Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity. 1. Optimization of the agonist "trigger"

Article abstract of DOI:10.1021/jm950626d

Directed screening of compounds selected from the Glaxo registry file for contractile activity on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with peripheral cholecystokinin (CCK) receptor agonist activity. Agonis

Full text of DOI:10.1021/jm950626d

562
J . Med. Chem. 1996, 39, 562-569
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. 1. Op tim iza tion of th e Agon ist “Tr igger ”
Christopher J . Aquino,† Duncan R. Armour,‡ J udd M. Berman,† Larry S. Birkemo,§ Robin A. E. Carr,‡
Dallas K. Croom,§ Milana Dezube,† Robert W. Dougherty, J r., Gregory N. Ervin,§ Mary K. Grizzle,§
J ulie E. Head,‡ Gavin C. Hirst,† Michael K. J ames,§ Michael F. J ohnson,§ Laurence J . Miller,|
Kennedy L. Queen, Thomas J . Rimele, David N. Smith,‡ and Elizabeth E. Sugg*,†
Departments of Medicinal Chemistry, Cellular Biochemistry, and Pharmacology, Glaxo Wellcome, 5 Moore Drive,
Research Triangle Park, North Carolina 27709, Center for Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905,
and Department of Medicinal Chemistry, Glaxo Wellcome Medicines Research Center, Gunnels Wood Road, Stevenage,
Herts, SG1 2NY U.K.
Received August 23, 1995X
Directed screening of compounds selected from the Glaxo registry file for contractile activity
on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with
peripheral cholecystokinin (CCK) receptor agonist activity. Agonist efficacy within this series
was modulated by variation of substituents on the N1-anilinoacetamide moiety. Remarkably,
a single methyl group confers agonist activity, with an N-isopropyl substituent providing optimal
efficacy. Hydrophilic substituents on the anilino nitrogen abolish agonist activity or produce
antagonists of CCK. In contrast, hydrophilic electron-donating groups at the para-position of
the anilino ring enhance or maintain in vitro and in vivo agonist activity. Despite decreased
affinity for the human CCK-A receptor, relative to CCK-8, some of these compounds are
equipotent to CCK as anorectic agents in rats following intraperitoneal administration.
In tr od u ction
The 1,4-benzodiazepine CCK-A (devazepide, MK-329,
L364,718)11 and CCK-B (L-365,260)12 receptor selective
antagonists were optimized from a natural product lead
which was originally identified by random screening in
a receptor binding assay.13 This same approach has
provided numerous nonpeptidyl antagonist ligands in
the past 10 years.8-10 Although receptor binding assays
are used routinely to screen for novel chemical leads,
these assays only measure ligand affinity and secondary
functional assays are required to fully characterize the
pharmacological profile of any given lead. Since our
goal was the identification of a CCK-A receptor agonist,
we chose a functional assay as our primary screen.
Compounds were selected for screening based on struc-
tural features of known CCK-A agonist peptides,6
particularly the unique phenylurea moiety of the tet-
rapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePheNH2 (A-
71623),7 and evaluated at a single concentration (30 µM)
for contractile activity on the isolated guinea pig gall-
bladder (GPGB).14 Reversal of contraction with MK-
329 (1 µM) was used to verify a CCK-A receptor
mediated response. Compounds which did not produce
contraction were evaluated for antagonist activity against
a concentration-response curve for CCK-8 (10-11-10-6
M). Of the 237 compounds screened, the majority were
either inactive or antagonists of CCK-8. However, a
series of 1,5-benzodiazepines was identified, 11c, 11d ,
and 11k (Table 1), in which agonist efficacy was
modulated by appropriate substitution of the N1-anili-
noacetamide moiety.
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.1 CCK-8
potently activates both peripheral (CCK-A) and central
(CCK-B) receptor subtypes.1 The utility of a CCK
receptor agonist for the treatment of obesity is suggested
by studies demonstrating that exogenous CCK can
shorten meal duration and reduce meal size in several
species, including lean2 and obese3 humans. Chronic
administration of CCK-8 to patients on total parenteral
nutrition has also demonstrated a role for CCK in the
prevention of gallstones.4 The relevant target for both
effects is the CCK-A receptor.5
The high molecular weight, acid lability of the Tyr-
(SO3H) residue, metabolic instability, lack of oral activ-
ity and lack of receptor selectivity appear to limit the
therapeutic potential of CCK-8. Despite remarkable
success in the design of metabolically stable, receptor
selective peptide ligands, no orally active agonist has
been reported.6,7 Because nonpeptide ligands have
historically offered greater opportunity for synthetic
manipulation of both pharmacodynamic (selectivity and
efficacy) and pharmacokinetic (oral bioavailability, du-
ration) parameters,8-10 we elected to search the Glaxo
registry file for novel nonpeptidyl CCK-A agonist leads.
We now report our efforts to optimize the agonist
“trigger” on these novel 1,5-benzodiazepines. In addi-
tion to exploring the potential for hydrophobic bulk at
the anilino nitrogen, we have also attempted to improve
the aqueous solubility of these compounds through the
introduction of hydrophilic groups on the anilino nitro-
gen or at the para-position of the aromatic ring.
* To whom correspondence should be addressed.
† Department of Medicinal Chemistry, Glaxo Wellcome.
‡ Department of Medicinal Chemistry, Glaxo Wellcome Medicines
Research Center.
§ Department of Pharmacology, Glaxo Wellcome.
Department of Cellular Biochemistry, Glaxo Wellcome.
| Center for Digestive Diseases, Mayo Clinic.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0022-2623/96/1839-0562$12.00/0 © 1996 American Chemical Society

Benzodiazepines with CCK-A Receptor Agonist Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 563
Ta ble 1. Functional Activity of Benzodiazepine Ligands on the
Isolated Guinea Pig Gallbladder
lation of N-phenyl-1,2-phenylenediamine with anisoyl chloride
in dichloromethane, followed by reduction with LiAlH4, pro-
vided the p-methoxybenzyl-substituted diamine 6 in overall
81% yield. Condensation of diamine 6 with 4 provided
benzodiazepine 7 in 83% yield. Following reduction of the
phenylhydrazone with Zn/acetic acid and oxidative cleavage
of the p-methoxybenzyl moiety, amine 9 was reacted with
phenyl isocyanate to give the intermediate urea 10. Where
protecting groups were employed, these were removed as the
final step by standard methods as described in the Experi-
mental Section. Compounds 11a -v were purified to homo-
geneity (>97%) by RP-HPLC chromatography and character-
1
ized by analytical RP-HPLC, H-NMR spectroscopy, low- and
high-resolution mass spectrometry, and combustion analysis.
Analogs were evaluated for in vitro functional efficacy on
the isolated guinea pig gallbladder at 30 or 1 µM concentra-
tions (Tables 1-4).14 The long incubation time (30-60 min)
required for each compound to achieve maximal response
precluded accurate measurement of potency through standard
concentration-response curves. The contractile activity of all
compounds reported was reversed completely with the CCK-A
receptor selective antagonist MK-329.11 This data is il-
lustrated for 11k , 11n -q, and 11s in Table 3. Compounds
which did not produce contraction were evaluated for antago-
nist activity against a concentration-response curve for CCK-8
(10-11-10-6 M). Receptor binding affinities were measured
on membrane preparations from CHO-K1 cell lines stably
transfected with cDNA from human CCK-A or CCK-B recep-
tors (Table 2).14 In vivo anorexia activity was assessed
following intraperitoneal (IP) or oral (PO) administration to
Long-Evans rats conditioned to a liquid diet and fasted for 2
h (Table 2).
GPGBa (30 µM)
compd
R1
%CCK-8
pKB
11a
11b
11c
11d
11e
11f
11g
11h
11i
H
CH3
ia
ia
6.33
6.27
CH3CH2
66
50
27
32
ia
CH3CH2CH2
CH3CH2CH2CH2
NCCH2CH2
HOOCCH2
EtOOCCH2
H2NCH2CH2
CBZHNCH2CH2
(CH3)2CH
6.33
16
7
ia
5.88
5.34
11j
11k
11l
86
26
43
c(C6H11
C6H5
)
11m
a
Functional activity in the isolated guinea pig gallbladder
following incubation with test ligand (30 µM) for 30 min at 37 °C;
%CCK-8, percent contraction normalized to the percent contraction
induced by 1 µM CCK-8; pKB, single dose pA2 calculated from the
fold-shift of the CCK-8 concentration response curve in the
presence of the test ligand (30 µM); ia, inactive.
