3-[2-(N-phenylacetamide)]-1,5-benzodiazepines: Orally active, binding selective CCK-A agonists

Article abstract of DOI:10.1021/jm960205b

A series of modifications were made to the C-3 substituent of the 1,5- benzodiazepine CCK-A agonist 1. Replacement of the inner urea NH and addition of a methyl group to generate a C-3 quaternary carbon resulted in acetamide 6, which showed CCK-A receptor

Full text of DOI:10.1021/jm960205b

3030
J . Med. Chem. 1996, 39, 3030-3034
Notes
3-[2-(N-P h en yla ceta m id e)]-1,5-ben zod ia zep in es: Or a lly Active, Bin d in g Selective
CCK-A Agon ists
Timothy M. Willson,*,† Brad R. Henke,† Tanya M. Momtahen,† Peter L. Myers,†,‡ Elizabeth E. Sugg,†
Rayomand J . Unwalla,§, Dallas K. Croom,|,∇ Robert W. Dougherty,#,∇ Mary K. Grizzle,| Michael F. J ohnson,|
Kennedy L. Queen,# Thomas J . Rimele,# J effrey D. Yingling,# and Michael K. J ames|,∇
Glaxo Wellcome Research and Development, Five Moore Drive, Research Triangle Park, North Carolina 27709
Received March 12, 1996X
A series of modifications were made to the C-3 substituent of the 1,5-benzodiazepine CCK-A
agonist 1. Replacement of the inner urea NH and addition of a methyl group to generate a
C-3 quaternary carbon resulted in acetamide 6, which showed CCK-A receptor binding
selectivity and sub-micromolar agonist activity in vitro. Benzodiazepine 6 was active in an in
vivo mouse gallbladder emptying assay and represents a novel orally active, binding selective
CCK-A agonist.
In tr od u ction
Cholecystokinin (CCK) is the major gastrointestinal
hormone that coordinates food digestion and satiety
signals in response to feeding. CCK circulates as a
series of C-terminal truncated peptides of 58, 33, 8, and
4 amino acids in length, with sulfated CCK-8 being the
minimum sequence for biological activity.1 The periph-
eral effects of CCK include contraction of the gallblad-
der, stimulation of enzyme secretion from the pancreas,
and contraction of the pyloric sphincter to slow gastric
emptying.1 In conjunction with these effects as a
digestive hormone, CCK functions as a satiety signal
F igu r e 1.
by direct stimulation of vagal afferents that, in turn,
signal feeding centers in the brain.2 These peripheral
activity, we set out to synthesize compounds with
effects of CCK are mediated through the seven trans-
binding selectivity for the CCK-A receptor subtype. We
membrane G-protein-coupled CCK-A receptor.1,3
A
report that systematic modification of the C-3 ureido
substituent has led to the identification of benzodiaz-
epine CCK-A agonists with both binding selectivity and
in vivo oral activity in mice.
