounds as claimed in Claim 1 or 2 wherein NR 1 R 2 represents pyrrolidino. L-l L P;WOPHR\iCX&5675.9,t06 51/93 -34-

4. Compounds as claimed in any one of Claims 1 to 3 wherein R 3 represents phenyl, 2- fluorophenyl or cyclohexyl. Compounds as claimed in any one of Claims to 4 wherein R 3 represents cyclohexyl.

6. Compounds as claimed in any one ofl Claims 1 to 5 wherein represents phenyl, 3-methyiphenyl, 3,5-dimethylphenyl, 3-dimelhylaminopheflyl, 4- fluorophenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3-nitrophenyl, 3- cyanomethyiphenyl, 3-carboxamidophenyl, 3-carboxyphenyl, 3 carboxymethylphenyl or 3-(1

7. Compounds as claimed in any one of claims i to 6 wherein R 4 represent 10 phenyl, 4-f luorophenyl, 3-dimethylaminophienyl, 3-carboxylphenyl, 3- carboxymethyiphenyl or 3-(1 B: 1 Cyclo hexyl-2,4-dioxo-5-(2 -oxo-2 -py rro lid -i1 -ylI-eth yl) -2,3 tetrahydro- 1 H-benzo[b][1 ,4]diazepin-3-yl]-3-(4-fluoro-phenyl)urea and enantiomers thereof. i s 9. A compound selected from -Cyclohexyl-2,4-dioxo-5-(2-oxo-2-pyrrolidifl-l -yl-ethyl)-2 13,4 ,5-tetrahyd ro- 1 H-benzo[b][1 ,4]diazepin-3-yl]-ureido)-beflzoic acid; -(2-Fluoro-phenyl)-2,4-dioxo-5-(2-oxo-2-PYrrolidin-l -yl-ethy!)-2,3 tetrahydro-1 H-benzo~b][l ,4]diazepin-3-yl]-u reido)-phely [)-acetic acid; -[2-(3,3-Dimethyl-piperidin-1 -yl)-2-oxo-ethyl]-5-(2-fluoro-pheflyl)-2,4- dioxo-2,3 ,4,5-tetrahydro-1 H-benzo[b][1 ,4]diazepin-3-ylI-ureido}-benzoic acid and enantiomers thereof. The use of a compound as defined in any one of Claims 1 to 9 in the manufacture of a medicament for the treatment of conditions where modifiCation 250f the effects of gastrin and/or CCK is of therapeutic benefit. M ~Y~ I'.\Of'P3R\MJC\C 6'759I 1. i6 ll./1

11. A pharmaceutical composition comprising a compound as defined in any one of Claims 1 to 9 in admixture with one or more physiologically acceptable carriers or excipients.

12. A method of treatment of a mammal including man for conditions where modifications of the effects of gastrin and/or CCK is of therapeutic benefit comprising administration of an effective amount of a compound as defined in any one of Claims 1 to 9.

