Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity (II): Optimization of the C3 amino substituent

Article abstract of DOI:10.1021/jm9601664

Analogs of the previously reported 1,5-benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonist 1 were prepared which explore substitution and/or replacement of the C-3 phenyl urea moiety. Agonist efficacy on the isolated guinea pig gallbladder (GPGB) was retained with a variety of substituted ureas and amide analogs. Three compounds were identified which were orally active in the mouse gallbladder emptying assay (MGBE). The 2-indolamide (52) and N-(carboxymethyl)-2-indolamide (54) derivatives had improved affinity for the human CCK-A receptor but reduced agonist efficacy on the GPGB. Neither indolamide was orally active in a rat feeding assay. In contrast, the (3-carboxyphenyl)urea derivative (29, GW7854) had moderately increased affinity for the human CCK-B receptor but was a potent full agonist on the GPGB and was orally active in both the MGBE and rat feeding assays. GW7854 was a full agonist (EC₅₀ = 60 nM) for calcium mobilization on CHO K1 cells expressing hCCK-A receptors and a potent antagonist of CCK-8 (pA₂ = 9.1) on CHO K1 cells expressing hCCK-B receptors. GW7854 is a potent mixed CCK-A agonist/CCK-B antagonist which is orally active in two in vivo models of CCK-A-mediated agonist activity.

Full text of DOI:10.1021/jm9601664

5236
J . Med. Chem. 1996, 39, 5236-5245
Discover y of 1,5-Ben zod ia zep in es w ith P er ip h er a l Ch olecystok in in (CCK-A)
Recep tor Agon ist Activity (II): Op tim iza tion of th e C3 Am in o Su bstitu en t
Gavin C. Hirst,† Christopher Aquino,† Lawrence Birkemo,‡ Dallas K. Croom,‡ Milana Dezube,
Robert W. Dougherty, J r.,§ Gregory N. Ervin,‡ Mary K. Grizzle,‡ Brad Henke,† Michael K. J ames,‡
Michael F. J ohnson,‡ Tanya Momtahen,† Kennedy L. Queen,§ Ronald G. Sherrill,† J erzy Szewczyk,†
Timothy M. Willson,† and Elizabeth E. Sugg*,†
Departments of Medicinal Chemistry, Cellular Biochemistry, and Pharmacology, Glaxo Wellcome Research and Development,
5 Moore Drive, Research Triangle Park, North Carolina 27709
Received March 1, 1996X
Analogs of the previously reported 1,5-benzodiazepine peripheral cholecystokinin (CCK-A)
receptor agonist 1 were prepared which explore substitution and/or replacement of the C-3
phenyl urea moiety. Agonist efficacy on the isolated guinea pig gallbladder (GPGB) was
retained with a variety of substituted ureas and amide analogs. Three compounds were
identified which were orally active in the mouse gallbladder emptying assay (MGBE). The
2-indolamide (52) and N-(carboxymethyl)-2-indolamide (54) derivatives had improved affinity
for the human CCK-A receptor but reduced agonist efficacy on the GPGB. Neither indolamide
was orally active in a rat feeding assay. In contrast, the (3-carboxyphenyl)urea derivative
(29, GW7854) had moderately increased affinity for the human CCK-B receptor but was a
potent full agonist on the GPGB and was orally active in both the MGBE and rat feeding
assays. GW7854 was a full agonist (EC50 ) 60 nM) for calcium mobilization on CHO K1 cells
expressing hCCK-A receptors and a potent antagonist of CCK-8 (pA2 ) 9.1) on CHO K1 cells
expressing hCCK-B receptors. GW7854 is a potent mixed CCK-A agonist/CCK-B antagonist
which is orally active in two in vivo models of CCK-A-mediated agonist activity.
In tr od u ction
(serotonergic and adrenergic) therapies for the treat-
ment of obesity.13
Cholecystokinin (CCK) is a gastrointestinal hormone
and neurotransmitter involved in nutrient assimilation,
including the secretion of bile and digestive enzymes
and the regulation of enteric transit.1 While a variety
of endogenous molecular forms of CCK have been
isolated, the C-terminal octapeptide (Asp-Tyr(SO3H)-
Met-Gly-Trp-Met-Asp-PheNH2, CCK-8) appears to be
the minimum sequence required for bioactivity.2-4 CCK
is released from intestinal I cells primarily in response
to fat and protein ingestion.5 The physiological actions
of CCK are mediated by two G-protein-coupled seven
transmembrane receptor subtypes. CCK-A receptors
predominate in the periphery (gallbladder, pancreas,
pyloric sphincter, and vagal afferent fibers) but are also
found in discrete regions of the brain.6 CCK-B or
gastrin receptors predominate in the brain and gastric
glands.7 Although CCK-A and CCK-B receptor se-
quences are highly conserved among species, consider-
able variation in receptor subtype distribution has been
reported.8 The utility of a CCK receptor agonist for the
treatment of obesity is suggested by studies demon-
strating that exogenous CCK can shorten meal duration
and reduce meal size in several species, including lean9
and obese10 humans. Chronic administration of CCK-8
to patients on total parenteral nutrition has also
demonstrated a role for CCK in the prevention of
gallstones.11 The relevant target for both effects is the
CCK-A receptor.12 An orally acting CCK-A agonist
which enhanced satiety and decreased meal size could
be a useful alternative to current centrally mediated
We recently reported the discovery of a series of 1,5-
benzodiazepines, exemplified by 1, which were CCK-A
receptor agonists in vitro (isolated guinea pig gallblad-
der) and in vivo (rat anorexia).14 Despite good CCK-A
agonist efficacy, these compounds had equivalent affin-
ity for both human CCK-A and CCK-B receptors and,
while some compounds were potent anorectics in the rat
following intraperitoneal administration, none were
orally active. We now report the synthesis and biologi-
cal profile of analogs of 1 which explore both substitu-
tion and/or replacement of the C3-position phenyl urea
moiety. The primary goal of this work was to identify
compounds which were orally active satiety agents in a
rat feeding model.
Ch em istr y
The key amine intermediate used in the preparation
of analogs reported in Tables 1-5 was prepared as
outlined in Scheme 1. N-Phenyl-1,2-phenylene diamine
was alklated with bromoacetanilide 214 and the substi-
tuted phenylene diamine 3 condensed with diacid
* To whom correspondence should be addressed.
† Department of Medicinal Chemistry.
‡ Department of Cellular Biochemistry.
§ Department of Pharmacology.
X Abstract published in Advance ACS Abstracts, December 1, 1996.
S0022-2623(96)00166-5 CCC: $12.00 © 1996 American Chemical Society

Orally Active 1,5-Benzodiazepines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5237
Sch em e 1. Synthesis of Intermediate 6a
Sch em e 2. Generalized Scheme for the Preparation of
C-3 Analogs of 1a
a
(a) RNCO, CH3CN; (b) RNHCOO2Ar; (c) RNH2, 1,1′-carbon-
yldiimidazole; (d) RNH2, (CCl3O)2CO; (e) EDC or DCC, HOBt,
RNH2.
a
(a) K2CO3, DMF; (b) THF; (c) Zn, CH3COOH.
Ta ble 2. In Vitro Functional Activity for Substituted
Phenylurea Analogs
Ta ble 1. In Vitro Functional Data for Unsubstituted Urea
Analogs
GPGBa
GPGB (30 µM)a
% CCK-8
no.
R
% CCK-8
no.
R
30 µM
1 µM
pKB
1
17
NHC6H5
86
ia
1
21
22
23
24
25
26
27
28
29
H
86
34
ND
20
38
14
5
78
89
100
ND
ND
16
ND
ND
ND
ND
ND
82
N
NH
2-CH3
2-OH
4-CH3
4-OCH3
4-COOH
3-CH3
3-OCH3
3-OH
5.5
18
19
20
NH2
NHCH2C6H5
NHCH(CH3)2
ia
1
22
a
Functional activity on the isolated guinea pig gallbladder
following incubation with test ligand at 30 µM for 30 min at 37
°C; % CCK-8, percent contraction normalized to that for CCK-8
(1 µM); ia, inactive.
3-COOH
95
a
Functional activity on the isolated guinea pig gallbladder
chloride 414 to afford hydrazone 5. Reduction of hydra-
zone 5 with zinc/acetic acid provided amine 6.
following incubation with test ligand at 30 µM for 30 min or 1 µM
for 60 min at 37 °C; % CCK-8, percent contraction normalized to
that for CCK-8 (1 µM); pKB, single dose pA2 calculated from the
fold shift of the CCK-8 concentration-response curve in the
presence of test ligand (30 µM); ia, inactive.
Most of the ureas listed in Tables 1-3 were prepared
by treatment of 6 with commercially available isocyan-
ates (Scheme 2). Alternatively, anilines were converted
to p-nitrophenyl carbamate derivatives15 or activated in
situ with triphosgene16 or carbonyldiimidazole17 prior
to reaction with benzodiazepine 6. Addition and re-
moval of protecting groups followed standard practice.18
Exceptions to these generalized approaches are noted
below.
