Discovery of new piperidine amide triazolobenzodiazepinones as intestinal-selective CCK1 receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2012.02.049

New cholecystokinin-1 receptor (CCK1R) agonist 'triggers' were identified using iterative library synthesis. Structural activity relationship studies led to the discovery of compound 10e, a potent CCK1R agonist that demonstrated robust weight loss in a di

Full text of DOI:10.1016/j.bmcl.2012.02.049

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2943–2947
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new piperidine amide triazolobenzodiazepinones
as intestinal-selective CCK1 receptor agonists
Kimberly O. Cameron a, , Elena E. Beretta a, Yue Chen b, Margaret Chu-Moyer a, Dilinie Fernando a,
⇑
Hua Gao a, Jeffrey Kohrt a, Sophie Lavergne a, Paul Da Silva Jardine a, Angel Guzman-Perez a,
Christopher Hoth a, David A. Perry a, John R. Hadcock a, Denise Gautreau a, Michael Makowski a,
Sylvie Perez a, Jana Polivkova a, Lucy Rogers a, Dennis O. Scott b, Andrew G. Swick a, Lucinda Thiede a,
Catherine E. Trebino a, Richard V. Trilles a, Julie Wilmowski a, Yingxin Zhang a
a Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, CT 06340, USA
b Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
New cholecystokinin-1 receptor (CCK1R) agonist ‘triggers’ were identified using iterative library synthe-
sis. Structural activity relationship studies led to the discovery of compound 10e, a potent CCK1R agonist
that demonstrated robust weight loss in a diet-induced obese rat model with very low systemic exposure.
Pharmacokinetic data suggest that efficacy is primarily driven through activation of CCK1R’s located
within the intestinal wall.
Received 4 January 2012
Revised 13 February 2012
Accepted 16 February 2012
Available online 23 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cholecystokinin-1 receptor
CCK1R
Obesity
Triazolobenzodiazepinone
The cholecystokinin-1 receptor (CCK1R) is located primarily
within the gastrointestinal tract and is a potential target for the
treatment of obesity as well as diabetes.1 Cholecystokinin (CCK)
is an endogenous peptidic hormone for the CCK1R and is released
from I-cells within the intestine in response to a meal. CCK acti-
vates the CCK1R and modulates a number of physiological func-
tions including gallbladder contraction, slowing of gastric
emptying and promotion of satiety resulting in suppression of food
intake.2 Numerous studies support the hypothesis that CCK medi-
ates 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
A number of small molecule CCK1R agonists have been reported
(Fig. 1)5 and we recently disclosed the structure of our prototype
CCK1R agonist, CE-326597, 4.6 Unfortunately, CE-326597 demon-
strated insufficient efficacy for the treatment of either diabetes
or obesity after 12 weeks of dosing in a Phase 2 clinical trial and
was discontinued. Similarly, GI181771X 1, after treatment for
6 months in a Phase 2 clinical trial, demonstrated no weight loss
efficacy.7 Dose-limiting gastrointestinal events including emesis
may have limited full assessment of the mechanism’s potential in
this trial. As we awaited results from our clinical trial, the team
continued to identify alternative CCK1R agonists as potential back-
ups to 4.
Our strategy for the discovery of CCK1R agonists was built
around the hypothesis that activation of the CCK1R’s located with-
in the intestinal wall was sufficient to drive robust weight loss effi-
cacy. Activation of the CCK1R’s located on the gallbladder was
deemed undesirable in order to avoid sustained gallbladder con-
traction and better mimic the gallbladder’s normal physiology of
contraction and refilling. CE-326597 (4) demonstrated robust food
intake effects in rodents with minimal systemic exposure.6 In addi-
tion, rapid gallbladder emptying was observed in mice along with
complete gallbladder refilling within 24 h at a minimally effica-
cious dose of 2 mg/kg. A combination of low systemic exposure
as well as low biliary clearance were hypothesized to enable gall-
bladder refilling by minimizing exposure of the agonist to the
CCK1R’s located on the gallbladder.6
The physical properties of CE-326597 (non-rule of five compli-
ant) provided the desired low systemic exposure which was driven
by poor absorption of drug into the intestinal wall for efficacy (rat
portal F = 9%) followed by moderate hepatic clearance (47 mL/min/
kg) and low biliary clearance (0.03 mL/min/kg). CE-326597 was
formulated as a spray-dried dispersion (SDD) formulation8 because
poor solubility (no detectable solubility in phosphate buffered
⇑
Corresponding author. Tel.: +1 617 551 3234.