related CCK-B receptor subtype is located primarily
within the central nervous system (CNS), where CCK
functions as a peptide neurotransmitter.4 CCK-A selec-
tive agonists have potential for use in obese patients
as satiety agents5 and for prevention of gallbladder
stasis on low fat diets.6
Ch em istr y
Therapeutic applications of CCK ligands have been
aggressively pursued following the identification of
benzodiazepine antagonists for CCK-A and CCK-B
receptors.7 By comparison, the development of CCK
agonists has been hampered by the lack of orally active
small molecule ligands. We recently reported the
identification of the first benzodiazepine CCK-A agonist
(1).8 However, benzodiazepine 1 shows relatively poor
selectivity toward CCK-A receptors and lacks oral
activity in vivo. With the aim of increasing the in vivo
Four benzodiazepines were synthesized in which the
urea nitrogens of 1 (X ) NH, Y ) NH, Figure 1) were
sequentially replaced with either carbon or oxygen. The
carbamate 2 (X ) NH, Y ) O) and the amide 4 (X )
NH, Y ) CH2) were synthesized from the 3-amino-1,5-
benzodiazepine 10 (Scheme 1). Conversion of 10 to the
alcohol 11 allowed for generation of the isomeric car-
bamate 3 (X ) O, Y ) NH). Synthesis of the amide 5
(X ) CH2, Y ) NH) started with the 1,5-benzodiazepine
12 (Scheme 2). The C-3 substituent was introduced by
allylation to 13a followed by oxidation to 14a and amide
bond formation to yield 5. Three analogs of 5 were
synthesized in which the 1,5-benzodiazepine contained
a quaternary carbon at C-3 (Scheme 2). Deprotonation
of 13a and reaction with alkyl halides generated the
C-3 dialkyled 1,5-benzodiazepines 13b-d . Oxidation to
acids 14b-d and amide bond formation yielded the
amides 6-8 (X ) CH2, Y ) NH, Z ) CH3, CH2CH3, or
CH2Ph). Addition of a C-3 substituent to the urea 1
was achieved by sequential methylation of 12, followed
* Address correspondence to: Timothy M. Willson, Glaxo Wellcome
Research and Development, NTH-M2134, Five Moore Drive, Research
Triangle Park, NC 27709. Tel: (919) 483-9875. FAX: (919) 315-5668.
E-mail: willson∼tm@glaxo.com.
† Department of Medicinal Chemistry.
‡ Current address: CombiChem Inc., La J olla, CA 92037.
§ Department of Structural Chemistry.
Current address: NIEHS, Research Triangle Park, NC 27709.
| Department of Pharmacology.
∇ Current address: Inspire Pharmaceuticals, Durham, NC 27703.
#
Department of Receptor Biochemistry.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
S0022-2623(96)00205-1 CCC: $12.00 © 1996 American Chemical Society

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 3031
Sch em e 1a
either the human CCK-A or human CCK-B receptors.8
IC50’s were determined using competitive radioligand
binding assays (Table 1). Urea 1 showed higher affinity
for the CCK-B receptor subtype compared to the CCK-A
receptor subtype. Changing the inner NH for CH2
resulted in a reversal in selectivity, with amide 5
showing a 5-fold higher affinity for the CCK-A receptor
subtype over the CCK-B receptor subtype. The reversal
in selectivity was due to a decrease in affinity for the
CCK-B receptor subtype. Addition of a methyl sub-
stituent at C-3, with amide 6, resulted in an additional
increase in binding selectivity, due primarily to a further
decrease in affinity for the CCK-B receptor subtype.
Significantly, amide 6 showed >100-fold selectivity for
the CCK-A receptor subtype over the CCK-B receptor
subtype. Amides 7 and 8, with ethyl and benzyl
substituents at C-3, had slightly lower selectivity for the
CCK-A receptor subtype. The C-3 methylated urea 9
showed higher affinity for the CCK-B receptor subtype,
and affinity for both receptor subtypes was diminished
compared to the unmethylated urea 1. Overall, substi-
tution of X ) NH to X ) CH2 resulted in an increase in
binding selectivity for the CCK-A receptor subtype. The
effect was most pronounced in the series with the C-3
quaternary carbon, where this substitution (comparison
of urea 9 with amide 6) resulted in a 500-fold increase
in selectivity for the CCK-A receptor subtype.
a
Reagents: (i) 2-bromo-N-isopropyl-N-phenylacetamide (ref 8),
K2CO3, DMF, 18 h; (ii) 2-(phenylhydrazono)propanedioyl dichloride
(ref 8), THF, 0 °C to room temperature, 18 h; Zn dust, AcOH, room
temperature, 3 h; (iii) malonyl dichloride, THF, 0 °C to room
temperature, 18 h; (iv) NaNO2, HCl, H2O, 50 °C, 1 h; Cu0, MeCN,
H2O, 80 °C, 1 h; (v) PhOC(O)Cl, Et3N, CH2Cl2, room temperature,
1 h; or PhNCO, pyridine, room temperature, 1 h; or PhCH2CO2H,
EDC, HOBT, DMF, room temperature, 18 h.
by electrophilic amination at C-3 to give the amine 15
(Scheme 3). Urea formation yielded the urea 9 (X )
NH, Y ) NH, Z ) CH3) with a methyl group attached
to the quaternary carbon at C-3.
