Substituted pyrazoles as novel sEH antagonist: Investigation of key binding interactions within the catalytic domain

Article abstract of DOI:10.1016/j.bmcl.2010.09.095

A novel series of pyrazole sEH inhibitors is reported. Lead optimization efforts to replace the aniline core are also described. In particular, 2-pyridine, 3-pyridine and pyridazine analogs are potent sEH inhibitors with favorable CYP3A4 inhibitory and microsomal stability profiles.

Full text of DOI:10.1016/j.bmcl.2010.09.095

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6379–6383
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted pyrazoles as novel sEH antagonist: Investigation of key binding
interactions within the catalytic domain
Ho Yin Lo a, , Chuk C. Man a, Roman W. Fleck a, , Neil A. Farrow a, Richard H. Ingraham b,à, Alison Kukulka c,
⇑
John R. Proudfoot a, Raj Betageri a, Tom Kirrane a, Usha Patel a, Rajiv Sharma a, Mary Ann Hoermann a,
Alisa Kabcenell b, Stéphane De Lombaert a
a Boehringer Ingelheim Pharmaceuticals Inc., Biomolecular Screening, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877, USA
b Medicinal Chemistry, Biomolecular Screening, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877, USA
c Cardiometabolic Diseases, Biomolecular Screening, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrazole sEH inhibitors is reported. Lead optimization efforts to replace the aniline core
are also described. In particular, 2-pyridine, 3-pyridine and pyridazine analogs are potent sEH inhibitors
with favorable CYP3A4 inhibitory and microsomal stability profiles.
Received 2 August 2010
Revised 14 September 2010
Accepted 16 September 2010
Available online 19 September 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
sEH
Pyrazoles
Aniline
EETs
Anti-hypertension
Epoxyeicosatrienoic acids (EETs) are metabolites generated
from the epoxidation of arachidonic acid by cytochrome P450
epoxygenases.1 Four isoforms of EETs, namely the 5,6-; 8,9-;
11,12- and 14,15-EET, can be produced depending on the site of
epoxidaton.2 They possess vasodilatory3 and anti-inflammatory
properties.4 A number of studies have suggested that EETs are
the potential endothelial derived hyperpolarizing factor (EDHF)5
involved in vascular, renal, and cardiac blood pressure regulation.
Leveraging the beneficial physiological characteristics of EETs rep-
resents a potential new avenue for the treatment of hypertension
and related end-organ damage. Towards this end, several ap-
proaches could be envisioned, including limiting EETs metabolism.
Soluble epoxide hydrolyase (Ephx2; sEH) is a cytosolic enzyme
found in mammalian tissues, including liver, kidney, intestine, and
the vasculature.6 An important enzymatic function of sEH is to
hydrolyze EETs to the corresponding dihydroxyeicosatrienoic acids
(DHETs).7 These diols are less potent vasodilators due to generally
lower intrinsic activity and rapid excretion from the body. sEH
serves as a major clearance mechanism for EETs and therefore is
anticipated to regulate vascular homeostasis.8 Indeed, salt-sensi-
tive male mice deficient in the sEH-expressing gene display lower
systolic blood pressure than wild type.9 In renal disease states de-
rived from diabetes and hypertension, endothelial dysfunction is
found to be the main cause for the condition. Inhibition of sEH
may improve renal function because of increasing EETs concentra-
tion in the body.10 AUDA, prototypical sEH inhibitor, has been re-
ported to lower mean arterial blood pressure and improve renal
function in a salt-sensitive rat model.11
We12 and others13,14 have been interested in designing sEH
inhibitors with a superior drug profile relative to AUDA and from a
distinct structural class to strengthen the proof-of-concept beyond
lipophilic ureas. We have previously reported benzamides12a,b and
more polar ureas12c,d as potent sEH inhibitors. We now report on a
novel structural series resulting from a focused high-throughput
screening campaign. The lead compound 1, with an IC50 of 41 nM
to human sEH, was readily co-crystallized with human sEH protein
(Fig. 1) to gain precise information on the key binding interactions of
the inhibitor with the enzyme. The X-ray structure reveals that com-
pound 1 resides within the catalytic domain of sEH: TYR383 and
TYR466 form H-bonds with the carbonyl group of the inhibitor
and the catalytic ASP335, responsible for the hydrolysis of epoxide,
engages an H-bond with the amide proton. This ‘triple’ interaction is
crucial for the inhibitor to achieve high affinity, since either replac-
ing the carbonyl or removing the amide proton (e.g., N-methylation)
resulted in a substantial loss of potency (data not shown).
⇑
Corresponding author. Tel.: +1 203 798 4923.
E-mail address: ho-yin.lo@boehringer-ingelheim.com (H.Y. Lo).
Present address: Index Ventures.
Present address: National Institutes of Health.
à
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.095