E-mail address: kimberly.o.cameron@pfizer.com (K.O. Cameron).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.049

2944
K. O. Cameron et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2943–2947
50
10
5
1
0.5
0.1
0.05
0.01
0.005
0.05 0.1
cCLb_CCK1
0.005 0.01
0.5
1
5
10
50
Figure 2. Rat IV biliary excretion versus calculated biliary excretion (cClb), both as
% drug excreted into the bile.
outside ‘rule-of-five’ space to meet our low systemic exposure
criteria (molecular weight >500 and <700, calculated logd >3 and
<6, and calculated biliary excretion <1%) to provide 82 amides for
synthesis.
Figure 1. Structures of CCK1R agonists.
Condensation of triazole 6 with indole-2-carboxaldehyde fol-
lowed by reduction and ester hydrolysis provided the key interme-
diate (7) for the amide library (Scheme 1). Compounds (71, 86%
success rate) were isolated as a mixture of diastereomers and
screened in quadruplicate at 2 concentrations (0.3 and 30 lM)
for their ability to stimulate release of calcium as compared to
saline) limited sufficient absorption for efficacy. To further improve
on the attributes of 4, increased hepatic clearance as well as
reduced absorption was desired in order to further minimize sys-
temic exposure, yet sufficient residency time in the gut wall to
achieve robust efficacy was required. Because CE-326597 had dem-
onstrated safety in both regulatory toxicology studies as well as in
humans, we chose to continue to work in the triazolobenzodiazepi-
none series but sought alternatives to the indole side-chain as well
as the amide portion of the molecule. Earlier SAR reports from
Glaxo describing their 1,5-benzodiazepine series indicated that
the urea in 1 could be replaced with a methyl indazole and main-
tain robust CCK1R agonism.9 In addition, they reported that the
isopropyl benzyl amide of 1 acted as a key ‘trigger’ for robust agon-
ism and functional activity was sensitive to modest changes.10 We
herein describe the identification of novel amide triggers which
provided robust CCK1R agonists and allowed us to further improve
on the pharmacokinetics of 4. Replacement of the methyl indole in
4 with methyl indazole allowed for further, albeit minor, structural
diversification.
Although in vitro assays were available to assess potency and
microsomal clearance, a bottleneck in our screening strategy was
the ability to efficiently assess biliary clearance, typically evaluated
in vivo using bile cannulated rats. For more efficient prospective
design of CCK1R agonists, a quantitative structure-pharmacoki-
netic relationship (QSPR) model was developed to estimate percent
of drug excreted into the bile and allow prioritization of com-
pounds for synthesis.11 This model was initially developed with lit-
erature compounds and was further trained using data from our
triazolobenzodiazepinones ( Fig. 2). The model generally agreed
well with generated data and suggested that when solubilizing
groups were incorporated into our triazolobenzdiazipinones,
increased biliary excretion was observed. We therefore anticipated
the need to fix solubility with a SDD formulation in our backup
program. In addition, we utilized a QSPR model to calculate logd
values for further triage of virtual compounds.
the CCK-8 peptide in a FLIPR functional assay using a CHO cell line
expressing the human CCK1R. Actives (>30% at 30 lM) were fur-
ther assessed for full dose–response in the functional assay as well
as in a binding assay using 125I-CCK-8 as the radiolabel.
We were excited to identify a number of potent CCK1R agonists
in this initial library and representative active amides (8a–f) are
provided in Table 1. Piperidine and pyrrolidine amides, especially
those with an ortho substituent, provided CCK1R agonists with
good to moderate potencies. Rapid follow-up with a second library
focused on available 2-substituted piperidine and pyrrolidine
amine monomers provided additional improvements in potency
and representative examples are provided (8g–k, Table 1). Piperi-
dine amides, especially those containing a substituted methylene
group at the 2 position (e.g., 8h) provided robust agonist leads
for more focused follow-up. Singleton chemistry was next used
to fine-tune the properties and potency of our leads. At this point,
we switched to the methyl indazole C-3 side-chain and the synthe-
sis is described in Scheme 2. The indazole moiety was introduced
at C3 using alkylation chemistry followed by double de-protection
to provide key intermediate 9 for the synthesis of amides (10a–e).
