Characterization of 3,3-dimethyl substituted N-aryl piperidines as potent microsomal prostaglandin E synthase-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.08.023

Here we report on novel, potent 3,3-dimethyl substituted N-aryl piperidine inhibitors of microsomal prostaglandin E synthases-1(mPGES-1). Example 14 potently inhibited PGE₂synthesis in an ex vivo human whole blood (HWB) assay with an IC_50<

Full text of DOI:10.1016/j.bmcl.2016.08.023

Accepted Manuscript
Characterization of 3,3-Dimethyl Substituted N-aryl Piperidines as Potent Mi-
crosomal Prostaglandin E Synthase-1 Inhibitors
Steven L. Kuklish, Stephen Antonysamy, Shobha N. Bhattachar, Srinivasan
Chandrasekhar, Matthew J. Fisher, Adrian J. Fretland, Karen Gooding, Anita
Harvey, Norman E. Hughes, John G. Luz, Peter R. Manninen, James E. McGee,
Antonio Navarro, Bryan H. Norman, Katherine M. Partridge, Steven J. Quimby,
Matthew A. Schiffler, Ashley V. Sloan, Alan M. Warshawsky, Jeremy S. York,
Xiao-Peng Yu
PII:
S0960-894X(16)30844-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.08.023
BMCL 24149
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 May 2016
2 August 2016
9 August 2016
Please cite this article as: Kuklish, S.L., Antonysamy, S., Bhattachar, S.N., Chandrasekhar, S., Fisher, M.J., Fretland,
A.J., Gooding, K., Harvey, A., Hughes, N.E., Luz, J.G., Manninen, P.R., McGee, J.E., Navarro, A., Norman, B.H.,
Partridge, K.M., Quimby, S.J., Schiffler, M.A., Sloan, A.V., Warshawsky, A.M., York, J.S., Yu, X-P.,
Characterization of 3,3-Dimethyl Substituted N-aryl Piperidines as Potent Microsomal Prostaglandin E Synthase-1
Inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.08.023
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Characterization of 3,3-Dimethyl Substituted N-aryl Piperidines as Potent Microsomal
Prostaglandin E Synthase-1 Inhibitors
a,
b
a
Steven L. Kuklish, * Stephen Antonysamy, Shobha N. Bhattachar, Srinivasan
a,c
a
a,d
a,c
a,c
a
Chandrasekhar, Matthew J. Fisher, Adrian J. Fretland, Karen Gooding, Anita Harvey,
a
b
a
a
Norman E. Hughes, John G. Luz, Peter R. Manninen, James E. McGee, Antonio Navarro,
a
a
a
a
Bryan H. Norman, Katherine M. Partridge, Steven J. Quimby, Matthew A. Schiffler, Ashley
a
a
a
a
V. Sloan, Alan M. Warshawsky, Jeremy S. York, Xiao-Peng Yu,
a
Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, Indiana 46285,
USA
b
Eli Lilly Biotechnology Center, San Diego, CA 92121, USA
c
Retired
d
Oncology Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive,
Waltham, MA 02451, USA
Abstract:
Here we report on novel, potent 3,3-dimethyl substituted N-aryl piperidine inhibitors of
microsomal prostaglandin E synthases-1(mPGES-1). Example 14 potently inhibited PGE
2
synthesis in an ex vivo human whole blood (HWB) assay with an IC50 of 7 nM. In addition, 14
had no activity in human COX-1 or COX-2 assays at 30 µM, and failed to inhibit human
mPGES-2 at 62.5 µM in a microsomal prep assay. These data are consistent with selective
mPGES-1-mediated reduction of PGE . In dog, 14 had oral bioavailability (74%), clearance
2

(
3.62 mL/(min*kg)) and volume of distribution (Vd,ss = 1.6 L/kg) values within our target ranges.
For these reasons, 14 was selected for further study.
Corresponding Author:
*
(S. L. K.) E-Mail: Kuklish_Steven@lilly.com; Tel: +1-317-433-6519.
Keywords: mPGES-1, Prostaglandin pathway, Dog bioavailability, Biomarker

