Note: Descriptions are shown in the official language in which they were submitted.
W093/02078 PCT/GB92/01~6
5 ~
. ~. , ,~
BENZODIAZEPINE DERIVATIVE~, COMPO8ITION~
CONTAINING THEM AND T~EIR ~8E IN ~E~APY
This invention relates to benzodiazepine compounds
which are useful as antagonists of cholecystokinin and
gastrin receptors.
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. Green, Ed., Raven
Press, N.Y., p.l69 and G. Nission, ibid. p.l27).
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 ;
carboxylterminal octapeptide, CCK-~ (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-NH2, which is the common
structural element shared by both CCK and gastrin. ~-
CCKs are believed to be physiological satiety ~;
, hormones, thereby possibly playing an important role in
appetite regulation (G. P. Smith, Eatina and Its
Disorders, A. J~ Stunkard and E. Stellar, Eds, Raven
Press, New York, 1984, p. 67), as well as stimulating -
coIonic 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 çt al., "Peptides in the Central Nervous
,. '~
WO 93/02078 PCr/GB92/01366
2112561
- 2 -
System", Ann. Repts. ~ed. Chem 17, 31, 33 tl982] and
references cited therein; J. A. Williams, Biomed Res. 3
107 ~1982~: and J.E. Morley, Life sci. 30, 479 tl982]).
The primary role of gastrin, on the other hand, ~
appears to be stimulation of the secretion of water and ~-
electrolytes from the stomach and, as such, 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.
There are at least two subtypes of choiecystokinin
receptors termed CCK-A and CCK-B (T.H. Moran et al., "Two - ~-
brain cholecystokinin receptors: implications for - -
behavioural actions", Brain Res., 362, 175-79 ~1986]).
Both subtypes are found both 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 (GI)
and central nervous (CNS) systems of animals, especially
mammals, and more 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 CCK-B receptors and gastrin receptors. Other
antagonists have activity at the CCK-A subtype.
8elective CCK antagonists are themselves useful in
treating CCK-related disorders of appetite regulatory
systems of animals as well as in potentiating and
prolonging opiate-mediated analgesia [see P. L. Faris et
al., Science 226, 1215 (1984)], thus having utility in
the treatment of pain. CCK-B and CCK-A antagonists have
also been shown to have a direct analgesic effect [M.F.
..
W093/0207~ PCT/GB92/01366
'2~t2-.~61 :~
- 3 -
O'Neill et al., Brain Re$earch, 534 2B7 (lsso)].
Selective CCK and gastrin antagonists are useful in the ~-~
modulation of behaviour mediated by dopaminergic and e
serotonergic neuronal systems and thus have utility in -~
the treatment of schizophrenia and depression (Rasmussen -
et. al., 1991, Eur. J. Pharmacol., 209, 135-138; Woodruff
et. al., 1991, Neuro~e~tides, 19, 45-46; Cervo et. al.,
1988, Eur. J. Pharmacol., 158, 53-59), 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. Patent
4,820,834. Certain CCK antagonists are useful anxiolytic
agents and can be used in the treatment of panic and
anxiety disorders. -
CCK has been reported to evoke the release of stress
hormones such as adrenocorticotrophic hormone, ~-
endorphin, vasopressin and oxytocin, CCX may function as
a mediator of responses to stress and as part of the
arousal system. CCK-A receptors are now known to be
present in a number of areas of the CNS and may be
, involved in modulating all of the above.
CCK may be involved in the regulation of stress and
its relationship with drug abuse e.g. alleviation of the ~`
benzodiazepine withdrawal syndrome (Singh et. al., 1992,
Br. J. Pharmacol., 105, 8-10) and neuroadaptive
processes.
Since CCK and gastrin also have trophic effects on `
certain tumours [K. Okyama, Hokkaido J. Med. Sci., 206-
216 (1985)], antagonists of CCK and gastrin are useful in
treating these tumours [see, R.D. Beauchamp et al., Ann.
~E~-, 202, 203 (1985)].
W093/02078 PCT/GB92/01366 ~
~.: ' . -
iiis6~ '
4 -
In the light of discussion in c. Xu et al.,
Peptides, ~, 1987, 769-772, CCK antagonists may also be `
effective in neuroprotection.
CCK receptor antagonists have been found to inhibit
the contractile effects of CCK on iris sphincter and
ciliary muscles of monkey and human eyes (Eur. J. -
Pharmacol., 211(2), 183-187; A. Bill et al., Acta - -
Physiol. Scand., 138, 479-485 tlg9o])~ thus having
utility in inducing miosis for therapeutic purposes.
A class of benzodiazepine antagonist compounds has
been reported which binds selectively to brain CCK (CCK-B
and CCK-A) and gastrin receptors tsee M. Bock et al.,J.
Med Chem., 32, 13-16 (1989)].
European patent application no. 0 167 919 discloses
benzodiazepine CCX and gastrin antagonists substituted in ~
the 3-position by, inter alia, a phenyl urea and at the -
5-position by an optionally substituted phenyl or pyridyl
group.
British patent application no. 1,034,872 discloses -~
benzodiazepines substituted at the 3-position by an
unsubstituted amino group or a substituted amino group
containing up to eight carbon atoms, and at the 5- -~
position by a monocyclic aryl moiety. The only 5- -- -
, substituents specifically disclosed are phenyl --
(substituted or unsubstituted) and thienyl. There is no -~
disclosure of a phenyl urea substituent at C-3.
British patent no. 1,309,947 discloses
benzodiazepines substituted at the 5-position by, inter
alia, a heterocyclic group, and at the 3-position by H,
alkyl, alkoxy, alkylthioalkyl, phenyl, benzyl or -~
hydroxybenzyl. The compounds are said to have - -
tranquilizer, muscle relaxant, antispasmodic,
anticonvulsant and hypnotic effects. There is no
suggestion of the phenyl urea substituent of--the -
W093/02078 PCT/GB92/01366
~1125~1 ~
.. .: .:
- 5 - - ~:
compounds of t~e present invention. Nor is there any
suggestion that the compounds are CCK or gastrin
antagonists.
