Alleviating CYP and hERG liabilities by structure optimization of dihydrofuran-fused tricyclic benzo[d]imidazole series – Potent, selective and orally efficacious microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors: Part-2

Article abstract of DOI:10.1016/j.bmcl.2018.02.048

In an effort to identify CYP and hERG clean mPGES-1 inhibitors from the dihydrofuran-fused tricyclic benzo[d]imidazole series lead 7, an extensive structure-activity relationship (SAR) studies were performed. Optimization of A, D and E-rings in 7 afforded many potent compounds with human whole blood potency in the range of 160–950 nM. Selected inhibitors 21d, 21j, 21m, 21n, 21p and 22b provided selectivity against COX-enzymes and mPGES-1 isoforms (mPGES-2 and cPGES) along with sufficient selectivity against prostanoid synthases. Most of the tested analogs demonstrated required metabolic stability in liver microsomes, low hERG and CYP liability. Oral pharmacokinetics and bioavailability of lead compounds 21j, 21m and 21p are discussed in multiple species like rat, guinea pig, dog, and cynomolgus monkey. Besides, these compounds revealed low to moderate activity against human pregnane X receptor (hPXR). The selected lead 21j further demonstrated in vivo efficacy in acute hyperalgesia (ED₅₀: 39.6 mg/kg) and MIA-induced osteoarthritic pain models (ED₅₀: 106 mg/kg).

Full text of DOI:10.1016/j.bmcl.2018.02.048

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Alleviating CYP and hERG liabilities by structure optimization of
dihydrofuran-fused tricyclic benzo[d]imidazole series – Potent, selective
and orally efficacious microsomal prostaglandin E synthase-1 (mPGES-1)
inhibitors: Part-2
Nagarajan Muthukaman a, Sanjay Deshmukh a, Macchindra Tambe a, Dnyandeo Pisal a, Shital Tondlekar a,
Mahamadhanif Shaikh a, Neelam Sarode a, Vidya G. Kattige b, Pooja Sawant b, Monali Pisat b,
Vikas Karande b, Srinivasa Honnegowda b, Abhay Kulkarni b, Dayanidhi Behera c, Satyawan B. Jadhav c,
Ramchandra R. Sangana c, Girish S. Gudi c, Neelima Khairatkar-Joshi b, Laxmikant A. Gharat a,
⇑
a Chemical Research, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra 400709, India
b Biological Research, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra 400709, India
c Drug Metabolism and Pharmacokinetics, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra 400709, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to identify CYP and hERG clean mPGES-1 inhibitors from the dihydrofuran-fused tricyclic
benzo[d]imidazole series lead 7, an extensive structure-activity relationship (SAR) studies were per-
formed. Optimization of A, D and E-rings in 7 afforded many potent compounds with human whole blood
potency in the range of 160–950 nM. Selected inhibitors 21d, 21j, 21m, 21n, 21p and 22b provided selec-
tivity against COX-enzymes and mPGES-1 isoforms (mPGES-2 and cPGES) along with sufficient selectivity
against prostanoid synthases. Most of the tested analogs demonstrated required metabolic stability in
liver microsomes, low hERG and CYP liability. Oral pharmacokinetics and bioavailability of lead com-
pounds 21j, 21m and 21p are discussed in multiple species like rat, guinea pig, dog, and cynomolgus
monkey. Besides, these compounds revealed low to moderate activity against human pregnane X recep-
tor (hPXR). The selected lead 21j further demonstrated in vivo efficacy in acute hyperalgesia (ED50: 39.6
mg/kg) and MIA-induced osteoarthritic pain models (ED50: 106 mg/kg).
Received 17 January 2018
Revised 26 February 2018
Accepted 26 February 2018
Available online xxxx
Keywords:
Rheumatoid arthritis
Bioavailability
Lipophilicity
PGE2
Thromboxane A
Hyperalgesia
Osteoarthritic pain
Ó 2018 Elsevier Ltd. All rights reserved.
Microsomal PGE synthase-1 (mPGES-1) is a dominant source of
biologically active PGE2, during the biosynthesis in the down-
stream of the COXs in arachidonic acid (AA) pathway. PGES is a ter-
minal enzyme, which is classified into three isoforms, namely
microsomal PGES-1 (mPGES-1), microsomal PGES-2 (mPGES-2)
and cytosolic PGES (cPGES).1 The role of prostaglandins (PGs) in
inflammatory pain is well established. Binding of PGs to prostanoid
receptors (EP1, EP2, EP3 and EP4) sensitizes pain specific neurons
to stimulate pain in central nociceptive systems and mPGES-1
expression was strongly up-regulated in the brain and spinal cord
during inflammation.2 An inducible enzyme mPGES-1, which is
functionally coupled to COX-2, is responsible for the release of
duction by LPS is completely suppressed in peritoneal macro-
phages derived from mPGES-1 knockout mice.3 This enabled the
use of mPGES-1 knockout mice as models for various diseases, such
as collagen induced arthritis, pain hypersensitivity and neuro-
pathic pain.4 An mPGES-1 knockout study in mice exhibits re-
diversion of the PGH2 substrate by a PG synthases into several
prostanoids (PGF2 , PGD2 and PGI2), including PGE2 and thrombox-
a
ane A2 (TXA2).4 Therefore, it is expected that mPGES-1 inhibitors
may not increase the risk of cardiovascular side effects associated
with COX-2 inhibitor, as they do not inhibit PGI2 production.5 Sim-
ilarly, additional knockout studies demonstrated devoid of gas-
trointestinal and renal toxicity, which are associated with COX-1
inhibitors.6 Further, these knockout animals revealed viable, fertile,
and normal phenotype, which signifies that mPGES-1 inhibitors
could possess anti-inflammatory potential with minimum or neg-
ligible side effect profile.4 Therefore, selective inhibition of
mPGES-1 might be a promising approach for the design of an effec-
tive anti-inflammatory drugs lacking NSAID related side effects.7
PGE2 in response to inflammatory stimuli, such as IL-1b, TNF-a,
and LPS. A previous study by Akira group has shown that PGE2 pro-
⇑
Corresponding author.
