A strategy of employing aminoheterocycles as amide mimics to identify novel, potent and bioavailable soluble epoxide hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.08.006

Distinct from previously reported urea and amide inhibitors of soluble epoxide hydrolase (sEH), a novel class of inhibitors were rationally designed based on the X-ray structure of this enzyme and known amide inhibitors. The structure-activity relationshi

Full text of DOI:10.1016/j.bmcl.2009.08.006

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5716–5721
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A strategy of employing aminoheterocycles as amide mimics to identify
novel, potent and bioavailable soluble epoxide hydrolase inhibitors
a
a
b
b
c
c
Hong C. Shen a, , Fa-Xiang Ding , Qiaolin Deng , Suoyu Xu , Xinchun Tong , Xiaoping Zhang , Yuli Chen ,
Gaochao Zhou c, Lee-Yuh Pai d, Magdalena Alonso-Galicia d, Sophie Roy d, Bei Zhang c, James R. Tata a,
Joel P. Berger c, Steven L. Colletti a
*
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
b Department of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
c Department of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
d Department of Cardiovascular Diseases, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Distinct from previously reported urea and amide inhibitors of soluble epoxide hydrolase (sEH), a novel
class of inhibitors were rationally designed based on the X-ray structure of this enzyme and known amide
inhibitors. The structure–activity relationship (SAR) study was focused on improving the sEH inhibitory
activity. Aminobenzisoxazoles emerged to be the optimal series, of which a potent human sEH inhibitor
7t was identified with a good pharmacokinetics (PK) profile. The strategy of employing aminoheterocy-
cles as amide replacements may represent a general approach to develop mimics of known hydrolase or
protease inhibitors containing an amide moiety.
Received 13 July 2009
Revised 28 July 2009
Accepted 3 August 2009
Available online 7 August 2009
Keywords:
Amide mimics
Aminoheterocycles
Inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
sEH
Soluble epoxide hydrolase
Soluble epoxide hydrolase, also referred to as sEH or EPHX2, is
an enzyme that converts epoxyeicosatrienoic acids (EETs) to dihy-
droxy eicosatrienoic acids (DHETs).1 As endogenous epoxides de-
rived from arachidonic acid by cytochrome P450 epoxygenases,2
EETs exhibit significant physiological effects leading to reduced
blood pressure and myocardial perfusion in animal models.3 These
effects presumably result from the well-known roles of EETs to in-
crease sodium renal excretion,4 relax conduit vessels, and dilate re-
nal afferent arterioles and coronary resistance vessels.5
Furthermore, there are indications that EETs may be anti-athero-
sclerotic.6 This hypothesis is based upon the observation that EETs
modify leukocyte adhesion, platelet aggregation, vascular smooth
muscle cell migration, and thrombolysis in preclinical animals.7
Lastly, EETs are anti-inflammatory as they reduce cytokine-in-
duced endothelial expression of several pro-inflammatory factors.8
Guided by the above observations, Arete Therapeutics recently
developed an sEH inhibitor which is now entering phase II clinical
trials for the metabolic syndromes.9 The sEH inhibitor most exten-
sively described in the literature is 12-(3-adamantan-1-yl-ureido)-
dodecanoic acid (AUDA), which appeared to have attained proof-
of-concept with regard to blood pressure reduction and renal pro-
tection in a salt-sensitive and hypertensive animal model.10 How-
ever, this class of disubstituted ureas is typically associated with
poor pharmacokinetics and physical properties. Amide sEH inhibi-
tors have also been reported. For example, a Taisho patent de-
scribed an amide sEH inhibitor that lowered blood pressure in
angiotensin II-induced hypertensive rats.11 In addition to amides,
chalcone oxides,12 carbamates,13 acyl hydrazones,14 and trans-3-
phenylglycidols15 have also been reported as sEH inhibitors.
The X-ray crystallographic structures of human sEH and an
inhibitor (4-(3-cyclohexyluriedo)-butyric acid) complex (PDB
code: 1ZD3) revealed the catalytic pocket and key structural ele-
ments required to inhibit this enzyme. The hydrolase catalytic
pocket of sEH consists of two tyrosine residues (Tyr381 and
Tyr465) which act as hydrogen bond donors to facilitate the epox-
ide ring opening by Asp333.16 It has been recognized that amide or
urea groups fit well in the hydrolase catalytic pocket. Specifically,
the carbonyl oxygen of the amide or urea is engaged in a hydrogen
bond interaction with Tyr381 and Tyr465, and the N–H acts as a
hydrogen bond donor to Asp333. Therefore, various ureas and
amides have been developed as reversible sEH inhibitors.17 How-
ever, novel sEH inhibitors beyond the scope of amides or ureas
are uncommon.12–15 Herein, we report our efforts in developing
* Corresponding author. Tel.: +1 732 594 1755; fax: +1 732 594 9473.