The present invention provides benzodiazepine
compounds of formula (I):
~ ~ N
Z
',~ '
wherein: ~:
one of W, X, Y or Z represents a nitrogen atom,
another of W, X, Y or Z is a nitrogen, oxygen or sulphur : -
atom or a group NR8 where R8 is H or Cl_6alkyl, and the
other two of W, X, Y and Z each independently represent ~-
nitrogen atoms or groups CR8, and the dotted circle
' represents two double bonds;
Rl represents Cl_6 alkyl, C3_7 cycloalkyl,
cyclopropylmethyl, (CH2)qimidazolyl, (CH2)qtetrazolyl,
(CH2)qtriazolyl, (where q is l, 2 or 3), CH2Co2R5 (where
R5 is Cl_4 alkyl) or a group CH2CoNR6R7 (where R6 ahd R7
each independently represents H or Cl_4alkyl, or R6 and
R7 together form a chain (CH2)p where p is 4 or 5);
R2 represents Cl_6 alkyl, halo, (CH2)rtetrazolyl,
optionally substituted in the tetrazole ring by
Cl_4alkyl, (CH2)rimidazolyl, CoNR6R7, SO(Cl_6alkyl),
SO2(Cl_6alkyl), CONHSO2R9, SO2NHCOR9, SONHRl0, cyano,
W093/02078 PCT/GB92/01366
-
~?,ll2~1
- 6 -
B(H)2 or (CH2)rcO2H~ where r is zero, 1 or 2, R9 is
C1_6alkyl, optionally substituted aryl, 2,2-
difluorocyclopropane or trifluoromethyl, and R10 is a
nitrogen containing heterocycle; ;~
R3 represents H, Cl_6 alkyl or halo;
m is 0, 1 or 2;
n is 0, 1, 2 or 3;
and salts or prodrugs thereof.
It will be appreciated that formul~ (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 ~ above. In
general, such prodrugs will be functional deri~atives 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. -- -
When Rl represents Cl_6alkyl, alkyl means --
linear or branched chain alkyl. Examples of suitable -
alkyl groups include methyl, ethyl, isopropyl and
, isobutyl groups. -~-
When Rl represents cycloalkyl, examples of ~-
suitable cycloalkyl groups include cyclopropyl,
cyclopentyl and cyclohexyl groups, preferably
cyclopropyl.
Halo includes fluoro, chloro and bromo. -~
Preferably halo will be fluoro or chloro. ~;
Suitable examples of the substituent
~3~ J ~ J.~ t
9 1~
~112S61
~, Z ' ';:.. -'
X~Y
include imidazolyl, N-me~hylimidazolyl and thiaz~lyl,
preferably thiazolyl.
In one subgroup of co~pounds of formula (I)
represents Cl_6 alkyl, C3_7 cycloalkyl,
cyclopropylmethyl, (CH2)qimidazolyl ~where q is 1, 2 or i -~
3), CH2Co2R5 (where R5 is cl_4 alkyl) or a group
CH2CoNR6R7 ~where R6 and R7 eac~ indepen~n~ly represent~
a hyd~ogen atom or a Cl_4 alkyl group, or R6 and R7 ~ -
together form a chain (CH2)p where p i~ 4 or 5~; R2
represents Cl_~ alkyl, halo, (CH2)stetrazolyl,
(CH2)simidazolyl or ~ group (CR2)s~02H, where s is zero,
1 or 2; and m and n each represent 1.
Preferably Rl is C1_6alkyl, more p~e~erably methyl
or iso-butyl~
When one substituent R2 is presen~, it will
preferably be located at the 3- or 4-position of the
phenyl ring, more preferably the 3-position. When ~wo
substituents R2 are present, they will p~eferably he
located at the 3- anq 4-positions.
5uitable values for R9 in~lude methyl, ethyl, i-
p~opyl, t-butyl, phenyl and trifluoromethyl.
When R9 is optionally substituted a~yl, this will ..
preferably be optionally su~stituted phenyl. Suitable
substituents include Cl_4alkyl, Cl_qalkoxy, ~alo an~ -
trifluoromethyl. Pre~erre~ ~re ~ompounds wherein R9 ie
unsubsti~uted aryl or aryl substituted by Cl_6alkyl, for
..... _ .. , ~ _ . .. ...
5 1;~, .; . ~
. ... ... . .
W093/02078 PCT/GB92/01366
'~ 11 2 ~ 61 ~
- 8 - -~
example phenyl substituted by Cl_6alkyl in the ortho
position.
When R9 is Cl_6alkyl, it will preferably represent -
C1_4alkyl. Particularly preferred are methyl and iso- ;-
propyl. -
When R2 is SO2NHR10, suitable values of R10 include,
for example, thiazole, thiadiazole and pyrazine. -~
Preferably R2 is tetrazolyl, methyl or COOH, more -~
preferably 5-tetrazolyl. ~ -
Preferably m is 1.
Preferably n is zero.
Preferably q is 1.
Preferably r is zero.
One subgroup of compounds according to the invention - ~-~
is represented by formula (II)~
~ H H
Z '~
( I l ) ,: .. ;
wherein ~- -
Z is a sulphur atom or a group NR18, where R18 is H
or methyl;
R20 is Cl_6alkyl; and -~
R21 is Cl_6alkyl, tetrazolyl or CO2H, preferably
tetrazolyl.