Resu lts a n d Discu ssion
Compounds were screened for functional CCK-A
agonist activity in inducing contraction of isolated
guinea pig gallbladder8 (Table 1). Contractions which
were reversed by addition of the receptor antagonist
MK-3297 were deemed to be CCK-A specific. In this
assay, the urea 1 gave a maximal contraction of 80%
relative to CCK-8. We chose to focus on the C-3 urea
substituent since previous studies in 1,4-benzodiaz-
epines had shown that this site affected CCK-A/CCK-B
selectivity,9 and initial studies with 1 had demonstrated
that the N-1 isopropylphenylacetamide was essential for
agonist activity.8 Replacement of either urea NH with
O, in carbamates 2 and 3, led to a loss in efficacy.
Similarly, replacement of the urea outer NH (Y, Figure
1) with CH2 in amide 4 led to a loss in efficacy. By
comparison, replacement of the urea inner NH (X,
Figure 1) with CH2 in amide 5 gave a partial agonist
with sub-micromolar potency. In an effort to build on
this lead, the effect of additional substitution at C-3 of
the benzodiazepine ring was studied. Addition of a
methyl group gave 6 which had increased efficacy. The
amide 7 with a larger ethyl substituent was a partial
agonist, and the amide 8 with a benzyl substituent
lacked measurable agonist activity. Thus, in 6, substi-
tution of the C-3 carbon with a combination of the
phenyl acetamide and the additional methyl group
generated a sub-micromolar CCK-A agonist with ef-
ficacy comparable to that of 1. To examine whether
incorporation of a quaternary carbon at C-3 increased
efficacy in other series, the C-3 methylated urea 9 was
synthesized. Unfortunately, the urea 9 showed a drop
in efficacy and a large drop in potency compared to those
in 1. Our initial studies suggest that differences in the
SAR between the acetamide and urea series were due
to conformational differences in the C-3 substituents.10
Compounds 1 and 5-9 were examined for their
binding selectivity between the human CCK-A11 and
CCK-B12 receptors. The binding assay employed CHO-
K1 cell lines that had been engineered to stably express
These studies identified compounds 5-7 as meeting
our in vitro criteria of CCK-A agonist activity with
increased CCK-A/CCK-B selectivity. To examine
whether these changes translated to increased in vivo
CCK-A agonist activity, we chose to test compounds 1
and 5-7 in a mouse gallbladder emptying assay.13 This
assay provided a robust measure of peripheral CCK
agonist activity mediated via CCK-A receptors13 and
avoided some of the problems associated with behavioral
feeding assays in rodents.14 The compounds were
administered to mice, and after 30 min the extent of
CCK-A-mediated gallbladder emptying was measured
(Table 2). All four compounds showed full agonist
activity when administered at a dose of 0.1 µmol kg-1
(∼0.05 mg kg-1) ip. However, when administered at a
dose of 1.0 µmol kg-1 (∼0.5 mg kg-1) po, only amides
5-7 showed significant agonist activity. Additional
testing at a dose of 10 µmol kg-1 (∼5 mg kg-1) po,
showed that 6 was a full agonist, but 7 was only a
partial agonist. Thus, with benzodiazepine 6 we have
identified an orally active, binding selective CCK-A
agonist.
Con clu sion
We have identified modifications of 1,5-benzodiaz-
epine 1 that lead to an increase in selectivity for the
CCK-A receptor while maintaining agonist activity. The
resulting 3-methyl-3-[(N-phenylcarbamoyl)methyl]-1,5-
benzodiazepine (6) is a novel orally active, binding
selective CCK-A agonist.15 This compound, GW 7178,
represents an important step toward development of
drug therapies for obesity that act through peripheral
CCK receptors.
