One-pot synthesis of novel tert-butyl-4-substituted phenyl-1H-1,2,3-triazolo piperazine/piperidine carboxylates, potential GPR119 agonists

Article abstract of DOI:10.1016/j.bmcl.2019.126707

We have synthesized a new series of 1,2,3-triazolo piperazine and piperidine carboxylate derivatives using a simple and one-pot click chemistry with significantly reduced reaction times (~5 min) and enhanced reaction yields (~95–98%). The fourteen novel compounds thus synthesized were tested for ability to target GPR119, a G-protein coupled target receptor that plays critical role in regulation of type-2 diabetes mellitus. Four analogs (3e, 3g, 5e and 5g) demonstrated similar or better EC₅₀ values over previously reported AR231453 activity towards GPR119.

Full text of DOI:10.1016/j.bmcl.2019.126707

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One-pot
synthesis
of
novel
tert-butyl-4-substituted
phenyl-1H-1,2,3-triazolo piperazine/piperidine carboxylates,
potential GPR119 agonists
Nagaraju Bashetti, J.V. Shanmukha Kumar, Nareshvarma Seelam,
B. Prasanna, Akiva Mintz, Naresh Damuka, Sriram Devanathan,
Kiran Kumar Solingapuram Sai
PII:
S0960-894X(19)30665-1
DOI:
https://doi.org/10.1016/j.bmcl.2019.126707
BMCL 126707
Reference:
To appear in:
Received date:
Revised date:
Accepted date:
3 July 2019
12 September 2019
14 September 2019
Please cite this article as: N. Bashetti, J.V. Shanmukha Kumar, N. Seelam, et al., One-pot
synthesis of novel tert-butyl-4-substituted phenyl-1H-1,2,3-triazolo piperazine/piperidine
carboxylates,
potential
GPR119
agonists,
(2019),
https://doi.org/10.1016/
j.bmcl.2019.126707
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One-pot synthesis of novel tert-butyl-4-substituted phenyl-1H-1,2,3-triazolo
piperazine/piperidine carboxylates, potential GPR119 agonists
Nagaraju Bashetti,a Shanmukha Kumar JV,a Nareshvarma Seelam,a Prasanna B,a Akiva Mintz,b
Naresh Damuka,c Sriram Devanathan,d Kiran Kumar Solingapuram Saic*
a. Department of Chemistry, Koneru Lakshmaiah Education Foundation, Andhra Pradesh, India
522502; b. Department of Radiology, Columbia University Medical Center, New York, NY,
USA; c. Department of Radiology, Wake Forest School of Medicine, Winston Salem, NC, USA;
d. Medical Guide llc, St. Louis, MO, USA.
Abstract
We have synthesized a new series of 1,2,3-triazolo piperazine and piperidine carboxylate
derivatives using a simple and one-pot click chemistry with significantly reduced reaction times
(~5 min) and enhanced reaction yields (~95-98%). The fourteen novel compounds thus
synthesized were tested for ability to target GPR119, a G-protein coupled target receptor that
plays critical role in regulation of type-2 diabetes mellitus. Four analogs (3e, 3g, 5e and 5g)
demonstrated similar or better EC50 values over previously reported AR231453 activity towards
GPR119.
Triazoles form an important class of heterocyclic compounds and have recently attracted
considerable attention amongst multiple pharmacological and medicinal chemistry groups for
their potential applications of therapeutics. The broad swathe of applicability and potency of
triazole nucleus stems from their ability to act as antiviral, antibacterial, antifungal,
anticonvulsant, antidepressant, anti-inflammatory, and anticancer molecules.1 Triazoles have also

