(3R)-N-(1-(tert-butylcarbonylmethyl)-2,3-dihydro-2-oxo-5-(2-pyridyl)- 1H-1,4-benzodiazepin-3-yl)-N'-(3-(methylamino)phenyl)urea (YF476): A potent and orally active gastrin/CCK-B antagonist

Article abstract of DOI:10.1021/jm960669+

A number of new 1,4-benzodiazepin-2-one-based gastrin/CCK-B receptor antagonists related to the archetypal analogue L-365,260, and more closely to the recently reported compound YM022, have been synthesized and evaluated for biological activity. The compounds were screened for their ability to inhibit the binding of [¹²⁵I]CCK-8 to gastrin/CCK-B receptors prepared from rat brains and that of [³H]L-364,718 to CCK-A receptors from rat pancreas, and were shown to be potent and selective ligands for the gastrin/CCK-B receptor. Functional studies in vivo demonstrated the compounds to be antagonists of the receptor as evidenced by their ability to inhibit pentagastrin-induced gastric acid secretion in anesthetized rats. More extensive evaluation in viva included determination of ED₅₀ values in the rat acid secretion model for selected compounds and an examination of the effect of these compounds on pentagastrin-induced gastric acid secretion in Heidenhain pouch dogs following oral and intravenous administration. Two compounds, i.e. (3R)-N- [1-[(tert-butylcarbonyl)methyl]-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4- benzodiazepin-3-yl]-N'-[3-(methylamino)phenyl]urea, 15c (YF476), and (3R)-N- [1-[(tert-Butylcarbonyl)methyl]-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4- benzodiazepin-3-yl]-N'-[3-(dimethylamino)phenyl]urea hydrochloride, 15d, showed potent dose-dependent effects in both models with the former showing excellent oral bioavailability and an ED₅₀ of 21 nmol/kg po in dogs. 15e is currently under clinical investigation for the treatment of gastro-oesophagal reflux disease (GORD).

Full text of DOI:10.1021/jm960669+

J . Med. Chem. 1997, 40, 331-341
331
(3R)-N-(1-(ter t-Bu tylca r bon ylm eth yl)-2,3-d ih yd r o-2-oxo-5-(2-p yr id yl)-1H-
1,4-ben zod ia zep in -3-yl)-N′-(3-(m eth yla m in o)p h en yl)u r ea (YF 476): A P oten t a n d
Or a lly Active Ga str in /CCK-B An ta gon ist
Graeme Semple,*,† Hamish Ryder,† David P. Rooker,† Andrzej R. Batt,† David A. Kendrick,† Michael Szelke,†
Mitsuaki Ohta,‡ Masato Satoh,‡ Akito Nishida,‡ Shinobu Akuzawa,‡ and Keiji Miyata‡
Ferring Research Institute, Chilworth Research Centre, Chilworth, Southampton SO16 7NP, U.K., and Institute for Drug
Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21, Miyukigaoka, Tsukuba, Ibaraki 305, J apan
Received September 25, 1996X
A number of new 1,4-benzodiazepin-2-one-based gastrin/CCK-B receptor antagonists related
to the archetypal analogue L-365,260, and more closely to the recently reported compound
YM022, have been synthesized and evaluated for biological activity. The compounds were
screened for their ability to inhibit the binding of [125I]CCK-8 to gastrin/CCK-B receptors
prepared from rat brains and that of [3H]L-364,718 to CCK-A receptors from rat pancreas,
and were shown to be potent and selective ligands for the gastrin/CCK-B receptor. Functional
studies in vivo demonstrated the compounds to be antagonists of the receptor as evidenced by
their ability to inhibit pentagastrin-induced gastric acid secretion in anesthetized rats. More
extensive evaluation in vivo included determination of ED50 values in the rat acid secretion
model for selected compounds and an examination of the effect of these compounds on
pentagastrin-induced gastric acid secretion in Heidenhain pouch dogs following oral and
intravenous administration. Two compounds, i.e. (3R)-N-[1-[(tert-butylcarbonyl)methyl]-2,3-
dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepin-3-yl]-N′-[3-(methylamino)phenyl]urea, 15c
(YF476), and (3R)-N-[1-[(tert-Butylcarbonyl)methyl]-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-
benzodiazepin-3-yl]-N′-[3-(dimethylamino)phenyl]urea hydrochloride, 15d , showed potent dose-
dependent effects in both models with the former showing excellent oral bioavailability and
an ED50 of 21nmol/kg po in dogs. 15c is currently under clinical investigation for the treatment
of gastro-oesophagal reflux disease (GORD).
In tr od u ction
Gastrin-17 is a heptadecapeptide hormone which acts
as a physiological mediator of acid secretion in response
to meals. It is closely related to cholecystokinin (CCK),
the C-terminal pentapeptide portion being identical.
Cloning of the gastrin and CCK receptors, designated
CCK-A (the peripheral receptor) and CCK-B (the central
receptor), has shown the gastrin and CCK-B receptors
to be identical.8 More recently there has been convinc-
ing evidence for a second gastrin receptor, which can
bind the glycine-extended form of gastrin-17 and which
appears to be responsible for the mitogenic effects of
gastrin.9
With the above in mind, we decided to investigate
inhibitors of the gastrin/CCK-B receptor as an alterna-
tive method of reducing gastric acid secretion, or indeed
as an adjunct to longer term treatment with known
inhibitors of acid secretion, which would avoid gastrin-
mediated side effects and hence provide a useful new
treatment for GORD and other gastrointestinal disor-
ders.
We have recently described the discovery of a potent
series of 1,4-benzodiazepin-2-one gastrin/CCK-B recep-
tor antagonists related to the archetypal analogue
L-365,260 (1, Figure 1) with sub-nanomolar affinities
for the receptor, the compound YM022 (2)10 being the
optimal structure in our series.11 Further improvement
of the in vivo activity and bioavailability of these
derivatives by incorporation of 1-alkylcarbonylmethyl
and 5-(2-pyridyl) substituents has been communicated
in preliminary form.12,13
Gastric acid secretion is controlled by the action of
(H+,K+)-ATPase (the proton pump) in response to
stimulation of muscarinic (M3), histamine (H2), or
gastrin receptors by their respective agonists. H2 recep-
tor antagonists and proton pump inhibitors are highly
effective in reducing acid secretion1-3 and have been the
treatments of choice for peptic ulcer disease for a
number of years. However, prolonged inhibition of
secretion with either of these agents causes continuous
stimulation of G-cells, resulting in hypergastrinaemia
which is believed to lead to hyperplasia of the oxyntic
mucosa4,5 and the so-called acid rebound phenomenon.
In addition, long-term administration of omeprazole, the
prototypical proton pump inhibitor, results in the
growth of gastric carcinomas in rats,6 although such an
effect has not been observed in humans.7
With the discovery of evidence for the involvement of
the pathogen Helicobacter pylori in the majority of
peptic ulcer cases, clinical prescribing regimens have
changed. The increasing use of combination therapies
has meant that antisecretory drugs are given for much
shorter periods of time and any rebound effect is less
important. However, Helicobacter pylori is not impli-
cated in the pathogenesis of gastro-oesophagal reflux
disease (GORD), and with longer treatment courses
required with existing antisecretory drugs for treatment
of this disorder, hypergastrinaemia, acid rebound, and
relapse again present a problem.
In this paper we would like to present the structure-
activity relationship studies which led us to the discov-
ery of (3R)-N-[1-[(tert-Butylcarbonyl)methyl]-2,3-dihydro-
† Ferring Research Institute.
‡ Yamanouchi Pharmaceutical Co., Ltd.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
S0022-2623(96)00669-3 CCC: $14.00 © 1997 American Chemical Society

332 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Semple et al.
F igu r e 2. Synthesis of 3-amino-1-[(tert-butylcarbonyl)-
methyl]-2,5-dihydro-5-(2-pyridyl)-1H-1,4-benzodiazepin-2-
one. Reagents and conditions: (i) BrCH2COBr, AcOH; (ii) (a)
t
NH3, (b) AcOH; (iii) (a) NaH, DMF, (b) BuCOCH2Br; (iv) (a)
i
KOtBu, (b) AmONO; (v) H2/5% Ru on C, 60 °C, 20 kg/cm2.
F igu r e 1. Structures of benzodiazepine-based gastrin/CCK-B
ligands.
2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepin-3-yl]-N′-[3-
(methylamino)phenyl]urea, (15c, YF476), a novel, potent
gastrin/CCK-B antagonist incorporating a combination
of these and other modifications and which is currently
undergoing clinical trials.
F igu r e 3. General strategy for benzodiazepine synthesis.
Ch em istr y
Two approaches have previously been applied suc-
cessfully to the synthesis of 3-substituted 1,4-benzodi-
azepines. The first involves construction of the parent
heterocycle, followed by functionalization. In the case
of 3-amino derivatives, this has been best achieved by
formation of an oxime at the 3-position of the 1-alkyl-
ated 1,4-benzodiazepine, followed by catalytic hydroge-
nation.14 As previously reported, YM022 was readily
prepared using this procedure.11 This method turned
out not to be generally applicable, however, as attempts
to adapt the chemistry for use in compounds with a 5-(2-
pyridyl) substituent on the benzodiazepine scaffold
failed at the final stage (Figure 2) when imine isomer-
ization to give 8 was observed in addition to the desired
oxime reduction.
gave variable yields, and large-scale use of thioethers
and highly toxic mercury salts is clearly undesirable.