Prostanoids are a class of 20-carbon fatty acid derivatives of arachidonic acid which
participate in a wide array of cellular signaling pathways. Transformation of arachidonic acid by
cyclooxygenase-1 or -2 (COX-1 or COX-2) yields prostaglandin H
modified by specific prostaglandin synthases to form biologically active products: prostaglandin
(PGE ), prostaglandin F2α (PGF2α), prostaglandin D (PGD ), prostacyclin (PGI ) and
thromboxane A Each of these products contributes to important cellular activities
for homeostasis and cell signaling.
significant attention, and PGE
2
(PGH
2
), which is further
E
2
2
2
2
2
1
, 2
2
(TxA ).
2
3
, 4
Specifically, the role of PGE in nociception has drawn
2
2
signaling has been hypothesized as the mechanism by which
5
COX inhibitors exert analgesic activity. In fact, recent analysis of COX inhibitors has revealed
a correlation between the reduction of PGE production in bacterial lipopolysaccharide (LPS)
2
stimulated human whole blood and the corresponding drug plasma concentrations need for
6
analgesic efficacy in humans.

Therapies that target the inhibition of both the COX-1 and COX-2 enzymes; e.g., aspirin
and ibuprofen, are effective at treating joint mobility issues and pain associated with chronic
7
inflammation. Unfortunately, with prolonged use, this class of drugs carries the risk of causing
gastric bleeding and ulcerations by inhibiting the synthesis of the normal gastroprotective PGE
2
8
produced through the COX-1 pathway. In an attempt to pave the way to safer therapeutics,
selective COX-2 inhibitors have been developed. While this approach proved efficacious,
increased adverse cardiovascular events led to the withdrawal of selective COX-2 drugs
4
, 9
rofecoxib and valdecoxib from the market.
remains available to patients.) The reduction of antithrombotic PGI
disrupting the pathway at COX-2, is speculated to contribute to the adverse cardiovascular
(One selective COX-2 inhibitor, celecoxib,
2
, a consequence of
1
0
outcomes. Because of the efficacy seen with the above therapies, selective reduction of PGE
2
1
1
levels continues to be an attractive option for development of therapeutic analgesics.
Three PGE
2
synthases have been identified to date: microsomal prostaglandin E
5
synthase-1 (mPGES-1), mPGES-2, and cytosolic PGES (cPGES). While mPGES-2 and cPGES
1
1
are constitutively expressed, only mPGES-1 is induced during the inflammatory response.
Moreover, recent evidence has shown that mPGES-2 does not contribute to the production of
1
2
PGE
2
in vivo. Studies with mPGES-1 knockout mice have demonstrated attenuated
nocifensive responding, reduced febrile response, and decreased severity to collagen antibody-
1
3
induced arthritis, each of which was accompanied by decreased PGE
reduction of PGE by mPGES-1 inhibition has been recognized as an attractive alternative to
COX-2 inhibitors, because through this mechanism, PGI
2
levels. Systemic
2
1
4
2
should not be suppressed.
Multiple mPGES-1 inhibitor chemotypes have been identified in the course of research
into this high value target (Figure 1). A group at NovaSAID published a series of substituted

benzimidazoles, exemplified by compound 1 with a reported IC50 of 25 nM in a microsomal
1
5
enzyme assay, and Daiichi Sankyo’s recent patent explored 5-phenylfuro[2,3-b]pyridines
1
6
17
exemplified by 2. Example 3 from Pfizer has a reported IC50 of 3 nM. In addition, we have
1
8
previously reported on a series of 2-(acylamino)imidazoles such as 4 (IC50 = 0.9 nM).
Figure 1. Structures of known mPGES-1 inhibitors.
In this manuscript, we describe our continued efforts toward identifying novel inhibitors
of human mPGES-1. To characterize the potency and selectivity of our ligands, we utilized a
series of three assays. First, enzyme inhibition was measured in vitro using a cell-free
microsomal mPGES-1 enzyme preparation where PGE concentration was evaluated by LC/MS.
2
Secondly, we tested compound efficacy and selectivity in hIL-1β stimulated A549 (epithelial
carcinoma) cells, which have previously shown to produce a variety of prostanoids, including
PGE
2
and PGI
2
. The prostanoids secreted into the medium were assayed with ELISA kits. In this
assay, coxibs inhibited both PGE
2
and PGI while mPGES-1 inhibitors blocked only the
2