Sulfone 36 was prepared by oxidation of the corre-
sponding thioether 35 with m-CPBA.
The phenoxy-substituted derivatives 40-43 were
prepared through the reaction of 6 with the anilines 12-
15, activated in situ with triphosgene. The anilines
were prepared as outlined in Scheme 3. Alkylation of
m-nitrophenol with tert-butyl bromoacetate afforded
intermediate 7. Deprotection of 7 provided phenoxy-
acetic acid 8, which was converted to the imidiazolide
in situ and treated with ammonia or morpholine to
provide amides 9 and 10. Direct alkylation of m-
nitrophenol with N,N-dimethylchloroacetamide pro-
Condensation of benzoxazolinone with benzodiazepine
6 at 125 °C provided 22 (Table 2).
Triflamide 33 (Table 3) was prepared from the cor-
responding aniline 34 by treatment with triflic anhy-
dride.

5238 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Hirst et al.
Ta ble 3. In Vitro Functional Activity for 3-Substituted
Phenylurea Analogs
Receptor binding affinities were measured on membrane
preparations from the stably transfected CHO-K1 cells
(Table 5).23 In vivo efficacy was assessed following
intraperitoneal (ip) or oral (po) administration in both
MGBE21 and rat feeding14 models (Table 5). The
reported ED50’s represent the half-maximal response for
test compound, relative to the highest dose tested.
Resu lts
The single concentration screening data used to
evaluate new compounds are provided in Tables 1-4.
Of the simple urea replacements in Table 1, the phenyl
moiety (1) was preferred over the piperazine derivative
17 or unsubstituted urea 18. Homologation to a benzyl
group (19) or replacement with an isopropyl moiety (20)
also reduced agonist efficacy.
Within the phenylurea series, substitution of a methyl
group at any position on the aromatic ring significantly
reduced agonist efficacy (Table 2). However, the 3-hy-
droxy (27), 3-methoxy (28), and 3-carboxyl (29) deriva-
tives were fully efficacious. These data suggested that
substitution at the 3-position was preferred over either
2- or 4-substitution. Therefore, additional analogs were
prepared to explore more fully the structure-activity
relationships of these substituted phenyl ureas (Table
3).
Full efficacy (g75%) was observed with a variety of
substitutents, including amines, acids, acid isosteres
and even the phenoxy derivatives 40-43. Partial
efficacy (>50%) was observed with the N,N-dimethyl
(30), thiomethyl (35), and methyl sulfoxide (36) deriva-
tives. Only the 3-methyl (26), 3-chloro (39), and (N,N-
dimethylamino)ethoxy (44) derivatives were poorly ef-
ficacious at the concentrations tested.
In contrast to the phenyl ureas, most of the C-3 amide
analogs were consistently less efficacious (e60%) at the
concentrations tested (Table 4). The phenyl (45), pyridyl
(47, 48), or substituted phenyl (49, 50) derivatives were
equally efficacious on the GPGB. While the 3-indola-
mide (51) was weakly active, the 2-indolamide (52) had
partial agonist efficacy. Only the N-(carboxymethyl)-
indolamide derivative 54 appeared fully efficacious.
Homologation to the N-carboxypropyl derivative (55)
reduced efficacy.
All analogs which induced at least a 40% contractile
response on the GPGB were evaluated at a single oral
dose (1 µmol/kg) in the MGBE assay. Of the 25
compounds which met this criteria, three compounds
(29, 52, and 54) were orally active in the mouse. These
compounds were characterized further for both in vitro
(Table 5) and in vivo (Table 6) potency and efficacy. Data
for 114 and CCK-8 are provided for comparative pur-
poses.
The (3-carboxyphenyl)urea (29), indolamide (52), and
N-(carboxymethyl)indolamide (54) derivatives were ap-
proximately 30-fold, 2.5-fold, and 100-fold more potent,
respectively, than 1 on the isolated GPGB (Table 5).
These analogs were 100-fold (29), 1260-fold (52), and
32-fold (54) less potent than CCK-8 on this tissue.
Again, only the ureas 1 and 29 were fully efficacious at
the highest concentration tested (30 µM). The human
CCK-A receptor binding affinities of 1 and 29 were 80-
fold and 200-fold less than those for CCK-8. Both ureas
had slightly better affinity for the human CCK-B
receptor subtype. The indoles 52 and 54 had 200-fold
GPGBa
% CCK-8
no.
R
30 µM
1 µM
1
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
H
86
58
90
79
89
76
58
53
89
76
ND
95
77
78
98
ND
ND
29
85
64
87
N(CH3)2
CH2OH
CN4H
NHTf
NH2
50
SCH3
SO2CH3
ND
ND
ND
ND
7
ND
ND
ND
85
CH2COOH
CONH2
Cl
OCH2CONH2
OCH2CONMe2
OCH2CO-morpholino
OCH2COOH
OCH2CH2NMe2
2
a
See Tables 1 and 2. ND, not determined.
vided intermediate 11. Catalytic hydrogenation of 7 and
9-11 provided anilines 12-15. Deprotection of the tert-
butyl ester of 16 afforded the phenoxyacetic acid 43.
The C-3 amide analogs listed in Table 4 were pre-
pared through the reaction of 6 with commercially
available carboxylic acids utilizing standard dehydrat-
ing protocols19 (Scheme 2). Alkylation of indole 52 with
tert-butyl bromoacetate or tert-butyl bromobutyrate,
followed by treatment with HCl/dioxane, provided 54
and 55, respectively.
All compounds reported in Tables 1-6 were purified
to homogeneity (>97%) by RP-HPLC, and the resultant
lyophile was characterized by analytical RP-HPLC, H
NMR spectroscopy, and high-resolution mass spectrom-
etry or combustion analysis.
1
Biology
Analogs were initially evaluated for in vitro functional
efficacy on the isolated guinea pig gallbladder (GPGB)
at 30 or 1 µM concentrations (Tables 1-4).14 The
contractile activity of all compounds reported was
reversed completely by addition of the CCK-A receptor
selective antagonist MK-32920 (0.5 µM). Compounds
which did not produce contraction were evaluated for
antagonist activity against a concentration-response
curve for CCK-8 (10-11 to 10-6 M).14
Compounds which induced g40% contraction of the
GPGB were subsequently evaluated for oral activity in
the mouse gallbladder emptying21 (MBGE) assay at a
single dose (1 µmol/kg, po). Only compounds which
demonstrated oral efficacy in the mouse were character-
ized in detail. Full concentration-response curves were
obtained on the GPGB, as well as for calcium mobiliza-
tion with CHO-K1 cells stably expressing the human
CCK-A (hCCK-A) or CCK-B (hCCK-B) receptors.22

Orally Active 1,5-Benzodiazepines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5239
Sch em e 3. Synthesis of Phenoxyaniline Derivatives 40-43a
a
(a) NaH, DMF, BrCH2COOtBu; (b) HCl, dioxane; (c) 1,1′-carbonyldiimidazole, THF, amine; (d) H2, Pd/C; (e) (CCl3O)2CO, (C2H5)3N,
THF, 6; (f) NaH, DMF, ClCH2CONH(CH3)2.
Ta ble 4. In vitro Functional Activity for C-3 Amide Analogs
Ta ble 5. In Vitro Profile of Lead Benzodiazepines
GPGBa
pEC50
plC50
b
no.
% max
h-CCK-A
h-CCK-B
A/B
1
29
52
54
5.9 ( 0.4 (2)
7.4 ( 0.3 (3)
6.3 ( 0.1 (2)
7.9 ( 0.1 (3)
86
102
49
65
100
7.3 ( 0.1 (3) 7.6 ( 0.1 (3)
6.9 ( 0.1 (4) 8.0 ( 0.1 (4)
0.4
0.1
7.8 ( 0.2 (3) 6.5 ( 0.3 (3) 20
8.0 ( 0.1 (3) 6.7 ( 0.1 (3) 20
CCK-8 9.1 ( 0.2 (4)
9.2 ( 0.1 (3) 9.5 ( 0.4 (9)
0.8
a
Potency and efficacy on the isolated guinea pig gallbladder.
pEC50, -log of the concentration that produces half-maximal
contractile response; % max, maximal contractile response at 30
µM, normalized to CCK-8 (1 µM), (SEM (number of determina-
GPGBa
% CCK-8
b
tions). pIC50, -log of the concentration displacing 50% of [125-
I]Bolton-Hunter CCK-8 from membrane preparations isolated
from CHO-K1 cells stably transfected with cDNA of human CCK-A
and CCK-B receptors, (SEM (number of determinations); A/B,
IC50 CCK-A/IC50 CCK-B.
no.