Final compounds were typically prepared as racemates and active
enantiomers or diastereomers was isolated by chiral chromatogra-
phy. Substituted piperidines that were not available commercially
or in our monomer stores were in general prepared following
known procedures12 and the active enantiomer was isolated by
chiral chromatography prior to amide bond formation. For compar-
ative purposes, the indazole version of 4 was prepared (10a) and
data are included in Table 2. At this point, more detailed in vitro
and in vivo screening in key assays was required to differentiate
compounds. Analogs were screened for unbound intrinsic human
microsomal clearance, rat biliary clearance, and mouse gallbladder
emptying (a well-accepted model used to assess in vivo functional
activity of CCK1R agonists).13,14 In addition, we generated free gut
exposure and free portal exposure at either 2 or 4 h and calculated
the ratio as a means of identifying compounds with improved gut
residency time along with low absorption (Table 2).
In order to identify novel agonist ‘triggers’, an amide library was
designed starting with a large, diverse set of secondary amines.
Due to the availability of starting materials, we opted to maintain
the indole side-chain for this initial library. A diverse set of amines
available in our compound stores was enumerated into a virtual
library. Compounds were filtered based on structural diversity as
well as physical properties where we sought compounds that fell

K. O. Cameron et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2943–2947
2945
Scheme 1. General methods for preparation of CCK1R agonists. Reagents and conditions: (a) (i) NaHMDS, 0 °C; (ii) Bromide, 0° to RT. (b) (i) cat. piperidine, toluene, reflux
with water removal; (ii) H2, Pd/C, NH4CO2H, EtOH, reflux; (iii) HCl, dioxane. (c) HNR1R2, HBTU, DIEA, DMA.
Table 1
hCCK1R in vitro results and calculated propertiesa
NR1R2
IC50 (nM)b
23.6
EC50 (nM) (% agonism vs CCK-8)b
123 (95)
celogd
cClb (% excreted to bile)
0.04
Human
199
lsomes Clint (mL/min/mg)
4
NBn, iso-Pr
4.9
8a
49.3
36.9
68.6
52.5 (97)
400 (78)
1310 (85)
3.8
3.8
4.9
0.063
0.058
0.558
>300
>300
148
N
OCH3
8b
8c
8d
N
Et
Ph
N
N
373
560 (86)
3.6
0.688
212
N
8e
8f
248
168
1670 (87)
4.9
4.6
0.076
0.151
NT
NT
N
N
Ph
Ph
2116.8 (83)
8g
80.1
494 (95)
4.8
0.139
220
N
8h
8i
44.7
4.24
93.3
340
84.2 (78)
256 (88)
31.9 (105)
421 (89)
5.1
6.2
3.8
3.6
0.050
0.011
0.098
0.064
146
76
Ph
N
N
N
N
8j
114
111
N
N
8k
a
NT = not tested; cClb = calculated % of drug excreted into the bile.
Geometric mean of at least 2 independent experiments.
b
Although the methoxyethyl piperidine analog (10b) displayed
ance. Compound 10c had only modest uptake into the gut as com-
pared to CE-326597. To further improve on clearance, methyl
groups were added as additional soft sites for oxidative metabo-
lism. Addition of a single methyl group to the meta position of
the phenyl group provided a loss in functional potency (10d) how-
superior potency in our in vitro assays, its microsomal and biliary
clearances were lower and higher, respectively, relative to 4. The 2-
benzyl substituted piperidine amide (10c), however, provided a
nice balance of potency, microsomal clearance, and biliary clear-

2946
K. O. Cameron et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2943–2947
Scheme 2. General methods for preparation of CCK1R agonists. Reagents and conditions: (a) (i) Sodium tert-butoxide, DMF, RT; (ii) Bromide, À50 °C to RT; (iii) TFA, DCM. (b)
(i) HNR1R2, DIEA, HBTU, DMF or HNR1R2, 4-methyl morpholine, CDMT, THF or HNR1R2, Et3N, TP3, THF; (ii) optional chiral chromatography.