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H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6379–6383
Table 1
Representative modifications
R1
N
O
R2
NH
N
N
Compound
R1
R2
Human sEH (IC50) (nM)a,b
41
*
1
CH2CH3
CF3
N
N
*
2
23
3
4
CF3
CF3
*
*
N
7.3
6.2
a
Figure 1. X-ray co-crystal structure of 1. Coordinates of the co-crystal structure of 1
with sEH are deposited in the RCSB protein data bank (ID: rcsb061561; PDB ID:
30TQ).
Values are means of a minimum of two experiments.
See Ref. 15 for details of the binding assay.
b
MN
Figure 2 illustrates that both pyridine moieties are facing open
channels pointing towards the solvent. The ethyl group of the
inhibitor resides in a deep hydrophobic pocket of the protein.
Some conservative structural modifications of 1, consistent with
the binding model, have provided additional information to guide
the lead optimization (Table 1). Replacement of the ethyl substitu-
tion in the pyrazole ring with trifluoromethyl moiety (e.g., 2) led
to a small improvement in potency. However, changing the right-
hand-side (RHS) 3-pyridine to a 4-pyridine or phenyl group resulted
in a threefold improvement in potency (3: IC50 = 7.3 nM).15
Although compound 3 showed an attractive potency level, the
potential generation of an aniline moiety in the molecule by met-
abolic hydrolysis was considered a potential liability for drug tox-
icity. It is well documented that aniline and related structures link
to idiosyncratic toxicity,16 mutagenesis,17 splenotoxicity18 and re-
nal toxicity19 in different xenobiotics. The proposed mechanism
for the toxicity involves N-hydroxylation and subsequent reactions
with macromolecular nucleophiles (such as DNA) in vivo to gener-
ate an aniline-MN (macromolecular nucleophile) adduct (Fig. 3)
and leads to adverse drug reactions as mentioned above.
MN
X
X
N-hydroxylation
NH2
NH
OH
NH
OH
Aniline
Aniline-MN adduct
MN: macromolecular nucleophile
X: nucleophilic nucleus; eg. O, S
Figure 3. Oxidative alkylation of aniline related structural feature.
amide analog 10 and benzyl amide 11 were first considered as
an aniline replacement. The syntheses of 3, 10 and 11 started with
aldol condensation of ethyl trifluoroacetate and 3-acetylpyridine to
generate the 1,4-diketone building block 5 (Scheme 1). Compound
5 was then reacted with 4-cyanophenylhydrazine, 4-hydrazino-
benzoic acid and 4-nitrophenylhydrazine to yield the correspond-
ing pyrazole intermediates 9, 8 and 7, respectively. In the case of
compounds 7 and 8, only the desired regioisomers were obtained.
However for compound 9, a pair of chromatographically separable
regioisomers was obtained in 1:1 ratio (structures assigned by
NOESY experiments). Reverse amide analog 10 and benzyl amide
analog 11 were obtained in moderate yields by simple functional
group transformations.
First, we investigated the possibility of replacing the aniline
moiety in the inhibitor while attempting to maintain the key bind-
ing interactions with the catalytic domain of sEH. The reverse
To our disappointment, when compared with the lead com-
pound 3, the activities of compound 10 and 11 were significantly
weaker (61- and 89-fold loss) (Table 2). From this result, the atom-
ic geometry of the aniline amide structure in compound 3 was be-
lieved to be optimal for interacting with the three key residues in
the catalytic domain. Therefore, instead of modifying the ‘amide
portion’ of the molecule, our attention shifted to the ‘aromatic
portion’ and aimed at replacing the core benzene ring with various
six-membered nitrogen containing heteroaromatics, since there
are reports showing that they are much less mutagenic than the
aniline.20 The reason behind could be the relatively low rate of
enzymatic N-hydroxylation for those heteroaromatics.
The syntheses of 2-pyridine 15, 3-pyridine 18, pyridazine 21
and pyrazine 24 analogs are described in Scheme 2. The formation
of pyrazole units in those analogs follows the procedure applied for
accessing compound 3 from the corresponding hydrazine interme-
diates. The synthesis of pyrimidine analog 28 proved to be more
challenging. As shown in Scheme 3, pyrazole intermediate 25
Figure 2. Surface representation of the X-ray co-crystal of sEH and 1.

