Benzodiazepine analogs of the formula: ##STR1## are disclosed which are antagonists of gastrin and cholecystokinin (CCK).

FIELD OF THE INVENTION 
This invention relates to the discovery of Benzodiazepine analogs of 
Formula I for use as antagonists of cholecystokinin (CCK) and gastrin when 
administered to animals, preferably humans. 
BACKGROUND OF THE INVENTION 
The Benzodiazepine analogs of Formula I of this invention are useful in 
treating various diseases caused by an excess of CCK or gastrin. 
Cholecystokinins (CCK) and gastrin are structurally related neuropeptides 
which exist in gastrointestinal tissue and in the central nervous system 
(see, V. Mutt, Gastrointestinal Hormones, G. B. J. Glass, Ed., Raven 
Press, New York, p. 169 and G. Nission, ibid, p. 127. 
Cholecystokinins include CCK-33, a neuropeptide of thirty-three amino acids 
in its originally isolated form (see, Mutt and Jorpes, Biochem. J. 125, 
678 (1971)), its carboxyl terminal octapeptide, CCK-8 (also a 
naturally-occurring neuropeptide and the minimum fully active sequence), 
and 39- and 12-amino acid forms. Gastrin occurs in 34-, 17- and 14-amino 
acid forms, with the minimum active sequence being the C-terminal 
tetrapeptide, Trp-Met-Asp-Phe-NH.sub.2, which is the common structural 
element shared by both CCK and gastrin. 
CCK's are believed to be physiological satiety hormones, thereby possibly 
playing an important role in appetite regulation (G. P. Smith, Eating and 
Its Disorders, A. J. Stunkard and E. Stellar, Eds, Raven Press, New York, 
1984, p. 67), as well as also stimulating colonic motility, gall bladder 
contraction, pancreatic enzyme secretion, and inhibiting gastric emptying. 
They reportedly co-exist with dopamine in certain mid-brain neurons and 
thus may also play a role in the functioning of dopaminergic systems in 
the brain, in addition to serving as neurotransmitters in their own right 
(see: A. J. Prange et al., "Peptides in the Central Nervous System", Ann. 
Repts. Med. Chem. 17, 31, 33 [1982] and references cited therein; J. A. 
Williams, Biomed. Res. 3 107 [1982]; and J. E. Morley, Life Sci. 30, 479, 
[1982]). 
The primary role of gastrin, on the other hand, appears to be stimulation 
of the secretion of water and electrolytes in the stomach, and, as such, 
it is involved in control of gastric acid and pepsin secretion. Other 
physiological effects of gastrin then include increased mucosal blood flow 
and increased antral motility. Rat studies have shown that gastrin has a 
positive trophic effect on the gastric mucosa, as evidenced by increased 
DNA, RNA and protein synthesis. 
Antagonists to CCK and to gastrin have been useful for preventing and 
treating CCK-related and/or gastrin-related disorders of the 
gastrointestinal (GI) and central nervous (CNS) systems of animals, 
preferably mammals, and especially those of humans. Just as there is some 
overlap in the biological activities of CCK and gastrin, antagonists also 
tend to have affinity for both receptors. In a practical sense, however, 
there is enough selectivity for the different receptors that greater 
activity against specific CCK- or gastrin-related disorders can often also 
be identified. 
Selective CCK antagonists are themselves useful in treating CCK-related 
disorders of the appetite regulatory systems of animals as well as in 
potentiating and prolonging opiate-mediated analgesia, thus having utility 
in the treatment of pain [see P. L. Faris et al., Science 226, 1215 
(1984)]. Selective gastrin antagonists are useful in the modulation of CNS 
behavior, as a palliative for gastrointestinal neoplasms, and in the 
treatment and prevention of gastrin-related disorders of the 
gastrointestinal system in humans and animals, such as peptic ulcers, 
Zollinger-Ellison syndrome, antral G cell hyperplasia and other conditions 
in which reduced gastrin activity is of therapeutic value. See e.g. U.S. 
Pat. No. 4,820,834. It is further expected that the CCK antagonists of 
Formula I are useful anxiolytic agents particularly in the treatment of 
panic and anxiety disorders. 
Since CCK and gastrin also have trophic effects on certain tumors [K. 
