Discovery of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as potent agonists of TGR5 via sequential combinatorial libraries

Article abstract of DOI:10.1016/j.bmcl.2012.12.076

Optimization of a high-throughput screening hit led to the discovery of a new series of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as highly potent agonists of TGR5. This novel chemotype was rapidly developed through iterative combinatorial library synthesis. It was determined that in vitro agonist potency correlated with functional activity data from human peripheral blood monocytes.

Full text of DOI:10.1016/j.bmcl.2012.12.076

Accepted Manuscript
Discovery of 5-Phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as Potent
Agonists of TGR5 via Sequential Combinatorial Libraries
Allyn T. Londregan, David W. Piotrowski, Kentaro Futatsugi, Joseph S.
Warmus, Markus Boehm, Philip A. Carpino, Janice E. Chin, Ann M. Janssen,
Nicole S. Roush, Joanne Buxton, Terri Hinchey
PII:
S0960-894X(12)01671-X
http://dx.doi.org/10.1016/j.bmcl.2012.12.076
BMCL 19961
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
9 November 2012
17 December 2012
21 December 2012
Please cite this article as: Londregan, A.T., Piotrowski, D.W., Futatsugi, K., Warmus, J.S., Boehm, M., Carpino,
P.A., Chin, J.E., Janssen, A.M., Roush, N.S., Buxton, J., Hinchey, T., Discovery of 5-Phenoxy-1,3-dimethyl-1H-
pyrazole-4-carboxamides as Potent Agonists of TGR5 via Sequential Combinatorial Libraries, Bioorganic &
Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2012.12.076
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Discovery of 5-Phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as Potent Agonists of TGR5 via Sequential
Combinatorial Libraries
Allyn T. Londregan,* David W. Piotrowski, Kentaro Futatsugi, Joseph S. Warmus, Markus Boehm, Philip A. Carpino, Janice
E. Chin, Ann M. Janssen, Nicole S. Roush, Joanne Buxton, Terri Hinchey
allyn.t.londregan@pfizer.com
Pfizer Worldwide Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States.
Abstract
Optimization of a high-throughput screening hit led to the discovery of a new series of 5-phenoxy-1,3-dimethyl-1H-
pyrazole-4-carboxamides as highly potent agonists of TGR5. This novel chemotype was rapidly developed through
iterative combinatorial library synthesis. It was determined that in vitro agonist potency correlated with functional
activity data from human peripheral blood monocytes.
TGR5 (Takeda G-protein-coupled receptor 5), is a class A G-protein-coupled receptor expressed in varying levels
in the liver, skeletal muscle, intestine, brown adipose tissues and monocytes.1 Activation of the TGR5 receptor by
endogenous bile acids triggers an intracellular increase in cAMP levels with subsequent upregulation of protein kinase A.
This activation cascade has multiple tissue-dependent effects important in metabolism and glucose homeostasis,
including increased secretion of GLP-1 (glucagon-like peptide-1) from intestinal cells,2 increased production of eNOS
(endothelial nitric oxide synthase) and nitric oxide in liver cells,3 and activation of type 2 iodothyronine deiodinase (D2)
in brown adipose and skeletal muscle.4 Respectively, these effects may potentiate glycemic control through enhanced
glucose-dependent insulin secretion, improve liver health, and increase energy expenditure. Recent studies also
demonstrate that TGR5 activation may ameliorate atherosclerosis through reduction of macrophage inflammation and
lipid loading.5 For these reasons, TGR5 agonists may function as useful agents for the control of metabolic diseases, such
as type II diabetes, and associated complications.
Figure 1: Selection of previously disclosed TGR5 agonists.

