Novel amide derivatives of 3-phenylglutaric acid as potent soluble epoxide hydrolase inhibitors

Article abstract of DOI:10.1007/s11030-019-10023-y

Abstract: Soluble epoxide hydrolase (sEH) enzyme plays an important role in the metabolism of endogenous chemical mediators, epoxyeicosatrienoic acids, which are involved in the regulation of blood pressure and inflammation. According to the pharmacophoric model suggested for sEH inhibitors, some new amide-based derivatives of 3-phenylglutaric acid were designed, synthesized and biologically evaluated. Docking study illustrated that the amide group as a primary pharmacophore had a suitable distance from the three amino acids of Tyr383, Tyr466 and Asp335 for effective hydrogen binding. Most of the compounds showed moderate to high sEH inhibitory activities in in vitro test in comparison with 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid, as a potent urea-based sEH inhibitor. Compound 6o with phenethyl in R position exhibited the highest activity with IC₅₀ value of 0.5?nM. Graphic abstract: In this study, some new amide-based derivatives of 3-phenylglutaric acid were designed, synthesized and biologically evaluated. Most of the synthesized compounds provided nanomolar range inhibition against sEH enzyme. The best observed IC₅₀ value was 0.5?nM. Incorporating a carboxylic moiety into these structures by forming carboxylate salts would increase the solubility and improving physicochemical properties.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-019-10023-y

Molecular Diversity
https://doi.org/10.1007/s11030-019-10023-y
ORIGINAL ARTICLE
Novel amide derivatives of 3‑phenylglutaric acid as potent soluble
epoxide hydrolase inhibitors
Elham Rezaee1 · Somayeh Minaei Amrolah1 · Maryam Nazari1 · Sayyed Abbas Tabatabai1
Received: 23 September 2019 / Accepted: 4 December 2019
© Springer Nature Switzerland AG 2019
Abstract
Soluble epoxide hydrolase (sEH) enzyme plays an important role in the metabolism of endogenous chemical mediators,
epoxyeicosatrienoic acids, which are involved in the regulation of blood pressure and infammation. According to the phar-
macophoric model suggested for sEH inhibitors, some new amide-based derivatives of 3-phenylglutaric acid were designed,
synthesized and biologically evaluated. Docking study illustrated that the amide group as a primary pharmacophore had
a suitable distance from the three amino acids of Tyr383, Tyr466 and Asp335 for efective hydrogen binding. Most of the
compounds showed moderate to high sEH inhibitory activities in in vitro test in comparison with 12-(3-Adamantan-1-yl-
ureido)-dodecanoic acid, as a potent urea-based sEH inhibitor. Compound 6o with phenethyl in R position exhibited the
highest activity with IC50 value of 0.5 nM.
Graphic abstract
In this study, some new amide-based derivatives of 3-phenylglutaric acid were designed, synthesized and biologically evalu-
ated. Most of the synthesized compounds provided nanomolar range inhibition against sEH enzyme. The best observed IC50
value was 0.5 nM. Incorporating a carboxylic moiety into these structures by forming carboxylate salts would increase the
solubility and improving physicochemical properties.
Keywords Biological evaluation · Phenylglutaric acid · Soluble epoxide hydrolase inhibitor · Synthesis
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-019-10023-y) contains
Introduction
supplementary material, which is available to authorized users.
Soluble epoxide hydrolase (sEH), a member of the α/β
hydrolase fold family, catalyzes the hydrolysis of epoxyeico-
satrienoic acids (EETs) to less active vicinal diol i.e., dihy-
droeicosatrienoic acids (DHETs) by the addition of water to
an epoxide [1–3]. EETs are the endogenous ligands derived
* Sayyed Abbas Tabatabai
sa_tabatabai@sbmu.ac.ir
1
Department of Pharmaceutical Chemistry, School
of Pharmacy, Shahid Beheshti University of Medical
Sciences, No. 2660, Vali-e-Asr, Tehran 1991953381, Iran
Vol.:(0123456789)
1 3

Molecular Diversity
from arachidonic acid by cytochrome (CYP) epoxygenases
in endothelial cells which can provide a wide range of physi-
ological efects [4, 5]. Among them, vasodilatory actions
in vascular conduit, renal aferent arterioles and coronary
vessels are more considerable due to blood pressure con-
trol and defensive efects on cardiovascular system [6, 7].
