Evaluation of novel TGR5 agonist in combination with Sitagliptin for possible treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmcl.2018.04.011

TGR5 is a member of G protein-coupled receptor (GPCR) superfamily, a promising molecular target for metabolic diseases. Activation of TGR5 promotes secretion of glucagon-like peptide-1 (GLP-1), which activates insulin secretion. A series of 2-thio-imidazole derivatives have been identified as novel, potent and orally efficacious TGR5 agonists. Compound 4d, a novel TGR5 agonist, in combination with Sitagliptin, a DPP-4 inhibitor, has demonstrated an adequate GLP-1 secretion and glucose lowering effect in animal models, suggesting a potential clinical option in treatment of type-2 diabetes.

Full text of DOI:10.1016/j.bmcl.2018.04.011

Accepted Manuscript
Evaluation of Novel TGR5 Agonist in combination with Sitagliptin for Possible
Treatment of Type 2 Diabetes
Sameer Agarwal, Santosh Sasane, Jeevan Kumar, Prashant Deshmukh, Hitesh
Bhayani, Poonam Giri, Suresh Giri, Shubhangi Soman, Neelima Kulkarni,
Mukul Jain
PII:
S0960-894X(18)30309-3
https://doi.org/10.1016/j.bmcl.2018.04.011
BMCL 25758
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 January 2018
31 March 2018
4 April 2018
Please cite this article as: Agarwal, S., Sasane, S., Kumar, J., Deshmukh, P., Bhayani, H., Giri, P., Giri, S., Soman,
S., Kulkarni, N., Jain, M., Evaluation of Novel TGR5 Agonist in combination with Sitagliptin for Possible Treatment
of Type 2 Diabetes, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.04.011
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Evaluation of Novel TGR5 Agonist in combination with Sitagliptin for Possible
Treatment of Type 2 Diabetes
Sameer Agarwala*, Santosh Sasanea,b, Jeevan Kumara,b, Prashant Deshmukha, Hitesh Bhayania, Poonam Giria,
Suresh Giria, Shubhangi Somanb, Neelima Kulkarnib and Mukul Jaina
b
aZydus Research Centre, Cadila Healthcare Ltd., Sarkhej-Bavla N.H. No. 8 A, Moraiya, Ahmedabad - 382210, India; Chemistry
Department, The M.S University, Baroda – 390 002, India
*E-mail: sameeragarwal@zyduscadila.com or sameer_ag@yahoo.com; Fax: +91-2717-665355. Tel: +91-2717-665555
ABSTRACT: TGR5 is a member of G protein-coupled receptor (GPCR) superfamily, a promising molecular
target for metabolic diseases. Activation of TGR5 promotes secretion of glucagonlike peptide-1 (GLP-1),
which activates insulin secretion. A series of 2-thio-imidazole derivatives were identified as novel,
potent and orally efficacious TGR5 agonists. Compound 4d, a novel TGR5 agonist, in combination with
Sitagliptin, a DPP-4 inhibitor, has demonstrated an adequate GLP-1 secretion and glucose lowering
effect in animal models, suggesting a potential clinical option in treatment of type-2 diabetes.
KEYWORDS: TGR5, TGR5 agonist, GPBAR1, GLP1 secretion, Type 2 diabetes, Sitagliptin, DPP-4 Inhibitor.
Type 2 diabetes is internationally recognized as one of the major threats to human health in 21st
century.1a It is characterized by reduced insulin sensitivity and elevated glucose levels.1b Hence, Type 2
diabetes is a major risk factor for microvascular and macrovascular complications including retinopathy,
nephropathy, neuropathy, and accelerated cardiovascular disease.2 Currently available oral therapies
include insulin secretagogues, such as sulfonylureas; activators of the peroxisome proliferator-activated
receptor-γ (PPAR-γ), like pioglitazone; glucose-lowering effectors for example metformin, DPP-IV
inhibitors; glucagon-like peptide-1 (GLP-1) analogues or mimetic; and alpha-glucosidase inhibitors.3
However, many patients are unable to achieve satisfactory glycemic control with these available
treatments. Therefore, yet there is a need for exploring novel therapies with distinct mechanism of
action, not dealing solely with reducing blood glucose or lipid but tackling the problem of metabolic
syndrome as a whole.
TGR5 (Takeda G-protein-coupled receptor 5), also known as GPBAR1 is a bile acid G protein-coupled
receptor primarily expressed in liver, gallbladder, intestine, spleen, and brain and activated by bile
acids.4 The activation of TGR5 receptor induce glucagon-like peptide-1 (GLP-1) secretion from primary
intestinal enteroendocrine cells by increasing intracellular cAMP levels. Moreover, it also increases
energy expenditure through the induction of type 2 iodothyronine deiodinase (D2).5 Thus, a novel TGR5
agonist may provide a treatment option for Type 2 diabetes with simultaneous management of glucose
levels, body weight, and associated complications.
Several structurally diverse chemical compounds are reported as TGR5 modulators (Figure 1).6 Towards
our research efforts for the identification of effective treatment for type 2 diabetes mellitus (T2DM),7 we
have recently reported discovery of an orally available potent TGR5 agonist.8 Combination therapy with
anti-diabetic drugs is often used for treatment of type 2 diabetes.9 Thus, we examined the combination
effects of novel TGR5 agonist and DPP-4 Inhibitor on glycemic control in animal models. TGR55 agonist
and DPP-410 have different but complimentary mechanism of action. TGR5 agonist stimulate GLP-1
1