Exp er im en ta l Section
Melting points were recorded on a Hoover capillary melting
point apparatus and are uncorrected. Elemental analyses
were performed by Atlantic Microlab Inc., Norcross, GA. 1H-
NMR spectra were recorded using a Varian VXR-300 or Varian

3032 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Notes
Sch em e 2a
a
Reagents: (i) NaH, DMF, 0 °C, 15 min; allyl bromide, DMF, 0 °C to room temperature, 18 h; (ii) NaH, DMF, 0 °C, 15 min; MeI, or
EtI, or PhCH2Br, DMF, room temperature 3 h; (iii) RuCl3, NaIO4, CCl4, H2O, room temperature, 1 d; (iv) PhNH2, PyBroP, Hunig’s base,
DMF, 50 °C, 2 d.
J ) 5, 22), 1.10 (d, 6 H, J ) 7); MS (FAB) m/ e 428 (MH+).
Anal. (C26H25N3O3) C, H, N.
Sch em e 3a
2-(3-Allyl-2,4-d ioxo-5-p h en yl-2,3,4,5-tetr a h yd r oben zo-
[b][1,5]d ia zep in -1-yl)-N-isop r op yl-N-p h en yla cet a m id e
(13a ). To a stirring solution of 4.43 g (10.4 mmol) of 12 in 20
mL of DMF at 0 °C was added in one portion 456 mg (11.4
mmol, 1.1 equiv) of sodium hydride (60% dispersion in oil).
The resulting solution was stirred at 0 °C for 20 min, during
which time gas evolution was observed, and then a solution
of 0.90 mL (10.4 mmol) of allyl bromide in 10 mL of DMF was
added dropwise over 20 min. The resulting brown solution
was stirred for 30 min at 0 °C and then at room temperature
for 18 h. The reaction was quenched by careful addition of 10
mL of H2O, and the solvent was removed in vacuo. The
residue was poured into 30 mL of H2O and extracted with
EtOAc (3 × 30 mL). The organic layers were washed with
brine (1 × 30 mL) and dried (MgSO4), and the solvent was
removed in vacuo. Purification of the brown residue by silica
gel flash column chromatography using petroleum ether/
EtOAc (7/3) as eluent afforded an off-white solid. Recrystal-
lization from EtOAc/petroleum ether (1/1) gave 2.33 g (48%)
of 13a as a white powder: mp 192-3 °C; 1H NMR (CDCl3,
300 MHz) δ 7.46-7.08 (m, 13 H), 6.94 (d, 1 H, J ) 8.1), 5.92
(m, 1 H), 5.02 (m, 2 H), 4.34 (d, 1 H), 4.04 (d, 1 H), 3.43 (t, 1
H), 2.78 (m, 2 H), 1.11 (m, 6 H); MS (FAB) m/ e 468 (MH+).
Anal. (C29H29N3O3) C, H, N.
a
Reagents: (i) NaH, DMF, 0 °C, 20 min; MeI, DMF, 0 °C, 1 h;
(ii) KHMDS, THF, 0 °C, 20 min; Ph2P(O)ONH2, THF, 0 °C to room
temperature, 18 h; (iii) PhNCO, pyridine, room temperature, 1 h.
Unity-300 spectrometer using tetramethylsilane as internal
standard. Chemical shifts are expressed as δ (ppm) values
for protons relative to the internal standard. Mass spectra
were recorded on a J EOL J MS-AX505HA or a J EOL SX-102
spectrometer.
2-(2,4-Dioxo-5-p h en yl-2,3,4,5-tetr a h yd r oben zo[b][1,5]-
d ia zep in -1-yl)-N-isop r op yl-N-p h en yla ceta m id e (12).
A
2-(3-Allyl-3-m e t h yl-2,4-d ioxo-5-p h e n yl-2,3,4,5-t e t r a -
h yd r oben zo[b][1,5]d ia zep in -1-yl)-N-isop r op yl-N-p h en y-
la ceta m id e (13b). To a stirring solution of 1.50 g (3.20 mmol)
of 13a in 15 mL of DMF at 0 °C was added 192 mg (4.81 mmol,
1.5 equiv) of sodium hydride (60% dispersion in mineral oil).