Table 2
hCCK1R in vitro and in vivo resultsa
NR1R2
IC50
EC50 (nM) (%
Logd
pH
7.4
% Dose
Human
l
somes free
Mouse % GBEe at Mouse % GBEf at
Rat free
(nM)b agonism vs CCK-
8)b
excreted into Clint (mL/min/mg)d
bilec
1 h 0.6 mg/kg
24 h (dose mg/kg) gut:portal
2 h, 4hg
4
NBn, iso-Pr
23.6
38.1
123 (95%)
380 (72%)
4.9
4.7
0.04
NT
597
NT
91h
NT
27.5 (2)h
NT
190, 65h
NT
10a NBn, iso-Pr
10b
9.9
36
2.7 (100)
12.9 (103)
669 (75)
3.1
4.6
5.1
0.26
0.32
NT
154
846
NT
85
91
NT
NT
NT
NT
NT
N
OCH
3
10c
7, 7
NT
Ph
N
N
10d
10e
87.6
CH
3
CH
3
20.3
25.4 (89)
5.6
0.044
2300
89h
19 (0.2)h
494, 371h
N
CH
3
a
b
c
All compounds are either achiral or the single active diasteromer; NT = not tested.
Geometric mean of at least 2 independent experiments.
Bile cannulated rats were iv dosed at 1 mg/kg and bile was collected over 24 h.
Intrinsic clearance corrected for microsomal binding.
d
e
f
GBE = gall bladder emptying as determined by GB weight as compared to vehicle; P <0.01 versus vehicle.
Dosed at minimal efficacious dose for food intake in mouse16
.
g
h
After oral administration of drug, gut, and portal exposures were measured and corrected for non-specific binding.
SDD formulation.
Table 3
PK parameters for 4 and 10e in male Sprague–Dawley ratsa
Ex
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 (%)
10e
10e
4
0.3
iv
po
iv
NA
0.46
NA
NA
0.25
NA
176
NA
41.7
NA
17.6
NA
3.70
NA
29.0
0.74
1190
42.5
2.04
NA
1.37
NA
NA
0.13
NA
6.0b
3.0
4
6.0b
po
14.6
1.33
1.0
a
NA = not applicable, mean of n = 3 rats.
SDD formulation.
b
ever the dimethyl benzyl piperidine (10e)15 provided a potent
functional agonist which demonstrated ꢀ4Â higher free intrinsic
clearance as compared to 4 and similar biliary clearance. In addi-
tion, the gut portal ratio was ꢀ2.5Â higher than that observed with
4 and this ratio was maintained at the 4 h time point. Compound
10e demonstrated complete gallbladder emptying in the mouse
model at 0.6 mg/kg, confirming functional activity. In addition,
complete mouse gallbladder refilling was achieved within 24 h
with this compound16 (Table 2). There was no detectable drug
exposure (<LLOQ) in the bile or systemic circulation after adminis-
tration of a gallbladder emptying dose of 0.6 mg/kg of 10e.
ance (0.08 mL/min/kg) contributing to its poor oral bioavailability
(0.13%) (Table 3). Portal exposure (rat portal F = 0.56%, free
Cmax = 0.063 nM) suggested that limited absorption also contrib-
uted to the overall poor systemic exposure of the compound (rat
F = 0.13%, free Cmax = 0.014 nM). Despite the low exposure ob-
served in both the systemic and portal circulation, compound
10e demonstrated ꢀ22% and ꢀ44% decrease in body weight gain
after 5-days of dosing in a diet-induced obese rat model relative
to vehicle at 2 and 6 mg/kg, respectively (Fig. 3). These weight loss
effects were attributed to a decrease in food intake.17 Unbound
intestinal exposure at 2 mg/kg were determined to be 1.07, 0.98,
and 0.65 nM at the 2, 4, and 8 h timepoints, respectively. Free
intestinal exposures were indeed lower than the rat CCK1 IC50
Pharmacokinetic studies with 10e in rat demonstrated high
plasma clearance (176 mL/min/kg, Table 3) and low biliary clear-

K. O. Cameron et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2943–2947
2947
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0.6 mg/kg
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W., Jr.; Ervin, G. N.; Grizzle, M. K.; Henke, B.; James, M. K.; Johnson, M. F.;
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-10
-15
-20
2 *m*g*/kg
6*m*g*/kg
Figure 3. Weight loss observed after 5 days of oral administration of 10e in a diet-
induced obese rat model as compared to vehicle (25% SDD, ⁄⁄⁄p <0.001).