R
30 µM
1 µM
45
46
47
48
49
50
51
52
53
54
55
phenyl
benzyl
3-pyridyl
4-pyridyl
57
ND
43
43
56
58
5
49
35
73
44
ND
28
ND
ND
ND
ND
ND
ND
ND
63
kg, 0.9 µg/kg), 29 was the most potent derivative
following po administration (ED50 ) 55 nmol/kg, 33 µg/
kg).
3-carboxyphenyl
2-carboxyphenyl
3-indolyl
Analogs 29, 52, and 54 were 10-100 times more
potent than 1 in the rat feeding assay following intra-
peritoneal (ip) administration. Since CCK-8 is 900-fold
less potent in the rat feeding model, relative to the
MGBE model, the potency of the benzodiazepine analogs
was more comparable to that of CCK-8 in this assay.
However, as seen with the isolated GPGB assay, only
the phenylurea derivatives (1 and 29) were fully effica-
cious in this assay. Neither indole achieved >50%
anorexia at the highest dose tested (10 µmol/kg, 6 mg/
kg). While we have no explanation for these efficacy
differences, which could reflect species, pharmacody-
namic, or pharmacokinetic differences, these data serve
to highlight the variability of animal models. When 29,
52, and 54 were screened for oral activity in the rat
2-indolyl
2-benzofuran
2-N-(carboxylmethyl)indolyl
2-N-(carboxypropyl)indolyl
ND
a
See Tables 1-3.
and 25-fold reduced affinity for the human CCK-A
receptor, compared to CCK-8. In contrast to the ureas,
both indoles had slightly better affinity (20-fold) for the
human CCK-A receptor subtype.
Analogs 29, 52, and 54 were potent, fully efficacious,
and orally active in the MGBE assay (Table 6). These
analogs were 40-200-fold less potent that CCK-8 fol-
lowing ip administration. While 54 was the most potent
compound following ip administration (ED50 ) 1.4 nmol/

5240 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Ta ble 6. In Vivo Profile of Lead Benzodiazepines
Hirst et al.
agonists 52 and 54 were not fully efficacious in all
MGBEa
anorexiab
ip
ED50 % max ED50 % max ED50 % maxc % maxd
models of CCK-A functional activity.
In contrast to the N1-anilidoacetamide substituent,
which primarily modulates the agonist efficacy14 of these
1,5-benzodiazepines, the C-3 substituent seems to influ-
ence both receptor subtype affinity and agonist efficacy.
That similar structural modifications at the C-3 position
of either the 1,4-benzodiazepine antagonists15,20,24,25 or
1,5-benzodiazepine agonists produced comparable
changes in receptor affinity and subtype selectivity
again suggests that these nonpeptide antagonists and
agonists may share similar structural determinants for
receptor recognition.14
ip
po
po
no.
1
ND
7.1
5.0
1.4
78
73
73
88
95
ia
55
300
600
ND
ia
77
85
950
60
95
96
87
56
44
95
ia
58
ia
29
52
54
74
10
ia
CCK-8 0.03
ND
27
ND
a
In vivo mouse gallbladder emptying assay; ED50, dose of test
compound that produces a half-maximal response of individual
compounds, nmol/kg; % max, maximal response at 1 nmol/kg, ip
for CCK-8, 0.1 µmol/kg ip or 1 µmol/kg po for test compounds.
b
Anorectic potency in rats conditioned to a palatable liquid diet
and fasted for 2 h; ED50,dose of test compound that produces a
half-maximal response, nmol/kg. c % max, maximal response at
0.5 µmol/kg, ip for CCK-8 and 10 µmol/kg, ip for test compound.
The identification of compounds within this series of
benzodiazepine agonists with improved CCK-A affinity
and selectivity compared to 1 has been the focus of two
recent reports.26,27 However, the functional character-
ization of 29 as a potent mixed CCK-A agonist/CCK-B
antagonist suggests that receptor selectivity based on
affinity may not be a critical feature for this class of
compounds. Since in vivo CCK-B agonist activity would
be unacceptable in a therapeutic entity because of the
potential for peripheral (enhanced gastric acid secretion)
or central (enhanced anxiety) side effects, the mixed
CCK-A agonist, CCK-B antagonist profile of 29 may
actually be ideal. GW 7854 (29) was orally active in
both in vivo models of gallbladder emptying and satiety.
Efforts to enhance the oral activity of this class of
compounds continue and will be reported at a later date.
d
% max, maximal response at 10 µmol/kg po for test compound;
ia, inactive; ND, not determined.
feeding model, only 29 was efficacious, inducing a 60%
reduction in food intake following a 10 µmol/kg (6 mg/
kg) dose.
The identification of 29 as an orally active analog in
two in vivo models of CCK-A-mediated activity prompted
us to explore further the functional selectivity of this
analog. The ability of 29 to influence intracellular
calcium was evaluated on the stably transfected CHO
cells expressing the hCCK-A or hCCK-B receptors and
loaded with FURA2-AM.22 CCK-8 was a full agonist
on both human CCK-A (EC50 ) 0.1 nM) and CCK-B
(EC50 ) 0.05 nM) cell lines.22 In contrast, analog 29
was a potent, full agonist on the CHO cells expressing
the hCCK-A receptor (EC50 ) 60 nM, n ) 2), but inactive
on the CHO cells expressing the hCCK-B receptor (up
to 10 mM). Remarkably, 29 potently blocked the CCK-8
concentration response curve on the CHO cells express-
ing the hCCK-B receptor (pA2 ) 9.1). Thus 29 is a
potent, mixed CCK-A agonist/CCK-B receptor antago-
nist which is orally active in two in vivo models of CCK-
A-mediated agonist activity.
Exp er im en ta l Section
Gen er a l. All chemicals and solvents are reagent grade
unless otherwise specified. CCK-8 was purchased from Sigma
(St. Louis, MO). MK-329 was obtained from Merck & Co.
(Rahway, NJ ). The following solvents and reagents have been
abbreviated: tetrahydrofuran (THF), ethyl ether (Et2O), di-
methyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichlo-
romethane (DCM), trifluoroacetic acid (TFA), dimethylforma-
mide (DMF). Reactions were monitored by thin-layer chro-
matography on 0.25 mm silica gel plates (60F-254, E. Merck)
and visualized with UV light. Final compounds were typically
purified by preparative reversed phase high-pressure liquid
chromatography (RP-HPLC) using a Waters Model 3000 Delta
Prep equipped with a Delta-pak radial compression cartridge
(C-18, 300 A, 15 m, 47 mm × 300 mm) as the stationary phase.
The mobile phase employed 0.1% aqueous TFA with acetoni-
trile as the organic modifier. Linear gradients were used in
all cases, and the flow rate was usually 100 mL/min (t0 ) 5
min). Appropriate fractions were combined and lyophilized
to obtain the target analogs. Analytical purity was assessed
by RP-HPLC using a Waters 600E system equipped with a
Waters 990 diode array spectrometer (l range 200-400 nm).
The stationary phase was a Vydac C-18 column (5 m, 4.6 mm
× 200 mm). The mobile phase was the same as above, and
the flow rate was 1.0 or 1.5 mL/min (t0 ) 3 min). Analytical
data is reported as retention time, tR , in minutes (% aceto-
nitrile, time, flow rate).
1H NMR spectra were recorded on either a Varian VXR-
300 or a Varian Unity-300 instrument. Chemical shifts are
reported in parts per million (ppm, δ units). Coupling
constants are reported in units of hertz. Splitting patterns
are designated as s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; b, broad. Low-resolution mass spectra (MS) were
recorded on a J EOL J MS-AX505HA, a J EOL SX-102, or a
SCIEX-APIiii spectrometers. High-resolution mass spectra
were recorded on a AMD-604 (AMD Electra GmbH) high-
resolution double-focusing mass spectrometer (Analytical In-
strument Group, Raleigh, NC). Mass spectra were acquired
in the positive ion mode under electrospray ionization (ESI)
Discu ssion
While it was not intuitively obvious that structure-
activity relationships developed for the 1,4-benzodiaz-
epine CCK-A and CCK-B receptor antagonists would be
applicable to these 1,5-benzodiazepine CCK-A agonists,
the literature served as a valuable starting point for the
optimization of 1. Indeed, some parallels were observed.
In both antagonist and agonist series, the C-3 phenyl-
urea derivatives were CCK-B receptor selective, based
on receptor affinity measurements.15,24 Within both
urea series, phenyl was preferred over benzyl- or alkyl-
ureas and substitution of the phenyl ring at the 3-posi-
tion was generally favored over the 2- or 4-position. One
striking difference between the agonist and antagonist
series was the loss of agonist efficacy with the 3-methyl
(26) or 3-chloro (39) derivatives. Both substitutions
were optimal in the 1,4-benzodiazepine antagonist
series.15
The 2-indolamide isomer was preferred over the
3-indolamide isomer in both agonist and antagonist
series.25 While the 2-indolamide substitution enhanced
CCK-A receptor affinity and selectivity in the 1,4-
benzodiazepine antagonists,25 the improvement of CCK-A
receptor affinity and selectivity of the 1,5-benzodiaz-
epine agonists was less dramatic. The indolamide

Orally Active 1,5-Benzodiazepines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5241
or fast atom bombardment (FAB) methods. Combustion
analyses were performed by Atlantic Microlabs, Inc., Norcross,
GA.
mL/min). Compounds 19, 20, 21, 23, 24, 25, 26, 27, 35, and
39 were made in an analogous manner.