The resulting solution was stirred 15 min, and then 0.36 mL
(5.76 mmol, 1.8 equiv) of methyl iodide was added. The
reaction mixture was stirred for 3 h at room temperature, and
then tje reactopm was quenched with 10 mL of H2O. The DMF
was removed in vacuo, and the residue was dissolved in 100
mL of Et2O and washed with 100 mL of H2O. The organic
layer was dried (MgSO4), and the solvents were removed in
vacuo to afford 1.61 g (99%) of 13b as a white solid which was
used without further purification: 1H NMR (DMSO-d6, 300
MHz, mixture of conformers) δ 7.56-7.11 (m, 13 H), 6.74 (m,
1 H), 5.85 (m, 0.34 H), 5.61 (m, 0.66 H), 5.10-4.69 (m, 2.34
H), 4.20 (m, 1.66 H), 1.94 (d, 2 H, J ) 7.3), 1.21 (s, 2 H), 0.98
(m, 6 H), 0.82 (s, 1 H); Rf ) 0.66 in hexane/EtOAc (1/1).
[1-[(Isop r op ylp h en ylca r ba m oyl)m eth yl]-3-m eth yl-2,4-
d io x o -5-p h e n y l-2,3,4,5-t e t r a h y d r o -1H -b e n zo [b ][1,4]-
d ia zep in -3-yl]a cetic Acid (14b). To a rapidly stirring
biphasic solution of 1.03 g (2.14 mmol) of 13b in 50 mL of CCl4
and 25 mL of H2O was added 44 mg (0.21 mmol, 0.1 equiv) of
ruthenium(III) chloride hydrate, followed by 4.58 g (21.40
mmol, 10.0 equiv) of sodium periodate. The resulting black
solution was stirred rapidly at room temperature for 24 h, then
diluted with 100 mL of H2O, and extracted with EtOAc (3 ×
200 mL). The organics were washed with brine (1 × 150 mL)
and saturated NaHSO3 (1 × 150 mL) and dried (MgSO4), and
6.9 g (5.0 mmol) sample of potassium carbonate was added to
a solution of 9.2 g (5.0 mmol) of N-phenyl-1,2-phenylenedi-
amine and 12.7 g (5.0 mmol) of 2-bromo-N-isopropyl-N-
phenylacetamide8 in DMF (200 mL), and the mixture was
allowed to stir overnight. The DMF was evaporated in vacuo,
and the residue was dissolved in ethyl acetate (400 mL) and
washed exhaustively with aqueous 1 N HCl (4 × 250 mL). The
organic layer was washed with water (2 × 200 mL), dried (Na2-
SO4), and evaporated to give 17.8 g of crude alkylated product.
The oil was purified by chromatography on silica gel (600 g)
using first CHCl3 (8000 mL) and then hexane/ethyl acetate
(2/1, 8000 mL) as eluents to give 10.0 g (56%) of N-isopropyl-
N-phenyl-2-[[2-(phenylamino)phenyl]amino]acetamide as
a
brown oil: 1H-NMR (300 MHz, CDCl3) δ 7.42-6.8 (m, 14 H),
6.36 (d, 1 H), 4.95 (m, 1 H), 3.22 (s, 2 H), 1.05 (d, 6 H); MS
(FAB) m/ e 360 (MH+); TLC Rf ) 0.18 (CHCl3).
To 20 mL of THF at 0 °C was simultaneously added
dropwise over 10 min a solution of 1.97 g (5.48 mmol) of
N-isopropyl-N-phenyl-2-[[2-(phenylamino)phenyl]amino]aceta-
mide in 20 mL of THF and 0.53 mL (5.48 mmol) of malonyl
dichloride in 20 mL of THF. The resulting red-brown solution
was stirred at room temperature for 5.5 h and the solvent
removed in vacuo. Purification of the resulting brown oil by
silica gel flash chromatography (50-75% ethyl acetate/
petroleum ether) followed by recrystallization from ethyl
acetate/petroleum ether gave 0.84 g (36%) of 12 as a white
1
powder: mp 199-200 °C; H NMR (CDCl3, 300 MHz) δ 7.6-
7.2 (m, 12 H), 7.09 (t, 1 H, J ) 8), 6.90 (d, 1 H, J ) 8), 5.05 (m,
1 H), 4.38 (d, 1 H, J ) 17), 4.04 (d, 1 H, J ) 17), 3.54 (dd, 2 H,

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 3033
Ta ble 1. In Vitro Activity of 1,5-Benzodiazepine CCK-A Agonists
structuresa
functional assayb
binding assayc
CCK-B (pIC50
no.
