Discovery of a potent G-protein-coupled receptor 119 agonist for the treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmc.2021.116071

The ever-growing prevalence of Type-2 diabetes in the world has an urgent need for multiple orally effective agents that can regulate glucose homeostasis. G-Protein coupled receptor 119 (GPR 119) agonists have demonstrated the glucose-dependent insulin secretion and showed beneficial effects on glycemic control in humans and/or relevant animal models. Herein, we describe our efforts towards identification of a potent and oral GPR 119 agonist 13c (ZY-G19), which showed in vitro potency in the cell-based assay and in vivo efficacy without exerting any significant signs of toxicity in relevant animal models.

Full text of DOI:10.1016/j.bmc.2021.116071

Bioorg. Med. Chem. 35 (2021) 116071
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a potent G-protein-coupled receptor 119 agonist for the
treatment of type 2 diabetes
,
b
,
,
Suresh Polaa *, Shailesh R. Shahb *, Harikishore Pingalia, Pandurang Zawarea, Baban Thubea,
Pankaj Makadiaa, Hoshang Patela, Debdutta Bandyopadhyaya, Akshyaya Ratha, Suresh Giria,
Jitendra H. Patela, R.K. Ranvira, S.R. Sundara, Harilal Patela, Jeevan Kumara, Mukul R. Jaina
a Zydus Research Centre, Sarkhej-Bavla N.H 8A Moraiya, Ahmedabad 382210, India
b Department of Chemistry, Faculty of Science, M. S. University of Baroda, Vadodara 390002, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
The ever-growing prevalence of Type-2 diabetes in the world has an urgent need for multiple orally effective
agents that can regulate glucose homeostasis. G-Protein coupled receptor 119 (GPR 119) agonists have
demonstrated the glucose-dependent insulin secretion and showed beneficial effects on glycemic control in
humans and/or relevant animal models. Herein, we describe our efforts towards identification of a potent and
oral GPR 119 agonist 13c (ZY-G19), which showed in vitro potency in the cell-based assay and in vivo efficacy
without exerting any significant signs of toxicity in relevant animal models.
GPR119 agonist
Thiazolidinediones (TZDs)
Type 2 diabetes mellitus (T2DM)
Insulin sensitizers
1. Introduction
(rhodopsin-type) GPCR family and is highly expressed in pancreatic
β-cells and the K and L cells of the gastrointestinal tract.13–15 It has also
Diabetes is one of the major causes of death worldwide in this cen-
tury after heart disease and cancer.1 According to the International
Diabetes Federation (IDF) data, 463 million individuals were suffering
from diabetes world over in 2019, which is expected to significantly
increase to 700 million by 2045.2 If untreated, diabetes also leads to
several other complications, such as cardiovascular disease, retinopathy,
nephropathy, neuropathy, dental diseases and kidney related dis-
eases.3–10 Type 2 diabetes mellitus (T2DM) is a complex chronic disease
characterized by metabolic disorder and hyperglycemia due to insulin
resistance, hepatic glucose overproduction and/or insufficient insulin
secretion. In addition to genetic predisposition, obesity is an added risk
factor associated with growing incidence of type 2 diabetes mellitus and
many patients with T2DM are obese. These patients are often accom-
panied by increased cardiovascular risk factors. Innovative new thera-
pies that could improve glucose metabolism and reduce body weight
and excess food intake will provide benefits to such patients. Although
there are a number of therapeutic options such as sulfonylureas, met-
formin, glitazones and glinides, they are unable to get satisfactory gly-
cemic control without adverse side effects. Thus, there is an urgent need
of novel therapeutic approaches for the treatment of T2DM by a good
glycemic control without side effects.11,12
been shown to be expressed in peripheral tissues including skeletal
muscles, cardiac muscles and liver. Coupling of GPR119 to Gα stimu-
latory proteins induces adenylate cyclase activity to increase intracel-
lular cyclic adenosine monophosphate (cAMP), which further regulates
GPR119-mediated release of GLP-1. This GPR119-mediated release of
GLP-1 from intestinal L-cells is shown in vitro to be protein kinase A
(PKA) dependent. Recent work has indicated a potential signaling axis
between GPR119 and 5-adenosine monophosphate-activated protein
kinase alpha (AMPKα), a key regulator of cellular energy state in
metabolically active tissues. The roles of GPR119 in regulating glucose
homeostasis and appetite are substantiated by its pattern of expression.
In pancreatic β-cell, activation of GPR119 is expected to potentiate
glucose-stimulated insulin secretion similar to GLP-1 and GIP. The
activation of GPR119 receptor expressed in enteroendocrine cells of
intestine is shown to modulate incretin hormone release, a key function
in maintaining glucose homeostasis. Thus the effects of GPR119 acti-
vation on glucose homeostasis are two fold, a direct action of glucose
stimulated insulin secretion in pancreatic β-cell and an indirect action of
stimulating the release of GLP-1 and GIP in enteroendocrine cells of
intestine.16–20 This glucose-dependent dual mechanism of action makes
GPR119 a promising target for discovery of anti-diabetic agents with
low risk of hypoglycemia and body weight gain. Several phospholipids
G Protein-coupled receptor 119 (GPR119) is a member of class A
* Corresponding authors at: Zydus Research Centre, Sarkhej-Bavla N.H 8A Moraiya, Ahmedabad 382210, India (S. Pola).
E-mail addresses: suresh.pola@zyduscadila.com (S. Pola), shailesh-chem@msubaroda.ac.in (S.R. Shah).
https://doi.org/10.1016/j.bmc.2021.116071
Received 7 December 2020; Received in revised form 30 January 2021; Accepted 4 February 2021
Available online 13 February 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
and lipid amides such as lysophosphatidylcholine (LPC), oleoylethano-
lamide (OEA) and oleoyldopamine (OLDA) have been identified as
endogenous ligands for GPR119. However, due to the low potency and
poor selectivity of these endogenous agonists towards GPR119 receptor,
many synthetic small molecule GPR119 agonists have been discovered
and disclosed by several research groups over the years.21–24 These
Knoevenagel condensation of benzaldehyde intermediates 6a-e, 7a-e,
11a-e and 12a-e with thiazolidine-2,4-dione in the presence of piper-
idiniumbenzoate in toluene afforded the test compounds 8a-e, 9a-e,
13a-e and 14a-e in good yield with high chemical purity.31
3.2. In-vitro evaluation
intense efforts led to the development of compounds till clinical phase as
25,26
shown in Fig. 1.
However, due to the tachyphylaxis, these com-
pounds failed to show desired efficacy in the clinical trials 27–29 and none
GPR119 agonistic activity of the synthesized compounds were
measured using a cAMP assay in the human GPR119 cell line. GPR119
agonist AR231453 compound was used as the positive control and the
results are shown in Table 1 and Table 2. cAMP stimulation is measured
for all the synthesized compounds and the activity is reported as EC50
values and %max of stimulation compared to maximal effect at 1 µM of
AR231453. As hypothesized, we synthesized compounds 8, 9, 13 and
14 by replacing pyridine ring of Arena compound with benzylidene
thiazolidinedione. To start with compounds 8a-e possessing methyl-
pyrimidine as central ring were synthesized and evaluated for GPR-119
agonistic activity. The results are represented in Table 1. Initial com-
pound 8a, methyl carbamate derivative showed weak GPR119 agonistic
activity with EC50 1.4 µM, while its homologue, the ethyl carbaamte 8b
showed improvement in agonistic activity (EC50: 157 nM; Emax: 141%).
This 9 fold difference in the potency encouraged us to study the effect of
various substitutions on piperidine nitrogen. We then synthesized
compound 8c and 8d with t-butyl and i-butyl carbamates respectively.
Both the compounds demonstrated significant improvement in potency
compared to their lower homologues 8a and 8b with EC50 of 88 nM (8c)
and 75 nM (8d). Further increase in bulk at this position with benzyl
carbamate found detrimental as evident from compound 8e with EC50 of
805 nM and Emax of 43.3%. Having done this, we then intended to know
the role of methyl group on central pyrimidine ring and hence synthe-
sized another series of compounds 9a-e without methyl group on py-
rimidine ring. Compound 9a with methyl carbamate was found inactive
whereas the ethyl carbamate 9b showed moderate potency with EC50
790 nM. As expected, compound 9c with t-butyl carbamate showed
potent GPR119 agonistic activity with EC50 of 130 nM and Emax of
121%. Surprisingly compound 9d with i-butyl carbamate group found
10 fold inferior to its methylated counterpart 8d. Interestingly a contrast
trend was observed with benzyl carbamates. Compound 9e with EC50 of
107 nM and Emax of 88% found 8 fold potent than its methylated
counterpart 8e. Our next task in this endeavour was to optimise the
position of thiazolidinedione on phenyl ring. To do so we have synthe-
sized meta-benzylidene thiazolidinedione derivatives 13a-e and 14a-e
with a similar substitution pattern as in compounds 8 and 9 and the
results are presented in Table 2. Methyl carbamates 13a and 14a are
found inactive and this is in line with our expectation as their corre-
sponding para-substituted analogues also failed to show any activity.
Compound 13b with ethyl carbamate not showing activity unlike its
para oriented counterpart 8b is surprising. However compound 14b
with EC50 of 819 nM found to be a weak agonist like its para substituted
could reach the market.
2. Designing
Arena compound (I) (Fig. 1) is reported to be a selective and potent
agonist of the GPR119 receptor across several species (EC50, melano-
phore: 2 nM, human; 1 nM, dog; 35 nM, cynomolgus monkey; 41 nM,
mouse; 44 nM, rat) and possesses aqueous solubility with no appreciable
inhibition of at least five cytochrome P450 enzymes (CYP2C9, 15 uM;
1A2 2D6 3A4, >40 uM; 2C19, 10 uM). Compound (I) also demonstrated
a dose-dependent inhibition of glucose excursion in the OGTT experi-
ment in male SD rats (22%, 0.3 mg/kg p.o.; 24%, 3 mg/kg p.o.; 70%, 30
mg/kg p.o.).29 However, further development of this compound has
been suspended for reasons not disclosed. In our efforts to discover
promising and safe GPR119 agonists, we replaced 2-methyl pyridine, a
polar head of Arena compound with clinically proven pharmacophore
benzylidenethiazolidinedione, as a novel heterocyclic head and identi-
fied this moiety as a pyridine surrogate (Fig. 2). Thiazolidinediones
(TZDs) are insulin sensitizers having a pleotropic pharmacology
including reduction of insulin resistance, a root cause of diabetes.
Importantly these agents also preserve pancreatic beta cell function or
mass better than insulin secretagogues such as sulfonylureas. However
The TZD class is reported to cause weight gain in animal models.30
Herein, we report the synthesis and biological evaluation of novel
thiazolidinedione derivatives as potent GPR119 agonists devoid of
adverse side effects.
3. Results and discussion
3.1. Chemistry
The general synthetic procedure leading to the compounds 8a-e, 9a-
e, 13a-e and 14a-e is outlined in Scheme1. Coupling reaction of
commercially available substituted 4,6-dichloropyrimidines 1a and 1b
with various N-substituted hydroxy piperidines 2a-e using t-BuOK as
base in N, N-dimethylformamide produced piperidine intermediate 3a-e
and 4a-e. Treatment of 3a-e and 4a-e with 4-hydroxybenzaldehyde 5 or
3-hydroxybenzaldehyde 10 in the presence of K2CO3 in N, N-dimethy-
lacetamide gave para substituted benzaldehyde 6a-e and 7a-e or meta
substituted benzaldehyde derivatives 11a-e and 12a-e respectively. The
Fig. 1. The synthetic ligands of GPR119 Agonists.