Preferably the salts of the compounds of formula (I)
are pharmaceutically acceptable, but non-pharmaceutically
acceptable salts may be used for the preparation of
pharmaceutically acceptable salts. The pharmaceutically
W093/02078 PCT/GB92/01366
2112~61 ~:
acceptable salts of the compounds of formula (I) include
the conventional non-toxic salts or the quaternary
ammonium salts of the compounds from formula (I) formed,
e.g., from inorganic or organic acids or bases. For
example, such conventional non-toxic salts include those
derived from inorganic acids such as hydrochloric,
hydrobromic, sulphuric, sulphamic, phosphoric, nitric and
the like; and the salts prepared from organic acids such
as acetic, propionic, succinic, glycolic, steric, lactic,
malic, tartaric, citric, ascorbic, palmoic, maleic,
hydroxymaleic, phenylacetic, glutamic, benzoic,
salicylic, sulphanilic, 2-acetoxy benzoic, fumaric,
toluenesulphonic, methanesulphonic, ethane disulphonic,
oxalic and isothionic.
The pharmaceutically acceptable salts of the present
invention can be synthesized from the compound 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.
For example, an acid of formula (I) may be reacted
, 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.
The present invention also encompasses a ~~-
pharmaceutical composition comprising a compound of
formula (1), or a salt or prodrug thereof and a
pharmaceutically acceptable carrier or diluent. ~ -
w093/0207X pcT/GBs2/ol~6
2~
-- 1 0 ~
.... `.
The compounds of formula (I~ and their salts ;
and prodrugs, may be administered to animals, preferably -~
to mammalsr and most especially to a human subject either -~
alone or, preferably, in combination with ~- -
S pharmaceutically acceptable carriers or diluents, - --
optionally with known adjuvants, such as alum, in a
pharmaceutical compostion, according to standard -
pharmaceutical practice. The compounds can be
administered orally, parenterally, including by ~
intravenous, intramuscular, intraperitoneal or - ~-
subcutaneous administration, or topically.
For oral use of an antagonist of CCK, according
to this in~ention, 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 addsd. 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
, flavouring 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. ~,~
For topical administration, a compound of
formula (I) may be formulated as, for example, a
suspension, lotion, cream or ointment.
W093/02078 PCT/GB92/0l366
.
~112~61
For topical administration, pharmaceutically
acceptable carriers are, for example, water, mixtures of
water and water-miscible solvents such as lower alkanols
or arylalkanols, vegetable oils, polyalkylene glycols,
S petroleum based jelly, ethyl cellulose, ethyl oleate,
carboxymethylcellulose, polyvinylpyrrolidone, isopropyl
myristate and other conventionally-employed non-toxic,
pharmaceutically acceptable organic and inorganic
carriers. The pharmaceutical prepara~ion may also
contain non-toxic auxiliary substances such as
emulsifying, preserving, wetting agents, bodying agents --
and the like, as for example, polyethylene glycols 200,
300, 400 and 600, carbowaxes 1,000, 1,500, 4,000, 6,000
and 10,000, antibacterial components such as quaternary
ammonium compounds, phenylmercuric salts known to have
cold sterilizing properties and which are non-injurious
in use, thimerosal, methyl and propyl paraben, benzyl
alcohol, phenyl ethanol, buffering ingredients such as
sodium chloride, sodium borate, sodium acetates,
gluconate buffers, and other conventional ingredients
such as sorbitan monolaurate, triethanolamine, oleate,
polyoxyethylene sorbitan monopalmitylate, dioctyl sodium ~ ~-
sulfosuccinate, monothioglycerol, thiosorbitol,
, ethylenediamine tetraacetic acid, and the like.
The compounds of formula (I) antagonise CCK and/or
gastrin and are useful for the treatment and prevention
of disorders including central nervous system disorders
wherein CCK and/or gastrin may be involved. Examples of
such disease states include gastrointestinal diseases,
including gastrointestinal ulcers, such as peptic and
duodenal ulcers, irritable bowel syndrome,
gastroesophagenal reflux disease or excess pancreatic or
gastrin secretion, acute pancreatitis, or motility
disorders; central nervous system disorders, including
W093/02078 PCT/GB92/01366 -~
`,
S 6 1
- 12 - -
,, . - ,
central nervous system disorders caused by CCK
interaction with dopamine, serotonin and other monoamine
neurotransmitters, such as neuroleptic disorders, tardive
dyskinesia, Parkinson's disease, psychosis or ~illes de
la Tourette syndrome; depression; schizophrenia;
disorders of appetite regulatory systems; Zollinger- ~
Ellison syndrome, antral and cell hyperplasia, or pain. ~ -
The compounds of formula (I) are particularly useful -
in the treatment or prevention of neurological disorders
involving anxiety disorders and panic disorders, wherein ;~
CCK and/or gastrin is involved. Examples of such
disorders include panic disorders, anxiety disorders,
panic syndrome, anticipatory anxiety, phobic anxiety,
panic anxiety, chronic anxiety and endogenous anxiety.
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
benzodiazepines, cocaine, alcohol and nicotine.
, The compounds of formula (I) may further by useful ~ ~
in the treatment of stress and its relationship with drug - -
abuse.
The compounds of formula (I) may further be useful
in the treatment of oncologic disorders wherein CCK may -
be involved. Examples of such oncologic disorders - ~-
include small cell adenocarcinomas and primary tumours of
the central nervous system glial and neuronal cells. - -
Examples of such adenocarcinomas and tumours include, but
are not limited to, tumours of the lower oesophagus,
, ~ .
' ~.",
W093/02078 PCT/~B92/01366
2112561
- 13 -
stomach, intestine, colon and lung, including small cell
lung carcinoma.
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, ~untington's chorea, Alzheimer's disease,
Amyotrophic Lateral Sclerosis, Parkinson's disease,
Olivo-ponto-cerebellar atrophy, anoxia such as from
drowning, spinal cord and head injury, and poisoning by
neurotoxins, including environmental neurotoxins.