Okyama, Hokkaido J. Med. Sci., 60, 206-216 (1985)], antagonists of CCK and 
gastrin are useful in treating these tumors [see, R. D. Beauchamp et al., 
Ann. Surg., 202, 303 (1985)]. 
Distinct chemical classes of CCK-receptor antagonists have been reported 
[R. Freidinger, Med. Res. Rev. 9, 271 (1989)]. The first class comprises 
derivatives of cyclic nucleotides, of which dibutyryl cyclic GMP has been 
shown to be the most potent by detailed structure-function studies (see, 
N. Barlas et al., Am. J. Physiol., 242, G 161 (1982) and P. Robberecht et 
al., Mol., Pharmacol., 17, 268 (1980)). 
The second class comprises peptide antagonists which are C-terminal 
fragments and analogs of CCK, of which both shorter 
(Boc-Met-Asp-Phe-NH.sub.2, Met-Asp-Phe-NH.sub.2), and longer 
(Cbz-Tyr(SO.sub.3 H)-Met-Gly-Trp-Met-Asp-NH.sub.2) C-terminal fragments of 
CCK can function as CCK antagonists, according to recent 
structure-function studies (see, R. T. Jensen et al., Biochem. Biophys. 
Acta., 757, 250 (1983), and M. Spanarkel et al., J. Biol. Chem., 258, 6746 
(1983)). The latter compound was recently reported to be a partial agonist 
[see, J. M. Howard et al., Gastroenterology 86(5) Part 2, 1118 (1984)]. 
The third class of CCK-receptor antagonists comprises the amino acid 
derivatives: proglumide, a derivative of glutaramic acid, and the N-acyl 
tryptophans including para-chlorobenzoyl-L-tryptophan (benzotript), [see, 
W. F. Hahne et al., Proc. Natl. Acad. Sci. U.S.A., 78, 6304 (1981), R. T. 
Jensen et al., Biochem. Biophys. Acta., 761, 269 (1983)]. All of these 
compounds, however, are relatively weak antagonists of CCK (IC.sub.50 : 
generally 10.sup.-4 M[although more potent analogs of proglumide have been 
recently reported in F. Makovec et al., Arzneim-Forsch Drug Res., 35 (II), 
1048 (1985) and in German Patent Application DE 3522506A1], but down to 
10.sup.-6 M in the case of peptides), and the peptide CCK-antagonists have 
substantial stability and absorption problems. 
In addition, a fourth class consists of improved CCK-antagonists comprising 
a nonpeptide of novel structure from fermentation sources [R. S. L. Chang 
et al., Science, 230, 177-179 (1985)] and 3-substituted benzodiazepines 
based on this structure [published European Patent Applications 167 919, 
167 920 and 169 392, B. E. Evans et al, Proc. Natl. Acad. Sci. U.S.A., 83, 
p. 4918-4922 (1986) and R. S. L. Chang et al, ibid, p. 4923-4926] have 
also been reported. 
No really effective receptor antagonists of the in vivo effects of gastrin 
have been reported (J. S. Morley, Gut Pept. Ulcer Proc., Hiroshima Symp. 
2nd, 1983, p. 1), and very weak in vitro antagonists, such as proglumide 
and certain peptides have been described [(J. Martinez, J. Med. Chem. 27, 
1597 (1984)]. Recently, however, pseudopeptide analogs of tetragastrin 
have been reported to be more effective gastrin antagonists than previous 
agents [J. Martinez et al., J. Med. Chem., 28, 1874-1879 (1985)]. 
A new class of Benzodiazepine antagonist compounds has further been 
reported which binds selectively to brain CCK (CCK-B) and gastrin 
receptors [see M. Bock et al., J. Med. Chem., 32, 13-16 (1989)]. One 
compound of interest reported in this reference to be a potent and 
selective antagonist of CCK-B receptors is 
(R)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N.su 
p.1 -(3-methylphenyl) urea (See U.S. Pat. No. 4,820,834.) One disadvantage 
of the new CCK-B compound reported in Bock et al., J. Med. Chem., 32, 
13-16 (1989) and U.S. Pat. No. 4,820,834, is that these CCK-B compounds 
are poorly water soluble. 