(14.7 nM) however, they were significantly higher than the portal
and plasma maximal concentrations observed at 6 mg/kg. The lim-
ited time-points used in these challenging pharmacokinetic studies
may in fact have missed Cmax within the intestinal wall. Regardless,
the data suggest that activation of intestinal CCK1R’s are primary
drivers for the weight loss efficacy observed.
6. Elliott, R. L.; Cameron, K. O.; Chin, J. E.; Bartlett, J. A.; Beretta, E. E.; Yue, Chen;
Jardine, P. D. S.; Dubins, J. S.; Gillaspy, M. L.; Hargrove, D. M.; Kalgutkar, A. S.;
LaFlamme, J. A.; Lame, M. E.; Martin, K. A.; Maurer, T. S.; Nardone, N. A.; Oliver,
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In summary, we have identified new piperidine amide ‘triggers’
in the triazolobenzodiazepinone series. Optimization of leads
based on SAR and physical properties has led us to the discovery
of compound 10e which demonstrated robust CCK1 receptor agon-
ism as measured by in vitro as well as in vivo studies. Pharmacody-
namic effects were attributed primarily to activation of intestinal
CCK1R’s due to low portal and systemic drug levels and relatively
higher intestinal drug levels measured pre-clinically. Compound
10e successfully completed regulatory safety studies but was sub-
sequently discontinued from further development due to disap-
pointing clinical data observed with our prototype CE-326597.
7. (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.
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8. (a) Compounds 4a and 10e were formulated as
a 25 wt % drug:75 wt %
hydroxypropyl methylcellulose acetate succinate-high granular (HPMCAS-
HG) SDD in 0.5% methylcellulose.; (b) Rasenack, N.; Müller, B. W. Pharm. Res.
1894, 2002, 19; (c) Friesen, D. T.; Shanker, R.; Crew, M.; Smithey, D. T.; Curatolo,
W. J.; Nightingale, J. A. S. Mol. Pharm. 2008, 5, 1003; (d) Curatolo, W.;
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K. J. Med. Chem. 1996, 39, 2655.
10. Aquino, C. J.; Armour, D. R.; Berman, J. M.; Birkemo, L. S.; Carr, R. A. E.; Croom,
D. K.; Dezube, M.; Dougherty, R. W., Jr.; Ervin, G. N.; Grizzle, M. K.; Head, J. E.;
Hirst, G. C.; James, M. K.; Johnson, M. F.; Miller, L. J.; Queen, K. L.; Rimele, T. J.;
Smith, D. N.; Sugg, E. E. J. Med. Chem. 1996, 39, 562.
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Acknowledgments
We thank Larry Miller for providing the CHO hCCK1R cell line,
Robert Corr for the preparation of library compounds, Robert Dep-
ianta, Ben Hritzko, Dan Virtue, and Sharon Matis for chiral separa-
tion of analogs, and Brenda Ramos, Samuel Bell, and Michael
Gumkowski for providing formulations.
14. All procedures were reviewed and approved by the Institutional Animal Care &
Use Committee.
Supplementary data
15. Stereochemistry of 10e was determined by single crystal X-ray analysis. The
crystal structure has been deposited at the Cambridge Crystallographic Data
Centre and was allocated the following deposition number: CCDC 855714.
16. Anorectic activity of 10e in mice was assessed utilizing an overnight fasting-re-
feeding paradigm. After an overnight fast, mice were orally given increasing
doses of 10e in a SDD formulation and food intake was measured. At the 2-h
time point, 0.2 mg/kg was determined to be the minimally efficacious dose,
significantly inhibiting food intake by 34% (p <0.05) as compared to vehicle-
treated mice. This dose was therefore used to assess gallbladder refilling.
17. Significant decreases in food intake relative to vehicle of 19% and 25% were
observed at 2 and 6 mg/kg, respectively. No gastrointestinal side-effects were
observed.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.02.049.
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