Several TGR5 agonists, including triterpenoid bile acid derivatives6 and small molecules7, have been described
recently in the peer-reviewed literature (Figure 1). Previous in vivo studies6,8 with bile acid-derived TGR5 agonist 1
demonstrated modulation of GLP-1 secretion as well as increased energy expenditure in corpulent mouse models.
Furthermore, compound 2 was shown to increase GLP-1 secretion in an in vivo canine model when dosed in an
intracolonic manner and co-administered with a glucose challenge.7a Despite these positive studies, systemic in vivo
evaluation of TGR5 agonism with ligands such as 1-4 remains a significant challenge. We discovered that many of the
reported TGR5 agonists lack the combined potency and pharmacokinetic properties to convincingly activate TGR5
receptors systemically. In recent reports7d,e, we described our efforts to obtain TGR5 agonists with improved profiles.
Two exemplars (5 and 6) from distinct chemical series were evaluated. In both cases, we obtained sufficient potency
and systemic exposure in canine models for evaluation of TGR5 agonism. While agents such as 5 and 6 represented
excellent tool compounds for us to study the effects of TGR5 activation in in vivo animal models, their profiles were not
sufficient for advancement into clinical trials.
This report describes the medicinal chemistry efforts associated with the discovery of a new class of TGR5
agonist, with the ultimate goal of finding an agent suitable to progress as a clinical candidate. For efficiency and speed,
we elected to advance this new chemotype exclusively with parallel synthetic methods. Improvements in lipophilic
efficiency9 (LipE) served as a general measure of progress.
In order to effectively evaluate TGR5 agonism, we developed two independent assays10—one with a high level of
receptor expression that would provide increased sensitivity and a large dynamic range for screening compounds with
widely varying potencies (induced assay), and another lower-expressing system that would provide a more conservative
estimate of agonist potency and intrinsic activity (uninduced assay). In the induced assay, a doxycycline-regulated
promoter system was used to over-express the human TGR5 receptor in a CHO cell line, with elevation of cAMP used as
the detection system. The lower-expressing uninduced system used the same cAMP detection methodology in cells
expressing basal TGR5 receptor levels cultured in the absence of doxycycline. We have found that the uninduced human
system correlates with data derived from other ex vivo human assays (vide infra); thus, we have determined that
optimization of this potency measure is most appropriate. 7d,e
MW
LogDa, PSA
LipEb
HLM Clintc (μL/min/mg)
hTGR5 induced EC50 (nM)d
hTGR5 uninduced EC50 (nM)d
429
3.1, 60.3
2.0
>320
120
5780
Figure 2: Profile of 7. aLogD determined by shake flask method. bLipE = -logEC50uninduced – logD. cHuman liver microsome intrinsic clearance. d Values are n>3 with a
standard deviation of < 0.5 log units.
High-throughput screening of the Pfizer compound collection with the induced-cAMP assay identified racemic
hit 7, a previously undisclosed 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamide, as an agonist of TGR5 (Figure 2).
Although the physical properties of the molecule (MW, LogD, PSA) were in an acceptable range, our previous experience
within the TGR5 program suggested that further attenuation of lipophilicity would most likely be required to improve

metabolic stability. This chemical series was highly enabled for parallel chemistry, with a synthetic modularity that made
7 an attractive starting point for further SAR development. Given the modest potency (5,780 nM, uninduced-cAMP) of 7,
our initial efforts were centered on rapid improvements measured by increased LipE.
Scheme 1. Reagents and conditions: (i) K2CO3, DMF, 100oC. (ii) KMnO4, acetone, 30oC. (iii) HNR1R2, HATU, NEt3, DMF, 30oC.
The robust nature of the synthetic chemistry used to assemble analogues of 7 meant that our program could be
driven exclusively by combinatorial libraries.
The synthesis11 of the 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-
carboxamides (12) is outlined in Scheme 1. Nucleophilic addition of a range of phenols (9) to commercially available 5-
chloro-1,3-dimethyl-1H-pyrazole-4-carboxaldehyde (8) under basic conditions, afforded phenoxy-carboxaldehyde (10).
Potassium permanganate-mediated oxidation of 10 to the corresponding carboxylic acid (11), was followed by coupling
with varied amines to give the desired 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamide (12). Using this three-step
protocol, it was possible to readily scan both the phenol (blue) and the amide regions (red). Interestingly, 5-chloro-1,3-
dimethyl-1H-pyrazole-carboxylic acid, -ester, or –alkylamide variants of 8, which may have allowed omission of the
aldehyde oxidation step, were inert under the phenol coupling conditions used here.
hTGR5 induced hTGR5 uninduced
LogDb
LipEc
Compound
A
R
EC50 (nM)a
EC50 (nM)a
> 10,000
> 10,000
1,460
13a
13b
13c
13d
13e
13f
C
C
C
C
C
C
C
C
C
C
C
C
C
N
2-F
3-Cl
337
2.5
3.0
2.9
3.3
2.6
3.3
3.3
3.4
3.3
2.2
2.5
1.3
2.4
1.4
-
-
349
2-Cl
15
2.7
3.1
3.0
2.7
-
2,3-diCl
2,3-diF
2,4-diCl
2,5-diCl
2-Cl, 4-CH3
2-CF3
7.9
231
65
3,180
12
549
13g
13h
13i
1,480
113
> 10,000
3,830 (+1020)
352
2.0
2.8
-
15
13j
2-CN
2,910
4,930
> 10,000
920
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
13k
13l
3-OCH3
4-CONH2
H
-
-
13m
13n
-
H
> 10,000
-
Table 1: SAR of Phenol Region. aUnless otherwise noted, values are n>3 with a standard deviation of < 0.5 log units. bLogD determined by shake flask
method. cLipE = -logEC50uninduced – logD.