E-mail address: Laxmikant.gharat@glenmarkpharma.com (L.A. Gharat).
https://doi.org/10.1016/j.bmcl.2018.02.048
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Muthukaman N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.02.048

2
N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Although development of novel mPGES-1 inhibitors has
received great attention recently, numerous inhibitors with variety
of chemo types are reported with only general SAR8 and the avail-
able in vivo efficacy data is still limited to very few compounds in
the literature. Some notable examples are, MF-63 (1) from Merck,9
pyridine-3-carboxamide (2) from Eli Lilly10 and PF-4693627 (3)
from Pfizer11 demonstrated in vivo efficacy in guinea pig
hyperalgesia model (Fig. 1). As well, we have recently reported
in vivo efficacy to few potent mPGES-1 inhibitors, such as quina-
zolinone (4), aminobenzimidazole-5-carboxamide (5) and 1,4-
dihydrochromeno[3,4-d]imidazole (6) in hyperalgesia pain
model.12,13 Further, mPGES-1 inhibitors from Eli Lilly (LY-
3023703)14 and our group (GRC27864)15 have completed Ph-I clin-
ical trials for the treatment of pain and an inflammatory diseases.
In the preceding communication,16 the design, synthesis and
initial SAR (structure-activity relationship) optimization of potent
dihydrofuran-fused benz[d]imidazole series, as exemplified by
lead 7 was described (Fig. 2). The mPGES-1 lead 7 and its analogs
were highly potent both in human and guinea pig enzymes, cell
permeable, selective against COX-enzymes with adequate PGE2
release human whole blood potency. In addition, it had adequate
brain penetration, orally bioavailable and was efficacious in animal
models of pain. However, compound 7 and other analogs from this
series suffered cytochrome P450 (CYP) enzyme (CYP3A4, CYP2C9
and CYP2C19) and hERG liability, which was expected to cause
adverse drug-drug interaction (DDI)17 and potential QTc prolonga-
tion safety issues18 if taken to clinical development. Overall, ana-
logs from this series with nitrogen incorporated in the E-ring
exhibited higher CYP3A4 liability, whereas compounds with meta-
and para-substituted phenyl as A-ring revealed CYP2C9 and
CYP2C19 liabilities.16a Further, compounds with substituted
pyridine as A-ring tested so far had shown decreased enzyme,
cell and human whole blood potency along with moderate to low
metabolic stability.16a Therefore, additional SAR optimization of
lead 7 is warranted in order to completely eliminate CYP and
hERG liabilities for further development.
Fig. 2. mPGES-1 lead 7. IC50: 3.9 nM; A549 cell IC50: 10.4 nM. Human whole blood
IC50: 275 nM. Guinea Pig whole blood IC50: 222 nM. CYP liability: 2C9, 2C19 (>50%
inhibition @10 mM). hERG liability: 56% inhibition @10 mM.
of molecule while performing lead optimization would provide
compounds with reduced hERG and CYP liability.19 With this
hypothesis, we initially synthesized two compounds by introduc-
ing 2, 6-difluorophenyl (21a) and 2-trifluoromethylphenyl (21b)
as A-ring along with 2, 6-dichlorophenyl group as E-ring without
any modification done on the other rings (Table 1). The biological
activity of 21a and 21b revealed comparable mPGES-1 enzyme
and cell potency (IC50s: <15 nM) similar to earlier lead 7 along with
human whole blood potency (HWB IC50s: 660 nM for 21a and 506
nM for 21b) similar to COX-2 inhibitor, Celecoxib (HWB IC50: 540
nM).8c,20 After having comparable human whole blood potency to
Celecoxib, compounds 21a and 21b were further evaluated for
CYP inhibition and unveiled low liability (<50%@ 10 mM) against
five major CYP isoforms tested, namely CYP1A2, CYP2D6,
CYP3A4, CYP2C9 and CYP2C19, respectively (Table 2).21 Next ana-
log 21c with 2-trifluoromethylphenyl as A-ring and 2-chloro-6-flu-
orophenyl as d-ring afforded single digit enzyme and, cellular
potency with a human whole blood IC50 of 536 nM, similar to com-
pound 21b. CYP inhibition study of 21c also revealed low CYP lia-
bility against all five major isoforms tested. Encouraged with these
results, additional SAR optimization was initiated to improve
human whole blood potency in this series. Therefore, combination
of 2-fluoro-5-trifluoromethylphenyl (21d), 2-fluoro-5-difluo-
romethylphenyl (21e), 2-fluoro-5-cyclopropyl phenyl (21f and
21g), 2-fluoro-4-trifluoromethylphenyl (21h) and 2-methyl-4-tri-
fluoromethyl phenyl (21i) analogs were synthesized and tested
for mPGES-1 potency. All these compounds demonstrated single
digit enzyme and A549 cell potency except 21g, which displayed
slightly lower enzyme potency (IC50: 16 nM) and single digit cell
potency (cell IC50: 3.2 nM). Among the analogs (21d–i) tested for
human whole blood potency, analogs 21d, 21e, 21g and 21i
afforded enhanced whole blood potency (HWB IC50s: <340 nM)
In this manuscript, we describe the synthesis and structure-
activity relationship (SAR) optimization based on lead 716a to
afford mPGES-1 inhibitors with low CYP and hERG liability
without altering the core scaffold. Therefore, we focused our
attention on the A, D and E-rings of lead 7 for optimization
(Fig. 2). It is well-known in the literature that reducing
lipophilicity (or increasing polarity) and disturbing the geometry
Fig. 1. Representative mPGES-1 inhibitors.
Please cite this article in press as: Muthukaman N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.02.048

N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Table 1
Dihydrofuran-fused benzo[d]imidazole SAR.