It is, therefore, an object of the present invention to provide antagonists 
of CCK and gastrin. If an antagonist compound could be prepared which 
would bind with the cell surface receptor of CCK or gastrin, then the 
antagonist compounds of this invention could be used to block the effect 
of CCK and gastrin. Another object of the present invention is to provide 
novel CCK and gastrin antagonist compounds which are water soluble. Other 
objects of the present invention are to provide methods of inhibiting the 
action of CCK and gastrin through the administration of novel 
benzodiazepine analog compounds. The above and other object are 
accomplished by the present invention in the manner more fully described 
below. 
SUMMARY OF THE INVENTION 
The present invention provides Benzodiazepine analogs of the formula: 
##STR2## 
for use as antagonists of CCK and gastrin. The above-mentioned compounds 
can be used in a method of acting upon a CCK and/or gastrin receptor which 
comprises administering a therapeutically effective but non-toxic amount 
of such compound to an animal, preferably a human. A pharmaceutical 
composition comprising a pharmaceutically acceptable carrier and, 
dispersed therein, an effective but non-toxic amount of such compound is 
another aspect of this invention.

DETAILED DESCRIPTION OF THE INVENTION 
Benzodiazepine analogs of Formula I provide antagonists of CCK and gastrin. 
The present invention further provides novel CCK and gastrin antagonist 
compound which are water soluble. The Benzodiazepine analogs of Formula I 
are useful in a method of antagonizing the binding of CCK to CCK receptors 
or antagonizing the binding of gastrin to gastrin receptors. The novel 
Benzodiazepine analogs of the present invention are illustrated by 
compounds having the formula: 
##STR3## 
wherein: R.sup.1 is H, --(CH.sub.2).sub.2 --CO.sub.2 CH.sub.3, or 
--(CH.sub.2).sub.2 --CO.sub.2 H; 
R.sup.2 is 
##STR4## 
R.sup.3 is absent, one or two of Halogen or CH.sub.3 ; R.sup.4 is absent, 
one or two of Halogen or CH.sub.3 ; 
R.sup.5 is 
##STR5## 
or the optical isomers, prodrugs or pharmaceutically acceptable salts 
thereof. 
Preferred compounds of this invention as set forth in the Examples are: 
N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-carbox 
yethyl-N'-{[3-methylphenyl]-urea}; and 
N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-carboxy 
ethyl-N'-{[3-methylphenyl]-urea}. 
It will be appreciated that formula (I) is intended to embrace all possible 
isomers, including optical isomers, and mixtures thereof, including 
racemates. 
The present invention includes within its scope prodrugs of the compounds 
of formula I above. In general, such prodrugs will be functional 
derivatives of the compounds of formula I which are readily convertible in 
vivo into the required compound of formula I. Conventional procedures for 
the selection and preparation of suitable prodrug derivatives are 
described, for example, in "Design of Prodrugs", ed. H. Bungaard, 
Elsevier, 1985. 
The pharmaceutically acceptable salts of the compounds of Formula I include 
the conventional non-toxic salts or the quarternary ammonium salts of the 
compounds of Formula I formed, e.g., from non-toxic inorganic or organic 
acids. For example, such conventional non-toxic salts include those 
derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, 
sulfamic, phosphoric, nitric and the like; and the salts prepared from 
organic acids such as acetic, propionic, succinic, glycolic, stearic, 
lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, 
phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, 
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, 
isethionic, and the like. 
The pharmaceutically acceptable salts of the present invention can be 
synthesized from the compounds of Formula I which contain a basic or 
acidic moiety by conventional chemical methods. Generally, the salts are 
prepared by reacting the free base or acid with stoichiometric amounts or 
with an excess of the desired salt-forming inorganic or organic acid or 
base in a suitable solvent or various combinations of solvents. 
The pharmaceutically acceptable salts of the acids of Formula I are also 
readily prepared by conventional procedures such as treating an acid of 
Formula I with an appropriate amount of a base, such as an alkali or 
alkaline earth metal hydroxide e.g. sodium, potassium, lithium, calcium, 
or magnesium, or an organic base such as an amine, e.g., 
dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine, 
benzylamine and the like, or a quaternary ammonium hydroxide such as 
tetramethylammonium hydroxide and the like. 
The compounds of Formula I antagonize CCK and/or gastrin and are useful as 
pharmaceutical agents for animals, preferably for mammals, and most 
especially for humans, for the treatment and prevention of 
gastrointestinal disorders and central nervous system disorders. 
