Design and synthesis of substituted nicotinamides as inhibitors of soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.bmcl.2009.08.074

A series of potent nicotinamide inhibitors of soluble epoxides hydrolase (sEH) is disclosed. This series was designed using structure-based deconstruction and a combination of two HTS hit series, resulting in hybrid analogs that retained the optimal poten

Full text of DOI:10.1016/j.bmcl.2009.08.074

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5864–5868
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of substituted nicotinamides as inhibitors
of soluble epoxide hydrolase
a
a
a
a
Steven J. Taylor a, , Fariba Soleymanzadeh , Anne B. Eldrup , Neil A. Farrow , Ingo Muegge ,
*
Alison Kukulka a, Alisa K. Kabcenell b, Stephane De Lombaert a
a Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc. 900 Ridgebury Rd. PO Box 368, Ridgefield, CT 06877-036, USA
b Department of Biotherapeutics and Integrative Biology, Boehringer Ingelheim Pharmaceuticals, Inc. 900 Ridgebury Rd. PO Box 368, Ridgefield, CT 06877-0368, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of potent nicotinamide inhibitors of soluble epoxides hydrolase (sEH) is disclosed. This series was
designed using structure-based deconstruction and a combination of two HTS hit series, resulting in
hybrid analogs that retained the optimal potency from one series, and acceptable in vitro metabolic sta-
bility from the other. Structure-guided optimization of these analogs gave rise to nanomolar inhibitors of
human sEH that had acceptable plasma exposure to qualify them as probes to determine the in vivo phe-
notypic consequences of sEH inhibition.
Received 22 July 2009
Revised 19 August 2009
Accepted 21 August 2009
Available online 26 August 2009
Keywords:
Soluble epoxide hydrolase
Nicotinamides
Ó 2009 Elsevier Ltd. All rights reserved.
Epoxyeicosatrienoic acids
Inhibitors
Soluble epoxide hydrolase (sEH) is an enzyme involved in the
metabolism of chemical mediators derived from arachidonic acid.1
In particular, sEH catalyzes the hydrolysis of epoxyeicosatrienoic
acids (EETs) (derived from oxidation of arachidonic acid by CYP2 J
and/or CYP2C) to the corresponding dihydroxyeicosatrienoic acids
(DHETs) (Figure 1). Inhibition of sEH is expected to increase local-
ized endogenous EETs levels, thereby potentiating their in vivo
pharmacological effects which include anti-inflammatory and
vasodilatory properties. For example, selective inhibition of soluble
epoxide hydrolase has been invoked to account for the antihyper-
tensive effect of dicyclohexyl urea (DCU) in the spontaneously
hypertensive rat.2 It is also reported that EETs elicit a vasodilatory
response by acting as an endothelium derived hyperpolarizing fac-
tor (EDHF) that mediates vasodilatation through the stimulation of
calcium-activated potassium channels in smooth muscle cells.3
Selective sEH inhibitors have also shown beneficial effects in an
angiotensin II-dependent model of hypertension in the Sprague–
Dawley rat,4 and protective action in models of hypertension-
induced renal damage and failure.5 An sEH inhibitor significantly
decreased the total bronchoalveolar lavage cell number in tobacco
smoke-exposed rats, with significant reductions noted in neutro-
phils, alveolar macrophages, and lymphocytes in a rat model of
airway inflammation.6 Combined, these reports suggest that
inhibition of sEH represents a potentially novel method for the
treatment of inflammatory and cardiovascular diseases.
To date, the majority of published sEH inhibitors can be de-
picted as urea derivatives, flanked on either side by lipophilic
groups.7 More recently, amide and carbamate inhibitors have also
been disclosed.8 The urea, amide, and carbamate moieties are
thought to mimic the transition-state for the hydrolysis of endog-
enous epoxides within the active site of sEH. This mechanism is
supported by the published crystal structure of sEH with a urea
inhibitor, where the urea pharmacophore is involved in direct
hydrogen bonding with the sEH catalytic triad of tyrosine 383,
O
CYP2Cs, CYP2Js
OH
Arachidonic Acid
O
OH
Soluble EpoxideHydrolase
(sEH)
O
O
Epoxyeicosatrienoic Acids (EETs)
OH
HO
OH
(DHETs)
Dihydroxyeicosatrienoic Acids
* Corresponding author. Tel.: +1 203 791 6371.
E-mail address: steven.taylor@boehringer-ingelheim.com (S.J. Taylor).