2

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
Fig. 2. Designing Strategy.
Scheme 1. Reagents and Conditions: (i) t-BuOK, THF, 0 ◦C, 30 min, (ii) K2CO3, DMA, 138 ◦C, 3 h, (iii) Thioazolidine-2,4-dione (TZD), Piperidine, benzoic acid,
Toluene, Reflux, 7 h.
Table 1
Table 2
GPR119 agonistic activities of compounds 8a-e and 9a-e.
GPR119 agonistic activities of compounds 13a-e and 14a-e.
Comp. No.
R
R1
hGPR119 agonist Activity
Comp. No.
R
R1
hGPR119 agonist Activity
% maxa
EC50(nM)
% maxa
EC50(nM)
13a
14a
CH3
H
IA
ND
8a
9a
CH3
H
1414.0
IA
53.3
ND
1870
33.7
13b
CH3
IA
ND
8b
CH3
157.4
141.3
14b
H
819
43.3
9b
H
790
51.84
13c
14c
CH3
H
43
160
8c
9c
CH3
H
88
68.8
125.3
78.3
130
121.6
13d
CH3
IA
ND
8d
CH3
75.1
70.18
14d
H
720
62.4
9d
H
745
51.0
13e
14e
CH3
H
105.7
153.4
137.5
98.3
8e
9e
CH3
H
805
43.3
88.3
107.4
AR231453
a
6.0
100
AR231453
a
6.0
100
%max: cAMP stimulation % compared to maximal effect at 1 µM AR231453.
%max: cAMP stimulation % compared to maximal effect at 1 µM AR231453.
in vitro GPR119 agonist structure activity relationship with different
carbamate substitutions on piperidine ring compounds 8c, 8d and 13c
were selected for evaluation of efficacy in relevant animal models.
analogue 9b. Interestingly, compound with t-butyl carbamate 13c was
found highly potent with EC50 of 43 nM and Emax of 160% and came out
as the most potent compound of this series. Compound 14c was also
found active but inferior to 13c in terms of potency as well as efficacy.
Surprisingly i-butyl carbamate 13d failed to show activity however
compound 14d showed moderate potency comparable to its para
analogue 9d. Among benzyl carbamates 13e found to be 8 fold potent
than its corresponding para analogue (8e) with EC50 of 105 nM and Emax
137% whereas compound 14e found equipotent to 9e. From the above
3.3. In-vivo studies
During oral glucose tolerance test (oGTT) an acute screening assay,
blood glucose levels were monitored for 120 min after oral adminis-
tration of glucose in C57 BL/N6 mice which are dosed with 50 mg/kg 8c,
3

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
Effect on Glucose Excursion
50
40
30
20
10
0
Compound 8c (50 mg/kg,p.o.)
Compound 8d (50 mg/kg,p.o.)
Compound 13c (50 mg/kg,p.o.)
Sitagliptin (50 mg/kg,p.o.)
Each bar represents mean ± s.e.m.
Fig. 3. The oGTT effect of compounds in C57 mice.
8d and 13c along with Sitagliptin 30 min prior to the glucose challenge
and data is presented in Figure 3. Compounds 8c and 13c significantly
reduced plasma glucose levels showing 34.6% and 32.4% improvement
in glucose excursion AUC respectively comparable sitagliptin, while
Compound 8d is found inferior with modest 24% improvement in
glucose excursion AUC.
all the tested doses dose as shown in Fig. 5. The effect of compound 13c
on glucose dependent insulin secretion was also evaluated where 50 mg/
kg/day dose of 13c showed significant increase in secretion of insulin at
10 and 30 min after glucose administration (Fig. 6).
The effect of compound 13c on plasma active GLP-1secretion at
various time points in male db/db mice after single dose oral adminis-
tration was also evaluated. Tretament with 13c at 50 mg/kg has
increased active GLP-1 levels by an impressive 2.6 fold at 10 min after
glucose administration. Further when co-administered with sitagliptin,
a DPP-4 Inhibitor, 13c showed a synergistic and significant elevation in
the active GLP-1 levels compared to that caused by either 13c or sita-
gliptin alone as shown in Fig. 7.
Having established primary in-vitro and in-vivo correlation, we then
intended to study the pharmacokinetic parameters of 8c and 13c in
Sprague Dawley rats at 25 mg/kg/day oral dose and the results are pre-
sented in Table 3. Based on impressive and superior pharmacokinetic
parameters over 8c, compound 13c (Cmax of 4956 ng/ml and AUC of
19,056 h.ng/ml) was chosen for evaluation of pharmacokinetic pa-
rameters in higher species.
In order to investigate the potential to reduce body weight gain, 13c
was dosed to diet induced obese Sprague Dawley rats for 28 days. No
significant effect on body weight gain was observed in the animals
treated at 25 and 50 mg/kg/day (Fig. 8). However, the graph shows a
trend of lowering in body weight towards the end of the treatment (50
mg/kg/day dose) which indicates that treatment if prolonged further
may result in further reduction in body weight gain.
13c, when dosed orally to Male Wistar rats and Beagle dogs at 10 mg/
kg exhibited oral bioavailability of 21.67% and 5.17% respectively with
decent exposure and good half-life in both the species as shown in
Table 4.
Subsequently in an oGTT experiment in db/db mice, a diabetic dis-
ease model, 13c showed a significant 69.1% improvement of in glucose
excursion AUC when dosed orally at 50 mg/kg along with sitagliptin as
positive control (Fig. 4).
Having achieved the primary goal of identifying potent and effica-
cious GPR119 agonist, our next end point of this endeavour was to study
the toxicity profile of the lead compound 13c with repeated dose
administration by oral gavage over the period of 28 days in Wistar rats.
Groups of 10 animals of each male female rats were dosed orally with
100, 200 and 400 mg/kg compound 13c once a day for 28 days. These
doses corresponds to 50×, 100× and 200× of the ED50 (considering
ED50 as ~2.0 mg) found from the efficacy study in db/db mice. There
were no significant treatment related clinical manifestations noted in
any of the treated group animals and there was no treatment mortality
Encouraged with the positive results, we then conducted an oGTT
experiment to study time and dose dependent effects of compound 13c
on glucose excursion in db/db mice. Compound 13c in doses ranging
from 1 to 50 mg/kg and sitagliptin at 50 mg/kg were administrated
orally for 14 days and blood glucose levels were measured for 0–120 min
after 30 min of the compound administration and AUC was calculated
for Day1 and Day14. Compound 13c showed significant and dose
dependent reduction of glucose excursion compared to day 1 vs day 14 at
Table 3
Pharmacokinetic parameters in fasted SD Rats at 25 mg/kg dose.
Compound
Tmax
Cmax
T1/2
Kel
AUC (0-t)
AUC (0-inf)
(h)
(ng/ml)
(h)
(h-1)
(h.ng/ml)
(h.ng/ml)
8c
2.25 ± 0.63
1.75 ± 0.25
834.78 ± 71.05
4.37 ± 0.33
3.09 ± 0.99
0.16 ± 0.01
0.33 ± 0.11
8108.40 ± 652.07
8310.72 ± 654.23
13c
4955.84 ± 738.16
19055.89 ± 1167.41
19709.18 ± 1370.42
Table 4
Pharmacokinetic parameters of compound 13c at 10 mg/kg oral dose.
Species
Tmax
Cmax
T1/2
Kel
AUC (0-t)
AUC (0-inf)
(h)
(ng/ml)
(h)
(h-1)
(h.ng/ml)
(h.ng/ml)
Wistar Rat
Beagle Dog
1.50 ± 0.55
3.00 ± 2.00
256.41 ± 106.37
1047.88 ± 46.28
12.30 ± 4.41
19.31 ± 5.66
0.07 ± 0.05
0.04 ± 0.01
1766.77 ± 299.26
1898.35 ± 352.46
14868.45 ± 6253.72
15195.01 ± 6311.96
4

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
500
450
400
350
300
250
200
150
100
50
Water Vehicle control
Vehicle control
Water Control
20
18
16
14
12
10
8
Vehicle control
Sitagliptin( 50 mg/kg)
Compound 13c (10 mg/kg)
Compound 13c ( 50 mg/kg)
Sitagliptin ( 50 mg/kg)
Compound 13c ( 50 mg/kg)
**
*
6
4
2
0
10 min
30 min
0
-30
0
30
60
90
120
150
Fig. 6. Effect on glucose dependent insulin secretion of compound 13c using
db/db model.* Significantly different from Vehicle control group (p < 0.05), **
(p < 0.01).
Time (min)
Fig. 4. The oGTT effect of compound 13c on serum glucose at various time
points in db/db mice after single oral dose administration. Each line represents
mean ± s.e.m.
liver enzymes (ALP, AST, and ALT), hemoglobin, albumin and creatinine
at any doses in both sex animals. Histopathological examination did not
reveal any treatment related adverse effects up to 400 mg/kg in both the
sexes. Although changes were noticed in liver, these were minimal and
could not be attributed to adverse effects of the compound treatment. No
evidence of local toxic effects noticed after repeated oral treatment with
Compound 13c in the gastrointestinal tract of the animals. Oral
administration of compound 13c for 28 days at the dose levels of 100,
200 and 400 mg/kg did not affect the survival of Wistar rats. No adverse
changes were noticed during biochemical estimations, organ weights
and histopathological examination. No observed adverse effect level
(NOAEL) of Compound 13c was found to be more than 400 mg/kg in
rats. These results clearly indicate that treatment with 13c on rodents
did not exert any significant side effects even at 200 times higher dose
than ED50 value.
occurred in this study up to the dose level of 400 mg/kg. No treatment
related adverse neurobehavioral changes were noticed in this study at
400 mg/kg. Food consumption was comparable to that of control groups
throughout the study period in both the treated groups of both male and
female animals. There were no findings of toxicological significance
during ophthalmic examination at 400 mg/kg in both the sexes. Animals
were sacrificed on day 29 and data analysis of blood biochemical pa-
rameters, organ weight ratios and histopathological findings.
No significant change in body weights of animals of both the sex was
observed. Analysis of organ to body weight ratios (Table 5) did not show
evidence of toxicity attributed to compound treatment at least at 100
mg/kg dose, which is 50× of ED50. There was no changes in relative
organ weight noted up to 400 mg/kg in male rats at the end of treatment
periods. Although statistically significant changes such as higher
weights of adrenals at 400 mg/kg, heart at 200 mg/kg, brain at 100 &
200 mg/kg, thymus at 400 mg/kg was noticed, none of these effects
were clearly associated with any histomorphological changes during
microscopic examination. No treatment related changes in relative
organ weights were evident in female rats either at the end of treatment
period or at post recovery termination. Similarly no significant alter-
ations were observed in biochemical parameters (Table 6) except the
decrease in urea levels in both sex animals and marginal decrease in
glucose levels in male animals. No significant changes were observed in
4. Docking study
To explain the GPR119 agonistic activity of compound 13c, docking
studies have been carried out using Glide,32 the automated docking
program implemented in the Schrodinger package. The geometry of
compound to be docked was subsequently optimized using the Lig-
Prep.33 The scoring function, binding mode and H-bonds were used to
assess the binding affinity of the compounds. A homology model of
GPR119 receptor was constructed based on template protein crystal
structure (PDB ID: 2R4R) with sequence identity 26.94% using Prime
module of Schrodinger, 34 which provided insight regarding the binding
site and binding site residues. The grid file has been generated by
specifying pocket identified by sitemap. The Arena molecule
(AR231453) was docked into the homology model using the induced fit
docking (IFD) protocol.34 The IFD is based on the docking program Glide
with the refinement module in Prime (Schrodinger, Inc.), which was
reported to accurately predict the ligand binding modes and concomi-
tant structural changes in the receptor. The results of docking have been
shown in Fig. 9. AR231453 molecule fits snugly to binding site and
adopts an extended conformation. The fluoro-phenyl ring forms pi-pi
interaction with Phe 249 and 1, 2, 4-oxadiazole ring forms a pi-pi
interaction with side chain of Tyr 271. This is also stabilized by
cation-pi interaction between nitro group of AR231453 and Phe 168.