T~e compounds of formula (I) may further be used to
induce miosis for therapeutic purposes after certain
types of examination and intraocular surgery. An example ~-
of intraocular surgery would include cateract surgery ;~
with implantation of an artificial lens. The CCK ~ `
antagonist compounds of this invention can be used to
prevent miosis occuring in association with iritis,
ureitis and trauma.
The present invention therefore provides a compound -~
, of formula (I) or a salt or prodrug thereof for use in
the preparation of a medicament. `-
The present invention also provides a compound of
formula (I) for use in therapy. `~
In a further or alternative embodiment the present
invention provides a method for the treatment or
prevention of a physiological disorder involvinq CCK
and/or gastrin which method comprises administration to a
patient in need thereof of a CCK and/or gastrin
antagonising amount of a compound of formula (I).
W093/02078 PCT/GB92/01366 ~
2 1 1 ~ ~ 6 1
- 14 - ~-~
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
prescibing 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 wll be in the range from about
0.005mg/kg to about lOOmg/kg of body weight, and
preferably, of from 0.05mg/kg to about 50mq/kg, such as
from about 0.5mg/kg to about 2Omg/kg of body weight,
administered in single or divided doses. In so~e cases,
however, it may be necessary to use dosages outside these
limits. For example, animal experiments have indicated
that doses as low as lng may be effective.
In effective treatment of panic syndrome, panic
disorder, anxiety disorder and the like, preferably about
0.05 mg/kg to about 0.5 mg/kg of CCK antagonist m~y be -
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, anaesthesia or loss
of pain sensation, the effective dosage preferably ranges
, from about 100 ng/kg to about lmg/kg by intravenous
administration. Oral administration is an alternative
route, as well as others. ---
In the treatment or irritable bowel syndrome, ~ -
preferably about O.l to 1~ 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 tumour
palliative for gastrointestinal neoplasma with gastrin
receptors, as a modulator of central nervous activity,
W093/0207~ PCT/GB92/01366
.~112561
- 15 -
treatment of Zollinger-Ellison syndro~e, or in the
treatment of peptic ulcer disease, an effective dosage of
preferably about O.l to about lO mg/kg administered one-
to-four times daily is indicated.
For use as neuroprotective agents the effective
dosage preferably ranges from about 0.5mg/kg to about
2Omg/kg.
Because these compounds antagonise the function of -
CCK in animals, they may also be used as feed additives
to increase the food intake of animals in daily dosage of
preferably about 0.05mg/kg to about 50mg/kg of body
weight.
The compounds of formula ~I) may be prepared by
processes analogous to those described in European Patent :~
Specification No. 0167919. For example, a compound of ::
formula (I) may be prepared from an intermediate of :~
formula ~TII) R l ` ~ `
~ R~ NH2
W~z
X`~~-'r '
wherein W, X, Y, Z, Rl, R3 and n are as defined for
formula (I); by reaction with an isocyanate of formula
(IV)
W093/02078 PCT/~B92/01366
21t2561
- 16 -
.
~ N C O -
( R2 )m~
( I V )
wherein R2 and m are as defined for formula (I). -~
The reaction is preferably conducted in a suitable
organic solvent, such as an ether, for example,
tetrahydrofuran, at room temperature.
The isocyanate of formula (IV) may be generated in - ~
situ from the corresponding amine by treatment with -
triphosgene. ~ `
Intermediates of formula (III) may be prepared from ~ `
compounds of formula ~V) -- -
(R~)~ ~ NHC
Z
( V )
wherein W, X, Y, Z, R3 and n are as defined for for~ula
(I) and G is a protecting group; by reactio~ with a
reagent suitable to introduce the group Rl, for example a
halide of formula RlHal where Hal represents halo such as
bromo or iodo, followed by deprotection.
The reaction is carried out in the presence of a
base, such as an alkali metal hydride or an alkaline
W093/02078 PCT/GB92/01366
~? I 1~
- 17 - :~
earth metal carbonate, for example sodium hydride or - .:
caesium carbonate. -; -
Compounds of formula (v) may be prepared from ~ .
compounds of formula (VI)
`~
H
( R ) n~
W~z '';'''-.''"''
X~ r
( V l ) ,~
wherein W, X, Y, Z, R3 and n are as defined for formula - -;
(I), by a reaction sequence comprising:
(i) reaction with a compound of formula (VII)
s ~ ~oo~
NHC
(Vll) :~
wherein G is as defined above, in the presence of a base,
such as a tertiary amine, for example triethylamine or N- :
methyl morpholine, and a coupling reagent. Any of the
: coupling reagents commonly used in peptide synthesis are ~;
suitable, for example, 1,3-dicyclohexylcarbodiimide (DCC) -
or isobutyl chloroformate.
(ii) Treatment with gaseous ammonia, preferably in
the presence of a mercury containing catalyst, such as
mercury(II) chloride. The reaction is conveniently
effected in a suitable organic solvent, such as an ether,
for example, tetrahydrofuran.
: '
W093~02078 PCT/GB92/01366
21~'~S61
- 18 -
(iii) Treatment with an organic acid, for example
acetic or propionic acid, optionally in the presence of
an ammonium salt, for example ammonium acetate.
Compounds of formula (VI) may be prepared aecording
S to the procedures described in Journal of Heterocyclic
Chemistry, 1975, 12, 49-57 and Journal of the Chemistry
societY, Perkin Transactions I, 1989, 1139-1145.