13. A process for the preparation of a compound as defined in Claim i which comprises. reacting a compound of formula (II), E NR 1 R 2 0I N N wherein RI 1 R2 R3, X and n have the meanings defined in formula with a compound R 4 Y selected from an isocyante (111), carbamoyl chloride (IV), imidazolide or phenyl carbamate (VI) wherein Ra is an optionally substituted phenoxy group, II N-R, CICONHR N^N1 N R H (III) (IV) RaCONHR 4 (VI) and R4 has the meanings defined in formula (II). WO 94/24151 PCT/EP94/01130 reacting a compound of formula (VII) NRIR 2 N S 0 (VII) wherein R 1 R 2 R 3 X and m have the meanings defined in formula and Y is a group selected from NCO, NHCOCI, or NHCORa wherein Ra is an optionally substituted phenoxy or imidazole group with the amine R 4 NH 2 (VIII) wherein R 4 has the meaning defined in formula reacting a compound of formula (IX) wherein R 3 R 4 X and m have the meanings defined in formula H O N H H R (X N N 7 4 0 (IX) R, with the compound R 2 RiNCOCH 2 Z wherein Z is a leaving group; Reacting an activated derivative of the compound of formula (XV) wherein R 3 R 4 X and m have the meanings defined in formula and Rb represents hydrogen CHCOR b (X)M NHCONHR 4 0 (XV) WO 94/74151 PCT/EP94/01130 with amine R 1 R 2 NH; and thereafter if necessary or desired either before or after separation into its stereochemical isomers converting one compound of formula into another compound of formula -4- INTERNATIONAL SEARCH R1EPORT n xal '.pplcison No ePCTE 94/01130 A. CLASSIFICAT110 .1 SUgtECr MATTR IPC 5 C07i. u6 A61K37/02 According to International Patent Classiicuion (IPC) or to both national classfication and IPC B. FIELDS SEARCHED Mindun documentation searched (dlassfication system followed by classification symbols) IPC 5 C07K C07D Documnentation searched, other than mnimum documentaion to the extent that such documeni-s Are .icluded in thc fields searched Electronic data base consulted during the internatzonal search (name of data base and, where practical, search term used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category' Citation of document, with indication, where appropriate, of the relevant paw-ges Relevant to claim No. PX WO,A,93 14074 (GLAXO SPA) 22 July 1993 1,4,6,7, 10-14 see claims; example X EP,A,0 487 207 (MERCK CO. INC.) 27 Mal, 1-3,6,7, 199ti 11-14 see claims; examples 6,7, 11, 13, 14 A WO,A,91 13862 (RHONE-POULENC RORER 1,10-13 19 September 1991 see claims; examples A EP,A,O 376 849 (ROUSSEL-UCLAF) 4 July 1990 1,10-13 cited in the application see claims Further documents are listed in the continuation of box C. MV Patent family members are listed in annex *Spec, categories of cited documents: later doa2ucnt published after the international filing dale A .oumet deinig te geera stae o th w isnotor piriority date and not in conflict with the application but considered to be of particular relevance 'netion eradthpinilorhoy ygte earlier document but published on or after the international document of particular relevance; the claimed invention fiing date cannot be considered novel or cannot be corisidered to WL document which may throw doubts on priority claim~s) or involve an inventive step when the document is tiaken aone which is cited to establish the publication date of another document of paricular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the O0' document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docte. other means merits, such combination being obvious to a person skilled document published prior to the international filing date brt in the art. later than the priority date claimed W' document member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report 2-2 June 1994 9 :a7; Name and mnailing addres of the ISA Authosized officer European Patent Office, P.B. 5818 Patentlanm 2 NL 2280 HYV Rijswijk Tel. (+31.70) 34D.2040,Mc, 31 651 epo nl,Fu r C Fax: 31.70) 340-3016 u r C F~orm PCT/ISA/210 (taccod sheet) (July 1992) INTERNATIONAL SEA RCHE REPORT Int, M A*~CaUOn No PCT/EP 94/01130 Patent document I Publication IPatent family I Publication cited in search report date member(s) -7date WO-A-9314074 22-07-93 AU-A- 3193593 22-07-93 AU-B- 3450193 03-08-93 CA-A- 2087672 22-07-93 CN-A- 1074678 28-07-93 EP-A- 0558104 01-09-93 EP-A-0487207 27-05-92 US-A- 5206234 27-04-93 JP-A- 5105667 27-04-93 WO-A-9113862 A-09-91 FR-A- 2659653 20-09-91 AU-B- 637220 20-05-93 AU-A- 7570491 10-10-91 EP-A- 0520016 30-12-92 JP-T- 5504970 29-07-93 EP-A-0376849 04-07-90 FR-A- 2641280 06-07-90 JP-A- 2215774 28-08-90 US-A- 4988692 29-01-91 Farm PCT15Ad210 (patent family annax) (July 1992) M .ernational applcation No. PCT/ EP 94/ 01130 INTERNATIONAL SEARCH REPORT I- I I Box I Observations where certain claims were found unsearchable (Continuation of item I of first sheet) This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1. Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: Remark: Although claim 13 is directed to a method of treatment of (diagnostic method practised on) the human/animal body the search has been carried out and based on the alleged effects of the compound/composition. 2. Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: 3. D Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of invention is la- ing (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: 1. D As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. O As all searchable claims could be searches without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.: 4. No required additional search fees were timely paid by the applicant Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest The additional search fees were accompanied by the applicant's protest. No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (continuation of first sheet (July 1992) I