These three interactions seem to stabilize the molecule in binding site.
Compound 13c also adopts an extended conformation similar to
AR231453 molecule in the binding site. 5-methyl pyrimidine forms a pi-
pi interaction with the side chain of Phe 168. In addition to this inter-
action, compound 13c forms Van der Waals interactions with Phe 172,
Phe 249, Arg 270, Ty2 271 and Gln 66. These interactions and similar
Vehicle control
30000
Comp 13c (1mg/kg)
Comp 13c (3mg/kg)
Comp 13c (10mg/kg)
20000
Comp 13c (25mg/kg)
Comp 13c (50 mg/kg)
Sitagliptin (50mg/kg)
*
10000
**
*
**
**
0
**
Day-1
Day-14
-10000
Fig. 5. The effect of compound 13c on AUC glucose (mg/dl.min) in male db/db
mice single oral dose administration (glucose load was 2gm/kg/10 mL, p.o.)
Each bar represents mean ± s.e.m. * Significantly different from Vehicle control
group (p < 0.05), ** (p < 0.01).
5

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
180
160
140
120
100
80
***
40
35
30
25
20
15
10
5
**
Vehicle + vehicle
Vehicle + Compound 13c (30)
Sita (0.5) + Vehicle
Sita (0.5) + Compound 13c (30)
Vehicle control
Compound 13c (10 mg/kg)
Compound 13c (50 mg/kg)
60
**
40
20
0
-30 min
10 min
30 min
0
Fig. 7. (i) Effect on plasma active GLP-1 of compound 13c using db/db model. Each bar represents mean ± s.e.m. ** significantly different from Vehicle control
group (p < 0.01), (ii) Plasma active GLP-1 (pg/ml) at 10 min after glucose load. ** Significantly different from Vehicle control group (p < 0.001), *** (p < 0.0001).
efforts on SAR generation lead to the discovery of compound 13c with
NIN vehicle
potent in vitro activity, in vivo efficacy in various animal models and
425
HFD vehicle
decent pharmacokinetic profile with no signs of toxicity at dose 200
times higher than efficacy dose. 13c (ZY-G19) has been identified as a
Compound 13c @25mg/kg
400
candidate for clinical development. Further follow-up of ZY-G19 will be
Compound 13c @50mg/kg
communicated in subsequent publication.
Sibutramine @10mg/kg
375
6. Experimental section
350
6.1. In vitro hGPR119 agonistic activity
325
Chinese Hamster Ovarian (CHO-K1) cells stably transfected with
◦
human GPR119 were grown at 37 C, 95% O2 and 5% CO2 in 75 cm
300
flasks containing DMEM/F12 (1:1) media with added 10% FBS (Gib-
coVR), Geneticin (GibcoVR) and grown until 90% confluent. Cells were
275
then washed (PBS), lifted with cell dissociation solution (InvtrogenVR),
counted and used for cAMP stimulation assays and/or passaging (1:10).
250
Following the manufacturer’s instructions for the LANCEVR Ultra cAMP
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Day of Treatment
assay (Perkin Elmer), cell transfected with hGPR119 were centrifuged
(1000 rpm, 5 min), re-suspended in cAMP assay buffer (HBSS, 0.1%
BSA, 0.5 mM IBMX and 5 mM HEPES) and seeded at 5000 cells/well in
optiplate-384 (Perkin Elmer). The cells were treated with compounds or
reference standard AR231453 over a range of concentrations (10 lM-0.6
lM) and incubated for 1 h. The cells were then lysed and cAMP was
quantified using Arbor Assay’s DetectX® Enzyme immuno Assay kit for
direct cAMP measurement. EC50 of the compounds were calculated
using Graph Pad Prism.
Fig. 8. The effect of compound 13c on body weight (gm) in 28 days of treat-
ment in DIO male SD Rats. Each line represents Mean ± SEM.
binding mode with respect to AR231453 molecule might be contrib-
uting to the potent agonistic activity of compound 13c.
5. Conclusion
6.2. Pharmacokinetic studies
In conclusion, we have identified benzylidenethiazolidinedione as a
novel polar head for discovering a new series of GPR119 agonists. The
All the animals used in the study were procured from the Animal
Table 5
Relative organ weightsa of Wistar rats administered orally with 13c for 28 days.
Dose (mg/kg) Heart
Liver
Kidneys
Spleen
Adrenals
Brain
Testes
Epididymides
Thymus
Male
Control
100
0.333 ± 0.036
0.347 ± 0.021
3.142 ± 0.231 0.787 ± 0.078 0.206 ± 0.026 0.020 ± 0.003
3.237 ± 0.160 0.831 ± 0.057 0.222 ± 0.010 0.023 ± 0.002
0.766 ± 0.038
1.248 ± 0.073 0.361 ± 0.028 0.148 ± 0.023
0.830* ± 0.074 1.228 ± 0.322 0.371 ± 0.056 0.167 ± 0.028
0.824* ± 0.038 1.289 ± 0.105 0.406 ± 0.058 0.175 ± 0.037
200
0.363* ± 0.024 3.304 ± 0.160 0.847 ± 0.067 0.220 ± 0.024 0.023 ± 0.002
400
0.338 ± 0.017
3.220 ± 0.253 0.845 ± 0.064 0.211 ± 0.010 0.023* ± 0.004 0.809 ± 0.038
1.230 ± 0.060 0.365 ± 0.027 0.196** ± 0.045
Female
Control
100
Ovaries
Uterus
0.380 ± 0.037
0.410 ± 0.030
0.394 ± 0.016
0.367 ± 0.044
3.219 ± 0.221 0.801 ± 0.071 0.244 ± 0.032 0.045 ± 0.007
3.243 ± 0.268 0.875 ± 0.125 0.241 ± 0.027 0.046 ± 0.007
3.285 ± 0.262 0.840 ± 0.057 0.242 ± 0.033 0.044 ± 0.010
3.172 ± 0.301 0.806 ± 0.093 0.228 ± 0.029 0.042 ± 0.006
1.100 ± 0.078
1.103 ± 0.066
1.114 ± 0.036
1.069 ± 0.051
0.091 ± 0.009 0.335 ± 0.108 0.260 ± 0.025
0.089 ± 0.013 0.345 ± 0.103 0.241 ± 0.022
0.089 ± 0.011 0.330 ± 0.142 0.257 ± 0.048
0.081 ± 0.007 0.429 ± 0.142 0.244 ± 0.043
200
400
*
Significant from control group at 5% level (p < 0.05).
Significant from control group at 1% level (p < 0.01).
Presented as organ-to-body weight percent ratio.
**
a
6

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
Table 6
Biochemical Parameters of Wistar rats administered orally with 13c for 28 days.
Dose (mg/kg)
Globulin (g/dl)
Glucose (mg/dl)
Creatinine (mg/dl)
ALP (U/L)
AST (U/L)
ALT (U/L)
Albumin (g.dl)
Urea (mg/dl)
Male
Control
100
2.62 ± 0.12
2.50 ± 0.11
2.64 ± 0.11
2.53 ± 0.15
89.02 ± 13.41
87.56 ± 12.75
75.19* ± 9.02
82.17 ± 10.39
0.61 ± 0.04
0.57 ± 0.03
0.57 ± 0.06
0.57 ± 0.05
174.77 ± 25.42
152.22 ± 24.91
164.68 ± 37.51
177.44 ± 46.84
119.40 ± 9.30
114.13 ± 24.86
116.98 ± 15.65
116.36 ± 16.65
30.47 ± 2.63
28.90 ± 3.98
30.15 ± 2.96
29.60 ± 4.46
3.56 ± 0.08
3.47 ± 0.08
3.61 ± 0.07
3.54 ± 0.07
58.14 ± 9.13
55.36 ± 10.27
55.62 ± 10.44
51.15 ± 6.00
200
400
Female
Control
100
2.44 ± 0.18
2.39 ± 0.11
2.42 ± 0.11
2.42 ± 0.13
78.40 ± 8.36
79.83 ± 8.27
82.10 ± 7.81
76.54 ± 8.67
0.60 ± 0.04
0.63 ± 0.05
0.66 ± 0.07
0.60 ± 0.07
100.14 ± 21.98
103.72 ± 28.12
91.73 ± 23.20
100.97 ± 26.55
128.84 ± 21.42
120.42 ± 23.17
128.19 ± 23.17
123.93 ± 14.28
21.28 ± 2.31
22.37 ± 1.82
22.20 ± 2.31
23.30 ± 3.22
3.57 ± 0.14
3.59 ± 0.11
3.65 ± 0.08
3.57 ± 0.14
48.09 ± 5.30
47.84 ± 4.89
46.42 ± 5.37
43.20 ± 7.38
200
400
*
Significant from control group at 5% level (p < 0.05).
Breeding Facility of Zydus Research Center. All animal studies were
conducted according to protocols reviewed and approved by the Insti-
tutional Animal Care and Ethics Committee at the Zydus Research
Centre.
6.3. In vivo experiments
6.3.1. Oral glucose tolerance tests (OGTT) in C57/BL6 mice
C57/BL6 mice of 6–8 week age were used for this experiment. Ani-
mals were grouped based on non-fasting serum glucose levels and were
kept on fasting overnight (day before OGTT). On the experiment day,
each animal received a single dose of vehicle/test compounds (50 mg/
kg) administered orally, 30 min post dosing animals were bled for basal
glucose level estimation and at the same time glucose load (3gm/kg) was
administered orally. Blood was collected at time points corresponding to
20, 40, 60 and 120 min after glucose load administration. Serum was
separated for determination of glucose levels and change in area under
curve for glucose was calculated and provided in Fig. 3.
The goal of these studies was to evaluate the Pharmacokinetic profile
of compounds 8c and 13c in Sprague-Dawley rats following a single oral
gavage (po; 25 mg/kg). Sprague Dawley (SD) rats (8 adult males), were
obtained from Zydus Research Centre, Ahmedabad India, AAALAC
Accreditation. All animals were housed in temperature-controlled rooms
with appropriate light/dark cycles. The animals were fasted overnight
before oral gavage dosing but were given access to water ad libitum. Food
was provided at 4 h after dosing. Compounds 8c and 13c were dosed as
solutions in 10% NMP and 10% solutol in normal Saline (i.v.) and ho-
mogenous suspension (p.o.) in 0.5% Tween 80 in 0.5% Na-CMC as a
single dose in rats. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6,
8, and 24 h post-dose in Na-heparin coated micro centrifuge tubes. Blood
samples were centrifuged to separate plasma and were then stored at
ꢀ 70 ◦C until analysis. PK parameters were calculated by non-
compartmental analysis using WinNonlin program, version 5.3 (Phar-
sight Corp., Mountain View, California). A model was selected based on
the vascular (i.v. bolus) or extravascular (p.o.) routes of administration.
For the p.o. route, the concentration at time zero was assumed to be
zero. Plasma concentrations below the limit of quantitation were treated
as zero concentration for the purpose of calculating the mean plasma
concentration values.