Where the above-described process for the
preparation of the compounds according to the invention
lo gives rise to mixtures of stereoisomers these isomers
may, if desired, be separated, suitably by conventional
techniques such as preparative chromatography. ~ -~
The novel compounds may be prepared in racemic form,
or individual enantiomers may be prepared either by
enantiospecific synthesis or by resolution. The novel
compounds may, for example, be resolved into their
component enantiomers by standard techniques, such as the
formation of diastereomeric pairs by salt formation with
an optically active acid, such as (-)-di-p-toluoyl-L-
tartaric acid and/or (+)-di-p-toluoyl-D-tartaric acid
followed by fractional crystallization and regeneration
of the free base. The novel compounds may also be
resolved by formation of diastereomeric esters or amides,
followed by chromatographic separation and removal of the
chiral auxiliary. Alternatively, enantiomers of the
novel compounds may be separated by HPLC using a chiral
column.
During any of the above synthetic sequences it may
be necessary and/or desirable to protect sensitive or
reactive groups on any of the molecules concerned. This
may be achieved by means of conventional protecting
groups, such as those described in Protective Groups in
Qraanic ChemistrY, ed. J.F.W. McOmie, Plenum Press, 1973;
and T.W. Greene and P.G.M. Wutts, Protective Groups in
W093/02078 PCT/GB92/01366
- 19 't1~.~61
organic Synthesis, John Wiley & Sons, 1991. The
protecting groups may be removed at a co~venient : : -
subsequent stage using methods known from the art. :
The following examples are provided 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 scope thereof.
' ''
,',~ ~"
'' . '"'
wo 93/02078 PCr/GB92/0l366 ~ ~ -
h)J I 1 2 5 6120 -
EXAMPLE 1: N-[3(R.S)-2.3-Dih~vdro-l-methYl-5-(1-
methylimidazol-2-~vl)-2-oxo-1H-1,4-benzodiazepin-3-vll-N/-[3-
,
methylphenyl]urea
Step 1: 1.3-Dihydro-3(R.S)-lbenzYlo~carbonylamino~5-(1- `
methvlimidazol-2-yl~2H-l,~benzodiazePin-2-one '' .- ':
To a stilTed, cooled (< 10C) solution of a-(isopropylthio)-
Na-(benzyloxycarbonyl)glycine ( 1 .4g) in anhydrous
dichloromethane (40ml), under a nitrogen atmosphere, was
o added dropwise N-methylmorpholine (O.~g) followed by
isobutylchloroformate (0.68g). The solution was stirred at 5C ~ -
for 10 minutes then heated to reflu~. 2-Aminophenyl~
methylimidazol-2-yl)methanone (1.Og, J. Het. Chem. 1975, 12,
49-57) in anhydrous dichloromethane (lOml) waæ added
dropwise and the resulting yellow solution heated at reflux for 1
hour then stirred at ambient temperature for 16 hours. The
reaction mixture was washed ~vith lN citric acid, brine then
dried (sodium sulphate) and evaporated to afford a yellow oil
(2.1g). Rf= 0.1 in ethyl acetate/n-hexane (1:3) on silica (trace of
. ~, .
20 amine, Rf- 0.2).
The crude (isopropylthio)glycinamide was dissolved in
anhydrous tetrahydrofuran (lOOml) and the ice cooled solution
was saturated with ammonia gas. Mercuric chloride (1.35g) .was
added and the passage of ammonia continued for 3 hours. The
25 mixture was filtered and the filtrate concentrated. The residue
was dissolved in glacial acetic acid (30ml), treated with
ammonium acetate (1.4g) and the resulting mixture stirred at
..
W O 93/02078 P ~ /GB92/01366 `:-
.
2S61 -~ ~
- 21 -
ambient temperature for 18 hours. The solvent was evaporated
and the residue partitioned between ethyl acetate (20ml) and lN
sodium hydroxide ~olution (25ml). The organic layer wa~
separated, dried (magnesium sulphate) then evaporated to give
5 a gummy solid (lg) which was purified by column
chromatography on silica using ethyl acetate/petroleum ether
(60-80) (1:1) to ethyl acetaWmethanol (9:1) as eluant. The title
compound was obtained as a colourless solid (0.38g). lH NMR ~ ~
(250MHz, CDC13) ~ 3.95 (3H, s); 5.12 (2H, m); 5.18 (lH, d, J = ` -
lo 8Hz); 6.65 (lH, d, J = 8Hz); 6.9 (lH, d, J = 8Hz); 7.0 (lH, s); 7.1-
7.5 (8H, m); 7.55 (lH, d, J = 8Hz).
Step 2- 1.3-Dihydro-3(R.S~-(benzvloxvcarbonYlamino3-1-
methyl-5-(1-methylimidazol-2-Y1~2H-1,4-benzodiazePin-2-one
To a stirred solution of 1,3-dihydro-3(R,S)- ~-
(benzylo~cycarbonylamino)-5-(1-methylimidazol-2-yl)-2H-1,4- -
benzodiazepin-2-one (348mg) in anhydrous dimethylfonnamide
(5ml), at ambient temperature, was added sodium hydride
(40mg of a 5~;% oil dispersion). After 30 minutes iodomethane
(142mg) was added and the mi~ture was stirred for 18 hours.
The solvent was evaporated then the residue was partitioned ; ~-
between ethyl acetate and water. The organic layer was ~-
separated and evaporated to give a gummy residue which was ~
purified by column chromatography o~ silica using ethyl ~ -
acetate/petroleum ether (60-80) (2:1) to ethyl acetate/methanol ~ :
(95:5j to afford the title compound as a yellow powder (140mg).
Rf = 0.2 in ethyl acetate on silica plates. lH NMR (250MHz, - `
CDC13) ~ 3.43 (3H, s); 3.94 (3H, s); 5.05-5.20 (2H, m); 5.29 (lH, - -;
.
WO 93/02078 PCI/GB92/01366
~112561
- 22 -
d, J = 8Hz); 6.71 (lH, d, J = 8Hz); 7.01 (lH, 8); 7.12 (lH, s); 7.25-
7.70 (9H, m~.