We were, however, able to make use of the same
strategy for the construction of the benzodiazepine ring
by using benzotriazole as the displaceable group (Figure
4) as reported recently by ourselves17 and others.18 The
benzyl carbamate of R-(1-benzotriazolyl)glycine19 (9) was
coupled to the requisite (2-aminobenzoyl)aryl derivative
in excellent yield, the benzotriazole moiety was then
displaced with ammonia under mild conditions, and the
ring synthesis was completed by brief acid treatment
to form the benzodiazepine 11. High yields of crystal-
lizable products were obtained in this way under mild
conditions and without the use of toxic reagents, and
this route became our method of choice.
The alternative strategy for the synthesis of 3-sub-
stituted 1,4-benzodiazepin-2-ones requires N-acylation
of a 2-aminobenzoyl derivative with a carboxylic acid
containing a suitable masked amine group X at the
R-position (Figure 3). The amine functionality is even-
tually revealed and the intermediate cyclized to give the
desired benzodiazepine.
This approach has been used to provide 3-amino-
substituted benzodiazepines in good yield by coupling
of 2-aminobenzophenone to R-(isopropylthio)-N-(benzyl-
oxycarbonyl)glycine15 (i.e. Figure 3, X ) isopropylthio),
followed by mercuric chloride-induced displacement of
the thioether group with ammonia and finally acid-
catalyzed cyclization to provide the desired benzodiaz-
epine.16 In our hands, this method proved very useful
for the small-scale preparation of a wide range of
protected 3-amino-5-arylbenzodiazepines and was highly
amenable to producing series of analogues with 1-, 3-,
and 5-position variations. Scale-up reactions, however,
Conversion of 11 to the key intermediate 13 was
accomplished by 1-alkylation with 1-bromopinacolone,
followed by removal of the carbamate protecting group.
In the 5-phenyl series the alkylated compound 12a could
be readily deprotected by hydrogenolysis of its benzyl
carbamate (Z) protecting group. However, attempted
removal of the Z-group from the 5-(2-pyridyl) analogue
12b under the same conditions resulted in concomitant
reduction and/or isomerization of the benzodiazepine
imine function, depending on the hydrogenation condi-
tions. The deprotection could, however, be effected in
this case by treatment of 12b with dry HBr in DCM.
With carbamate protecting groups other than Z, a
cleaner conversion to 13b was achieved by treatment
with TMSI.17
It is well documented that the opposite enantiomers
of benzodiazepine-based gastrin/CCK-B ligands behave
quite differently towards the receptor, with the (3R)-
isomer being consistently the more active species.11,20

A Potent and Orally Active Gastrin/ CCK-B Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 333
F igu r e 4. Benzotriazole-mediated synthesis of 5-phenyl- and 5-(2-pyridyl)-1,4-benzodiazepin-2-ones. Reagents and conditions:
t
(i) EDC, 9, 0 °C to room temperature; (ii) (a) NH3/MeOH, (b) AcOH; (iii) (a) NaH, DMF, (b) BuCOCH2Br; (iv) ArdPh, H2/5% Pd
on C; Ard2-Pyr, HBr in DCM, 0 °C; (v) 3-R′-PhNCO.
F igu r e 5. Interconversion of isomeric benzodiazepines via 3,5-dichlorosalicyl imines.
Where appropriate, we prepared the more active enan-
tiomers of 14 and 15 by resolution of the 3-amino
derivatives 13 followed by reaction of the resultant
homochiral amine intermediate with the required iso-
cyanate. The amine 13a was readily resolved using a
resolution-racemization method,21 but under the same
conditions, the analogue 13b was converted to the
isomeric 3-amino-2,5-dihydro-5-(2-pyridyl)-1H-1,4-ben-
zodiazepin-2-one (8) obtained previously, and we were
only able to resolve 13b by classical fractional crystal-
lization of its (R)- and (S)-mandelate salts.17
The resolution-racemization process operates through
the interconversion of a series of imine intermediates
which reversibly epimerize the 3-amino center (Figure
5, X ) CH). However it appears that when the 5-sub-
stituent is a 2-pyridyl group (Figure 5, X ) N) the
favored isomerization is for the imino function within
the benzodiazepine ring to move into conjugation with

334 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Semple et al.
the external imine and not vice versa. This process does
not appear to be reversible when X ) N, and so the
eventual hydrolysis of the salicyl imine in this case gave
the mandelic acid salt of 8 as the only isolable product.
As with the differences in the hydrogenolysis products
between the two series, the formation of this alternative
product can only be ascribed to the differing electronic
properties of the 5-phenyl and 5-(2-pyridyl) substituents
adjacent to the benzodiazepine imine function.
carboxyphenyl)urea derivatives.29 We can now report
that when combinations of the above modifications are
incorporated into the same molecule, the improvements
in the in vivo effects are essentially additive, resulting
eventually in the identification of 15c which has potent
oral antisecretory activity in Heidenhain pouch dogs.
We initially examined the effect of incorporating
neutral, acidic, or basic groups in the 3-position of the
phenylurea substituent of the 1-[(tert-butylcarbonyl)-
methyl]-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-
one core structure as shown in Table 1. We observed
that the incorporation of a 3-carboxy substituent, to give
14b, did not improve binding to the rat gastrin/CCK-B
receptor, although some in vivo antisecretory activity
was still observed at 0.1 µmol/kg iv in our rat screening
model. It is important to note that basal levels of acid
secretion in this model are typically around 25-30% of
the pentagastrin-induced peak; hence a figure of 75-
80% inhibition represents the return of acid secretion
to basal levels or below.
Biology
The methods used for measuring binding of [125I]CCK-
8 to rat brains and that of [3H]L-364,718 to rat pancreas
were essentially identical to those described previ-
ously.22 Specific binding was defined as the difference
between total binding and nonspecific binding in the
presence of 1 mM CCK-8 or L-364,718.
For in vivo screening studies, gastric acid secretion
was measured in anesthetized rats as reported previ-
ously.10 Acid secretion was measured at pH 7.0 using
the pH-stat method with the addition of 0.025 N NaOH
to the reservoir. Approximately 30 min after basal
secretion had stabilized, pentagastrin at a rate of 20
nmol/kg/h was infused through the femoral vein. Test
compounds were dissolved with polyethylene glycol 300
and injected iv 1 h after the start of pentagastrin
infusion.
In the secondary in vivo test system, male beagle dogs
with a Heidenhain pouch were used. One month after
preparation of the pouch, secretory experiments were
performed once a week in each animal throughout the
experiments. Acidity of the gastric juice was measured
by automatic titration of the gastric juice with 0.05 M
NaOH to pH 7.0. Pentagastrin was infused at a rate of
8 µmol/kg/h through the femoral vein. Test compounds
were administered po or iv at 3 h after the start of
pentagastrin infusion.
The (3-aminophenyl)urea derivative 14f was essen-
tially equipotent with the parent 3-tolylurea derivative
14a both in vitro and in vivo in rats. However, when
alkylated amino groups were incorporated into the same
position (14g-j), there was a significant improvement
in the compounds’ affinities for the gastrin/CCK-B
receptor, which was accompanied in most cases by
improved receptor selectivity. In addition, all of the
[3-(alkylamino)phenyl]urea analogues showed signifi-
cant inhibition of pentagastrin-induced gastric acid
secretion in vivo in rats at the screening dose of 0.1
µmol/kg iv.
We next examined the effect of introducing the 5-(2-
pyridyl) substituent, which we had previously shown
conferred improved bioavailability when incorporated
as a single change to the parent structure,13 into the
[3-(alkylamino)phenyl]urea series. As can be seen in
Table 1, all of the resulting compounds with the excep-
tion of the 3-(1-piperidyl)phenyl urea analogue 15h
showed comparable or improved affinity and selectivity
for the gastrin/CCK-B receptor when compared to the
parent 3-tolylurea derivative 15a . In addition, the
active compounds in this second [3-(alkylamino)phenyl]-
urea series again showed potent inhibition of pentagas-
trin-induced gastric acid secretion in vivo in rats at the
screening dose of 0.1 µmol/kg iv. No significant advan-
tage was observed by either increasing the size of the
alkyl groups on the amino nitrogen or incorporating
them into a ring.
Resu lts a n d Discu ssion
A major drawback associated with the early benzo-
diazepine-based gastrin/CCK-B receptor antagonists
was their lack of oral efficacy. This is exemplified by
L-365,260, which is only sparingly soluble in water and
has very limited oral bioavailability unless dosed as a
solution in PEG 600.23 We have previously shown that
incorporation of a (tert-butylcarbonyl)methyl group at
the 1-position12 or a 2-pyridyl group at the 5-position13
of the parent benzodiazepine structure provides a
significant increase in absorption. Similar results have
been achieved by incorporation of either a cyclohexyl
group24 or a cyclic amine to form an amidino functional-
ity in the 5-position.25 Other attempts to improve
aqueous solubility have included introducing acidic
groups,26 or lipophilic surrogates thereof,27 into the
3-position of the aryl urea portion of either the 1,4-
benzodiazepin-2-one parent system or closely related
structures.28 We have recently shown that the opposite
strategy, introducing basic amino substituents into the
same region of the YM022 series, can provide an
improvement in selectivity for the gastrin/CCK-B recep-
tor over the CCK-A receptor. More significantly per-
haps, increased inhibition of pentagastrin-induced gas-
tric acid secretion in rats following intraduodenal
administration was also observed for these compounds
when compared to either the (3-methylphenyl)- or (3-
Several compounds from the two series were selected
for more extensive evaluation in vivo (Table 2) according
to the methods previously described.30 ED50 values
were determined for the inhibition of pentagastrin-
induced gastric acid secretion in rats following iv
administration of the test compound, and as expected,
all of the analogues tested showed potency comparable
to that of the original parent compound YM022 in this
model.
