Controlled modification of acidity in cholecystokinin B receptor antagonists: N-(1,4-benzodiazepin-3-yl)-N′-[3-(tetrazol-5-ylamino)phenyl]ureas

Article abstract of DOI:10.1021/jm9506736

The design, synthesis, and biological activity of a novel series of CCK-B receptor antagonists (1) which incorporate a tetrazol-5-ylamino functionality attached to the phenyl ring of the arylurea moiety of L-365,260 are described. In these compounds, the

Full text of DOI:10.1021/jm9506736

842
J . Med. Chem. 1996, 39, 842-849
Con tr olled Mod ifica tion of Acid ity in Ch olecystok in in B Recep tor An ta gon ists:
N-(1,4-Ben zod ia zep in -3-yl)-N ′-[3-(tetr a zol-5-yla m in o)p h en yl]u r ea s
J ose´ L. Castro,*,† Richard G. Ball,§ Howard B. Broughton,† Michael G. N. Russell,† Denise Rathbone,†
Alan P. Watt,† Raymond Baker,† Kerry L. Chapman,‡ Alan E. Fletcher,‡ Smita Patel,‡ Alison J . Smith,‡
George R. Marshall,| Wayne Ryecroft,| and Victor G. Matassa†
Chemistry, Biochemistry, and Pharmacology Departments, Merck Sharp & Dohme Research Laboratories, Neuroscience
Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K., and Biophysical Chemistry Department,
Merck Research Laboratories, Rahway, New J ersey 07065
Received September 12, 1995X
The design, synthesis, and biological activity of a novel series of CCK-B receptor antagonists
(1) which incorporate a tetrazol-5-ylamino functionality attached to the phenyl ring of the
arylurea moiety of L-365,260 are described. In these compounds, the acidity of the tetrazole
was gradually modified by utilization of simple conformational constraints, and X-ray
crystallographic data were obtained to support the conformational dependence of the pKa of
the aminotetrazoles. Compounds to emerge from the present work such as 1f and 2c,d are
among the highest affinity and, in the case of 1f, most selective (CCK-A/CCK-B, 37 000)
antagonists so far reported for this receptor. The C5-cyclohexyl compound 2c (L-736,380) dose-
dependently inhibited gastric acid secretion in anesthetized rats (ID50, 0.064 mg/kg) and ex
vivo binding of [125I]CCK-8S in BKTO mice brain membranes (ED50, 1.7 mg/kg) and is one of
the most potent acidic CCK-B receptor antagonists yet described.
Ch a r t 1
In tr od u ction
The polypeptide hormone cholecystokinin (CCK) is a
member of a family of peptides which was first isolated
from the gastrointestinal tract and subsequently identi-
fied in the central nervous system (CNS). Several
biologically active forms of CCK exist, with CCK-58,
CCK-33, CCK-8, and CCK-4 predominating in the
periphery and CCK-8 being the prevailing form in the
CNS.1 Two CCK receptor subtypes, CCK-A and CCK-
B, which mediate the diverse biological functions of CCK
have been identified so far. The human CCK-A2 and
CCK-B3-5 receptors have been cloned and shown to
belong to the G-protein-coupled receptor (GPCR) super-
family. CCK-A (alimentary) receptors are primarily
located in the gut where they mediate pancreatic
enzyme secretion, gallbladder contraction, gastric emp-
tying, and intestinal motility.6 Peripheral CCK-A re-
ceptors have also been implicated in satiety,7 gastrointes-
tinal cancer,6 and neuroprotection.8 Additionally, CCK-A
receptors have been identified in discrete areas of the
CNS9 and might play a significant role in neuropsychi-
atric disorders.10 CCK-B (brain) receptors are more
widely distributed in the CNS, and they are thought to
be involved in the modulation of anxiety,11 panic dis-
order,12 depression,13 nociception,14 and satiety.10,15
CCK-B receptors have also been found in the periphery
and might be identical with the gastrin receptor.5
This wealth of potential therapeutic utilities for CCK
receptor agonists and antagonists has provided the
impetus for intensive research in the area, and over the
past decade, a variety of selective nonpeptidic CCK-A
and CCK-B receptor antagonists have been disclosed by
several laboratories.16,17 From a pioneering program of
work at Merck,18 sparked by the isolation of the natural
product asperlicin, series of benzodiazepine-based CCK-A
and CCK-B receptor antagonists were developed, and
first-generation compounds such as MK-329 (CCK-A)
and L-365,260 (CCK-B) have been evaluated in the
clinic. The latter compound, however, had to be spe-
cially formulated in order to achieve adequate levels of
oral bioavailability, mainly as a consequence of the low
aqueous solubility of its crystalline form (<0.002 mg/
mL, pH 7.4).19 Comprehensive structure-affinity re-
lationship (SAR) studies on L-365,26020 revealed regions
of the molecule which permitted the modulation of its
physicochemical properties and often resulted in im-
proved CCK-B receptor affinity and selectivity. Most
notably, the evolution of second-generation benzodiaze-
pine-based CCK-B receptor antagonists which incorpo-
rate basic21 or acidic22 solubilizing functionalities has
been recently disclosed. Prominent compounds to emerge
from the latter series include the tetrazole L-368,730
and the 1,2,4-oxadiazolone L-369,466 (Chart 1) which,
despite excellent potency, selectivity, and solubility, not
† Chemistry Department.
‡ Biochemistry Department.