Resu lts
A directed functional screen of phenylurea-containing
compounds from the Glaxo registry files identified a
series of 1,5-benzodiazepines in which agonist efficacy
was mediated by substitution of the N1-anilinoaceta-
mide functionality (Table 1). Remarkably, while com-
pounds with a hydrogen (11a ) or methyl (11b) substit-
uent were weak antagonists of CCK-8 on the isolated
GPGB, the ethyl (11c), n-propyl (11d ), n-butyl (11e),
and cyanoethyl (11f) derivatives were agonists. Hydro-
philic substituents produced either weak antagonists or
inactive compounds (11g-j). Branching of the ethyl
moiety to the isopropyl substituent (11k ) further en-
hanced agonist efficacy. However, there was a limit to
the hydrophobic bulk tolerated, as seen with the n-butyl
(11e) or cyclohexyl (11l) derivatives. Finally, the diphe-
nyl derivative (11m ) retained reasonable agonist ef-
ficacy. The preferred substituent for maximal efficacy
on the GPGB was the N-isopropyl derivative 11k . This
analog had equivalent affinity for the human CCK-A
and CCK-B receptors (Table 2) and was anorectic in rats
(ED50 ) 0.95 µmol/kg) following ip administration (Table
2). Unfortunately, 11k was not active following oral
administration (up to 10 µmol/kg).
Sch em e 1a
a
(a) NaB(O2CCH3)3H, ClCH2CH2Cl; (b) BrCH2COBr, TEA,
DCM (c) NaH, DMF, 2.
Meth od s
The poor aqueous solubility of 11k and lack of
tolerance for substitution of hydrophilic moieties on the
anilino nitrogen prompted us to investigate substitution
on the aromatic moiety. Related compounds identified
in the original screen suggested that para-substitution
would be preferred for maintaining agonist efficacy
(data not shown). A range of substituents were pre-
pared (Table 2). Except for the p-carboxyl derivative
(11r ), which was a weak antagonist of CCK-8, all of
these compounds retained agonist efficacy. These para-
substituted compounds had 60-3000-fold reduced af-
finity for the human CCK-A receptor, compared to CCK-
8, with essentially equal affinity for both human CCK-A
Compounds of general structure 11 were prepared by
alkylation of intermediate phenylurea 10 with various bro-
moacetamides 2 (Scheme 1). Bromoacetamides 2 were pre-
pared in high yield from the reaction of the corresponding
secondary amines 1 with bromoacetyl bromide. Where suit-
able secondary amines could not be purchased from com-
mercial suppliers, they were synthesized from the primary
amine and aldehyde or ketone via reductive amination with
sodium triacetoxyborohydride15 in excellent yields and purity.
The convergent synthesis of intermediate phenylurea 10 is
illustrated in Scheme 2. Condensation of ketomalonic acid
with phenylhydrazine provided phenylhydrazone 3, which was
subsequently converted to the diacid chloride 4 by treatment
with phosphorus pentachloride in carbon tetrachloride. Acy-

564 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Aquino et al.
Sch em e 2a
a
(a) C6H5NHNH2, EtOH, H2O; (b) PCl5, CCl4; (c) p-CH3OC6H4COCl, TEA, DCM; (d) LiAlH4, THF; (e) Zn dust, AcOH; (f) (NH4)2Ce(NO3)6,
CH3CN, H2O; (g) C6H5NCO, DCM.
Ta ble 2. Biological Profile of Functionalized Isopropylaniline Benzodiazepines
%CCK-8a
pIC50
anorexiac
b
compd
R2
30 µM
1 µM
pKB
hCCK-A
hCCK-B
A/B
ED50
% max.
11k
11n
11o
11p
11q
11r
H
OH
OCH3
N(CH3)2‚TFA
morpholino
COOH
86
81
93
75
ND
10
ND
ND
ND
82
46
74
ND
53
100
7.3 ( 0.3 (4)
7.4 ( 0.1 (4)
7.1 ( 0.1 (3)
7.2 ( 0.2 (4)
5.7 ( 0.1 (6)
5.6 ( 0.01 (3)
7.0 ( 0.1 (4)
9.2 ( 0.1 (3)
7.6 ( 0.04 (3)
7.8 ( 0.4 (3)
7.3 ( 0.1 (3)
6.6 ( 0.1 (3)
6.8 ( 0.1 (3)
7.4 ( 0.1 (2)
7.5 ( 0.5 (3)
9.5 ( 0.4 (9)
0.4
0.5
0.7
3.4
0.1
0.02
0.3
0.8
0.95
0.05
0.07
0.09
0.20
ND
48
74
62
54
26
6.14
ND
56
11s
CCK-8
F
0.20
0.03
96d
a
Functional activity in the isolated guinea pig gallbladder following incubation with test ligand (30 µM) for 30 min and/or (1 µM) for
60 min at 37 °C; %CCK-8, percent contraction normalized to the percent contractrion induced by 1 µM CCK-8; pKB, single dose pA2
b
calculated from the solid-shift of the CCK-8 concentration-response curve in the presence of the test ligand (30 µM). 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 ( SD (number of determinations); B/A, CCK-A receptor selectivity, IC50 (B)/IC50 (A).
c Anorectic potency in rats conditioned to a palatable liquid diet and fasted for 2 h, ED50, µmol/kg; % max, maximal response at 1 µmol/kg
d
ip, t ) 30 min. % max., maximal response at 0.5 µmol/kg ip, t ) 30 min; ND, not determined.
and CCK-B receptors. The functional activity (agonist
or antagonist) associated with the CCK-B receptor
affinity is unknown and needs to be evaluated.
and more efficacious than 11k at the 1 µmol/kg dose.
Again, none of these compounds were orally active (up
to 10 µmol/kg) in the rat.
Despite decreased CCK-A receptor affinity, some
(11n -p ) of these compounds were potent anorectics in
the rat following ip administration, with potency and
efficacy comparable to CCK-8. The p-OH (11n ), p-OCH3
(11o), and p-N(CH3)2 (11p ) derivatives were more potent
Finally, an additional subset of compounds was
identified which demonstrate that the anilino moiety
is not required for agonist activity (Table 4). Substitu-
tion of the N1-acetamide moiety with simple aliphatic
substituents also provided agonist leads. To date, no

Benzodiazepines with CCK-A Receptor Agonist Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 565
Ta ble 3. GPGB Contraction in the Absence or Presence of
MK-329
Discu ssion
The mechanism for the remarkable conversion from
antagonist to agonist activity in this series of compounds
is unknown. Incorporation of small, bulky, lipophilic
groups on the anilino nitrogen can introduce steric,
electronic, and conformational effects. In addition to
simply binding a lipophilic pocket within the receptor,
these groups might also perterb the E to Z conforma-
tional distribution of the amide bond or affect the
interplanar angle between the phenyl ring and amide
group.16 Both the spacial geometry of the acetamide as
well as the ability of the phenyl ring to conjugate with
the amide could be influenced by substitution. Like-
wise, incorporation of para-substituents on the phenyl
ring might not merely optimize a direct spacial or
electronic interaction with the receptor, but could also
inductively alter the hydrogen-bonding ability of the
amide carbonyl or the partial double bond character of
the N-C bond.