Figure 1. Role of soluble epoxide hydrolase in the conversion of EETs to DHETs.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.074

S. J. Taylor et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5864–5868
5865
tyrosine 466, and aspartic acid 335.9 In October 2007, a phase 1
clinical trial for hypertension was initiated with a sEH inhibitor
from the urea class, AR9281.10
In this report, we disclose our efforts towards designing novel
synthetic inhibitors of sEH. Our goal was to identify a small,
drug-like inhibitor of sEH that could be administered orally once
daily. High-throughput screening (HTS) of a selection of our corpo-
rate library against sEH revealed three distinct chemical series, one
of which is exemplified by the amide (1) shown in Figure 2. Initial
SAR focused around the nicotinamide 1 which, albeit displaying
excellent intrinsic potency using an assay that measures the
displacement of a rhodamine-labeled probe from sEH,15 suffered
from a poor in vitro metabolic half-life. In vivo and in vitro metab-
olite identification revealed that oxidative metabolism was pre-
dominantly occurring on the benzhydryl motif,11,12 most likely
accounting for the rapid clearance of these analogs when dosed
intravenously. Partial optimization of another of our HTS leads
resulted in a class of urea inhibitors, exemplified by 2, which
displayed extended half life in the presence of human and rat
microsomes, but resulted in lower potencies than the correspond-
ing benzhydryl amides 1.12 We hypothesized that a combination of
the right-hand side (RHS) dichlorobenzylamine present in the urea
series coupled with the left-hand side substituents featured in the
nicotinamides could provide inhibitors that retained the optimal
properties from each of the parent series.
A crystal structure of inhibitor 1 bound to human sEH showed
that the arylamide moiety occupies a large pocket that opens
towards solvent (Figure 3, S1 pocket).12 One aryl group of the
benzhydryl motif is located in a deep pocket (S2) while the other
occupies a narrow polar channel to solvent (S3). Docking (GLIDE)
of compound 3 into the crystal structure of human sEH suggested
that the dichlorobenzyl amine moiety of 3 could be used as an
alternative to the benzhydryl motif of 1. Figure 3 illustrates that
both chloro groups of the dichlorobenzyl amine moiety were opti-
mally positioned to allow for the addition of surrogate moieties to
the two RHS aryl groups of 1. The pocket occupied by the 2-chloro
moiety was deeply buried in the protein matrix, and therefore
severely limited the optimization in this area. In contrast, the 4-
chloro vector allows access to solvent and more space for further
structural modifications.
Figure 3. Compound 3 (cyan) containing a 2, 4-dichlorobenzyl motif docked into
the co-crystal structure of human soluble epoxide hydrolase and compound 1
(yellow) PDB code 3I1Y.
eroaromatic ring. Synthesis of the amides was performed under
standard amide bond forming conditions, as shown in Scheme 1.
These hybrid compounds, and the initial HTS lead series analogs
were profiled for their ability to inhibit human and rat sEH as well
as their propensity to undergo in vitro oxidative metabolism.
Achieving potency against the rat enzyme was also important, as
the pharmacological effects of sEH inhibition were going to be as-
sessed in this species. The baseline pyridone, amino pyridine, and
pyrrolidine pyridine analogs were all significantly less potent
(>50-fold), than the corresponding diphenyl propyl amine analogs
(Table 1, entries 1–6).13 This potency trend was observed for both
the human and the rat isoforms of sEH. Despite their lack of intrin-
sic potency, the compounds in the dichlorobenzylamide series
were, in general, less susceptible to in vitro oxidative metabolism
compared to the benzhydryl analogs, supporting our initial
hypothesis. We theorized that the potency of the compounds in
this series could be increased by substitution of the nicotinamide
moiety with lipophilic groups that would better fit the large,
non-polar S1 pocket of sEH. Gratifyingly, the alkylated pyridone
12 was significantly more potent than the previously tested hybrid
molecules 6, 8, and 10, and retained the improved h-LM/r-LM data
of these initial, less potent analogs (Table 1, entry 7). Furthermore,
substitution of the pyridyl ring with a trifluoroethoxy group
yielded compound 14, that demonstrated high potency in both
the human and rat molecular sEH assays and displayed a decreased
susceptibility towards in vitro oxidative metabolism in the h-LM
assay (Table 1 entry 9 and 10). However, albeit displaying a
significant increase in potency, the dichlorobenzyl amides were
still not as potent as the analogous compounds from the benzhy-
dryl amide series.