X
Y
Z
ED50 (µM)
RE
CCK-A (pIC50
)
)
sel
0.3
CCK-8
AspTyr(SO3H)MetGlyTrpMetAspPheNH2
0.002 ( 0.001 (5)
1.0
0.8
0.2
0.2
0.3
0.5
0.7
0.6
ns
8.88 ( 0.22 (8)
7.26 ( 0.05 (4)
9.46 ( 0.04 (8)
7.62 ( 0.02 (3)
1
2
3
4
5
6
7
8
9
NH
NH
O
NH
CH2
CH2
CH2
CH2
NH
NH
O
H
H
H
H
1.6 ( 0.9 (2)
0.4
-
NH
CH2
NH
NH
NH
NH
NH
-
-
H
0.43 (1)
0.20 ( 0.11 (5)
0.78 (1)
-
6.81 ( 0.08 (3)
7.12 ( 0.02 (3)
7.44 ( 0.04 (2)
6.41 ( 0.06 (2)
5.88 ( 0.02 (2)
6.08 ( 0.04 (3)
5.08 ( 0.04 (3)
6.10 ( 0.19 (2)
5.05 ( 0.09 (2)
6.55 ( 0.27 (2)
5
110
22
23
0.2
CH3
CH2CH3
CH2Ph
CH3
>10 (1)
0.5
a
b
Figure 1. Functional activity in the isolated guinea pig gallblader following incubation with the test ligand; ED50, concentration at
which 50% of the maximal contraction was observed ( SE (number of determinations); -, an ED50 could not be determined; RE, relative
efficacy as determined by the maximal contraction observed at 30 µM standardized to CCK-8 (1 µM) ) 1.0, all values ( 0.1, n g 3; ns, no
significant contraction observed. c Binding affinity for human CCK-A and CCK-B receptors; pIC50, -log of the concentration that displaced
50% of [125I]Bolton-Hunter CCK-8 from membrane preparations isolated from CHO-K1 cells stably transfected with cDNA of human
CCK-A and CCK-B receptors ( SE (number of determinations); sel, CCK-A receptor selectivity calculated from IC50 (B)/IC50 (A).
Ta ble 2. In Vivo Activity of 1,5-Benzodiazepine CCK-A
Ack n ow led gm en t. We gratefully acknowledge Dr.
Agonists
Laurence J . Miller (Mayo Clinic, Rochester, MN) for
providing the CHO-K1 cells stably transfected with
mouse gallbladder emptyinga
human CCK-A and CCK-B receptors.
0.1 µmol
1.0 µmol
10 µmol
no.
kg-1 ip (%)
kg-1 po (%)
kg-1 po (%)
CCK-8
95b
78
63
73
68
-
ns
34
36
45
-
-
Refer en ces
1
5
6
7
(1) Crawley, J . N.; Corwin, R. L. Biological Actions of Cholecysto-
kinin. Peptides 1994, 4, 731-755.
(2) Schwartz, G. J .; McHugh, P. R.; Moran, T. H. Gastric Loads and
Cholecystokinin Synergistically Stimulate Rat Vagal Afferents.
Am. J . Physiol. 1993, 265, R872-R876.
-
73
42
a
Following overnight food deprivation, male CD-1 mice (10
animals per dose) were treated (ip or po) with vehicle (ethanol/
propylene glycol/water, 2/3/5, 1 mL kg-1) or test compound
dissolved in vehicle (1 mL kg-1). Thirty minutes after drug
treatment, animals were sacrificed (CO2) and the gallbladders
were dissected out and weighed. Gallbladder wet weights of the
treated animals were normalized to the vehicle control group.