Examples of such gastrointestinal disorders include ulcers, such as peptic 
and gastrointestinal ulcers, irritable bowel syndrome, gastroesophagenal 
reflux disease or excess pancreatic or gastrin secretion, acute 
pancreatitis, or motility disorders, Zollinger-Ellison syndrome, and 
antral and cell hyperplasia. 
Examples of central nervous system disorders include central nervous system 
disorders caused by CCK interaction with dopamine, such as neuroleptic 
induced tardive dyskinesia, Parkinson's disease, schizophrenia, other 
psychosis or Gilles de la Tourette syndrome, and disorders of appetite 
regulatory systems. 
The compounds of Formula I may further be useful in the treatment or 
prevention of additional central nervous system disorders including 
neurological and psychiatric disorders. Examples of such central nervous 
system disorders include anxiety disorders and panic disorders, wherein 
CCK and/or gastrin is involved. Additional examples of central nervous 
system disorders include panic syndrome, anticipatory anxiety, phobic 
anxiety, panic anxiety, chronic anxiety, and endogenous anxiety. 
The compounds of Formula I may further be useful in the treatment of 
oncologic disorders wherein CCK or gastrin may be involved. Examples of 
such oncologic disorders include small cell adenocarcinomas and primary 
tumors of the central nervous system glial and neuronal cells. Examples of 
such adenocarcinomas and tumors include, but are not limited to, tumors of 
the lower esophagus, stomach, intestine, colon and lung, including small 
cell lung carcinoma. 
The compounds of Formula I may further be used to control pupil 
constriction in the eye. The compounds may be used for therapeutic 
purposes during eye examinations and intraocular surgery in order to 
prevent miosis. The compounds may further be used to inhibit miosis 
occurring in association with iritis, uveitis and trauma. 
The compounds of Formula I are also useful for directly inducing analgesia, 
opiate or non-opiate mediated, as well as anesthesia or loss of the 
sensation of pain. 
The compounds of Formula I may further be useful for preventing or treating 
the withdrawal response produced by chronic treatment or abuse of drugs or 
alcohol. Such drugs include, but are not limited to cocaine, alcohol or 
nicotine. 
The compounds of formula (I) may also be useful as neuroprotective agents, 
for example, in the treatment and/or prevention of neurodegenerative 
disorders arising as a consequence of such pathological conditions as 
stroke, hypoglycaemia, cerebral palsy, transient cerebral ischaemic 
attack, cerebral ischaemia during cardiac pulmonary surgery or cardiac 
arrest, perinatal asphyxia, epilepsy, Huntington's chorea, Alzheimer's 
disease, Amyotrophic Laterial Sclerosis, Parkinson's disease, 
Olivo-pontocerebellar atrophy, anoxia such as from drowing, spinal cord 
and head injury, and poisoning by neurotoxins, including environmental 
neurotoxins. 
The present invention also encompasses a pharmaceutical composition useful 
in the treatment of CCK and/or gastrin disorders comprising the 
administration of a therapeutically effective but non-toxic amount of the 
compounds of Formula I, with or without pharmaceutically acceptable 
carriers or diluents. 
The compounds of Formula I, may be administered to animals, preferably to 
mammals, and most especially to a human subject either alone or, 
preferably, in combination with pharmaceutically-acceptable carriers or 
diluents, optionally with known adjuvants, such as alum, in a 
pharmaceutical composition, according to standard pharmaceutical practice. 
The compounds can be administered orally or parenterally, including 
intravenous, intramuscular, intraperitoneal, subcutaneous and topical 
administration. 
For oral use of an antagonist of CCK, according to this invention, the 
selected compounds may be administered, for example, in the form of 
tablets or capsules, or as an aqueous solution or suspension. In the case 
of tablets for oral use, carriers which are commonly used include lactose 
and corn starch, and lubricating agents, such as magnesium stearate, are 
commonly added. For oral administration in capsule form, useful diluents 
include lactose and dried corn starch. When aqueous suspensions are 
required for oral use, the active ingredient is combined with emulsifying 
and suspending agents. If desired, certain sweetening and/or flavoring 
agents may be added. For intramuscular, intraperitoneal, subcutaneous and 
intravenous use, sterile solutions of the active ingredient are usually 
prepared, and the pH of the solutions should be suitably adjusted and 
buffered. For intravenous use, the total concentration of solutes should 
be controlled in order to render the preparation isotonic. 
