Discovery of N-benzyl-2-[(4S)-4-(1H-indol-3-ylmethyl)-5-oxo-1-phenyl-4,5- dihydro-6H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-6-yl]-N-isopropylacetamide, an orally active, gut-selective CCK1 receptor agonist for the potential treatment of obesity

Article abstract of DOI:10.1016/j.bmcl.2010.08.115

We describe the design, synthesis, and structure-activity relationships of triazolobenzodiazepinone CCK1 receptor agonists. Analogs in this series demonstrate potent agonist activity as measured by in vitro and in vivo assays for CCK1 agonism. Our efforts

Full text of DOI:10.1016/j.bmcl.2010.08.115

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6797–6801
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-benzyl-2-[(4S)-4-(1H-indol-3-ylmethyl)-5-oxo-1-phenyl-4,5-
dihydro-6H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-6-yl]-N-isopropylacetamide,
an orally active, gut-selective CCK1 receptor agonist for the potential treatment
of obesity
Richard L. Elliott a, Kimberly O. Cameron a, , Janice E. Chin a, Jeremy A. Bartlett b, Elena E. Beretta a,
⇑
Yue Chen c, Paul Da Silva Jardine a, Jeffrey S. Dubins a, Melissa L. Gillaspy a, Diane M. Hargrove a,
Amit S. Kalgutkar c, Janet A. LaFlamme a, Mary E. Lame c, Kelly A. Martin a, Tristan S. Maurer c,
Nancy A. Nardone a, Robert M. Oliver a, Dennis O. Scott c, Dexue Sun a, Andrew G. Swick a,
Catherine E. Trebino a, Yingxin Zhang a
a Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, CT 06340, United States
b Department of Pharmaceutical Sciences, Pfizer Global Research and Development, Groton, CT 06340, United States
c Department of Drug Metabolism and Disposition, Pfizer Global Research and Development, Groton, CT 06340, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the design, synthesis, and structure–activity relationships of triazolobenzodiazepinone CCK1
receptor agonists. Analogs in this series demonstrate potent agonist activity as measured by in vitro and
in vivo assays for CCK1 agonism. Our efforts resulted in the identification of compound 4a which signif-
icantly reduced food intake with minimal systemic exposure in rodents.
Received 29 June 2010
Revised 22 August 2010
Accepted 24 August 2010
Available online 28 September 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
CCK
Cholecystokinin
CCK1R
Gut selective
Triazolobenzodiazepinone
Cholecystokinin (CCK) is an endogenous gastrointestinal
hormone that is released from the gut in response to ingestion of
a meal. CCK has numerous physiological functions, including stim-
ulation of gallbladder contraction, slowing of gastric emptying and
promotion of satiety resulting in suppression of food intake.1 CCK
interacts with two G protein-coupled receptor subtypes, CCK1R
and CCK2R. The CCK1R is localized primarily in the periphery (gas-
trointestinal tract) whereas the CCK2R is predominantly located in
the brain and gastric mucosa.2 Numerous studies support the
hypothesis that CCK mediates its satiety effect through the CCK1R3
which relays the post-prandial satiety signal via the vagal afferent
neurons to the central nervous system (CNS).4 These data suggest
that CCK1R agonists may be effective anorectic agents for the treat-
ment of obesity. In addition, recent reports suggest that activation
of the CCK1R may also be beneficial for the management of
diabetes.5
Small molecule CCK1R agonists (e.g., 1,5-benzodiazepine
GI181771X (1),6 thiazole SR-146131 (2),7 and imidazole carboxam-
ide (3))8 have been disclosed (Fig. 1). The results of phase I and IIA
clinical trials conducted with 1 have also been reported.9 Despite
statistically significant reduction in food intake following oral
administration of 1, no weight loss was discerned in subsequent
Phase II trials.9b,c Dose-limiting gastrointestinal events including
emesis were reported which may have precluded a thorough anal-
ysis of the CCK1 mechanism in the clinic. Herein we describe our
efforts to design a novel series of triazolobenzodiazepinone-based
CCK1R agonists, leading to the discovery of our clinical candidate
4a, recently evaluated in Phase IIA clinical trials for the treatment
of obesity and management of glycemic control.10
Our strategy for the design of an orally active, small molecule
CCK1R agonist was based on the premise that systemic exposure
is not required for anorectic activity and that robust efficacy could
be achieved solely through activation of receptors within the en-
teric nervous system (ENS).4 This hypothesis is supported by pre-
clinical studies with both 1 and 3, wherein despite poor systemic
⇑
Corresponding author. Tel.: +1 860 441 3410; fax: +1 860 441 4734.