E-mail address: hong_shen@merck.com (H.C. Shen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.006

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5716–5721
5717
Tyr381
quickly demonstrated that aminobenzisoxazoles (5) appeared to
be optimal and hence it was selected as a lead structure for further
optimization. Herein we report the systematic and rational medic-
inal chemistry efforts that led to the discovery of a novel, potent,
and bioavailable sEH inhibitor.
B
O
N
C
A
Tyr465
R1
R2
R1
R2
N
H
H
Initially, a scaffold hopping strategy19 was employed to identify
a hit from monocyclic aminoheterocycles shown in Table 1. The
first round of screening of various amino-pyridine, pyrazine, oxadi-
azole and pyrimidine derivatives did not yield any potent inhibi-
tors of the human sEH.
2
1
Asp333
Tyr381
B
A
O
FG
O
Tyr465
N
FG
N
PG
N
H
H
N
Subsequently, a reasonably comprehensive list of bicyclic amino-
heterocyclic analogs was interrogated to identify a hit (Table 2).
Among compounds including benzisoxazole 7a and 7d, benzisothiaz-
ole7b, indazole7c, benzothiazole7e, benzoxazole7f, andquinoxaline
7g, only benzisoxazole 7a provided good activity (IC50 = 25 nM)
against human sEH. Therefore, 7a was selectived as a hit for sEH
and further SAR was based upon the aminobenzoxazole scaffold.
To introduce more polarity to this scaffold, pyridine-fused isox-
azoles were prepared (7h–k). Distinct enzyme inhibitory activities
were observed for the four pyridine-fused isoxazole regioisomers,
of which 7h and 7i demonstrated better sEH inhibition than 7j
and 7k. Aniline-derived analogs such as 7a provided superior activ-
ity to benzylamine derivative 7d. Other benzyl amine-derived ana-
logs 7e–7g were inactive against sEH.
Taking benzisoxazole 7a as the hit, the SAR was also explored in
regard to the substitution of aniline (Table 3) and benzisoxazole
(Table 4), respectively. Among various substitutions of the aniline,
the para-CF3 group gave the best sEH inhibition (7a). ortho- and
meta-Substituted compounds were generally less active than the
para-substituted analogs (7a vs 7l, 7m vs 7n, and 7p vs 7o, 7q).
In addition, biphenyl or naphthylamine analogs 7p–s were 8- to
50-fold less active against human sEH than compound 7a.
Ar
Asp333
H
4
5
3
Figure 1. Pharmacophore of aminoheterocyclic sEH inhibitors.
aminobenzisoxazole derivatives as a novel class of sEH inhibitors.
This bioisostere approach18 may lead to potential advantages in
intellectual property, target potency and pharmacokinetics (PK).
Based on known amide inhibitors of sEH such as general struc-
tures 1 and 3,17 the design of aminoheterocycle derivatives
stemmed from the tethering strategy of the carbonyl of a benzam-
ide with the adjacent side chain of 1 or benzene ring of 3 (Fig. 1). As
a result, scaffolds 2 and 4 could be envisioned in which heteroatom
A mimics the oxygen of the amide carbonyl, and the N–H group in
2 and 4 overlaps with the N–H of amides 1 and 3, respectively.