§ Biophysical Chemistry Department.
| Pharmacology Department.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
0022-2623/96/1839-0842$12.00/0 © 1996 American Chemical Society

Modification of Acidity in CCK-B Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 843
Ta ble 1. CCK Receptor Binding Affinities and Physicochemical Properties of N-(5-Phenyl-1,4-benzodiazepin-3-yl)-N′-
[3-(tetrazol-5-ylamino)phenyl]ureas
IC50 (nM)a
solubility
(mg/mL, pH 7.4)
d
compd
1a
1b
1c
1d
1e
1f
n
R
R1
CCK-B
CCK-Ab
log Dc
pKa
1
0
0
0
0
0
H
H
H
Me
Me
H
H
Me
H
Me
7.5
5.7
0.58
1.1
20
0.11
3000 (16)
3000 (32)
3000 (20)
3000 (27)
3000 (4)
4080
1.47
0.89
1.14
1.37
1.58
1.38
5.9
5.1
e
5.4
5.7
4.8
0.02
0.10
0.16
0.88
0.15
0.87
-CH2-CH2-
L-365,260
8.5
736
<0.002
a
Receptor binding is expressed as IC50, the concentration of compound required for half-maximal inhibition of the binding of [125I]BH
CCK-8S to receptors in pancreatic tissue (CCK-A) or guinea pig cortical membranes (CCK-B). The results represent the geometric mean
b
of two to three separate experiments. Full IC50 not obtained, percentage of inhibition at a concentration of 3000 nM given in parentheses.
d
c Log P measured at pH 7.4. Determined by nonlinear regression analysis of pH-dependent partition measurements.32 e This pKa could
not be reliably determined due to poor solubility of the compound at low pHs.
Sch em e 1a
Ta ble 2. CCK Receptor Binding Affinities and Solubilities of
N-(5-Cyclohexyl-1,4-benzodiazepin-3-yl)-N′-[3-(tetrazol-5-
ylamino)phenyl]ureas
IC50 (nM)a
solubility
compd
n
R
R1
CCK-B
CCK-A (µg/mL, pH 7.4)
2a
2b
2c
2d
2e
1
0
0
0
0
H
H
H
Me
Me
H
H
Me
H
Me
0.28
0.20
0.054
0.074
0.30
153
200
400
802
1600
3.7
1.8
60
57
0.49
a
See Table 1, footnote a. The results represent the geometric
mean of two to five separate experiments.
a
Reagents: (a) triphosgene, Et3N, THF.
unexpectedly showed a much reduced ability to cross
the blood-brain barrier (BBB) when compared to the
uncharged lead L-365,260. It proved, therefore, of
interest to investigate the possibility of influencing the
brain penetrability of related compounds by modulating
the pKa of a constant acid moiety. Here we report on
the design, synthesis, and biological activity of a series
of CCK-B receptor antagonists (1) that incorporate a
5-aminotetrazole unit, the pKa of which could be gradu-
ally modified by rationally controlling the torsion angles
around bonds a and b (see structure 1) using simple
conformational constraints. X-ray crystallographic evi-
dence was obtained to support the conformational
dependence of the pKa of the aminotetrazoles.
one (3),23 or its C5-cyclohexyl analogue 4,24 and the
corresponding 3-(tetrazol-5-ylamino)anilines utilizing a
triphosgene-promoted coupling (Scheme 1). 3-[(Tetra-
zol-5-ylamino)methyl]aniline (6) was prepared by reduc-
tive alkylation of 5-aminotetrazole with 3-nitrobenz-
aldehyde followed by catalytic hydrogenation (Scheme
2). Synthesis of the remaining anilines was achieved
as shown in Scheme 3. Thus, reaction of 3-nitroaniline
or 2-methyl-5-nitroaniline with cyanogen bromide using
a modified literature procedure25 afforded cyanamides
7 and 8, respectively, which were converted to the
corresponding tetrazoles 9 and 10 by reaction with
sodium azide.26 Alternatively, cyanamides 7 and 8 were
N-methylated to give 13 and 14 and subsequently
Syn th etic Ch em istr y
transformed into tetrazoles 15 and 16, as above.
similar sequence was followed to prepare 6-nitro-1-
tetrazol-5-ylindoline (19). The anilines were obtained
A
The compounds shown in Tables 1 and 2 were
synthesized from the previously described (3R)-amino-
1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-

844 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Castro et al.
Sch em e 2a
Ta ble 3. Solid State Conformations and pKas of Substituted
5-Aminotetrazoles
angle (deg)b
a
Reagents: (a) EtOH, AcOH, reflux; (b) NaBH4, EtOH; (c) H2,
Pd-C, MeOH-H2O.
PhPlane - HetPlane -
a
compd
n
R
R1
AmPlane
AmPlane
pKa
Sch em e 3a
5
9
10
15
16
19
1
0
0
0
0
0
H
H
H
H
H
Me
Me
5.53
4.23
4.46
4.46
5.02
4.02
Me
H
Me
40
34
70
13
9
13
-CH2CH2-
a
b
Determined by potentiometric titration.28 PhPlane: plane
containing the carbon atoms of the phenyl ring. AmPlane: plane
containing the linking nitrogen atom and its three substituents.
HetPlane: plane containing the non-hydrogen atoms of the tetra-
zole.