GPGB tension, g
compd
concn, µm
compd
compd + MK329
% reversal
11k
11n
11o
11p
11q
11s
30
30
30
1
1
1
2.53
2.25
2.88
2.55
1.88
2.14
-0.43
0
-0.2
0.01
-0.43
-0.34
117
100
107
100
123
116
Ta ble 4. Agonist Activity of Atypical Benzodiazepine Ligands
on the Isolated Guinea Pig Gallbladder
While efforts continue to optimize these benzodiaz-
epines as potential drug candidates, an equally impor-
tant goal is to understand how they bind and activate
the CCK-A receptor. Historically, the design of non-
peptidyl agonist ligands for peptide receptors has fo-
cused on iterative structural modifications of the en-
dogenous peptide directed toward stabilizing the bioactive
conformation.17 This approach assumes that receptor
activation depends on a single unique interaction with
the agonist ligand. Recent efforts to identify the amino
acid residues required for receptor recognition of non-
peptide antagonists of the neurokinin,18,19 angiotensin
II (AII),18,19 opioid,20-22 and CCK23,24 receptor families,
however, suggest that the binding site(s) for nonpeptide
antagonists are distinct from that for peptide ligands.
Moreover, the few nonpeptide agonists reported to date
resemble nonpeptide antagonists more than the endog-
enous peptide they mimic, with minor structural modi-
fications providing the “trigger” for interconversion
between antagonist and agonist functional activity. This
GPGBa (30 µM)
compd
R1
R3
C6H5
CH2C6H5
CH(CH3)2
CH2CH3
%CCK-8
11k
11t
11u
11w
(CH3)2CH
(CH3)2CH
(CH3)2CH
CH3CH2
86
34
21
38
a
Functional activity in the isolated guinea pig gallbladder
following incubation with test ligand (30 µM) for 30 min at 37 °C;
%CCK-8, percent contraction normalized to the percent contraction
induced by 1 µM CCK-8.
attempt has been made to optimize the efficacy of this
series of compounds and the data are presented here
only to demonstrate the nonrigid requirements for
agonist activity in this series of compounds and to
underline the general necessity for bulk and lipophilicity
at this position.
Ta ble 5. Analytical Data for Target Analogs
HRMS molecular ionc
compd
yield, %
method
formulaa
C30H25N5O4
% purityb
theory
found
analysis
11a
11b
11c
11d
11e
11f
11g
11h
11i
95
59
56
47
34
47
45
59
35
72
45
68
69
59
47
57
64
54
87
39
38
50
A
A
A
A
A
A
B
A
C
A
A
A
A
D
A
A
A
E
95
>99
98
>99
>99
520.1985
534.2141
548.2298
562.2454
576.2511
520.1987
534.2143
548.2299
562.2451
576.2612
C
C
C
C
C
31H27N5O4
32H29N5O4‚0.4H2O
33H31N5O4
34H33N5O4‚0.6H2O
33H28N6O4‚0.6H2O
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Nd
C32H27N5O6
98
>99
98
>99
98
96
96
95
>99
95
96
96
97
97
>99
99
578.2040
606.2353
563.2407
697.2775
562.2454
602.2767
596.2298
578.2403
592.2560
605.2876
647.2982
606.2354
580.2360
576.2611
528.2611
500.2298
578.2045
606.2348
563.2407
697.2764
562.2456
602.2769
596.2296
578.2401
592.2563
605.2875
647.2982
606.2354
580.2366
576.2617
528.2607
500.2298
C
C
C
C
C
34H31N5O6‚0.5H2O
32H30N6O4‚TFA‚2H2O
40H36N6O6‚1.5H2O
33H31N5O4
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Ne
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
11j
11k
11l
36H35N5O4‚1.2H2O
11m
11n
11o
11p
11q
11r
11s
11t
C36H29N5O4‚0.5EtOH
C
C
C
C
C
C
C
33H31N5O5‚H2O
34H33N5O5
35H36N6O4‚0.5TFA‚0.5H2O
37H38N6O5‚H2O
34H31N5O6
33H30N5O4F
34H33N5O4‚1.6H2O
A
A
A
A
C, H, N
C, H, Nf
C, H, N
C, H, N
11u
11v
C30H33N5O4‚0.4TFA
C
28H29N5O4
a
b
Satisfactory 1H-NMR and low-resolution MS were obtained for all compounds. Purity as determined by reversed phase HPLC. c FAB
MS, [M + H]+. H: calcd 5.04, found 4.73; N: calcd 14.40, found 13.93. e H: calcd 6.05, found 6.56; N: calcd 11.24, found 10.04. f H:
calcd 6.04, found 5.60.
d

566 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Aquino et al.
(m, 1H), 5.00 (s, 2H), 6.65 (d, 2H, J ) 9.0 Hz), 6.86 (d, 2H, J
) 9.0 Hz), 7.31-7.43 (m, 5H); TLC Rf ) 0.77 (CH2Cl2/CH3OH,
19:1).
was first demonstrated in the opioid peptide family with
the conversion of the potent agonist 14-hydroxydihy-
dromorphinone into the antagonists naloxone or naltr-
exone through simple replacement of a methyl group
with allyl or cyclopropylmethyl, respectively.25 More
recently, substitution of an isobutyl moiety for hydrogen
in a series of potent nonpeptidyl angiotensin II (AII)
antagonists provided a partial AII agonist.26,27 Finally,
with the 1,5-benzodiazepine CCK-A agonists reported
here, a minor chemical modification (CH3 to CH3CH2,
Table 1) converts an antagonist into an agonist. Thus,
although multiple structural alignments between 11k
and CCK-8 could be proposed, it remains to be deter-
mined whether these structurally diverse ligands share
similar receptor binding determinants.
Gen er al P r ocedu r e for Acylation of Secon dar y Am in es
To Give 2-Br om oa ceta m id es of Gen er a l F or m u la 2.
A
solution of amine (152 mmol) in DCM (250 mL) and triethy-
lamine (152 mmol) was cooled with stirring in an ice bath (<3
°C). Bromoacetyl bromide (152 mmol) dissolved in DCM (100
mL) was added dropwise over 45 min with stirring. The
reaction mixture was stirred overnight at ambient tempera-
ture, washed successively with 0.3 N HCl (300 mL) and brine
(300 mL), dried over sodium sulfate, filtered, and evaporated
in vacuo. The resultant oil was filtered through a pad of silica
gel (150 g), the silica was washed with EtOAc/hexanes (1:1,
900 mL), and the combined filtrates were evaporated in vacuo
to give the corresponding 2-bromoacetamide in good yield
(>85% typical) and high purity. Spectral data for representa-
tive compounds are as follows.
2-B r o m o -N -is o p r o p y l-N -(4-m e t h o x y p h e n y l)a c e t a -
m id e: 1H NMR (300 MHz, CDCl3) δ 1.04 (d, 6H, J ) 6.8 Hz),
3.53 (s, 2H), 3.84 (s, 3H), 4.93 (m, 1H), 6.93 (d, 2H, J ) 9.1
Hz), 7.10 (d, 3H, J ) 9.1 Hz); TLC Rf ) 0.18 (EtOAc/hexane,
3:17).
N-(4-(Ben zyloxy)p h en yl)-2-b r om o-N-isop r op yla cet a -
m id e: 1H NMR (300 MHz, CDCl3) δ 1.05 (d, 6H, J ) 6.7 Hz),
3.55 (s, 2H), 4.94 (m, 1H), 5.08 (s, 2H), 7.01 (d, 2H, J ) 8.7
Hz), 7.11 (d, 2H, J ) 8.6 Hz), 7.36-7.47 (m, 5H); TLC Rf )
0.16 (EtOAc/hexane, 3:17).
2-(P h en ylh yd r a zon o)m a lon ic Acid (3). Phenylhydra-
zine (23.3 g, 215 mmol) was added dropwise over 40 min to a
vigorously stirred solution of ketomalonic acid monohydrate
(29.33 g, 215 mmol) in ethanol (140 mL) and water (300 mL)
at ambient temperature. The resultant slurry was stirred
overnight at ambient temperature. The precipitate was
separated by filtration, washed sucessively with cold water
(100 mL) and ethanol (25 mL), air-dried, and dried at 75 °C
overnight in a vacuum oven to give 3 (42.38 g, 204 mmol, 95%)
as a yellow solid: 1H NMR (300 MHz, DMSO-d6) δ 7.12 (t,
1H), 7.35-7.48 (m, 4H); mp 155-157 °C dec.
2-(P h en ylh yd r a zon o)p r op a n ed ioyl Dich lor id e (4).