6.3.1.1. Oral glucose tolerance tests (OGTT) in db/db mice. db/db Mice of
5–7 week age were used for this experiment. Animals were kept on
fasting and were grouped based on fasting serum glucose levels and after
grouping OGTT was performed. Each animal received a single dose of
vehicle/test compounds (50 mg/kg) administered orally, 30 min post
dosing animals were bled for basal glucose level estimation and at the
same time glucose load (2 g/kg) was administered per orally. Blood was
collected at time points corresponding to 30, 60 and 120 min after
glucose load administration. Serum is separated for determination of
glucose levels and change in area under curve for glucose and glucose
excursion is calculated from Vehicle control with glucose load vs water
control group without glucose load and provided in the above figure as
% reduction in AUC glucose excursion vs vehicle control is calculated.
6.3.1.2. Body weight changes in diet induced obesity (DIO) rats. The high
fat diet induced obesity (DIO) in rats exhibits various features of meta-
bolic syndrome in humans. The metabolic syndrome is characterized by
abdominal obesity, high triglycerides, impaired fasting glucose and
hyperinsulinemia. Animals with uniform body weight were divided into
two groups. The first group was provided with normal chow diet (NIN)
and the 2nd group was provided with High Fat diet (HFD) for a period of
12 weeks. After 12 weeks diet feeding, the HFD fed animals were divided
into 4 treatment groups. Formulations were prepared using vehicle
{PG450, Twin 80 and 0.1% sodium CMC (5:5:90)} and body weight was
recorded daily for 28 days.
6.4. Chemistry experiments
6.4.1. Synthetic materials and methods
All the solvents and reagents were obtained from commercial sup-
pliers and were used without further purification or were prepared ac-
cording to published procedures. The melting points were recorded on a
scientific melting point apparatus and are uncorrected. NMR spectra
were measured on a Bruker’s 400 UltraSheild NMR spectrometer and the
chemical shifts are reported in parts per million (ppm) relative to tet-
ramethylsilane (TMS). Mass spectra were recorded on Perkin-Elmer
Sciex API 3000. ESI-Q-TOF-MS measurements were performed on a
Fig. 9. AR 231453 (green) superposed with 13C (grey) in the GPR119 binding
site. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
7

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
micrOTOF-Q II (Bruker Daltonics) mass spectrometer. IR spectra were
recorded as neat (for oils) or as KBr pellet (for solid) on FT-IR 8300
Shimadzu spectrophotometer and the values are reported in wave-
6.4.3.2. Ethyl 4-((6-chloro-5-methylpyrimidin-4-yl) oxy) piperidine-1-
carboxylate (3b). The title compound was synthesized from 2b and
commercially available 1a, according to the general procedure
described above; Yellow Oil; Yield: 52%; Purity by UPLC: 98.57%; IR:
3018, 2933, 2872, 1739, 1685, 1554, 1435, 1274, 1136 cmꢀ 1. 1HNMR
(CDCl3, 400 MHz): δ 1.27 (t, J = 7.2 Hz, 3H), 1.77–1.80 (m, 2H),
1.96–2.04 (m, 2H), 2.22 (s, 3H), 3.39–3.45 (m, 2H), 3.73–3.75 (m, 2H),
4.14 (q, J = 8.4 & d 7.2 Hz, 2H), 5.30–5.36 (m, 1H), 8.40 (s, 1H); ESI/MS
m/z: 299.9 (M+H)+.
numbers
ν
(cmꢀ 1). Purity was determined by UPLC (Acquity Waters) via
the following conditions. Column: YMC-Triart C18 (100 × 2.0 mm).
Flow: 0.4 mL/min. Mobile phase: 0.05% TFA in Water: ACN (Gradient).
Detector: UV at 254 nm. Reactions were monitored using thin layer
silica gel chromatography (TLC) using 0.25 mm silica gel 60F plates
from Merck. Plates were visualized by treatment with UV, acidic p-
anisaldehyde stain, KMnO4 stain with gentle heating. Products were
purified by column chromatography using silica gel 100–200 mesh and
the solvent systems indicated. All final compounds were obtained with
over 95% purity as indicated by UPLC.
6.4.3.3. t-butyl 4-((6-chloro-5-methylpyrimidin-4-yl) oxy) piperidine-1-
carboxylate (3c). The title compound was synthesized from commer-
cially available 2c and 1a, according to the general procedure described
above; off white solid; Yield: 73%; m.p: 84–85 ◦C; Purity by UPLC:
99.79%; IR: 3433, 3416, 2997, 1691, 1560, 1421, 1253, 771 cmꢀ 1
.
6.4.2. General procedure for the preparation of carbamates 2a–e
To a solution of 4-hydroxy piperidine (1.0 equivalent) in dichloro-
methane, triethylamine (1.5 equivalent) was added at 0 ◦C followed by
dropwise addition of chloroformates (methyl, ethyl, isobutyl and
benzyl) (1.2 equivalent). After stirring for 30 min at 0 ◦C, the reaction
mixture was poured into water and extracted with dichloromethane.
The organic extracts were washed with water, brine and dried over so-
dium sulfate. Then organic extract was concentrated under reduced
pressure to yield product with quantitative yield.
1HNMR (CDCl3, 400 MHz): δ 1.47 (s, 9H), 1.73–1.80 (m, 2H), 1.94–2.0
(m, 2H), 2.22 (s, 3H), 3.32–3.39 (m, 2H), 3.68–3.74 (m, 2H), 5.31–5.35
(m, 1H), 8.38 (s, 1H); ESI/MS m/z: 328.0 (M+H).+
6.4.3.4. Isobutyl 4-((6-chloro-5-methylpyrimidin-4-yl) oxy) piperidine-1-
carboxylate (3d). The title compound was synthesized from 2d and
commercially available 1a, according to the general procedure
described above; Pale yellow oil; Yield: 61%; Purity by UPLC: 96.21%;
1HNMR (CDCl3, 400 MHz): δ 0.94 (d, J = 6.8 Hz, 6H), 1.74–1.80 (m,
2H), 1.93–2.00 (m, 2H), 2.23 (s, 3H), 3.40–3.46 (m, 2H), 3.73–3.79 (m,
2H), 3.88 (d, J = 6.4 Hz, 2H), 5.32–5.37 (m, 1H), 8.38 (s, 1H); ESI/MS
m/z: 327.9 (M+H)+.
6.4.2.1. Methyl 4-hydroxypiperidine-1-carboxylate (2a). Colorless oil;
Yield: 73%; 1H NMR (CDCl3, 400 MHz): δ 1.45–1.54 (m, 2H), 1.86–1.91
(m. 3H), 3.28–3.39 (m, 2H), 3.79–3.84 (m, 2H), 4.63 (s, 3H), 4.85 (d, J
= 8.0 Hz, 1H); ESI/MS m/z: 160.1 (M+H)+.
6.4.3.5. Benzyl 4-((6-chloro-5-methylpyrimidin-4-yl) oxy) piperidine-1-
carboxylate (3e). The title compound was synthesized from 2e and
commercially available 1a, according to the general procedure
described above; Yellow oil; Yield: 40%; Purity by UPLC: 90.84%; IR:
3433, 3416, 3018, 2958, 1691, 1554, 1433, 1205, 680 cmꢀ 1; 1H NMR
(CDCl3, 400 MHz): δ 1.99 (s, 2H), 2.04 (d, J = 3.6 Hz, 2H), 2.22 (s, 3H),
3.75–3.81 (m, 2H), 4.09–4.15 (m, 2H), 5.15 (s, 2H), 5.32–5.38 (m, 1H),
7.26–7.39 (m, 5H), 8.38 (s, 1H); ESI/MS m/z: 384.0 (M+Na)+.
6.4.2.2. Ethyl 4-hydroxypiperidine-1-carboxylate (2b). Pale yellow oil;
1
Yield: 84%; H NMR (CDCl3, 400 MHz): δ 1.13 (t, J = 11.2 Hz, 3H),
1.43–1.52 (m, 2H), 1.84–1.89 (m. 3H), 3.07–3.13 (m, 2H), 3.26–3.36
(m, 2H), 3.85–3.90 (m, 2H), 4.84–4.88 (m, 1H); ESI/MS m/z: 173.8
(M+H)+.
6.4.2.3. Isobutyl 4-hydroxypiperidine-1-carboxylate (2d). Pale yellow
oil; Yield: 81%; 1H NMR (CDCl3, 400 MHz): δ 0.97 (d, J = 8.0 Hz, 6H),
1.44–1.53 (m, 2H), 1.59–1.64 (m, 2H), 1.85–1.98 (m. 3H), 3.08–3.14
(m, 2H), 3.84–3.92 (m, 3H); ESI/MS m/z: 201.9 (M+H)+.
6.4.3.6. Methyl 4-((6-chloropyrimidin-4-yl) oxy) piperidine-1-carboxylate
(4a). The title compound was synthesized from 2a and commercially
available 1b, according to the general procedure described above; Thick
oil; Yield: 73%; Purity by UPLC: 97.37%; IR: 3016, 2958, 2870, 1745,
6.4.2.4. Benzyl 4-hydroxypiperidine-1-carboxylate (2e). Colorless oil;
Yield: 65%; 1H NMR (CDCl3, 400 MHz): δ 1.47–1.49 (m, 2H), 1.55–1.62
(m, 2H), 3.11–3.17 (m, 2H), 3.83–3.92 (m, 3H), 5.12 (s, 2H), 7.30–7.38
(m, 5H); ESI/MS m/z: 258.1 (M+Na)+.
1697, 1568, 1452, 1276, 1238, 1141, 1085, 985, 756 cmꢀ 1
;
1HNMR
(CDCl3, 400 MHz): δ 1.75–1.79 (m, 2H), 1.97–2.01 (m, 2H), 3.29–3.39
(m, 2H), 3.71 (s, 3H), 3.73–3.77 (m, 2H), 5.30–5.36 (m, 1H), 6.76 (s,
1H), 8.55 (s, 1H); ESI/MS m/z: 271.8 (M+H)+.
6.4.3. General procedure for the preparation of 3a-e and 4a-e
Potassium tert-butoxide (0.9 equivalents) was added to a solution of
intermediate 2a-e (1 equivalent) and 4, 6-dichloropyrimidine 1a-b (1
equivalent) in dry Tetrahydrofuran at 0 ◦C and the reaction mixture was
stirred for 15–20 h at 30 ◦C. The reaction mixture was poured into ice
cold water and extracted with ethyl acetate. The organic extract was
washed with water, brine, dried over sodium sulfate and concentrated
under reduced pressure to yield the desired product with good yield and
high purity.
6.4.3.7. Ethyl 4-((6-chloropyrimidin-4-yl) oxy) piperidine-1-carboxylate
(4b). The title compound was synthesized from 2b and commercially
available 1b, according to the general procedure described above; White
solid; Yield: 67%; Purity by UPLC: 99.54%; IR: 3014, 2978, 2868, 1685,
1568, 1460, 1425, 1352, 1234, 1130, 1085, 1028, 981, 775 cmꢀ 1
;
1HNMR (CDCl3, 400 MHz): δ 1.27 (t, J = 7.2 Hz, 3H), 1.71–1.79 (m, 2H),
1.96–2.01 (m, 2H), 3.32–3.39 (m, 2H), 3.78–3.81 (m, 2H), 4.16 (q, J =
14.2 & 7.2 Hz, 2H), 5.30–5.36 (m, 1H), 6.76 (s, 1H), 8.55 (s, 1H); ESI/
MS m/z: 285.9 (M+H)+.