We elected to explore variation of the amide and phenol moieties separately in two sequential libraries. The
SAR of the phenol region was scanned first using the above protocol, with selected examples shown in Table 1. Both the
substituent and the substitution pattern of the phenol proved to be important. In general, lipophilic substituents were
better tolerated than polar variants. Of the substituents investigated, 2-chloro was preferred, either exclusively (13c) or
with other lipophilic substituents (13f and 13h). Gratifyingly, the 2,3-dichloro analogue (13d), showed an approximately
25-fold improvement in potency (231 nM, uninduced-cAMP) compared to the original lead. Since this was achieved
with only a minor increase in LogD, the overall LipE improved significantly relative to lead 7 (3.1 vs. 2.0). 2-
Trifluoromethyl (13i) also was well tolerated (352 nM, uninduced-cAMP).
hTGR5 induced
EC50 (nM)a
hTGR5 uninduced
EC50 (nM)a
LipEb
3.1
Compound
R
14a
6.8
159
14b
14c
14d
4,680
9,530
3,140
> 10,000
> 10,000
> 10,000
-
-
-
14e
14f
14g
1,400
393
> 10,000
8,460
-
0.9
-
6,400
>10,000
14h
14i
>10,000
>10,000
>10,000
>10,000
-
-
14j
1.2
105
3.5
Table 2: SAR of Amide Region. aValues are n>3 with a standard deviation of < 0.5 log units. bLipE = -logEC50uninduced – logD.
Due to its improved potency and LipE characteristics, we elected to incorporate the 2,3-dichlorophenyl moiety
into a subsequent library in order to explore the SAR of the amide region. A subset of those results is summarized in
Table 2. The enantiopure homologues of 13d (compounds 14a and 14b) had widely disparate potencies, with the S-
enantiomer (14a) slightly improved (159 nM, uninduced-cAMP) when compared to the racemate. A number of isomeric
pyridyl-piperidines, pyridyl-pyrrolidines, and phenyl-piperidines (representative examples 14c-14f) were also