E-mail address: kimberly.o.cameron@pfizer.com (K.O. Cameron).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.115

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R. L. Elliott et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6797–6801
N
HO2C
H3C
COOH
O
EtO
S
O
O
O
N
N
N
N
HN
N
OCH3
Cl
NH
O
N
O
H3C
N
NH
O
OH
CH3O
N
CH3
3
GI181771X, 1
SR-146131, 2
Figure 1. Structures of CCK1R agonists.
exposure, significant suppression of food intake was observed in
rodents.6c,8 Therefore we desired a gut-selective CCK1R agonist
with the appropriate balance of solubility and membrane perme-
ability to achieve enough free drug exposure to stimulate CCK1R
activation within the ENS, yet rapid hepatic clearance to minimize
plasma exposure. This approach offers a key advantage in that po-
tential off-target pharmacology associated with mechanisms that
require peripheral or CNS exposure would be avoided. In addition,
a CCK1R agonist with this profile has the potential to better mimic
the normal gallbladder physiology of contraction and refilling.
To achieve this profile, we deliberately diverged from Lipinski’s
‘rule of 5’ for orally bioavailable drugs11 seeking high molecular
weight, lipophilic compounds to enhance intestinal permeability
and increase hepatic clearance. Given the encouraging reports on
suppression of food intake by 1, we focused on the 1,5-benzodiaz-
epine template as a starting point for design.6,9a Compound 1 is a
CCK1 in vitro partial agonist with molecular weight 605, moderate
rat clearance driven primarily by biliary clearance, and poor oral
bioavailability. Shifting the clearance mechanism from biliary to
hepatic was desirable to avoid the potential for enterohepatic
recirculation. Because of their physical properties, 1,5-benzodiaze-
pine CCK1 agonists generally suffered from poor oral absorption.
Attempts to correlate efficacy as a function of absorptive perme-
ability using the Caco-2 in vitro assay were not informative6c
although solubility limitations may have confounded interpreta-
tion of these in vitro data. We utilized log D in the design of analogs
to drive to a physicochemical property space appropriate for
enhancing absorptive permeability and hepatic clearance. Relative
to 1, our focus for design was to maximize hepatic clearance by
increasing lipophilicity and to improve permeability by decreasing
the number of hydrogen bond donors and acceptors while main-
taining CCK1R potency and selectivity to achieve the desired phar-
macological profile.
chose to conserve this motif and focused on structural modifica-
tions to the benzodiazepine core. Key to our success was the dis-
covery that a fused triazolobenzodiazepinone core could mimic
the hydrogen bond acceptor properties of the carbonyl group pres-
ent at C-4 in the 1,5-benzodiazepinone core while maintaining
CCK1R binding and functional activity (Fig. 2).12
Initial synthetic attempts to access the core failed; our break-
through came with the discovery of a novel, one-step condensation
approach, thus enabling the development of this series. The syn-
thesis of triazolobenzodiazepinone analogs is illustrated in Scheme
1. Acid-catalyzed condensation of 1,2-phenylenediamine (5) with
3,3-diethoxy-acrylic acid ethyl ester (6) followed by condensation
of 7 with benzoyl hydrazine provided the triazolobenzodiazepi-
none scaffold 8. N-Alkylation with the appropriate a-bromo-acet-
amide (9) afforded key intermediate 10. Oximation at the C-3
position in 10 with isoamyl nitrite followed by reduction of the
oxime provided the amine intermediate 11 which was readily
functionalized to generate the corresponding amide, urea or carba-
mate analogs 4b–f. Alternatively, condensation of 10 with an aro-
matic aldehyde followed by reduction, or alkylation with the
appropriate arylmethyl halide afforded compounds 4a,13 4g, and
4h. Chiral separation of either the racemic products or amine inter-
mediates (11a and 11b) provided the desired active enantiomers.