We next examined the effect of the compounds on
pentagastrin-induced gastric acid secretion in Heiden-
hain pouch dogs. As we were searching for compounds
with significant oral activity, for our first screening
point we selected an oral dose of 3 µmol/kg and all of
the compounds tested showed a significant effect at this
dose. There was already an indication that the incor-

A Potent and Orally Active Gastrin/ CCK-B Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 335
Ta ble 1. Structure-Activity Relationships of 3-Substituted Phenylurea Derivatives of 2,3-Dihydro-1H-1,4-benzodiazepin-2-ones 14
and 15
in vivo screening
(% inhibition of H+
compd
no.
CCK-Ba
(nM)
CCK-Ab
(nM)
ratio
(CCK-A/CCK-B)
secretion in rats at 0.1
config
R′
µmol/kg iv)c
2
0.11
(0.10-0.11)
0.52
146
(120-170)
111
1327
213
ED50 ) 8.3 nmol/kg
(6.1-10.9)
77d
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
15a
15b
15c
15d
15e
15f
15g
15h
R
Me
(0.43-0.63)
3.29
(2.41-4.50)
0.16
(0.10-0.28)
1.29
(0.25-6.70)
0.59
(0.28-1.21)
0.50
(0.38-0.67)
0.11
(0.08-0.15)
0.21
(0.18-0.29)
0.14
(0.11-0.18)
0.17
(0.13-0.22)
0.44
(0.33-0.59)
0.43
(0.35-0.54)
0.10
(0.08-0.13)
0.20
(0.15-0.26)
1.26
(0.83-1.91)
0.11
(0.07-0.19)
0.11
(85-146)
2059
(1594-2659)
589
(527-658)
700
(631-776)
561
(521-605)
953
(807-1125)
120
(88-164)
89
(73-108)
558
(421-741)
157
(145-171)
470
(361-611)
1820
(1699-1949)
502
(434-581)
113
(82-156)
572
(486-674)
301
(230-395)
253
RS
RS
RS
RS
R
CO2H
626
56
76
65
74
75
80
65
82
79
78
62
87
65d
nd
74
75
55
NO2
3681
542
CN
NHCHO
NH2
951
1906
1091
423
R
NHMe
NMe2
R
R
NEt2
3986
923
R
1-pyrrolidyl
Me
R
1068
4233
5020
565
R
NH2
R
NHMe
NMe2
R
S
NMe2
454
R
NEt2
2736
2300
1124
R
1-pyrrolidyl
1-piperidyl
(0.10-0.12)
0.84
(188-342)
944
R
(0.79-0.88)
(759-1173)
a
b
IC50 value for displacement of [125I]CCK-8 from gastrin/CCK-B receptors from rat brain (95% confidence limits). IC50 value for
displacement of [3H]L-364,718 from CCK-A receptors from rat pancreas (95% confidence limits). c Inhibition of pentagastrin-induced gastric
acid secretion in anethsetized rats (0.1 µmol/kg iv). Percent inhibition at a dose of 0.03 µmol/kg.
d
Ta ble 2. Further in Vivo Evaluation of New Gastrin/CCK-B Antagonists. Inhibition of Pentagastrin-Induced Gastric Acid Secretion
in Rats and Heidenhain Pouch Dogs
inhibition of pentagastrin-induced gastric acid inhibition of pentagastrin-induced gastric acid
compd
SD rat (iv) ED50 (µmol/kg)
secretion in Heidenhain pouch dogs (po)
secretion in Heidenhain pouch dogs (iv)
YM022 (2)
0.0078
72% at 3 µmol/kg
ED50 ) 0.026 µmol/kg
(0.008-0.051)
26% at 1 µmol/kg
ED50 ) 1.9 µmol/kg (1.0-2.9)a
53% at 3 µmol/kg
14a
14h
15a
0.0057
0.012
0.016
nd
nd
nd
99% at 3 µmol/kg
97% at 3 µmol/kg
7% at 0.3 µmol/kg
15c
0.008
0.011
100% at 0.1 µmol/kg
65% at 0.03 µmol/kg
ED50 ) 0.021 µmol/kg (0.013-0.029)
98% at 0.1 µmol/kg
77% at 0.03 µmol/kg
ED50 ) 0.018 µmol/kg
(0.013-0.027)
15d
ED50 ) 0.014 µmol/kg
(0.009-0.033)
a
Figures in parentheses represent 95% confidence limits. nd ) not determined.
poration of an alkylated amino group conferred benefi-
cial effects on oral absorption, as 14h was considerably
more potent than 14a at this preliminary oral dose level.
All three analogues which incorporated a 5-(2-pyridyl)
substituent showed complete inhibition of acid secretion
at the first screening dose. Lowering the dose allowed
us to show that the combination of a 5-(2-pyridyl) group
and a 3-(alkylamino) substituent on the urea portion
provided highly potent orally active compounds. 15c
and 15d were essentially equipotent in this dog model,
and an ED50 value of 21 nmol/kg (∼0.01 mg/kg) was
determined for 15c,30 thus showing this compound to
be about 90 times more potent than YM022 after oral
administration. In addition, the inhibiton curves (Fig-
ure 6) showed that both compounds had an excellent
duration of action, maintaining complete inhibiton of
acid secretion for more than 6 h following oral admin-
istration at the 100 nmol/kg dose level.

336 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Semple et al.
uncorrected. Optical rotations were measured using a Perkin-
Elmer 241 polarimeter and are uncorrected. 1H NMR spectra
were obtained using either a J EOL FX-90Q, a J EOL EX-270,
or a J EOL J NN-EX-400 spectrometer. 13C NMR spectra were
obtained using a J EOL EX-270 spectrometer. Carbon atom
types were determined by 90- and 135-DEPT experiments.
Chemical shifts are reported in ppm (δ) downfield of tetra-
methylsilane as an internal reference (δ 0.0). Positive FAB
mass spectra were recorded in glycerol or thioglycerol matrix
either at the Michael Barber Centre for Mass Spectrometry,
UMIST, Manchester, U.K., or by the Analytical Department
of Yamanouchi Pharmaceutical Co. Elemental analyses were
determined either by Elemental Microanalysis Ltd., Oke-
hampton, Devon, U.K. or by the Analytical Department of
Yamanouchi Pharmaceutical Co. and are within (0.4% of
calculated values. All reagents were obtained from commercial
sources and used without further purification unless otherwise
noted.
2-[2-[2-Br om oa cet yl)a m in o]b en zoyl]p yr id in e (4)31
.
3b17,32 (2.5 g, 12.63 mmol) was taken up in acetic acid (25 mL)
and the mixture stirred at 4 °C. Bromoacetyl bromide (1.1
mL, 12.8 mmol) was added dropwise over 20 min, and stirring
continued for a further 15 min at 4 °C. The mixture was then
evaporated, and the residue was partitioned between EtOAc
(50 mL) and 0.5 M NaOH (50 mL). The organic portion was
washed with water and brine, dried over Mg2SO4, and evapo-
rated. The title compound was crystallized from EtOAc/
hexane as a pale brown solid (3.25 g, 81%): mp 92-94 °C; 1H
NMR (400 MHz, CDCl3) δ 11.67 (br s, 1H), 8.73 (d, 1H, J )
5Hz), 8.64 (d, 1H, J ) 8Hz), 7.96-7.90 (m, 2H), 7.82 (dd, 1H,
J 1 ) 8 Hz, J 2 ) 2 Hz), 7.61 (m, 1H), 7.51 (ddd, 1H, J 1 ) 7 Hz,
J 2 ) 5 Hz, J 3 ) 2 Hz), 7.17 (t, 1H, J ) 8 Hz), 4.03 (s, 2H) ppm.
Anal. (C14H11N2O2Br) C, H, N, Br.
F igu r e 6. Inhibition of pentagastrin-induced gastric acid
secretion in Heidenhain pouch dogs by oral administration of
15c and 15d . Pentagastrin 8 µmol/kg/h was infused from 3 h
before, until 6 h after drug administration (t ) 0), and acid
secretion was measured by automatic titration. Each point is
represented as a percentage of values observed immediately
before drug administration and is the mean ( SEM (five
animals per group).
2,3-Dih yd r o-5-(2-p yr id yl)-1H -1,4-b e n zod ia ze p in -2-
on e (5). The title compound was prepared directly from 3b
without purification of the bromoacetylated intermediate 4 as
1
described:31 mp 245-246 °C (lit.31 mp 232-234 °C); H NMR
(400 MHz, CDCl3) δ 9.08 (br s, 1H), 8.64 (dd, 1H, J 1 ) 5 Hz,
J 2 ) 1 Hz), 7.95 (d, 1H, J ) 8 Hz), 7.81 (dt, 1H, J t ) 8 Hz, J d
) 2Hz), 7.37 (dd, 1H, J 1 ) 7 Hz, J 2 ) 5 Hz), 7.33 (dd, 1H, J 1
) 8 Hz, J 2 ) 1 Hz), 7.15 (t, 1H, J ) 8 Hz), 7.08 (d, 1H, J ) 8
Hz), 4.37 (s, 2H) ppm. Anal. (C14H11N3O) C, H, N.