secretion while DPP-4 inhibitors would protect these compounds from GLP-1 degradation. Hence,
combination of TGR5 agonist with DPP4 inhibitor has a strong therapeutic rational for potential
treatment of type 2 diabetes mellitus.
Figure 1. Selected bile acid and non-bile acid TGR5 receptor agonist reported in literature.
Several modeling studies of TGR5 have been reported.11 A TGR5 receptor homology model was
generated, which imparted insight regarding the binding site, and the binding site residues.8a Our
computer aided docking studies suggested that the introduction of linker would be tolerated.
Compounds having incorporated an alkyl chain with O-benzyl linker substituted with various aryl or
heteroaryl groups were synthesized and evaluated as novel TGR5 agonist. Fairly straight forward
synthesis allowed rapid access to analogs of 4 as outlined in Scheme 1. The key intermediate 3 was
obtained from commercially available 2-(substituted-phenyl)acetonitrile using protocol reported by our
laboratories.8 The alkylation of 2-mercapto imidazole derivative with excess of bromo-ethanol followed
by further alkylation with substituted benzyl bromide provided test compounds 4a-4l in good yields and
high chemical purity.8
Scheme 1. Synthesis of Compound 4. Reagent and Conditions: (a) 1-bromoethanol, K2CO3, acetone, r.t., 2 h; (b)
Substituted benzyl bromide, K2CO3, DMF, 90 ᵒC, 3-5 h.
The ability of test compounds to activate TGR5 were measured in luciferase based reporter gene assay.
Overviews of all the compounds that have been synthesized and tested for hTGR5 activity are listed in
Table 1. Compounds 4a and 4b having 2,6-difluoro and 2-chloro-6-fluoro substituents respectively with
an extended linker fits well and shows excellent TGR5 agonistic activity with hTGR5 EC50 of 14.4 nM and
1.6 nM respectively. Substitution with electron withdrawing groups on the terminal phenyl ring was
preferred and compound 4d substituted with 4-cyano exhibited EC50 values of 2.3 nM. However,
increasing polarity by oxidation of the 2-S group on the imidazole ring into corresponding sulfone (4f)
resulted in significant decrease in TGR5 agonistic potency, suggesting polarity was not tolerated on the
central imidazole ring. Interestingly, 4-cyano substituted compound 4d12 was less lipophilic (Log P = 7.2)
compared to compounds 4a and 4b and reference compound 2. The pharmacokinetic properties of 4d
were investigated after single oral administration in female C57BL/6 mice, receiving 30 mg/kg
2