Gallbladder emptying was inhibited by the CCK-A receptor
antagonist MK-329 (0.5 µmol kg-1 ip); %, percent emptying p <
(3) Dourish, C. T.; Ruckert, A. C.; Tattersall, F. D.; Iversen, S. D.
Evidence that Decreased Feeding Induced by Systemic Injection
of Cholecystokinin is Mediated by CCK-A Receptors. Eur. J .
Pharmacol. 1989, 173, 233-234.
(4) Dourish, C. T.; Hill, D. R. Classification and Function of CCK
Receptors. Trends Pharmacol. Sci. 1987, 8, 207-208.
(5) Pi-Sunyer, F. X.; Kissileff, H. R.; Thornton, J .; Smith, G. P.
C-Terminal Octapeptide of Cholecystokinin Decreases Food
Intake in Obese Men. Physiol. Behav. 1982, 29, 627-630.
(6) (a) Froehlich, F.; Gonvers, J . J .; Fried, M. Role of Nutrient Fat
and Cholecystokinin in Regulation of Gallbladder Emptying in
Man. Dig. Dis. Sci. 1995, 40, 529-533. (b) Roslyn, J . J .;
DenBesten, L.; Pitt, H. A.; Kuchenbecker, S.; Polarek, J . W.
Effects of Cholecystokinin on Gallbladder Stasis and Cholesterol
Gallstone Formation. J . Surg. Res. 1981, 30, 200-204.
(7) Freidinger, R. M. Cholecystokinin and Gastrin Antagonists. Med.
Res. Rev. 1989, 9, 271-290.
(8) Aquino, C. J .; Armour, D. R.; Berman, J . M.; Birkemo, L. S.;
Carr, R. A. E.; Croom, D. K.; Dezube, M.; Dougherty, R. W.;
Ervin, G. N.; Grizzle, M. K.; Head, J . E.; Hirst, G. C.; J ames, M.
K.; J ohnson, M. F.; Miller, L. J .; Queen, K. L.; Rimele, T. J .;
Smith, D. N.; Sugg, E. E. Discovery of 1,5-Benzodiazepines with
Peripheral Cholecystokinin (CCK-A) Receptor Agonist Activity
I. Optimization of the Agonist “Trigger”. J . Med. Chem. 1996,
39, 562-569.
b
0.05; ns, not statistically significant; -, not determined. CCK-8
at 1 nmol kg-1 ip.
the solvent was removed in vacuo to afford 0.98 g (92%) of
14b as a dark gray foam: 1H NMR (CDCl3, 300 MHz, mixture
of conformations) δ 7.53-7.09 (m, 13 H), 6.86 (m, 1 H), 5.02
(m, 1 H), 4.34 (d, 1 H), 4.41 (m, 1 H), 4.08 (m, 1 H), 3.27 (d, 1
H), 3.03 (d, 1 H), 1.24 (s, 3 H), 1.11 (m, 6 H); MS (FAB) m/ e
500 (MH+). The crude product was used directly in the next
reaction.
Purification of a portion of the crude product by C-18 reverse
phase MPLC using methanol/0.1% TFA-H2O (13/2) as eluent
(9) Bock, M. G.; DiPardo, R. M.; Evans, B. E.; Rittle, K. E.; Whitter,
W. L.; Veber, D. F.; Anderson, P. S.; Freidinger, R. M. Benzo-
diazepine Gastrin and Brain Cholecystokinin Receptor Ligands;
L-365,260. J . Med. Chem. 1989, 32, 13-16.
gave 14b as
a white powder: mp 100-105 °C. Anal.
(C29H29N3O5‚CF3CO2H‚H2O) C, H, N.
(10) Unwalla, R. J . Unpublished results.