To obtain an indication of the magnitude of the
advantage provided by our new analogues in terms of
oral bioavailability, we determined the ability of YM022,
15c, and 15d to inhibit pentagastrin-induced gastric
acid secretion in Heidenhain pouch dogs following iv
administration, and as expected all three compounds
showed potent effects via this route (Table 2). When
compared to their effects following oral dosing, we
observed that compounds 15c and 15d showed only a
modest difference in activity between oral and iv
administration, whereas YM022 was around 70 times
less potent following oral administration than by the iv
route. These data indicated that both 15c and 15d , but
not YM022, have excellent oral bioavailibility in dogs,
and this together with their long duration of action in
vivo suggested that we had identified two possible
clinical candidates for the treatment of GORD and other
gastrointestinal disorders.
Finally we conducted physicochemical studies on the
two potential clinical candidates to determine their
stability in the crystalline form. Gratifyingly, 15c was
obtained in a single-crystal form when isolated as its
free base and remained stable even after 3 months at
40 °C and 75% relative humidity (no imine bond
tautomerization was observed under these conditions).
This compound, given the code number YF476, was
selected for further development and is currently under
clinical investigation for the treatment of GORD.
1-[(ter t-Bu tylcar bon yl)m eth yl]-2,3-dih ydr o-5-(2-pyr idyl)-
1H-1,4-ben zod ia zep in -2-on e (6). 5 (8.3 g, 35 mmol) was
azeotroped with DMF and the residue dissolved in dry DMF
(300 mL) at 4 °C under nitrogen. Sodium hydride (1.4 g, 80%
dispersion in oil, 46.6 mmol) was added portionwise with
stirring, and the mixture was stirred for 40 min at 4 °C.
1-Bromopinacolone (6.0 mL, 46 mmol) was added, and the
mixture was stirred at 4 °C to room temperature over 2 h.
The mixture was evaporated to dryness and partitioned
between EtOAc and water. The organic portion was washed
with brine, dried (MgSO4), and evaporated. The residue was
crystallized from EtOAc/hexane to provide a colorless solid
1
(8.65 g, 74%): mp 180-183 °C; H NMR (400 MHz, CDCl3) δ
8.63 (dd, 1H, J 1 ) 5 Hz, J 2 ) 1 Hz), 8.08 (d, 1H, J ) 8 Hz),
7.81 (dt, 1H, J t ) 8 Hz, J d ) 2 Hz), 7.48 (dt, 1H, J t ) 8 Hz, J d
) 2 Hz), 7.38-7.33 (m, 2H), 7.21 (m, 1H), 7.08 (d, 1H, J )
8Hz), 5.09 (d, 1H, J ) 18 Hz), 4.86 (d, 1H, J ) 10 Hz), 4.36 (d,
1H, J ) 18 Hz), 3.98 (d, 1H, J ) 10 Hz), 1.28 (s, 9H) ppm;
FAB-MS (M + H)+ ) 336. Anal. (C20H21N3O2) C, H, N.
1-[(ter t-Bu tylca r bon yl)m eth yl]-2,3-d ih yd r o-3-oxim id o-
5-(2-p yr id yl)-1H-1,4-ben zod ia zep in -2-on e (7). The title
compound was prepared using a modified version of the
previously described procedure.14,11 6 (2.00 g, 6.0 mmol) was
suspended in dry toluene (72 mL) at -20 °C under nitrogen.
KOtBu (2.68 g, 23.9 mmol) was added portionwise to the
stirring mixture so that the internal temperature did not rise
above -10 °C, and stirring continued for a further 30 min at
-20 °C. Isoamyl nitrite (2.10 g, 3equiv) was added, and the
mixture was stirred at -20 to -5 °C over 3 h and then poured
into a mixture of ice (100 g), AcOH (5 mL), and EtOAc (100
mL). The aqueous portion was basified and extracted with
EtOAc (50 mL), and the combined organic portions were
washed with brine, dried (Mg2SO4), and evaporated. The
residue was crystallized from hot EtOAc (70 mL) as a pale
Exp er im en ta l Section
Analytical and spectroscopic data for test compounds are
included in the Supporting Information. Melting points were
determined using a Yanaco MP-500D instrument and are

A Potent and Orally Active Gastrin/ CCK-B Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 337
yellow solid (1.10 g, 51%): mp 262-264 °C dec; 1H NMR (400
MHz, DMSO-d6) δ 11.17 (1H, s), 8.65 (d, 1H, J ) 8 Hz), 8.22
(d, 1H, J ) 8 Hz), 8.05 (dt, 1H, J t ) 8 Hz, J d ) 2 Hz), 7.63-
7.58 (m, 2H); 7.40 (d, 1H, J ) 6 Hz), 7.30-7.24 (m, 2H), 5.03
(d, 1H, J ) 18 Hz), 4.91 (d, 1H, J ) 18 Hz), 1.17 (s, 9H) ppm;
FAB-MS (M + H)+ ) 365. Anal. (C20H20N4O3) C, H, N.
3-Am in o-1-[(ter t-bu tylca r bon yl)m eth yl]-2,5-d ih yd r o-5-
(2-p yr id yl)-1H-1,4-ben zod ia zep in -2-on e (8). 7 (500 mg,
1.36 mmol) was taken up in MeOH (25 mL). The solution was
degassed and treated with 5% Ru/C (Lancaster Synthesis, 150
mg). The mixture was hydrogenated in a Parr apparatus (20
kg/cm2 H2, 60 °C, 24 h) and then filtered and the catalyst well
washed with methanol. The solvent was removed by evapora-
tion, and the title compound was obtained by chromatography
on silica (eluant 5% MeOH in CHCl3) as a colorless solid (446
mg, 93%): 1H-NMR (400 MHz, CDCl3) δ 8.65 (d, 1H, J ) 4
Hz), 7.97 (d, 1H, J ) 8 Hz), 7.81 (m, 1H), 7.26 (m, 1H), 7.19
(m, 1H), 7.01 (m, 2H), 6.44 (d, 1H, J ) 8 Hz), 5.98 (s, 1H),
5.15 (d, 1H, J ) 18 Hz), 4.89 (br s, 2H), 4.70 (d, 1H, J ) 18
Hz), 1.31 (s, 9H) ppm; FAB-MS (M + 1)+ ) 351.
the mixture was evaporated, azeotroped with toluene, and
crystallized from AcOH/water/dioxane to provide a pale brown
solid (1.26 g, 80%) which was dried in vacuo over P2O5: 1H
NMR (270 MHz, CDCl3) δ 7.4-7.2 (m, 3H), 6.78 (m, 1H), 3.35
(m, 4H), 2.02 (m, 4H) ppm.
Meth yl 3-(4-P en ten oyla m in o)ben zoa te. Methyl 3-ami-
nobenzoate (4.5 g, 29.8 mmol) was taken up in DCM (10 mL)
and pyridine (1 mL) at 0 °C. 4-Pentenoyl chloride (freshly
prepared from 4-pentenoic acid (3.0 g, 29.97 mmol) and thionyl
chloride (6.6 mL) at room temperature for 1 h, evaporated and
azeotroped with DCM) was added dropwise in DCM (3 mL).
The mixture was allowed to warm to room temperature, stirred
overnight, and then evaporated. The residue was partitioned
between EtOAc and 1 M HCl. The organic portion was washed
with 5% KHCO3 and brine, filtered (Whatman 1PS phase
separator), and evaporated. The residue was chromato-
graphed (eluant 30% EtOAc in hexanes) to provide a colorless
oil (2.20 g, 32%): 1H NMR (270 MHz, CDCl3) δ 8.05 (t, 1H, J
) 1.5 Hz), 7.93 (d, 1H, J ) 8 Hz), 7.79 (d, 1H, J ) 8 Hz), 7.56
(br s, 1H), 7.42 (t, 1H, J ) 8 Hz), 5.92 (m, 1H), 5.2-5.0 (m,
2H), 3.93 (s, 3H), 2.51 (m, 4H) ppm.
P r ep a r a tion of 3-Su bstitu ted Ca r boxylic Acid s a n d
Isocya n a tes. 3-(F or m yla m in o)ben zoic Acid . Acetic an-
hydride (76 mL) was added to 98% formic acid (130 mL), and
the mixture was stirred at room temperature for 30 min.
3-Aminobenzoic acid (15 g, 109.5 mmol) was then added. The
mixture was stirred at room temperature for 1 h and then
treated with water (1.3 L) and stirring continued overnight.
The resultant white precipitate was collected, washed with
water, and dried in vacuo over P2O5 (15.4 g, 85%): 1H NMR
(270 MHz, MeOH-d4) δ 8.45-8.3 (m, 2H), 8.0-7.5 (m, 4H) ppm.
3-(N-F or m ylm eth yla m in o)ben zoic Acid . A solution of
3-(formylamino)benzoic acid (2.28 g, 13.8 mmol) in DMF (25
mL) was added dropwise to a suspension of sodium hydride
(1.05 g, 80% dispersion in oil) in DMF (15 mL) at 0 °C. The
mixture was allowed to warm to room temperature over 1 h,
and then iodomethane (0.95 ml) was added. A second portion
of iodomethane (0.95 mL) was added after 1 h, and the mixture
was stirred at room temperature overnight. The solvent was
removed by evaporation, and the residue was partitioned
between ethyl acetate and 1 M HCl. The organic layer was
washed with brine, filtered (Whatman 1 PS phase separator),
and evaporated. The residue was chromatographed on silica
(eluant 60% EtOAc in hexane) to provide the methyl ester of
the title compound as a colorless solid (2.30 g, 86%). A portion
of this ester (900 mg, 4.66 mmol) was taken up in dioxane/
water (2/1, v/v, 30 mL) and treated with LiOH‚H2O (378 mg,
9 mmol) at room temperature with stirring overnight. The
mixture was acidified with 1 M HCl and extracted twice with
EtOAc. The combined extracts were washed with brine,
filtered (Whatman 1 PS phase separator), and evaporated. The
title compound (420 mg, 50%) was used in the next step
without further purification: 1H NMR (270 MHz, CDCl3) δ 8.52
(s, 1H), 8.0-7.85 (m, 2H), 7.5-7.35 (m, 2H), 3.36 (s, 3H) ppm.
