Discovery of a Potent and Orally Efficacious TGR5 Receptor Agonist

Article abstract of DOI:10.1021/acsmedchemlett.5b00323

TGR5 is a G protein-coupled receptor (GPCR), activation of which promotes secretion of glucagon-like peptide-1 (GLP-1) and modulates insulin secretion. The 2-thio-imidazole derivative 6g was identified as a novel, potent, and selective TGR5 agonist (hTGR5

Full text of DOI:10.1021/acsmedchemlett.5b00323

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Potent and Orally Efficacious TGR5 Receptor Agonist
Sameer Agarwal,* Amit Patil, Umesh Aware, Prashant Deshmukh, Brijesh Darji, Santosh Sasane,
Kalapatapu V. V. M. Sairam, Priyanka Priyadarsiny, Poonam Giri, Harilal Patel, Suresh Giri, Mukul Jain,
and Ranjit C. Desai
Zydus Research Centre, Cadila Healthcare Ltd., Sarkhej-Bavla N.H. No. 8 A, Moraiya, Ahmedabad 382 210, India
S
* Supporting Information
ABSTRACT: TGR5 is a G protein-coupled receptor
(GPCR), activation of which promotes secretion of
glucagon-like peptide-1 (GLP-1) and modulates insulin
secretion. The 2-thio-imidazole derivative 6g was identified
as a novel, potent, and selective TGR5 agonist (hTGR5 EC50 =
57 pM, mTGR5 = 62 pM) with a favorable pharmacokinetic
profile. The compound 6g was found to have potent glucose
lowering effects in vivo during an oral glucose tolerance test in
DIO C57 mice with ED50 of 7.9 mg/kg and ED90 of 29.2 mg/
kg.
KEYWORDS: TGR5, TGR5 agonist, GPR 131, GPBAR1, GLP1 secretion, diabetes
iabetes is increasing globally at an alarming state.1 Type 2
Ddiabetes is characterized by reduced insulin sensitivity
combined with impaired insulin secretion resulting in higher
blood glucose levels.2−4 Although a range of therapies are
available such as sulfonylureas, metformin, and glinides,5,6 they
are unable to achieve satisfactory glycemic control. Therefore,
there is an urgent need for research exploring therapies with
distinct unprecedented mechanisms of action.
Takeda G-protein-coupled receptor 5 (TGR5), also known
as GPR 131, GPBAR1, or M-BAR is G protein-coupled
receptor primarily expressed in monocytes and macrophages,
lung, spleen, and the intestinal tract, and it is activated by bile
acids.7,8 It has been suggested that bile acids induce glucagon-
like peptide-1 (GLP-1) secretion from primary intestinal
enteroendocrine cells by increasing intracellular cAMP levels
via the TGR5 receptor.9,10 Additionally, activation of TGR5
receptors in brown adipose tissue has been proposed to
increase energy expenditure through the induction of type 2
iodothyronine deiodinase (D2).11 Thus, a small molecule
TGR5 agonist may be beneficial for the treatment of type 2
diabetes with simultaneous management of glucose levels, body
weight, and associated complications.
Figure 1. (a) Selected bile acid and nonbile acid TGR5 receptor
agonist reported in literature. (b) Schematic representation of ligand
optimization.
A wide range of structurally diverse modulators of TGR5
have been reported in the literature by various pharmaceutical
companies (Figure 1a).12−22 Most of these reported TGR5
agonists, however, possess insufficient potency and/or lack
metabolic stability. This communication describes the discovery
of 2-((2-(4-(1H-imidazol-1-yl)phenoxy)ethyl) thio)-5-(2-(3,4-
dimethoxyphenyl) propan-2-yl)-1-(4-fluoro phenyl)-1H-imida-
zole (6g), a potent, selective, and orally efficacious TGR5
agonist.