In addition, the EETs possess protective roles on vascular
system by inhibition of oxygen radicals produced by hypoxi-
areoxygenation [8]. Therefore, inhibition of sEH enzyme for
the purpose of increasing EET levels has been considered
as a hopeful treatment of some cardiovascular disorders,
infammation and pain [9–11]. The sEH enzyme possesses
homodimeric structure that each monomer containing two
domains with phosphatase and epoxide hydrolase activities
[12–14]. The sEH enzyme converts epoxide ring to vici-
nal diol through an SN2-type reaction. The partial positive
charge of epoxide ring would be increased via hydrogen
bonding of epoxide with two amino acids of Tyr381 and
Tyr465 and then nucleophilic attack by Asp333 would lead
to an alkyl enzyme intermediate. The diol product results
from the addition of a water molecule activated by His523 to
the alkyl intermediate, consequently [15, 16]. It can be con-
cluded from structure activity relationship (SAR) studies of
sEH inhibitors, a molecule possessing the carbonyl oxygen
and the N–H group as amide or urea moieties for hydrogen
bond interactions with tyrosine and aspartate, respectively,
can inhibit the catalytic activity of sEH enzyme [17, 18].
Thus, various urea and amide derivatives have been devel-
oped as reversible or slowly reversible sEH inhibitors such
as N-adamantyl-N′-cyclohexylurea (ACU) as the frst gen-
eration and 12-(3-adamantan-1-yl-ureido)-dodecanoic acid
(AUDA) as the second generation of sEH inhibitors [19, 20].
During eforts to improve the potency and physiochemical
properties, Kim et al. proposed the completest pharmacoph-
oric model for these amide and urea-based sEH inhibitors
included three pharmacophores connecting with two linkers.
However, these inhibitors sufered from poor pharmacoki-
netic properties and short half-lives [21–23]. In this work,
by studying the previous well-known inhibitors a series of
the novel 3-phenylglutaric acid amide-based structures were
designed, synthesized and assessed in vitro.
Fig. 1 General SAR for amide and urea-based sEH inhibitors
Fig. 2 General scafold of the designed derivatives
pharmacophore (P2) like ester, ketone or alcohol would
be in favor of the activity. The L2P3 region of the model
is not necessary for the inhibitory activity and could be
useful as a solubilizing group [24]. By studying the previ-
ous well-known inhibitors and based on the general SAR
for sEH inhibitors, a series of novel 3-phenylglutaric acid
amide-based structures were designed, synthesized and
assessed in vitro (Fig. 2). The amide group and the car-
boxylic acid in the represented structures were consid-
ered as the primary pharmacophore (P1) and secondary
pharmacophore (P2), respectively, which were linked by
2-phenyl propylene (L1) as a lipophilic spacer. Moreo-
ver, several hydrophobic or hydrophilic groups in the R
position were added to the main structure for improving
inhibitory potency. Additionally, the carboxylic moiety
was inserted to the designed structures for increasing the
solubility and improving physicochemical properties by
forming carboxylate salt.
Results and discussion
Drug design strategy
Figure 1 displays general SAR for amide and urea-based
sEH inhibitors. Adamantyl or trifluoromethylphenyl
as the R group and aromatic linkers (L1) such as phe-
nyl or ortho-fluorophenyl would afford more potent
compounds. Moreover, a polar group for the secondary
1 3

Molecular Diversity
According to the obtained results, substitutes in the R
position would increase inhibitory activity in the order of
phenethyl > benzyl > phenyl. This could be due to higher
lipophilicity of the potent compounds and also the suf-
cient distance of phenyl ring from the amide group as the
frst pharmacophore allowing better ftting of the phenyl
ring into the lipophilic pocket of the enzyme. It appears
that amide group, lipophilic rings in the R position and
carboxylic acid of this series play an essential role in ef-
cacy and involvement of the para substitutes in the phenyl
ring did not signifcantly afect inhibitory activity. The
other parts of the structures may be more important for
the inhibitory activity, and these results are in line with
the docking study which confrms the importance of the
P1 moiety in the binding of the designed structures to
the enzyme. Since most of the sEH inhibitors have low
solubility, high melting point and weak pharmacokinetic
properties [23], great attention is paid to the development
of the analogues with enhanced solubility which can be
provided by the addition of polar groups such as carbox-
ylic moiety through the formation of carboxylate salts.