When a compound according to Formula I is used as an antagonist of CCK or 
gastrin in a human subject, the daily dosage will normally be determined 
by the prescribing physician with the dosage generally varying according 
to the age, weight, and response of the individual patient, as well as the 
severity of the patient's symptoms. However, in most instances, an 
effective daily dosage will be in the range of from about 0.005 mg/kg to 
about 50 mg/kg of body weight, and preferably, of from about 0.05 mg/kg to 
about 50 mg/kg of body weight, and most preferably, of from about 0.5 
mg/kg to about 20 mg/kg of body weight administered in single or divided 
doses. 
In some cases, however, it may be necessary to use dosage levels outside 
these limits. For example, doses as low as about 1 ng/kg, about 0.005 
.mu.g to about 0.05 .mu.g, or about 100 ng to about 100 .mu.g/kg may be 
administered. 
In the effective treatment of panic syndrome, panic disorder, anxiety 
disorder and the like, preferably about 0.05 mg/kg to about 1.0 mg/kg of 
CCK antagonist maybe administered orally (p.o.), administered in single or 
divided doses per day (b.i.d.). Other routes of administration are also 
suitable. 
For directly inducing analgesia, anesthesia or loss of pain sensation, the 
effective dosage range is preferably from about 100 ng/kg to about 1 mg/kg 
by intraperitoneal administration. Oral administration is an alternative 
route, as well as others. 
In the treatment of irritable bowel syndrome, preferably about 0.1 to 10 
mg/kg of CCK antagonist is administered orally (p.o.), administered in 
single or divided doses per day (b.i.d.). Other routes of administration 
are also suitable. 
The use of a gastrin antagonist as a tumor palliative for gastrointestinal 
neoplasma with gastrin receptors, as a modulator of central nervous 
activity, treatment of Zollinger-Ellison syndrome, or in the treatment of 
peptic ulcer disease, an effective dosage is preferably from about 0.1 to 
about 10 mg/kg administered one-to-four times daily is indicated. 
Because these compounds antagonize the function of CCK in animals, they may 
also be used as feed additives to increase the food intake of animals in 
daily dosage preferably from about 0.05 mg/kg to about 50 mg/kg of body 
weight. 
The compounds of Formula I may be prepared according to the reaction 
schemes as set forth below. 
##STR6## 
1. CCK Receptor Binding (Pancreas) 
CCK-8 sulphated was radiolabelled with .sup.125 I-Bolton Hunter reagent 
(2000 Ci/mmole). Receptor binding was performed according to Chang and 
Lotti (Proc. Natl. Acad. Sci. 83, 4923-4926, 1986) with minor 
modifications. 
Male Sprague-Dawley rats (150-200 g) were sacrificed by decapitation. The 
whole pancreas was dissected free of fat tissue and was homogenized in 25 
volumes of ice-cold 10 mM Hepes buffer with 0.1% soya bean trypsin 
inhibitor (pH 7.4 at 25.degree. C.) with a Kinematica Polytron. The 
homogenates were centrifuged at 47,800 g for 10 min. Pellets were 
resuspended in 10 volumes of binding assay buffer (20 mM Hepes, 1 mM EGTA, 
5 mM MgCl.sub.2, 150 mM NaCl, bacitracin 0.25 mg/ml, soya bean trypsin 
inhibitor 0.1 mg/ml, and bovine serum albumin 2 mg/ml, pH 6.5 at 
25.degree. C.) using a teflon homogenizer, 15 strokes at 500 rpm. The 
homogenate was further diluted in binding assay buffer to give a final 
concentration of 0.5 mg original wet weight/1 ml buffer. For the binding 
assay, 50 .mu.l of buffer (for total binding) or unlabeled CCK-8 sulfated 
to give a final concentration of 1 .mu.M (for nonspecific binding) or the 
compounds of Formula I (for determination of inhibition of .sup.125 I-CCK 
binding) and 50 .mu.l of 500 pM .sup.125 I-CCK-8 (i.e. 50 pM final 
concentration) were added to 400 .mu.l of the membrane suspensions in 
microfuge tubes. All assays were run in duplicate. The reaction mixtures 
were incubated at 25.degree. C. for 2 hours and the reaction terminated by 
rapid filtration (Brandell 24 well cell harvester) over Whatman GF/C 
filters, washing 3.times.4 mls with ice-cold 100 mM NaCl. The 
radioactivity on the filters was counted with a LKB gamma counter. 