suspension prepared in 0.5% Tween 80 + 99.5% (0.5%) Na-CMC in water. Mean maximum plasma
concentration of 4d was reached at 0.2 h (Tmax). The compound 4d achieved a maximum plasma
concentration of 2.1 µM with an AUC of 2.5 µM∙h and terminal half-life of 1 h.
Table 1. In vitro activity of reference compound and compounds 4a-4l.
hTGR5
EC50 (nM)
CLogPa
Compound
2
R1
R2
X
Ar
2, Exelixis
17.08
7.96
4a
4b
4c
OMe
OMe
Cl
OMe
OMe
Cl
S
S
S
14.4
1.6
8.51
8.56
9.38
35.7
4d
4e
4f
OMe
Cl
OMe
Cl
S
2.3
67.5
7.2
S
8.03
4.88
5.63
OMe
Cl
OMe
Cl
SO2
SO2
94.7
4g
>1000
4h
4i
OMe
OMe
OMe
OMe
OMe
OMe
S
S
S
21.6
17.2
>100
7.27
8.82
6.53
4j
4k
4l
Cl
Cl
S
S
>1000
>100
7.27
6.49
OMe
OMe
a Calculated from QikProp, Schrödinger Software, Release 2017-4.
Next, compound 4d was accelerated for assessment in in vivo GLP-1 secretion study in C57 mice.
Compound 4d alone shows minimal 1.3 fold increase in GLP-1 secretion. However, the GLP-1 stimulation
effect of 4d was enhanced considerably by Sitagliptin (a dipeptidyl peptidase (DPP)-4 inhibitor that
3

prevents GLP-1 degradation) at suboptimal dose (0.1 mg/kg). Total effect of GLP-1 stimulation was 3 fold
over that in the control group (Figure 2). Since GLP-1 has short half-life, it is likely that the TGR5
stimulated GLP-1 is degraded by DPP-4. Hence, concombinant inhibition of DPP-4 showed significant
GLP-1 elevation. Further, we examined the combination effects of compound 4d and Sitagliptin on
plasma glucose levels in C57 mice. Single oral dose of 4d in combination with suboptimal dose of
Sitagliptin significantly lowered the blood glucose excursion, with 48% improvement in AUC glucose,
consistent with our finding of elevated plasma GLP-1 levels. These results highlighted the considerable
therapeutic potential of combining TGR5 agonist 4d with a DPP-4 inhibitor in treatment of type 2
diabetes.
Figure 2. In vivo GLP-1 secretion study of 4d in C57 mice.a
a n = 4 animals/group; C57 mice; po 30 mg/Kg; Formulation: 5% Tween 20 + 0.5% sodium CMC (95%). * P < 0.05; **
P < 0.005 versus control. Error bar indicates SEM.
Molecular docking study was performed to understand the interaction between the TGR5 and the
synthetic agonist 4d (Figure 3), using Glide 5.4, in the generated homology model.8a Tyr 240 appeared to
be one of the significant amino acid that confers stability to the ligand with respect to its H-bond
interactions and aromatic stacking interactions. Meta-methoxy group forms hydrogen bond with side
chain of Tyr 240. The –CN group of compound 4d forms hydrogen bond with the side chain of Asn 147.
he stacking interaction by Trp 237 was observed with fluoro-phenyl group which fits well in the
bottom pocket. In addition to these interactions, compound 4d forms Van der Waals inter actions with
Phe 141, Leu 97, Leu 99, Leu 100 and Ala 182. All these interactions might be contributing to the potent
agonistic activity of this compound.
4