Compd
R2 (A-ring)
R1
X
mPGES-1 IC50 (nM)a,b
A549, 2% FBS PGE2 IC50 (nM)a,c
hWBA IC50 (nM)a,d
21a
21b
21c
21d
21e
21f
21g
21h
21i
2,6-diF-Phenyl
2CF3-Phenyl
2CF3-Phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
F
F
F
F
Cl
F
F
F
Me
Me
Me
Me
Me
Cl
F
Me
F
F
6.4
4.9
5.1
3.0
9.2
3.6
16
3.1
6.1
5.0
14
5.4
3.3
10
10
8.4
25
15
5.2
9.7
9.6
6.3
6.0
3.2
9.6
6
14
11
10
30
–
661
506
536
256
336
1377
197
570
270
208
337
876
222
162
447
261
955
–
2F, 5CF3-phenyl
2F, 5CHF2-phenyl
2F, 5Cy-phenyl
2F, 5Cy-phenyl
2F, 4CF3-phenyl
2CH3, 4CF3-Phenyl
4F, 3CF3-phenyl
4CF3-phenyl
4F, 3CF3-phenyl
2F, 5CF3-phenyl
2F, 5CHF2-phenyl
2F, 5Cy-phenyl
5CF3-Pyridin-2-yl
5CF3-Pyridin-2-yl
5CF3-Pyridin-2-yl
4CF3-Phenyl
21j
21k
21l
21m
21n
21o
21p
21q
21r
22a
22b
22c
22d
22e
–
4.1
14
27
2.6
3.0
4.3
3.1
4
H
H
32
Me
Me
Me
Me
Me
3.3
5.7
4.9
16
897
405
350
177
673
3CF3-Phenyl
4Cy-Phenyl
2F, 4CH3-Phenyl
5CF3-Pyridin-2-yl
F
F
F
12
–: not determined.
a
MF-63 (1) was used as a positive control here and the in-house data of MF-63 (1) for enzyme IC50: 1.9 nM; A549 cell IC50: 56 nM; hWB IC50: 1.36 mM. For the literature
reported potency of MF-63, see Ref. 9.
b
IC50 values are derived from graphs plotted with data from a minimum of two experiments in duplicates.
IC50 values represent the concentration to inhibit 50% of PGE2 relative to vehicle control and derived from the graphs plotted with data from a minimum of two
c
experiments in duplicates.
d
Lipopolysaccharide (LPS) stimulated human whole blood (hWB) assay. IC50 values represent the concentration to inhibit 50% of PGE2 relative to vehicle control and
minimum of two experiments in duplicates.
over 21a–c. In contrast, 21h (HWB IC50: 570 nM) and 21f (HWB
IC50: 1377 nM) provided lower human whole blood potency.21
Analogs 21d–i were further assessed for potential CYP inhibition
and exhibited least liability against all five major CYP isoforms
studied (Table 2). Then, 4-fluoro-3-trifluoromethylphenyl as A-ring
analog 21j (no 2-substituted phenyl as A-ring) was prepared and
tested for CYP activity, which revealed moderate liability against
CYP3A4, CYP2C9 and CYP2C19 isoforms (45–55%@10 mM) despite
having suitable mPGES-1 enzyme, cell and human whole blood
potency (Tables 1 and 2). The CYP data of 21j further reconfirm
our hypothesis that 2-substituted phenyl as A-ring is essential in
this series to afford compounds with low or negligible CYP liability.
Next, we focused our attention on the E-ring modification in
order to afford additional analogs with low CYP liability (<50%@
10 mM). In the earlier study, compounds with 2, 6-dichlorophenyl,
2-chloro-6-fluorophenyl and 3, 5-dichloropyridin-4-yl as E-ring of
lead 7 delivered mPGES-1 inhibitors with CYP liability (>50%@ 10
mM).16a In order to overcome CYP liability, 4-trifluoromethylphenyl
(21k) and 4-fluoro-3-trifluoromethylphenyl (21l) as A-ring analogs
having 2-chloro-6-methylphenyl as E-ring were synthesized and
tested for mPGES-1 potency and CYP liability. Analog 21k
provided higher human whole blood potency (IC50: 337 nM) than
Celecoxib along with robust enzyme and cell potency, whereas
such as 2-fluoro-5-trifluoromethylphenyl (21m), 2-fluoro-5-
difluoromethylphenyl (21n) and 2-fluoro-5-cyclopropylphenyl
(21o) as A-ring along with 2-chloro-6-methylphenyl as common
E-ring analogs were synthesized and tested for CYP liability and
mPGES-1 potency. As projected, analogs 21m–o also displayed
diminished CYP inhibition for all major CYP isoforms tested along
with noteworthy enzyme, cellular and human whole blood
potency (Tables 1 and 2).21
Having several potent mPGES-1 inhibitors in hand with low CYP
liability (<50%@ 10 mM), additional SAR was generated by introduc-
ing N-methyl group in the d-ring along with 2-chloro-6-
fluorophenyl as E-ring intact for further optimization. Therefore,
new analogs having 4-trifluoromethylphenyl (22a), 3-trifluo-
romethylphenyl (22b), 4-cyclopropylphenyl (22c) and 2-fluoro-4-
methylphenyl (22d) were synthesized and tested for mPGES-1
potency and CYP inhibition studies. Among these, analogs 22b–d
unveiled human whole blood potency ꢀ405 nM IC50s along with
single digit enzyme and A549 cellular potency, whereas, 22a
(HWB IC50: 897 nM) exhibited fivefold lower human whole blood
potency in comparison to 22d (HWB IC50: 177 nM), despite having
ꢀ15 nM enzyme and cell potency (Table 1). Further CYP inhibition
study on N-methyl d-ring analogs 22a–c provided low CYP liability
for all five major isoforms tested, while 22d revealed modest
CYP2C9 liability (52% inhibition @10 mM) and other CYP isoforms
gave low liability (<50%@ 10 mM). In order to improve the physic-
ochemical properties in our scaffold,22 substituted pyridine A-ring
was introduced in the place of phenyl ring. Therefore, analogs
21l provided > 2-fold reduced whole blood potency (hWB IC50
:
876 nM) even though it had <10 nM enzyme and cellular
potency. Gratifyingly, low CYP liability (<50%@ 10 mM) were
observed to 21k and 21l as foreseen. Hence, additional analogs
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4
N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Table 2
In vitro DMPK data of selected mPGES-1 inhibitors.