The more active enantiomer was identified by preparing and test-
ing both in vitro. As an example, data for the enantiomers of the in-
dol-3-ylmethyl derivative, 4a and 4h, are provided (Table 1).
Binding, using 125I-CCK-8 as the radiolabel, and functional activity
were assessed using a CHO cell line expressing either the human
(Table 1) or rat CCK1R (Table 2). Compounds were determined to
be agonists by their ability to stimulate the release of calcium
using a FLIPR assay.
Preparation of 4b where the N1-isopropylphenylacetamide
moiety and the urea side-chain found in 1 were maintained pro-
vided a CCK1R partial agonist with improved binding (ꢀ6Â) and
comparable functional activity relative to 1, thus confirming the
Given the importance of the N1-isopropylanilidoacetamide
moiety for CCK1R agonism in 1,5-benzodiazepine analogs,6b we
R1
N
R1
N
N
O
O
N
O
O
N
N
O
O
O
N
N
N
COOH
NH
O
NH
O
R2
NH
O
R2
X
NH
N
Y
Ph
GI181771X, 1
4b-f
Figure 2. Design principle.

R. L. Elliott et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6797–6801
6799
H
N
O
N
C-3
H
N
O
NH2
CO2Et
a
b
c
+
N
NH2 OEt OEt
R1
N
N
R1 = Ph
9b: R1 = Bn
N
OEt
9a:
5
6
7
8
O
Br
R1
R1
R1
N
N
N
O
O
O
O
O
O
N
N
N
N
d
e
NH2
NH
R2
N
N
O
N
N
N
N
N
N
10a: R1 = Ph
11a: R1 = Ph
10b:
11b:
R1 = Bn
R1 = Bn
4b-f
R1
f or g
N
O
O
N
4a, 4g-4h
R2
N
N
N
Scheme 1. Synthesis of CCK1R Agonists. Reagents: (a) AcOH, xylenes, reflux; (b) AcOH, benzoyl hydrazine, reflux; (c) NaHMDS, DMF, 9a or 9b; (d) (i) NaH, isoamyl nitrite; (ii)
H2, Pd/C, NH4CO2H; (iii) optional chiral separation; (e) (i) CDI, DMAP, R2NH2; or pyridine, R2COCl; or NaHCO3, R2OCOCl; (ii) optional chiral separation; (f) (i) R2CHO, cat.
piperidine, toluene, reflux; (ii) H2, Pd/C, NH4CO2H; (iii) Chiral separation; (g) (i) NaH, R2CH2X; (ii) Chiral separation.
bioactivity of our new template. Replacement of the aniline func-
tionality with benzyl provided 4c, which led to 2.5Â and 4Â losses
in binding and functional potencies as compared to 4b. Removal of
the acid (4d) and alternatives to the urea (4e and 4f) were explored
for improved potency as well as to increase log D and microsomal
clearance. With the exception of 4e, these analogs maintained
moderate to good in vitro potency at the CCK1R. As expected, high-
er intrinsic clearance and decreased solubility were observed with
these non-acidic, more lipophilic analogs. Replacement of the C-3
amine derivatives with indol-3-ylmethyl or indazol-3-ylmethyl
tryptophan mimetics14 (4a and 4g) provided further increases in
log D and clearance while maintaining in vitro potency. In addition,
we observed good in vitro CCK1R agonist selectivity15 with both
analogs; 4a also demonstrated very high intrinsic clearance, albeit
at a solubility cost. Interestingly, the 3(R) antipode 4h had de-
creased (ꢀ8Â) binding affinity as compared to 4a with no detect-
able agonism.
compounds (4a, 4f, and 4g; log D >3) exhibited robust efficacy in
this model. The added lipophilicity presumably enhanced intesti-
nal permeability of these compounds, providing sufficient drug
exposure at the site of action. Interestingly, no gastrointestinal
side-effects were observed in this model with any of the CCK1R
agonists tested. Confidence in CCK1R mediated efficacy observed
in the feeding model was established by pre-treating rats with a
CCK1R antagonist followed by administration of 4a, resulting in
the loss of food intake efficacy.17 Because compound 4a demon-
strated robust efficacy along with high intrinsic clearance in micro-
somes, we progressed this compound forward to more detailed PK
studies to support our gut-selective hypothesis.