(3-Nitr op h en oxy)a cetic Acid ter t-Bu tyl Ester (7). So-
dium hydride (1.42 g, 35.9 mmol) was added to a 0 °C solution
of 3-nitrophenol (5.00 g, 35.9 mmol) in DMF (20 mL), and the
resultant solution stirred at 0 °C for 10 min. tert-Butyl
bromoacetate (5.80 mL, 35.9 mmol) was added dropwise, and
the resultant mixture allowed to attain room temperature
overnight. The reaction mixture was poured into water (100
mL), and the resultant mixture extracted into ethyl acetate
(3 × 50 mL). The combined organics were washed with
saturated aqueous sodium carbonate (50 mL), water (2 × 50
mL), and brine (20 mL), dried (MgSO4), and concentrated in
vacuo to afford the crude intermediate 3: 1H NMR (300 MHz,
CDCl3) δ 1.50 (s, 9H), 4.60 (s, 2H), 7.26 (m, 1H), 7.47 (t, J )
7.1 Hz, 1H), 7.69 (m, 1H), 7.83 (dd, J ) 7.1, 0.8 Hz, 1H).
Intermediate 7 was made in analogous manner.
(3-Nitr op h en oxy)a cetic Acid (8). A mixture of the above
intermediate 7 (3.00 g, 11.8 mmol) and 4 N HCl in dioxane
(10 mL) was stirred at room temperature for 2 h. Diethyl ether
(50 mL) was added, and the solvents were decanted to leave a
pale brown gum. Trituration with diethyl ether gave the
desired product as a cream-colored solid (1.49 g): 1H NMR (300
MHz, DMSO-d6): δ 4.82 (s, 2H), 7.40 (dd, J ) 7.1, 0.8 Hz, 1H),
7.57 (t, J ) 7.1 Hz, 1H), 7.69 (t, J ) 0.8 Hz, 1H), 7.83 (dd, J
) 7.1, 0.8 Hz, 1H).
(3-Nitr op h en oxy)a ceta m id e (9). A mixture of intermedi-
ate 8 (0.245 g, 1.24 mmol) and 1,1-carbonyldiimidazole (0.201
g, 1.24 mmol) in THF (5 mL) was stirred at room temperature
for 3 h, ammonia (2.0 M in methanol, 2.48 mL, 4.96 mmol)
was added, and the resultant solution was stirred at room
temperature for 6 h. The solvents were removed in vacuo, and
the residual solid was triturated with diethyl ether to afford
the crude product which was recrystallized from ethyl acetate
to afford the desired product (0.123 g) as a white solid: 1H
NMR (300 MHz, CDCl3) δ 4.58 (s, 2H), 7.28 (dd, J ) 7.1, 0.8
Hz, 1H), 7.51 (t, J ) 7.1 Hz, 1H), 7.80 (t, J ) 0.8 Hz, 1H), 7.83
(dd, J ) 7.1, 0.8 Hz, 1H). Intermediate 10 was also made in
this manner.
N-Isopr opyl-N-ph en yl-2-[[2-(ph en ylam in o)ph en yl]am i-
n o]a ceta m id e (3). Potassium carbonate (6.9 g, 50 mmol) was
added to a solution of N-phenylphenylenediamine (9.2 g, 50
mmol) in DMF and 2-bromo-N-isopropyl-N-phenylacetamide,
2 (12.7 g, 50 mmol), in DMF (200 mL), and the mixture was
stirred 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 (Na2SO4),
and evaporated to give the crude 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 the titled compound (10.0 g) as an oil: 1H NMR (300
MHz, CDCl3) δ 1.05 (d, 6H), 3.22 (s, 2H), 4.95 (m, 1H), 6.36
(d, 1H), 7.42-6.8 (m, 14 H); LRMS (FAB) m/ z 360 (M + H)+;
TLC Rf ) 0.18 (CHCl3).
2-[2,4-Dioxo-5-p h en yl-3-(p h en ylh yd r a zon o)-2,3,4,5-tet-
r a h yd r o ben zo[b][1,4]d ia zep in -1-yl]-N-isop r op yl-N-p h en -
yla ceta m id e (5). N-Isopropyl-N-phenyl-2-[[2-(phenylamino)-
phenyl]amino]acetamide, 3 (10 g, 27.8 mmol), and 2-(phen-
ylhydrazono)propanedioyl dichloride, 4 (6.83 g, 27.8 mmol),
were each dissolved in THF (100 mL) and added simulta-
neously, with stirring, to a flask containing THF (100 mL) at
0 °C, under nitrogen. The reaction mixture was allowed to
warm to room temperature and stirred for 4 h. The THF was
evaporated in vacuo, and the residue was dissolved in ethyl
acetate (200 mL). The ethyl acetate solution was washed with
10% aqueous sodium carbonate (2 × 200 mL) and water (2 ×
200 mL), dried (Na2SO4), and concentrated in vacuo. The
residual foam was treated with diethyl ether (50 mL) to
precipitate the title compound as a bright yellow solid (7.5 g).
1H NMR (300 MHz, CDCl3) δ 1.05 (m, 6H), 4.4 (m, 2H), 5.05
(m, 1H), 7.6-6.8 (m, 19 H), 11.4 and 10.85 (s, 1H); LRMS
(FAB) m/ z 532 (M + H)+; TLC Rf ) 0.19 (hexane:ethyl acetate,
2:1).
2-(3-Am in o-2,4-dioxo-5-ph en yl-2,3,4,5-tetr ah ydr oben zo-
[b][1,4]diazepin -1-yl)-N-isopr opyl-N-ph en ylacetam ide (6).
Zinc powder (9.1 g, 139 mmol) was added in portions to a slurry
of 2-[2,4-dioxo-5-phenyl-3-(phenylhydrazono)-2,3,4,5-tetrahy-
drobenzo[b][1,4]-diazepin-1-yl]-N-isopropyl-N-phenylaceta-
mide, 16 (7.5 g, 14.1 mmol), in glacial acetic acid (30 mL), and
the mixture was cooled to 0 °C. The reaction mixture was
allowed to warm to room temperature and stirred for an
additional hour. The solids were removed by filtration through
a Celite pad, and the glacial acetic acid was evaporated in
vacuo. The residue was dissolved in ethyl acetate (200 mL),
washed with 10% aqueous sodium carbonate (2 × 100 mL) and
water (2 × 100 mL), dried (Na2SO4), and evaporated to a tan
oil. Trituration with hexane/ethyl acetate (2:1) provided the
title compound as a light tan powder (6.3 g): 1H NMR (300
MHz, CDCl3) δ 1.05 (d, 6H), 4.3-4.0 (m, 3H), 5.05 (m, 1H),
7.6-6.8 (m, 14H); LRMS (FAB) m/ z 448 (M + H)+ ; TLC Rf )
0.25 (chloroform:methanol, 9:1).
(3-Am in op h en oxy)a ceta m id e (12). A mixture of inter-
mediate 9 (0.123 g, 0.63 mmol) and 10% palladium on carbon
(0.012 g) in ethyl acetate (10 mL) was stirred under an
atmosphere of hydrogen for 1 h. The solids were removed by
filtration, and the filtrate was concentrated in vacuo to afford
the desired intermediate (0.100 g) as a white foam: 1H NMR
(300 MHz, CDCl3) δ 4.48 (s, 2H), 6.22 (dd, J ) 7.1, 0.8 Hz,
1H), 6.37 (m, 2H), 7.08 (t, J ) 0.8 Hz, 1H). Intermediates 13,
14, and 15 were made in this manner.
2-(2,4-Dioxo-5-p h en yl-3-u r eid o-2,3,4,5-tetr a h yd r o-1H-
ben zo[b][1,4]d ia zep in -1-yl)-N-isop r op yl-N-p h en yla ceta -
m id e (18). To a stirring solution of intermediate 6 (200 mg,
0.45 mmol) in DCM (5 mL) was added (S)-(-)-R-methylbenzyl
isocyanate (0.064 mL, 0.45 mmol). The reaction mixture was
stirred for 12 h and then concentrated to dryness. The residue
was triturated with methanol to give 131 mg of desired
product. This crude urea (131 mg, 0.22 mmol) was dissolved
in acetic acid (10 mL) and treated with 10% Pd/C (131 mg)
under 50 psi of hydrogen. After 12 h the solids were removed
by filtration through a pad of Celite, concentrated, and purified
by RP-HPLC (42-60% acetonitrile, 30 min) to give the titled
compound (0.036 g) as a white lyophile: 1H NMR (CDCl3, 300
MHz) δ 1.08 (t, J ) 6.6 Hz, 6H); 4.12 (d, J ) 17.0 Hz, 1H),
4.44 (d, J ) 17.0 Hz, 1H), 4.83 (bs, 2H), 5.00 (m, 1H), 5.21 (d,
J ) 7.6 Hz, 1H), 6.14 (m, 1H), 6.97 (d, J ) 7.9 Hz, 1H), 7.3 (m,
8H), 7.5 (m, 5H); MS C22H27N5O4 (M + H)+ ) 486.2141,
observed (M + H)+ ) 486.2133; tR) 26.5 min (42-60%
acetonitrile, 30 min, t0) 3 min).