6.4.3.1. Methyl 4-((6-chloro-5-methylpyrimidin-4-yl) oxy) piperidine-1-
carboxylate (3a). The title compound was synthesized from 2a and
commercially available 1a, according to the general procedure
described above; White solid; Yield: 72%; Purity by UPLC: 99.29%; IR:
2993, 2864, 1716, 1556, 1442, 1359, 1313, 1228, 1049, 1022, 823, 765
cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 1.76–1.82 (m, 2H), 1.97–2.02 (m,
2H), 2.29 (s, 3H), 3.40–3.46 (m, 2H), 3.71 (s, 3H), 3.73–3.77 (m, 2H),
5.33–5.37 (m, 1H), 8.38 (s, 1H); ESI/MS m/z: 285.8 (M)+.
6.4.3.8. tert-butyl
4-((6-chloropyrimidin-4-yl)
oxy)
piperidine-1-
carboxylate (4c). The title compound was synthesized from commer-
cially available 2c and 1b, according to the general procedure described
above; Thick oil; Yield: 30%; Purity by UPLC: 99.74%; IR: 3392, 3019,
2979, 2935, 1680, 1569, 1216, 758 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ
1.47 (s, 9H), 1.70–1.76 (m, 2H), 1.95–2.00 (m, 2H), 3.25–3.31(m, 2H),
3.75 (t, J = 12.4 Hz, 2H), 5.28–5.32 (m, 1H), 6.75 (s, 1H), 8.54 (s, 1H);
ESI/MS m/z: 313.0 (M)+.
8

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
6.4.3.9. Isobutyl
4-((6-chloropyrimidin-4-yl)
oxy)
piperidine-1-
general procedure described above; Thick oil; Yield: 63%; Purity by
UPLC: 92.56%; 1HNMR (CDCl3, 400 MHz): δ 0.94 (d, J = 6.8 Hz, 6H),
1.77–1.85 (m, 2H), 1.92–2.04 (m, 3H), 2.18 (s, 3H), 3.42–3.48 (m, 2H),
3.75–3.81 (m, 2H), 3.89 (d, J = 6.8 Hz, 2H), 5.30–5.38 (m, 1H), 7.28 (d,
J = 6.8 Hz, 2H), 7.94 (d, J = 9.2 Hz, 2H), 8.25 (s, 1H), 9.99 (s, 1H); ESI/
MS m/z: 413.9 (M+H)+.
carboxylate (4d). The title compound was synthesized from 2d and
commercially available 1b, according to the general procedure
described above; Thick oil; Yield: 65%; Purity by UPLC: 97.38%; IR:
3012, 2962, 2874, 1691, 1568, 1454, 1350, 1230, 1085, 1030, 756
cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 0.96 (d, J = 7.6 Hz, 6H), 1.71–1.80
(m, 2H), 1.89–2.03 (m, 3H), 3.33–3.40 (m, 2H), 3.77–3.83 (m, 2H), 3.88
(d, J = 6.8 Hz, 2H), 5.30–5.36 (m, 1H), 6.76 (s, 1H), 8.55 (s, 1H); ESI/
MS m/z: 314.0 (M+H)+.
6.4.4.5. Benzyl 4-((6-(4-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (6e). The title compound was synthesized from
intermediate 3e and p-hydroxybenzaldehyde (5), according to the
general procedure described above; Thick oil; Yield: 46%; Purity by
UPLC: 93.54%; 1HNMR (CDCl3, 400 MHz): δ 1.77–1.83 (m, 2H),
2.01–2.04 (m, 2H), 2.17 (s, 3H), 3.45–3.52 (m, 2H), 3.77–3.83 (m, 2H),
5.15 (s, 2H), 5.35–5.38 (m, 1H), 7.27–7.38 (m, 7H), 7.94 (d, J = 9.2 Hz,
2H), 8.25 (s, 1H), 9.99 (s, 1H); ESI/MS m/z: 447.9 (M+H)+.
6.4.3.10. Benzyl
4-((6-chloropyrimidin-4-yl)
oxy)
piperidine-1-
carboxylate (4e). The title compound was synthesized from 2e and
commercially available 1b, according to the general procedure
described above; Thick oil; Yield: 54%; Purity by UPLC: 98.57%; IR:
3016, 2960, 2872, 1685, 1570, 1541, 1452, 1350, 1226, 1217, 1087,
1030, 985, 756 cmꢀ 1; 1H NMR (CDCl3, 400 MHz): δ 1.75–1.77 (m, 2H),
1.98–2.04 (m, 2H), 3.37–3.43 (m, 2H), 3.79–3.85 (m, 2H), 5.14 (s, 2H),
5.30–5.36 (m, 1H), 6.75 (s, 1H), 7.30–7.37 (m, 5H), 8.54 (s, 1H); ESI/
MS m/z: 347.9 (M+H)+.
6.4.4.6. Methyl 4-((6-(4-formylphenoxy) pyrimidin-4-yl) oxy) piperidine-
1-carboxylate (7a). The title compound was synthesized from interme-
diate 4a and p-hydroxybenzaldehyde (5), according to the general
procedure described above; Thick oil; Yield: 81%; Purity by UPLC:
97.23%; IR: 3375, 2955, 2862, 1701, 1685, 1566, 1411, 1259, 1215,
1109, 1026, 844, 763 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 1.72–1.80 (m,
2H), 1.98–2.02 (m, 2H), 3.33–3.40 (m, 2H), 3.71 (s, 3H), 3.78–3.81 (m,
2H), 5.30–5.35 (m, 1H), 6.24 (s, 1H), 7.30 (d, J = 6.8 Hz, 2H), 7.95 (d, J
= 9.2 Hz, 2H), 8.42 (s, 1H), 9.99 (s, 1H); ESI/MS m/z: 380.0 (M+Na)+.
6.4.4. General procedure for preparation of 6a-e, 7a-e, 11a-e and 12a-e
To a solution of intermediate 3a-e and 4a-e (1 equivalent) and p-
hydroxybenzaldehyde (5) or m-hydroxybenzaldehyde (10) (1 equiva-
lent) in N,N-dimethylformamide, cesium carbonate (2 equivalent) was
added at 30 ◦C and then reaction mixture was stirred at 70 ◦C for 24 h.
Then reaction mixture was cooled to 30 ◦C and poured to ice cold water
and extracted with ethyl acetate. Then organic layer was washed with
water, brine, dried over sodium sulfate and concentrated under reduced
pressure to obtained crude product. Then crude product was purified
through column chromatography using 15–20% ethyl acetate in hexane
to give the desired intermediates 6a-e, 7a-e, 11a-e and 12a-e.
6.4.4.7. Ethyl 4-((6-(4-formylphenoxy) pyrimidin-4-yl) oxy) piperidine-1-
carboxylate (7b). The title compound was synthesized from the inter-
mediate 4b and p-hydroxybenzaldehyde (5), according to the general
procedure described above; Off-white solid; Yield: 72%; Purity by UPLC:
96.89%; IR: 3429, 2989, 2864, 1687, 1581, 1438, 1246, 1211, 1134,
1028, 854, 767 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 1.27 (t, J = 7.4 Hz,
3H), 1.71–1.80 (m, 2H), 1.97–2.04 (m, 2H), 3.32–3.38 (m, 2H),
3.78–3.83 (m, 2H), 4.15 (q, J = 14.2 and 6.8 Hz, 2H), 5.30–5.35 (m, 1H),
6.24 (s, 1H), 7.30 (d, J = 6.8 Hz, 2H), 7.96 (d, J = 7.2 Hz, 2H), 8.43 (s,
1H), 10.01 (s, 1H); ESI/MS m/z: 372.1 (M+H)+.
6.4.4.1. Methyl 4-((6-(4-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (6a). The title compound was synthesized from
3a and p-hydroxybenzaldehyde (5), according to the general procedure
described above; Thick oil; Yield: 92%; Purity by UPLC: 96.55%; IR:
3375, 2955, 2862, 1701, 1685, 1566, 1411, 1259, 1215, 1109, 1026,
844, 763 cmꢀ 1
;
1HNMR (CDCl3, 400 MHz): δ 1.77–1.85 (m, 2H),
6.4.4.8. tert-butyl
4-((6-(4-formylphenoxy)
pyrimidin-4-yl)
oxy)
1.98–2.04 (m, 2H), 2.18 (s, 3H), 3.42–3.48 (m, 2H), 3.71 (s, 3H),
3.75–3.77 (m, 2H), 5.33–5.39 (m, 1H), 7.29 (d, J = 6.8 Hz, 2H), 7.94 (d,
J = 8.8 Hz, 2H), 8.25 (s, 1H), 9.99 (s, 1H); ESI/MS m/z: 371.9 (M+H)+.
piperidine-1-carboxylate (7c). The title compound was synthesized in-
termediate 4c and p-hydroxybenzaldehyde (5), according to the general
procedure described above; Off-white solid product; Yield: 57%; Purity
by UPLC: 98.84%; IR: 3383, 2970, 2868, 1687, 1581, 1469, 1421, 1253,
1215, 1161, 1024, 846, 765 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 1.47 (s,
9H), 1.71–1.77 (m, 2H), 1.96–2.01 (m, 2H), 3.25–3.31(m, 2H),
3.76–3.79 (m, 2H), 5.28–5.32 (m, 1H), 6.24 (s, 1H), 7.30 (d, J = 6.4 Hz,
2H), 7.95 (d, J = 6.8 Hz, 2H), 8.42 (s, 1H), 10.01 (s, 1H); ESI/MS m/z:
400.0 (M+H)+.
6.4.4.2. Ethyl 4-((6-(4-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (6b). The title compound was synthesized from
3b and p-hydroxybenzaldehyde (5), according to the general procedure
described above; Thick Oil; Yield: 36%; Purity by UPLC: 96.51%;
1HNMR (DMSO‑d6, 400 MHz): δ 1.27 (t, J = 7.2 Hz, 3H), 1.63–1.69 (m,
2H), 1.92–1.97 (m, 2H), 2.11 (s, 3H), 3.32–3.38 (m, 2H), 3.61–3.67 (m,
2H), 4.05 (q, J = 10.0 Hz & 2.8 Hz, 2H), 5.30–5.32 (m, 1H), 7.38 (d, J =
6.8 Hz, 2H), 7.95 (d, J = 4.4 Hz, 2H), 8.29 (s, 1H), 9.98 (s, 1H); ESI/MS
m/z: 385.9 (M+H)+.
6.4.4.9. Isobutyl 4-((6-(4-formylphenoxy) pyrimidin-4-yl) oxy) piperidine-
1-carboxylate (7d). The title compound was synthesized from the in-
termediate 4d and p-hydroxybenzaldehyde (5), according to the general
procedure described above; colorless thick oil; Yield: 78%; Purity by
UPLC: 97.71%; IR: 3464, 3018, 2964, 1687, 1581, 1460, 1251, 1217,
1161, 1035, 756 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 0.94 (d, J = 6.8 Hz,
6H), 1.72–1.80 (m, 2H), 1.91–1.96 (m, 1H), 1.98–2.04 (m, 2H),
3.33–3.39 (m, 2H), 3.79–3.85 (m, 2H), 3.88 (d, J = 6.4 Hz, 2H),
5.30–5.36 (m, 1H), 6.24 (s, 1H), 7.30 (d, J = 9.2 Hz, 2H), 7.95 (d, J =
9.2 Hz, 2H), 8.42 (s, 1H), 10.01 (s, 1H); ESI/MS m/z: 400.1 (M+H)+.
6.4.4.3. tert-butyl 4-((6-(4-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (6c). The title compound was synthesized from
intermediate 3c and p-hydroxybenzaldehyde (5), according to the gen-
eral procedure described above; Thick oil; Yield: 66%; Purity by UPLC:
98.27%; IR: 3018, 2980, 2870, 1685, 1570, 1425, 1261, 1217, 1112,
1026, 758 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 1.48 (s, 9H), 1.75–1.81
(m, 2H), 1.96–2.01 (m, 2H), 2.18 (s, 3H), 3.34–3.41 (m, 2H), 3.70–3.76
(m, 2H), 5.32–5.36 (m, 1H), 7.28 (d, J = 6.4 Hz, 2H), 7.94 (d, J = 6.8 Hz,
2H), 8.25 (s, 1H), 9.99 (s, 1H); ESI/MS m/z: 414.0 (M+H)+.