Based upon the docking data and the co-structure of the nico-
tinamide inhibitor (R = H), a panel of compounds were synthesized
that combined the dichlorobenzyl scaffold 4 with a diverse selec-
tion of aromatic moieties that had previously provided potent
sEH inhibitors with the benzhydryl RHS 5 (Table 1).12 The selection
criteria for the groups was driven by diversity of chemical struc-
ture with respect to polarity and substituent patterns on the het-
We then focused our optimization on the benzylamine portion
of the molecule while keeping the (2,2,2)-trifluoroethoxy substi-
tuted pyridine constant. Based upon modeling of 3, we explored
substitution at the 2- and 4-position of the aromatic ring of the
benzylamide, as these substituents would be engaging the sEH
S2 pocket and S3 channel, respectively (Fig. 3).
Synthesis of differentially substituted benzyl amine reagents is
shown in Scheme 2. Generation of the 2-chloro 4-methylsulfona-
mide benzylamine (18) was accomplished by treatment of
3-chloro-4-methylbenzenesulfonyl chloride (16) with methyl
amine followed by bromination with NBS. The bromide 17 was
subsequently converted to the benzylic azide which was then re-
duced under Staudinger conditions to furnish the benzylamine
18. 2-Chloro-4-methylsulfonyl-benzylamine (21) was synthesized
by conversion of 2-chloro-4-methylsulfonyl-benzoic acid 19 to
the primary amide 20, by generation of the mixed anhydride
with BOC2O, followed by displacement with hydroxylamine
O
N
H
1
O
Cl
N
benzhydryl nicotinamides
N
H
R
h-sEH 7 nM
N
Cl
h/r LM t1/2 7/<5 min
3
hybrid inhibitors
potency and
O
O
Cl
metabolic stability?
N
N
H
HN
Cl
2
O
ureas
h-sEH180 nM
h/r LM t1/2 300/104 min
Figure 2. Representative examples of urea and benzhydryl nicotinamide series of
sEH inhibitors.

5866
S. J. Taylor et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5864–5868
Table 1
Human and rat soluble epoxide hydrolase inhibition, and in vitro microsomal stability for hybrid compounds in comparison to compounds from the initial nicotinamide hit class
O
Cl
O
N
H
N
H
Cl
4
5
a
a
Entry
Compd
R
RHS
h-sEH IC50 (nM)
r-sEH IC50 (nM)
h-LM/r-LM (t min)
½
1
2
6
7
4
5
400
8.1
330
10
78/191
96/7
O
N
H
3
4
8
9
4
5
520
9.1
430
8.5
46/44
33/5
H2N
N
N
5
6
10
11
4
5
1500
18
500
9
nt
46/41
N
7
8
12
13
4
5
16
2.6
6
4
24/19
4/5
O
N
EtO
9
10
14
15
4
5
6.6
5.4
7.3
7.8
178/7
38/<3
F3C
O
N
a
Values are means of a minimum of two experiments (nt = not tested).
h-LM (in vitro human liver microsomal half life), r-LM (in vitro rat liver microsomal half life)
b
12,16
.
O
O
environment (Table 2), as well as in the human and rat sEH enzyme
assays.12,17 They were also profiled in human and rat microsomal
preparations (h-LM/r-LM), as a predictor of in vivo oxidative
metabolism. Sequential removal of the 2-and 4-chlorine atoms
from the benzyl ring resulted in a 3- and 10-fold loss in potency,
respectively, against the human isoform of sEH (Table 2, entries
2 and 3). This observation is consistent with the chlorine atoms
occupying the large lipophilic S2 pocket of human sEH. Interest-
ingly, removal of the halogen at the 2-position of the benzylamine
resulted in a significant loss of inhibition of the human but not the
rat isozyme (Table 2, entry 3). Complete removal of all substitution
off the benzyl ring led to a much weaker inhibitor, especially of the
human sEH (Table 2, entry 4). Based upon previous studies with
the benzhydryl analogs 1, we elected to replace the 4-chloro sub-
stituent with more polar functional groups in hope that this would
give rise to additional protection against oxidative metabolism
while maintaining acceptable potency. We hypothesized that sub-
stitution with a polar functional group would be tolerated at this
site as the directionality of substitution off the para position en-
gages solvent, and a number of hydrophilic amino acid side chains
(i.e., serine 415). To this end, replacement of the para-chloro sub-
stituent with a methylsulfone (34) resulted in a slight decrease
in potency, but this analog had a significantly longer half-life in
the presence of rat liver microsomes (Table 2, entry 5). Replacing
the 2-chlorine substituent of 14 with a methylsulfonyl group
resulted in a 10-fold decrease in potency (Table 2, entry 6). The
possibility that the methylsulfone in this position could compete
+
a
OH
H2N
N
H
R
R
R
R
N
N
Scheme 1. General procedure for the synthesis of differentially substituted
benzylamines. Reagents and conditions: (a) EDC-HCl (1.2 equiv), HOBT (1.2 equiv),
DIEA (1.2–2.2 equiv), anhyd. DMF (0.2 M), rt 16 h.