Therefore, the hydrogen bond interactions between amides and
Tyr381, Tyr465 and Asp333 of sEH should be preserved in new
scaffolds such as 2 and 4. The structure–activity relationship
(SAR) of such compounds was initially explored regarding different
choices of monocyclic and fused bicyclic heterocycles. It was
Table 1
In vitro SAR of human sEH inhibition by monocyclic aminoheterocyclesa
Compds
sEH IC50 (h)
lM
Compds
sEH IC50 (h) lM
CF3
CF3
H
N
N
O
>10
>10
N
N
Ph
N
N
H
Ph
6a
6f
Ph
O
CF3
N
O
N
4.7
>10
>10
>10
2
N
N
H
H
Ph
Ph
6g
6b
O
N
N
H
N
>10
>10
>10
N
N
H
H
6c
6h
O
N
N
N
N
H
Ph
N
6d
6i
N
F3C
O
N
H
N
N
N
N
H
6e
6j
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
a

5718
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5716–5721
Table 2
In vitro SAR of human sEH inhibition by bicyclic aminoheterocyclesa
Compds
sEH IC50 (h),
lM
Compds
sEH IC50 (h), lM
O
N
N
N
0.025
>10
N
H
N
H
H
CF3
7g
7a
S
N
O
N
N
2.5
0.027
0.031
N
CF3
N
H
CF3
7b
7h
H
O
N
N
N
N
>10
N
N
CF3
CF3
H
CF3
H
H
7c
7i
O
O
N
N
N
1.2
0.34
N
N
H
O
CF3
7d
7j
S
N
N
N
N
N
H
>10
>10
1.1
N
H
CF3
7e
7k
O
N
H
7f
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
a
Substituting benzisoxazole with different functional groups
It is conceivable that by avoiding the potential in vivo amide
hydrolysis by proteases, aminoheterocycle mimics of amides may
improve PK properties especially bioavailability and clearance. In-
deed, the rat PK profile of compound 7t (Table 5) was characterized
effectively improved the sEH inhibitory activity of compounds.
For example, CF3-substituted analog 7t had an IC50 of 4 nM for hu-
man sEH. The docking of 7t in the binding pocket of sEH reveals a
hydrogen bonding interaction between Tyr381 and the oxygen and
nitrogen atoms of the benzisoxazole (Fig. 2). However, no direct
hydrogen bond was identified between 7t and Tyr465. The pres-
ence of a –CF3 group dramatically enhanced its inhibitory activity
against sEH versus 7a, which is likely due to the additional favor-
able interaction between the –CF3 fluoro atom and N–H of
Gln382. In contrast, two other –CF3 regioisomers, 7u and 7v, were
less active. Regarding the other side of the scaffold, two aminopyr-
idine-derived analogs (7ee and 7ff) lost sEH inhibitory activity
significantly.
by excellent bioavailability (98%), good oral exposure (1.6 lM h kg/
mg), moderate clearance (30 mL/min/kg), and good half-life (5.9 h).
It was later determined that the DHET inhibition of 7t was
shifted 300-fold in the presence of 10% human serum
(IC50 = 1.2 lM) with respect to its human sEH enzyme inhibition
(IC50 = 4 nM). Furthermore, 7t was much less active against rat
sEH (IC50 = 127 nM). Plasma protein binding data in human and
rat plasma were also obtained. The free fraction of the radiolabeled
7t was less than 1% in both species (0.9% for rat and 0.7% for human
at compound concentrations of 0.5–25 lM). The collective data
In further exploratory studies, various 6- and 7-aryl substi-
tuted benzisoxazoles were prepared. These compounds provided
moderate to excellent sEH inhibitory activities (Table 4). Of par-
ticular interest were analogs 7w with an IC50 of 8 nM, and 7z
and 7cc with balanced human and rat enzyme activity
(IC50 = 20–30 nM). With low nanomolar activities against human
sEH, 7-arylbenzisoxazoles were typically more active than the
corresponding 6-arylbenzisoxazoles (7gg vs 7w, 7hh vs 7x, and
7ii vs 7dd). The introduction of a nitrogen atom into the ring in
trifluoromethylpyridine analog 7jj resulted in a fourfold loss of
human sEH activity with respect to 7t.
suggested that it was unlikely that 7t would demonstrate robust
sEH engagement in vivo, in contrast to several previously described
sEH inhibitors.17t
The synthesis of 7t commenced with benzoic acid 8 (Scheme 1).
The amide formation provided intermediate 9, which then reacted
with phosphorus pentachloride to produce chloroimine 10. The
subsequent reaction of compound 10 with trimethylsilyl hydroxy-
amine generated hydroxy amidine 11, which upon a desilylation
and a base-mediated cyclization, resulted in the formation of 7t
with excellent overall yield.20 The same chemistry was readily ap-
plied to prepare all analogs described in Tables 3 and 4.