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3 4
been reported to inhibit glycogen synthase kinase-3,2 acts as antagonists of GABA receptors,
agonists of muscarine receptors,5 and as a neuroleptic,6 and these class of compounds also show
anti-HIV-1,7 cytotoxic,8 antihistaminic,9 and antiproliferative activities.10-13 Although several
synthetic methods have been reported to design and synthesize pharmacologically active triazole
14-16
derivatives, they are either too tedious and/or arduous and have low reaction yields.
Thus,
advancements in design and synthesis aspects of novel triazole derivatives would significantly
enhance the broader pharmacological applications.
GPR119, a G-protein coupled target receptor plays a critical role in the potential therapeutic
applications of type 2-diabetes mellitus, cancer and obesity.17 Triazole derivatives were recently
investigated for their agonistic activity on GPR119, based on the hypothesis that such agonists
will advance understandings of (a) molecular mechanisms and (b) structural contributions of
novel derivatives for GPR119, especially in pathogenesis and treatment of obesity and type-2
diabetes mellitus (T2DM). Though multiple approaches are being investigated to synthesize
triazole entities for use against GPR119, a simple one-step synthetic route will significantly
advance the field to identify more biologically active GPR119 analogs. Herein we describe a
simple one-pot synthetic method for novel tert-butyl-4-subsituted phenyl-1H-1,2,3-triazolo
piperazine/piperidine carboxylate derivatives using Cu(I)-assisted click chemistry resulting in
high chemical yields and purities. Furthermore, we have demonstrated their preliminary in vitro
biological potency towards binding of GPR119 in human embryonic kidney (HEK) 293 cells
transfected with GPR119.
AR231543 is one of the early reported, highly potent small-molecule GPR119 agonist (Arena
Pharmaceuticals).18 Oral demonstration of AR231543 in rats significantly improved the
circulating levels of insulin, and glucagon-like peptide 1 (GLP-1). AR231543 lowered the blood

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glucose concentrations in mouse islets in murine models of T2DM.19 ZSY-13 is a [1,2,4] triazole
[4,3-b] pyridazine-based analog reported to be a potent GPR agonist that activates GPR119-
mediated signaling pathway in T2DM.17 We used the core structure of AR231543 and ZSY-13
as our leads to design and synthesize 14 derivatives involving both piperazine- and piperidine-
based triazole units (Figure 1).
Tert-butyl 4-propioloylpiperazine-1-carboxylate (1) was reacted with aryl/alkyl substituted
azides (1.0 eq) (2) in the presence of CuI (10 mol%) , DIPEA (1.5 eq) in DMF (5 mL) at 0 °C
for 5 min. The crude reaction mixture was quenched with ice cold water and the resultant solid
was filtered, dried under vacuum and washed with anhydrous diethyl ether to produce final tert-
butyl-1H-1,2,3-triazole-4-carbonyl piperazine-1-carboxylates (3a-g) in >95% purity and ~90-
97% isolated yields. Similarly, tert-butyl 4-(propioloyloxy)piperidine-1-carboxylate (4) was
reacted with alkyl/aryl-azides (2) under similar conditions as above to obtain the corresponding
tert-butyl-1H-1,2,3-triazole-4-carbonyloxy piperidine-1-carboxylates (5a-g) in >96% purity and
~92-97% isolated yields as shown in Scheme 1. Both the reactions follow typical click chemistry
reaction mechanisms.20

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Scheme.1: One-pot synthesis of 1, 2, 3-triazolo piperazine and piperidine carboxylates
The fourteen new compounds and their isolated yields were shown in Table.1. Structures of the
newly synthesized compounds were completely characterized using (a) elemental analyses [C, H,
1
N], (b) Infra-red spectroscopy, and (c) H and 13C NMR, and (d) Mass spectral studies (see
supplementary material).
Compound Isolated EC50 (nM)
Compound Isolated
yield (%)
EC50 (nM)
12.31±2.11
R
yield
(%)
95
12.34±3.12
13.56±2.45
92
3a
3b
5a
94
94
10.43±2.11
5b

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95
8.78±1.23
93
11.12±3.54
3c
5c
97
92
7.12±1.12
4.21±0.13
97
93
8.17±2.11
3.59±1.01
3d
3e
5d
5e
91
97
12.43±1.63
3.12±0.42
95
96
13.98±2.76
3.67±1.01
3f
5f
3g
5g
Table 1: Library of novel triazole analogs synthesized from the one-pot synthesis, shown in
Scheme 1 and their EC50 values
All the fourteen synthesized derivatives were tested for their efficacy to facilitate glucose-
stimulated insulin secretion (GSIS) using human embryonic kidney HEK-293 cells transfected
with plasmids encoding GPR119 and pCRE-Luc, a reporter designed to monitor cAMP-mediated
signal transduction (HEK-293/GPR119/pCRE-luc). Previous work established that after
treatment with agonists, HEK-293/GPR119/pCRE-luc cells demonstrated enhanced
bioluminescence, which corresponded to increased levels of cAMP on western blot analyses, as
GPR119 stimulates insulin secretion in
a
glucose-dependent manner.17 HEK-
293/GPR119/pCRE-luc cells was therefore used a cell-based screening assay for our library of
17
compounds. AR231453 was used as the reference standard. Briefly, cells expressing GPR119
and CRE-luc were plated at a density of 10,000 cells per well in a 96-well plate. After 24 h in
culture, all 14 compounds at various concentrations (0.1 nM to 1mM) were added. DMSO was
used as a negative control. After an additional 24 h, luciferase activities were measured using the