3-(1-P yr r olid yl)ben zoic Acid . m-Aminobenzoic acid (13.7
g, 0.1 mol) was taken up in methanol (150 mL) and cooled to
0 °C. Acetyl chloride (10 mL) was added dropwise, and then
the mixture was heated at reflux under nitrogen for 1 h. The
mixture was cooled, evaporated, and partitioned between
EtOAc and 5% KHCO3. The organic portion was washed with
brine, filtered (Whatman 1PS phase separator) and evaporated
to provide methyl m-aminobenzoate as a brown oil which
crystallized on standing (13.2 g, 88%). A portion of this amino
ester (5.45 g, 36.1 mmol) was taken up in dry DMF (70mL)
and treated with sodium hydride (3.78 g, 80% dispersion in
oil, 126 mmol) at 0 °C under nitrogen for 14 h. 1,4-Dibro-
mobutane (14.05 g, 65 mmol) and potassium iodide (0.6 g, 3.7
mmol) were added, and the mixture was heated at 80 °C for
72 h. The mixture was cooled, evaporated, and partitioned
between EtOAc and 5% KHCO3. The organic portion was
washed with brine, filtered (Whatman 1PS phase separator),
and evaporated. The residue was chromatographed on silica
(eluant 8% EtOAc in hexane) to provide methyl 3-(1-pyr-
rolidyl)benzoate as a pale yellow solid (1.70 g, 23%). The solid
was taken up in dioxane/water (40 mL) and treated with
LiOH‚H2O (1.75 g, 5 equiv) at room temperature for 10 min,
then at 40 °C for 30 min. Acetic acid (10 mL) was added, and
Meth yl 3-[(5-Br om op en ta n oyl)a m in o]ben zoa te. Meth-
yl 3-(4-pentenoylamino)benzoate (1.7 g, 7.3 mmol) was taken
up in dry THF (25 mL) at room temperature under nitrogen.
9-BBN (20 mL, 0.5M solution) was added, and the mixture
was stirred at room temperature for 3 h. NaOH 1 M, (8 mL)
was then added followed by 27% hydrogen peroxide (2.5 mL,
dropwise). Stirring was continued at 40 °C for 1 h, and then
the mixture was evaporated, taken up in EtOAc, and washed
with 5% KHCO3 and brine. The organic portion was filtered
(Whatman 1PS phase separator), evaporated, and chromato-
graphed (eluant 95% EtOAc in hexanes) to provide methyl
3-[(5-hydroxypentanoyl)amino]benzoate as a mixture with
borates. The crude product was taken up in DCM (120 mL)
and treated with triphenylphosphine (5 g) and carbon tetra-
bromide (6.2 g) at room temperature for 2 h with stirring. The
mixture was then evaporated and chromatographed (eluant
40% EtOAc in hexanes) to provide the title compound as a
colorless oil which solidified to a wax on standing (1.54 g, 67%).
1H NMR (270 MHz, CDCl3) δ 9.62 (br s, 1H), 8.26 (t, 1H, J )
1.5 Hz), 7.95-7.8 (m, 2H), 7.4 (m, 1H), 3.96 (s, 3H), 3.44 (m,
2H), 2.66 (m, 2H), 1.98 (m, 4H) ppm.
Meth yl 3-(2-Oxo-1-p ip er id in yl)ben zoa te. Methyl 3-[(5-
bromopentanoyl)amino]benzoate (1.50 g, 4.88 mmol) was taken
up in dry DMF (40 mL) and treated with NaH (160 mg, 80%
dispersion in oil, 5.33 mmol) at 0 °C. The mixture was stirred
at room temperature under nitrogen for 10 min, KI (80 mg)
was added, and the mixture was heated at 70 °C for 4 h. The
mixture was evaporated and partitioned between EtOAc and
1 M HCl. The organic portion was washed with 5% KHCO3
and brine, filtered (Whatman 1 PS phase separator), and
evaporated. The residue was chromatographed (eluant 2%
MeOH in EtOAc) to provide the title compound as a colorless
oil (640 mg, 56%): 1H NMR (270 MHz, CDCl3) δ 8.1-7.95 (m,
2H), 7.60-7.50 (m, 2H), 3.98 (s, 3H), 3.75 (m, 2H), 2.64 (m,
2H), 2.03 (m, 4H) ppm.
3-(1-P ip er id in yl)ben zoic Acid . Methyl 3-(2-oxo-1-pip-
eridinyl)benzoate (640 mg, 2.75 mmol) was dissolved in dry
THF (30 mL), and borane-tetrahydrofuran complex (5 mL, 1
M solution in THF) was added. The mixture was stirred under
nitrogen at reflux for 1 h, then cooled, and evaporated. The
residue was taken up in MeOH/acetic acid (6/1, v/v, 70 mL)
and heated at reflux for 3 h, then evaporated, and chromato-
graphed (eluant 10% EtOAc in hexane) to provide the methyl
ester of the title compound as a colorless oil (540 mg, 90%):
1H NMR (270 MHz, CDCl3) δ 7.59 (t, 1H, J ) 1.5 Hz), 7.46
(dd, 1H, J 1 ) 8 Hz, J 2 ) 1.5 Hz), 7.28 (t, 1H, J ) 8 Hz), 7.11
(m, 1H), 3.89 (s, 3H), 3.18 (m, 4H), 1.75-1.62 (m, 4H), 1.61-
1.55 (m, 2H) ppm. The oil was dissolved in dioxane (12 mL)
and water (8 mL). LiOH‚H2O (300 mg, 7.143 mmol) was
added, and the mixture was stirred at 40 °C for 1 h, then
acidified with acetic acid, evaporated, and azeotroped with
toluene. The residue was chromatographed on silica (eluant
60:40:2, EtOAc/hexanes/AcOH, v/v/v) to provide the title
compound as a colorless solid (450 mg, 90%): 1H NMR (270
MHz, CDCl3) δ 7.66 (s, 1H), 7.54 (d, 1H, J ) 8 Hz), 7.32 (t,

338 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Semple et al.
1H, J ) 8 Hz), 7.17 (dd, 1H, J 1 ) 8 Hz, J 2 ) 1.5 Hz), 3.22 (m,
4H), 1.72 (m, 4H), 1.61 (m, 2H) ppm.
was poured into a mixture of 5% KHCO3 (100 mL) and EtOAc
(150 mL). The organic portion was washed with 5% KHCO3,
water, and brine, dried, and evaporated. (The intermediate
10b could be isolated at this stage by chromatography on silica
(eluant 55% EtOAc in hexanes) to provide the pure compound
as a yellow oil in 93% yield: 1H NMR (CDCl3, 270 MHz) δ
11.65 (br s, 1H), 8.54 (d, 1H, J ) 5.5 Hz), 8.50 (d, 1H, J ) 8
Hz), 8.03 (d, 1H, J ) 8 Hz), 7.75 (m, 3H), 7.5-7.08 (m, 12H),
6.98 (br m, 1H),; 5.05 (m, 2H) ppm.) The resultant crude
product was treated with an ice-cold saturated solution of
ammonia in methanol (30 mL), and the mixture was stoppered
and stirred at room temperature for 1 h, then cooled, and
evaporated. The residue was treated with a solution of
ammonium acetate in acetic acid (0.1 g/mL, 30 mL) at room
temperature for 1 h. The mixture was evaporated and
partitioned between CHCl3 and 1 M NaOH. The organic
portion was washed with brine, dried, and evaporated, and
the product was crystallized from EtOAc/hexane. The product
was recrystallized from EtOAc/hexane to afford the title
compound as a colorless solid (1.01 g, 79%): 1H NMR (CDCl3,
270 MHz) δ 9.0 (br s, 1H), 8.60 (d, 1H, J ) 6 Hz), 8.07 (d, 1H,
J ) 8 Hz), 7.82 (dt, 1H, J t ) 8 Hz, J d ) 1 Hz), 7.4-7.2 (m,
9H), 6.98 (d, 1H, J ) 8 Hz), 6.65 (d, 1H, J ) 8 Hz), 5.37 (d,
1H, J ) 8 Hz), 5.16 (m, 2H) ppm.
(RS)-3-[(Ben zyloxyca r bon yl)a m in o]-1-[(ter t-bu tylca r -
bon yl)m eth yl]-2,3-d ih yd r o-5-(2-p yr id yl)-1H-1,4-ben zod i-
a zep in -2-on e (12b). 11b (7.3 g, 18.9 mmol) was taken up in
dry DMF (73 mL) at 0 °C. Sodium hydride (740 mg, 80%
dispersion in oil, 1.3 equiv) was added portionwise keeping
the internal temperature below 5 °C, and the mixture was
stirred at room temperature 1 h. The mixture was recooled
to 0 °C, and 1-bromopinacolone (10.21 g, 57 mmol) was added
dropwise keeping the internal temperature below 10 °C, and
stirring was continued at room temperature for a further 1 h.