Unfortunately, no inhibition of food intake was observed in rats
dosed with crystalline 4a, likely due to inadequate absorption of
the less soluble crystalline form (no detectable solubility in phos-
phate buffered saline). Reformulation of crystalline 4a as a solid
spray dried dispersion (SDD)18 led to significant improvements in
solubility (ꢀ20Â) and consequently robust food intake efficacy
over 12 h (Fig. 3). Independent PK studies with 4a assessed intesti-
nal and plasma exposure. Despite the solubility enhancement of
the SDD formulation, oral exposure of 4a in rats remained poor
(F = 1%, Table 3). Unbound plasma concentrations remained well
below the CCK1 rat IC50 of 12 nM (Cmax = 0.98 0.30 nM). In addi-
tion, exposure in the small intestine (normalized for non-specific
binding) was determined. A maximal unbound gut exposure of
136 nM was measured at 0.25 h post-dose, after which time, gut
concentrations steadily declined (95 nM, 14 nM and 3.0 nM at 1,
2 and 4 h post-dose, respectively). Overall, these data indicated
that sufficient free gut concentrations (PCCK1R binding IC50) of
The mouse gallbladder emptying model16 was used to rapidly
assess in vivo functional activity. With the exception of 4c, com-
pounds tested in this assay demonstrated complete gallbladder
emptying at oral doses of 6 mg/kg, confirming target activity (Table
1). By 24 h, 4a demonstrated nearly complete refilling of the gall-
bladder, whereas sustained gallbladder contraction was observed
after dosing with 1.
Food intake efficacy was assessed at 6 mg/kg in a rat spontane-
ous feeding model during the normal, nighttime feeding cycle (Ta-
ble 2). No significant efficacy was observed with our more polar
analogs (4c–d) despite having good in vitro potency and functional
activity at the rat receptor. On the other hand, the more lipophilic

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R. L. Elliott et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6797–6801
Table 1
hCCK1R in vitro and mouse in vivo results for 1 and triazolobenzodiazepinone analogs 4a–ha
c
R1
R2
log Db
IC50 (nM)
EC50 (nM)
(% agonism
vs CCK-8)c
Human microsomes
Clint (mL/min/kg)
pH 6.5 kinetic
Mouse % GBEd
at 1 h 6 mg/kg
Mouse % GBE
at 24 h 2 mg/kg
solubility (
lM)
1
See Figure 1
1.09
131 7.7
283 7.3
<8.0
499
82
91.4d
(62.8 0.5)
315 34.9
(53 2)
4ae
Bn
3.39
31.7 4.3
199
0.94
91f
27.5f,g
N
H
O
O
H
160 30.6
(67.8 3.0)
N
4b
4c
Ph
0.92
0.44
19.7 0.9
49.3 8.4
<8.0
33.3
395
390
NT
64
NT
NT
OH
OH
H
645 158
(55.5 4.4)
N
Bn
4d
4e
4f
Bn
Ph
Bn
–NHPh
2.82
3.13
3.08
24.3 2.3
201 32
91.3 34.1
(73.7 2.6)
1810 451
(40.1 2.5)
628 138
85.4
259
29.0
6.0
90.4
NT
NT
NT
NT
–Bn
4.6
–OPh
15
4
12.1
90
(57.7 6.4)
522 187
(72.3 2.1)
4g
Bn
Bn
3.16
3.39
48.5 11
246 39
39.5
NT
2.26
NT
86.3
NT
NT
NT
N
N
H
4hh
>10,000
N
H
a
b
c
h = Human; NT = not tested; NS = not significant;
Measured at pH 7.4.