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Steady-Glo luciferase assay system and an EnVision microplate reader according to the reported
17
assays and manufacturer's instructions. Resultant EC50 values through the luciferase activities
were calculated (Table 1). From the fourteen novel compounds tested, we prioritized four
analogs i.e., 3e, 3g: piperazine- and 5e, 5g: piperidine-based carboxylates, based on the fact that
they exhibit a >3-fold increase in the luciferase activity for further validation. The four analogs,
i.e., 3e, 3g, 5e and 5g displayed EC50 values in the range of standard AR231543’s value of 4.78
nM (Figure 2). This initial screening suggests that (a) triazole substituted piperidine and
piperazine carboxylate derivatives could serve as a potential avenue for GPR119 units and (b)
among those substituents, –PhCN and –PhSO2CH3 groups may enhance the binding affinity of
the analogs. Additionally, these analogs synthesized by one-pot click chemistry in short time,
demonstrate comparable agonistic activity as AR231543 towards GPR119. We are currently
conducting additional in vitro and ex vivo assessments on these derivatives with respect to their
efficacy on activating GLP-1, including in vitro analysis for cGMP activations, desensitization,
and insulin-regulated induction experiments.
8
6
4
2
0
3e
3g
5e
5g
A R 231453
Figure 2: EC50 values of 3e, 3g, 5e and 5g in HEK-293 cells (triplicate) with AR231543 as
reference standard, with p*>0.05 as statistically significant

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In the present study, we have demonstrated a simple, one-pot synthetic strategy to synthesize
novel tert-butyl-4-subsituted phenyl-1H-1,2,3-triazolo piperazine/piperidine carboxylate
derivatives in high reaction yields and chemical purities. We also identified four novel potential
GPR119 agonists and further biological evaluations including, extensive GLP-1 binding
characterizations are currently explored in the lab. The four analogs with strong binding affinity
could serve as promising GLP-1 candidates for the treatment of T2DM.
Acknowledgements:
The authors acknowledge financial support for these studies provided by the Translational Imaging
Program at the Wake Forest School of Medicine, CTSA ULTR001420 (pilot funds to K.K.S.S), P30
ADRC grant 5P30AG049638-03 (to S. Craft), and startup funds from Wake Forest School of Medicine
(to K.K.S.S).
References
1.
Inhibition and Medicinal Chemistry. 2011;26(1): 1-21.
2. Olesen PH, Sørensen AR, Ursø B, et al. Synthesis and in Vitro Characterization of 1-(4-
Kharb R, Sharma PC, Yar MS. Pharmacological significance of triazole scaffold. Journal of Enzyme
Aminofurazan-3-yl)-5-dialkylaminomethyl-1H-[1,2,3]triazole-4-carboxylic Acid Derivatives. A New Class
of Selective GSK-3 Inhibitors. Journal of Medicinal Chemistry. 2003;46(15): 3333-3341.
3.
Holden-Dye L, Krogsgaard-Larsen P, Nielsen L, Walker RJ. GABA receptors on the somatic muscle
cells of the parasitic nematode, Ascaris suum: stereoselectivity indicates similarity to a GABAA-type
agonist recognition site. British journal of pharmacology. 1989;98(3): 841-850.
4.
Biagi G, Giorgi I, Livi O, Lucacchini A, Martini C, Scartoni V. Studies on specific inhibition of
benzodiazepine receptor binding by some C-benzoyl-1,2,3-triazole derivatives. Journal of
Pharmaceutical Sciences. 1993;82(9): 893-896.