Step 3: N-[3(R.S)-2.3-Dih~dro-1-methyl-5-(1-
metbYlimidazol-2-vl)-2-oxo- lH- 1 .4-benzodiazePin-3-~vll-Nt-[3-
meth~lPhenvl]urea
To a solution of 1,3-dihydro-1-methyl-3~R,S)-
~benzylo cycarbonylamino)-5-(1-methylimidazol-2-yl)-2H-1,4-
benzodiazepin-2-one (O.llg) in methanol (30ml) was added 90%
formic acid (2ml~ and this mixture was added to a stirred
o suspension of 10% palladium on carbon (0.2g) in methanol
(lOml). The reactio~ mixture was stirred at ambient
temperature for 30 minutes then filtered and the solvent
evaporated. The residue was partitioned between ethyl acetate
(20ml) and 109~ sodiu~n carbonate solution ~20ml). The organic
phase was separated, dried (magIlesium sulphate) then
evaporated to give 1,3-dihydro-3(R,S)-amino-l-methyl-5-(1-
methylimidazol-2-yl~2H-1,4-benzodiazepin-2-one (50mg) whi~
was used without fi~rther purification.
A solution of this crude amine (50mg) in anhydrous
tetraliydrofuran (3ml) was treated with m-tolylisocyanate
(30mg). After 1 hour the resul~ng colourless precipitate was
collected by filtration and washed with diethyl ether to a~ord
the tit le compound (40mg) as a colourless solid of mp 222-223C.
lH NMR (360MHz, CDC13) ~ 2.30 (3H, s); 3.44 (3H, s); 3.98 (3H,
s); 5.52 (lH, d, J = 8Hz); 6.85-7.70 (llH, m). (Found: C, 63.96;
H~ 5.54; N~ 20-41- C22H22N62--5H20 requires C, 64.22; H,
5.63; N, 20.43%).
WO 93102078 PCI/GB92/01366
- 23 - ~ 2 ~ 6 1 ~-
EXAMPLE 2: N-r3(R,S)-2,3-Dihydro-1-(2-methylproPY1~2-oxo-5-
(thiazol-2-yl)-lH-1,4-benzodiazepin-3-vll-N/-~3-
methvlphenyllurea
Step 1: 1.3-Dihydro-3(R.S)-(benzyloxycarbonYlamino)-5-
(thiazol-2-Yl)-2H-1~4-benzodiazepin-2-one ..
The title compound (2.03g, 46%) was obtained from c~
(i~opropylthio)-Na-(benzyloxycarbonyl)glycine and 2-
aminophenyl-(thiazol-2-yl)methanone (J. Het. Chem. 1975, ~, ~ ~-
lo 49-57) as described in E~ample 1. mp 205C (dec.)
(dichloromethane/n-hexane). Rf = 0.25 in ethyl
acetaWpetroleum ether (60-80) (1:1) on silica plates; lH NM~
(360MHz, d6 DMSO) ~ 5.07 (2H, s); 5.17 (lH, d, J = 9Hz); 7.26-
7.39 (7H, m); 7.64 (lH, dd, J1 = 7Hz, J2 = 8Hz); 7.88 (lH, d, J = -
7Hz); 7.95-8.00 (2H, m); 8.50 (lH, d, J = 7Hz); 10.92 (lH, broad -~
s); MS, FAB+ ~ntz 393 for (M+H)+. (Found: C, 60.61; H, 4.15; N,
14-20- C20H16~43S- 0-2H20 requires C, 60.66; H, 4.17; N, - -
14.15%).
Step 2: 1.3-Dihydro-3(R.S)-(benzYloxycarbonYlamino)-1-(2- -
methYlpropYl~5~thiazol-2-Y1)-2H-1.4-benzodiazepin-2-one
The title compound (500mg) was obtained firom 1,3-dihydr~
3(R,S)-(benzyloxycarbonylamino)-5-(thiazol-2-yl)-2H- 1,4- ~ -
benzodiazepin-2-one and 1-iodo-2-methylpropane using a ~ -
procedure similar to that described in Example 1. mp 115- - --
117C. Rf = 0.45 in ethyl acetate/petroleum ether (60-80) (1~
on silica plates; lH NMR (360MHz, CDCl3) ~ 0.58 (3H, d, J =
7Hz); 0.71 (3H, d, J = 7Hz); 1.66-1.77 (lH, m); 3.43 (lH, dd, J1 =
6Hz, J2 = 14Hz); 4.29 (lH, dd, Jl = 9Hz, d2 = 14Hz); 5.09-5.18
WO 93/0207X PCr/GB92/01366
211~S61
- 24-
(2H, m); 5.41 (lH, d, J = 8Hz); 6.67 (lH, d, J = 8Hz); 7.30-7.93
(llH, m).
Step 3: N-r3(R.S)-2.3-DihYdro-1-(2-methvlpropvl)-2-oxo-5-
.
(thiazol-2-Yl)-1H-1,4-benzodiazePin-3-Yll-N/-r3
meth~lphenvllurea
Crude 1,3-dihydro-3(1R,S)-amino-5-(thiazol-2-yl)-2H-1,4-
benzodiazepin-2-one (22Gmg) was obtained from 1,3-dihydro-
3(R,S)-(benzyloxycarbonylamino)-5-thiazol-2-yl)-2H-1,4-
benzodiazepin-2-one as described in ExamPle 1, except that tlle
o reaction time was 36 hours.