hydrochloride. Selective reduction of the corresponding amide to
amine 21 was accomplished with borane in THF. 2-Chloro-4-cya-
nobenzylamine (24) was synthesized by bromination of 3-chloro-
4-methylbenzonitrile (22) with NBS, followed by displacement of
the bromide with potassium phthalimide. Deprotection with
hydrazine revealed the benzyl amine 24. Synthesis of C-(40-meth-
ylsulfonyl-biphenyl-4-yl)methylamine 27 proceeded via Suzuki–
Miyaura coupling14 of 4-bromobenzonitrile 25 with 4-methylsul-
fonyl boronic acid. Subsequent hydrogenation and deprotection
of the intermediate carbamate provided compound 27. 4-Bromo-
2-trifluoromethoxy-benzylamine (30) was generated by selective
lithium-halogen exchange of 28, followed by in situ trapping of
the aryl lithium species with morpholine-4-carbaldehyde. The
resulting aldehyde (29) was converted to the benzylamine 30 via
a reductive amination. These amines (and commercially available
benzylamines) were coupled with 6-(2,2,2-trifluoroethoxy)-nico-
tinic acid using the general conditions outlined in Scheme 1.
The resulting nicotinamides were profiled in an sEH cellular
assay to determine their activity in a more physiological relevant

S. J. Taylor et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5864–5868
5867
Replacement of the 4-chlorine in 14 with a nitrile (40) or a N-
methylsulfonamide (38) gave similar results to the methylsulfone
analog 34 (Table 2, entries 9 and 10). Likewise replacement of
the 4-chlorine atom with a tetrazole yielded a potent inhibitor of
human sEH, but not rat sEH (Table 2, entry 14). Introduction of a
trifluoromethoxy group in the 2-position, even when lacking a
complimentary substituent in the 4-position of the aryl ring, gave
the most potent sEH inhibitor (4.6 nM), however 39 displayed a
short in vitro microsomal half life (Table 2, entry 10). This short
half life was attenuated by placement of a halogen atom at the
4-position (Table 2, entry 12), essentially doubling the in vitro
half-life of the compound 41, while maintaining acceptable intrin-
sic potency. Finally adding an additional aryl ring to the benzyl-
amine (Table 2, entry 13), was tolerated as well.
Based upon the in vitro profile of these compounds we then as-
sessed the in vivo exposure of a few representative analogs prior to
initiating a full pharmacokinetic profile and evaluating these series
further (Fig. 4).14 Individual compounds were dosed orally as a sus-
pension (5 mg/kg) in Sprague–Dawley rats (n = 3). Plasma samples
were collected at 1, 2, 4, and 6 h post dosing. Compound 14 showed
Cl
Br Cl
Cl
c, d
H2N
a, b
O
Cl
O
O
S
S
S
N
N
O
O
O
18
16
17
H
H
O
Cl
Cl
O
Cl
e
f
H2N
H2N
21
HO
O
S
O
O
S
S
20
O
O
19
O
Cl
Cl
Cl
O
N
i
g, h
N
H2N
O
23
N
N
O
22
24
N
H2N
N
I
j
k
O
S
Br
S
25
26
27
O
O
a 2.5 lM plasma level at 1 hour that was essentially maintained
over 6 h. This result is in contrast to the relatively rapid predicted
H
OCF3
Br
OCF3
OCF3
Br
m
l
O
H2N
Br
Plasma Exposure of Select
30
28
29
Nicotinamide
12
Scheme 2. Reagents and conditions: (a) methylamine, CH2Cl2, rt, 97%; (b) NBS, CCl4,
AIBN, reflux, 24 h, 27%; (c) NaN3, DMF, 40 °C, 16 h, 50%; (d) PPh3, THF/water, 24 h,
30%; (e) BOC2O, NH4HCO3, pyridine, 84%; (f) BH3 THF, reflux, 88%; (g) NBS, CCl4,
AIBN, reflux, 24 h, 86%; (h) potassium phthalimide, DMF, rt, 2 h, 71%; (i) N2H2,
ethanol, reflux, 1 h, 63%; (j) 4-(methylsulphonyl)phenylboronic acid, Pd2(dba)3, KF,
[(t-Bu)3PH]BF4, THF 80 °C, 18 h, 44%; (k) (i) BOC2O, 50 psi H2, 10% Pd/C, 18 h, 85%,
(ii) HCl/dioxane, rt, 4 h, 55%; (l) n-BuLi, morpholine N-carbaldehyde ꢀ78 °C, 79%;
(m) NaBH4, NH3, MeOH, rt, 16 h, 83%.