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5716–5721
5719
Table 3
In vitro SAR of human sEH inhibition by aminobenzisoxazolesa
Compds
sEH IC50 (h, r),
lM
Compds
sEH IC50 (h, r),
0.17, 0.74
lM
O
O
N
N
N
0.025, 0.076
N
H
H
CF3
7a
7p
O
O
N
CF3
N
0.96, —
0.96, —
N
N
H
H
7l
7q
O
O
N
N
0.06, 0.17
0.16, 0.16
1.2, —
N
N
OCF3
H
H
7m
7r
O
O
N
N
OCF3
0.84, 4.6
N
N
H
H
7n
7s
O
N
N
5.2, —
H
F3CO
7o
a
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
Table 4
In vitro SAR of human and rat sEH inhibition by substituted aminobenzisoxazolesa
Compds
sEH IC50 (h, r),
0.004, 0.127
lM
Compds
sEH IC50 (h, r),
0.019, 0.030
lM
CF3
7
O
O
N
N
N
N
CF3
N
CF3
H
H
7cc
7t
H
N
6
F3C
O
O
N
N
0.018, —
0.07, 0.077
N
CF3
N
CF3
H
H
7u
7dd
O
CF3
N
O
F3C
N
N
CF3
0.040, 1.5
>10, >10
H
N
CF3
H
N
7ee
7v
CF3
O
O
N
N
CF3
0.008, 0.07
0.074, 3.4
N
CF3
N
CF3
H
N
H
7w
7ff
(continued on next page)

5720
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5716–5721
Table 4 (continued)
Compds
sEH IC50 (h, r),
lM
Compds
sEH IC50 (h, r),
0.004, 0.079
lM
CF3
CF3
O
O
N
0.47, 0.57
N
N
CF3
N
CF3
H
H
7gg
7x
CF3
O
N
F
O
N
0.091, 0.055
0.023, 0.020
0.058, 0.019
0.07, 0.29
0.01, 0.13
0.008, 0.041
0.019, 0.3
N
CF3
H
N
CF3
H
7hh
7y
NH
N
O
N
O
N
CF3
CF3
CF3
N
H
N
CF3
H
7z
7ii
CF3
N
O
O
N
N
N
N
N
H
H
CF3
7jj
7aa
O
N
N
N
H
7bb
a
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
In conclusion, aminobenzisoxazole analogs were discovered as
novel and potent sEH inhibitors. An optimized compound 7t dis-
played a good PK profile and excellent human sEH inhibitory activ-
ity (IC50 = 4 nM). The success of this amide bioisostere approach
may find utility in protease inhibitor programs in which the amide
moiety of such inhibitors might be replaced with aminoheterocy-
cles. In addition to intellectual property considerations, the obvi-
ous advantages include improved bioavailability and reduced
clearance as the potentially metabolically labile amide is removed.
Due to its modest rat sEH activity and significant serum shift, the
sEH engagement of 7t in rat is assumed to be rather poor. Future
studies on the aminoheterocyclic inhibitor series should be direc-
Table 5
Rat PK of 7ta
Compds F% Cl (mL/min/kg) Vdss (L/kg) T1/2 (h) AUCNpo
7t 98 30 5.9 1.6
(lM h kg/mg)
Figure 2. The minimized docking model of 7t (green) in human sEH. The dashed
lines illustrate atom pairs within hydrogen bond distance. The pictures were
prepared by PyMOL (Delano Scientific LLC, South San Francisco, CA). The model is
based on the x-ray crystallographic structure of 1ZD3 (PDB code): human soluble
epoxide hydrolase 4-(3-cyclohexyluriedo)-butyric acid complex.
7
a
Formulations: 1 mg/mL ethanol/PEG/water (20:40:40). IV dose: 1 mg/kg (n = 2).
PO dose: 2 mg/kg (n = 3). Blood concentration was determined by LC/MS/MS fol-
lowing protein precipitation with acetonitrile.

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5716–5721
5721
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2006, 15, 58.
CF3
CF3
CF3
F
F
F
b
a
H
N
OH
N
17. (a) Delombaert, S.; Eldrup, A. B.; Kowalski, J. A.; Mugge, I. A.; Soleymanzadeh,
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Ingraham, R. H.; Proudfoot, J. R. WO2003002555, 2003.; (i) Cywin, C. L.; De
Lombaert, S.; Eldrup, A. B.; Ingraham, R. H.; Taylor, S.; Soleymanzadeh, F.