The Exelixis compound 5 showed good potency, although
with a modest pharmacokinetic profile, and thus represented an
Received: August 10, 2015
Accepted: November 20, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00323
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Letter
attractive starting point for generating novel TGR5 agonists.
Table 1. In Vitro Activity of Reference Compound (5) and
Compounds 6a−6l
Several modeling studies of TGR5 have been published.23,24
A
a
TGR5 receptor homology model was generated, which
provided insight regarding the binding site and binding site
residues. Modeling studies show that the van der Waals surface
of the active site of TGR5 receptor has a narrow channel
extending from the terminal aryl group of 5 to a hollow
opening near the extracellular loop between TM4 and TM5
(refer Supporting Information). Thus, it was hypothesized that,
a linker, perhaps with a hydrogen bond acceptor such as oxygen
atom, that fits suitably in the narrow channel and provides
conformational flexibility and positional adaptability, would be
beneficial. Keeping this in view a linker was attached to 5,
which provided 6 (Figure 1b). Moreover, further substitution
on the terminal phenyl ring of 6 may also provide an
opportunity for potential hydrogen bonds or π−π interactions.
Accordingly, several compounds were designed and synthe-
sized, and their activity data was used to build a structure−
activity relationship (SAR) profile (Table 1). The 2-thio-
imidazole derivatives (6) were synthesized as depicted in
Scheme 1.
Synthesis of 6 began with the dimethylation of commercially
available 2-(3,4-dimethoxyphenyl) acetonitrile (7) followed by
selective reduction of cyano group with diisobutylaluminum
hydride (DIBAL) to produce the aldehyde 9.25 Treatment of 9
with methylmagnesium bromide followed by oxidation of the
resulting alcohol 10 under Swern condition afforded 11, which
in turn was brominated using tetrabutyl ammoniumtribromide
́
to afford 12. Further transformation of 12 under Delepine
reaction conditions gave amine 13 as its corresponding
hydrobromide salt, which on treatment with 1-fluoro-4-
isothiocyanatobenzene provided the thiourea derivative 14.
The construction of the imidazole skeleton is achieved via
cyclization of 14 in boiling acetic acid to give the 2-mercapto
imidazole derivative 15. Finally, alkylation with excess of 1,2-
dibromoethane gave the advanced intermediate 16, which on
further alkylation with substituted phenol afforded the test
compounds 6a−6l in good yields and high chemical purity.26
The compounds were evaluated for their ability to activate
TGR5 using luciferase-based reporter gene assays. Consistent
with our hypothesis, extension of the side chain was helpful and
the compound 6a was found to be potent (EC50 = 38 nM). We
then turned our attention to optimize substitution on the
terminal phenyl ring. The compounds 6b exhibited EC50 of 2.3
nM, indicating that substitution on the terminal phenyl ring was
preferred. However, 6b was very lipophilic (cLog P = 7.2).
Thus, incorporation of polar heterocycles at C-4 position was
investigated. The compound 6c, having pyrrole group,
displayed potent TGR5 agonistic activity as compared to 6a.
The 4-pyrrole derivative 6c was an agonist with EC50 of 7.8 nM,
indicating that introduction of nitrogen containing heterocycle
may induce a positive influence on the activity. Therefore,
pyrazole and triazole rings were substituted at C-4 position to
further probe the SAR. The pyrazole (6d) and triazole (6e)
derivatives were found to be nearly equipotent. The thiazole
analogue 6f (EC50 = 31.5 nM) was found to be less potent than
compounds 6c, 6d, and 6e. These results indicated that
carbon−nitrogen bond for introduction of terminal heterocyclic
group might be beneficial to increase the binding affinity of the
compounds; however, carbon−carbon linkage might not favor
this. Further optimization led to the discovery of many potent
compounds and compound 6g with a polar C-4 imidazole
substituent was identified as most potent TGR5 agonist (EC50
a
EC50 values given are expressed as mean
SEM of three
independent experiments; mean standard deviation is 16%.
b
Calculated from ChemBioDraw Ultra 12.0 by CambridgeSoft.