synthesis of PGE
a basis for excluding compounds. Finally, compounds were incubated in LPS-stimulated single-
donor human whole blood (HWB), which was then monitored for levels of PGE , TxB , and
2
. Therefore, this assay served to determine selectivity of action and was used as
2
2
PGF2α by LC/MS/MS. This assay provided a direct measurement of target engagement in a
relevant human tissue, obviating the need to factor in additional drug concentration corrections
such as fraction unbound in the determination of potency. The relationship between PGE
2
reduction (as measured in patient plasma samples) and clinical benefit, which was known from
previous work with COX-2 inhibitors, was used in combination with PK data to project an
6
efficacious oral dose. We chose an efficacious dose of celecoxib as the benchmark for both
PGE
2
reduction and duration of coverage by which we would compare our inhibitors.
Celecoxib, a widely prescribed and efficacious COX-2 inhibitor, is reported to have a
HWB IC50 of 540 ± 70 nM (monitored by PGE
therapeutic dose (200 mg) that affords plasma drug concentrations exceeding the HWB IC50 for
–12 hours. We therefore set out to develop inhibitors capable of delivering a celocoxib-like
2
levels) and an oral exposure in humans after a
8
profile, but whose PK properties would potentially maintain plasma concentrations which
exceeded their HWB IC50 values for 24 hours after oral dosing.
As part of ongoing efforts, we sought ligands structurally distinct from those found in our
previous work. Here we report on novel, potent 3,3-dimethyl substituted N-aryl piperidine
1
9
inhibitors of mPGES-1 (Table 1). The piperidine stereocenter was assigned based on X-ray co-
crystal data (vide infra) and the less potent isomer was assigned the opposite configuration by
2
0
deduction. Initial evaluation of stereoisomeric pairs 5–6 and 7–8 revealed a significant
preference for the (S) configuration at the piperidine stereocenter. Inhibition of PGE production
generally was consistent across the three screening assays, and for the reasons given above, our
2

focus was ultimately on potency in the HWB assay. Replacement of the 8-chloroquinoline of 5
with an 8-methylquinoline (9) improved the HWB activity to 2 nM. Changing the 4-
hydroxymethylcyclohexane of 9 to a tetrahydropyran (10) or tetrahydrofuran (11) decreased
potency by more than an order of magnitude in the HWB assay. Acyclic analog 12 was
approximately as potent as its cyclic congeners (11–12) in HWB (IC50 77 nM). The combination
of the 3-hydroxymethylcyclohexane from 7 and the 8-methylquinoline from 9 produced the most
potent compound (13) in the HWB assay, with an IC50 of 1 nM. Replacing a methylene in the
cyclohexyl ring of 13 with an oxygen atom resulted in a potency loss in HWB (14).
HWB
1
2
Piperidine
Chirality
mPGES-1 A549 PGE
2
Compd
R
R
a
PGE
2
ICb 50
IC50 (µM)
IC50 (µM)
(
µM)
5
S
R
S
0.004
0.03 (1)
0.023
0.005
0.002
6
7
8
9
3.72
0.003
0.504
0.001
>10.0 (1)
0.011 (1)
4.5 (1)
R
S
0.002 (1)

1
1
1
0
1
2
S
S
S
0.007
0.006
0.012
0.072
0.036
0.051
0.077
0.023 (1)
0.089 (1)
1
1
3
4
S
S
0.001
0.002
0.003
0.005
0.001
0.007
Table 1. Values are reported as geometric means with a minimum of three replicates unless noted by the trailing
number in parentheses. All geometric sample standard deviations (×/÷) were less than 2.0 for mPGES-1 enzyme
and A549 assay data and less than 3.0 for HWB data.
To better understand the SAR and interactions observed with our ligands, we generated
an X-ray co-crystal structure of 14 with human mPGES-1. The structure revealed a water-
mediated hydrogen bond network between w26, Gln36, Ile32, and the amide carbonyl of 14
(
Figure 2A). Additionally, His53 shares a hydrogen bond with the hydroxyl group of 14. The
space above the glutathione cofactor is occupied by the tetrahydropyran with interactions
dominated by van der Waals contacts in a similar manner to previously reported cocrystal
2
1, 22
structures of mPGES-1 inhibitors.
A surface map reveals a significant contact area with
Leu39 and Ile32, forming the top of a cleft in which the ligand sits. Notably, the methyl
substituent of the quinoline ring fits deeply in the protein surface, displacing a conserved water
molecule seen in previously reported cocrystal structures (PDB codes 4YK5, 4YL0, 4YL1 and
2
1
4
YL3).