2. CCK Receptor Binding (Brain) 
CCK-8 sulphated was radiolabelled and the binding was performed according 
to the description for the pancreas method with minor modifications. 
Male Hartley guinea pigs (300-500 g) were sacrificed by decapitation and 
the cortex was removed and homogenized in 25 mL ice-cold 0.32M sucrose. 
The homogenates were centrifuged at 1000 g for 10 minutes and the 
resulting supernatant was recentrifuged at 20,000 g for 20 minutes. The 
P.sub.2 pellet was resuspended in binding assay buffer (20 mM 
N-2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid (HEPES), 5 mM 
MgCl.sub.2, 0.25 mg/ml bacitracin, 1 mM ethylene 
glycol-bis-(.beta.-aminoethylether-N,N'-tetraacetic acid) (EGTA)pH 6.5 at 
25.degree. C., using a teflon homogenizer (5 strokes at 500 rpm) to give a 
final concentration of 10 mg original wet weight 11.2 mls buffer. For the 
binding assay, 50 .mu.l of buffer (for total binding) or unlabeled CCK-8 
sulfate to give a final concentration of 1 .mu.M (for nonspecific binding) 
or the compounds of Formula I (for determination of inhibition of .sup.125 
I-CCK-8 binding) and 50 .mu.l of 500 pM .sup.125 I-CCK-8 (i.e. final 
concentration of 50 pM) were added to 400 .mu.l of the membrane 
suspensions in microfuge tubes. All assays were run in duplicate. The 
reaction mixtures were incubated at 25.degree. C. for 2 hours and then the 
reaction was terminated on Whatman GF/C filters by rapid filtration 
(Brandell 24 well cell Harvester) with 3.times.5 ml washes of cold 100 mM 
NaCl. The radioactivity on the filters was then counted with a LKB gamma 
counter. 
5. Gastrin Antagonism 
Gastrin antagonist activity of compounds of Formula I is determined using 
the following assay. 
A. Gastrin Receptor Binding in Guinea Pig Gastric Glands 
Preparation of guinea pig gastric mucosal glands 
Guinea pig gastric muscosal glands were prepared by the procedure of Chang 
et al., Science 230, 177-179 (1985) with slight modification. Gastric 
mucosa from guinea pigs (300-500 g body weight, male Hartley) were 
isolated by scraping with a glass slide after washing stomach in ice-cold, 
aerated buffer consisting of the following: 130 mM NaCl, 12 mM 
NaHCO.sub.3, 3 mM NaH.sub.2 PO.sub.4, 3 mM Na.sub.2 HPO.sub.4, 3 mM 
K.sub.2 HPO.sub.4, 2 mM MgSO.sub.4, 1 mM CaCl.sub.2, 5 mM glucose and 4 mM 
L-glutamine, 50 mM HEPES, 0.25 mg/ml bacitracin, 0.10 mg/ml soya bean 
trypsin inhibitor, 0.1 mg/ml bovine serum albumin, at pH 6.5, and then 
incubated in a 37.degree. C. shaking water bath for 40 minutes in buffer 
containing 1 mg/ml collagenase and bubbled with 95% O.sub.2 and 5% 
CO.sub.2. The tissues were passed twice through a 5 ml syringe to 
liberate the gastric glands, and then filtered through Nitex #202 gauge 
nylon mesh. The filtered glands were centrifuged at 272 g for 5 minutes 
and washed twice by resuspension in 25 ml buffer and centrifugation. 