H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6379–6383
6381
F
F
HN
H2N
NO2
F
O
O
O
O
a
F
F
F
F
F
F
+
O
N
NO2
b
N
N
N
5
6
N
single regioisomer
HN
H2N
N
O
HN
H2N
e
c
g
OH
F
F
F
F
F
F
F
F
F
O
N
N
N
N
NH2
N
N
N
OH
9
8
7
N
N
N
1:1 ratio of regioisomers
O
single regioisomer
O
f
d
N
NH2
Cl
Cl
h,i
N
N
F
F
F
F
F
F
F
F
F
O
O
HN
O
N
N
N
N
N
NH
N
N
HN
N
N
11
10
3
N
N
N
Scheme 1. Synthetic route for amide, reverse amide and benzyl amine analogs. Reagents and conditions: (a) NaOMe, MeOH, reflux, 50%; (b) (i) 4-nitrophenylhydrazine, EtOH,
reflux; (ii) acetic acid, 100 °C, 46%; (c) Pd/C, H2, 1 atm., EtOH, rt, 97%; (d) isonicotinoyl chloride, Et3N, CH2Cl2, 50%; (e) (i) 4-hydrazinobenzoic acid, EtOH, reflux; (ii) acetic acid,
100 °C, 25%; (f) 4-aminopyridine, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, rt, 32%; (g) (i) 4-cyanophenylhydrazine, EtOH, reflux; (ii) acetic acid, 100 °C, 12%; (h)
LiAlH4, Et2O, 0 °C, 70%; (i) isonicotinoyl chloride, Et3N, CH2Cl2, 27%.
CF3
N
CF3
N
CF3
N
O
b
a
d
100%
c
F
NH2
NH
H2N
F
HN
H2N
F
N
N
N
N
N
N
N
N
45%
43%
85%
12
14
15
13
N
N
N
N
6:4 ratio of regioisomers
CF3
N
CF3
N
CF3
N
O
e
d
H2N
HN
b
NO2
NO2
NH2
NH
N
N
N
N
N
N
N
N
70%
86%
70%
16
17
18
N
N
single regioisomer
CF3
CF3
N
CF3
N
O
c
d
H2N
HN
b
Cl
N
Cl
NH2
NH
N
N
30%
N
N
N
N
N
N
N
N
N
N
57%
67%
19
20
21
N
N
N
6:4 ratio of regioisomers
CF3
N
O
O
O
b
f
N
NH
N
H2N
HN
N
N
d
Br
NH2
Br
NH
NH
73%
N
85%
N
N
25%
23
22
24
N
single regioisomer
Scheme 2. Synthetic route for 2-pyridine, 3-pyridine, pyridazine and pyrazine analogs. Reagents and conditions: (a) NaNO2, SnCl2, HCl; (b) (i) 5, EtOH, reflux; (ii) acetic acid,
100 °C; (c) NH3/MeOH, 150 °C; (d) benzoyl chloride, pyridine; (e) Pd/C, H2, 1 atm., EtOH; (f) N2H4, EtOH, 100 °C, microwave.
was obtained as a pair of separable regioisomers by condensation
reaction of 5 with ethyl hydrazinoacetate. The desired regioisomer
25 was then hydrolyzed and reacted with DMF under POCl3 and
NaPF6 condition to generate the corresponding vinamidinium salt
26.21 The pyrimidine core 27 was formed by a condensation
reaction of 26 with guanidine carbonate. The target compound
28 was obtained by an acylation reaction of 27 with benzoyl
chloride.
The biological activities of these heterocycles are summarized
in Table 3. The binding affinities of the 2-pyridine 15, 3-pyridine