Ta ble 4. X-ray Crystallographic Data for
5-[(3-Nitrophenyl)amino]tetrazoles 10, 15, and 16
compound
10
15
16
formula
fw
a (Å)
b (Å)
c (Å)
Z
C8H8N6O2
220.19
6.082(3)
4.932(2)
15.864(2)
2
C9H10N6O2
234.21
11.634(1)
9.738(2)
9.749(2)
4
C8H8N6O2
220.19
18.333(1)
30.222(2)
7.066(1)
16
space group
radiation
P21
P21
Fdd2
Cu KR
Cu KR
Cu KR
temperature (K)
reflections measured
reflections used
R
294
294
294
1142a
853
2347a
2347
939a
876
0.038b
0.02
0.047c
0.18
0.043b
0.20
residual peak (e Å-3
)
a
b
Difractometer used: AFC5R. Solved with SHELXS and re-
fined with SDP-PLUS on F. c Solved with SHELXS and refined
with SHELXL on F2.
a
Reagents: (a) BrCN, NaOH, AcOH-H2O; (b) BrCN, NaOH,
AcOH-EtOH-H2O; (c) NaN3, NH4Cl, DMF, 165 °C; (d) H2, Pd-
C, MeOH-H2O; (e) NaH, MeI, THF-DMF.
methylene group of 5 to give 9 leads to a substantial
decrease in pKa (4.23), presumably as a direct conse-
quence of electron delocalization from the amino group
to both the tetrazole nucleus and the phenyl ring. This
would effectively reduce the amount of electron density
that the amino group is able to donate to the tetrazole
moiety resulting in a reduction in pKa compared to 5-[(3-
nitrobenzyl)amino]tetrazole (5). It would, therefore, be
reasonable to anticipate that by utilizing conformational
constraints to adjust the degree of the delocalization of
the amino group electron lone pair to these two rings,
it should be possible to further modulate the pKa. In
this context, introduction of a methyl group ortho to the
aminotetrazole as in 10 should reduce the delocalization
into the phenyl ring and increase its pKa compared to
9. Incorporation of a second methyl group to give 16
should continue this deconjugation process and raise the
pKa even further. This is, indeed, the case, and the
measured pKas for 10 and 16 were 4.46 and 5.02,
respectively. Structural evidence in support of the
above notion was obtained from single-crystal X-ray
analysis of the nitro compounds 10 and 16 (Tables 3
and 4). In both of these compounds the exocyclic amino
group has a planar configuration and is in full conjuga-
tion with the tetrazole ring (N lone pair almost orthogo-
from the appropriate nitro compounds by catalytic
hydrogenation.
Resu lts a n d Discu ssion
Mod u la tion of p Ka . The data presented in Tables
1 and 3 clearly show that the pKa of this novel series of
CCK-B receptor antagonists can be controlled through
the use of minor structural modifications around the
aminotetrazole group. This acid moiety was chosen
because its pKa (6.0)27 is substantially higher than that
for tetrazole itself (4.9),27 and it was anticipated that
the acidity of the former could be gradually increased
by modifying the degree of charge delocalization from
the amino group into the tetrazole ring. Here we
describe in detail the implementation of this idea
utilizing the model compounds 5, 9, 10, 15, 16, and 19,
which are themselves intermediates in the synthesis of
the final products 1a -f and 2a -e. In this regard,
attachment of 5-aminotetrazole through its exocyclic
nitrogen atom to a 3-nitrophenyl ring using a methylene
spacer results in a compound (5) which has a measured
pKa of 5.53 (Table 3).28 Removal of the insulating

Modification of Acidity in CCK-B Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 845
F igu r e 1. Computer-generated stereoview of the superimposed crystal conformations for 5-[(3-nitrophenyl)amino]tetrazoles 10
(light gray), 15 (medium gray), and 16 (black).
nal to the plane of the tetrazole) (Figure 1 and Table
3). Both compounds differ, however, in the degree of
conjugation with the phenyl ring, which can be numeri-
cally expressed by considering the angles between the
plane containing the carbon atoms of the phenyl ring
(PhPlane) and the plane containing the linking amino
group and its three substituents (AmPlane). As can be
seen in Table 3 and Figure 1, this angle is much larger
for 16 (70°) than for 10 (40°), and it is reflected in the
higher pKa of the former. Gratifyingly, the planarity
of the nitrogen atom and the conformations seen in the
solid state for 10 and 16 could be reproduced in ab initio
calculations at the 6-31G* level29-31 and would, there-
fore, not appear to be a consequence of crystal packing.
Translocation of the methyl group from the ortho
position of 10 to the adjacent exocyclic nitrogen atom,
to give 15, had no effect on the pKa, and not surprisingly,
the conformation of this compound in the crystal was
very similar to that of 10 (Figure 1 and Table 3).
Having been able to modulate the pKa of 9 from 4.23 to
5.02, it was now of interest to increase the acidity of 9.