A
B
Figure 2. (A) Monomer 1 is colored orange. Monomer 2 is colored cyan. Protein side chains and backbone atoms, shown as
sticks, are colored by atom (O, red; N, blue; C, same as respective monomer). 14 is represented by sticks and colored by atom (O,
red; N, blue; C, yellow). GTT is represented by sticks and colored by atom (O, red; N, blue; C, magenta; S, tan). Waters are
represented by spheres (red). Hydrogen bonds are indicated by green dashes. (B) The binding site is represented as a surface with
the color scheme as is in (A). GTT is depicted by spheres. Polar areas are represented by blue (N) and red (O), and nonpolar
surfaces are represented by cyan in monomer 1 and orange in monomer 2. The 8-methyl of the quinolone points inwards into a
water channel at the back of the active site.
For compounds 5–14, PGI
2
production in A549 cells was not decreased at up to 10 µM
were not decreased at up to 30 µM compound
test concentrations. Similarly, PGF2α and TxB
2
concentrations in the HWB assay. These observations suggest that the compounds do not inhibit
the COX enzymes. In line with these observations, 14 had no activity in human COX-1 or COX-
2
assays at 30 µM, and examples 10, 13, and 14 failed to inhibit human mPGES-2 at 62.5 µM in
a microsomal prep assay. These data are consistent with selective mPGES-1-mediated reduction
of PGE in stimulated whole blood.
2
Compounds 9, 13, and 14 were selected for additional characterization (Table 2).
Microsomal metabolism in rat, dog, and human hepatocytes was moderate for 9 and 13, while 14
was less metabolized. In addition, 14 exhibited less CYP2C9 and CYP3A4 inhibition than did 9
and 13 (Table 2).

CYP2C9% CYP2D6% CYP3A4%
a
b
b
b
Cmpd
clogP
Rat%
Dog%
Human%
c
c
c
Inh
Inh
Inh
9
4.31
4.31
2.91
64
73
16
60
57
13
46
50
20
54
64
15
4
35
43
10
13
4
0
d
d
d
d
d
d
1
0
Table 2. a) Calculated using Marvin and calculator plugin freewar (www.chemaxon.com, ChemAxon Kft.
Budapest, Hungary); b) Percent metabolized in hepatic microsomes at 4 µM test concentration; c) Percent inhibition
of probe substrate metabolism in human hepatic microsomes at 10 µM test concentration; d) Data is an average of
two separate runs.
Next, we evaluated the pharmacokinetic (PK) properties for these ligands in rats (Table 3).
Compounds were dosed orally at 5 mg/kg or intravenously at 1 mg/kg. Steady state volumes of
distribution ranged between 1.3 and 2.3 L/kg. While 9 and 13 both had bioavailability greater
than our target of 20%, 14 proved superior. The clearance and Cmax values were within ranges
we considered desirable.
1
mg/kg IV
(h)
5 mg/kg PO
CL
Cmpd
t
½
Vd,ss (L/kg)
C
max (nM)
%F
(
mL/(min*kg))
9
21 ± 1.56
34 ± 2.49
14 ± 4.07
2.6 ± 0.19
0.6 ± 0.01
2.3 ± 0.22
1.4 ± 0.05
1.3 ± 0.07
2.3 ± 0.19
1450 ± 125
1430 ± 193
2050 ± 176
54
30
100
1
1
3
4
Table 3. Pharmacokinetics in rats. Data are reported as mean of observations from three animals with standard
deviation.
Further profiling of 9, 13, and 14 revealed a measurable time dependent inhibition (TDI)
of CYP3A4 when tested at a 10 µM concentration in human hepatic microsomes. Inhibition of
7
5% and 82%, for 9 and 13 respectively, represented a potential risk factor for drug-drug
interactions with prolonged drug exposure. With a corresponding value of 39%, compound 14
appeared to differentiate. At this point, our focus turned to 14 for further study.
To investigate possible species differences, we evaluated 14 in rat (IC50 = 7.87 µM ×/÷
1.16) and dog (IC50 = 11 nM ×/÷ 1.17) mPGES-1 enzyme assays. The species selectivity

1
7, 18, 23
difference is consistent with previously reported mPGES-1 inhibitors.
The dog enzyme
and whole blood (IC50 = 28 nM ×/÷ 1.53) potencies were similar to those observed in the
corresponding human assays. This suggested the possibility of exploring preclinical
pharmacology in dogs and prompted us to evaluate the pharmacokinetic profile of 14 in that
species.
In dog, the clearance (CL = 3.62 ± 0.36 mL/(min*kg)) and volume of distribution (Vd,ss
=
1
.6 ± 0.15 L/kg) of 14 after IV (1 mg/kg) administration and the bioavailability (%F = 74%) after
a 15 mg/kg oral dose were within our target ranges. In a separate dog PK study (Figure 3), after
a 3 mg/kg oral dose, drug plasma levels exceeded the DWB IC50 for 24 hr. Thereby, compound
1
4 met our exposure criterion in a preclinical species.
DWB IC 23nM
5
0
Time (h)
Figure 3. PK of 14 in beagle after 3 mg/kg po dosing (n = 3). Diamonds indicate means and error bars
represent standard deviation of measured values. Dog whole blood potency noted for comparison purposes.