Phosphorus pentachloride (36.84 g, 177 mmol) was added
portionwise over a 20 min period to a stirred slurry of 3 (14.73
g, 70.8 mmol) in chloroform (90 mL) at 5 °C. After complete
addition, the solution was warmed to room temperature,
stirred for 1 h, and then heated to reflux for 3 h. The solution
was cooled in an ice bath and the resultant preciptate filtered,
washed with cold hexane (50 mL), and dried under high
vacuum overnight to give 4 (13.4 g, 54.7 mmol, 77%) as a bright
yellow solid: 1H NMR (300 MHz, DMSO-d6) δ 7.12 (t, 1H),
7.20-7.56 (m, 4H); mp 135-138 °C dec.
4-Meth oxy-N-(2-(p h en yla m in o)p h en yl)ben za m id e (5).
p-Anisoyl chloride (18.66 g, 109 mmol) in dichloromethane (100
mL) was added dropwise with stirring over 20 min to a solution
of N-phenyl-1,2-phenylenediamine (20.15 g, 109 mmol) and
triethylamine (11.07 g, 109 mmol) in dichloromethane (325
mL). The reaction mixture was maintained at < 5 °C with
an ice/acetone bath during the addition, then warmed to
ambient temperature, and stirred 2 h. The organic mixture
was washed successively with water (200 mL), 2 N HCl (80
mL), and brine (160 mL), dried over sodium sulfate, and
filtered through a pad of silica (150 g), further eluting with
ethyl acetate (1 L). The eluants were combined and concen-
trated in vacuo. The resultant solid was triturated overnight
with Et2O (350 mL), cooled, filtered, and dried in vacuo to give
5 (21.67 g, 88.4 mmol, 81%) as a light pink solid: 1H NMR
(300 MHz, CDCl3) δ 3.82 (s, 3H), 5.75 (br s, 1H), 6.80-6.91-
(m, 5H), 7.12-7.29 (m, 5H), 7.62 (d, 2H, J ) 8.8 Hz), 8.15 (dd,
1H, J ) 1.7, 7.8 Hz), 8.36 (s, 1H); TLC Rf ) 0.24 (EtOAc/Hex,
1:4); mp 148-150 °C.
Exp er im en ta l Section
Gen er a l. All chemical and solvents are reagent grade
unless otherwise specified. CCK-8 was purchased from Sigma
(St. Louis, MO). MK-32911 was obtained from Merck & Co.
(Rahway, NJ ). The following solvents and reagents have been
abbreviated: tetrahydrofuran (THF), ethyl ether (Et2O), dim-
ethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichloromethane
(DCM), trifluoroacetic acid (TFA), dimethylformamide (DMF).
Reactions were monitored by thin-layer chromatography 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 chromatog-
raphy (RP-HPLC) using a Waters Model 3000 Delta Prep
equipped with a Delta-pak radial compression cartridge (C-
18, 300 A, 15 µm, 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 100 mL/min (to ) 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 (λ range 200-400 nm). The
stationary phase was a Vydac C-18 column (5 µm, 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 (to ) 3 min). Analytical data
is reported as retention time, tR, in minutes (% acetonitrile,
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 (Hz). 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
Instrument Group, Raleigh, NC). Mass spectra were acquired
in the positive ion mode under electrospray ionization (ESI)
or fast atom bombardment (FAB) methods. Combustion
analyses were performed by Atlantic Microlabs, Inc., Norcross,
GA.
Gen er a l Meth od for Red u ctive Am in a tion of P r im a r y
Am in es w ith Keton es/Ald eh yd es To Obta in Com p ou n d s
of Gen er a l F or m u la 1. Where appropriate secondary amines
could not be procured from commercial sources, a primary
amine was converted to the desired secondary amine via
reductive amination with an appropriate aldehyde or ketone
employing the method of Abdel-Magid.15 Spectral data for
representative compounds are as follows.
Isop r op yl(4-m eth oxyp h en yl)a m in e: 1H NMR (300 MHz,
CDCl3) δ 1.18 (d, 6H, J ) 6.1 Hz), 2.92 (br s, 1H), 3.55 (m,
1H), 3.75 (s, 3H), 6.57 (d, 2H, J ) 9.1 Hz), 6.78 (d, 2H, J ) 8.8
Hz); TLC Rf ) 0.72 (EtOAc/hexanes, 2:3).
(4-(Ben zyloxy)p h en yl)isop r op yla m in e: 1H NMR (300
MHz, CDCl3) δ 1.21 (d, 6H, J ) 6.4 Hz), 2.84 (br s, 1H), 3.54
N-(4-Meth oxyben zyl)-N-ph en ylben zen e-1,2-diam in e (6).
A solution of 5 (5.0 g, 15.7 mmol) in THF (30 mL) was added
over 45 min to a stirred solution of lithium aluminum hydride
(1.0 g, 25.1 mmol) in THF (40 mL) cooled to 5 °C and then
heated to reflux for 1.5 h. The solution was cooled to room
temperature, and excess lithium aluminum hydride was
quenched with ethanol until hydrogen evolution ceased.

Benzodiazepines with CCK-A Receptor Agonist Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 567
Saturated aqueous sodium hydrogen carbonate (100 mL) was
added and the resultant solution extracted with ethyl acetate
(3 × 100 mL). The combined organic extracts were dried with
sodium sulfate, filtered through a pad of silica gel, and further
eluted with ethyl acetate (500 mL), and the combined filtrates
were concentrated in vacuo to give 6 (4.78 g, 15.7 mmol, 100%)
as a brown oil which solidified on standing: 1H NMR (300
MHz, CDCl3) δ 3.79 (s, 3H), 4.27 (s, 2H), 4.52 (br s, 1H), 5.08
(s, 1H), 6.67-6.74 (m, 4H), 6.79-6.86 (m, 3H), 7.04-7.24 (m,
6H); TLC Rf ) 0.57 (EtOAc/Hex, 1:4).
1-(4-Met h oxyb en zyl)-5-p h en yl-3-(p h en ylh yd r a zon o)-
1,5-d ih yd r oben zo[b][1,4]d ia zep in e-2,4-d ion e (7). Solu-
tions of 6 (4.86 g, 15.7 mmol) in THF (40 mL) and 4 (5.58 g,
22.8 mmol) in THF (40 mL) were added concomitantly drop-
wise with cooling in an ice/methanol bath over 30 min. The
solution was allowed to warm to room temperature and stirred
for 16 h. A yellow precipitate was separated by filtration,
washed with cold THF (40 mL), and dried under high vacuum
overnight to give the HCl salt of 7 (6.23 g, 13.1 mmol, 83%) as
a yellow solid: 1H NMR (300 MHz, CDCl3) δ 3.78 (s, 3H), 4.69
(d, 1H, J ) 14.7 Hz), 5.76 (d, 1H, J ) 14.9 Hz), 6.80-6.87 (m,
3H), 7.02-7.12 (m, 4H), 7.19-7.40 (m, 11H), 11.19 (s, 1H);
LRMS (FAB) m/ z 477.0 [M + H]+; TLC Rf ) 0.18 (EtOAc/
Hex, 1:4).
3-Am in o-1-(4-m eth oxyben zyl)-5-ph en yl-1,5-dih ydr oben -
zo[b][1, 4]d ia zep in e-2,4-d ion e (8). To a vigorously stirred
slurry of zinc dust (6.49 g, 99.2 mmol) in acetic acid (50 mL)
cooled to 10 °C was added a slurry of 7 (5.75 g, 12.1 mmol) in
acetic acid (30 mL) over 15 min. After complete addition, the
solution was warmed to room temperature and stirred for 3
h. The zinc was separated by filtration and the cake washed
with ethyl acetate (75 mL). The filtrate was concentrated in
vacuo and partitioned between water (60 mL) and ethyl acetate
(100 mL). The pH was adjusted to 9 with saturated sodium
hydrogen carbonate, and the phases were separated. The
aqueous phase was extracted with ethyl acetate (2 × 75 mL),
the organic layers were combined, dried with magnesium
sulfate, filtered, and concentrated in vacuo to give a yellow
oil which was dried under high vacuum to give 8 (4.59 g, 11.9
mmol, 98%): 1H NMR (300 MHz, CDCl3) δ 3.05 (s, 2H), 3.75
(s, 3H), 4.35 (s, 1H), 4.64 (d, 1H, J ) 14.7 Hz), 5.82 (d, 1H, J
) 14.7 Hz), 6.59-6.85 (m, 6H), 7.06-7.29 (m, 6H), 7.51 (d,
1H, J ) 7.4 Hz); LRMS (FAB) m/ z 388.2 [M + H]+; TLC Rf )
0.50 (CH2Cl2/CH3OH, 9:1).