synthesized, but were primarily inactive in the uninduced cAMP assay. Further analogues, such as 14g-i, which
contained ring-constrained and/or polar heterocyclic amides, did not have measureable activity in the uninduced cAMP
assay. Pleasingly, ring-opened pyridine variant 14j was quite potent (105 nM, uninduced-cAMP). Combined, these data
suggested the correct positioning of the pyridyl nitrogen (i.e. 14a and 14j) would be important for critical receptor
interactions and that incorporation of alternative polar groups in this region of the molecule would likely be challenging.
The ring-opened analogue (14j) was chosen for further development since it showed improved lipophilic efficiency,
offered the opportunity for dual vector exploration about the amide nitrogen, and reduced complexity through the
removal of a the stereocenter.
Scheme 2. Reagents and conditions: (i) K2CO3, MeOH, 30oC; NaBH4. (ii) 10, HATU, NEt3, DMF, 30oC.
In order to fully expand the amide region of this chemotype, we developed a modified two-step library
protocol12 that explored two substituents simultaneously on the amide nitrogen (Scheme 2). Reductive amination of an
aryl aldehyde (15) with an alkylamine (16), afforded a diverse set of in situ monomers (18) akin to the N-(pyridin-3-
ylmethyl)ethanamine of 14j. A simple amide coupling with acid 17, yielded the desired analogues (19). The diversity of
the amine monomers (18) was significantly enhanced in this library by utilizing abundantly available aryl aldehydes and
alkylamines as synthetic precursors.
hTGR5 induced hTGR5 uninduced
LipEb
3.8
Compound
R1
Ar
EC50 (nM)a
EC50 (nM)a
19a
0.30
33
19b
19c
1.8
1.2
60
49
3.8
3.5
19d
19e
19f
2.4
14
70
3.7
3.2
3.0
3.6
922
7.9
18
1,020
1,080
19d
Table 3: Selection of New TGR5 Agonists. aValues are n>3 with a standard deviation of < 0.5 log units. bLipE = -logEC50uninduced – logD.

Using this new library procedure, we synthesized a series of analogues. Selected examples are shown in Table 3.
The R1-nitrogen substituent consisted of 1-6 carbon alkanes and ethers (acyclic and cyclic) while the aromatic (Ar)
component was comprised of 3-pyridyl and 3-pyridyl isosteres. Several new compounds were discovered with
exceptional potencies (33-70 nM, 19a-19d), each with LipE values >3.50. The R1-nitrogen substituent had a large impact
on the measured potency, with branched N-alkanes such as N-isopropyl (19a-c) and N-sec-butyl (19d) preferred. As
expected, the 3-pyridyl moiety was essential, with a close-in thiazole isostere (19b) also tolerated.
With multiple examples of highly potent TGR5 agonists in hand, we chose to characterize select compounds in
more detail (Table 4). First, we wished to verify the correlation between the cAMP assay (induced or uninduced) and
functional activity for this new series. As such, compound 14a was assessed in human peripheral blood mononuclear
cells (PBMCs) isolated from whole human blood, that natively express TGR5.13 In accordance with other independent
series, there appeared to be a greater correlation between the functional potency derived from the ex vivo human
PBMC cAMP assay1 and cAMP-uninduced assay compared to the cAMP-induced assay.
Secondly, we chose to evaluate and compare the metabolic stability of our new leads. High intrinsic human liver
clearance (HLM CLint) persisted among the actives presented here, with 19b (76 L/min/mg) only modestly improved
when compared to the original HTS hit 7 (>320 L/min/mg). During the evaluation of this and other chemical series in
these laboratories, we observed a direct correlation between in vitro TGR5 agonism and intrinsic liver clearance, which
may be exacerbated by the apparent necessity for lipophilic moieties in lead structures. In this work, we were able to
efficiently use lipophilicity (LipE↑) to improve potency, but we were unable to satisfactorily lower the LogD values of
our leads while maintaining activity. Such liabilities are potentially significant hurdles to the development of clinically
viable systemic agents.
Ex vivo PBMC (cAMP) hTGR5 uninduced EC50 hTGR5 induced EC50
HLM Clintc
(μL/min/mg)
> 320
LogDd
Compound
EC50 (nM)a
(nM)b
159
33
(nM)b
14a
19a
19b
2
379
-
6.8
3.5
3.2
3.2
3.6
3.0
2.5
0.30
1.8
> 320
-
60
76
600
110
457
75
23
> 320
5
2.3
184
6
212
5.5
98
Table 4: Additional Characterization of TGR5 Agonists. a(n=2). b Values are n>3 with a standard deviation of < 0.5 log units. cHuman liver microsome
intrinsic clearance. dLogD determined by shake flask method.
In conclusion, a new series of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides have been identified as
highly potent in vitro agonists of TGR5. Several exemplars are among the most potent TGR5 agonists reported to date.
Each design cycle was advanced through improvements in lipophilic efficiency. This novel series was rapidly developed
through iterative combinatorial library synthesis due in part to its modular nature. It was determined that in vitro
agonist potency correlated with functional activity data from ex vivo peripheral blood monocyte assay. Thus far, high
metabolic clearance has hindered further advancement of this series. Future lead optimization efforts to improve
systemic exposure will be necessary prior to evaluation of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides in vivo.
More advanced studies with other TGR5 agonists within our portfolio have been reported in the literature, with further
results expected to be published in due course.
Acknowledgments: We thank Dr. David Hepworth for his advice and support in this research.