Compd
Metabolic stability (%
remaining)a
% CYP inhibition @10 mM concentrationb
PPB (% bound)c
hERG inh. @ 10 mMd
PAMPA Papp (*10ꢁ6 cm/s)e
HLM
RLM
GPLM
1A2
2D6
3A4
2C9
2C19
H
GPig
21a
21b
21c
21d
21e
21f
72
97
93
89
88
100
95
100
82
100
100
95
100
83
93
91
100
98
97
68
58
–
–
–
80
86
89
79
68
93
74
80
72
100
62
80
46
60
55
71
68
51
51
58
69
14
18
17
22
31
36
14
–
26
ꢁ6
25
–
24
28
14
4
34
5
n.i
n.i
4
12
ꢁ1
7
5
–
16
19
6
24
17
11
2
35
16
29
35
42
42
26
39
1
48
46
47
15
28
12
56
44
19
47
52
52
41
30
46
33
22
22
10
16
38
55
19
31
16
14
24
15
ꢁ7
36
24
24
10
–
–
–
–
–
–
–
5.8
–
–
–
–
–
–
0.12
0.62
–
–
–
–
0.31
0.33
–
–
1.34
–
–
0.63
–
–
–
> 99.5
99.96
99.82
–
>99.5
99.65
99.5
–
96
86
93
92
–
91
91
–
97
97
87
89
88
68
89
96
71
66
4
ꢁ12
21
37
20
46
34
30
ꢁ4
16
ꢁ13
40
11
8
21g
21h
21i
–
–
>99.5
–
99.46
>99.5
–
99.97
99.92
–
99.96
–
>99.5
–
98.64
>99.5
–
99.96
98.42
–
99.84
–
16.2
3.8
–
c21j
21k
21l
–
–
21m
21n
21o
21p
22a
22b
22c
22d
22e
ꢁ2
14
31
9
ꢁ5
29
25
14
3
12.3
11.8
–
5.4
–
26.5
–
12.8
–
99.71
–
>99.5
99.85
99.72
–
>99.5
99.65
41
26
14
19
23
17
–
–
–: not determined.
a
Percentage of test compound remaining after 60 min incubation with liver microsomes (human, rat and guinea Pig) at 37 °C. MS experiment was conducted in triplicates
(see supporting info (SI) for details. HLM: human liver microsomes; RLM: rat liver microsomes; GPLM: guinea pig liver microsomes.
b
Cytochrome P450 (CYP)% inhibition as compared to control (no inhibitor) and conducted in triplicates (see SI for details).
Plasma protein binding (PPB) was determined using equilibrium dialysis method. 10 mM concentrations were used (see SI for details) and the data provided are% bound.
c
PPB data of 21j: 99.1 (dog); 99.48 (cynomolgus monkey); PPB data of 21m: 99.7 (dog); PPB data of 21p: 99.2 (dog); 99.5 (cynomolgus monkey).
d
For hERG assay details (patch clamp), see Ref. 23.
PAMPA permeability assay was carried out using 2% lecithin dodecane mixture to determine the passive permeability and the study was conducted in triplicates.
e
having 5-trifluoromethyl-pyridine-2-yl as common A-ring along
with 2,6-dichlorophenyl (21p), 2-chloro-6-fluorophenyl (21q), 2-
chloro-6-methylphenyl (21r) and 2-chloro-6-fluoro- phenyl as
common E-ring, in combination with N-methyl as d-ring (22e)
were tested for mPGES-1 potency. Among these, compounds 21p
and 22e retained potency similar to other analogs in the Table 1,
whereas 21q and 21r revealed substantial drop in hWB potency,
slightly lower enzyme and cell potency in comparison to other
analogs.21 Further CYP inhibition study of pyridine analogs 21p
and 22e revealed moderate CYP2C9 liability (52–56% inhibition
@10 mM, Table 2).
Next, most of the analogs from Table 1 were further assessed for
metabolic stability in liver microsomes (human, rat and guinea
pig), plasma protein binding (PPB), hERG channel activity and
PAMPA permeability. As illustrated in Table 2, most of the tested
mPGES-1 inhibitors (21a–p and 22a–e) revealed >50% metabolic
stability in across species, > 98% plasma protein binding (PPB,%
bound) in both human and guinea pig, and low PAMPA permeabil-
ity (Table 2).21 Further, few selected mPGES-1 inhibitors (21d, 21i,
21j, 21m, 21n, 21p, 22b and 22d) demonstrated low activity in the
hERG channel assay (ꢀ26% inhibition @10 mM in patch clamp).23
The notable differences in hERG potency between current series
and earlier compound 7 series analogs16a were not explainable.
However, we believe that the low hERG potency in the current
series is due to the introduction of various ortho-substituents in
A and E-rings, respectively.19
the selected six showed potent PGF2 inhibition at 10 mM test con-
a
centration and the PGF2 potency of 21d (IC50: 2.47 mM), 21j (IC50
:
a
2.32 mM) and 22b (IC50: 3.18 mM) were >450-fold margin over their
corresponding mPGES-1 cellular potency (Tables 1 and 3).
Since, most of the reported mPGES-1 inhibitors were not active
against rodent enzyme (rat and mouse) except NovaSaid
inhibitor,24 we further assessed chosen compounds (21d, 21j,
21m, 21n, 21p and 22b) for rat, mouse and guinea pig enzyme
potency. As disclosed in Table 4, though the lead compounds were
not active against rat and mouse mPGES-1 enzymes, these are
highly active in guinea pig enzymes (Guinea pig IC50s: <45 nM
and WB IC50s: <465 nM), which is a pre-clinical efficacy species.21
The observed species selectivity difference in rodents are consis-
tent with previously reported mPGES-1 inhibitors.9–11,13a,16a Next,
analogs 21j, 21m and 21p among the six leads were further evalu-
ated for dog and cynomolgus monkey whole blood potency and to
our surprise, dog exemplified modest whole blood potency (IC50s:
3 to 6 mM range), whereas a lower% inhibition in monkey whole
blood (ꢀ50% inhibition @10 mM) was observed as shown in Table 4.