DMF (2 mL) was added sodium hydride (0.0104 g, 60%
suspension in mineral oil, 0.256 mmol) with stirring and
cooling in an ice bath (<3 °C). The mixture was stirred for 30
min followed by addition of the appropriate 2-bromoacetamide
(general formula 2) (0.256 mmol). The reaction mixture was
stirred at ambient temperature for 16 h, the solvent removed
in vacuo, and the crude product purified by preparative RP-
HPLC chromatography on a C-18 column with linear gradient
elution typically 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 products of general formula 11 as white
lyophiles. Spectral data and HPLC conditions for compounds
from Tables 1-3 are given below.
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-p h en yla cet a -
m id e (11a ): 1H NMR (250 MHz, DMSO-d6) δ 4.76 (d, 1H, J
) 16 Hz), 5.08 (d, 1H, J ) 16 Hz), 5.18 (d, 1H, J ) 8 Hz),
6.90-7.73 (m, 20H), 9.20 (s, 1H), 10.40 (s, 1H); HPLC tR ) 18
min (30-60% CH3CN, 30 min, 1.5 mL/min); mp 250-252 °C.
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-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-m eth yl-N-p h e-
n yla ceta m id e (11b): 1H NMR (250 MHz, DMSO-d6) δ 3.25
(s, 3H), 4.48 (d, 1H, J ) 16 Hz), 4.76 (d, 1H, J ) 16 Hz), 5.13
(d, 1H, J ) 8 Hz), 6.90-7.70 (m, 20H), 9.28 (s, 1H); HPLC tR
) 16.5 min (30-60% CH3CN, 30 min, 1.5 mL/min); mp 248-
252 °C.
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-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-eth yl-N-p h en y-
la ceta m id e (11c): 1H NMR (250 MHz, DMSO-d6) δ 1.05 (t,
3H, J ) 7 Hz), 3.72 (q, 2H, J ) 7 Hz), 4.40 (d, 1H, J ) 16 Hz),
4.70 (d, 1H, J ) 16 Hz), 5.11 (d, 1H, J ) 8 Hz), 6.90-7.70 (m,
20 H), 9.22 (s, 1H); HPLC tR ) 22 min (30-60% CH3CN, 30
min, 1.5 mL/min); mp 242 °C.
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-p r op yl-N-p h e-
n yla ceta m id e (11d ): 1H NMR (250 MHz, CDCl3) δ 0.85 (t,
3H, J ) 7 Hz), 1.50 (m, 2H), 3.65 (m, 2H), 4.25 (d, 1H, J ) 16
Hz), 4.58 (d, 1H, J ) 16 Hz), 5.40 (d, 1H, J ) 8 Hz), 6.40 (d,
1H, J ) 8 Hz), 6.89 (s, 1H), 6.95-7.50 (m, 19H); HPLC tR
)
27.5 min (30-60% CH3CN, 30 min, 1.5 mL/min); mp 237 °C.
N-Bu tyl-2-[2,4-dioxo-5-ph en yl-3-(3-ph en ylu r eido)-2,3,4,5-
t et r a h yd r ob en zo[b][1,4]d ia zep in -1-yl]-N-p h en yla cet a -
1
m id e (11e): H NMR (300 MHz, CDCl3) δ 0.85 (m, 3H), 1.25
3-Am in o-1-p h en yl-1,5-d ih yd r oben zo[b][1,4]d ia zep in e-
2,4-d ion e (9). Ceric ammonium nitrate (1.84 g, 3.35 mmol)
was added portionwise over 10 min to a stirred solution of 8
(0.50 g, 1.29 mmol) in acetonitrile/H2O (9:1, 12 mL) at ambient
temperature and the solution stirred overnight at room
temperature. The reaction mixture was concentrated in vacuo
and the resultant solid partitioned between saturated aqueous
potassium carbonate (40 mL) and ethanol (60 mL). The phases
were separated and the aqueous phase extracted with ethanol
(4 × 50 mL). The ethanol portions were combined, dried over
sodium sulfate, and concentrated in vacuo to a tan solid. This
solid was extracted exhaustively with boiling DCM (10 × 60
mL), and the organics were combined, dried over sodium
sulfate, filtered, and concentrated to give 9 (0.30 g, 1.12 mmol,
87%) as a tan solid: 1H NMR (300 MHz, DMSO-d6) δ 1.98 (br
s, 2H), 4.08 (s, 1H), 6.86 (d, 1H, J ) 8.4 Hz), 7.11-7.46 (m,
8H), 10.78 (br s, 1H); 13C (75.429 MHz, DMSO-d6) δ 56.98,
123.41, 126.22, 126.51, 127.34, 128.30, 128.89, 130.15, 132.29,
134.42, 142.36, 168.13, 169.39; LRMS (FAB) m/ z 268.10 [M
+ H]+; TLC Rf ) 0.21 (CH2Cl2/CH3OH, 15:1).
1-(2,4-Dioxo-1-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)-3-p h en ylu r ea (10). To a slurry of 9
(0.398 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 2 h,
and the cream precipitate was separated by filtration to afford
10 (0.413 g, 1.07 mmol, 72%) as a white solid: 1H NMR (300
MHz, DMSO-d6) δ 4.97 (d, 1H, J ) 7.5 Hz), 6.88-6.97 (m, 3H),
7.13-7.47 (m, 12H), 9.16 (s, 1H), 10.78 (br s, 1H); TLC Rf )
0.21 (CH2Cl2/CH3OH, 19:1).
(m, 2H), 1.45 (m, 2H), 3.66 (m, 2H), 4.26 (d, 1H, J ) 16.8 Hz),
4.55 (d, 1H, J ) 17.0 Hz), 5.38 (d, 1H, J ) 7.6 Hz), 6.43 (d,
1H, J ) 7.3 Hz), 7.00 (m, 3H), 7.13-7.49 (m, 17H); LRMS
(FAB) m/ z 576.1 [M + H]+; TLC Rf ) 0.23 (EtOAc/hexanes,
2:3); HPLC tR ) 24.0 min (47-57% CH3CN, 30 min, 1.0 mL/
min).
N-(2-Cyan oeth yl)-2-[2,4-dioxo-5-ph en yl-3-(3-ph en ylu r e-
id o)-2,3,4,5-tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-p h e-
n yla ceta m id e (11f): 1H NMR (250 MHz, DMSO-d6) δ 2.75
(t, 2H, J ) 6 Hz), 3.95 (t, 2H, J ) 6 Hz), 4.45 (d, 1H, J ) 16
Hz), 4.72 (d, 1H, J ) 16 Hz), 5.13 (d, 1H, J ) 8 Hz), 6.90-7.70
(m, 20H), 9.22 (s, 1H); mp 291 °C dec.
[[2-[2,4-Dioxo-5-p h e n yl-3-(3-p h e n ylu r e id o)-2,3,4,5-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]a cetyl]p h en yla m i-
n o]a cetic a cid (11g). A solution of 11h (22 mg, 0.036 mmol),
methanol (5 mL), and 1 N NaOH (0.190 mL, 0.190 mmol) was
stirred for 16 h at ambient temperature. The solvent was
removed in vacuo and the residue dissolved in aqueous sodium
hydrogen carbonate (25 mL) and washed with EtOAc (25 mL).