Chemistry
The synthetic route for the desired compounds is shown
in Scheme 1. Firstly, benzaldehyde 1 as a starting material
was treated with ethyl acetoacetate in the presence of piperi-
dine in anhydrous ethanol under refux to give the diethyl
2,4-diacetyl-3-phenylpentanedioate 3. The anhydride 5 was
obtained by the hydrolysis of compound 3 with NaOH 50%
followed by reaction with acetic anhydride under refux
condition. Finally, the fnish products 6a–o were aforded
by reaction of intermediate 5 with diferent aliphatic and
aromatic amines in dry dioxane [25].
In vitro sEH inhibitory activity
Synthesized compounds were tested for sEH inhibitory
activity of sEH, and AUDA was used as a positive control
agent. Results are summarized in Table 1. It is interest-
ing to note that most synthesized compounds had potent
inhibitory activity against sEH with reasonable IC50 values
(0.5–28.7 nM) and that the most potent compound was 6o
with IC50 value of 0.5 nM equal to AUDA (IC50 =0.5 nM).
Scheme 1 Reagents and conditions: a Piperidine, anhydrous EtOH, refux, 10 min, rt, 12 h, 63%; b NaOH(aq)50%, EtOH, refux, 3 h, rt, 18 h,
73%; c Ac2O, refux, 2 h, 67%; d Proper amine, dry dioxane, rt, 24 h, 51-91%
1 3

Molecular Diversity
Table 1 Inhibitory activities of the amide-based derivatives (6a–6o)
compound
6a
R
IC50a nM compound
R
IC50a nM
NDb
6i
28.7
6b
25
6j
5
6c
24
25
27
6k
6l
5.8
5.2
6d
6e
6m
5.15
6f
26
6n
5.8
6g
6h
25
23
6o
0.5
0.5
AUDAc
-
aInhibition activities were measured using a Cayman fuorescence-based human soluble epoxide hydrolase assay kit (Item Number 10011671).
IC50 values represent the inhibitor concentrations required to decrease enzyme activity by 50%
bND: not determined
c12-(3-Adamantan-1-yl-ureido)-dodecanoic acid: a positive control compound
between carboxylic group and Met419. The spacer phenyl
ring had hydrophobic interaction with Lue397. Furthermore,
the phenyl moiety in R position was observed to adopt a
parallel π–π interaction with Trp336.
Modeling study
In order to illustrate possible interactions between 5-(phene-
thyl amino)-5-oxo-3-phenyl pentanoic acid (6o) and the
enzyme, a molecular modeling study was conducted based
on the available crystal structure of sEH. According to
Fig. 3a, the docking study revealed that the analogue ftted
well in the hydrolase catalytic pocket of the sEH. Appar-
ently, the amide group of 6o was correctly located into the
active site pocket and had a proper distance from the three
amino acids of Tyr383, Tyr466 and Asp335 for impressive
hydrogen bonding. Extra hydrogen bond could be formed
Also, to confrm the impact of phenyl ring distance from
the amide group on inhibitory activity, the docking study
of compound 6c with phenyl ring in R position and IC50 of
24 nM was performed. Figure 3b represents the superimpo-
sition of compound 6o and 6c in the catalytic pocket of sEH
enzyme. Accordingly, the phenyl rings of both compounds
in R position had the same directions but the amide group
as P1 moiety of 6c, unlike 6o, did not have proper distance
1 3

Molecular Diversity
Fig. 3 a Compound 6o (salmon pink sticks) in the catalytic pocket
of sEH (amino acid residues in blue PDB: 3ANS). The amide group
has suitable distance from the three amino acids Tyr383, Tyr466 and
Asp335 for hydrogen bonding. Additional hydrogen bond could be
formed between carboxylic group and Met419. The phenyl ring in
spacer had hydrophobic interaction with Lue397. Furthermore, the
phenyl moiety in R position was observed to adopt a parallel π–π
interaction with Trp336. b The superimposition of compound 6o
(salmon pink sticks) and 6c (green sticks) in the catalytic pocket of
sEH enzyme
from three amino acids of Tyr383, Tyr466 and Asp335 to
form hydrogen bond.
