Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides as inhibitors of soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.bmcl.2013.04.019

1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified from high through-put screening using encoded library technology. The triazine heterocycle proved to be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution was important for reducing clearance, and establishing good oral exposure. Based on this lead optimization work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N- {[[4-bromo-2-(trifluoromethoxy)]-phenyl]methyl}-4-piperidinecarboxamide (27) was identified as a useful tool compound for in vivo investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement and the suitability of 27 as a tool compound for study in various disease models.

Full text of DOI:10.1016/j.bmcl.2013.04.019

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3584–3588
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides
as inhibitors of soluble epoxide hydrolase
Reema K. Thalji a, Jeff J. McAtee a, Svetlana Belyanskaya d, Martin Brandt d, Gregory D. Brown a,
Melissa H. Costell b, Yun Ding d, Jason W. Dodson a, Steve H. Eisennagel c, Rusty E. Fries c,
Jeffrey W. Gross d, Mark R. Harpel b, Dennis A. Holt a, David I. Israel d, Larry J. Jolivette c, Daniel Krosky b,
Hu Li d, Quinn Lu d, Tracy Mandichak d, Theresa Roethke c, Christine G. Schnackenberg b,
Benjamin Schwartz d, Lisa M. Shewchuk d, Wensheng Xie d, David J. Behm b,
Stephen A. Douglas b, Ami L. Shaw d, Joseph P. Marino Jr. a,
⇑
a Department of Chemistry, Heart Failure Disease Performance Unit, Metabolic Pathways and Cardiovascular Therapeutic Area Unit, GlaxoSmithKline,
709 Swedeland Road, King of Prussia, PA 19406, United States
b Department of Biology, Heart Failure Disease Performance Unit, Metabolic Pathways and Cardiovascular Therapeutic Area Unit, GlaxoSmithKline, 709 Swedeland Road,
King of Prussia, PA 19406, United States
c Department of Drug Metabolism and Pharmacokinetics, Heart Failure Disease Performance Unit, Metabolic Pathways and Cardiovascular Therapeutic Area Unit,
GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, United States
d Platform Technology and Science, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 February 2013
Revised 5 April 2013
Accepted 8 April 2013
Available online 16 April 2013
1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified
from high through-put screening using encoded library technology. The triazine heterocycle proved to
be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution
was important for reducing clearance, and establishing good oral exposure. Based on this lead optimiza-
tion work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N-{[[4-bromo-2-(trifluoromethoxy)]-phe-
nyl]methyl}-4-piperidinecarboxamide (27) was identified as
a useful tool compound for in vivo
Keywords:
investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide
derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement
and the suitability of 27 as a tool compound for study in various disease models.
Published by Elsevier Ltd.
Soluble epoxide hydrolase
Epoxyeicosatrienoic acid (EET)
Dihydroxyeicosatrienoic acid (DHET)
9,10-Epoxyoctadec-12(Z)-enoic acid (LTX)
Leukotoxin (LTX)
Triazine
Inhibition of the enzyme soluble epoxide hydrolase (sEH) has
recently emerged as a new approach to suppressing maladaptive
functional and structural changes that are associated with cardio-
vascular disease.1 Soluble epoxide hydrolase is a ubiquitously ex-
pressed enzyme responsible for the hydrolysis of the epoxides,
epoxyeicosatrienoic acids (EETs), to their corresponding diols,
dihydroxyeicosatrienoic acids (DHETs). In mammalian cells epox-
yeicosanoids are formed through the cytochrome P450 oxidative
metabolism (primarily CYP2C and CYP2J)1 of arachidonic acid. EETs
are known to function as autocrine and paracrine effectors produc-
ing a variety of cardiovascular and inflammatory effects.2 In mes-
enteric and renal vessel beds EETs produce vasodilatory effects,
and various reports suggest EETs may function as the endothe-
lium-derived hyperpolarizing factor playing an integral role in
the hyperpolarization of smooth muscle.3 Based on the biochemi-
cal pathways affected by EETs, it is possible that EETs are cardio
protective by preventing apoptosis (PI3K/AKT)4,5, reducing inflam-
mation (NF-
B).4,6,7
To fully investigate the utility of a sEH inhibitor for cardiovascu-
j
B)6, and suppressing oxidative stress (KATP, NF-
j
lar and other disease indications our initial objective was to iden-
tify a tool compound with appropriate potency, selectivity, and
oral pharmacokinetics. The first step on our critical path to a tool
compound focused on cellular potency. Since sEH is an intracellu-
lar target we developed a cell assay overexpressing sEH to assess
cell permeability and inhibition of EET to DHET enzymatic hydro-
lysis by sEH. Achieving good cardiac ion channel selectivity was
also critical since chemical agents which block the L-type calcium
channel (CaV1.2) or cardiac sodium channel (NaV1.5) would likely
lower blood pressure, decrease cardiac output, and thus confound
the interpretation of in vivo cardiovascular results. Selectivity
⇑
Corresponding author.