The pH of the aqueous phase was adjusted to 1.6 with 6 N
HCl and extracted with EtOAc (2 × 25 mL). The organic
phases were dried over sodium sulfate, filtered, and concen-
trated. The crude product was purified by preparative RP-
HPLC with linear gradient elution from 32 to 42% CH3CN in
H2O with 0.1% TFA buffer over 30 min with a flow rate of 100
mL/min. The fraction containing the desired material was
frozen and lyophilized to give 11g (6.6 mg, 11.4 mmol, 31%)
as a white lyophile: 1H NMR (300 MHz, DMSO-d6) δ 4.29 (s,
2H), 4.48 (d, 1H, J ) 16.8 Hz), 4.73 (d, 1H, J ) 16.8 Hz), 5.05
(d, 1H, J ) 7.8 Hz), 6.92 (m, 3H), 7.18-7.55 (m, 17H), 9.15 (s,
1H); LRMS (FAB) m/ z 578.0 [M + H]+; TLC Rf ) 0.32 (CH2-
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
of Gen er a l F or m u la 11. To a solution of 10 (0.256 mmol) in

568 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Aquino et al.
Cl2/CH3OH, 9:1); HPLC tR ) 27.0 min (32-42% CH3CN, 30
min, 1.0 mL/min).
as a white lyophile: 1H NMR (300 MHz, DMSO-d6) δ 0.94 (m,
6H), 4.20 (d, 1H, J ) 16.9 Hz), 4.50 (d, 1H, J ) 16.9 Hz), 4.74
(m, 1H), 5.04 (d, 1H, J ) 7.8 Hz), 6.86-6.97 (m, 4H), 7.11 (m,
2H), 7.21-7.52 (m, 12H), 9.15 (s, 1H), 9.80 (s, 1H); LRMS
(FAB) m/ z 578.1 [M + H]+; TLC Rf ) 0.48 (CH2Cl2/CH3OH,
9:1); HPLC tR ) 14.0 min (42-52% CH3CN, 30 min, 1.0 mL/
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-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-isop r op yl-N-(4-
m eth oxyp h en yl)-a ceta m id e (11o): 1H NMR (300 MHz,
CDCl3) δ 1.00 (m, 6H), 3.83 (s, 3H), 4.17 (d, 1H, J ) 16.8 Hz),
4.45 (d, 1H, J ) 16.8 Hz), 4.95 (m, 1H), 5.35 (m, 1H), 6.97 (m,
3H), 7.16 (m, 5H), 7.30 (m, 6H), 7.41 (m, 4H); LRMS (FAB)
m/ z 592.0 [M + H]+; TLC Rf ) 0.15 (EtOAc/hexanes, 2:3);
HPLC tR ) 27.5 min (42-60% CH3CN, 30 min, 1.5 mL/min).
N-[4-(Dim eth yla m in o)p h en yl]-2-[2,4-d ioxo-5-p h en yl-3-
(3-ph en ylu r eido)-2,3,4,5-tetr ah ydr oben zo[b][1,4]diazepin -
1-yl]-N-isop r op yla ceta m id e (11p ): 1H NMR (300 MHz,
CDCl3) δ 1.01 (m, 6H), 3.01 (s, 6H), 4.20 (d, 1H, J ) 16.4 Hz),
4.41 (d, 1H, J ) 16.4 Hz), 4.93 (m, 1H), 5.38 (d, 1H, J ) 7.6
Hz), 6.61 (d, 1H, J ) 7.3 Hz), 6.96-7.40 (m, 19H); LRMS (FAB)
m/ z 605.1 [M + H]+; TLC Rf ) 0.13 (EtOAc/hexanes, 2:3);
HPLC tR ) 14.5 min (38-48% CH3CN, 30 min, 1.0 mL/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-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-isop r op yl-N-(4-
m or p h olin -4-ylp h en yl)a ceta m id e (11q): 1H NMR (300
MHz, CDCl3) δ 0.98 (m, 6H), 3.12 (s, 2H), 3.84 (s, 2H), 4.18 (d,
1H, J ) 16.1 Hz), 4.42 (d, 1H, J ) 16.1 Hz), 4.92 (m, 1H, J )
7.1 Hz), 5.38 (d, 1H, J ) 7.8 Hz), 6.51 (d, 1H, J ) 7.8 Hz),
6.80-7.50 (m, 18H); LRMS (FAB) m/ z 647.3 [M + H]+; TLC
Rf ) 0.22 (CH2Cl2/CH3OH, 9:1); HPLC tR ) 22.5 min (30-60%
CH3CN, 30 min, 1.5 mL/min).
4-[[2-[2,4-Dioxo-5-p h en yl-3-(3-p h en ylu r eid o)-2,3,4,5-
t et r a h yd r ob en zo[b][1,4]d ia zep in -1-yl]a cet yl]isop r op y-
la m in o]ben zoic Acid (11r ). A mixture of of 4-[[2-[2,4-dioxo-
5-ph en yl-3-(3-ph en ylu r eido)-2,3,4,5-t et r a h ydr oben zo[b]-
[1,4]diazepin-1-yl]acetyl]isopropylamino]benzoic acid benzyl
ester (30 mg, 0.043 mmol) and 10% Pd/C (35 mg) in ethanol/
EtOAc (20 mL, 3:1) was stirred under hydrogen (1 atm) at
ambient temperature for 16 h. The catalyst was separated
by filtration and the solvent removed in vacuo. The crude
product was purified by preparative RP-HPLC with linear
gradient elution from 40 to 50% CH3CN in H2O with 0.1% TFA
buffer over 30 min with a flow rate of 100 mL/min. The
fraction containing the desired material was frozen and
lyophilized to give 11r (14.1 mg, 0.023 mmol, 54%) as a white
lyophile: 1H NMR (300 MHz, CD3OD) δ 1.08 (m, 6H), 4.26 (d,
1H, J ) 16.6 Hz), 4.60 (d, 1H, J ) 16.6 Hz), 4.80 (m, 1H), 6.96
(t, 1H), 7.04 (dd, 1H, J ) 8.2, 1.1 Hz), 7.20-7.51 (m, 16H),
8.19 (d, 2H, J ) 8.5 Hz); LRMS (FAB) m/ z 606.2 [M + H]+;
HPLC tR ) 12.0 min (40-50% CH3CN, 30 min, 1.0 mL/min).
[[2-[2,4-Dioxo-5-p h e n yl-3-(3-p h e n ylu r e id o)-2,3,4,5-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]a cetyl]p h en yla m i-
n o]a cetic a cid eth yl ester (11h ): 1H NMR (300 MHz, CDCl3)
δ 1.23 (t, 3H), 4.16 (q, 2H), 4.30 (d, 1H, J ) 17.4 Hz), 4.42 (d,
2H, J ) 18.3 Hz), 4.61 (d, 1H, J ) 16.9 Hz), 5.41 (d, 1H, J )
7.8 Hz), 6.60 (d, 1H, J ) 7.8 Hz), 6.97 (m, 2H), 7.14-7.48 (m,
20H); LRMS (ESI) m/ z 606.1 [M + H]+; TLC Rf ) 0.25 (CH2-
Cl2/CH3OH, 19:1); HPLC tR ) 15.5 min (47-57% CH3CN, 30
min, 1.0 mL/min).
N-(2-Am in oeth yl)-2-[2,4-Dioxo-5-ph en yl-3-(3-ph en ylu r e-
id o)-2,3,4,5-tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-p h e-
n yla ceta m id e (11i). A mixture of 11j (202 mg, 0.290 mmol)
and 10% Pd/C (40 mg) in ethanol/EtOAc (35 mL, 5:2) was
stirred under hydrogen (1 atm) at ambient temperature for
16 h. The catalyst was separated by filtration and the solvent
removed in vacuo. The crude product was purified by pre-
parative RP-HPLC with linear gradient elution from 30 to 37%
CH3CN in H2O with 0.1% TFA buffer over 30 min with a flow
rate of 100 mL/min. The fraction containing the desired
material was frozen and lyophilized to give 11i (57 mg, 0.101
mmol, 35%) as a white lyophile: 1H NMR (300 MHz, DMSO-
d6) δ 2.88 (m, 2H), 3.87 (m, 2H), 4.34 (d, 1H, J ) 16.8 Hz),
4.66 (d, 1H, J ) 16.8 Hz), 5.06 (m, 1H), 6.86-6.98 (m, 3H),
7.18-7.60 (m, 15H), 7.75 (m, 2H), 9.15 (m, 1H); LRMS (ESI)
m/ z 563.0 [M + H]+; HPLC tR ) 15.0 min (30-37% CH3CN,
30 min, 1.0 mL/min).