The mixture was evaporated, taken up in DCM (73 mL), and
3-[(ter t-Bu tyloxyca r bon yl)a m in o]ben zoic Acid . 3-Ami-
nobenzoic acid (24.69 g, 180 mmol) was taken up in 2 M KOH
(180 mL) and dioxane (180 mL) and treated with di-tert-butyl
dicarbonate (53.03 g, 243 mmol) at room temperature over-
night. Dioxane was removed by evaporation, and the solution
was diluted with 1 M KOH (300 mL). The aqueous portion
was washed with ether (200 and 300 mL) and acidified to pH
4 with concentrated HCl. The resultant white precipitate was
collected by filtration, washed with water, and dried in vacuo
(P2O5) to give the title compound (38.97 g, 91%): 1H NMR (90
MHz, DMSO-d6) δ 9.52 (br s, 1H), 8.13 (m, 1H), 7.7-7.3 (m,
3H), 1.49 (s, 9H) ppm.
3-[N-(ter t-Bu tyloxycar bon yl)m eth ylam in o]ben zoic Acid.
Sodium hydride (18.33 g, 60% dispersion in oil, 458.3 mmol)
was added portionwise to a solution of 3-[(tert-butyloxycarbo-
nyl)amino]benzoic acid (43.48 g, 183.3 mmol) in DMF (600 mL)
below 10 °C, and the mixture was allowed to warm to room
temperature with stirring over 1 h. MeI (84.54 g, 595.6 mmol)
was added dropwise to the solution over 30 min at 5 °C, and
the mixture was stirred at room temperature for 2 h. The
mixture was evaporated, and the residue was partitioned
between EtOAc (1.2 L) and water (600 mL). The organic
portion was washed with saturated NaHCO3 (100 mL) and
water (5 × 200 mL), dried (Mg2SO4), and evaporated. The
residual oil was taken up in methanol (1 L), 1 M LiOH (185
mL) was added to the solution at 5 °C, and the mixture was
stirred at room temperature for 12 h. A further portion of 1
M LiOH (90 mL) was added, and the mixture was stirred for
1 h. The solution was concentrated to remove methanol,
diluted with water and washed with EtOAc/hexane (1:2, v/v,
300 and 150 mL). The aqueous portion was acidified to pH 4
with concentrated HCl and extracted with EtOAc (400 and 200
mL). The combined organic portions were washed with brine,
dried (MgSO4), and evaporated. The residue was recrystallized
from EtOAc/hexane (1:20 v/v, 420 mL) to give the title
compound (36.08 g, 78%): 1H NMR (90 MHz, CDCl3) δ 8.0-
7.9 (m, 2H), 7.5-7.4 (m, 2H), 3.31 (s, 3H), 1.47 (s, 9H) ppm.
3-[N-(ter t-Bu tyloxyca r bon yl)m eth yla m in o]p h en yl Iso-
cya n a te. Et3N (3.71 g, 36.7 mmol) and a solution of ethyl
chloroformate (4.31 g, 39.7 mmol) in acetone (10 mL) were
successively added dropwise to a solution of 3-[N-(tert-butyl-
oxycarbonyl)methylamino]benzoic acid (8.0 g, 31.8 mmol) in
acetone (64 mL) below 5 °C. After the solution was stirred
for 30 min, a solution of NaN3 (3.1 g, 47.7 mmol) in water (10
mL) was added below 5 °C. The mixture was stirred at the
same temperature for a further 1 h and then poured into
toluene (80 mL) and water (160 mL). The organic portion was
washed with brine, refluxed for 2 h, and evaporated. The
residue was distilled (100-105 °C/0.9-1.0 mmHg) to afford
the title compound (6.2 g, 78%) as a yellow oil.
3-(Dim eth yla m in o)p h en yl Isocya n a te. 3-(Dimethylami-
no)benzoic acid (350 g, 2.12 mol) was dissolved in acetone (2.8
L). Et3N (249 g, 2.46 mol) was added dropwise to the solution
below 10 °C, followed by the addition of a solution of ethyl
chloroformate (287 g, 2.65 mol) in acetone (875 mL) below 5
°C. After the mixture was stirred for 30 min, a solution of
NaN3 (201 g, 3.18 mol) in water (570 mL) was added dropwise
below 5 °C. The reaction mixture was stirred at 0-5 °C for a
further 1 h and then poured into toluene-ice water (2:3, 11
L). The aqueous portion was extracted with a small amount
of toluene, and the combined organic portions were washed
with water and brine and dried (MgSO4). After MgSO4 was
removed by filtration, the filtrate was added dropwise to hot
toluene (1.5 L). The mixture was refluxed for 1 h and then
evaporated, and the residue was distilled (0.6-0.8 mmHg, 74-
77 °C) to afford the isocyanate (252 g, 74%) as a pale yellow
oil.
washed with 5% NaHCO3
and brine. The organic portion was
evaporated and recrystallized from EtOAc/hexane (1:1 v/v, ca.
150 mL) to provide the title compound as a white solid (7.31
g, 80%): 1H NMR (CDCl3, 270 MHz) δ 8.60 (d, 1H, J ) 4 Hz),
8.14 (d, 1H, J ) 8 Hz), 7.80 (dt, 1H, J t ) 7.5 Hz, J d ) 1.5 Hz),
7.50 (dt, 1H, J t ) 8.5 Hz, J d ) 1.5 Hz), 7.4-7.2 (m, 8H), 7.11
(d, 1H, J ) 8 Hz), 6.72 (d, 1H, J ) 8 Hz), 5.51 (d, 1H, J ) 8
Hz), 5.12 (m, 2H), 5.0 (d, 1H, J ) 17.8 Hz), 4.48 (d, 1H, J )
17.8 Hz), 1.25 (s, 9H) ppm; 13C NMR (CDCl3, 67.8 MHz) δ 208.2
(q), 166.7 (q), 166.2 (q), 155.7 (q), 148.6 (CH), 142.1 (q), 136.7
(CH), 136.2 (q), 132.0 (CH), 130.9 (CH), 128.4 (CH), 128.0 (CH),
124.8 (CH), 124.7 (CH), 124.3 (CH), 124.2 (q), 121.6 (CH), 68.8
(CH), 66.8 (CH2), 54.2 (CH2), 43.4 (q), 26.3 (CH3) ppm.
(RS)-3-Am in o-1-[(ter t-bu tylca r bon yl)m eth yl]-2,3-d ih y-
d r o-5-(2-p yr id yl)-1H-1,4-ben zod ia zep in -2-on e (13b). 12b
(2.45 g, 5.12 mmol) was taken up in DCM (100 mL) at 0 °C,
and the solution was saturated with dry HBr gas. The mixture
was then stoppered and stirred at 0 °C for 4 h. EtOAc (100
mL) was then added, and the ice-cold mixture was filtered.
The resultant hygroscopic pale yellow solid was washed well
with EtOAc and then taken up in water (200 mL) and washed
with ether (50 mL). The aqueous portion was basified with
5% KHCO3 to pH 8 and extracted with chloroform (3 × 100
mL). The combined chloroform extracts were washed with
brine, dried, and evaporated to give a near colorless foam (1.68
g, 93%): TLC (E. Merck; Kieselgel silica plates) single spot,
Rf ) 0.25 (eluant CHCl3/MeOH/AcOH, 20:2:1, v/v/v); 1H NMR
(CDCl3, 270 MHz) δ 8.62 (d, 1H, J ) 5 Hz), 8.18 (d, 1H, J ) 8
Hz), 7.80 (dt, 1H, J t ) 8 Hz, J d ) 1 Hz), 7.48 (dt, 1H, J t ) 8
Hz, J d ) 1 Hz), 7.37 (m, 2H), 7.20 (t, 1H, J ) 8 Hz), 7.09 (d,
1H, J ) 8 Hz), 5.07 (d, 1H, J ) 18 Hz), 4.66 (s, 1H), 4.45 (d,
1H, J ) 18 Hz) ppm; 13C NMR (CDCl3, 67.8 MHz) δ 208.9 (q),
169.9 (q), 155.9 (q), 148.8 (CH), 142.5 (q), 136.7 (CH), 131.8
(CH), 130.5 (CH), 128.7 (q), 124.5 (CH), 124.3 (CH), 124.1 (CH),
124.4 (CH), 70.4 (CH), 54.1 (CH2), 44.0 (q), 26.4 (CH3) ppm.
Attem p ted Ra cem iza tion -Resolu tion of 13b. Crude
13b (380 mg, 1.056 mmol) was taken up in MeCN (4 mL) and
treated with (S)-mandelic acid (170 mg, 1.2 mmol). 3,5-
Dichlorosalicaldehyde (10 mg) was added to the stirring
mixture, and the mixture was cooled to 0 °C. Precipitation
was observed, and stirring was continued at 0 °C overnight.
(RS)-3-[(Ben zyloxyca r b on yl)a m in o]-2,3-d ih yd r o-5-(2-
p yr id yl)-1H-1,4-ben zod ia zep in -2-on e (11b). 3b17 (660 mg,
3.33 mmol) and 919 (1.63 g, 5 mmol) were mixed together in
DCM (30 mL) at 0 °C under nitrogen. Water soluble carbo-
diimide (EDC, 1 g, 5 mmol) and DMAP (30 mg) were added,
and the mixture was stirred at 0 °C for 10 min and at room
temperature for 10 min. The resulting pale brown solution

A Potent and Orally Active Gastrin/ CCK-B Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 339
The resultant white solid was collected by filtration and
washed with small portions of cold MeCN to provide the
mandelate salt of 8 (304 mg, 58%). A portion of the salt (156
mg, 0.311 mmol) was partitioned between 5% KHCO3 and
CHCl3 and the organic portion was washed with brine, filtered
(Whatman 1 PS phase separator), and evaporated to give the
free amine of 8 (110 mg, 98%). The analytical data for the
free amine was identical to that described above for 8.