Preparation of 14 was achieved by a convergent synthesis as shown in Schemes 1–3. The
chirally pure acid 21 was prepared as follows: ketone 15 was converted to vinyl triflate 16
followed by palladium mediated carbonylation in the presence of methanol to provide the methyl
ester 17. Attempts to hydrogenate the olefin stereoselectively were unsuccessful; therefore, we
continued with racemic 18. Deprotection of 18 followed by nucleophilic aromatic substitution
on 2-chloro-8-methylquinoline gave rac-20, which was subjected to chiral chromatography to
provide methyl ester (S)-20 as a single enantiomer. Acidic hydrolysis of the ester gave rise to
the advanced intermediate (S)-21. The acidic conditions were employed because they did not
epimerize the stereocenter alpha to the carbonyl.
Scheme 1. Synthesis of 21. Reagents and conditions: (a) Tf
dppf, Et N, 88%; (c) H , Pd/C, CH OH, 85%; (d) HCl, dioxane, 92%; (e) 2-chloro-8-methylquinoline, K
DMSO, chiral separation, 43%; (f) H SO (aq), 96%.
2
NPh, BuLi, THF, 75%; (b) CO, CH
3
OH, Pd(OAc)
2
,
3
2
3
2
3
CO ,
2
4

Synthesis of the 4-aminotetrahydropyran 22 (Scheme 2) began with a hetero-Diels–Alder
cycloaddition between aldehyde 23 and diene 24 to provide dihydropyranone 25. Palladium
catalyzed hydrogenation of the olefin moiety of 25 gave ketone 26, which was reduced with
LAH to afford rac-27. The racemate was resolved by chiral chromatography to give chirally
pure (2S,4R)-27. Mesylation to furnish (2S,4R)-28, followed by S
azide, yielded (2S,4R)-29, which was hydrogenated to provide (2S,4R)-22. Finally, (S)-21 and
2S,4R)-22 were coupled to give the desired amide 14 (Scheme 3).
N
2 displacement with sodium
(
Scheme 2. Synthesis of intermediate 22. Reagents and conditions: (a) ZnCl
EtOAc, Et N, 84%; (c) LAH, THF; (d) Chiral Chromatography, 42% two steps; (e) MsCl, DIEA, CH
NaN , DMF; (g) H , PtO2, EtOAc, 85%.
2
, THF/Toluene, 73%; (b) H
2
, Pd/C,
3
2
Cl
2
, 97%; (f)
3
2

3
Scheme 3. a) Synthesis 14. Reagents and conditions: (a) PyBOP, Et N, DMF, 76%
In summary, we have identified a novel series of 3,3-dimethylpiperidine-1-quinolines as
inhibitors of mPGES-1. Importantly, these compounds are capable of selectively inhibiting
PGE production at low nanomolar concentrations in HWB. The potency of 14, taken along with
2
its pharmacokinetic profile, suggests that this compound would be amenable to clinical
investigation for anti-inflammatory therapy.
Accession Codes
Atomic coordinates and structure factors for compound 14 with mPGES-1 are available from the
RSCB Protein Data Bank with accession code 5K0I.
Acknowledgements
We express our sincere appreciation to Dr. Daniel Mudra and Jennifer Weller for ADME support
and critical review of this manuscript. Use of the Advanced Photon Source, an Office of Science
User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne
National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-
0
6CH11357. The beamline staff, Stephen Wasserman, John W. Koss, David Smith, and Laura
Morisco are gratefully acknowledged.
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Compounds 5–8 were synthesized in an analogous method to 14 but carried
through as 1:1 mixtures at the piperidine center, and ultimately separated by
chiral chromatography as the final step. We desired a more efficient route
and chose to synthesize advanced intermediate 21 as a single enantiomer
(
Scheme 3). Protein Co-crystal structure of thereby prepared 14 revealed the

configuration of the preferred stereo center to be S (figure 2). The remaining
products (9–13) were prepared from the preferred isomer.
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1
2
2
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Graphical Abstract
Dog PK
X-Ray Cocrystal
DWB IC 23nM
5
0
Time (h)