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C. US20030119900, 2003.; (k) Kroetz, D.; Zeldin, D. C.; Hammock, B. D.;
Morisseau, C. US20040092487, 2004.; (l) Hammock, B. D.; Kim, I.-H.;
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Erickson, D.; Hoffman, A. F.; Warren, T. C. WO200023060, 2000.; (n) Hwang, S.
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Pai, L.-Y.; Alonso-Galicia, M.; Chen, X.; Zhang, B.; Tata, J. R.; Berger, J. P.; Colletti,
S. L. Bioorg. Med. Chem. Lett. 2009, 19, 3398; (t) Shen, H. C.; Ding, F.-X.; Wang, S.;
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Mitra, K.; Kumar, S.; Tsai, C.; Stevenson, A. S.; Pai, L.-Y.; Alonso-Galicia, M.;
Chen, X.; Soisson, S. M.; Roy, S.; Zhang, B.; Tata, J. R.; Berger, J. P.; Colletti, S. L. J.
Med. Chem. 2009, 52, in press; (u) Shen, H. C.; Ding, F.-X.; Deng, Q.; Xu, S.; Chen.
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Cl
O
O
CF3
CF3
8
9
10
CF3
CF3
OH
N
d
F
c
O
N
N
CF3
N
CF3
H
H
11
7t
Scheme 1. Reagents and conditions: (a) (COCl)2, Et3N, DMF, then 4-trifluorometh-
ylaniline, CH2Cl2, 1 h, 85%; (b) PCl5, 65 °C, 2 h, 44%; (c) NH2OTMS, THF, rt, 16 h, then
1 N HCl, 5 min, 61%; (d) KOt-Bu, NMP, 100 °C, 1.5 h, 95%.
ted towards reducing the impact of serum upon their potency by
introducing polar groups. The potential indications of sEH inhibi-
tors, including hypertension, diabetes, atherosclerosis, inflamma-
tion, and pain, are all worth pursuing once a potent sEH inhibitor
in rat with good ex vivo (or in vivo) target engagement is
identified.
References and notes
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20. The procedures of preparation of 7t: To the solution of 8 (500 mg, 2.4 mmol) in
20 mL DCM was added DMF (10 lL) and oxalyl chloride (3 mL, 2 M, 6 mmol) at
0 °C, and the resulting solution was stirred at rt for 1 h. After removing the
solvent, the residue was dissolved in 20 mL DCM and added to a solution of 4-
trifluromethyl aniline (387 mg, 2.4 mmol) and triethylamine (338 lL,
2.4 mmol) in 20 mL DCM at 0 °C. The resulting solution was stirred at rt for
1 h. The reaction mixture was washed with 3 N HCl (2 Â 10 mL) and satd
NaHCO3 (100 mL), dried over Na2SO4, concentrated to give 9 (0.72 g) as white
solid. The solution of 9 (0.72 g, 2.05 mmol) and PCl5 (0.43 g, 2.05 mmol) in
thionylchloride (3 mL) was heated at 65 °C for 2 h. After concentration, the
residue was treated diethylether, the solid was filtered, and the filtrated was
concentrated to give 10 (0.33 g, 44% yield) as a colorless oil. To the solution of
10 (0.33 g, 0.89 mmol) in THF (20 mL) was added trimethylsilyloxyamine
(0.88 mL, 7.14 mmol) dropwise and the resulting solution was stirred at rt
overnight. To the reaction solution was added 1 N HCl (10 mL) and stirred for
5 min before was neutralized to pH 8 by NaHCO3. The solution was extracted
with EtOAc (3 Â 50 mL), the combined organic phase was washed with brine
(2 Â 100 mL) and dried over Na2SO4. After concentration, the residue was
purified on silica gel using 35% EtOAc /hexane to give 11 (200 mg, 61% yield) as
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a
white solid. The solution 11 (200 mg, 0.54 mmol)) and potassium tert-
butoxide (74 mg, 0.63 mmol) in 8 mL of NMP was heated at 100 °C for 1.5 h.
After cooling to rt, the solution was diluted with EtOAc (100 mL), washed with
water (3 Â 100 mL), dried over Na2SO4, purified on silica gel using 35% of EtOAc
in hexanes to give 7t (180 mg, 95% yield) as a white solid. 1H NMR (CDCl3,
500 MHz): d 7.90–7.87 (2H, m), 7.75 (d, 2H), 7.69 (d, 2H), 7.49 (t, 1H), 6.71 (s,
1H); LCMS m/z: 347 (M++1).