In order to achieve such a decrease in pKa, the degree
of delocalization of the nitrogen lone pair should be
maximized toward the phenyl ring and minimized
toward the tetrazole. Both of these requirements could
be realized by constraining the nitrogen atom in a rigid
5-membered ring to afford a 1-(tetrazol-5-yl)indoline
(19), which had a measured pKa of 4.02. The pKa
modulation discussed above was corroborated in the
final compounds 1a ,b,d -f (Table 1) which, indeed,
followed a very similar acidity pattern to the model
system.32
Str u ctu r e-Affin ity Rela tion sh ip s. The affinities
of the compounds 1a -f and 2a -e for CCK receptors
(Tables 1 and 2) were assessed by radioligand binding
techniques as previously reported.21 The 3R-enanti-
omers were synthesized and evaluated because this
absolute stereochemistry usually confers CCK-B over
CCK-A receptor selectivity.20-22,24 It can be seen from
the data in Table 1 that an aminotetrazole functionality
is well tolerated at the CCK-B receptor, either directly
attached to the phenyl ring (1b) or linked through a
methylene spacer (1a ). The CCK-B receptor affinity of
1b (IC50, 5.7 nM) was increased 1 order of magnitude
by introduction of an ortho methyl group to give 1c (IC50,
0.58 nM). This agrees with previous SAR studies on
L-365,260 which showed that methylation of the phenyl
ring at position 4′ or 3′ results in some 5-fold increase
in receptor affinity compared to the unsubstituted
phenyl.20a A similar (5-fold) improvement in affinity
was observed by N-methylation of the exocyclic nitrogen
atom (1d ; IC50, 1.1 nM). These two contributions to
lipophilic binding are not additive in improving the
CCK-B receptor affinity, and in fact, the combination
of ortho and N-methylation (1e; IC50, 20 nM) gives rise
to a 34-fold decrease in affinity compared to the most
active compound 1c. The lipophilic advantage of both
substitutions could be exploited, however, in the con-
strained indoline analogue 1f, the most active compound
in this series (IC50, 0.11 nM). This remarkable 180-fold
difference in affinity between 1e and 1f might suggest
that the preferred bioactive conformation of these
molecules has a more coplanar arrangement of the
aminotetrazole moiety with respect to the phenyl ring
(1f),33 rather than the highly twisted disposition seen
in the crystal structure of 16 (model system analogue
of 1e).34
Compounds 1c,d ,f (Table 1) showed a much improved
in vitro profile (CCK-B affinity, CCK-B/CCK-A receptor
selectivity) and aqueous solubility compared to that of
L-365,260. In agreement with previously described
findings,24 the more lipophilic cyclohexyl derivatives
2a -e (Table 2) all showed improved affinity for CCK-B
receptors compared to the C5-phenyl analogues, but
their aqueous solubility at physiological pH was sub-
stantially reduced. The 10-50-fold increase in affinity
for 2a -e compared to 1a -e has been speculated to arise
from improved interactions between the cyclohexyl ring
and Val349 in the human and rat CCK-B receptors. The
indolinyltetrazole 1f (L-738,425) and the C5-cyclohexyl
compounds 2c (L-736,380) and 2d (L-737,481) are
extremely high affinity ligands for the CCK-B receptor,
and 1f has exceptional selectivity (CCK-A/CCK-B, 37 000)
over the CCK-A receptor. Compounds such as 1c and
2c were also shown to be devoid of affinity for the
GABAA benzodiazepine receptor ([3H]Ro15-1788: IC50,
>10 000; rat cortical membranes). Not surprisingly,5
however, 1b,c and 2c were unable to differentiate
between CCK-B and gastrin receptors, and indeed, their
affinities for these two receptors were almost identical
([125I]gastrin: IC50s, 3.9, 0.47, and 0.13 nM, respectively;
guinea pig gastric glands).35
F u n ction a l Activity a n d Br a in P en etr a tion . The
similar affinities of the compounds for CCK-B and
gastrin receptors were utilized to assess their functional
antagonist activity in vivo. Thus, L-736,380 (2c) showed
a dose-related inhibition of pentagastrin-induced gastric
acid secretion in anesthetized rats,35 with an ID50 of
0.064 mg/kg, following dosing by the intraperitoneal
route. L-365,260 is also active in this model, but it is

846 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Castro et al.
tion by ex vivo binding has also been described.36 Radioligand
binding to guinea pig cortical membranes was performed using
50 pM 125I-labeled Bolton Hunter CCK-8S in 20 mM HEPES
buffer, pH 6.5, containing 150 mM NaCl, 5 mM MgCl2, 1 mM
EGTA, and 0.025% bacitracin. For rat pancreatic membranes,
assay buffer was supplemented with 0.01% trypsin inhibitor
and 0.2% BSA. Guinea pig cortical membranes were prepared
by homogenization in 0.32 M sucrose, centrifugation, and
resuspension of the P2 pellet in assay buffer at 1 g of wet
weight in 120 mL. Rat pancreatic membranes were prepared
in 10 mM HEPES/0.01% trypsin inhibitor, pH 7.4, and
centrifuged, and the pellet was resuspended in assay buffer
at a 1:2000 dilution. Specific binding in all cases was defined
using 1 µM CCK-8S, and the reaction was terminated by
filtration through Whatman GF/C filters, using a Brandel cell
harvester with 3 × 3 mL washes in ice-cold 100 mM saline
wash buffer. Filters were counted on a LKB γ counter.
some 15-fold weaker (ID50, 1.0 mg/kg). Because
L-365,260 is known to be a CCK-B receptor antagonist,
and CCK-B and gastrin receptors appear to be identical,
it is reasonable to conclude that the compounds de-
scribed above are also CCK-B antagonists. Further
evidence to support this conclusion was gained from an
in vitro electrophysiological model of CCK-B receptor
activation carried out in rat brain slices.35,21 Thus,
compound 1c blocked the pentagastrin-induced excita-
tion of single neurons in an in vitro slice preparation of
the rat ventromedial hypothalamic nucleus (VMH slice)
with a Kb of 0.52 ( 0.06 nM (n ) 5), indicating that it
is a potent and selective CCK-B antagonist.