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H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6379–6383
Table 2
Modification of amide
18, and pyridazine 21 to sEH are equivalent to aniline analog 4. The
positive results from this modification indicated that alterations of
the electronic property of the central aromatic core had minimal
impact on the binding activity.
However, there was a slight drop in potencies for pyrazine ana-
log 24 and pyrimidine analog 28. The reason for the loss of binding
activity is not fully understood at this point. It could be speculated
that there might be a subtle alteration of the torsional angle be-
tween the left hand side pyrazole and the core aromatic system
in analog 24 and 28, which leads to less favorable binding and re-
sults in lost of potencies.
The encouraging binding potencies of compounds 15, 18 and 21
prompted us to further profile them in various in vitro assays
(Table 4). In a human sEH DPPO cellular assay,22 2-pyridine analog
15 showed superior activity over 3-pyridine 18 and pyridazine 21.
The deep drop in cell activity of 18 might result from its inferior
cell permeability (consistent with its very poor caco-2 result). Both
Compound
Human sEH (IC50) (nM)a,b
3
10
11
7.3
450
650
a
Values are means of a minimum of two experiments.
See Ref. 15 for details of the binding assay.
b
CF3
CF3
N
O
N
a
b
H
N
N
O
NH2
N
N
O
O
N
N
N
25
26
1:1 ratio of regioisomers
c
15 and 21 have good selectivity over CYP3A4 (>30 lM) and moder-
ate human liver microsomal stabilities (36% and 31%, respectively).
Overall, 2-pyridine analog 15 processes a very attractive profile for
further lead optimization.
CF3
N
CF3
N
O
N
N
d
NH
NH2
N
N
N
N
In conclusion, we have discovered a novel series of small
molecules sEH inhibitors featuring pyrazole and aniline structural
moieties. Successful replacement of the aniline with 2-pyridine,
3-pyridine or pyridazine rings maintains high inhibitory potency
and favorable CYP3A4 inhibitory and microsomal stability profiles.
The optimization of this pyrazole series toward potential drug
candidates and their pharmacological characterization will be re-
ported in due course.
28
27
N
N
Scheme 3. Synthetic route for pyrimidine analog. Reagents and conditions: (a) 5,
HCl, EtOH, 80 °C, 58%; (b) (i) LiOH, MeOH; (ii) POCl3, NaPF6, DMF, 52%; (c) NaH,
guanidine carbonate, EtOH, 80 °C, 48%; (d) benzoyl chloride, iPr2NEt, 48%.
Table 3
Modification of central aromatic ring
CF3
O
References and notes
R
N
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N
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a
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Profiles of representative compounds
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Compound
Human sEH
Human sEH DPPO
(IC50) (nM)a,c
CYP3A4
h-LM
(IC50) (nM)a,b
(IC50) (
l
M)a
(%Qh)a
4
15
18
21
6.2
4.7
9.9
8.8
1
5
397
83
NTd
>30
NTd
>30
NTd
36
NTd
31
a
b
c
Values are means of a minimum of two experiments.
See Ref. 15 for details of the binding assay.
See Ref. 22 for details of the cell assay.
NT: not tested.
d

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15. Details for the binding assay: Test compounds were dissolved and serially
diluted in DMSO, with final dilution in assay buffer (20 mM TES, 200 mM NaCl,
0.05% w/v CHAPS, 1 mM TCEP, pH 7.0) to achieve 1% DMSO in the assay. Test
compounds were dispensed into a black, flat-bottom 96-well plate. Positive
controls were reaction mixtures containing no test compound; negative
controls (blanks) were reaction mixtures containing a reference inhibitor.
The reaction was started with the addition of a mixture of either human or rat
sEH (final assay concentration is 10 nM) and probe (final assay concentration is
2.5 nM). The reaction was mixed by briefly shaking the plate on an orbital
shaker, and the plates were incubated in the dark for 30 min at room
temperature. Fluorescence polarization is measured on an LJL Analyst using a