The poor potency in dog and monkey perhaps attributed to high
plasma protein binding and lipophilicity of lead compounds which
may cause serum shift and therefore might have led to lower
whole blood potency (see Table 2 Footnote for dog and monkey
PPB data).8a,8g
After assessing in vitro pharmacology, pharmacokinetics and
prostanoid synthases selectivity (Tables 2–4), chosen analogs 21j,
21m and 21p were subjected to pharmacokinetic (PK) study in
rat, guinea pig, dog, and cynomolgus monkey in 10 mg/kg oral dose
(Table 5).21,25 The plasma PK of compound 21j was determined in
rat, dog and cynomolgus monkey following intravenous (i.v) and
oral administration.25 The intravenous clearance was 11.2 mL/
min/kg in rats, 1.61 mL/min/kg in dogs and 3.35 mL/min/kg in
monkeys. The volume of distribution (Vz) was 3.85, 1.94 and
1.83 L/kg in rat, dog and monkey, respectively. The oral half-life
Additionally, the chosen analogs 21d, 21j, 21m, 21n, 21p and
22b were profiled against a battery of in vitro and cellular assays
to evaluate their selectivity over other prostanoid synthases.21 As
represented in Table 3, selected mPGES-1 inhibitors demonstrated
selectivity over COX-1, COX-2, mPGES-2, cPGES, PGDS, PGIS, TXAS
synthases and further these compounds did not inhibit cytosolic
phospholipase A2 (cPLA2), an enzyme upstream of arachidonic acid
(AA) pathway. However, few compounds (21d, 21j and 22b) among
Please cite this article in press as: Muthukaman N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.02.048

N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Table 3
COX-1/2 and prostanoid synthases selectivity of selected mPGES-1 inhibitors.
Compd
% inhibition @ 10 mM test concentrationa
COX-1
COX-2
mPGES-2
cPGES
PGDS
PGIS
TXAS
PGF2
cPLA2
a
21d
21j
21m
21n
21p
22b
14.7
8.27
14.6
38.2
7.8
28.7
23.1
31.0
43.2
35.9
6.5
13.5
11.6
23.5
3.8
7.8
7.9
4.2
0.0
21.1
11.0
0
5.8
0.0
19.5
27.6
15.1
16.5
5.6
2.5
7.5
20.1
4.3
3.9
4.6
99.2b
100c
17.9
22.9
ꢁ14.9
5.1
25.5
0.87
17.2
1.3
ꢁ20.1
ꢁ30.7
15.4
4.3
19.7
24
22.1
61.4d
10.6
a
b
c
% inhibition values are means of at least two experiments in duplicates, see SI for experimental details.
PGF2 IC50: 2.47 mM (740-fold over A549 cell potency).
a
PGF2 IC50: 2.32 mM (461-fold over A549 cell potency).
a
d
PGF2 IC50: 3.18 mM (556-fold over A549 cell potency).
a
Table 4
Cross-species activity of selected mPGES-1 inhibitors.
Compd hIC50 (nM)a A549 cell IC50 (nM)b hWBA IC50 (nM)c GPig IC50 (nM)d,e GPigWBA IC50 (nM)d,f Dog WBA IC50 (nM)g % inhibition @10 mM
concentrationh
h
h
i
Rat
Mouse
Monkey
21d
21j
21m
21n
21P
22b
3.0
5.0
3.3
10
8.4
5.7
9.6
14
30
–
4.1
3
256
208
222
162
261
405
14
24
19
46
12
4.3
204
465
340
222
259
188
–
11.7
10.5
0.0
0.7
7.1
14.4
22.1
8.2
2.5
10.9
5.2
–
6221
3029
–
3538
–
50.4
48.2
–
37
–
3.4
–: not determined.
a,b,c
See Table 1 foot note for details.
Same positive control was used for guinea pig enzyme and WBA potency as shown in the foot note of Table 1.
IC50 values are derived from graphs plotted with data from a minimum of two experiments in duplicates.
d
e
f
Lipopolysaccharide (LPS) stimulated guinea pig (GPig) whole blood assay (WBA). IC50 values represent the concentration to inhibit 50% of PGE2 relative to vehicle control
and minimum of two experiments in duplicates.
g
Lipopolysaccharide (LPS) stimulated dog whole blood assay. IC50 values represent the concentration to inhibit 50% of PGE2 relative to vehicle control (Celecoxib) and
minimum of two experiments in duplicates. Study was conducted at Vimta Labs, Hyderabad, India.
h
Study protocol was done similar to human and guinea pig enzyme assay (see Table 1 foot note for details).
Lipopolysaccharide (LPS) stimulated Rhesus monkey whole blood assay. IC50 values represent the concentration to inhibit 50% of PGE2 relative to vehicle control
i
(Celecoxib) and minimum of two experiments in duplicates. Study was conducted at Lonza India Ltd, Hyderabad, India.
Table 5
Oral Pharmacokinetics (PK)a of selected mPGES-1 inhibitors- 21j, 21m and 21p.
Example
Speciesb
Cmax (ng/mL)
AUC0-24 (ng.h/mL)
Tmax (h)
T1/2 (h)
CL (mL/min/kg)
Vz (L/kg)
% F
CNS b/pg
21j
Ratc
995 82
361 91
492 183
680 326
210 24
80 21
843 111
499 11
344 52
91 78
11020 2289
4125 2337
8236 3235
9669 3623
2579 296
408 164
12933 2507
7507 1937
4025 365
639 410
2
2
12
24
4
4
–
0.5
11.2 0.4
–
1.61 0.3
3.35 0.87
8.2 1.3
–
3.5 0.5
1.6 0.3
16.2 2.4
–
3.9 1.5
8.5 1.1
3.85 0.6
–
1.94 0.13
1.83 0.41
4.84 0.15
–
9.17 1.8
2.1 0.3
3.77 0.8
–
5.4 0.79
4.1 3.3
91
–
15
44
14
–
40
11
43
–
25
21
0.18
0.44
0.72
0.32
0.04
BQL
ND
0.16
0.16
0.58
0.32
0.37
G.Pig*,d
Doge
14.4 3.6
6.45 1.13
7.0 0.9
–
Monkeyf
Ratc
21m
21p
G.Pigd
Doge
1
8
20
3
Monkeyf
12
4
12
12
6
12 2.6
2.7 0.8
–
17
5.3 3.6
c
Rat
G.Pigd
Doge
336 106
323 195
4432 1543
3669 2180
5
Monkeyf
–: not applicable; ND: not determined; BQL: Below quantification level.