E-mail address: joseph.p.marino@gsk.com (J.P. Marino).
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.04.019

R. K. Thalji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3584–3588
3585
CF3
against microsomal epoxide hydrolase (mEH), an a/b-hydrolase fold
O
i
.
CF3
O
O
enzyme with sequence and structural similarity to sEH was priori-
tized since mEH plays a key role in xenobiotic and extrahepatic
metabolism.8 Also, of concern was selectivity against the cyto-
chrome P450 monooxygenases since these metabolic enzymes con-
vert arachidonic acid to EET.1a Inhibition of P450 enzymes could
reduce EET levels, and thus suppress the desired pharmacological ef-
fect gained by blocking sEH. In terms of the desired pharmacokinetic
profile for a tool compound dosed chronically, good oral bioavail-
ability was critical since drug exposure from chow is often targeted.
Chow dosing was desired in order to maintain stable drug levels and
avoid labor intensive oral gavage dosing on large groups of animals
over multiple days. This article will describe the lead optimization of
screening hit 4, the developability profile of in vivo sEH tool com-
pound 27, and in vivo biomarker study of 27 in the rat.
NH2
HO
N
H
N
ii.
NH
Boc
Cl
6
5
H
N
N
CF3
N
N
CF3
O
N
H
7
Cl
H
N
iii, iv
N
N
R
N
H
8a-b
NH
N
N
6
v, vi
CF3
O
Cl
N
Cl
N
H
R1
N
N
N
N
N
R2
R
10a-h
N
N
Recently several structural types of N-benzyl amide inhibitors
(1; Boehringer, 2; Taisho, and 3; Hammock and Landry labs) of
sEH have been reported to possess good potency, aqueous solubil-
ity, and metabolic stability (Fig. 1).9 Our own efforts in this area be-
gan with the discovery of lead 4 (Fig. 1) derived from high through-
put screening employing encoded library technology (ELT).10 Like
previous amide inhibitors of sEH, it was anticipated that the amide
functionality contained in 4 interacts with the catalytic residues
(Tyr381/465 and Asp333) in the sEH active site.
The unique triamino-triazine functionality represented a good
starting point for optimization of developability parameters such
as bioavailability and clearance. Our goal for this series was to
identify a tool compound with good cellular potency, selectivity,
and oral PK for study in chronic models of cardiovascular and
respiratory disease.
Soluble (human and rat) and microsomal (human) epoxide
hydrolase enzyme activity was measured using a fluorescence-
based detection assay employing 3-phenyl-oxiranyl-acetic acid cy-
ano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME) de-
signed by Hammock and coworkers.11 Cellular activity was
determined by assessing a compounds ability to inhibit the conver-
sion of EETs to DHETs in human embryonic kidney (HEK) cells
over-expressing human sEH. This was accomplished using an ELISA
readout which employed a radiolabeled DHET antibody.12 In terms
of oral PK, in general, early analogs from this series displayed good
oral bioavailability however clearance was moderate to high. Lead
optimization focused on identifying new leads with nanomolar
sEH potency, greater than 1000-fold selectivity over CYP2C9
P45013, and low iv clearance (with good oral %F).
The impact of substitution on the triazine ring was initially
probed. Functionalization of the piperidine group was accom-
plished through N-benzyl-piperidine 6, readily available from
piperidine-4-carboxylic acid (Scheme 1). Reaction of piperidine 6
with the appropriate dichloro-triazine intermediate, followed by
chloride displacement with the requisite amine gave rise to targets
9
R
Scheme 1. Preparation of triamino-triazines. Reagents and conditions: (i) EDC,
DIEA, CH2Cl2/DMF; (ii) TFA, CH2Cl2; (iii) 7, Hunigs base, CH3CN, RT, 24 h; (iv) R-NH2,
Hunigs base, CH3CN, RT, 24 h; (v) 9 (R = Me, Et, c-hexyl, phenyl), DIEA, CH2Cl2, rt.
(vi) MeNH2 or NHR1R2, MeOH, 40 °C.