The prevalent CNS distribution of CCK-B receptors
makes almost mandatory that clinically useful antago-
nists for this receptor should have the ability to cross
the BBB. In this regard, the CNS penetration of several
of these novel aminotetrazolyl-based CCK-B receptor
antagonists, after systemic (iv) administration, was
assessed using an ex vivo binding assay in BKTO mice.36
In this model 1c,f, 2c, and the racemate of 2a (IC50, 0.82
nM) dose-dependently inhibited ex vivo binding of
p Ka Deter m in a tion s. Potentiometric pKa determinations
for aminotetrazoles 5, 9, 10, 15, 16, and 19 were performed
using a Sirius PCA-101 titrator (Sirius Analytical Instruments
Ltd., East Sussex, England) equipped with
a Ross type
combination glass electrode calibrated for mixed solvent ti-
trations. The mixed solvent approach was employed because
of the limited aqueous solubility of the compounds across the
pH range. A cosolvent of 1,4-dioxane/water (60:40, v/v), ionic
strength adjusted with 0.15 M KCl, was used. Three separate
titrations were performed for each compound with different
water/cosolvent ratios to obtain pKas in the presence of
cosolvent (psKa values). Aqueous pKa values were calculated
by extrapolation to 0% cosolvent using the Yasuda-Shedlovsky
relationship:28 a linear plot of psKa + log [H2O] versus 1/ꢀ,
where ꢀ is the dielectric constant of the water cosolvent
mixture. pKas for final compounds 1a ,b,d -f were determined
by nonlinear regression analysis of pH-dependent partition
measurements.32
[
125I]BH-CCK-8S to mouse brain membranes with ED50s
of 14, 10, 1.7, and 16 mg/kg, respectively.37 The extent
of inhibition of ex vivo binding for 1d was only 19% at
10 mg/kg iv. For reference, L-365,260 has an ED50 of
13 mg/kg in this assay. Because the CCK-B affinity of
L-365,260 is 15-, 77-, and 157-fold lower than that of
1c,f and 2c, respectively, the above data show that these
acidic compounds are significantly less brain penetrant
than the former. Additionally, the increase in lipophi-
licity on going from the C5-phenyl compound 1c (log D,
1.14) to its cyclohexyl analogue 2c (log D, 1.85) did not
affect brain penetration, and the 8-fold difference in
ED50s (14 vs 1.7) simply reflects the 10-fold improved
affinity of 2c. To date, 2c is one of the most potent
acidic CCK-B receptor antagonists reported. The present
data suggest that pKa in this series of compounds does
not influence their brain uptake.38
Ch em ica l Meth od s. Gen er a l Dir ection s. Unless other-
wise stated, all 1H NMR spectra were recorded at 360 MHz
on a Bruker AM 360 spectrometer or at 250 MHz on a Bruker
AC250 instrument. Mass spectra were obtained with a VG70-
250 spectrometer. Melting points are uncorrected. Anhydrous
THF, DMF, Et2O, MeOH, and toluene were purchased from
Aldrich Chemical Co., Sureseal. Et3N was distilled from CaH2.
All solutions were dried over Na2SO4 or MgSO4 and concen-
trated on a Bu¨chi rotary evaporator. Flash chromatography
was performed on silica gel (Fluka Art. No. 60738). Log Ds
were determined using 1-octanol and pH 7.4 buffer by the
shake flask method.
Con clu sion s
A novel series of CCK-B receptor antagonists (1)
which incorporate an aminotetrazole unit attached to
the phenylurea moiety of L-365,260 was designed and
synthesized. In these compounds, the acidity of the
tetrazole functionality was gradually modified by uti-
lization of simple constraints, and X-ray crystallographic
data were obtained to support the conformational
dependence of the aminotetrazole pKas. The structural
changes introduced for the modulation of the pKa affect
CCK-B receptor affinity and selectivity and appear to
suggest that the preferred bioactive conformation of
these molecules has a rather coplanar arrangement of
the aminotetrazole and the phenyl ring of the arylurea
moiety. Compounds such as 1c,f and 2c,d showed a
much improved in vitro profile compared to that of
L-365,260 and are among the most potent and, in the
case of 1f, most selective (CCK-A/CCK-B, 37 000) CCK-B
receptor antagonists so far reported.
Gen er a l P r oced u r e for th e P r ep a r a tion of (1,4-Ben -
zod ia zep in -3-yl)u r ea s 1a -f a n d 2a -e: (+)-N-[(3R)-5-Cy-
cloh e xyl-2,3-d ih yd r o-1-m e t h yl-2-oxo-1H -1,4-b e n zod i-
a ze p in -3-yl]-N ′-[3-(N -m e t h yl-N -t e t r a zol-5-yla m in o)-
p h en yl]u r ea (2c). To a cooled (0 °C) and stirred milky
solution of 3-(N-methyl-N-tetrazol-5-ylamino)aniline (17) (230
mg, 1.21 mmol) in anhydrous THF (30 mL) was added solid
triphosgene (120 mg, 0.40 mmol) in one portion, under a
nitrogen atmosphere. After 5 min of stirring, anhydrous Et3N
(505 µL, 3.63 mmol) was added dropwise, and the mixture was
allowed to warm to 17 °C over 15 min. The reaction mixture
was recooled to 0 °C, and a solution of 424 (225 mg, 0.83 mmol)
in anhydrous THF (5 mL) was added dropwise via cannula
over 4 min. After the mixture was stirred at 0 °C for 15 min
and at room temperature for 2 h, a white precipitate was
removed by filtration and the solvent was evaporated under
reduced pressure. The remaining residue was dissolved in
EtOAc (200 mL), washed with 10% aqueous citric acid (2 ×
40 mL) and brine (40 mL), dried, and concentrated. Flash
chromatography of the crude material (CH2Cl2-MeOH, 90:
10) afforded 336 mg (83%) of 2c as a white solid: mp 222-
1
225 °C (MeOH); [R]23 15.9° (c 0.71, DMF); H NMR (DMSO-
D
Exp er im en ta l Section
d6) δ 15.2 (1H, br s), 9.11 (1H, s), 7.74 (1H, d, J ) 7.7 Hz),
7.63 (1H, t, J ) 8.4 Hz), 7.54 (1H, d, J ) 8.2 Hz), 7.48 (1H, br
s), 7.37 (1H, t, J ) 7.2 Hz), 7.29 (1H, d, J ) 8.4 Hz), 7.25 (1H,
t, J ) 8.0 Hz), 7.12 (1H, br d, J ) 8.9 Hz), 6.94 (1H, br d, J )
8.0 Hz), 5.06 (1H, d, J ) 8.4 Hz), 3.39 (3H, s), 3.34 (3H, s),
2.93 (1H, m), 1.90 (1H, m), 1.78 (1H, m), 1.68-1.08 (7H, m),
Biologica l Meth od s. Detailed procedures for the assess-
ment of antagonist properties of CCK-B ligands using in vitro
pentagastrin-induced excitation of VMH neurons and by in
vivo pentagastrin-induced gastric acid secretion in rats have
been previously reported.21,35 Measurement of CNS penetra-

Modification of Acidity in CCK-B Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 847
Ta ble 5. Physical Data for
N-(1,4-Benzodiazepin-3-yl)-N′-[3-(tetrazol-5-ylamino)phenyl]-
ureas
was added to give 880 mg (81%) of 13 as pale yellow needles:
1H NMR (DMSO-d6) δ 7.98 (1H, m), 7.85 (1H, t, J ) 2.2 Hz),
7.73 (1H, t, J ) 7.7 Hz), 7.60 (1H, m), 3.45 (3H, s); MS (CI)
m/ z 177 (M-).