6.4.4.10. Benzyl
4-((6-(4-formylphenoxy)
pyrimidin-4-yl)
oxy)
piperidine-1-carboxylate (7e). The title compound was synthesized from
the intermediate 4e and p-hydroxybenzaldehyde (5), according to the
general procedure described above and the crude product was directly
used for the next step.
6.4.4.4. Isobutyl 4-((6-(4-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (6d). The title compound was synthesized from
intermediate 3d and p-hydroxybenzaldehyde (5), according to the
9

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Bioorganic & Medicinal Chemistry 35 (2021) 116071
6.4.4.11. Methyl 4-((6-(3-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (11a). The title compound was synthesized
from the intermediate 3a and m-hydroxybenzaldehyde (10), according
to the general procedure described above and the crude product was
directly used for the next step.
6.4.4.19. Isobutyl
4-((6-(3-formylphenoxy)
pyrimidin-4-yl)
oxy)
piperidine-1-carboxylate (12d). The title compound was synthesized
from the intermediate 4d and m-hydroxybenzaldehyde (10), according
to the general procedure described above; Thick oil; Yield: 83%; Purity
by UPLC: 98.96%; IR: 3016, 2962, 2874, 1701, 1581, 1460, 1450, 1251,
1230, 1170, 1035, 837, 756 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 0.94 (d,
J = 6.8 Hz, 6H), 1.73–1.79 (m, 2H), 1.98–2.04 (m, 3H), 3.33–3.39 (m,
2H), 3.79–3.85 (m, 2H), 3.88 (d, J = 6.4 Hz, 2H), 5.29–5.34 (m, 1H),
6.21 (s, 1H), 7.41 (d, J = 6.8 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.66 (s,
1H), 7.78 (d, J = 7.6 Hz, 1H), 8.40 (s, 1H), 10.02 (s, 1H); ESI/MS m/z:
400.1 (M+H)+.
6.4.4.12. Ethyl 4-((6-(3-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (11b). The title compound was synthesized
from the intermediate 3b and and m-hydroxybenzaldehyde (10), ac-
cording to the general procedure described above and the crude product
was directly used for the next step.
6.4.4.20. Benzyl
4-((6-(3-formylphenoxy)
pyrimidin-4-yl)
oxy)
6.4.4.13. tert-butyl
4-((6-(3-formylphenoxy)-5-methylpyrimidin-4-yl)
piperidine-1-carboxylate (12e). The title compound was synthesized
from the intermediate 4e and m-hydroxybenzaldehyde (10), according
to the general procedure described above and the crude product was
directly used for the next step.
oxy) piperidine-1-carboxylate (11c). The title compound was synthesized
from the intermediate 3c and and m-hydroxybenzaldehyde (10), ac-
cording to the general procedure described above; Thick oil; Yield: 81%;
Purity by UPLC: 98.57%; IR: 3010, 2978, 2866, 1701, 1585, 1570, 1425,
1365, 1261, 1232, 1143, 1026, 756 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ
1.48 (s, 9H), 1.76–1.81 (m, 2H), 1.97–2.01 (m, 2H), 2.18 (s, 3H),
3.35–3.41 (m, 2H), 3.70–3.76 (m, 2H), 5.32–5.36 (m, 1H), 7.39–7.42
(m, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 4.0 Hz, 1H), 7.75 (d, J =
7.6 Hz, 1H), 8.23 (s, 1H), 10.01 (s, 1H); ESI/MS m/z: 414.0 (M+H)+.
6.4.5. General procedure for the preparation of 8a-e, 9a-e, 13a-e and 14a-e
To a solution of intermediate 6a-e, 7a-e, 11a-e and 12a-e (1
equivalent), thiazolidine-2, 4-dione (0.9 equivalent) and benzoic acid
(0.1 equivalent) in toluene, piperdine (1.5 equivalent) was added at
30 ◦C and the reaction mixture was refluxed for 7 h. Then reaction
mixture was cooled to 30 ◦C and solid product was filtered, washed with
ice cold toluene and dried to give desired product. In all final compounds
6.4.4.14. Isobutyl 4-((6-(3-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (11d). The title compound was synthesized
from the intermediate 3d and m-hydroxybenzaldehyde (10), according
to the general procedure described above and the crude product was
directly used for the next step.
–
–
–
8a-e, 9a-e, 13a-e and 14a-e, the CH proton from the newly intro-
duced benzylidene moiety appeared as a sharp singlet at 7.70–7.90 ppm.
This value indicates that all final compounds 8a-e, 9a-e, 13a-e and 14a-
e are in Z conformation which being the most thermodynamically stable
isomer according to the literature.35–38
6.4.4.15. Benzyl 4-((6-(3-formylphenoxy)-5-methylpyrimidin-4-yl) oxy)
piperidine-1-carboxylate (11e). The title compound was synthesized
from the intermediate 3e and m-hydroxybenzaldehyde (10), according
to the general procedure described above and the crude product was
directly used for the next step.
6.4.5.1. Methyl-4-((6-(4-((2,
4-dioxothiazolidin-5-ylidene)
methyl)
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (8a). The
title compound was synthesized from the intermediate 6a according to
the general procedure described above; Yellow solid; Yield: 66.6%; m.p:
>200 ◦C; Purity by UPLC: 97.65%; IR: 3194, 3072, 2862, 2748, 1755,
6.4.4.16. Methyl
4-((6-(3-formylphenoxy)
pyrimidin-4-yl)
oxy)
1703, 1687, 1572, 1413, 1220 cmꢀ 1
;
1H NMR (CDCl3, 400 MHz): δ
piperidine-1-carboxylate (12a). The title compound was synthesized
from the intermediate 4a and m-hydroxybenzaldehyde (10), according
to the general procedure described above; Thick oil; Yield: 61%; Purity
by UPLC: 98.25%; 1HNMR (CDCl3, 400 MHz): δ 1.74–1.78 (m, 2H),
1.97–2.01 (m, 2H), 3.33–3.42 (m, 2H), 3.71 (s, 3H), 3.78–3.80 (m, 2H),
5.30–5.33 (m, 1H), 6.21 (s, 1H), 7.40–7.43 (m, 1H), 7.60 (t, J = 7.6 Hz,
1H), 7.66 (d, J = 4.0 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 8.40 (s, 1H),
10.08 (s, 1H); ESI/MS m/z: 357.9 (M+H)+.
1.79–1.83 (m, 2H), 1.98–2.03 (m, 2H), 2.19 (s, 3H), 3.43–3.48 (m, 2H),
3.72 (s, 3H), 3.73–3.79 (m, 2H), 5.35–5.39 (m, 1H), 7.21–7.26 (m, 2H),
7.53 (dd, J = 6.8 and 2.0 Hz, 2H), 7.77 (s, 1H), 8.25 (s, 1H); 13C NMR
(100 MHz, DMSO‑d6): δ 7.92, 30.63, 66.72, 71.72, 102.76, 122.86,
123.44, 128.19, 128.90, 130.31, 131.78, 137.43, 154.59, 155.00,
167.48, 167.81, 168.24, 168.30; HRMS ESI/MS m/z calcd for
C
22H22N4O6S 471.1260 (M+H)+, found 471.1251.
6.4.5.2. Ethyl-4-((6-(4-((2,
4-dioxothiazolidin-5-ylidene)
methyl)
6.4.4.17. Ethyl 4-((6-(3-formylphenoxy) pyrimidin-4-yl) oxy) piperidine-
1-carboxylate (12b). The title compound was synthesized from the in-
termediate 4b and m-hydroxybenzaldehyde (10), according to the
general procedure described above; Thick oil; Yield: 57%; Purity by
UPLC: 97.29%; 1HNMR (CDCl3, 400 MHz): δ 1.27 (t, J = 7.0 Hz, 3H),
1.71–1.79 (m, 2H), 1.97–2.01 (m, 2H), 3.32–3.38 (m, 2H), 3.80–3.83
(m, 2H), 4.16 (q, J = 14.2 & 6.8 Hz, 2H), 5.29–5.35 (m, 1H), 6.21 (s,
1H), 7.41 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.66 (d, J = 3.6
Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 8.41 (s, 1H), 10.02 (s, 1H); ESI/MS m/
z: 372.0 (M+H)+.
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (8b). The
title compound was synthesized from the intermediate 6b according to
the general procedure described above; Yellow solid; Yield; 37.7%; m.p;
>200 ◦C; Purity by UPLC: 97.19%; IR: 3109, 2953, 2933, 2852, 2750,
1743, 1701, 1589, 1572, 1222, 1111 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ
1.81 (t, J = 7.0 Hz, 3H), 1.62–1.69 (m, 2H), 1.91–1.96 (m, 2H), 2.11 (s,
3H), 3.31–3.37 (m, 2H), 3.60–3.66 (m, 2H), 4.03 (q, J = 7.2 Hz, 2H),
5.27–5.33 (m, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H),
7.80 (s, 1H), 8.26 (s, 1H), 12.6 (s, 1H); HRMS ESI/MS m/z calcd for
C
23H24N4O6S 485.1417 (M+H)+, found 485.1411.
6.4.4.18. tert-butyl 4-((6-(3-formylphenoxy) pyrimidin-4-yl) oxy)
piperidine-1-carboxylate (12c). The title compound was synthesized
from the intermediate 4c and m-hydroxybenzaldehyde (10), according
to the general procedure described above; Thick oil; Yield: 58%; Purity
by UPLC: 98.45%; 1HNMR (CDCl3, 400 MHz): δ 1.47 (s, 9H), 1.71–1.76
(m, 2H), 1.96–2.01 (m, 2H), 3.24–3.31 (m, 2H), 3.76–3.79 (m, 2H),
5.28–5.32 (m, 1H), 6.20 (s, 1H), 7.40–7.43 (m, 1H), 7.60 (t, J = 7.8 Hz,
1H), 7.66 (d, J = 4.0 Hz, 1H), 7.78 (d, J = 6.4 Hz, 1H), 8.41 (s, 1H),
10.02 (s, 1H); ESI/MS m/z: 400.2 (M+H)+.
6.4.5.3. Tert-butyl-4-((6-(4-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (8c). The
title compound was synthesized from the intermediate 6c according to
the general procedure described above; White solid; Yield: 47%; m.p:
207–208 ◦C; Purity by UPLC: 99.48%; IR: 3500, 3070, 2928, 2750,
1743, 1705, 1685, 1589, 1566, 1506, 1415, 1220, 1020, 900; 1H NMR
(CDCl3, 400 MHz): δ 1.46 (s, 9H), 1.76–1.83 (m, 2H), 1.97–2.02 (m, 2H),
2.18 (s, 3H), 3.35–3.41 (m, 2H), 3.71–3.75 (m, 2H), 5.33–5.37 (m, 1H),
10

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
7.22–7.25 (m, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.77 (s, 1H), 8.25 (s, 1H);
13C NMR (100 MHz, DMSO‑d6): δ 7.91, 28.53, 30.74, 71.93, 79.25,
102.73, 121.86, 122.84, 123.50, 130.32, 130.82, 131.52, 131.96,
154.35, 154.99, 167.46, 167.88, 168.30; HRMS ESI/MS m/z calcd for
180–182 ◦C; Purity by UPLC: 97.52%; IR: 2976, 2858, 2758, 1743,
1701, 1608, 1589, 1465, 1411, 1251, 1174, 1033 cmꢀ 1
;
1H NMR
(CDCl3, 400 MHz): δ 1.47 (s, 9H), 1.70–1.78 (m, 2H), 1.97–2.01 (m, 2H),
3.25–3.31 (m, 2H), 3.71–3.80 (m, 2H), 5.29–5.33 (m, 1H), 6.24 (d, J =
8.0 Hz, 1H), 7.23–7.26 (m, 2H), 7.53–7.56 (m, 2H), 7.81 (s, 1H), 8.42 (s,
1H); 13C NMR (100 MHz, DMSO‑d6): δ 28.51, 30.77, 72.48, 79.28,
92.78, 122.90, 123.71, 130.77, 131.44, 132.16, 154.30, 158.33, 167.81,
168.28, 170.54, 170.91; HRMS ESI/MS m/z calcd for C24H26N4O6S
499.1573 (M+H)+, found 499.1568.