N-[2-[(Ca r b ob en zyloxy)a m in o]et h yl]-2-[2,4-Dioxo-5-
ph en yl-3-(3-ph en ylu r eido)-2,3,4,5-tetr ah ydr oben zo[b][1,4]-
d ia zep in -1-yl]-N-p h en yla ceta m id e (11j): 1H NMR (300
MHz, CDCl3) δ 3.36 (m, 2H), 3.81 (m, 2H), 4.23 (d, 1H, J )
16.6 Hz), 4.53 (d, 1H, J ) 16.6 Hz), 5.02 (m, 2H), 5.36 (m, 2H),
6.44 (d, 1H, J ) 7.6 Hz), 6.96-7.46 (m, 25H); LRMS (FAB)
m/ z 697.2 [M + H]+; TLC Rf ) 0.24 (CH2Cl2/CH3OH, 19:1);
HPLC tR ) 19.0 min (47-57% CH3CN, 30 min, 1.0 mL/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-
tetr ah ydr oben zo[b][1,4]diazepin -1-yl]-N-isopr opyl-N-ph e-
1
n yla ceta m id e (11k ): H 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); LRMS (FAB) m/ z 562.0 [M + H]+;
TLC Rf ) 0.21 (CH2Cl2/CH3OH, 19:1); HPLC tR ) 16.5 min
(51-60% CH3CN, 30 min, 1.0 mL/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-cycloh exyl-N-
p h en yla ceta m id e (11l): 1H NMR (300 MHz, DMSO-d6) δ
0.81-1.01 (m, 3H), 1.22-1.33 (m, 2H), 1.49 (m, 1H), 1.64-
1.79 (m, 4H), 4.18 (d, 1H, J ) 16.6 Hz), 4.37 (m, 1H), 4.44 (d,
1H, J ) 16.6 Hz), 5.03 (d, 1H, J ) 7.8 Hz), 6.88-6.96 (m, 3H),
7.18-7.56 (m, 17H), 9.14 (s, 1H); LRMS (FAB) m/ z 602.1 [M
+ H]+; HPLC tR ) 18.0 min (47-57% CH3CN, 30 min, 1.0 mL/
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,N-d ip h en yla c-
eta m id e (11m ): 1H NMR (300 MHz, DMSO-d6) δ 4.58 (d, 1H,
J ) 16.6 Hz), 4.81 (d, 1H, J ) 16.6 Hz), 5.08 (d, 1H, J ) 7.9
Hz), 6.86-6.99 (m, 1H), 7.19-7.64 (m, 22H), 9.16 (s, 1H);
LRMS (ESI) m/ z 596.5 [M + H]+; TLC Rf ) 0.23 (EtOAc/
hexanes, 2:3); HPLC tR ) 9.5 min (51-60% CH3CN, 30 min,
1.5 mL/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-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-(4-h yd r oxyp h e-
n yl)-N-isop r op yla ceta m id e (11n ). A mixture of 2-[2,4-
dioxo-5-ph en yl-3-(3-ph en ylu r eido)-2,3,4,5-t et r a h ydr o-
benzo[b][1,4]diazepin-1-yl]-N-isopropyl-N-(4-phenoxyphenyl)-
acetamide (170 mg, 0.254 mmol) in ethanol/EtOAc/methanol
(25 mL, 3:1:1) and 10% Pd/C (100 mg) was stirred under
hydrogen (1 atm) at ambient temperature for 16 h. The
catalyst was separated by filtration and the solvent removed
in vacuo. The crude product was purified by preparative RP-
HPLC with linear gradient elution from 42 to 52% CH3CN in
H2O with 0.1% TFA buffer over 30 min with a flow rate of 100
mL/min. The fraction containing the desired material was
frozen and lyophilized to give 11n (44.4 mg, 0.077 mmol, 30%)
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-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N-(4-flu or o-p h en -
yl)-N-isop r op yla ceta m id e (11s): 1H NMR (300 MHz, DMSO-
d6) δ 0.96 (m, 6H), 4.20 (d, 1H, J ) 16.6 Hz), 4.47 (d, 1H, J )
16.6 Hz), 4.79 (m, 1H), 5.04 (d, 1H, J ) 7.6 Hz), 6.86-6.97 (m,
3H), 7.21-7.54 (m, 16H), 9.15 (s, 1H); LRMS (FAB) m/ z 580.2
[M + H]+; TLC Rf ) 0.20 (EtOAc/hexanes, 2:3); HPLC tR
28.0 min (30-60% CH3CN, 30 min, 1.5 mL/min).
)
N -Be n zyl-2-[2,4-d ioxo-5-p h e n yl-3-(3-p h e n ylu r e id 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 y-
la ceta m id e (11t): 1H NMR (300 MHz, acetone-d6) δ 1.06-
1.22 (m, 6H), 4.40-5.35 (m, 6H), 6.56 (m, 1H), 6.89-7.72 (m,
19H), 8.56 (m, 1H); LRMS (FAB) m/ z 576.1 [M + H]+; TLC Rf
) 0.12 (EtOAc/hexanes, 2:3); HPLC tR ) 24.5 min (43-53%
CH3CN, 30 min, 1.0 mL/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,N-d iisop r op y-
1
la ceta m id e (11u ): H NMR (300 MHz, CDCl3) δ 1.25-1.37
(m, 12H), 3.55 (m, 1H), 3.91 (m, 1H), 4.67 (d, 1H, J ) 15.8
Hz), 4.77 (d, 1H, J ) 15.8 Hz), 5.40 (d, 1H, J ) 7.1 Hz), 6.54
(d, 1H, J ) 7.1 Hz), 7.03 (m, 2H), 7.15-7.43 (m, 13H); LRMS
(FAB) m/ z 528.0 [M + H]+; TLC Rf ) 0.24 (CH2Cl2/CH3OH,
19:1); HPLC tR ) 17.0 min (45-55% CH3CN, 30 min, 1.0 mL/
min).

Benzodiazepines with CCK-A Receptor Agonist Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 569
(11) Evans, B. E.; Bock, M. G.; Rittle, K. E.; Dipardo, R. M.; Whitter,
W. L.; Veber, D. L.; Anderson, P. S.; Freidinger, R. M. Design of
Potent, Orally Effective, Nonpeptidyl Antagonists of the Peptide
Hormone Cholecystokinin. Proc. Natl. Acad. Sci. U.S.A. 1986,
83, 4918-4922.
(12) Bock, M. G.; Dipardo, R. M.; Evans, B. E.; Rittle, K. E.; Whitter,
W. L.; Veber, D. F.; Anderson, P. S.; Freidinger, R. M. Benzo-
diazepine Gastrin and Brain CCK Receptor Ligands: L365,260.
J . Med. Chem. 1989, 32, 13-16.
(13) Chang, R. S.; Lotti, V. J .; Monaghan, R. L.; Birnbaum, J .;
Stapley, E. O.; Goetz, M. A.; Albers-Schonberg, G.; Patchett, A.
A.; Liesch, J . M.; Hensens, O. D. A Potent Nonpeptide Choly-
cystokinin Antagonist Selective for Peripheral Tissues Isolated
from Aspergillus Alliaceus. Science 1985, 230, 177-179.
(14) Sugg, E. E.; Kimery, M. J .; Ding, J . M.; Kenakin, D. C.; Miller,
L. J .; Queen K. L.; Rimele, T. J . CCK-A Receptor Selective
Antagonists Derived from the CCK-A Receptor Selective Tet-
rapeptide Agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623).
J . Med. Chem. 1995, 38, 207-211.
(15) Abdel-Magid, A. F.; Maryanoff, C. A.; Carson, K. G. Reductive
Amination of Aldehydes and Ketones by Using Sodium Triac-
etoxyborohydride. Tetrahedron Lett. 1990, 31, 5595-5598.