mp, °C
[R]D, deg
compd
empirical formula
(solvent)a
(c, g/100 mL)b
Gen er a l P r oced u r e for th e P r ep a r a tion of Tetr a zoles
9, 10, 15, a n d 16: 3-(N-Meth yl-N-tetr a zol-5-yla m in o)-
n itr oben zen e (15). To a stirred suspension of NaN3 (352 mg,
5.4 mmol) and NH4Cl (2.37 g, 44.3 mmol) in anhydrous DMF
(2 mL) was added dropwise, via cannula, a solution of 13 (800
mg, 4.5 mmol) in anhydrous DMF (4 mL). The resulting yellow
mixture was heated at 165 °C for 3 h 45 min, under nitrogen.
After cooling, water (60 mL) and 2 N NaOH (8 mL) were
added, and the clear solution was extracted once with Et2O
(30 mL). The aqueous phase was acidified with 5 N HCl, and
the pale yellow precipitate was collected, washed with water
(2 × 15 mL), and recrystallized from H2O-EtOH (3:1, 30 mL)
to give 863 mg (86.9%) of 15 as pale yellow needles: mp 196-
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
C25H23N9O2‚H2O
190-195 (A)
233-243 (A)
205-210 (A)
198-201 (A)
217-225 (B)
215-220 (C)
175-178 (D)
190-193 (A)
222-225 (A)
190-193 (B)
223-225 (B)
+75.4 (0.50)
+69.6 (0.50)
+65.2 (0.50)
+63.4 (0.50)
+68.6 (0.50)
+64.8 (0.50)
+17.4 (0.50)
+8.4 (0.50)
+15.9 (0.71)
+12.2 (0.29)
+20.3 (0.34)
C
C
C
C
C
C
C
C
C
C
24H21N9O2‚0.1H2O
25H23N9O2‚0.5H2O
25H23N9O2‚0.5H2O
26H25N9O2‚0.2H2O
26H23N9O2‚1.6H2O
25H29N9O2‚0.05H2O
24H27N9O2‚0.6H2O
25H29N9O2
25H29N9O2
26H31N9O2
a
A, MeOH; B, MeOH-CH2Cl2; C, MeOH-H2O; D, MeOH-
1
198 °C; H NMR (DMSO-d6) δ 8.43 (1H, t, J ) 2.3 Hz), 7.94
b
EtOAc. DMF solutions.
(2H, m), 7.68 (1H, t, J ) 8.3 Hz), 3.56 (3H, s); MS (CI) m/ z
220 (M-).
0.89 (1H, m); MS (CI) m/ z 488 (M- + 1). Anal. (C25H29
N9O2‚0.25H2O) C, H, N.
-
1
9: mp 228-230 °C (EtOH-H2O, 2:1); H NMR (DMSO-d6)
δ 15.8 (1H, br s), 10.43 (1H, s), 8.57 (1H, t, J ) 2.1 Hz), 7.90
(1H, m), 7.80 (1H, m), 7.61 (1H, t, J ) 8.2 Hz); MS (CI) m/ z
206 (M-).
The enantiomeric purity of 2c was shown to be >99% ee by
HPLC analysis using a PIRKLE (S)-DNBL column (250 mm
× 4.6 mm i.d., 5 µm particle size) and eluting with CH2Cl2-
MeOH-AcOH (94:2.5:0.8) (flow, 1 mL/min; retention time, 5.66
min for the R-enantiomer and 10.21 min for the S-enantiomer).
3-[(Tetr azol-5-ylam in o)m eth yl]n itr oben zen e (5). A mix-
ture of 3-nitrobenzaldehyde (1.51 g, 10 mmol) and 5-amino-
tetrazole monohydrate (1.03 g, 10 mmol) in absolute EtOH (30
mL) and glacial AcOH (0.57 mL, 10 mmol) was stirred at room
temperature for 40 min and then refluxed for 5.5 h under
nitrogen. Solvents were removed under high vacuum, the
remaining solid was suspended in absolute EtOH (50 mL), and
NaBH4 (1.2 g) was added at room temperature over 20 min.