Figure 3. Docking pose of 4d in homology model of TGR5 receptor.
In summary, we have identified a potent series of 2-thio imidazole derivatives as novel class of TGR5
agonist. Furthermore, compound 4d, in combination with Sitagliptin (DPP-4 inhibitor), exhibited an
adequate GLP-1 secretion and glucose lowering effect in animal models. The beneficial therapeutic
potential of combining TGR5 agonist with a DPP-4 inhibitor in treatment of type 2 diabetes has been
demonstrated. Our results indicate that the combination of these agents is a possible therapeutic option
for management type 2 diabetes mellitus.
Acknowledgement
Authors thank management of Zydus Research Centre, Cadila Healthcare Ltd., for support and
encouragement. ZRC Communication No.: 568
Supporting Information
Supplementary data associated with this article can be found, in the online version.
References and notes
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(12) Spectral data of 4-((2-((5-(2-(3,4-dimethoxyphenyl)propan-2-yl)-1-(4-fluorophenyl)-1H-imidazol-
2-yl)thio)ethoxy)methyl)benzonitrile (4d): Waxy solid. IR (KBr, max cm-1): 3020, 2972, 2937 (Ar C-
H-str), 2230 (C≡N), 1510 (C=N), 1462, 1440, 1410 (Ar C=C), 1217 (C−O ether), 758 (C−H); 1H NMR
(400 MHz, DMSO-d6): δ = 7.80 (d, J = 8.4 Hz, 2H, Ar-H), 7.47 (d, J = 8.4 Hz, 2H, Ar-H), 7.07 (s, 1H,
imidazole-4H), 7.04 (t, J = 8.8 Hz, 2H, Ar-H), 6.74 (d, J = 9.2 Hz, 1H, Ar-H), 6.62 – 6.59 (m, 2H, Ar-
H), 6.42 – 6.40 (m, 2H, Ar-H), 4.54 (s, 2H, OCH2Ar), 3.71 (s, 3H, OCH3), 3.64 (t, J = 6.4 Hz, 2H,
OCH2), 3.55 (s, 3H, OCH3), 3.20 (t, J = 6.4 Hz, 2H, SCH2), 1.41 (s, 6H, 2CH3); 13C NMR and DEPT:
DMSO-d6, 100 MHz): 163.35 (C), 160.90 (C), 148.73 (C), 147.72 (C), 144.67 (C), 143.59 (C), 143.24
(C), 139.97 (C), 132.65 (C), 132.65 (CH), 131.61 (CH), 128.26 (CH), 126.10 (CH), 119.30 (C),
118.73 (CH), 115.87 (CH), 115.65 (CH),112.08 (CH), 110.60 (CH), 110.53 (CH), 71.19 (CH2), 69.25
(CH2), 56.10 (OCH3), 55.88 (OCH3), 38.07 (C), 32.20 (CH2), 29.87 (CH3); MS (ESI): m/z (%) = 532.15
(100%) (M+H)+; ESI-Q-TOF-MS: m/z [M+H]+ calcd for [C30H30FN3O3S]+: 531.6464; found:
531.8973; UPLC (% Purity) = 98.87%.
6

 Novel 2-thio-imidazole derivatives have been identified as potent TGR5 agonist
 Compound 4d in combination with Sitagliptin, shows GLP-1 secretion in animal models
 Combination of these agents shows glucose lowering effect in animal models
 TGR5 agonist & DPP-4 Inhibitor combination is a possible option for type-2 diabetes
7

Graphical Abstract
Evaluation of Novel TGR5 Agonist in combination with Sitagliptin for Possible
Treatment of Type 2 Diabetes
Sameer Agarwala*, Santosh Sasanea,b, Jeevan Kumara,b, Prashant Deshmukha, Hitesh Bhayania, Poonam Giria, Suresh Giria,
Shubhangi Somanb, Neelima Kulkarnib and Mukul Jaina
aZydus Research Centre, Cadila Healthcare Ltd. Sarkhej-Bavla N.H. No. 8 A, Moraiya, Ahmedabad - 382210, India; bChemistry
Department, The M.S University, Baroda – 390 002, India;
*E-mail: sameeragarwal@zyduscadila.com or sameer_ag@yahoo.com; Fax: +91-2717-665355. Tel: +91-2717-665555
A series of novel 2-thio-imidazole derivatives were
identified as potent and orally efficacious TGR5 agonist.
Compound 4d, a novel TGR5 agonist, in combination with
Sitagliptin, a DPP-4 inhibitor, has demonstrated an
adequate GLP-1 secretion and glucose lowering effect in
animal models, suggesting a potential clinical option in
treatment of type-2 diabetes.
8