4, 8a–b, and 10a–h (Table 1). Early analogs (8a and 8b), in which
the N,N-4-methyl-piperazine was replaced with other amine
groups, did not show an improvement in iv clearance or potency,
relative to 4. However when the piperazine group was replaced
with an alkyl or aryl substituent (4 ? 10a–h), potency was im-
proved by an order of magnitude. The most substantial change in
clearance in the rat (iv) occurred where R = phenyl (10d). However
the 10-fold reduction in clearance for 10d may be due to the extre-
mely high plasma protein binding observed for this analog (rat
Fu = 1.5%). In most cases the CYP2C9 selectivity was 100-fold or
better which indicates that inhibition of the CYP2C family may
not be an issue for this chemical class.13 SAR exploration around
the methyl-amine group of lead 10a identified several substitu-
tions (10g and 10h) that gave rise to improved cellular potency,
however P450 selectivity was reduced.
The triazine core was another area that was examined to deter-
mine if this core offered advantages over other six-membered aro-
matic rings. Phenyl, pyridine, and pyrimidine core substitutions
were synthesized in straight-forward fashion according to known
procedures. In general, pyridines, pyrimidines, and phenyl core
replacements led to a decrease in activity relative to triazine (Ta-
ble 2). Examination of the iv clearance for the most potent pyrim-
idine (13) and pyridine (16) did not suggest a clear advantage for
these two heterocycles over the triazine 10a. Additionally, 13 dis-
played reduced CYP2C9 selectivity, indicating no advantage over
triazine 10a.
The crystal structure of 10a bound in the active site of human
sEH,19 highlighted in Figure 2, suggested that there was space
Cl
O
O
OMe
O
OMe
OMe
OMe
F
F
N
H
N
N
N
H
MeO2S
N
O
F
MeO
CF3
1
2
O
N
H
H
N
N
N
Cl
4
N
N
N
H
N
N
N
Cl
S
O
3
O
Figure 1. N-Benzylamide inhibitors of sEH.

3586
R. K. Thalji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3584–3588
Table 1
Activity profile for substituted amino-triazine analogs 8 and 10
Ex.
R
R1/R2
Hu sEH Enz, HEK IC50 (nM)
Rat Clp iv (ml/min/kg) (1.0 mpk)
P450 CYP2C9 IC50 (nM)
4
8a
8b
4-Me-1-piperazinyl
NHMe
NMe(CH2)2-NMe2
Me
Et
c-Hexyl
Ph
Me
Me
H/Me
H/Me
H/Me
H/Me
H/Me
H/Me
H/Me
Me/Me
40, NT
20, 100
80, NT
3, 100
4, 80
1, NT
1, 63
1, 16
1, 10
1, 3
46
79
140
51
NT
NT
3
NT
NT
NT
27
16,000
25,000
17,000
25,000
9600
63
1800
10,000
400
10a
10b
10c
10d
10e
10f
10g
10h
H/i-Pr
Me
Me
–(CH2)2O–(CH2)2À
400
1800
H/–(CH2)2ÀOMe
1, 1
NT – not tested; mpk – mg/kg.
Table 2
Table 3
Triazine core replacements
Benzyl substitutions
CF3
O
O
N
H
N
H
R3
H
H
N
X
N
N
N
N
Z
N
N
y
Ex.
R3
Hu sEH Enz Hu sEH HEK Rat Clp iv
P450
CYP2C9
Ex.
x, y, z
Hu sEH Enz,
HEK IC50 (nM)
Rat Clp iv 1.0 mg/kg
(ml/min/kg)
P450 CYP2C9
IC50 (nM)
IC50 (nM)
IC50 (nM)
1.0 mg/kg
(ml/min/kg) IC50 (nM)
10a
11
12
13
14
15
16
17
N, N, N
3, 100
51
NT
NT
73
NT
NT
61
NT
25,000
10a 2-CF3
3
1
100
3
51
44
NT
NT
NT
NT
NT
NT
NT
7
25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>33,000
N, CH, N
N, N, CH
CH, N, N
CH, CH, N
CH, N, CH
N, CH, CH
CH, CH, CH
40, NT
160, NT
5, 80
126, NT
1585, NT
10, 100
32, NT
>25,000
>25,000
1000
>25,000
>25,000
11,000
2000
18
19
20
21
22
23
24
25
26
2-OCF3
2-CH3
2-OCH3
3-OCF3
4-OCF3
H
2-CN
2-CF3-4-F
2-CF3-4-OCH3
200
1600
630
400
200
80
3
1
0.5
NT
NT
NT
NT
NT
NT
50
2
27a 2-OCF3-4-Br
3
12
a
Rat enzyme sEH IC50 for 27 = 2 nM.