B. Binding Studies 
The washed guinea pig gastric glands prepared as above were resuspended in 
25 ml of standard buffer. For binding studies, to 250 .mu.l of gastric 
glands, 30 .mu.l of buffer (for total binding) or gastrin (3 .mu.M final 
concentration, for nonspecific binding) or test compound and 20 .mu.l of 
.sup.125 I-gastrin (NEN, 2200 Ci/mmole, 0.1 nM final concentration) were 
added. AV assays were run in triplicate. The tubes were aerated with 95% 
O.sub.2 and 5% CO.sub.2 and capped. The reaction mixtures after incubation 
at 25.degree. C. for 30 minutes in a shaking water bath were rapidly 
filtered (Brandell 24 well cell harvester) over Whatman and G/F B filters 
presoaked in assay buffer and immediately washed further with 3.times.4 ml 
of 100 mM ice cold NaCl. The radioactivity on the filters was measured 
using a LKB gamma counter. 
In Vitro Results 
Effect of The Compounds of Formula I on .sup.125 I-CCK-8 receptor binding 
The preferred compounds of Formula I are those which produced 
dose-dependent inhibition of specific .sup.125 I-CCK-8 binding as defined 
as the difference between total and non-specific (i.e. in the presence of 
1 .mu.m CCK) binding. 
Drug displacement studies were performed with at least 10 concentrations of 
compounds of formula 1 and the IC.sub.50 values were determined by 
regression analysis. IC.sub.50 refers to the concentration of the compound 
required to inhibit 50% of specific binding of .sup.125 I-CCK-8. 
The data in Table I were obtained for compounds of Formula I. 
TABLE I 
______________________________________ 
CCK RECEPTOR BINDING RESULTS 
IC.sub.50 (.mu.M) 
Compound .sup.125 I-CCK 
.sup.25 I-CCK 
.sup.25 I-Gastrin 
of Ex # Pancreas Brain Gastric Glands 
______________________________________ 
1 &gt;3 3.68 N.D. 
2 &gt;3 0.51 N.D. 
______________________________________ 
N.D. = NO DATA 
EXAMPLES 
Examples provided are intended to assist in a further understanding of the 
invention. Particular materials employed, species and conditions are 
intended to be further illustrative of the invention and not limitative of 
the reasonable scope thereof. 
EXAMPLE 1 
Synthesis of 
N-{1,3-Dihyro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-carbox 
yethyl-N'-{[3-methylphenyl]-urea} 
A. 
N-{1,3-Dihyro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-methyl 
carboxyethyl-N'-{[3-methylphenyl]-urea} 
To a solution of 
1,3-dihyro-3(R,S)-amino-5-phenyl-2H-1,4-benzodiazepin-2-one (100 mg, 0.377 
mmole) in 2 ml of toluene was added 56 mg (0.189 mmole) triphosgene and 56 
.mu.L of triethylamine (0.377 mmole). After 10 minutes, 100 mg (0.517 
mmole) of N-methoxycarbonylethyl-m-toluidine and triethylamine (56 .mu.L, 
0.377 mmole) were added and stirring was continued for 2 additional hours. 
The reaction mixture was concentrated to dryness. Ethyl acetate and water 
were added to the residue. The layers were separated and the aqueous layer 
was extracted with ethyl acetate. The combined organic extracts were dried 
(sodium sulfate) and concentrated to give approximately 150 mg of crude 
product. Preparative thick layer chromatography on 0.5 mm.times.20 
cm.times.20 cm precoated silica gel plates (ethyl acetatehexane, 1:1 v/v 
elution) afforded 60 mg of the analytical product after it was 
crystallized from petroleum ether: m.p. 197.degree.-198.degree. C. 
HPLC=99.3% pure at 214 nm; TLC R.sub.f =0.30 (EtOAc-hexane, 1:1). 
NMR (DMSO-D.sub.6): Consistent with structure assignment and confirms 
presence of solvent. 
FAB MS: 485 (M.sup.+ +1). 
Analysis for C.sub.28 H.sub.28 N.sub.4 O.sub.4 : Calculated: C, 69.40, H, 
5.82, N, 11.56. Found: C, 69.19, H, 5.96, N, 11.52. 
B. 
N-}1,3-Dihyro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-carbox 
yethyl-N'-{[3-methylphenyl]-urea} 
N-{1,3-Dihyro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-methylc 
arboxyethyl-N'-{[3-methylphenyl]-urea}, (40 mg) was mixed with 7 mg of 
lithium hydroxide in 2 ml of dimethoxyethane and 0.5 ml of water. The 
reaction mixture was stirred for 8 hours and concentrated in vacuo. Ethyl 
acetate was added to the residue and 1N HCl solution was added until the 
mixture was neutral. The ethyl acetate extracts were dried (sodium 
sulfate) and concentrated to give 30 mg of crude product. The crude 
product was triturated with ethyl acetate and petroleum ether to give the 
title compound: m.p. &gt;175.degree. C. (d). 