10
8
Cpd.
6
4
2
0
34
14
40
12
39
with the central amide pharmacophore for binding with the cata-
lytic tyrosines 383 and 466 may explain the decrease in potency,
as this substituent can be accommodated by the sheer size of the
pocket. Mono substitution of the aromatic ring with a methylsulf-
one in either the 2- or 4-positions, resulted in a significant drop in
potency compared to the monochloro derivatives 31 and 32 (Table
2, entries 7 and 8).
0
1
2
3
4
5
6
Time (hours)
Figure 4. Rapid assessment of compound exposure (RACE) for select hybrid
molecules. Average plasma concentration in three discrete animals per compound
after 5 mg/kg dose, dosed PO as a homogeneous suspension in CMC-Tween.
Table 2
Potency and in vitro metabolic stability of compounds with differentially substituted benzylamine P2-3 motifs
R1
O
F
N
H
F
F
O
N
R2
R1
R2
h-sEH IC50 (nM)
r-sEH IC50 (nM)
h-LM/r-LM (t min)
h-sEH HepG2 (nM)
a
a
Entry
Compd
½
1
2
3
4
5
6
7
8
9
10
11
12
13
14
14
31
32
33
34
35
36
37
38
39
40
41
42
43
Cl
Cl
H
H
Cl
SO2Me
H
SO2Me
Cl
OCF3
Cl
Cl
H
Cl
H
SO2Me
Cl
SO2Me
H
SO2NMe
H
CN
Br
4-SO2Me (phenyl)
tetrazole
6.6
21
77
1200
15
66
110
270
14
4.6
14
7.3
22
7
50
16
11
25
160
19
6
178/7
nt
nt
18
nt
nt
nt
28
nt
nt
96/75
300/99
156/173
nt
81/25
78/7
188/105
300/17
nt
nt
nt
31
21
12
0.1
24
38
8
7
4.3
180
OCF3
H
Cl
12
12
21
300/227
a
b
c
Values are means of a minimum of two experiments (nt = not tested), compound synthesis and purity reported in Ref. 16.
h-LM (in vitro human liver microsomal half life), r-LM (in vitro rat liver microsomal half life)12,16
.
12
Cellular assay for inhibition of sEH in human Hep G2 Cells, ELISA readout
.

5868
S. J. Taylor et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5864–5868
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driven clearance could be a problem for this class of inhibitors.
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15. Ingraham, R. H.; Cardozo, M. G.; Grygon, C. Anne; Kroe, R. Rebecca; Proudfoot, J.
R. WO 02/082082.
These studies also demonstrated that combination of the
dichlorobenzylamine with the arylamide of 1 resulted in low
molecular weight, potent sEH inhibitors, that show extended plas-
ma exposure in rats when dosed orally. A variety of substituents
were tolerated on the RHS of the sEH inhibitor, whereas extension
off the phenylamide with large lipophilic groups was required for
potency against human and rat sEH. By combining a potent aryl
amides with a metabolically stable dichlorobenzyl amines, hybrid
molecules were generated that retained the optimal properties
associated with each series. While low nanomolar potencies were
achieved, the compounds in this hybrid series were still not as po-
tent as the analogous compounds within the benzhydrylamide ser-
ies. These studies serve as the foundation for extended chemical
elaboration of the phenyl moiety to further increase potency, and
to address additional target-independent profiles. However, these
compounds, structurally distinct from the urea-based inhibitors,
still have acceptable profiles such that they can be used as alterna-
tive tools to assess the in vivo pharmacological effect of sEH inhi-
bition in appropriate animal models of cardiovascular and
inflammatory diseases.
Acknowledgements
We thank John Proudfoot and Derek Cogan with their help in
preparation of the graphics for this Letter, and Michael August
and Lori Patnaude for running the HT screen.
16. Eldrup, A. B.; Farrow, N. A.; Kowalski, J. A.; De Lombaert, S.; Muegge, I. A.;
Soleymanzadeh, F.; Swinamer, A. D.; Taylor, S. J. WO 07/098352 August 30,
2007.
References and notes
17. An alignment of the rat and human protein sequences reveals that there are
several amino-acid differences within the ligand binding site. Many of these
changes are close to the region of the protein that would influence the SAR
described in Table 2.
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