10a. The amide group of 10a is located in the center of the pocket,
and makes hydrogen bond contacts with Tyr381/465 and Asp333.
The phenyl ring of the benzyl substituent is positioned near the
opposite opening (left in the picture), with the trifluoromethyl
group positioned in a small hydrophobic pocket. The most fertile
region of the molecule for improving cellular potency and reducing
clearance proved to be the benzyl moiety. A series of analogs syn-
thesized to explore the impact of benzyl ring substitution on po-
tency and oral PK are highlighted in Table 3. Substituted benzyl
analogs of 10a were synthesized in a similar fashion as 10a, origi-
nating from commercially available or custom synthesized
benzylamines.
The preference for an electron withdrawing substituent on the
benzyl ring is evident if one compares the activity for leads 10a
and 18 to analogs which contain electron donating groups such
as 19 and 20 (Table 3). A 2–3 order of magnitude loss in potency
is observed with ortho-methyl and ortho-methoxy compared to
ortho-trifluoromethyl or ortho-trifluoromethoxy. ortho-Substitu-
tion on the phenyl ring of the benzyl group, relative to meta- or
para-positioning proved to be a critical discovery as evidenced by
the 100-fold reduction in potency for 21 and 22 relative to 18
(Table 3). The crystal structure of 10a (Fig. 2) indicates that there
is a hydrophobic pocket adjacent to the ortho-position which
serves as an anchoring point for the ligand and likely explains
the ortho-substitution preference over analogs 21, 22, and also
unsubstituted benzyl 23 (Table 3). Polarized, electron-withdrawing
Figure 2. X-ray crystallographic structure of human soluble epoxide hydrolase-1-
(1,3,5-triazin-2-yl)piperidine-4-carboxamide complex (PDB code 4JNC).
surrounding the benzyl and triazine groups for additional substitu-
tion. The crystal structure revealed that molecule 10a is oriented in
the center of a highly lipophilic, tube-like active site, specially
designed to accommodate a linear EET molecule. The triazine
group is positioned at the opening (right of the picture) of the ac-
tive site, with the triazine substituents directed out into solvent.
This may explain why larger substitutions such as morpholine
(Ex. 10g) are accommodated without a loss in potency relative to

R. K. Thalji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3584–3588
3587
groups were also found to be less optimal compared to non-polar-
ized electron-withdrawing groups. For example, cyano-analog 24
displayed a 10-fold loss in activity relative to trifluoromethyl
(10a) or trifluoromethoxy (18). Combining a 2-trifluoromethyl-
or 2-trifluoromethoxy substituent with a variety of 4-substituents
(F, 25; OCH3, 26; Br, 27) resulted in no loss of enzyme activity at
human sEH. In the case of 25, a 2-trifluoromethyl-4-fluoro-analog,
activity in the human cellular assay did decrease 10-fold relative to
the other para-substituted analogs (26 and 27). However more
importantly, the addition of a para-substituent led to a significant
reduction in iv clearance. Compound 27 (previously published as
GSK2188931B)18 was progressed forward as an in vivo tool com-
pound based on the low nanomolar potency it displayed across
all sEH assays. It also exhibited low clearance in the rat, and dem-
onstrated good CYP2C8 and 2C9 selectivity(Table 4). In terms of a
broader selectivity analysis, 27 was highly selective over human
microsomal epoxide hydrolase (mEH IC50 > 50 lM), cardiac ion
channels (CaV1.2, hERG, and NaV1.5), and a diversity of targets
in a CerepÓ panel (Table 4).14
Prior to evaluating tool compound 27 in chronic models of car-
diovascular disease in the rat it was critical to confirm that in vivo
inhibition of sEH could be achieved. Since circulating cells such as
leukocytes and neutrophils in mammalian plasma contain high
levels of sEH15, it was postulated that a plasma sEH biomarker as-
say could be readily established. It is also worth noting that leuko-
toxin or 9,10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived
from linoleic acid) is present at relatively high concentrations
(compared to epoxyeicosatrienoic acids) in circulating neutro-
phils.16 Since sEH is also responsible for the conversion of leuko-
toxin to leukotoxin diol, an increase in the systemic leukotoxin/
leukotoxin diol (LTX/LTX diol) ratio upon treatment with inhibitor
was used as confirmation of in vivo target engagement. Treatment
of rats with 27 at three doses, 0.2, 2, and 20 mg/kg (po gavage), re-