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10) TGR5 is a Gαs-protein coupled that, when stimulated, induces the activation of Adenylate Cyclase (AC), thereby resulting in increases of intracellular cAMP.
Agonists of TGR5 were identified using an in-vitro HTRF® (Homogeneous Time-Resolved Fluorescence) competitive immunoassay (HTRF® cAMP dynamic 2 Assay
Kit; Cis Bio cat # 62AM4PEC) which compares basal cAMP level in whole cells with cAMP levels reached after stimulation with compound. A tracer molecule, d2-
labeled cAMP, acts as an acceptor for a Europium (Eu3+) cryptate donor. A monoclonal anti-cAMP antibody has been labeled with Eu-cryptate so that when d2-
cAMP binds to the Mab, energy is transferred from the donor to the acceptor. The complex is excited by light at a wavelength of 340 nm and d2-cAMP binding
is detected by emission at wavelength 665 nm. In this assay, intracellular cAMP generated by TGR5 activation competes with the d2-cAMP tracer molecule for
binding to the labeled antibody, thus resulting in a change in fluorescence due to the prevention of energy transfer from the donor to the tracer. The
fluorescent signal is therefore inversely proportional to the concentration of cellular cAMP resulting from TGR5 activation. Detecting and calculating the ratio of
665 nm/620 nm emissions allowed sources of interference to be minimized (e.g. medium, colored compounds). The two cell line used in this assay, Flp-In™-
CHO-TO-humanTGR5 was constructed using the Flp-In™ T-REx™ System (pcDNA™5/FRT/TO Vector Kit Invitrogen cat# V6520-20). This expression system utilized
the tetracycline repressor gene to tightly regulate transcription of the gene of interest (GOI). In the absence of tetracycline, transcription is blocked and
therefore little or no TGR5 is expressed. However, when cells are induced with doxycycline (an analog of tetracycline), transcription of our GOI occurs and very
high levels of TGR5 are expressed. The screening cascade we developed utilized both the induced and uninduced cell lines. Test compounds were serially diluted
in 100 % dimethysulfoxide (DMSO) and spotted 0.5 μL/well to an empty, 384-well, polypropylene plate (Costar # 3654). A reported TGR5 agonist (S)-1-(6-fluoro-
2-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-(isoquinolin-5-yloxy)ethanone (JP 2006063064) was used as a high control while DMSO was used as the low control.
Reference compounds were also used in the assay. All wells of the spotted compound plate were diluted 1:120 with 60 μL/well of assay media. 5 μL was