2-[2,4-D io x o -5-p h e n y l-3-(3-p h e n y lu r e id o )-2,3,4,5-
t et r a h yd r ob en zo[b][1,4]d ia zep in -1-yl]-N-isop r op yl-N-
p h en yla ceta m id e (1). To a slurry of 6 (0.667 g, 1.49 mmol)
in dichloromethane (5 mL) was added phenyl isocyanate (0.177
g, 1.49 mmol) with stirring at ambient temperature. The
reaction mixture was stirred for 2 h, and the cream-colored
precipitate was separated by filtration to afford the crude
product which was purified by preparative RP-HPLC chroma-
tography on a C-18 column with linear gradient elution from
40 to 60% acetonitrile in water with 0.1% TFA buffer over 30
min at a rate of 100 mL/min. Fractions containing the desired
material were combined, frozen, and lyophilized to afford the
titled product (0.181 g) as a white lyophile: 1H NMR (300 MHz,
DMSO-d6) δ 0.95 (d, 3H, J ) 7.3 Hz), 0.98 (d, 3H, J ) 7.3 Hz),
4.19 (d, 1H, J ) 16.6 Hz), 4.48 (d, 1H, J ) 16.9 Hz), 4.79 (m,
1H), 5.04 (d, 1H, J ) 7.8 Hz), 6.87-6.92 (m, 1H), 6.95 (d, 1H,
J ) 7.6 Hz), 7.18-7.57 (m, 17H), 9.14 (s, 1H); HRMS calculated
for C33H31N5O4 (M + H)+ 562.2454, observed (M + H)+
562.2456; HPLC tR ) 16.5 min (51-60% CH3CN, 30 min, 1.0
2-[3-[3-(2-Hyd r oxyp h en yl)u r eid o]-2,4-d ioxo-5-p h en yl-
2,3,4,5-tetr a h yd r o-1H-ben zo[b][1,4]d ia zep in -1-yl]-N-iso-
p r op yl-N-p h en yla ceta m id e (22). A solution of intermediate
6 (0.221 g, 0.5 mmol) and 2-benzoxazolinone (0.070 g, 0.5
mmol) in dry DMF (2 mL) was stirred at 125 °C for 48 h. The
reaction mixture was poured into an ice-1 N HCl mixture,
and the product was extracted with AcOEt. The solvent was
removed in vacuo, and the product was purifed by column
chromatography on silica gel (MeOH:CHCl3, 1:99), providing

5242 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Hirst et al.
the crude product. Trituration with methanol afforded the
title compound (0.040 g) as a white amorphous solid. 1H NMR
(300 MHz, DMSO-d6) δ 0.96 (2d, J ) 7 Hz, 6H), 4.17 (d, J )
16 Hz, 1H), 4.46 (d, J ) 16 Hz, 1H), 4.79 (m, 1H), 5.07 (m,
1H), 6.60-7.00 (m, 3H), 7.21-7.82 (m, 16H), 8.65 (s, 1H), 9.72
(s, 1H); MS C33H32N5O5 (M + H)+ ) 578.2403, observed (M +
H)+ ) 578.2388; RP-HPLC column Dynamax C-8, 2 mL/min
(70% acetonitrile), tR ) 4.3 min.
neutralized and triturated with 1 N HCl and water to give
the crude product. The crude product was dissolved in ethyl
acetate (20 mL), heated to reflux for 3 h and then cooled. The
resulting precipitate was separated by filtration and dried
under vacuum to provide the title compound (225 mg) as a
white solid: 1H NMR (300 MHz, DMSO-d6): δ 0.96 (m, 6H),
4.18 (d, J ) 15.8 Hz, 1H), 4.48 (d, J ) 16 Hz, 1H), 4.8 (m, 1H),
5.05 (d, J ) 9 Hz, 1H), 7.6-6.9 (m, 18H), 8.05 (s, 1H), 9.4 (s,
1H); MS (FAB) m/ z 606 (M + H)+. Anal. (C34H31N5O6‚0.3TFA)
C, H, N.
N-Isopr opyl-2-[3-[3-(3-h ydr oxyph en yl)u r eido]-2,4-dioxo-
5-p h en yl-2,3,4,5-tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-
p h en yla ceta m id e (28). A solution of 3-(benzyloxy)aniline
(0.50 g, 2.51 mmol) and 1,1′-carbonyldiimidazole (0.434 g, 2.67
mmol) in methylene chloride (25 mL) was stirred at room
temperature for 16 h, and the solvents were then removed in
vacuo. A mixture of this crude product (0.245 g, 0.678 mmol)
and 2-(3-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo[b]-
[1,4]diazepin-1-yl)-N-isopropyl-N-phenylacetamide, 6 (0.150 g,
0.339 mmol), in DMF (15 mL) was heated at 90 °C for 6h. The
reaction mixture was poured into water (30 mL), and the
resultant mixture was extracted into ethyl acetate (3 × 30 mL).
The combined organics were washed with water (2 × 20 mL)
and brine (20 mL), dried (MgSO4), and concentrated in vacuo
to afford the crude intermediate. Purification by flash column
chromatography (CH2Cl2:MeOH, 50:1) gave the pure interme-
diate N-isopropyl-2-[3-[3-[3-(benzyloxy)phenyl]ureido]-2,4-di-
oxo-5-phenyl-2,3,4,5-tetrahydrobenzo[b][1,4]diazepin-1-yl]-N-
phenylacetamide (101 mg) as a white powder: 1H NMR (300
MHz, CDCl3) δ 1.07 (2d, J ) 7.4 Hz, 6H), 4.15 (d, J ) 14.4 Hz,
1H), 4.45 (d, J ) 14.4 Hz, 1H), 5.00 (m, 3H), 5.40 (d, J ) 7.4
Hz, 1H), 6.40 (br, 1H), 6.61 (dd, J ) 7.4, 1.1 Hz, 1H), 6.8-7.4
(m, 24H). Compounds 30 and 37 were also made in this
manner.
A mixture of N-isopropyl-2-[3-[3-[3-(benzyloxy)phenyl]ure-
ido]-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo[b][1,4]diazepin-
1-yl]-N-phenylacetamide (0.100 g, 0.150 mmol) and 10%
palladium on carbon (0.010 g) in ethanol (10 mL) was stirred
under an atmosphere of hydrogen at 50 psi for 2 h. The solids
were removed by filtration, and the filtrate wasconcentrated
in vacuo to afford the crude product. This was purified by
preparative RP-HPLC (42-68% acetonitrile, 30 min) at a rate
of 100 mL/min. Fractions containing the desired material were
combined, frozen, and lyophilized to provide the titled com-
pound as a white powder (26.2 mg): 1H NMR (300 MHz,
CDCl3) δ 1.06 (s × d, J ) 7.3 Hz, 6H), 4.18 (d, J ) 15.3 Hz,
1H), 4.52 (d, J ) 15.3 Hz, 1H), 4.97 (sept, J ) 7.3 Hz, 1H),
5.42 (d, J ) 6.8 Hz, 1H), 6.42 (d, J ) 6.7 Hz, 1H), 6.62 (d, J )
6.7 Hz, 1H), 6.85-7.0 (m, 2H), 7.14-7.55 (m, 17H); HRMS
calculated for C33H31N5O5 (M + H)+ ) 578.2397, observed (M
+ H)+ ) 578.2383; RP-HPLC (42-68% CH3CN, 30 min; 1 mL/
min) tR) 11.6 min.