3‑Phenylpentanedioic acid [4] A mixture of intermediate
3 (35 g, 0.1 mol), NaOH 50% (100 mL) and ethanol 96%
(150 mL) was refuxed for 3 h. Then, it was stirred at room
temperature for 18 h and monitored with TLC. After add-
ing 50 mL distilled water followed by acidifying with HCl
37%, the mixture was extracted with diethyl ether, dried over
MgSO4 and concentrated in vacuum. Finally, the light yel-
low powder (15.2 g, 73% yield) was obtained without puri-
fcation. mp: 145–147 °C; IR: (KBr) ν (cm−1): 2515–3368
(OH), 1698, 1713 (C=O); LC–MS: m/z 231 (M+23), 247
(M+39).
Methods and materials
General
All laboratory grade reagents were supplied commercially
from Aldrich or Merck Company and were used without
further purifcation. Melting points were obtained using an
Electrothermal 9100 apparatus and are uncorrected. Mass
spectra were obtained using a HPLC Agilent 1100 spec-
4‑Phenyldihydro‑2H‑pirane‑6,2(3H)‑dione [5] A mixture
of intermediate 4 (13 g, 62.5 mmol) in acetic anhydride
(70 mL) was refuxed for 2 h. Then, the mixture was con-
centrated in vacuum and washed with diethyl ether to obtain
a light yellow powder (8 g, 67% yield). mp. 103–105 °C;
IR: (KBr) ν (cm−1): 1748, 1805 (C=O); LC–MS: m/z 191
(M+1).
1
trometer. H NMR and 13C NMR spectra were recorded
at 400 and 125 MHz, respectively, on a Bruker Avance II
spectrophotometer using DMSO-d6 as a solvent. Chemi-
cal shifts were expressed in parts per millions (ppm), and
tetramethylsilane was used as an internal standard. Infrared
spectra were run on a Perkin Elmer 834 spectrometer. The
absorptions were reported on the wavenumber (cm−1) scale,
in the range 400–4000 cm−1.
General procedure for the synthesis
of 3‑phenylglutaric acid amide derivatives (6a–6o)
A mixture of intermediate 5 (1 equiv) and proper amine (1
equiv) in dry dioxane (10 mL) was stirred at room tempera-
ture for 24 h. The medium was acidifed with HCl 2 M, and
the formed precipitate was fltered and washed with water
to achieve fnal products.
Synthesis
Diethyl 2,4‑diacetyl‑3‑phenylpentanedioate [3] A mixture
of benzaldehyde 1 (0.27 mol, 27 mL), ethyl acetoacetate
2 (0.58 mol, 73 mL), in the presence of piperidine (4 mL)
in anhydrous ethanol (200 mL) was stirred under refux
for 10 min and then stirred at room temperature for 12 h.
The solvent was evaporated, and the residue was washed
with cold ethanol 96% to obtain a white crystalline powder
(60 g, 63% yield). mp: 150–152 °C; IR: (KBr) ν (cm−1):
2946–3038 (OH-enol), 1688, 1698 (C=O); LC–MS: m/z
371(M+23), 387 (M+39).
5‑(Adamantly amino)‑5‑oxo‑3‑phenyl pentanoic acid (6a) 6a
(0.28 g, 2.6 mmol) as white powder was synthesized by
general procedure using a mixture of intermediate 5 (0.5 g,
2.6 mmol) and amantadine hydrochloride (0.49 g, 2.6 mmol)
in the presence of anhydride sodium carbonate (0.54 g, 60%
1
yield); mp. 207–210 °C; H NMR (400 MHz, DMSO-d6)
1 3

Molecular Diversity
δ p.p.m.: 1.54 (br s, 3H, adamantly), 1.65 (br s, 3H, ada-
mantly), 1.79 (s, 6H, adamantly), 1.94 (br s, 3H, adamantly),
2.44 (m, 4H, CH2CH2), 3.38 (quin, 1H, CH-benzyl), 7.21
(m, 5H, phenyl); 13C NMR (125 MHz, DMSO-d6) δ p.p.m.:
29.609, 36.884, 39.427, 39.825, 39.992, 40.159, 40.326,
40.493, 40.659, 40.826, 41.713, 43.657, 51.408, 127.028,
128.280, 128.894, 144.570, 170.593, 173.707; LC–MS: m/z
342 (M+1), 705 (2 M+23); Anal. Calcd. For C21H27NO3:
C, 73.87; H, 7.97; N, 4.10; Found: C, 74.03; H, 7.90; N,
3.97; IR: (KBr) ν max (cm−1): 3337 (N–H), 2528–3064
(O–H), 1699, 1732 (C=O).