transferred from the compound plate to the assay plate containing 5 μL/well of cells (final compound dilution = 1:240). After 30 minutes incubation at 22 ºC, 5
μL of d2-labeled cAMP and 5 μL of anti-cAMP antibody (both diluted 1:20 in cell lysis buffer as described in the manufacturers assay protocol) were dispensed to
each well of the assay plate using a Thermo Multidrop Combi. The plates were incubated at room temperature for 60 minutes and then read with a Perkin-
Elmer Envision™ 2104 multilabel plate reader using excitation wavelength of 330 nm and emission wavelengths of 615 nm and 665 nm to detect changes in the
HTRF® signal. EC50 determinations were made from an agonist response curves analyzed with a curve fitting program using a 4-parameter logistic dose response
equation. The intrinsic activity was calculated as the percent of maximal activity of the test compound relative to the activity of a reported TGR5 agonist (S)-1-
(6-fluoro-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-(isoquinolin-5-yloxy)ethanone (JP 2006063064). For further details see reference 7e.
11) General Experimental Procedures. All reactions were carried out in vials under nitrogen atmosphere. Step 1: A solution of compound 8 (100 mol, 1.0 equiv)
and compound 9 (150 mol, 1.5 equiv) in DMF (0.1 M) was treated with K2CO3 (300 mol, 3.0 equiv) and stirred at 110oC for 16 hours (LCMS check). The
reaction was filtered and the filtrate concentrated to afford 10. The crude aldehyde 10 (100 mol, 1.0 equiv) was dissolved in acetone:water (2:1, 0.1 M) and
treated with KMnO4 (600 mol, 6 equiv) and stirred at 30oC for 16 hours (LCMS check). The reaction was filtered and the filtrate concentrated to afford 11. The
crude acid 11 (100 mol, 1.0 equiv) was treated with HATU (120 mol, 1.20 equiv) followed by the crude amine (100 mol, 1.0 equiv) and NEt3 (300 mol, 3.0
equiv). The reaction was stirred at 30oC for 16 hours (LCMS check). The reactions were concentrated and purified directly by reversed phase preparative HPLC
using a C18 column and eluting with acetonitrile-water (0.225% formic acid or pH=10 NH4OH) gradient. All compounds were deemed greater than 95% purity by
1
LCMS and HPLC. Data for compound 14a: H NMR (400 MHz, CDCl3) 8.50 - 8.40 (m, 2H), 7.30 - 7.05 (m, 4H), 6.82 (dd, J=1.4, 8.4 Hz, 1H), 5.92 - 5.64 (m, 1H),
4.09 - 3.84 (m, 1H), 3.63 (s, 3H), 2.97 - 2.85 (m, 1H), 2.39 (br. s., 1H), 2.36 (s, 4H), 1.82 (br. s., 1H), 1.73 - 1.19 (m, 5H). m/z = 445.1 (M+H)+. LCMS = 100% purity.
12) General Experimental Procedures. All reactions were carried out in vials under nitrogen atmosphere. Step 1: A solution of compound 15 (125 mol, 1.0 equiv)
and compound 16 (125 mol, 1.0 equiv) in MeOH (0.3 M) was treated with NaHCO3 (250 mol, 2.0 equiv) and stirred for 16 hours at 30oC. The reaction was
then treated with NaBH4 (125 mol, 1.0 equiv) and stirred for 3 hours (LCMS check). The reaction mixture was filtered and concentrated. The crude amine was
suspended in 1N NaOH (1 mL) and extracted with dichloromethane (3 x 1 mL). The organics were pooled, dried (Na2SO4) and evacuated to afford 18. Step 2: A
solution of 17 (75 mol, 0.60 equiv) in DMF (0.1 M) was treated with HATU (75 mol, 0.60 equiv) followed by the crude amine 18 (125 mol, 1.0 equiv) and
iPr2NEt (225 mol, 1.8 equiv). The reactions were concentrated and purified directly by reversed phase preparative HPLC using a C18 column and eluting with
acetonitrile-water (0.225% formic acid or pH=10 NH4OH) gradient. All compounds were deemed greater than 95% purity by LCMS and HPLC.
13) Whole blood from human donors was treated with heparin (1 mL heparin added to 50 mL blood) and mononuclear cells were collected using Sigma-Aldrich
Accuspin tubes (catalog number A-7054), following the manufacturer’s instructions. Remaining red blood cells were lysed by resuspending the pellet in 9 mL
water, then mixed with 1 mL 10X HBSS (Hank’s Balanced Salt Solution, Invitrogen catalog number 14065-056). After Phosphate Buffered Saline was added to
bring the total volume to 45 mL, tubes were centrifuged at 1200 rpm at room temperature for 10 min. The pellet was resuspended in 5 mL RPMI with 0.1% BSA
(bovine serum albumin, Sigma catalog number A7888) then filtered through a 100 micron cell strainer (BD catalog number 352360). The resulting PBMCs were
plated at 50,0000 cells in 45 L RPMI with 0.1% BSA, 0.5 mM IBMX per well in Matrix 96 well plates. Test compounds were serially diluted in RPMI with 0.5 mM
IBMX and the PBMCs were treated with compounds for 1 h at 37oC. cAMP accumulation was measured and analyzed as described for the recombinantly
expressed human and canine TGR5 cAMP assays.
Graphical Abstract:

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