A solution of this crude amine (127mg) in anhydrous
tetrahydrofuran (2ml) was treated with m-tolyl isocyanate
(45.5mg). After 2 hours the solvent was evaporated and the -
product purified by column chromatography (HPLC) on silica
using ethyl acetate/petroleum ether (60-80) (1:1) as eluant to
afford the title product (8mg). mp 222C (dec.). Rf = 0.35 in
ethyl acetate/petroleum ether (60-80) (1:1) on silica plates; ~H
NMR (360MHz, CDCl3) ~ 0.59 (3H, d, J = 6Hz); 0.71 (3H, d, J =
6Hz); 1.69-1.78 (lH, m); 3.45 (lH, dd, J1 = 6Hz, J2 = 14Hz); 4.30
(lH, dd, J1 = 9Hz, J2 = 14Hz); 5.63 (lh, broad s); 6.74-7.92 (llH,
m); MS, FAB+ m/z 448 for (M+H)+.
EXAMPLE 3: N-[3(R.S~2.3-DihYdro-1-(2-methYlProPYl)-2-oxo-5-
(thiazol-2-vl)-lE-1,4-benzodiazepin-3-vll-N/-[3-(tetrazol-5-
Yl)phenyllurea
Step 1: 5-(3-NitrophenYl)tetrazole
To a solution of 3-cyanonitrobenzene (20g) in 1-methyl-2-
wo 93/02078 PCI/GB92/01366
~`~112561 :
- 25 -
pyrrolidinone (200ml) was added triethylamine hydrochloride
(27.9g) followed by sodium azide (26.4g). The mixture was
heated at 160C for 1.5 hours, then cooled to ambient
temperature, poured into ice water (1OOOml) and acidified using -
5M HCl. The solid which precipitated from the mi2ture was ~ -~
filtered, washed with water and dried under vacuum at 50C to
afford the title tetrazole (22.1g, 86%) as a beige powder. mp ~ ~ -
154-156C. lH NMR (360MHz, CDCl3) ~ 7.59 (lH, dd, J = 8Hz);
8.19 (lH, d, J = 8Hz), 8.36 (lH, d, J = 8Hz); 8.86 (lH, s).
0 Step 2: 5-(3-Aminophenvl)tetrazolehydrochloride
To a solution of 5-(3-nitrophenyl)tetrazole (22g) in ethanol ~ ~
(500ml) was added 10% palladium on carbon (1.5g, 7% (W/w)) in ~ ~-
hydrochloric acid (23ml of a 5M solution). The mi2ture was
hydrogenated at 40 psi for 10 minutes then the catalyst filtered ~ ~;
of f and washed with water. The solvents were evaporated in - ~ -
vacuo and the brown solid azeotroped with toluene (4 x 100ml).
The resulting solid was triturated with hot ethanol to give 5-(3- -;
aminophenyl)tetrazole hydrochloride (16.3g) as a beige powder. ~ -
mp 203-205C. lH NMR (360MHz, D20) o 7.63 (lH, d, J = 8Hz), -
7.75 (lH, dd, J = 8Hz), 8.00 (2H, m).
SteP 3: N-[3(R,S)-2,3-DihYdro-1-(2-meth~rlpropvl)-2-oxo-5-
(thiazol-2-yl)-lH-1,4-benzodiazepin-3-Yll-N/-~3-(tetrazol-5-
yl)phenyllurea
Triethylamine (193mg) was added to a stirred, cooled (0C)
suspension of 5-(3-aminophenyl)tetrazole hydrochloride (188mg)
in anhydrous tetrahydrofuran (2ml). Triphosgene (93mg) was
WO 93/02078 PCI /GB92/01366
7112~61
- 26- -
added followed by a further quantity of triethylamine (96mg)
ensuring the pH > 7. The reaction mixture was stirred at
ambient temperature for 30 minutes. A solution of 1,3-dihydro-
3(R,S)-amino-5-(thiazol-2-yl)-2H-1,4-benzodiazepin-2-one
(254mg) m anhydrous tetIahydrofilran (lml) was added and the
mixture s~rred for 2 hollrs. The reaction mi~cture was diluted
with ethyl acetate (15ml) then acidified using 20% acetic acid.
The organic layer was separated and the aqueous re-extracted
with ethyl acetate (15ml). The combined organics were washed
lo with brine, dried (sodium sulphate) then filtered and e vaporated
to dryness. The crude product was purified by column
chromatography on silica using
dichloromethane/methanoVacetic acid (95:5:0.5) to af~ord the
title compound (12mg). mp 192C (dec.). Rf = 0.65 in ethyl
acetate/acetic acid (50:1) on silica plates; lH NMR (360MHz, d~
DMSO) ~ 0.47 (3E~, d, J = 6Hz); 0.68 (3H, d, J = 6Hz); 1.50-1.62
(lH, m); 3.64 (lH, dd, Jl = 6Hz, J2 = 15 H z); 4.17 (lH, dd, Jl =
8Hz, J2 = 15Hz); 5.38 (lH, d, J = 8 H z); 7.44-8.18 (llH, m); 9.34
(lH, s); MS, FAB+ m/z 502 for (M+H)+. (Found: C, 55.71; H,
4.69; N, 24.15. C24H23NgO2S.H20 requires C, 55.48; H, 4.86; -
N, 24.26%).
W093/02078 pcTJGs92/ol366 ~
~ 1 1 2 j 61
- 27 -
':
EXAMPLE_4A Tablets containina 1-25mq of comnound
Amount mg
Compound of formula (I) l.o 2.0 25.0
Microcrystalline cellulose20.0 20.0 20.0 -~
Modified food corn starch 20.0 20.0 20.0 - -~ -
Lactose 58.5 57.5 34.5
Magnesium Stearate 0.5 0.5 0.5
EXAMPLE 4B Tablets containinq 26-lOOmq of compound -
Amount mg
Compound of formula (I) 26.0 50.0 100.0
Microcrystalline cellulose80.0 80.0 80.0 ;~
Modified food corn starch 80.0 80.0 80.0
Lactose 213.5 189.5 139.5
~agnesium Stearate 0.5 0.5 0.5
The compound of formula (I), cellulose, lactose and a
portion of the corn starch are mixed and granulated with
10% corn starch paste. The resulting granulation is
sieved, dried and blended with the remainder of the corn
starch and the magnesium stearate. The resulting ~-
granulation is then compressed into tablets containing
l.Omg, 2.0mg, 25.0mg, 26.0mg, 50.0mg and lOOmg of the
, active compound per tablet. --~
EXAMPLE 5 Parenteral in~ection
Amount mg
Compound of formula (I) 1 to lOOmg
Citric Acid Monohydrate 0.75mg `
Sodium Phosphate 4.5mg
Sodium Chloride 9mg -~
Water for injection to lml
The sodium phosphate, citric acid monohydrate and sodium
chloride are dissolved in a portion of the water. The
W093/02078 PCT/GB92/01366 ~-
2561
- 28 -
compound of formula (I) is dissolved or suspended in the
solution and made up to volume.