13C NMR (125 MHz, DMSO-d6) δ p.p.m.: 39.029, 40.426,
43.512, 115.931, 116.107, 121.630, 121.691, 121.729,
121.791, 127.262, 128.254, 129.092, 136.211, 144.366,
158.752, 170.104, 173.665; LC–MS: m/z 324 (M+23), 625
(2 M+23), 942 (3 M+39); Anal. Calcd. For C17H16FNO3:
C, 67.76; H, 5.35; N, 4.65; Found: C, 67.50; H, 5.48; N,
4.79; IR: (KBr) ν max (cm−1): 3341 (N–H), 2467–3209
(O–H), 1662, 1706 (C=O).
5‑(4‑Chlorophenyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6e) 6e as white powder (0.76 g, 91% yield) was synthe-
sized by general procedure using a mixture of intermediate
5 (0.5 g, 2.6 mmol) and 4-chloroaniline (0.34 g, 2.6 mmol);
mp. 164–166 °C; 1H NMR (400 MHz, DMSO-d6) δ p.p.m.:
2.6 (m, 4H, CH2CH2), 3.56 (quin, 1H, CH-benzyl), 7.17
(m, J=3.87 Hz, 1H, H4-phenyl), 7.29 (m, 6H, H2, H3, H5,
H6-phenyl, H3, H5-aniline), 7.53 (d, J = 8.23 Hz, 2H, H2,
H6-aniline), 9.98 (s, 1H, NH), 12.06 (br s, 1H, COOH);
13C NMR (125 MHz, DMSO-d6) δ p.p.m.: 38.993, 39.824,
40.962, 121.431, 121.523, 127.266, 127.513, 128.254,
129.093, 129.381, 138.732, 138.835, 144.351, 170.370,
173.690; LC–MS: m/z 318 (M + 1), 340 (M + 23); Anal.
Calcd. For C17H16ClNO3: C, 64.26; H, 5.08; N, 4.41; Found:
C, 64.38; H, 5.02; N, 4.34; IR: (KBr) ν max (cm−1): 3340
(N–H), 2400–3283 (O–H), 1669, 1725 (C=O).
5‑(Cyclohexyl amino)‑5‑oxo‑3‑phenyl pentanoic acid (6b) 6b
as white powder (0.39 g, 51% yield) was synthesized by
general procedure using a mixture of intermediate 5 (0.5 g,
2.6 mmol) and cyclohexyl amine (0.26 g, 2.6 mmol); mp.
1
170–173 °C; H NMR (400 MHz, DMSO-d6) δ p.p.m.:
1.05 (m, 6H, H3, H4, H5-cyclohexyl), 1.56 (m, 4H, H2,
H6-cyclohexyl), 2.46 (m, 4H, CH2CH2), 3.42 (quin, 1H, CH-
benzyl), 3.56 (s, 1H, H1-cyclohexyl), 7.2 (m, 5H, H2, H3, H4,
H5, H6-phenyl), 7.57 (d, J = 7.62 Hz, 1H, NH); 13C NMR
(125 MHz, DMSO-d6) δ p.p.m.: 25.316, 25.371, 26.059,
33.152, 33.224, 39.372, 39.835, 40.834, 48.009, 127.076,
127.235, 128.244, 128.292, 128.918, 129.059, 144.459,
170.004, 173.736; LC–MS: m/z 290 (M+1), 312(M+23),
601 (2 M+23); Anal. Calcd. For C17H23NO3: C, 70.56; H,
8.01; N, 4.84; Found: C, 70.71; H, 7.94; N, 4.72; IR: (KBr)
ν max (cm−1): 3335 (N–H), 2529–2945 (O–H), 1640, 1724
(C=O).