3-[3-[1-[(Isopr op ylp h en ylca r ba m oyl)m eth yl]-2,4-dioxo-
5-p h en yl-2,3,4,5-tetr a h yd r o-1H-ben zo[b][1,4]d ia zep in -3-
yl]u r eid o} ben zoic Acid (29). A solution of 3-(ethoxycar-
bonyl)phenyl isocyanate (124 mg) in dichloromethane (3 mL)
was added to a solution of 2-(3-amino-2,4-dioxo-5-phenyl-
2,3,4,5-tetrahydrobenzo[b][1,4]diazepin-1-yl)-N-isopropyl-N-
phenylacetamide, 6 (288 mg), in dichloromethane (3 mL). The
reaction mixture was allowed to stir at room temperature for
30 min. The dichloromethane was evaporated in vacuo, and
the residue was suspended in acetonitrile and heated at reflux
for 1 h with stirring. The product precipitated upon cooling
to 0 °C and was washed with cold acetonitrile to give 3-[3-[1-
[(Isopropylphenylcarbamoyl)methyl]-2,4-dioxo-5-phenyl-2,3,4,5-
tetrahydro-1H-benzo[b][1,4]diazepin-3-yl]ureido]benzoic acid
ethyl ester as a white solid (312 mg): 1H NMR (300 MHz,
DMSO-d6): d 0.96 (m, 6H), 1.27 (t, J ) 7.2 Hz, 3H), 4.18 (d, J
) 15.8 Hz, 1H), 4.3 (dd, J ) 6.8 Hz, 2H), 4.48 (d, J ) 16 Hz,
1H), 4.8 (m, 1H), 5.05 (d, J ) 9 Hz, 1H), 7.6-6.9 (m, 18H),
8.05 (s, 1H), 9.4 (s, 1H); MS (FAB) m/ z 634 (M + H)+.
A solution of 3-[3-[1-[(Isopropylphenylcarbamoyl)methyl]-
2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-
3-yl]ureido] benzoic acid ethyl ester (312 mg, 0.493 mol) in
methanol (23 mL) and tetrahydrofuran (10 mL) was heated
at reflux. Aqueous 5% potassium carbonate (6.5 mL) was
added, and the reflux was maintained for 2.5 h. The reaction
mixture was concentrated in vacuo, and the residue was
2-[3-[3-[3-(Hyd r oxym eth yl)p h en yl]u r eid o]-2,4-d ioxo-5-
p h en yl-2,3,4,5-t et r a h yd r o-1H -b en zo[b][1,4]d ia zep in -1-
yl]-N-isop r op yl-N-p h en yla ceta m id e (31). To a stirring
solution of m-nitrobenzyl alcohol (2.0 g, 13.5 mmol) and
triethylamine (2.0 mL, 14.3 mmol) in chloroform (20 mL) was
added acetyl chloride (2 mL, 28.1 mmol) in chloroform (20 mL)
at 0 °C. Stirring was continued at room temperature for 1 h,
and the reaction mixture was washed successively with 1 N
HCl, water, and saturated NaHCO3 and dried, andthe solvent
was evaporated in vacuo. The residue was dissolved in acetic
acid (100 mL), and Zn powder (6 g, 91.7 mmol) was added.
The resulting mixture was stirred for 3 h, the solids were
removed by filtration, and the filtrate was concentrated in
vacuo. The residue was dissolved in ethyl acetate and washed
with saturated NaHCO3, and the solvent was evaporated in
vacuo. The residue was dissolved in chloroform (30 mL) at 0
°C, and pyridine (1 mL, 12.3 mmol) and p-nitrophenyl chlo-
roformate (1 g, 4.97 mmol) in chloroform (10 mL) were added
successively. The reaction mixture was stirred at room
temperature for 1 h, and the solvent was evaporated in vacuo.
The residue was dissolved in ethyl acetate and washed
successively with 1 N HCl and saturated NaHCO3. The
product was recrystallized from ethyl acetate:hexane mixture
providing 3-acetoxyaniline p-nitrophenyl carbamate (1.09 g):
1H NMR (400 MHz, CDCl3) δ 2.10 (s, 3H), 5.09 (s, 2H), 7.00-
7.47 (m, 7H), 8.27 (d, J ) 9 Hz, 2H).
A solution of intermediate 6 (663 mg, 1.5 mmol), 3-acetoxy-
aniline p-nitrophenyl carbamate (447 mg, 1.5 mmol), and
triethylamine (0.21 mL) in acetonitrile (10 mL) was stirred at
room temperature for 3 h. The product was removed by
filtration and washed with methanol, providing the product
(0.650 g) as a white amorphous solid. 1H NMR (400 MHz,
DMSO-d6) δ 0.94 (d, J ) 7 Hz, 3H), 0.96 (d, J ) 7 Hz, 3H),
2.01 (s, 3H), 4.17 (d, J ) 16 Hz, 1H), 4.46 (d, J ) 16 Hz, 1H),
4.78 (m, 1H), 4.96 (s, 3H), 5.01 (d, J ) 7.7 Hz, 1H), 6.84-6.96
(m, 3H), 7.16-7.58 (m, 16H), 9.19 (s, 1H); HRMS calculated
for C36H36N5O6 (M + H)+ ) 634.2665, observed (M + H)+
)
634.2650; RP-HPLC column, dynamax C-8, 2 mL/min (60%
acetonitrile) tR ) 9.46 min.
The above intermediate (300 mg, 0.47 mmol) was suspended
in methanol (50 mL) and 1 N aqueous NaOH (2 mL) added.
The reaction mixture was stirred overnight, and the solvent
was evaporated in vacuo. The residue was triturated with
water, and the product was collected by filtration, affording
the titled compound (250 mg). 1H NMR (400 MHz, DMSO-
d6) δ 0.94 (d, J ) 7 Hz, 3H), 0.96 (d, J ) 7 Hz, 3H), 4.17 (d, J
) 16 Hz, 1H), 4.37 (s, 3H), 4.46 (d, J ) 16 Hz, 1H), 4.78 (m ,
1H), 5.01 (m, 1H), 6.80-6.96 (m, 3H), 7.10-7.56 (m, 16H), 9.09
(s, 1H); HRMS calculated for C34H34N5O5 (M + H)+ ) 592.2559,
observed (M + H)+ ) 592.2556; RP-HPLC column, dynamax
C-8, 2 mL/min (60% acetonitrile) tR ) 4.89 min.
2-[2,4-Dioxo-5-p h en yl-3-[3-[3-(1H-tetr a zol-5-yl)p h en yl]-
u r eid o]-2,3,4,5-tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-
isop r op yl-N-p h en yla ceta m id e (32). To a vigorously stir-
ring solution of 2-(3-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetra-
hydrobenzo[b][1,4]diazepin-1-yl)-N-isopropyl-N-phenylaceta-
mide, 6 (0.070 g, 0.156 mmol), in tetrahydrofuran (3 mL) at
ambient temperature was added 1,1′-carbonyldiimidazole
(0.025 g, 0.169 mmol). The resulting mixture was stirred for
1.5 h at ambient temperature. 3-(2H-Tetrazol-5-yl)pheny-
lamine hydrochloride (31.3 mg, 0.157 mmol) was added, and
the reaction mixture was heated at reflux overnight. The
solids were removed by filtration and the filtrate concentrated
to a yellow oil. The oil was purified by preparative RP-HPLC
(43-53% acetonitrile, 30 min) at a rate of 100 mL/min.
Fractions containing the desired material were combined,

Orally Active 1,5-Benzodiazepines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5243
frozen, and lyophilized to provide the title compound as a white
powder (50 mg): 1H NMR (300 MHz, DMSO-d6) δ 0.96 (d, J )
7.3 Hz, 3H), 0.98 (d, J ) 7.3 Hz, 3H), 4.20 (d, J ) 16.8 Hz,
1H), 4.49 (d, J ) 17.1 Hz, 1H), 4.79 (m, 1H), 5.06 (d, J ) 7.3
Hz, 1H), 6.98 (m, 2H), 7.24-7.55 (m, 17H), 8.17 (s, 1H), 9.44
(s, 1H); HRMS calculated for C34H32N9O4 (M + H)+ ) 630.2582,
observed (M + H)+ ) 630.2577; RP-HPLC (43-53% CH3CN,
30 min; 1 mL/min) tR ) 15 min (t0) 3 min). Compounds 17
and 18 were prepared in this manner.
2-[3-[3-(3-Am in op h e n yl)u r e id o]-2,4-d ioxo-5-p h e n yl-
2,3,4,5-tetr ah ydr oben zo[b][1,4]diazepin -1-yl]-N-isopr opyl-
N-p h en yla ceta m id e (34). To a solution of 6 (0.300 g, 0.678
mmol) in dichloromethane (5 mL) under nitrogen was added
3-nitrophenyl isocyanate (0.111 g, 0.678 mmol) at ambient
temperature. After 20 min of stirring, the reaction mixture
was evaporated in vacuo to a tacky solid. The residue was
combined with 10 mL of acetonitrile and brought to reflux.