Op tica l Resolu tion of 3-Am in o-1-[(ter t-bu tylca r bon yl)-
m eth yl]-2,3-d ih yd r o-5-(2-p yr id yl)-1H-1,4-ben zod ia zep in -
2-on e (13b) by F r a ction a l Cr ysta lliza tion . Crude 13b (14
g, 40 mmol) was taken up in acetonitrile (50 mL) at -5 °C.
(R)-Mandelic acid (3.2 g, 21 mmol) was added, and the mixture
was stirred at -5 °C. A thick precipitate was formed, and
acetonitrile (20 mL) was added dropwise to enable easier
filtration. After 1 h at -5 °C the mixture was filtered and
washed with small portions of cold MeCN. The resultant white
precipitate was recrystallized from MeCN. (Anal. for mande-
late salt (C28H30N4O5‚0.5H2O) C, H, N.) The salt was parti-
tioned between 5% KHCO3 and CHCl3, and the organic portion
was washed with brine and filtered (Whatman 1 PS phase
Gen er a l P r oced u r es for th e P r ep a r a tion of 3-Am in o-
1-[(ter t-bu tylca r bon yl)m eth yl]-2,3-d ih yd r o-1H-1,4-ben zo-
d ia zep in -2-on e Ur ea Der iva tives (14 or 15). P r oced u r e
A. Rea ction of th e Am in e a n d th e P u r e Isocya n a te. The
required 3-amino-2,3-dihydro-1H-1,4-benzodiazepin-2-one (1
mmol) was taken up in DCM (25 mL) at 0 °C. To this solution
was added the pure isocyanate (1.02 mmol, either from
commercial sources or prepared as described above), and
stirring was continued at room temperature for 2 h. The
solvent was evaporated in vacuo, and the crude product was
purified by flash chromatography on silica. Compounds 14a ,
14c, 14d , 14h , 15a , 15d , and 15e were prepared using this
general method.
P r oced u r e B. Rea ction of th e Am in e a n d th e Isocy-
a n a te P r ep a r ed in Situ fr om th e Ben zoic Acid . To a
solution of the requisite benzoic acid (3 mmol, either from
commercial sources or prepared as described above) in toluene
(5 mL) was added diphenyl phosphoazidate (825 mg, 3 mmol)
and Et3N (303 mg, 3 mmol). The mixture was stirred at room
temperature for 2 h, heated at reflux for 3 h, and cooled to 0
°C. A solution of the required 3-amino-2,3-dihydro-1H-1,4-
benzodiazepin-2-one (1 mmol) in toluene (5 mL) was added,
and the mixture was stirred at room temperature overnight,
then evaporated, taken up in EtOAc, washed with 5% KHCO3,
H2O, and brine, filtered (Whatman 1PS phase separator), and
evaporated, and the crude product was purified by flash
chromatography on silica. Compounds 14e, 14i, 14j, 15f, 15g,
and 15h were prepared using this general method.
Sp ecific P r oced u r es for th e P r ep a r a tion of Ur ea
Der iva tives (14 or 15). N-[(3RS)-1-[(ter t-bu tylca r bon yl)-
m et h yl]-2,3-d ih yd r o-2-oxo-5-p h en yl-1H -1,4-b en zod ia z-
ep in 3-yl]-N′-(3-ca r boxyp h en yl)u r ea (14b). (RS)-13a (600
mg, 1.72 mmol) was taken up in dry THF (8 mL) and Et3N
(0.26 mL, 1.9 mmol) and the solution cooled to 0 °C. The
mixture was treated with a solution of p-nitrophenyl chloro-
formate (0.38 g, 1.9 mmol) in THF (4 mL), stirred at room
temperature for 1 h, evaporated, and chromatographed (eluant
EtOAc/hexane, 60:40, v/v) to provide an off-white solid (670
mg, 76%). The solid was taken up in DMF (10 mL) and
m-aminobenzoic acid (245 mg, 1.75 mmol) added. The mixture
was stirred at 45 °C for 18 h, cooled, and evaporated, and the
residue was chromatographed (eluant EtOAc/hexane/AcOH,
60:40:2 v/v/v). The title compound was isolated as a colorless
solid by recrystallization from acetonitrile (328 mg, 49%).
(3R)-N-[1-[(ter t-Bu tylca r bon yl)m eth yl]-2,3-d ih yd r o-2-
oxo-5-ph en yl-1H-1,4-ben zodiazepin -3-yl]-N′-(3-am in oph e-
n yl)u r ea (14f). 14e (700 mg, 1.37 mmol) was taken up in
acetone (10 mL) and treated with 4 M HCl at room temper-
ature for 62 h. The mixture was partially evaporated to
remove the acetone and partitioned between DCM and 5%
KHCO3. The organic portion was washed with brine, dried
(MgSO4), and evaporated. The residue was chromatographed
on silica (eluant CHCl3/MeOH/AcOH, 100:2:1, v/v/v), and the
title compound was recrystallized from acetonitrile to provide
a colorless solid (320 mg, 48%).
(3R)-N-[1-[(ter t-Bu tylca r bon yl)m eth yl]-2,3-d ih yd r o-2-
oxo-5-p h en yl-1H -1,4-b en zod ia zep in -3-yl]-N′-[3-(m et h yl-
a m in o)p h en yl]u r ea (14g). (3R)-N-[1-[(tert-Butylcarbonyl)-
methyl]-2,3-dihydro-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-
yl]-N′-[3-(N-formylmethylamino)phenyl]urea was prepared from
(R)-13a and 3-(N-formylmethylamino)benzoic acid using gen-
eral procedure B, and the product was partially purified by
chromatography (eluant 75% EtOAc in hexane; 960 mg, 65%).
The residue was taken up in acetone (15 mL) and the stirring
solution treated with 4 M HCl at room temperature for 3 days.
The mixture was partially evaportated to remove acetone, and
the residue was partitioned between 5% KHCO3 and DCM.
The organic portion was washed with brine, filtered (Whatman
1PS phase separator), and evaporated, and the crude product
was purified by flash chromatography on silica (eluant CHCl3/
MeOH/AcOH, 120:2:1, v/v/v). The title compound was isolated
as a colorless solid by recrystallization from MeCN (400 mg,
45%).
separator) to give the free (R)-amine (4.42 g, 32%): [R]D
)
+212.6° (c ) 0.715, CHCl3); 1H NMR and 13C NMR data were
identical to those for the racemic compound.
The filtrate from above was washed with base and taken
up in MeCN (30 mL). Addition of (S)-mandelic acid afforded
the (S)-amine salt in a similar manner. This was recrystal-
lized from MeCN and washed with base to provide the free
(S)-amine (3.87 g, 28%): [R]D ) -213.4° (c ) 0.671, CHCl3).
(RS)-3-[(Ben zyloxycar bon yl)am in o]-2,3-dih ydr o-5-ph en -
yl-1H-1,4-ben zod ia zep in -2-on e (11a ). The title compound
was prepared from 2-aminobenzophenone and (benzyloxycar-
bonyl)(1-benzotriazolyl)glycine as described above for 11b in
79% yield. Analytical data were identical to literature val-
ues.14
P r ep a r a tion of (RS)-3-[(Ben zyloxyca r bon yl)a m in o]-1-
[(ter t-b u t ylca r b on yl)m et h yl]-2,3-d ih yd r o-5-p h en yl-1H -
1,4-ben zod ia zep in -2-on e (12a ). The title compound was
prepared by alkylation of 11a with 1-bromopinacolone as
described above for 12b in 86% yield: 1H NMR (CDCl3, 270
MHz) δ 7.8-7.2 (m, 14H), 6.74 (d, 1H, J ) 8 Hz), 5.53 (d, 1H,
J ) 8 Hz), 5.23 (s, 2H), 5.05 (d, 1H, J ) 18 Hz), 4.77 (d, 1H,
J ) 18 Hz), 1.33 (s, 9H) ppm.
P r ep a r a tion of (RS)-3-Am in o-1-[(ter t-bu tylca r bon yl)-
m et h yl]-2,3-d ih yd r o-5-p h en yl-1H -1,4-b en zod ia zep in -2-
on e (8a ). The title compound was prepared from 12a by
hydrogenolysis of the benzyl carbamate group in essentially
quantitative yield as previously described:16 1H NMR (CDCl3,
270 MHz) δ 7.60 (d, 2H, J ) 8 Hz), 7.5-7.0 (m, 7H), 4.96 (d,
1H, J ) 17 Hz), 4.55 (m, 3H), 2.82 (br s, 2H), 1.18 (s, 9H) ppm.