After a further 15 h of stirring, the solvent was evaporated,
and the remaining residue was dissolved in H2O (100 mL) and
extracted with Et2O (2 × 30 mL). The basic aqueous phase
was acidified to pH 2 with 2 N HCl, and the precipitated solid
was collected by filtration, washed with water and Et2O, and
finally recrystallized from absolute EtOH to give 420 mg (19%)
1
10: mp 245-249 °C (MeOH-H2O); H NMR (DMSO-d6) δ
9.34 (1H, s), 8.80 (1H, d, J ) 2.4 Hz), 7.82 (1H, dd, J ) 8.3,
2.4 Hz), 7.49 (1H, d, J ) 8.3 Hz), 2.41 (3H, s); MS (CI) m/ z
220 (M-).
16: mp 201-205 °C (H2O); 1H NMR (DMSO-d6) δ 8.24 (1H,
d, J ) 2.4 Hz), 8.15 (1H, dd, J ) 8.4, 2.4 Hz), 7.65 (1H, d, J )
8.4 Hz), 3.40 (3H, s), 2.24 (3H, s); MS (CI) m/ z 234 (M-).
6-Nitr o-1-(tetr a zol-5-yl)in d olin e (19). To a cooled (4 °C)
and stirred solution of 6-nitroindoline (3.04 g, 18.5 mmol) in a
mixture of glacial AcOH (22 mL), H2O (11 mL), and absolute
EtOH (23 mL) was added solid cyanogen bromide (2.93 g, 27.7
mmol) followed by 1 N NaOH (25 mL), over 3 min. The
mixture was allowed to warm to room temperature and stirred
for 21 h, and the solid was collected by filtration, washed with
water, and dried over P2O5 to give 2.94 g (84%) of 1-cyano-6-
nitroindoline as a yellow solid: 1H NMR (DMSO-d6) δ 7.90
(1H, dd, J ) 8.1, 2.2 Hz), 7.55-7.50 (2H, m), 4.23 (2H, t, J )
8.4 Hz), 3.30 (2H, t, J ) 8.4 Hz); MS (CI) m/ z 189 (M-).
Reaction of 1-cyano-6-nitroindoline with sodium azide, using
a similar method to that described for 15, afforded 19 in 33%
isolated yield: mp 262-270 °C (MeOH-H2O); 1H NMR
(DMSO-d6) δ 8.60 (1H, d, J ) 2.2 Hz), 7.83 (1H, dd, J ) 8.1,
2.2 Hz), 7.50 (1H, d, J ) 8.1 Hz), 4.21 (2H, t, J ) 8.5 Hz), 3.40
(2H, t, J ) 8.5 Hz); MS (CI) m/ z 232 (M-).
Gen er a l P r oced u r e for th e P r ep a r a tion of An ilin es 6,
11, 12, 17, 18, a n d 20: 3-(Tetr a zol-5-yla m in o)a n ilin e (11).
A solution of 9 (410 mg) in a mixture of MeOH (50 mL) and
H2O (5 mL) was hydrogenated at 35 psi over 10% Pd-C (170
mg) for 4 min. The catalyst was filtered off and washed with
MeOH (2 × 10 mL), and solvents were removed under vacuum.
The residue was azeotroped with MeOH (20 mL) and further
dried under high vacuum to give 324 mg (93%) of 11 as a
brown solid: 1H NMR (DMSO-d6) δ 9.44 (1H, s), 6.92 (1H, t, J
) 8.0 Hz), 6.76 (1H, s), 6.57 (1H, d, J ) 8.0 Hz), 6.17 (1H, d,
J ) 8.0 Hz).
1
of 5 as white needles: mp 208-10 °C; H NMR (DMSO-d6) δ
8.21 (1H, br s), 8.12 (1H, br d, J ) 9 Hz), 7.80 (1H, d, J ) 8.0
Hz), 7.70 (1H, br t, J ) 6.3 Hz), 7.64 (1H, t, J ) 8.0 Hz), 4.53
(2H, d, J ) 6.3 Hz); MS (CI) m/ z 220 (M-).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Cya n -
a m id es 7 a n d 8: (2-Meth yl-5-n itr op h en yl)cya n a m id e (8).
To a cooled (4 °C) and stirred suspension of 2-methyl-5-
nitroaniline (15.2 g, 100 mmol) in a mixture of AcOH (145 mL)
and water (47 mL) was added cyanogen bromide (15.9 g, 150
mmol) followed by 1 N NaOH (110 mL), over 10 min. After 2
h, the mixture was diluted with EtOH (160 mL) and stirred
for a further 1.5 h before 2 N NaOH (10 mL) was added, and
stirring was continued at 15 °C for 16 h. Solvents were
removed under vacuum (bath temperature, <45 °C), 2 N
NaOH (500 mL) was added, and the resulting mixture was
filtered to remove a fine precipitate. The clear filtrate was
acidified to pH 1 with 5 N HCl, and the solid was collected,
washed with water, and dried over P2O5 to give 15.5 g (88%)
of 8: 1H NMR (DMSO-d6) δ 7.84 (1H, dd, J ) 8.3, 2.4 Hz),
7.77 (1H, d, J ) 2.4 Hz), 7.49 (1H, d, J ) 8.3 Hz), 2.30 (3H, s);
MS (CI) m/z 177 (M-).
Ack n ow led gm en t. The authors wish to thank Dr.
Gen er a l P r oced u r e for t h e Met h yla t ion of Cya n -
a m id es 7 a n d 8: N-Met h yl-(3-n it r op h en yl)cya n a m id e
(13). To a cooled (-20 °C) and stirred solution of (3-nitro-
phenyl)cyanamide (7) (1 g, 6.1 mmol) in a mixture of anhy-
drous THF and anhydrous DMF (3:1, 20 mL) was added NaH
(60% dispersion in oil, 294 mg) in one portion, under nitrogen.