After 30 min of stirring, the slurry was cooled to 0-5 °C and
filtered, washing with cold acetonitrile. The product was dried
under high vacuum overnight under high vacuum to provide
N-isopropyl-2-[3-[3-(3-nitrophenyl)ureido]-2,4-dioxo-5-phenyl-
2,3,4,5-tetrahydrobenzo[b][1,4]diazepin-1-yl]-N-phenyl-aceta-
mide as a white crystalline solid (335 mg). 1H NMR (300 MHz,
DMSO-d6) δ 0.96 (m, 6H), 4.19 (d, 1H, J ) 16.8 Hz), 4.49 (d,
1H, J ) 16.8 Hz), 4.79 (m, 1H); 5.03 (m, 1H), 6.95 (d, 1H, J )
8.4 Hz), 7.03 (d, 1H, J ) 7.3 Hz), 7.26 (m, 1H), 7.36 (m, 4H),
7.50 (m, 10H), 7.76 (m, 1H), 8.46 (s, 1H), 9.69 (s, 1H); LRMS
(FAB) m/ z 607.1 (M + H)+ ; HPLC tR ) 23.24 min (48-60%
CH3CN, 30 min, 1.5 mL/min).
d ia zep in -1-yl]-N-isop r op yl-N-p h en yla ceta m id e (35). To
a stirring solution of intermediate 6 (221 mg, 0.50 mmol) in
DCM (5 mL) was added 3-(methylthio)phenyl isocyanate (83
mg, 0.50 mmol). The reaction mixture was stirred for 12 h,
concentrated to dryness, and triturated with methanol (3 × 5
mL) to yield the titled compound (220.6 mg): 1H NMR (300
MHz, DMSO-d6) δ 0.91 (t, J ) 7.4 Hz, 6H), 2.34 (s, 3H), 4.13
(d, J ) 16.6 Hz, 1H), 4.43 (d, J ) 16.6 Hz, 1H), 4.74 (m, 1H),
4.97 (d, J ) 7.8 Hz, 1H), 6.73 (m, 1H), 6.89 (m, 3H), 7.13 (t, J
) 8.0 Hz, 1H), 7.20 (m, 1H), 7.34 (m, 5H), 7.5 (m, 8H), 9.16 (s,
1H); MS C34H34N5O4S (M + H)+ ) 608.2331; observed (M +
H)+ ) 608.2325; tR ) 30.5 min (42-60% acetonitrile, 30 min),
then 60-100% acetonitrile, 10 min, t0) 3 min.
N-Isop r op yl-2-[3-[3-[3-(m eth ylsu lfon yl)p h en yl]u r eid o]-
2,4-d ioxo-5-p h en yl-2,3,4,5-t et r a h yd r o-1H -b en zo[b][1,4]-
d ia zep in -1-yl]-N-isop r op yl-N-p h en yla ceta m id e (36). To
a stirring solution of 35 (86 mg, 0.14 mmol) in DCM (5 mL)
was added 50% m-CPBA (100 mg, 0.28 mmol). The reaction
mixture stirred for 12 h at room temperature, then concen-
trated to dryness, and purified by RP-HPLC (42-60% aceto-
nitrile, 30 min) to give the title product (63.4 mg) as a white
lyophile: 1H NMR (DMSO-d6, 300 MHz) δ 0.91 (t, J ) 7.4 Hz,
6H), 2.34 (s, 3H), 4.17 (d, J ) 16.6 Hz, 1H), 4.47 (d, J ) 16.6
Hz, 1H), 4.78 (m, 1H), 5.02 (d, J ) 7.8 Hz 1H), 6.96 (m, 2H),
7.24 (m, 1H), 7.34 (m, 14H), 7.89 (m, 1H), 8.06 (s, 1H), 9.57 (s,
1H); HRMS calculated for C34
H33N5O6S (M + H)+ ) 640.2230,
observed (M + H)+ ) 640.2246; tR ) 31.75 min (42-60%
acetonitrile, 30 min then 60-100% acetonitrile, 10 min, t0
3 min).
)
A suspension of N-isopropyl-2-[3-[3-(3-nitrophenyl)ureido]-
2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo[b][1,4]diazepin-1-
yl]-N-phenylacetamide (0.280 g, 0.462 mmol) in absolute
ethanol (25 mL) was combined with 10% palladium on carbon
(100 mg) and hydrogenated on a Parr shaker at 50 psi for 88
h. The reaction mixture was filtered and then evaporated in
vacuo to a residue. The crude product was purified on flash
silica gel using 40-100% ethyl acetate in hexane in 100 mL
increments of 20%. Fractions containing the desired product
were combined and evaporated in vacuo to a solid (172 mg). A
portion of the solid (122 mg) was purified on flash silica gel
using 0-2% methanol in dichloromethane in 200 mL incre-
ments of 0.5%. Fractions containing the product were com-
bined and evaporated in vacuo to a solid. The product was
dried under high vacuum to provide the title compound (107
mg) as a white solid: 1H NMR (300 MHz, DMSO-d6) δ 0.96
(m, 6H), 4.17 (d, 1H, J ) 16.7 Hz), 4.47 (d, 1H, J ) 16.7 Hz),
4.78 (m, 1H), 4.93 (bs, 2H), 5.00 (d, 1H, J ) 7.9 Hz), 6.10 (d,
1H, J ) 7.9 Hz), 6.48 (d, 1H, J ) 7.8 Hz), 6.58 (bs, 1H), 6.80
(m, 2H), 6.93 (d, 1H, J ) 7.9), 7.24 (m, 1H), 7.35 (m, 4H), 7.48
(m, 8H), 8.81 (s, 1H); LRMS (FAB) m/ z (M + H)+ ) 576.9;
HPLC tR ) 20.3 min (30-48% CH3CN in water with 0.1% TFA,
30 min, 1.5 mL/min).
2-[2,4-Dioxo-5-p h en yl-3-[3-[3-[[(t r iflu or om et h yl)su l-
fon yl]a m in o)]p h en yl]u r eid o]-2,3,4,5-tetr a h yd r oben zo[b]-
[1,4]d ia zep in -1-yl]-N-isop r op yl-N-p h en yla ceta m id e (33).
A solution of 34 (0.190 g, 0.329 mmol) and triethylamine (50.6
mL, 0.362 mmol) in dichloromethane (5 mL) was cooled under
nitrogen to -78 °C with a dry ice/acetone bath. Triflic
anhydride (55.5 mL, 0.329 mmol) was added via micropipet,
and the reaction mixture was allowed to warm to ambient
temperature. Triethylamine was added (101.2 mL, 0.724
mmol), and the reaction mixture was cooled again to -78 °C.
Triflic anhydride (111 mL, 0.658 mmol) was added, and the
reaction was warmed again to ambient temperature forming
2-[3-[3-[1-[(Isop r op ylp h en ylca r ba m oyl)m eth yl]-2,4-d i-
oxo-5-ph en yl-2,3,4,5-tetr ah ydr o-1H-ben zo[b][1,4]diazepin -
3-yl]u r eido]ph en oxy]acetam ide (40). Intermediate 12 (0.100
g, 0.602 mmol) was dissolved in THF (20 mL), triethylamine
(0.182 mL, 1.19 mmol) was added, the resultant solution was
cooled to 0 °C, and then triphosgene (0.0594 g, 0.2 mmol) was
added. This mixture was stirred at 0 °C for 0.5 h prior to the
addition of intermediate 6 (0.265 g, 0.602 mmol), and the
resultant mixture was allowed to attain room temperature over
16 h. The solvents were concentrated in vacuo and the residue
partitioned between ethyl acetate (50 mL) and 0.5 N hydro-
chloric acid (3 × 20 mL). The organics were washed with
water (2 × 20 mL) and brine (20 mL), dried (MgSO4), and
concentrated in vacuo to afford the crude product (0.309 g).
This was purified by preparative RP-HPLC (40-63% aceto-
nitrile, 30 min) at a rate of 100 mL/min. Fractions containing
the desired material were combined, frozen, and lyophilized
to provide the title compound as a white powder (177 mg): 1H
NMR (300 MHz, DMSO-d6) δ 0.96 (2d, J ) 7.3 Hz, 6H), 4.18
(d, J ) 15.3 Hz, 1H), 4.30 (s, 2H), 4.48 (d, J ) 15.3 Hz, 1H),
4.80 (sept, J ) 7.3 Hz, 1H), 5.02 (d, J ) 6.8 Hz, 1H), 6.46 (d,
J ) 6.7 Hz, 1H), 6.85-7.0 (m, 4H), 7.14-7.55 (m, 17H), 9.20
(s, 1H); HPLC tR ) 17.9 min (30-70% CH3CN, 30 min, 1.0
mL/min); HRMS calculated for C35H34N6O6 (M + H)+
)
635.2761, observed (M + H)+
) 635.2741. Compounds 41-
44 and intermediate 16 were made in this manner.
1H-In d ole-2-ca r boxylic Acid [1-[(Isop r op ylp h en ylca r -
b a m oyl)m et h yl]-2,4-d ioxo-5-p h en yl-2,3,4,5-t et r a h yd r o-
1H-ben zo[b][1,4]d ia zep in -3-yl]a m id e 52. To a vigorously
stirred solution of 2-(3-amino-2,4-dioxo-5-phenyl-2,3,4,5-tet-
rahydrobenzo[b][1,4]diazepin-1-yl)-N-isopropyl-N-phenylaceta-
mide, 6 (0.116 g, 0.262 mmol), in N,N-dimethylformamide (5
mL) at ambient temperature was added indole-2-carboxylic
acid (0.0423 g, 0.262 mmol), N-hydroxybenzotriazole (0.0354
g, 0.262 mmol), and 1-[3-(dimethylamino)propyl]-3-ethylcar-
bodiimide hydrochloride (0.0503 g, 0.262 mmol) successively.