Mean standard error of at least n of three independent experiments.
P <0.001 versus vehicle.
3(S)-isomer.
Dosed in SDD formulation.
P <0.003 versus 1.
3(R)-isomer.
lsomes = liver microsomes; GBE = gallbladder emptying; compounds tested in vivo were amorphous unless noted.
d
e
f
g
h
Table 2
125
100
75
50
25
0
vehicle
SDD
crystalline
amorphous
Rat CCK1R in vitro and in vivo resultsa
b
b
IC50 (nM)
EC50 (nM)
Rat SPFI %c reduction at 6 mg/kg
1
52.6 10.4
12.2 3.8
17.0 3.0
2.0 1.0
NT
63.8 8.8
445.8 43.6
428 23
À52
À56d
NS
4a
4c
4d
4f
4g
4h
294
NT
6
NS
À41
À43
NS
21
NT
3
481.2 154.9
NT
a
NT = not tested; NS = not significant; SPFI = spontaneous food intake; com-
4 h
12 h
pounds tested in vivo were amorphous unless noted.
b
Mean standard error of at least two independent experiments.
% Food intake at 4 h post-dose; P <0.05 versus vehicle unless noted.
Dosed in SDD formulation.
Figure 3. The effects of 4a (6 mg/kg) on spontaneous rat food intake at 4 h and 12 h
post-dose.
c
d
min/kg generated from rat liver microsomes19 and suggested that
the primary clearance mechanism of 4a was via oxidative metabo-
lism in the liver. Published data on compound 1 indicated that bil-
iary clearance significantly contributed to the overall clearance of
the compound in rat (65% of parent is excreted into the bile after
iv administration).6c Differences in the clearance mechanisms be-
tween 1 and 4a may explain the observed disparate rates of gall-
bladder refilling. The presence of a CCK1R agonist in the bile may
trigger activation of receptors present on the gallbladder leading
to sustained contraction.
4a were achieved for up to 2 h following oral administration and
likely accounted for the oral food intake activity observed in rat.
Food intake effects were observed for up to 12 h (Fig. 3) suggesting
that sufficient receptor activation was achieved to trigger down-
stream events leading to sustained efficacy.
Additional PK studies in rat indicated that 4a has moderate
plasma clearance (41.7 mL/min/kg, Table 3) and low biliary clear-
ance (0.0277 mL/min/kg). The in vivo rat clearance data was in
good agreement with the predicted blood clearance of 47 mL/

R. L. Elliott et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6797–6801
6801
Table 3
PK parameters of 4a in male Sprague-Dawley ratsa
Dose (mg/kg)
Route
Cmax (ng/mL)
Tmax (h)
Clp (mL/min/kg)
Vdss (L/kg)
AUC(0-tlast) (ng h/mL)
t1/2 (h)
F (%)
3.0
iv
po
NA
14.6 4.4
NA
1.33 0.58
41.7 8.8
NA
3.70 0.62
NA
1190 253
42.5 15.7
1.37 0.13
NA
NA
1.0 0.4
6.0b
a
NA = not applicable, mean standard deviation, n of 3.
SDD formulation.
b
L. J.; Queen, K. L.; Rimele, T. J.; Smith, D. N.; Sugg, E. E. J. Med. Chem. 1996, 39,
562; (c) Sugg, E. E.; Birkemo, L.; Gan, L. S.; Tippin, T. K. Pharm. Biotechnol. 1998,
11, 507.
In summary, the design and synthesis of CCK1R agonists with
physicochemical properties suited for limited systemic exposure
has led to the identification of our clinical candidate, 4a. Com-
pound 4a demonstrated sustained food intake efficacy in our pre-
clinical model with complete gallbladder refilling 24 h after dosing.
Additionally, PK studies with 4a to assess intestinal and systemic
exposure suggest that efficacy is primarily derived from activation
of the intestinal CCK1 receptors. Unfortunately, after 12 weeks of
dosing in overweight and obese subjects with type-2 diabetes, 4a
demonstrated inadequate efficacy (HbA1c and body weight) and
was discontinued from clinical development. Additional details
will be reported in subsequent publications.