EXAMPLE 6 Topical formulation
Amount ma
Compound of formula (I) l-lOg
Emulsifying Wax 30g
Liquid paraffin 20g
White Soft Paraffin to lOOg
10 The white soft paraffin is heated until molten. The
liquid paraffin and emulsifying wax are incorporated and
stirred until dissolved. The compound of formula (I) is
added and stirring continued until dispersed. The -
mixture is then cooled until solid.
BIOLOGICAL ACTIVITY
1. CCK Receptor Bindina fPancreas)
CCK-8 sulphated was radiolabelled with 125I-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 (150-200g) were sacrificed ~-
, by decapitation. The whole pancreas was dissected free
of fat tissue and was homogenized in 25 volumes of ice-
cold 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethane
sulphonic acid (HEPES) buffer with 0.1% soya bean trypsin
inhibitor (pH 7.4~at 25~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 (20mM (HEPES)), lmM ethylene glycol-bis-(~-
aminoethylether-N,N'-tetraacetic acid) (EGTA), 5mM MgC12,
150 mM NaCl, bacitracin 0.25 mg/ml, soya bean trypsin
inhibitor 0.1 mg/ml, and bovine serum albumin 2 mg/ml pH
W093/02078 PCT/GB92~01366
~ 1 t 2 a 6 1
~ ~.
, .
- 29 - `~
6.s at 25C) using a Teflon (trademark) homogenizer, 15
strokes at 500 rpm. The homogenate was further diluted ~-
in binding assay buffer to qive a final concentration of ~-
0.5 mg original wet weight/l ml buffer. For the binding
assay, 50 ~1 of buffer (for total binding) or unlabelled
CCK-8 sulphated to give a final concentration of 1 ~M
(for nonspecific binding) or the compounds of Formula I
(for determination of inhibition of 125I-CCK-8 binding) -~
and 50 ~1 of 500 pM 125I-CC~-8 (i.e. 50 pM final ~ --
concentration) were added to 400 ~1 of the membrane
suspensions in microfuge tubes. All assays were run in --
duplicate. The reaction mixtures were incubated at 25~C
for 2 hours and the reaction terminated by rapid
filtration (Brandell 24 well cell harvester) over Whatman
GF/C filters, washing 3 x 4 mls with ice-cold 100 Mm ~-~
NaCl. The radioactivity on the filters was counted with
a LKB gamma counter.
2. CCK Receptor Bindina tBrain) i~
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 (300-500g) were sacrificed
. by decapitation and the cortex was removed and
homogenized in 25 mL ice-cold 0.32 M 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 P2 pellet was resuspended in binding
assay buffer (20mM HEPES, 5 mM MgC12, 0.25 mg/ml
bacitracin, 1 mM EGTA pH 6.5 at 25 C), using a Teflon --
(trademark) homogenizer (5 strokes at 500 rpm) to give a ;~
final concentration of 10 mg original wet weight/1.2 ml
buffer. For the binding assay, 50 ~1 of buffer (for
total binding) or unlabelled CCK-8 sulphated to give a ~-
W093/02078 PCT/GB92/01366
25'61
- 30 -
final concentration of 1 ~M ( for nonspecific binding) or
the compounds of Formula I (for determination of
inhibition of 125I-CCK-8 binding) and 50 ~1 of 500 pM
125I-CCK-8 (i.e. final concentration of 50 pM) were added
to 400 ~1 of the membrane suspensions in microfuge tubes.
All assays were run in duplicate. The reaction mixtures
were incubated at 25 C for 2 hours and then the reaction
was terminated by rapid filtration (Brandell 24 well cell
harvester) on Whatman GF/C filters with 3 x 5 ml washes
of cold 100 mM NaCl. The radioactivity on the filters
was counted with a LKB gamma counter.
In Vitro Results
Effects of the Compounds of Formula I
~ I-CCK-8 rece~tor bindina -~
The preferred compounds of Formula I are those which
produced dose-dependent inhibition of specific 125I-CCK-8 ~-
binding as defined as the difference betweén total and
non-specific (i.e. in the presence of 1 ~M CCK) binding.
Drug displacement studies were performed with at
least 10 concentrations of compounds of Formula I and the
ICsO values were determined by regression analysis IC50
refers to the concentration of the compound required to
, inhibit 50% of specific binding of 125I-CCK-8.
The data in Table I were obtained for compounds of ~-~
Formula I.
-~
' ~'' "..
`''.`-`'','''~
- WO 93/02078 PCr/CB92/01366
~!1 12 5 61
,;..
- 3 1 -
TABLE I
CCK RECEPTOR BINDING RESULTS
IC5 o ( nM) , ~ ;
Compound 1~5I-CCK 125I-CCK
of Ex # Pancreas Brain
4~30 600
2 1300 78 ;~
3 1300 6.9
, ~, ,,
,"','~
. ~.
-' . ~, '',
'.~- ..
'-`', ''
'''` ~ "`'.
; ~'
.