(16) Koetsch, C. F.; Britain, J . W. Effect of N-Alkyl on Formation of
Quinolines from N-Alkylbenzoylacetanilides. J . Org. Chem.
1959, 24, 1551-1553.
(17) Marshall, G. R. A Hierarchical Approach to Peptidomimetic
Design Tetrahedron 1993, 17, 3547-3558.
(18) Strader, C. D.; Fong, T. M.; Tata, M. R.; Underwood, D. Structure
and Function of G Protein-Coupled Receptors. Ann. Rev. Bio-
chem. 1994, 63, 101-132.
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-
tetr a h yd r oben zo[b][1,4]d ia zep in -1-yl]-N,N-d ieth yla ceta -
m id e (11v): 1H NMR (250 MHz, CDCl3) δ 1.09 (t, 3H, J ) 7
Hz), 1.25 (t, 3H, J ) 7 Hz), 3.38 (m, 4H), 4.62 (d, 1H, J ) 15
Hz), 4.94 (d, 1H, J ) 15 Hz), 5.45 (d, 1H, J ) 7 Hz), 6.45 (d,
1H), 6.95-7.48 (m, 14 H); HPLC tR ) 22 min (30-60% CH3-
CN, 30 min, 1.5 mL/min).
An or exia Assa ys. Male Long-Evans rats (225-300 g)
were conditioned for 2 weeks to consume a palatable liquid
diet (Bio-Serve F1657, Frenchtown, NJ ) after a 2 h fast. On
pretreatment day, rats were fasted (100 min) and injected ip
with drug vehicle (propylene glycol, PG, 1 mL/kg) and an oral
preload of saline (0.9% NaCl, 8 mL/kg). Liquid diet access was
provided 20 min later, and consumption was measured at 30,
90, and 180 min. To qualify for the drug treatment study, rats
had to consume at least 8 mL of liquid diet within the first 30
min on the pretreatment day. The next day, following the 100
min deprivation, rats (8-10 animals per dose) were treated
ip or po with vehicle (PG, 1 mL/kg) or various doses (0.01-10
µmol/kg) of test compound dissolved in PG (1 mL/kg), im-
mediately followed by the saline oral preload. Food access was
again provided 20 min later, and food intake was measured
at 30, 90, and 180 min. All food intake data were normalized
for each rat to the respective values from the pretreatment
day. Potency was determined at 30 min and efficacy at the
30 min, 1 µmol/kg dose.
(19) Schwartz, T. W. Locating Ligand-Binding Sites in 7TM Receptors
by Protein Engineering. Curr. Opin. Biotech. 1994, 5, 434-444.
(20) Heerding, J .; Raynor, K.; Kong, H.; Yu, L.; and Reisine, T.
Mutagenesis Reveals that Agonists and Peptide Antagonists
Bind in Fundamentally Distinct Manners to the Rat mu Recep-
tor than Do Nonpeptide Antagonists. Regulatory Peptides 1994,
54, 119-120.
Ack n ow led gm en t. The authors gratefully acknowl-
edge the analytical support of Larry Shampine and
Roderick Davis.
Refer en ces
(21) Wang, J . B.; Imai, Y.; J ohnson, P. S.; Walther, D.; Stein, C.;
Schaefer, M.; Wu, J . M.; Wang, W. F.; Moriwaki, A.; Uhl, G. R.
Human µ Receptor: Gene Structure, Expression, and µ/κ chi-
meras That Define Nontransmembrane Domains Influencing
Peptide Binding Affinities. Ibid. 1994, 54, 317-320.
(22) Onogi, T.; Minami, M.; Katao, Y.; Nakagawa, T.; Aoki, Y.; Toya,
T.; Katsumata, S.; and Satoh, M. DAMGO, A µ-Opioid Receptor
Selective Agonist, Distinguishes Between µ- and δ-opioid recep-
tors Around their First Extracellular Loops. FEBS Lett. 1995,
357, 93-97.
(23) Beinborn, M.; Lee, Y. M.; McBride, E. W.; Quinn, S. M.; Kopin,
A. S. A Single Amino Acid of the Cholecystokinin-B/Gastrin
Receptor Determines Specificity for Non-Peptide Antagonists.
Nature 1993, 362, 348-350.
(24) Mantamadiotis, T.; Baldwin, G. S. The Seventh Transmembrane
Domain of Gastrin/CCK Receptors Contributes to Nonpeptide
Antagonist Binding. Biochem. Biophys. Res. Commun. 1994, 201,
1381-1389.
(25) Furst, S.; Hosztafi, S; Friedmann, T. Structure Activity Relation-
ships of Synthetic and Semisynthetic Opioid Agonists and
Antagonists. Curr. Med. Chem. 1995, 1, 423-440.
(1) Crawley, J . N.; Corwin, R. L. Biological Actions of Cholecysto-
kinin. Peptides 1994, 15, 731-755.
(2) Kissileff, H. R.; Pi-Sunyer, F. X.; Thornton, J .; Smith, G. P.
C-Terminal Octapeptide of Cholecystokinin Decreases Food
Intake in Man. Am. J . Clin. Nutr. 1981, 34, 154-160.
(3) Pi-Sunyer, X.; Kissileff, H. R.; Thornton, J .; Smith, G. P.
C-Terminal Octapeptide of Cholecystokinin Decreases Food
Intake in Obese Men. Physiol. Behav. 1982, 29, 627-630.
(4) Sitzmann, J . V.; Pitt, H. A.; Steinborn, P. A.; Pasha, Z. R.;
Sanders, R. C. Cholecystokinin Prevents Parenteral Nutrition
Induced Biliary Sludge in Humans Surg. Gynacol. Obstetrics
1990170, 25-31
(5) Dourish, C. T.; Ruckert, A. C.; Tattersall, F. D.; Iversen, S. D.
Evidence That Decreased Feeding Induced by Systemic Injection
of Cholecystokinin is Mediated by CCK-A Receptors. Eur. J .
Pharmacol. 1989, 173, 233-234.
(6) Hermkens, P. H. H.; Ottenheijm, H. C. J .; van der Werf-Pieters,
J . M. L.; Broekkamp, C. L. E.; de Boer, T.; van Nispen, J . W.
CCK-A Agonists: Endeavours Involving Structure-Activity Re-
lationship Studies. Recl. Trav. Chim. Pays-Bas 1993, 112, 95-
106.
(7) Shiosaki, K.; Lin, C. W.; Kopecka, H.; Craig, R.; Wagenaar, F.
L.; Bianchi, B.; Miller, T.; Witte, D.; Nadzan, A. M. Development
of CCK-Tetrapeptide Analogues as Potent and Selective CCK-A
Receptor Agonists. J . Med. Chem. 1990, 33, 2950-2952.
(8) Freidinger, R. M. Toward Peptide Receptor Ligand Drugs:
Progress on Nonpeptides. Prog. Drug Res. 1985, 40, 33-98.
(9) Giannis, A.; Kolter T. Peptidomimetics for Receptor Ligands
Discovery, Development, and Medicinal Perspectives. Angew.
Chem., Int. Ed. Engl. 1993, 32, 1244-1267.
(26) Perlman, S.; Schambye, J . T.; Rivero, R. A.; Greenlee, W. J .;
Hjorth, S. A.; Schwartz, T. W. Nonpeptide Angiotensin Agonist.
J . Biol. Chem. 1995, 270, 1493-1496.
(27) Kivlign, S. D.; Huckle, W. R.; Zingaro, G. J .; Rivero, R. A.; Lotti,
V. J .; Chang, R. S. L.; Schorn, T. W.; Kevin, N.; J ohnson, R. G.,
J r.; Greenlee, W. J .; Siegl, P. K. S. Discovery of L-162,313: A
Nonpeptide That Mimics the Biological Actions of Angiotensin
II. Am. J . Physiol., 1995, 268, R820-R823.
(10) Rees, D. C. Non-Peptide Ligands for Membrane-Bound Peptide
Receptors. Curr. Med. Chem. 1994, 1, 145-158.
J M950626D