*
– Female guinea pig was used.
a
Cmax, AUC0-24, Tmax, and T1/2 were determined based on oral dosing and clearance (CL), volume of distribution (Vz) were determined based on i.v dosing. Bioavailability (%
F) was determined based on both oral and i.v dosing. This is applicable to all species.
b
Rat, guinea pig, dog and cynomolgus monkey i.v dose is 1 mg/kg and p.o dose is 10 mg/kg.
Vehicle for rat Oral dosing- 0.5% methylcellulose (MC) suspension; Vehicle for rat i.v- 20% NMP + 20% Ethanol + 60% PEG 200).The data represented is mean SD (n = 3).
c
Male Sprague-Dawley rats were used.
d
Vehicle for guinea pig oral dosing- 20% NMP + 0.5% MC suspension. The data represented is mean SD (n = 3).
Vehicle for beagle dog oral dosing- 2.5 lL/mL Tween 80 + 0.5 (w/v) MC suspension. Vehicle for dog i.v- 20% NMP + 20%EtOH + 10%PG + 50% premix solvent (PEG 200:
e
Milli-Q water, 3:2). See Ref. 25.
f
Vehicle for cynomolgus monkey oral dosing- 2.5 mL/mL Tween 80 + 0.5 (w/v) MC suspension. Vehicle for cynomolgus monkey i.v dosing- 10% NMP + 10% ethanol + 10%
PEG + 70% premix solvent (v/v mixture of 3:2 PEG200: Milli-Q water). See Ref. 25.
g
See supporting info for the experimental details.
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6
N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
(T1/2) is ranged from approximately 4–14 h, while absorption was
moderate to slow (Tmax = 2–24 h) in all three species. The oral
bioavailability of 21j was relatively higher in rat and monkey (%
F = 91 and 44), whereas dog exemplified poor oral bioavailability
(%F = 15).25b Plasma Cmax was higher to moderate in rat, dog and
monkey, whereas AUC0-24 was comparable in across species. The
guinea pig PK of 21j demonstrated lower Cmax and AUC0-24 in
comparison to rat, dog and monkey PK values. Another lead 21m
demonstrated moderate to low clearance (CL = 8.2 –1.6 mL/min/
kg), low volume of distribution (Vz = 9.2–2.1 L/kg) in rat, dog and
monkey, and longer oral half-life (T1/2 = 7–20 h) in across species
compared to analog 21j. The oral bioavailability of 21m in rat
and monkey was poor (%F = 14–11%), whereas dog demonstrated
40% oral bioavailability. In addition, third lead 21p divulged
comparable CL, Vz and oral T1/2 in across species similar to
earlier leads 21j and 21m, respectively. The oral bioavailability of
21p was as comparable to 21m and inferior to 21j. Plasma Cmax
and AUC0-24 of 21m and 21p was lower compared to 21j in
across species. The guinea pig oral PK of 21m and 21p also
revealed inferior to compound 21j as shown in Table 5. Based on
the oral PK data, compound 21j appeared better than other two
analogs. mPGES-1 is functionally coupled to COX-2 in the
arachidonic acid (AA) pathway and it is well known that
peripheral injury elicits a predominant increase in mPGES-1
expression and PGE2 levels in the CNS, which plays a role in pain
mitigation.2d Therefore, mPGES-1 inhibitor should probably have
CNS penetration for its in vivo efficacy similar to Celecoxib (COX-
2 inhibitor).20,26 Among the compounds (21j, 21m and 21p) evalu-
ated for CNS penetration in across species (rat, guinea pig, dog and
monkey), compounds 21j and 21p revealed comparable brain to
plasma ratio (B/P) in across species, whereas 21m unveiled lower
brain to plasma ratio (Table 5). Next, the advanced leads (21j,
21m and 21p) were further evaluated for human PXR (pregnane
X receptor) activation. Among this, 21m and 21p were found to
be moderate PXR activator, whereas 21j exhibited low PXR activity
relative to the effect of rifampicin at 10 mM test concentration (see
reference section for data).27
Finally, lead 21j was further selected for in vivo efficacy study
(acute hyperalgesia and monosodium iodoacetate (MIA)-induced
osteoarthritis pain models)21 over other leads 21m and 21p due
to the following reasons: (i) a better whole blood potency, (ii) a
lower hERG and PXR activity, and (iii) a better oral PK and CNS pen-
etration in across species. In the acute hyperalgesia model, the
antihyperalgesic effect of compound 21j was studied at 10, 30
and 100 mg/kg in guinea pig and the results are summarized in
Fig. 3 and Table 6. Injection of LPS into the plantar region of right
paw of guinea pig caused a significant thermal hyperalgesic
response as compared to saline injected animals. When adminis-
tered orally at 2 h before LPS injection, the compound 21j signifi-
cantly inhibited the hyperalgesic response in a dose-dependent
manner with maximum inhibition of 66% at 100 mg/kg (Fig. 1).
The calculated ED50 was found to be 39.6 mg/kg. As depicted in
Table 6, the plasma and brain concentrations of 21j for 100 mg/
kg, at 7 h post compound treatment (pharmacodynamics time
Fig. 3. Analgesic effects of 21j in the guinea pig hyperalgesia pain model. Data are
expressed as percentage of hyperalgesia, with the naive group (injected intraplan-
tarly with saline) as 0% and the vehicle-treated LPS-injected group as 100%. Results
are shown as Mean S.E.M. (n = 6–9/group).
Fig. 4. Analgesic effects of 21j in the guinea pig MIA induced OA pain model. Data
are expressed as percentage of incapacitance, with the naive group (injected with
saline in shoulder joint) as 0% and the vehicle-treated MIA-injected group as 100%.
Results are shown as Mean S.E.M. (n = 6–8/group).
Table 6
Plasma concentration of 21j at PD time point (HA model).