C
25H28N4O6S 513.1730 (M+H)+, found 513.1728.
6.4.5.4. Isobutyl-4-((6-(4-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (8d). The
title compound was synthesized from the intermediate 6d according to
the general procedure described above; Yellow Solid; Yield: 40%; m.p:
>200 ◦C; Purity by UPLC: 98.77%; IR: 2962, 2750, 1745, 1705, 1589,
1570, 1417, 1219, 1112, 1022 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ 0.89
(d, J = 6.8 Hz, 6H), 1.62–1.69 (m, 2H), 1.83–1.90 (m, 1H), 1.93–1.97
(m, 2H), 2.11 (s, 3H), 3.30–3.35 (m, 2H), 3.60–3.66 (m, 2H), 3.79 (d, J
= 6.4 Hz, 2H), 5.30–5.33 (m, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.65 (d, J =
8.8 Hz, 2H), 7.81 (s, 1H), 8.27 (s, 1H), 12.61 (s, 1H); HRMS ESI/MS m/z
calcd for C25H28N4O6S 513.1730 (M+H)+, found 513.1725.
6.4.5.9. Isobutyl-4-((6-(4-((2, 4-dioxothiazolidin-5-ylidene) methyl) phe-
noxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (9d). The title com-
pound was synthesized from the intermediate 7d according to the
general procedure described above; Yellow Solid; Yield: 61.7%; m.p:
168–170 ◦C; Purity by UPLC: 97.56%; IR: 2958, 2754, 1745, 1707,
1587, 1475, 1222, 1153, 1030 cmꢀ 1; 1H NMR (CDCl3, 400 MHz): δ 0.94
(d, J = 6.4, 6H), 1.72–1.81 (m, 2H), 1.90–2.03 (m, 3H), 3.34–3.40 (m,
2H), 3.81–3.85 (m, 2H), 3.88 (d, J = 6.8 Hz, 2H), 5.30–5.36 (m, 1H),
6.25 (d, J = 0.8 Hz, 1H), 7.23–7.26 (m, 2H), 7.53–7.56 (m, 2H), 7.80 (s,
1H), 8.42 (s, 1H); 13C NMR (100 MHz, DMSO‑d6): δ 19.35, 28.02, 30.71,
41.24, 71.17, 72.33, 92.80, 122.89, 123.70, 130.77, 131.44, 132.16,
154.25, 155.11, 158.33, 167.80, 168.27, 170.55, 170.90; HRMS ESI/MS
m/z calcd for C24H26N4O6S 499.1573 (M+H)+, found 499.1570.
6.4.5.5. Benzyl-4-((6-(4-((2,4-dioxothiazolidin-5-ylidene) methyl) phen
oxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (8e). The
title compound was synthesized from the intermediate 6e according to
the general procedure described above; Yellow Solid; Yield: 58.3%; m.p:
>200 ◦C; Purity by UPLC: 97.59%; IR: 3435, 2951, 2748, 1745, 1708,
1587, 1570, 1504, 1419, 1220, 1112 cmꢀ 1; 1HNMR (CDCl3, 400 MHz): δ
1.63–1.71(m, 2H), 1.93–1.98 (m, 2H), 2.11 (s, 3H), 3.31–3.37 (m, 2H),
3.60–3.66 (m, 2H), 5.08 (s, 2H), 5.30–5.33 (m, 1H), 7.31 (d, J = 8.8 Hz,
2H), 7.35–7.39 (m, 5H), 7.63–7.65 (d, J = 8.8 Hz, 2H), 7.81 (s, 1H), 8.27
(s, 1H), 12.61 (s, 1H); 13C NMR (100 MHz, DMSO‑d6): δ 7.92, 30.63,
41.12, 66.72, 71.72, 102.76, 122.86, 123.44, 128.19, 130.31, 131.58,
131.98, 137.43, 154.59, 155.00, 167.65, 168.24, 168.30; HRMS ESI/MS
m/z calcd for C28H26N4O6S 547.1573 (M+H)+, found 547.1568.
6.4.5.10. Benzyl-4-((6-(4-((2, 4-dioxothiazolidin-5-ylidene) methyl) phe-
noxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (9e). The title com-
pound was synthesized from the intermediate 7e according to the
general procedure described above; Yellow Solid; Yield: 39.5%; m.p:
190–192 ◦C; Purity by UPLC: 96.39%; IR: 3435, 3064, 2931, 2748,
1745, 1693, 1504, 1255, 1220 cmꢀ 1
;
1H NMR (CDCl3, 400 MHz): δ
1.78–1.85 (m, 2H), 2.00–2.04 (m, 2H), 3.37–3.43 (m, 2H), 3.82–3.85
(m, 2H), 5.15 (s, 2H), 5.30–5.36 (m, 1H), 6.24 (s, 1H), 7.23–7.26 (m,
2H), 7.30–7.39 (m, 5H), 7.53–7.56 (m, 2H), 7.81 (s, 1H), 8.41 (s, 1H);
13C NMR (100 MHz, DMSO‑d6): δ 30.69, 41.34, 66.73, 72.28, 92.80,
122.90, 123.72, 128.20, 128.90, 130.77, 131.45, 132.17, 137.41,
154.25, 154.88, 158.33, 167.81, 168.29, 170.55, 170.90; HRMS ESI/MS
m/z calcd for C27H24N4O6S 533.1417 (M+H)+, found 533.1411.
6.4.5.6. Methyl-4-((6-(4-((2, 4-dioxothiazolidin-5-ylidene) methyl) phe-
noxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (9a). The title com-
pound was synthesized from the intermediate 7a according to the
general procedure described above; Yellow Solid; Yield: 69.7%; m.p:
228–230 ◦C; Purity by UPLC: 95.31%; IR: 3427, 3109, 3041, 2958,
2764, 1745, 1705, 1583, 1460, 1253, 1174 cmꢀ 1; 1H NMR (CDCl3, 400
MHz): δ 1.68–1.80 (m, 2H), 1.98–2.03 (m, 2H), 3.34–3.40 (m, 2H), 3.71
(s, 3H), 3.73–3.79 (m, 2H), 5.30–5.36 (m, 1H), 6.25 (s, 1H), 7.23–7.26
(m, 2H), 7.53–7.56 (m, 2H), 7.81 (s, 1H), 8.42 (s, 1H); 13C NMR (100
MHz, DMSO‑d6): δ 30.54, 41.05, 52.75, 71.93, 93.00, 122.42, 122.63,
130.42, 131.96, 132.56, 137.43, 154.31, 157.75, 166.82, 167.25,
170.33, 170.96; HRMS ESI/MS m/z calcd for C21H20N4O6S 457.1104
(M+H)+, found 457.1101.
6.4.5.11. Methyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (13a).
The title compound was synthesized from the intermediate 11a ac-
cording to the general procedure described above; Off White Solid;
Yield: 50%; m.p: 120–122 ◦C; Purity by UPLC: 97.43%; IR: 3435, 3064,
2958, 2754, 1753, 1705, 1589, 1568, 1413, 1234, 1112 cmꢀ 1; 1H NMR
(CDCl3, 400 MHz): δ 1.79–1.85 (m, 2H), 1.98–2.03 (m, 2H), 2.19 (s, 3H),
3.44–3.48 (m, 2H), 3.72 (s, 3H), 3.75–3.76 (m, 2H), 5.35–3.38 (m, 1H),
7.18–7.21 (m, 2H), 7.26–7.28 (m, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.51 (t,
J = 5.2 Hz, 1H), 7.80 (s, 1H), 8.24 (s, 1H); 13C NMR (100 MHz,
DMSO‑d6): δ 7.91, 22.68, 52.83, 71.71, 102.50, 123.48, 124.08, 125.12,
126.71, 129.72, 130.97, 135.08, 153.99, 154.54, 155.55, 167.61,
168.18; HRMS ESI/MS m/z calcd for C22H22N4O6S 471.1260 (M+H)+,
found 471.1256.
6.4.5.7. Ethyl-4-((6-(4-((2, 4-dioxothiazolidin-5-ylidene) methyl) phe-
noxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (9b). The title com-
pound was synthesized from the intermediate 7b according to the
general procedure described above; Yellow Solid; Yield: 63.0%; m.p:
200–202 ◦C; Purity by UPLC: 99.62%; IR: 3429, 3119, 2949, 2748,
1745, 1701, 1601, 1589, 1554, 1502, 1475, 1435, 1253, 1174, 1155,
1132 cmꢀ 1
;
1H NMR (CDCl3, 400 MHz): δ 1.27 (t, J = 7.2 Hz, 3H),
1.73–1.80 (m, 2H), 1.98–2.03 (m, 2H), 3.32–3.39 (m, 2H), 3.80–3.83
(m, 2H), 4.15 (q, J = 7.2 Hz, 2H), 5.30–5.35 (m, 1H), 6.25 (s, 1H),
7.24–7.26 (m, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.80 (s, 1H), 8.42 (s, 1H);
13C NMR (100 MHz, CDCl3): δ 14.70, 30.55, 40.97, 61.54, 72.09, 93.09,
122.45, 130.35, 132.03, 132.64, 154.37, 155.61, 157.68, 166.64,
167.05, 170.29, 171.02; HRMS ESI/MS m/z calcd for C22H22N4O6S
471.1260 (M+H)+, found 471.1254.
6.4.5.12. Ethyl-4-((6-(3-((2,
4-dioxothiazolidin-5-ylidene)
methyl)
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (13b).
The title compound was synthesized from the intermediate 11b ac-
cording to the general procedure described above; Off White Solid;
Yield: 48%; m.p: 138–139 ◦C; Purity by UPLC: 99.34%; IR: 3169, 3068,
2947, 2754, 1753, 1708, 1585, 1570, 1425, 1236, 1157 cmꢀ 1; 1H NMR
(CDCl3, 400 MHz): δ 1.26 (t, J = 10.4 Hz, 3H), 1.77–1.85 (m, 2H),
1.98–2.04 (m, 2H), 2.19 (s, 3H), 3.42–3.48 (m, 2H), 3.75–3.79 (m, 2H),
4.17 (q, J = 6.8 Hz, 2H), 5.34–3.39 (m, 1H), 7.18–7.21 (m, 1H),
7.26–7.28 (m, 1H), 7.36 (d, J = 8 Hz, 1H), 7.51 (t, J = 5.2 Hz, 1H), 7.79
(s, 1H), 8.24 (s, 1H), 9.15 (s, 1H); 13C NMR (100 MHz, DMSO‑d6): δ 7.90,
14.43, 15.07, 22.54, 30.64, 31.43, 61.21, 71.75, 102.50, 123.47,
6.4.5.8. Tert-butyl-4-((6-(4-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (9c). The title
compound was synthesized from the intermediate 7c according to the
general procedure described above; Yellow Solid; Yield: 43.0%; m.p:
11

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
124.08, 125.08, 126.71, 130.97, 131.33, 135.07, 153.99, 154.54,
155.11, 167.61, 167.67, 168.17; HRMS ESI/MS m/z calcd for
C23H24N4O6S 485.1417 (M+H)+, found 485.1412.