HPLC=95% pure at 214 nm; TLC R.sub.f =0.45 (EtOAc). 
NMR (DMSO-D.sub.6): Consistent with structure assignment and confirms 
presence of solvent. 
FAB MS: 471 (M.sup.+ +1). 
Analysis for C.sub.27 H.sub.26 N.sub.4 O.sub.4. 0.35 EtOAc.0.15 H.sub.2 O: 
Calculated: C, 67.67, H, 5.82, N, 11.12. Found: C, 67.70, H, 5.97, N, 
11.14. 
EXAMPLE 2 
Synthesis of 
N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-carbox 
yethyl-N'-{[3-methylphenyl]-urea} 
A. 
1,3-Dihydro-1-methyl-3(R,S)-(methylcarboxyethyl)amino-5-phenyl-1H-1,4-benz 
odiazepin-2-one 
1,3-Dihydro-1-methyl-3(R,S)-amino-5-phenyl-2H-1,4-benzodiazepin-2-one (1 g, 
377 mole) was dissolved in 10 ml of dry N,N-dimethylformamide and treated 
with 1.04 g of solid sodium carbonate at room temperature. To this 
suspension was added 810 mg (3.77 mmole) of methyl 3-iodopropionate and 
the reaction mixture was stirred overnight. An additional 800 mg of methyl 
3-iodopropionate and 500 mg of sodium carbonate were added and the 
reaction mixture was then heated to 50.degree. C. After 24 hours the 
reaction mixture was filtered and concentrated under reduced pressure. The 
residue was partitioned between ethyl acetate and water. The organic phase 
was washed twice more with water, then dried (sodium sulfate), and 
concentrated to yield 720 mg of crude product. The title compound was 
obtained as an oil which crystallized on standing after flash silica gel 
chromatography employing ethyl acetate-hexane (1:1 v/v). Recrystallization 
from ether afforded the analytical material which had m.p. 
136.degree.-137.degree. C. 
B. 
N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-methyl 
carboxyethyl-N'-{[3-methylphenyl]-urea} 
To a solution of 130 mg (0.37 mmole) of 
1,3-dihydro-1-methyl-3(R,S)-(methylcarboxyethyl)-amino-5-phenyl-1H-1,4-ben 
zodiazepin-2-one in 2 ml of tetrahydrofuran was added 48 .mu.L of 
m-toluidine isocyanate at room temperature. The resulting solution was 
protected from moisture and stirred for 1 hour. The solvent was removed 
under reduced pressure and the residual solid was recrystallized from a 
methanol-ethyl acetate-hexane solvent mixture to give 110 mg of the title 
compound with m.p. 198.degree. C. 
C. 
N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-carbox 
yethyl-N'-{[3-methylphenyl]-urea} 
N-[1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-N-methylc 
arboxyethyl-N'-{[3-methylphenyl]urea} (70 mg) was mixed with 25 mg of 
lithium hydroxide in 3 ml of dimethoxyethane and 0.5 ml of water. The 
reaction mixture was stirred for 4 hours and concentrated in vacuo. Ethyl 
acetate and water were added to the residue and the resulting mixture was 
neutralized with 1N HCl solution. The layers were separated and the 
aqueous layer was extrated with ethyl acetate. The combined organic 
extracts were dried (sodium sulfate) and concentrated to give the crude 
product. The crude product was recrystallized from a methanol-ethyl 
acetate-hexane solvent mixture to give the title compound: m.p. 
172.degree.-174.degree. C. (d). 
HPLC=99.4% pure at 214 nm; TLC R.sub.f =0.48 (EtOAc). 
NMR (DMSO-D.sub.6): Consistent with structure assignment and confirms 
presence of solvent. 
FAB MS: 471 (M.sup.+ +1). 
Analysis for C.sub.27 H.sub.26 N.sub.4 O.sub.4. 0.40 EtOAc.0.20 H.sub.2 O: 
Calculated: C, 67.43, H, 5.86, N, 11.00. Found: C, 67.44, H, 5.48, N, 
10.98.