Dose (mg/kg, po, od)
ED50 (mg/kg)
% Hyperalgesia inhibition
Plasma concentration @PD
time point (ng/ml)a
Brain concentration @PD time
point (ng/g)a
Brain to Plasma
Ratio (B/P)
10
30
100
20
44
66
94
145
581
96
87
616
1.02
0.60
1.06
39.6
a
Concentrations are means of n = 6–9 animals per dose group. The data represented is from a single experiment. Study protocol is provided in the SI.
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N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
7
point)21 was found to be 581 ng/mL (equivalent to 1084 nM in
plasma) and 616 ng/mL (equivalent to 1149 nM in brain), respec-
tively with brain to plasma (B/P) ratio of 1.06. The plasma concen-
tration was found to be greater than 2-fold over guinea pig whole
blood potency and roughly 5-fold higher than its human whole
blood potency at steady state and also unveiled more than ade-
quate concentration in the brain.
The analgesic property of compound 21j was further evaluated
in a guinea pig model of MIA-induced osteoarthritic pain.21 Anal-
gesic effect of compound 21j was studied at 100, 250 and 500
mg/kg and the results are summarized in Fig. 4 and Table 7.
Intra-articular injection of monoiodo acetate (MIA) in the right
shoulder joint of guinea pig on day 0 caused inflammation and
pain, resulting a decreased weight bearing or incapacitance on
the injected forelimb by day 3 as compared to saline injected ani-
mals. When tested on day 3, compound 21j dose-dependently
reversed incapacitance with maximum reversal of 83% at 6 h after
dosing at 500 mg/kg (Fig. 2). The calculated ED50 was found to be
106 mg/kg. As depicted in Table 7, the plasma concentration at
PD time point (2h post compound treatment) of 21j for 500 mg/
kg was found to be 1896 ng/mL (equivalent to 3537 nM in plasma),
which was 7-fold higher than its guinea pig whole blood potency
and 17-fold higher than its human whole blood potency. Overall,
compound 21j showed efficient analgesic activity in both LPS-
induced hyperalgesia and MIA-induced osteoarthritic pain models
when administered orally and thus suppresses both peripheral and
centrally mediated PGE2 synthesis9,28
The synthetic strategies employed in the preparation of com-
pounds exemplified in Tables 1–4 are described in Scheme 1..16,21
The synthetic route began with acetylation of commercially avail-
able 4-aminosalicylic acid 8 using Ac2O, followed by nitration in
TFA using sodium nitrite (NaNO2) to afford nitrated compound 9.
Esterification of compound 9 with conc.H2SO4 in methanol at 90
°C gave N-deacetylated ester product 10 in good yield.29 The inter-
mediate 10 was alkylated using methallyl chloride in the presence
of K2CO3 in DMF at 80 °C to afford compound 11 in 55% yield. Clai-
sen rearrangement of 11 in N, N-dimethylaniline and subsequent
treatment with formic acid at 100 °C gave dihydrobenzofuran
derivative 12.30 Compound 12 was methylated in parallel, using
MeI in NaH/DMF to afford compound 13. Nitro reduction of com-
pounds 12 and 13 in Fe/aq.HCl afforded diamine compounds 14
and 15. Aryl isothiocyanate 16,31 prepared from the corresponding
aryl aniline using thiophosgene and diisopropylethyl amine,
reacted with diamines 14 and 15 independently in the presence
of N,N0-diisopropylcarbodiimide to afford tricyclic dihydrobenzo-
furoaminoimidazole derivatives 17 and 18 in 40–70% yield (for
two steps).32 The ester derivatives 17 and 18 were hydrolyzed to
carboxylic acid derivatives 19 and 20 in the presence of 10% NaOH
solution, suitable for amide formation. The conversion of car-
boxylic acids (19 and 20) to acid chlorides using thionyl chloride,
followed by reaction with substituted anilines afforded aryl amides
21a–o and 22a–d in 20–37% yield. Standard coupling reagents
(TBTU/HOBT, EDCI/HOBT, T3P/DIPEA and SOCl2 etc.) did not work
Table 7
Plasma concentration of 21j at PD time point (MIA model).
Dose
(mg/kg, po, od)
ED50
(mg/kg)
% Incapacitance
reversal
Plasma concentration
@PD time point (ng/ml)a
100
250
500
48
73
83
678
1508
1896
106
a
Concentrations are means of n = 6–8 animals per dose group. The data repre-
sented is from a single experiment. Study protocol is provided in the SI.
Scheme 1. Reagents and conditions: (a) Ac2O, EtOH, reflux; (b) NaNO2, TFA, ꢁ5 to 0 °C, 2 h; (c) MeOH, conc.H2SO4, reflux; (d) methallyl chloride, K2CO3, DMF, 80 °C, 3 h, 55%;
(e) N,N-dimethylaniline, reflux, 1.5 h, then formic acid, 100 °C, 3–4 h, 25%; (f) MeI, 60% NaH in mineral oil, DMF, 0 °C to rt, 3 h, 60%; (g) Fe, aq.HCl, 0 °C to rt, 1 h, 45%; (h)
thiophosgene, DIPEA, DCM, 0 °C to rt, 4–6 h; 50–70%; (i) N,N0-diisopropylcarbodiimide, Ar-NCS, CH3CN, rt, 24 h, 40–70%; (j) 10% NaOH, MeOH, 60 °C, 8–10 h, 65–80%; (k)
SOCl2, DCM, reflux, 2 h, then DIPEA, DCM, aryl amine, 8–12 h, 20–37% - used for 21a–o and 22a–d; (l) COMU, DIPEA, 1 h, then 5-(trifluoromethyl)pyridin-2-amine-N-oxide
(A), rt, 12–16 h; (m) Iron power, AcOH, 70 °C, 3 h, 50–60% (for two steps) – used for 21p–r and 22e.
Please cite this article in press as: Muthukaman N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.02.048

8
N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Franco, F. M.; hanau, C. E.; Scholten, J. A.; Springer, J. R.; Fobian, Y. M.; Carter, J.