= 57 pM) with reduced cLog P value compared to compounds
6c−6e. To explore possibilities for further structural
optimization, we focused our investigation to the substituents
of 6g. Introduction of several substituents at the terminal part
of the molecule indicated that a variety of groups were well
tolerated at this position. However, these substituents did not
improve potency compared to 6g (compounds 6h−6j). These
data suggest that possibly both lipophilicity and electronics play
key roles in the activity for this class of compounds.
Compounds with fused heterocyclic systems (6k and 6l)
were poor agonists suggesting that fused systems hindered the
receptor compound interaction.
The most potent compound 6g was docked into the putative
binding pocket in the TGR5 homology model (Figure 2). The
ligand binding site in TGR5 is hydrophobic, and Tyr 240
appears to be an important amino acid that stabilizes the ligand
B
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Letter
a
π−π stacking interaction with Trp 237, which nicely fits in the
bottom pocket and perhaps adds to the stability. The distance
between sulfur and the farthest aromatic carbon is ∼5.5 Å in
compound 5 and is ∼10.5 Å in compound 6g. The high
potency of 6g suggests that extension of side chain into the
channel leading to the exterior might play an important role in
modulating biological activity. Notably, it was found that the N-
3 nitrogen atom of the terminal C-4 imidazole group of 6g is
involved in hydrogen-bonding interactions with Asn 147 of the
extra cellular loop between TM4 and TM5. This may stabilize
the conformations and may be crucial in the activation of
TGR5. However, such hydrogen-bonding interaction was not
observed with compounds 6c, 6d, and 6e. This modeling study
proposed that 6g exerts its TGR5 activity by effectively utilizing
a number of different interactions.
Scheme 1. Synthesis of Compound 6
Furthermore, compound 6g showed dose-dependent activa-
tion of a cAMP responsive element-driven luciferase reporter in
TGR5 overexpressing CHO K1 cells (EC50 = 14 nM).
Additionally, it modulated GLP-1 secretion in vitro at 10 and
30 μM concentration in NCI-H716 cells (refer Supporting
Information). Many bile acid-derived TGR5 agonists are
known to activate the nuclear bile acid receptor FXR
(Farnesoid X Receptor);13 compound 6g exhibited good
selectivity against FXR (FXR coactivator functional assay
EC50 = 6.4 μM). Recent reports have shown that TGR5
activation decreases pro-inflammatory cytokine production by
cyclic AMP-mediated inhibition of NF-κB signaling.27 Con-
sistent with this, 6g showed a dose-dependent inhibition of
TNF-α in an LPS-induced cytokine release assay in human
whole blood, similar to prednisolone (IC50 = 251 nM). It has
long been a challenge to discover a TGR5 agonist that is potent
against both the human and mouse receptor,15 albeit, recent
literature19 described potent compounds active in both
receptors. Importantly here, compound 6g displayed excellent
potency on mTGR5 (EC50 = 62 pM), similar to its potency on
hTGR5; this enabled its evaluation in an in vivo mouse model
of efficacy.
a
Reagent and conditions: (a) MeI, NaH, THF, 0 °C to r.t., 3 h, 93%;
(b) DIBAL, toluene, −78 °C, 2 h, 93%; (c) MeMgBr, dry Et2O, 0 °C
to r.t., 80%; (d) oxalyl chloride, DMSO, Et3N, DCM, −78 °C, 2 h,
82%; (e) Bu4NBr3, DCM, MeOH, 0 °C to r.t., 7 h, 100%; (f) HMTA,
DCM, r.t., 48 h; (g) EtOH, HCl, 80 °C, 3 h; (h) 1-fluoro-4-
isothiocyanatobenzene, Et3N, DCM, 0 °C to r.t., 1 h, 42%; (i) AcOH,
118 °C, 3 h, 73%; (j) 1,2-dibromoethane, K2CO3, acetone, r.t., 2 h,
73%; (k) substituted phenol, K2CO3, DMF, 90 °C, 3−5 h.