After 8 min of stirring at -20 °C, iodomethane (1.14 mL, 18.4
mmol) was added, the red mixture was allowed to warm to
room temperature, and it was diluted with anhydrous DMF
(5 mL). After a further 45 min, H2O (75 mL; CAUTION!
hydrogen evolution) was added and products were extracted
with EtOAc (2 × 100 mL). The combined organic solutions
were washed with brine (50 mL), dried, and concentrated. The
residue was dissolved in EtOAc (30 mL), and hexane (100 mL)
Richard Herbert and Mr. Steven Thomas for the acqui-
1
sition of H NMR and MS spectra.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR and mi-
croanalytical data for novel compounds and X-ray crystal-
lographic data for 10, 15, and 16 (16 pages). Ordering
information is given on any current masthead page.
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1951, 73, 1792-1795.
(28) (a) Yasuda, M. Dissociation Constants of Some Carboxylic Acids
in Mixed Aqueous Solvents. Bull. Chem. Soc. J pn. 1959, 32,
429-432. (b) Shedlovsky, T.; Kay, R. L. The Ionisation Constant
of Acetic Acid in Water-Methanol Mixtures at 25 °C from
Conductance Measurements. J . Am. Chem. Soc. 1956, 60, 151-
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(15) Dethloff, L. A.; de la Iglesia, F. A. Cholecystokinin Antagonists:
A Toxicological Perspective. Drug Metab. Res. 1992, 24, 267-
293.
(16) For reviews, see, for example: (a) Makovec, F. CCK-B/Gastrin-
Receptor Antagonists. Drugs Future 1993, 18, 919-931. (b)
Trivedi, B. K. Cholecystokinin Receptor Antagonists: Current
Status. Curr. Med. Chem. 1994, 1, 313-327. (c) Trivedi, B. K.
Ligands for Cholecystokinin Receptors: Recent Developments.
Curr. Opin. Ther. Pat. 1994, 4, 31-44. (d) Lowe, J . A., III.
Cholecystokinin-B Receptor Antagonists. Exp. Opin. Ther. Pat.
1995, 5, 231-237.

Modification of Acidity in CCK-B Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 849
(29) Semiempirical methods30 failed to reproduce even qualitatively
the conformations seen in the crystal for 10 and 16, while ab
initio calculations using Gaussian31 only gave satisfactory results
when optimization at the Hartree-Fock level on the neutral
forms was carried out using the 6-31G* basis set.
(30) MOPAC 6 (QCPE Program 455) was used with the AM1, PM3,
or MNDO Hamiltonian using either Eigenvector following or
BFGS optimization.
(31) Frisch, M. J .; Head-Gordon, M.; Trucks, G. W.; Foresman, J . B.;
Schlegel, H. B.; Raghavachari, K.; Robb, M.; Binkley, J . S.;
Gonzalez, C.; Defrees, D. J .; Fox, D. J .; Whiteside, R. A.; Seeger,
R.; Melius, C. F.; Baker, J .; Martin, R. L.; Kahn, L. R.; Stewart,
J . J . P.; Topiol, S.; Pople, J . A. Gaussian 90, Revision J .
Gaussian, Inc.: Pittsburgh, PA, 1990.
(32) Schaper, K.-J . Simultaneous Determination of Electronic and
Lipophilic Properties [pKa, P(Ion), P(Neutral)] of Acids and Bases
by Nonlinear Regression Analysis of pH-Dependent Partition
Measurements. J . Chem. Res. (Miniprint) 1979, 4480-4493.
(33) Although we have been unable to obtain a crystal structure for
indoline 19, ab initio calculations (6-31G* basis set) carried out
on this compound predicted a minimun energy conformation
which has a fairly coplanar arrangement of the indoline and the
tetrazole rings: PhPlane - AmPLane angle, 10°; HetPlane -
AmPlane angle, 17°.
most obviously conformation, are clearly involved which make
definite conclusions hazardous.
(35) Patel, S.; Smith, A. J .; Chapman, K. L.; Fletcher, A. E.; Kemp,
J . A.; Marshall, G. R.; Hargreaves, R. J .; Ryecroft, W.; Iversen,
L. L.; Iversen, S. D.; Baker, R.; Showell, G. A.; Bourrain, S.;
Neduvelil, J . G.; Matassa, V. G.; Freedman, S. B. Biological
Properties of the Benzodiazepine Amidine Derivative L-740,093,
a Cholecystokinin-B/Gastrin Receptor Antagonist with High
Affinity In Vitro and High Potency In Vivo. Mol. Pharmacol.
1994, 46, 943-948.
(36) Patel, S.; Chapman, K. L.; Heald, A.; Smith, A. J .; Freedman,
S. B. Measurement of Central Nervous System Activity of
Systemically Administered CCK-B Receptor Antagonists by Ex
Vivo Binding. Eur. J . Pharmacol. 1994, 253, 237-244.
(37) In this ex vivo binding assay, other acidic benzodiazepine-based
CCK-B receptor antagonists such as oxadiazolone L-369,466,
tetrazole L-368,730,22a and the recently disclosed acylsulfon-
amide L-736,30922b had ED50 values of 6.5, 18, and 4.8 mg/kg,
respectively. The ED50/IC50 ratios for these compounds (25, 18,
and 17, respectively) were comparable to that observed for 2c
(30).38
(38) Comparisons of the above brain penetration data have to be
taking cautiously, however, because in this model the extent of
binding to central CCK-B receptors is measured after 30 min of
dosing, and different pharmacokinetic behavior of the compounds
might influence the end result.
(34) In Table 1, there is some suggestion of a relationship between
pKa and receptor affinity (compare 1c-f). However, other factors,
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