S.; Xing, L.; Yang, S.; Shaffer, A. F.; Jerome, G. M.; Baratta, M. T.; Moore, W. M.;
Vazquez, M. L. Bioorg Med Chem Lett. 2013;23:1120;
in our hand for the direct synthesis of pyridine amide derivatives
21p–r and 22e (Table 1). Therefore, amide derivatives 19aa–cc
and 20aa were prepared by coupling of 5-(trifluoromethyl)pyri-
din-2-amine N-oxide (A)33a with corresponding aryl carboxylic
acids 19 and 20 using COMU/DIPEA based coupling reagent,
followed by N-oxide cleavage33b with Fe powder in acetic acid to
afford aryl amides 21p–r and 22e in 50–60% yield over two steps.
In summary, SAR optimization of A, D and E-rings of dihydrofu-
ran-fused tricyclic benzo[d]imidazole 7 afforded many potent
mPGES-1 inhibitors with low CYP and hERG liability. Several iden-
tified lead compounds (21d, 21j, 21m, 21n, 21p and 22b) exhibited
selectivity over COX-enzymes, mPGES-2 and prostanoid synthases.
The chosen pre-clinical lead 21j demonstrated enviable oral phar-
macokinetics, adequate CNS penetration in multiple species like
rat, guinea pig, dog and monkey, and exhibited oral bioavailability
in rat and monkey. Besides, 21j exemplified in vivo efficacy in acute
hyperalgesia and MIA-induced osteoarthritis pain models, and fur-
ther revealed low PXR activity. Detailed toxicology and in vivo
study of all lead compounds will be the subject of future disclosure.
(c) Kablaoui N, Patel S, Shao J, et al. Bioorg Med Chem Lett. 2013;23:907.
12. Banerjee A, Pawar MY, Patil S, et al. Bioorg Med Chem Lett. 2014;24:4838.
13. (a) Muthukaman N, Deshmukh S, Sarode N, et al. Bioorg Med Chem Lett.
2016;26:5977;
(b) Muthukaman N, Tambe M, Shaikh M, et al. Bioorg Med Chem Lett.
2017;27:2594.
14. (a)
A
Single Dose Study of LY3023703 in Healthy Participants.
http://clinicaltrials.gov/ct2/show/NCT01632579;
(b) Jin Y, Smith CL, Hu L, et al. Clin Pharmacol Ther. 2016;99:274.
15. (a) The structure of GRC-27864 has not been disclosed yet. See: GRC27864 First
in Man, Single Ascending Dose Study in Healthy Volunteers.
http://clinicaltrials.gov/ct2/show/NCT02179645; (b) GRC-27864 has moved to
Ph-IIb clinical trials. For more details see: http://www.indian-commodity.
com/top-news/glenmark-initiates-phase-IIb-dose-range-finding-study-for-
grc-27864.aspx.
16. (a) Muthukaman N, Tambe M, Deshmukh S, et al. Bioorg Med Chem Lett.
2017;27:5131(b) Gharat L.A., Muthukaman N., Narayana L., Khairatkar-Joshi N.,
Kattige V.G. PCT Int Appl WO2012/055995, 2012.
17. (a) Prueksaritanont T, Chu X, Gibson C, et al. AAPS J. 2013;15:629–645;
(b) Nettleton DO, Einolf HJ. Curr Top Med Chem. 2011;11:382–403.
18. (a) Nachimuthu S, Assar MD, Schussler JM. Ther Adv Drug Saf. 2012;3:241;
(b) Pollard CE, Valentin J-P, Hammond TG. Br J Pharmacol. 2008;154:1538;
(c) Gintant G, Sager PT, Stockbridge N. Nat Rev Drug Discovery. 2016;15:457.
19. (a) Waring MJ. Expert Opin Drug Discovery. 2010;5:235–248;
(b) Lovering F, Bikker J, Humblet C. J Med Chem. 2009;52:6752;
(c) Lewis DFV, Dickins M. Drug Metab Rev. 2003;35:1–18;
(d) Jamieson C, Moir EM, Rankovic Z, Wishart G. J Med Chem. 2006;49:5029;
(e) Waring MJ, Johnstone CA. Bioorg Med Chem Lett. 2007;17:1759.
20. Huntjens DRH, Danhof M, Della Pasqua OE. Rheumatology. 2005;44:846.
21. See supporting information for in vitro, in vivo and DMPK experimental details
and analytical data.
Acknowledgements
We thank Dr. Sanjib das for the review of this manuscript and
valuable inputs. We also thank scientists from the analytical sup-
port group for their help in compound characterization.
22. (a) Meanwell NA. Chem Res Toxicol. 2011;24:1420;
A. Supplementary data
(b) Arnott JA, Planey SL. Expert Opin Drug Discovery. 2012;7:863;
(c) Morphy R. J Med Chem. 2006;49:2969.
23. hERG channel assay study (patch clamp) was conducted at Eurofins Lab- USA
(http://www.eurofinsus.com).
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.02.048.
24. (a) Wannberg J.; Alterman M.; Malm J. PCT Intl. Appl. WO2012/117062, 2012.
(b) Chemical structure of NovaSaid mPGES-1 inhibitor is disclosed in Ref. 13.
25. (a) Monkey oral PK study was done at Maccine Pte Ltd, Singapore.(b) Dog oral
PK study was conducted at Vimta Labs, Hyderabad, India. The low oral
bioavailability (%F) observed to 21j in dog was due to emesis with one dog out
of three dogs used in this study.
26. Dembo G, Park SB, Kharasch ED. Anesthesiology. 2005;102:409.
27. PXR activation is 34% (EC50: 2.46 mM) for 21j, 57% for 21m and 46% for 21p
relative to Rifampicin, at 10 mM test concentration and the study was
conducted at PuraCYP, USA.
28. (a) Yaksh TL, Kokotos G, Svensson CI, et al. Pharmacol Exp Ther. 2006;316:466;
(b) Futaki DN, Harada M, Sugimoto M, et al. Pharm Pharmacol. 2009;61:607.
29. For the conversion of 8 to 10, the following literature procedure was followed.
See related references: (a) Martin RF, White J, Lobachevsky P, et al., PCT Intl.
Appl. WO2011/123890, 2011; (b) Lee A-R, Huang W-H, Chu C-H, et al., US2012/
101157, 2012.;
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Please cite this article in press as: Muthukaman N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.02.048