The physicochemical properties of 6g suggested that it had
suitable drugability properties and potential for in vivo
validation (MW 559; experimental cLog P = 4.7; good
solubility profile). A comparison of the in vivo pharmacokinetic
properties of the compound 5 and our lead candidate 6g (Table
2) shows that 6g has substantially better oral exposure and
bioavailability compared to compound 5. Introduction of the
C-4 imidazole group was not only beneficial for potency but
a
Table 2. Pharmacokinetics Properties of 5 and 6g
parameters
species
5
6g
mouse
mouse
rat
PO dose, mg/kg
Cmax (ng/mL)
AUC (ng·h/mL)
i.v. dose, mg/kg
Vdss (L/kg)
3
3
3
51.75
73.48
1
788.84
754.21
1
147.71 72.53
207.13 98.15
1
1.54
45.52
0.76
7
0.83
47.28
0.33
71
2.05 0.97
79.73 30.92
0.32 0.05
31
Cl (mL/min/kg)
Figure 2. Binding mode of 6g in homology model of TGR5 receptor.
T1/2, iv (h)
%F
through H-bond interactions and aromatic stacking inter-
actions. 3,4-Dimethoxyphenyl group of ligand has OH···O
interaction between the oxygen of the meta-methoxy and the
hydroxy of Tyr 240. This group also forms π−π stacking
interaction with Phe 96. The fluoro-phenyl group also shows
a
Rat PK data is mean
SD, n = 4. Mouse PK data is mean data
because of composite study design, n = 3/time point. Formulation:
PO, 0.5% Tween 80 + 99.5% (0.5%) sodium-CMC in water; IV, 10%
NMP + 10% solutol + 80% normal saline.
C
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Letter
also improved pharmacokinetic profile relative to 5, probably
due to better permeability and solubility (Supporting
Information). The plasma protein binding (PPB) of compound
6g was determined to be very high (99.5 to 99.9% in
rodents).28
Having identified a compound with high potency and
excellent pharmacokinetic profile, 6g was evaluated for in vivo
GLP-1 secretion in C57 mice. A single oral administration of 6g
at 30 mg/kg dose increased plasma GLP-1 levels by 3.8-fold
with respect to vehicle control (Figure 3a). It is noteworthy to
pharmacodynamics and safety evaluations of 6g will be reported
in due course.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00323.
Experimental procedures and analytical data (PDF)
Spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*Fax: +91-2717-665355. Tel: +91-2717-665555. E-mail:
sameeragarwal@zyduscadila.com or sameer_ag@yahoo.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Authors thank Mr. Jeevan Kumar for modeling studies, Mr.
Vishwanath Pawar for safety pharmacology studies, and the
management of Zydus Research Centre, Cadila Healthcare Ltd.
for support and encouragement. ZRC Communication No.
471.
ABBREVIATIONS
■
GPCR, G protein-coupled receptor; BAs, bile acids; CA, cholic
acid; LCA, lithocholic acid; FXR, farnesoid X receptor; cAMP,
cyclic adenosine monophosphate; CMC, carboxymethylcellu-
lose; NMP, N-methyl-2-pyrrolidone; T2D, type II diabetes;
SAR, structure−activity relationship; PPB, plasma protein
binding; MW, molecular weight; TM, trans membrane
Figure 3. (a) In vivo GLP-1 secretion study of 6g in C57 (n = 4
animals/group; female C57 mice; po 30 mg/kg; formulation, 5%
Tween 20 + 0.5% sodium CMC (95%); ***P < 0.001 versus control;
error bar indicates SEM). (b) In vivo efficacy of 6g in DIO C57 mice
(n = 4 animals/group; formulation, 5% Tween 20 + 0.5% sodium
CMC (95%); *P < 0.05; **P < 0.005 versus control; error bar
indicates SEM).
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