Journal Pre-proof
5.
Moltzen EK, Pedersen H, Boegesoe KP, et al. Bioisosteres of Arecoline: 1,2,3,6-Tetrahydro-5-
pyridyl-Substituted and 3-Piperidyl-Substituted Derivatives of Tetrazoles and 1,2,3-Triazoles. Synthesis
and Muscarinic Activity. Journal of Medicinal Chemistry. 1994;37(24): 4085-4099.
6.
Chakrabarti JK, Hotten T, Pullar I, Steggles D. Synthesis and pharmacological evaluation of CNS
activities of [1, 2, 3] triazolo [4, 5-b][1, 5]-, imidazolo [4, 5-b][1, 5]-, and pyrido [2, 3-b][1, 5]
benzodiazepines. 10-Piperazinyl-4H-1, 2, 3-triazolo [4, 5-b][1, 5] benzodiazepines with neuroleptic
activity. Journal of medicinal chemistry. 1989;32(10): 2375-2381.
7.
Alvarez R, Velazquez S, San-Felix A, et al. 1, 2, 3-Triazole-[2, 5-Bis-O-(tert-butyldimethylsilyl)-.
beta.-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'', 2''-oxathiole 2'', 2''-dioxide)(TSAO) Analogs: Synthesis
and Anti-HIV-1 Activity. Journal of medicinal chemistry. 1994;37(24): 4185-4194.
8.
Sanghvi YS, Bhattacharya BK, Kini GD, et al. Growth inhibition and induction of cellular
differentiation of human myeloid leukemia cells in culture by carbamoyl congeners of ribavirin. Journal
of medicinal chemistry. 1990;33(1): 336-344.
9.
Buckle DR, Rockell CJ, Smith H, Spicer BA. Studies on 1, 2, 3-triazoles. 13.(Piperazinylalkoxy)-[1]
benzopyrano [2, 3-d]-1, 2, 3-triazol-9 (1H)-ones with combined H1-antihistamine and mast cell
stabilizing properties. Journal of medicinal chemistry. 1986;29(11): 2262-2267.
10.
Hupe DJ, Boltz R, Cohen C, et al. The inhibition of receptor-mediated and voltage-dependent
calcium entry by the antiproliferative L-651,582. Journal of Biological Chemistry. 1991;266(16): 10136-
10142.
11.
dopamine D3 receptor selective ligands. Bioorganic & medicinal chemistry. 2013;21(11): 2988-2998.
12. Hayatshahi HS, Xu K, Griffin SA, et al. Analogues of Arylamide Phenylpiperazine Ligands To
Li A, Mishra Y, Malik M, et al. Evaluation of N-phenyl homopiperazine analogs as potential
Investigate the Factors Influencing D3 Dopamine Receptor Bitropic Binding and Receptor Subtype
Selectivity. ACS Chemical Neuroscience. 2018;9(12): 2972-2983.
13.
Peng X, Wang Q, Mishra Y, et al. Synthesis, pharmacological evaluation and molecular modeling
studies of triazole containing dopamine D3 receptor ligands. Bioorganic & medicinal chemistry letters.
2015;25(3): 519-523.
14.
Wang B-L, Zhang L-Y, Zhan Y-Z, et al. Synthesis and biological activities of novel 1,2,4-triazole
thiones and bis(1,2,4-triazole thiones) containing phenylpyrazole and piperazine moieties. Journal of
Fluorine Chemistry. 2016;184: 36-44.
15.
Anil Kumar BSP, Harsha Vardhan Reddy K, Satish G, Uday Kumar R, Nageswar YVD. Synthesis of
β-hydroxy-1,4-disubstituted-1,2,3-triazoles catalyzed by copper ferrite nanoparticles in tap water using
click chemistry. RSC Adv., 2014;4(105): 60652-60657.
16.
via Click Chemistry. The Journal of Organic Chemistry. 2008;73(1): 323-326.
17. Zhang S-y, Li J, Xie X. Discovery and characterization of novel small molecule agonists of G
protein-coupled receptor 119. Acta pharmacologica Sinica. 2014;35(4): 540-548.
18. Shah U, Kowalski TJ. Chapter Sixteen - GPR119 Agonists for the Potential Treatment of Type 2
Punidha S, Sinha J, Kumar A, Ravikanth M. First Triazole-Bridged Unsymmetrical Porphyrin Dyad
Diabetes and Related Metabolic Disorders. In: Litwack G, ed. Vitamins & Hormones. Vol 84. Academic
Press; 2010:415-448.
19.
molecules for GPR119. Biochemical Pharmacology. 2016;119: 66-75.
20. Pachón LD, van Maarseveen JH, Rothenberg G. Click Chemistry: Copper Clusters Catalyse the
Cycloaddition of Azides with Terminal Alkynes. Advanced Synthesis & Catalysis. 2005;347(6): 811-815.
Hassing HA, Fares S, Larsen O, et al. Biased signaling of lipids and allosteric actions of synthetic

Figure 1

Figure 2