7. Bignon, E.; Bachy, A.; Boigegrain, R.; Brodin, R.; Cottineau, M.; Gully, D.;
Herbert, J. M.; Keane, P.; Labie, C.; Mollimard, J. C.; Olliero, D.; Oury-Donat, F.;
Petereau, C.; Prabonnaud, V.; Rockstroh, M. P.; Schaeffer, P.; Servant, O.;
Thurneyssen, O.; Soubrié, P.; Pascal, M.; Maffrand, J. P.; Le Fur, G. J. Pharmacol.
Exp. Ther. 1999, 289, 742.
8. Zhu, C.; Hansen, A. R.; Bateman, T.; Chen, Z.; Holt, T. G.; Hubert, J. A.; Karanam,
B. V.; Lee, S. J.; Pan, J.; Qian, S.; Reddy, V. B.; Reitman, M. L.; Strack, A. M.; Tong,
V.; Weingarth, D. T.; Wolff, M. S.; MacNeil, D. J.; Weber, A. E.; Duffy, J. L.;
Edmondson, S. D. Bioorg. Med. Chem. Lett. 2008, 8, 4393.
9. (a) Castillo, E. J.; Delgado-Aros, S.; Camilleri, M.; Burton, D.; Stephans, D.;
O’Connor-Semmes, R.; Walker, A.; Shachoy-Clark, A.; Zinsmeister, A. R. Am. J.
Physiol. Gastrointest. Liver Physiol. 2004, 287, G363; (b) Jordon, J.; Greenway, F.
L.; Leiter, L. A.; Li, Z.; Jacobson, P.; Murphy, K.; Hill, J.; Kler, L.; Aftring, R. P. Clin.
Pharmacol. Ther. 2008, 83, 281; (c) Roses, A. D. Clin. Pharmacol. Ther. 2009, 85,
362.
Acknowledgments
10. Additional details regarding pharmacology and clinical trial results of 4a will
be reported in future publications.
We thank Andrew Butler, Jon Bordner, Richard Hank, Sharon
Matis, Daniel Virtue, Robert DePianta, Marlys Hammond, Terrell
Patterson, Li She, Jim Coutcher, and Lucy Rogers, for their contribu-
tions to this work. We thank Larry Miller for providing the CHO
hCCK1R cell line.
11. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
2001, 46, 3.
12. Elliott, R. L.; Cameron, K. O.; Hammond, M. U.S. Patent 7,358,242, 2005.
13. Stereochemistry at C-3 for the active enantiomer 4a was determined to be in
the
S configuration based on the following experiment: (1) single X-ray
analysis of N-benzyl-2-{(4S)-4-[(5-bromo-1H-indol-3-yl)methyl]-5-oxo-1-
phenyl-4,5-dihydro-6H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-6-yl}-N-
isopropylacetamide; (2) removal of bromide via hydrogenation provided 4a.
The crystal structure has been deposited at the Cambridge Crystallographic
Data Centre and was allocated the following deposition number: CCDC 789675.
14. (a) Evans, B. E.; Bock, M. G.; Rittle, K. E.; DiPardo, R. M.; Whitter, W. L.; Veber, D.
F.; Anderson, P. S.; Freidinger, R. M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 4918;
(b) Henke, B. R.; Aquino, C. J.; Birkemo, L. S.; Croom, D. K.; Dougherty, R. W., Jr.;
Ervin, G. N.; Grizzle, M. K.; Hirst, G. C.; James, M. K.; Johnson, M. F.; Queen, K. L.;
Sherrill, R. G.; Sugg, E. E.; Suh, E. M.; Szewczyk, J. W.; Unwalla, R. J.; Yingling, J.;
Willson, T. M. J. Med. Chem. 1997, 40, 2706.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.115.
References and notes
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anxiety,2b CCK2R stimulation was evaluated for 4a and 4g in
a
calcium
mobilization assay (FLIPR) while CCK2R inhibition was examined by GTP 35S
binding. Compounds 4a and 4g had no significant activity at 10,000 nM in
either assay. full account of the triazolobenzodiazepinone SAR will be
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