Resolu tion of 3-Am in o-1-[(ter t-bu tylca r bon yl)m eth yl]-
2,3-d ih yd r o-5-p h en yl-1H-1,4-ben zod ia zep in -2-on e (13a )
by Resolu tion -Ra cem iza tion P r oced u r e. 13a was re-
solved using minor modifications to the resolution-racemiza-
tion procedure previously described.11,21 Racemic 13a (7.7 g,
22.95 mmol) was taken up in acetonitrile (20 mL) at -5 °C,
and (S)-mandelic acid (3.44 g, 22.63 mmol) was added to the
stirred solution, followed 30 min later by 3,5-dichlorosalical-
dehyde (65 mg). After the mixture was stirred overnight at
-5 °C, the resultant precipitate was collected by suction
filtration, washed with small portions of cold acetonitrile and
ether, and recrystallized from acetonitrile to give the (S)-
mandelate salt of the (R)-isomer title compound as a white
solid (6.70 g, 59%). A portion of this solid (460 mg, 0.897
mmol) was partitioned between CHCl3 and 0.5 M NaOH. The
organic portion was washed with brine, filtered (Whatman 1PS
phase separator), and evaporated to provide the title compound
as a colorless foam (1H NMR identical to racemate). The chiral
integrity of the amine was examined by coupling to Boc-Phe-
OH using water soluble carbodiimide/HOBT in DMF. Analyti-
cal HPLC (Spherisorb C-18, 4.6 × 100 mm, 5 µm column,
linear gradient of 40-90% 0.1%TFA/acetonitrile in 0.1%TFA/
water, flow rate 0.8 mL/min) of the crude product showed only
a single diasteromer peak at 17.7 min, whereas the racemic
amine produced a 1:1 mixture of diastereomers under the same
conditions (retention times 17.4 and 17.7 min).
(3R)-N-[1-[(ter t-Bu tylca r bon yl)m eth yl]-2,3-d ih yd r o-2-
oxo-5-(2-p yr id yl)-1H -1,4-b en zod ia zep in -3-yl]-N′-(3-a m i-
n oph en yl)u r ea (15b). (3R)-N-[1-[(tert-Butylcarbonyl)methyl]-

340 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Semple et al.
2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepin-3-yl]-N′-
[3-[(tert-butyloxycarbonyl)amino]phenyl]urea was prepared
from (R)-13b (850 mg, 2.43 mmol) and 3-[(tert-butyloxycarbo-
nyl)amino]benzoic acid (1.19 g, 5 mmol) using general proce-
dure B. The crude urea was taken up in DCM (10 mL) and
treated with TFA (30 mL) at room temperature for 30 min
under nitrogen. The mixture was evaporated and azeotroped
with CHCl3. The residue was partitioned between DCM and
5% KHCO3, and the organic portion was washed with brine,
dried (MgSO4), and evaporated. The residue was chromato-
graphed on silica (eluant 6% MeOH in EtOAc). The residue
was taken up in 1:1 EtOAc/ether (40 mL) at -10 °C and
treated dropwise with 4 M HCl in dioxane (0.5 mL). The
mixture was stirred at -10 °C for 30 min, and the resultant
precipitate was collected by filtration, washed with ether, and
dried over NaOH to provide the hydrochloride salt of the title
compound as a colorless solid (845 mg, 67%).
fold volume of 50 mM Tris-HCl buffer (pH 7.7) by the use of a
Polytrone-type homogeniser, the homogenate was twice cen-
trifuged for 10 min at 50000g by the use of an ultracentrifuge,
the pellet thus obtained was suspended in a 40-fold volume of
50 mM Tris-HCl buffer (containing 0.2% BSA, 5 mM MgCl2,
0.1 mg/mL bacitracin and 5 mM DTT; pH 7.7), and the
suspension was stored at -80 °C until the membrane prepara-
tions were required. The membrane preparations were then
warmed to room temperature, diluted 1:10 with the buffer, and
incubated at 37 °C for 30 min in the presence of [3H]L-364,
718, and the test compound was then separated by suction
filtration. Nonspecific binding was determined in the presence
of 1 mM L-364,718. Test compounds were dissolved in DMSO.
The amount of labeled ligand bound to the receptor was
measured by the use of a liquid scintillation counter; IC50
values were determined, being that concentration of test
compound required to inhibit specific binding by 50%.
Mea su r em en t of In h ibition of P en ta ga str in -Stim u la t-
ed Ga str ic Acid Secr etion in Ra ts. A cannula was inserted
into the trachea of a rat anesthetized with urethane (intrap-
eritoneally administered, 1.25 g/kg), the abdominal wall was
incised to expose the gastric and duodenal portions, and a
polyethylene cannula was set in the anterior stomach after
ligation of the cardia. The duodenum was then subjected to
slight section, a polyethylene cannula was inserted from the
incised portion toward the stomach, and the pylorus was
ligated to fix the cannula. Physiological saline (with pH
adjusted to 7.0) was perfused from the anterior stomach
toward the pylorus at a rate of 3 mL/min, and the gastric acid
secretion was measured by continuous titration of the perfu-
sate by the use of a pH-stat (AUT-201; Toa Electronics, Ltd.).
The continuous titration was carried out by using 25 mM
NaOH solution until the pH reached 7.0, and the result was
expressed as the amount of gastric acid secreted for every 10
min (mequiv/10 min). Pentagastrin was intravenously ad-
ministered at a rate of 15 mg/kg/h. The secretion of gastric
acid increased upon administration of pentagastrin, reaching
the maximum level after 60 min, after which time the level
was stably maintained. A test drug was then intravenously
administered, and the secretion of gastric acid was measured.
ED50 values were determined for some examples, this being
the amount of the drug required to reduce the amount of
secreted gastric acid down to 50% of the maximum level.
Mea su r em en t of In h ibition of Ga str ic Acid Secr etion
in Heid en h a in P ou ch Dogs. Male beagle dogs with Heiden-
hain pouch were used.33 One month after preparation of the
pouch, secretory experiments were performed once a week in
each animal throughout the course of the investigation. Dogs
were deprived of food for 18 h prior to experiments but allowed
free access to water. A polyethylene tube was intubated
through the femoral vein to infuse pentagastrin at a rate of 8
µmol/kg/h. Test compounds were administered po or iv at 3 h
after the start of pentagastrin infusion. Gastric juice was
collected every 15 min and the volume measured. The acidity
of the gastric juice was measured by automatic titration with
0.05 N NaOH to pH 7.0. In the case of iv injection, test
compounds were dissolved in DMF.
(3R)-N-[1-[(ter t-Bu tylca r bon yl)m eth yl]-2,3-d ih yd r o-2-
oxo-5-(2-p yr id yl)-1H -1,4-b en zod ia zep in -3-yl]-N′-[3-(m e-
th ylam in o)ph en yl]u r ea (15c). 3-[N-[(tert-Butyloxycarbonyl)-
methyl]amino]phenyl isocyanate (19.30 g, 77.7 mmol) was
added dropwise to a solution of (R)-13b (27.19 g, 77.6 mmol)
in DCM (200 mL) below 20 °C, and the mixture was stirred at
room temperature for 30 min and then evaporated. The
residue was taken up in EtOAc (200 mL) and water (100 mL),
concentrated HCl (120 mL) was then added dropwise to the
mixture below 20 °C, and the mixture was stirred for 3 h. The
aqueous phase was separated, added to DCM (500 mL), and
then basified to pH 10 with 20% NaOH while the temperature
was maintained below 20 °C. The resultant organic portion
was washed with brine (300 mL), and evaporated, and the
residue was crystallized from ethanol. The resultant solid was
recrystallized from ethanol (1.8 L) to provide the title com-
pound as a white crystalline solid (27.30 g, 72%), mp 243-
246 °C.
(3R)-N-[1-[(ter t-Bu tylca r bon yl)m eth yl]-2,3-d ih yd r o-2-
oxo-5-(2-p yr id yl)-1H -1,4-b en zod ia zep in -3-yl]-N′-[3-(d i-
m eth yla m in o)p h en yl]u r ea Hyd r och lor id e (15d ‚HCl). 15d
(70 g, 136.6 mmol, prepared as described in general method
A but not chromatographed) was taken up in ethanol (1 L),
and 2.26 M HCl/ethanol (63.5 mL, 143.4 mmol) was added
dropwise. The mixture was warmed to 50 °C to afford a clear
solution, which was seeded, cooled to 0 °C, and stirred at the
same temperature overnight. The resultant precipitate was
collected by filtration to provide the title compound as a white
crystalline solid (62.4 g, 82%), mp 181-184 °C.
Mea su r em en t of Bin d in g Affin ity for CCK-B/Ga str in
Recep tor s. About 100 Sprague-Dawley (SD) rats were
decapitated without anesthesia, the whole brain was im-
mediately excised from each of the rats and homogenized in a
10-fold volume of 0.32 M aqueous solution of sucrose by the
use of a Teflon-coated homogenizer, the homogenate thus
obtained was centrifuged for 10 min at 900g by the use of a
cooled centrifuge, and the supernatant was further centrifuged
for 15 min at 11500g. The pellet thus obtained was dispersed
in 50 mM Tris-HCl buffer (pH 7.4) containing 0.08% Triton
X-100. This suspension was allowed to stand for 30 min and
again centrifuged for 15 min at 11500g. The precipitate thus
obtained was washed twice with 5 mM Tris-HCl buffer and
twice with 50 mM Tris-HCl buffer in that order with centrifu-
gal separation. The washed precipitate was suspended in 50
mM Tris-HCl buffer, and the suspension thus obtained was
stored at -80 °C until the membrane preparation was re-
quired. For the assay the membrane preparations were
warmed to room temperature, diluted with 10 mM HEPES
buffer (containing 130 mM NaCl, 5 mM MgCl2, 1 mM EDTA,
and 0.25 mg/mL bacitracin; pH 6.5), incubated at 25 °C for
120 min in the presence of [125I]BH-CCK-8 and the test
compound (dissolved in DMSO), and then separated by suction
filtration. Nonspecific binding was determined in the presence
of 1 mM CCK-8. The amount of labeled ligand bound to the
receptor was measured by the use of a γ-counter. IC50 values
were determined, being that concentration of test compound
required to inhibit specific binding by 50%.
Su p p or tin g In for m a tion Ava ila ble: Spectroscopic data
for test substances (1 page). Ordering information is given
on any current masthead page.
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