Triethylamine (8 drops) was added dropwise to maintain the
basicity (pH ) 9) of the solution. The resultant mixture was
stirred at ambient temperature for 5 h. The solvent was
removed in vacuo and the crude product purified by prepara-
tive RP-HPLC (40-60% acetonitrile, 30 min) at a rate of 100
mL/min. Fractions containing the desired material were
combined, frozen, and lyophilized to afford the titled product
(0.141 g) as a white lyophile. 1H NMR (300 MHz, CDCl3) δ
1.06 (d, J ) 7.3 Hz, 3H), 1.09 (d, J ) 7.3 Hz, 3H), 4.22 (d, J )
16.6 Hz, 1H), 4.40 (d, J ) 16.4 Hz, 1H), 5.02 (m, 1H), 5.50 (d,
J ) 7.1 Hz, 1H), 7.02 (d, J ) 8.1 Hz, 1H), 7.10-7.47 (m, 16H),
a
slurry. The slurry was filtered, washed with dichlo-
romethane, and dried under high vacuum to provide the title
compound as a white solid (0.069 g): 1H NMR (300 MHz,
DMSO-d6) δ 0.96 (m, 6H), 4.18 (d, 1H, J ) 16.6 Hz), 4.48 (d,
1H, J ) 16.6 Hz), 4.78 (m, 1H), 5.02 (d, 1H, J ) 7.8 Hz), 6.78
(d, 1H, J ) 7.7 Hz), 6.89 (d, 1H, J ) 7.2 Hz), 6.95 (d, 1H, J )
8.0), 7.13 (d, 1H, J ) 8.1 Hz), 7.24 (m, 2H), 7.35 (m, 4H), 7.48
(m, 10H), 9.34 (s, 1H); LRMS (FAB) m/ z 709.2 (M + H)+;
HPLC tR ) 27.1 min (42-60% CH3CN, 30 min, 1.5 mL/min).
Anal. (C34H31N6O6S1F3‚0.3H2O) C, H, N.
N-Isop r op yl-2-[3-[3-[3-(m eth ylth io)p h en yl]u r eid o]-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]-

5244 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Hirst et al.
7.57 (d, J ) 6.8 Hz, 1H), 7.67 (d, J ) 7.8 Hz, 1H), 9.29 (br s,
1H); HRMS calculated for C35H31N5O4 (M + H)+ ) 586.2464,
observed (M + H)+ ) 586.2461, TLC (EtOAc:hexane (2:3)) Rf
) 0.16; RP-HPLC tR ) 19.5 min (51-60% CH3CN 30 min; 1
mL/min). Compounds 45-51 were prepared in this manner.
Ack n ow led gm en t. The authors gratefully acknowl-
edge the analytical support of Larry Shampine and
Roderick Davis.
Refer en ces
[2-[[[1[[-(Isopr opylph en ylcar bam oyl)m eth yl]-2,4-dioxo-
5-p h en yl-2,3,4,5-tetr a h yd r o-1H-ben zo[b][1,4]d ia zep in -3-
yl]ca r ba m oyl]in d ol-1-yl]a cetic Acid (54). To a vigorously
stirred solution of 1H-indole-2-carboxylic acid [1-[(isopropyl-
phenylcarbamoyl)methyl]-2,4-dioxo-5-phenyl-2,3,4,5-tetrahy-
dro-1H-benzo[b][1,4]diazepin-3-yl]amide, 52 (0.101 g, 0.172
mmol), in N,N-dimethylformamide (3 mL) cooled to 0 °C was
added sodium hydride (60% suspension in mineral oil) (0.0083
g, 0.172 mmol). After 20 min, tert-butyl bromoacetate (0.0336
g, 0.172 mmol) was added. The resultant mixture was stirred
at 0 °C for 90 min followed by slow warming to ambient
temperature and stirring overnight. The solvent was concen-
trated in vacuo to give a brown oil which was dissolved in
dichloromethane (30 mL) and washed successively with satu-
rated aqueous sodium bicarbonate (20 mL) and brine (20 mL).
The resultant solution was dried over sodium sulfate, filtered,
and concentrated in vacuo to give a yellow oil (0.142 g) which
was purified by flash chromatography on silica gel (9 g) with
an eluant of a mixture of ethyl acetate and hexane (1:2, 200
mL) to give [2-[[1-[(isopropylphenylcarbamoyl)methyl]-2,4-
dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3-
yl]carbamoyl]indol-1-yl]acetic acid tert-butyl ester (0.092 g) as
a white foam: 1H NMR (300 MHz, CDCl3) δ 1.07 (d, J ) 4.9
Hz, 3H), 1.09 (d, J ) 4.6 Hz, 3H), 1.37 (s, 9H), 4.17 (d, J )
16.6 Hz, 1H), 4.44 (d, J ) 16.9 Hz, 1H), 5.01 (m, 1H), 5.18 (d,
J ) 17.1 Hz, 1H), 5.24 (d, J ) 18 Hz, 1H), 5.47 (d, J ) 7.6 Hz,
1H), 7.01 (dd, J ) 1.2, 8.3 Hz, 1H), 7.13-7.51 (m, 17H), 7.57
(d, J ) 7.3 Hz, 1H), 7.67 (d, J ) 7.8 Hz, 1H); LRMS (FAB)
m/ z 700.2 (M + H)+; RP-HPLC (60-70% CH3CN; 30 min; 1
mL/min) tR ) 17.5 min.
(1) Crawley, J . N.; Corwin, R. L. Biological Actions of Cholecysto-
kinin. Peptides 1994, 15, 731-755.
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geneity of Cholocystokinin in the Central and Peripheral Ner-
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Lee, C. M. Gastrin and Cholecystokinin in Central and Per-
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To a solution of [2-[[1-[(isopropylphenylcarbamoyl)methyl]-
2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-
3-yl]carbamoyl]indol-1-yl]acetic acid tert-butyl ester (0.072 g,
0.103 mmol) in dichloromethane (4 mL) at ambient temper-
ature was added trifluoroacetic acid (1.5 mL) gradually with
stirring. After 30 min, the solvents were concentrated in vacuo
to afford a clear glass. The glass was purified by preparative
RP-HPLC (45-55% acetonitrile, 30 min) at a rate of 100 mL/
min. Fractions containing the desired material were com-
bined, frozen, and lyophilized to afford the title compound
(0.050 g) as a white powder: 1H NMR (300 MHz, CDCl3) δ
1.07 (d, J ) 4.4 Hz, 3H), 1.10 (d, J ) 4.4 Hz, 3H), 4.23 (d, J )
16.6 Hz, 1H), 4.40 (d, J ) 16.6 Hz, 1H), 5.01 (m, 1H), 5.06 (s,
2H), 5.44 (d, J ) 7.1 Hz, 1H), 7.03 (dd, J ) 8.1, 1.2 Hz, 1H),
7.17-7.52 (m, 17H), 7.67 (d, J ) 8.1 Hz, 1H), 7.74 (d, J ) 7.1
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ceptor Agonist Activity I. Optimization of the Agonist “Trigger”.
J . Med. Chem. 1996, 39, 562-569.
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Freeman, S. B.; Amith, A. J .; Patel, S.; Anderson, P. S.;
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Hz, 1H); HRMS calculated for C37H33N5O6 (M + H)+
)
644.2653, observed (M + H)+ ) 644.2646; RP-HPLC (45-55%
CH3CN; 30 min; 1 mL/min) tR) 22 min. Compound 55 was
prepared in a similar manner.
In tr a cellu la r Ca lciu m Mea su r em en ts. CHO-K1 cells
stably transfected with hCCK-A or hCCK-B receptors were
grown on glass coverslips to 75-90% confluency. The cells
were loaded for 50 min in serum-free culture medium contain-
ing 5 mM FURA2-AM and 2.5 mM probenecid. A J ASCO
CAF-102 calcium analyzer was used to measure changes in
intracellular calcium concentration by standard ratiometric
techniques using excitation wavelengths of 340 and 380 nm.
Cells were perfused with increasing concentrations of CCK-8
(n ) 2)22 or compound 29 (n ) 2) until a plateau in the 340/
380 ratio was achieved. A washout/recovery period of at least
10 min was allowed between successive stimulations. The
maximal response was normalized to the maximal response
induced by CCK-8. EC50’s were calculated at the concentration
required to induce half-maximal response. In addition to the
agonist concentration-response curves, the CHO-K1 cells
expressing the human CCK-B receptor were perfused for 1 h
with three concentrations of compound 29 (10-8, 10-7, 10-6 M,
n ) 2), concentration-reponse curves were acquired for CCK-8
(10-12 to 10-6M), and the pA2 for 29 was calculated by a Schild
analysis.28
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J M9601664