sulted in a 50–100% increase in the leukotoxin/leukotoxin diol ra-
tio over baseline thus confirming in vivo inhibition of sEH by 27
(Fig. 3). Cmax drug levels for the 0.2, 2.0, and 20 mg/kg doses were
8, 112, and 750 ng/mL or 16, 223, and 1490 nM, respectively
(Fig. 3). The comparable LTX/LTX diol ratios observed at the four
hour time point across a ꢀ100-fold concentration range (three
doses) suggests maximal sEH inhibition was achieved. Similarly,
analogs from a structurally distinct series also exhibit a similar
magnitude of effect over a comparable concentration/dose range
suggesting that the effect on LTX/LTX diol ratio is maximal. These
data also suggest plasma protein binding (rat % unbound is 1.5)
does not play a role in suppressing sEH activity at least at the 4-
h time point. While the 2 and 20 mg/kg doses deliver Cmax free
drug concentrations (3 and 24 nM) which are at or eightfold above
the cellular IC50 for sEH (3 nM), the free Cmax concentration asso-
ciated with the 0.2 mg/kg dose (0.25 nM) is almost 10-fold below
the cellular IC50 for 27. At earlier time points, 1 and 2 h, it appears
as though the 0.2 mg/kg response is attenuated compared to the
response at 4 h. The robust LTX/LTX diol response at 4 h for the
0.2 mg/kg dose is curious, given the low drug concentrations. It is
possible that a slow off-rate is responsible for the enhanced phar-
macodynamic effect once drug levels have declined. Analogs from
this chemical series have been shown to have off-rates in the min-
utes to hours range.17
Figure 3. Plasma levels and leukotoxin/leukotoxin diol ratio in 27 treated rats.
SAR exploration around the high through-put screening hit 4,
an sEH inhibitor, revealed that the triazine functionality was criti-
cal for maintaining potency, selectivity, and exposure. Substitution
at the para-position of the benzyl moiety was found to be impor-
tant for reducing clearance and improving exposure in rodents.
X-ray crystallography helped rationalize SAR and provided guid-
ance for future chemical group modifications. Tool compound 27
was discovered which displayed excellent cellular potency, good
cardiac ion channel/broad-based selectivity, and promising oral
exposure. Tool compound 27 represents one of the first sEH
compounds disclosed in the literature with extensive selectivity
characterization data (cardiac ion channels and Cerep screening)
which underscores its suitability for rigorous target validation
study.20 In addition, 27 demonstrated robust in vivo sEH enzyme
activity as evidenced by changes observed in plasma leukotoxin/
leukotoxin diol ratios in rats. These robust effects on serum sEH
biomarkers validate 27 as a useful in vivo tool compound for
exploring the utility of sEH inhibitors for the treatment of chronic
diseases.18
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
019.
References and notes
Table 4
Profile for tool compound 27
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Rat Clp iv 2.0 mg/kg T1/2 iv Vdss iv %F DNAUC iv P450 2C8, CaV1.2,
(ml/min/kg)
(h)
(L/kg)
(h-kg/L) 2C9 (lM) hERG, NaV1.5
(IC50
; lM)
12
4.6
2.1
100 1.3
>30
>10

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13. Most analogs were screened against CYP2C8, CYP2C19, and CYP2J2 as well.
Little or no inhibition was observed at 10
lM concentration.
14. Less than 50% inhibition observed at 1 M in the following binding assays: A1
l
(human), A2A (human), A3 (human), B2 (human), Alpha 1 (human), Alpha 2
(human), Beta 1 (human), AT1 (human), BZD (human), CCK1 (human), D1
(human), D2S (human), ETA (human), GABA (human), GAL2 (human), CXCR2
(human), H1 (human), H2 (human), MC4 (human), MT1 (human), M1 (human),
M2 (human), M3 (human), M4 (human), NK2 (human), NK3 (human), Y1
(human), Y2 (human), NTS1 (human), Delta 2 (human), Kappa Opioid (human),
Mu Opioid (human), P2X (human), 5-HT1A (human), 5-HT1B (human), 5-HT2A
(human), 5-HT3 (human), 5-HT5A (human), 5-HT6 (human), 5-HT7 (human),
Sigma (human), VPAC1 (human), V1a (human), Kv (human), SKCa (human), Cl-
channel (human), Norepinephrine transporter (human), and Dopamine
transporter (human).
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17. Data not obtained for 27, however structurally analogous compounds possess
half-lives up to 60 min.
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19. The X-ray coordinates have been deposited in the RCSB Protein Data Bank
(PDB). Code is 4JNC.
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