2-[2,4-Dioxo-5-p h en yl-3-m eth yl-3-[(p h en ylca r ba m oyl)-
m eth yl]-2,3,4,5-tetr a h yd r oben zo[b][1,5]d ia zep in -1-yl]-N-
isop r op yl-N-p h en yla ceta m id e (6). A solution of 250 mg
(0.50 mmol) of 14b, 0.091 mL (1.0 mmol) of aniline, 0.47 g (1.0
mmol) of PyBroP, and 0.35 mL (2.0 mmol) of N,N-diisopropyl-
N-ethylamine in 2 mL of DMF was stirred at 50 °C for 2 d.
The reaction mixture was diluted with 50 mL of 1 N HCl and
extracted with EtOAc (3×). The organic extract was washed
with 1 N HCl, brine, saturated NaHCO3, and brine, dried over
MgSO4, and concentrated in vacuo to a yellow oil. Purification
by silica gel flash chromatography (50% EtOAc/petroleum
ether) followed by recrystallization from MeCN/H2O (2/1) gave
0.125 g (44%) of 6 as a white powder: mp 228-9 °C; 1H NMR
(DMSO-d6, 300 MHz, mixture of conformations) δ 9.73 (s, 1
H), 7.6-7.2 (m , 16 H), 7.05 (m, 2 H), 6.65 (d, 1 H, J ) 8), 4.85
(m, 1 H), 4.22 (m, 2 H), 2.33 (m, 2 H), 1.20 (s, 3 H), 1.00 (m, 6
H); MS (FAB) m/ e 575 (MH+). Anal. (C35H34N4O4) C, H, N.
(11) (a) Ulrich, C. D.; Ferber, I.; Holicky, E.; Hadac, E.; Buell, G.;
Miller, L. J . Molecular Cloning and Functional Expression of
the Human Gallbladder Cholecystokinin A Receptor. Biochem.
Biophys. Res. Commun. 1993, 193, 204-211. (b) de Weerth, A.;
Pisegna, J . R.; Huppi, K.; Wank, S. A. Molecular Cloning,
Functional Expression and Chromasomal Localization of the
Human Cholecystokinin Type A Receptor. Biochem. Biophys.
Res. Commun. 1993, 194, 811-818.
(12) (a) Pisegna, J . R.; de Weerth, A.; Huppi, K.; Wank, S. A.
Molecular Cloning of the Human Brain and Gastric Cholecys-
tokinin Receptor: Structure, Functional Expression and Cho-
masomal Localization. Biochem. Biophys. Res. Commun. 1992,
189 , 296-303. (b) Lee, Y. M.; Beinborn, M.; McBride, E. W.;
Lu, M.; Kolakowski, L. F., J r.; Kopin, A. S. The Human Brain
Cholecystokinin-B/Gastrin Receptor. Cloning and Characteriza-
tion. J . Biol. Chem. 1993, 268, 8164-8169.
(13) Makovec, F.; Bani, M.; Cereda, R.; Chiste, R.; Pacini, M. A.;
Revel, L.; Rovati, L. C. Antispasmodic Activity on the Gallblad-
der of the Mouse of CR 1409 (Lorglumide), a Potent Antagonist
of Peripheral CCK. Pharmacol. Res. Commun. 1987, 19, 41-
51.

3034 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Notes
(14) In rodents, CCK may affect feeding behavior by both peripheral
and centrally mediated pathways: (a) Della-Fera, M. A.; Cole-
man, B. D.; Baile, C. A. CNS injection of CCK in rats: effects
on real and sham feeding and gastric emptying. Am. J . Physiol.
1990, 258, R1165-R1169. (b) Crawley, J . Clarification of the
behavioral functions of peripheral and central cholecystokinin:
two separate peptide pools. Peptides 1985, 6, 129-36.
(15) Subsequent to the completion of our work, a patent application
describing 1,4-benzodiazepines as orally active CCK agonists has
been published. Badorc, A.; Despeyroux, P.; Gully, D.; De
Cointet, P.; Frehel, D.; Maffrand, J -P. Preparation of 3-acy-
lamino-5-phenyl-1,4-benzodiazepin-2-ones as CCK receptor ago-
nists. EP 95 400293, 1995; Chem. Abstr. 1995, 124, 8858.
J M960205B