169–170 ◦C; Purity by UPLC: 96.27%; IR: 3455, 3126, 2929, 2777,
1743, 1691, 1599, 1570, 1462, 1330, 1247, 1178, 1134, 1024, 846
cmꢀ 1; 1H NMR (CDCl3, 400 MHz): δ 1.27 (t, J = 9.2 Hz, 3H), 1.72–1.80
(m, 2H), 1.98–2.04 (m, 2H), 3.33–3.39 (m, 2H), 3.80–3.83 (m, 2H),
4.13–4.18 (q, J = 7.2 Hz, 2H), 5.30–5.35 (m, 1H), 6.21 (s, 1H),
7.21–7.24 (m, 1H), 7.28–7.29 (m, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.53 (t,
J = 8.0 Hz, 1H), 7.81 (s, 1H), 8.42 (s, 1H), 8.77 (s, 1H); 13C NMR (100
MHz, DMSO‑d6): δ 15.05, 30.68, 41.19, 61.23, 72.32, 92.55, 123.51,
123.98, 125.40, 127.03, 131.10, 131.22, 135.36, 153.32, 155.06,
158.31, 167.84, 168.23, 170.72; HRMS ESI/MS m/z calcd for
6.4.5.13. Tert-butyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (13c).
The title compound was synthesized from the intermediate 11c ac-
cording to the general procedure described above; Yellow Solid; Yield:
60%; m.p: 188–190 ◦C; Purity by UPLC: 98.27%; IR: 3429, 3138, 2964,
2848, 2773, 1741, 1695, 1585, 1564, 1417, 1288, 1240, 1112 cmꢀ 1; 1H
NMR (CDCl3, 400 MHz): δ 1.48 (s, 9H), 1.76–1.83 (m, 2H), 1.97–2.03
(m, 2H), 2.19 (s, 3H), 3.36–3.42 (m, 2H), 3.70–3.75 (m, 2H), 5.33–5.37
(m, 1H), 7.19–7.21 (m, 2H), 7.26–7.28 (m, 1H), 7.36 (d, J = 8 Hz, 1H),
7.51 (t, J = 5.2 Hz, 1H), 7.80 (s, 1H), 8.24 (s, 1H); 13C NMR (100 MHz,
DMSO‑d6): δ 7.91, 28.53, 30.75, 71.89, 79.25, 102.49, 123.47, 124.07,
125.13, 125.78, 126.71, 128.66, 129.36, 130.97, 131.29, 135.08,
153.99, 154.40, 167.65, 168.19; HRMS ESI/MS m/z calcd for
C
22H22N4O6S 471.1260 (M+H)+, found 471.1261.
6.4.5.18. Tert-butyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (14c). The title
compound was synthesized from the intermediate 12c according to the
general procedure described above; White Solid; Yield: 55%; m.p:
170–171 ◦C; Purity by UPLC: 99.63%; IR: 3433, 3117, 3036, 2974,
2762, 1745, 1707, 1593, 1574, 1460, 1321, 1176, 1033 cmꢀ 1; 1H NMR
(CDCl3, 400 MHz): δ 1.47 (s, 9H), 1.70–1.78 (m, 2H), 1.97–2.01 (m, 2H),
3.25–3.32 (m, 2H), 3.76–3.80 (m, 2H), 5.28–5.32 (m, 1H), 6.20 (s, 1H),
7.21–7.24 (m, 1H), 7.28–7.29 (m, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.53 (t,
J = 8.0 Hz, 1H), 7.82 (s, 1H), 8.41 (s, 1H); 13C NMR (100 MHz,
DMSO‑d6): δ 28.52, 30.52, 72.45, 79.29, 92.56, 123.50, 123.96, 125.57,
127.03, 130.99, 131.23, 135.41, 153.33, 154.36, 158.41, 168.16,
170.80; HRMS ESI/MS m/z calcd for C24H26N4O6S 499.1573 (M+H)+,
found 499.1568.
C
25H28N4O6S 513.1730 (M+H)+, found 513.1726.
6.4.5.14. Isobutyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (13d).
The title compound was synthesized from the intermediate 11d ac-
cording to the general procedure described above; Yellow Solid; Yield:
50%; m.p: 112–113 ◦C; Purity by UPLC: 98.94%; IR: 3435, 2960, 2756,
1753, 1707, 1589, 1566, 1431 cmꢀ 1; 1H NMR (CDCl3, 400 MHz): δ 0.95
(d, J = 6.4 Hz, 6H), 1.78–1.86 (m, 2H), 1.90–2.04 (m, 3H), 2.19 (s, 3H),
3.43–3.49 (m, 2H), 3.75–3.81 (m, 2H), 3.89 (d, J = 6.8 Hz, 2H),
5.34–5.40 (m, 1H), 7.19 (m, 1H), 7.26–7.35 (m, 1H), 7.36 (d, J = 8.0 Hz,
6.4.5.19. Isobutyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (14d). The title
compound was synthesized from the intermediate 12d according to the
general procedure described above; Yellow Solid; Yield: 33.6%; m.p:
136–138 ◦C; Purity by UPLC: 98.24%; IR: 3429, 3155, 3063, 2960,
1743, 1705, 1600, 1460,1178 cmꢀ 1; 1H NMR (CDCl3, 400 MHz): δ 0.95
(d, J = 6.4 Hz, 6H), 1.78–1.86 (m, 2H), 1.90–2.04 (m, 3H), 2.19 (s, 3H),
3.43–3.49 (m, 2H), 3.75–3.81 (m, 2H), 3.89 (d, J = 6.8 Hz, 2H),
5.34–5.40 (m, 1H), 7.19 (m, 1H), 7.26–7.35 (m, 1H), 7.36 (d, J = 8.0 Hz,
1H), 7.51 (t, J = 8.0 Hz, 1H), 7.80 (s, 1H), 8.24 (s, 1H), 8.78 (s, 1H); 13
C
NMR (100 MHz, DMSO‑d6): δ 7.91, 19.36, 28.03, 30.67, 71.17, 71.74,
102.50, 123.48, 124.09, 125.07, 126.71, 130.97, 131.33, 135.07,
153.99, 154.54, 155.14, 167.61, 168.17; HRMS ESI/MS m/z calcd for
C
25H28N4O6S 513.1730 (M+H)+, found 513.1731.
6.4.5.15. Benzyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl)
phenoxy)-5-methylpyrimidin-4-yl) oxy) piperidine-1-carboxylate (13e).
The title compound was synthesized from the intermediate 11e ac-
cording to the general procedure described above; Yellow Solid; Yield:
20%; m.p: 128–129 ◦C; Purity by UPLC: 98.48%; IR: 3433, 2951, 2764,
1H), 7.51 (d, J = 8.0 Hz, 1H), 7.80 (s, 1H), 8.24 (s, 1H), 8.78 (s, 1H); 13
C
NMR (100 MHz, DMSO‑d6): δ 19.35, 28.02, 30.73, 41.25, 71.17, 72.30,
92.57, 115.67, 123.52, 124.01, 125.28, 127.04, 129.82, 131.24, 131.24,
135.34, 153.33, 155.12, 158.32, 167.68, 168.15, 170.80; HRMS ESI/MS
m/z calcd for C24H26N4O6S 499.1573 (M+H)+, found 499.1570.
1751, 1705, 1587, 1568, 1431 cmꢀ 1
;
1H NMR (CDCl3, 400 MHz): δ
1.87–1.95 (m, 2H), 2.08–2.16 (m, 2H), 2.16 (s, 3H), 2.49–2.60 (m, 2H),
2.79–2.81 (m, 2H), 3.63 (s, 2H), 5.20–5.24 (m, 1H), 7.17–7.20 (m, 1H),
7.26–7.30 (m, 2H), 7.31–7.38 (m, 5H), 7.51 (d, J = 8.0 Hz, 1H), 7.79 (s,
1H), 8.22 (s, 1H); 13C NMR (100 MHz, DMSO‑d6): δ 7.92, 22.69, 30.63,
66.72, 71.67, 102.50, 123.43, 123.97, 125.68, 126.68, 128.02, 128.32,
128.90, 130.88, 130.94, 135.21, 137.43, 153.98, 154.54, 154.92,
167.61, 168.16, 168.40, 168.53; HRMS ESI/MS m/z calcd for
6.4.5.20. Benzyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl) phe-
noxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (14e). The title
compound was synthesized from the intermediate 12e according to the
general procedure described above; Yellow Solid; Yield: 40.7%; m.p:
178–180 ◦C; Purity by UPLC: 97.08%; IR: 3176, 3057, 2951, 2359,
C
28H26N4O6S 547.1573 (M+H)+, found 547.1569.
1743, 1589, 1570, 1465, 1172 cmꢀ 1
;
1H NMR (CDCl3, 400 MHz): δ
1.78–1.85 (m, 2H), 2.00–2.04 (m, 2H), 3.37–3.43 (m, 2H), 3.82–3.85
(m, 2H), 5.15 (s, 2H), 5.30–5.36 (m, 1H), 6.21 (s, 1H), 7.21–7.23 (m,
1H), 7.26–7.30 (m, 2H), 7.31–7.40 (m, 5H), 7.53 (d, J = 8.0 Hz, 1H),
7.81 (s, 1H), 8.41 (s, 1H), 8.61 (s, 1H); 13C NMR (100 MHz, DMSO‑d6): δ
30.74, 66.72, 72.24, 92.57, 123.51, 123.99, 125.38, 127.04, 128.08,
128.90, 131.13, 131.23, 135.36, 137.41, 153.32, 154.88, 158.31,
167.80, 168.21, 170.75; HRMS ESI/MS m/z calcd for C27H24N4O6S
533.1417 (M+H)+, found 533.1420.
6.4.5.16. Methyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl) phe-
noxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (14a). The title
compound was synthesized from the intermediate 12a according to the
general procedure described above; Yellow Solid; Yield: 30%; m.p:
200–202 ◦C; Purity by UPLC: 92.97%; IR: 3433, 3130, 3026, 2958,
1747, 1695, 1593, 1456, 1255, 1182 cmꢀ 1; 1H NMR (CDCl3, 400 MHz):
δ 1.74–1.80 (m, 2H), 1.98–2.02 (m, 2H), 3.34–3.40 (m, 2H), 3.71 (s,
3H), 3.75–3.81 (m, 2H), 5.30–5.34 (m, 1H), 6.20 (s, 1H), 7.18–7.23 (m,
1H), 7.26–7.28 (m, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz,
1H), 7.80 (s, 1H), 8.41 (s, 1H); HRMS ESI/MS m/z calcd for
Declaration of Competing Interest
C
21H20N4O6S 457.1104 (M+H)+, found 457.1098.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
6.4.5.17. Ethyl-4-((6-(3-((2, 4-dioxothiazolidin-5-ylidene) methyl) phe-
noxy) pyrimidin-4-yl) oxy) piperidine-1-carboxylate (14b). The title
compound was synthesized from the intermediate 12b according to the
general procedure described above; Yellow Solid; Yield: 66%; m.p:
12

S. Pola et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116071
18 Hansen HS, Rosenkilde MM, Holst JJ, Schwartz TW. Trends Pharmacol Sci. 2012;33:
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