5‑(4‑Bromophenyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6f) 6f as white powder (0.87 g, 91% yield) was synthe-
sized by general procedure using a mixture of interme-
diate 5 (0.5 g, 2.6 mmol) and 4-bromoaniline (0.45 g,
2.6 mmol); mp. 178–179 °C; 1H NMR (400 MHz, DMSO-
d6) δ p.p.m.: 2.59 (m, 4H, CH2CH2), 3.56 (quin, 1H, CH-
benzyl), 7.18 (m, 1H, H4-phenyl), 7.27 (m, 4H, H2, H3, H5,
H6-phenyl), 7.43 (d, J=7.99 Hz, 2H, H3, H5-aniline), 7.48
5‑(Phenyl amino)‑5‑oxo‑3‑phenyl pentanoic acid (6c) 6c
as white powder (0.52 g, 70% yield) was synthesized by
general procedure using a mixture of intermediate 5 (0.5 g,
2.6 mmol) and aniline (0.24 mL, 2.6 mmol); mp. 145–
147 °C; 1H NMR (400 MHz, DMSO-d6) δ p.p.m.: 2.63 (m,
4H, CH2CH2), 3.58 (quin, 1H, CH-benzyl), 7 (t, J=7.05 Hz,
1H, H4-aniline), 7.17 (m, 1H, H4-phenyl), 7.25 (m, 6H, H2,
H3, H5, H6-phenyl, H3, H5-aniline), 7.51 (d, J = 8.15 Hz,
2H, H2, H6-aniline), 10 (s, 1H, NH), 12.07 (s, 1H, COOH);
LC–MS: m/z 284 (M+1), 306 (M +23); Anal. Calcd. For
C17H17NO3: C, 72.07; H, 6.05; N, 4.94; Found: C, 72.20;
H, 5.98; N, 4.85; IR: (KBr) ν max (cm−1): 3359 (N–H),
2562–3259 (O–H), 1666, 1691 (C=O).
(d, J=8.56 Hz, 2H, H2, H6-aniline), 9.98 (s, 1H, NH); 13
C
NMR (125 MHz, DMSO-d6) δ p.p.m.: 38.987, 39.830,
40.966, 115.507, 121.821, 121.914, 127.269, 128.255,
129.096, 132.293, 139.147, 139.253, 144.348, 170.442,
173.690; LC–MS: m/z 385 (M+23), 747 (2 M+23); Anal.
Calcd. For C17H16BrNO3: C, 56.37; H, 4.45; N, 3.87; Found:
C, 56.51; H, 4.38; N, 3.79; IR: (KBr) ν max (cm−1): 3322
(N–H), 2619–3179 (O–H), 1659, 1715 (C=O).
5‑(4‑Nitrophenyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6 g) 6 g as yellow powder (0.47 g, 55% yield) was synthe-
sized by general procedure using a mixture of intermediate 5
(0.5 g, 2.6 mmol) and 4-nitroaniline (0.36 g, 2.6 mmol); mp.
107–108 °C; 1H NMR (400 MHz, DMSO-d6) δ p.p.m.: 2.66
(m, 2H, CH2), 3.01 (m, 2H, CH2), 3.57 (m, 1H, CH-benzyl),
7.17 (m, 1H, H4-phenyl), 7.28 (m, 2H, H2, H6-phenyl), 7.36
(t, J=7.68 Hz, 2H, H3, H5-phenyl), 7.75 (d, J=9.11 Hz, 2H,
H2, H6-aniline), 8.17 (d, J =9.12 Hz, 2H, H3, H5-aniline),
10.48 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6) δ
5‑(4‑Fluorophenyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6d) 6d as white powder (0.43 g, 54% yield) was syn-
thesized by general procedure using a mixture of inter-
mediate 5 (0.5 g, 2.6 mmol) and 4-fuoroaniline (0.29 g,
2.6 mmol); mp. 147–149 °C; 1H NMR (400 MHz, DMSO-
d6) δ p.p.m.: 2.6 (m, 4H, CH2CH2), 3.56 (quin, 1H, CH-
benzyl), 7.08 (t, J=8.71 Hz, 2H, H3, H5-aniline),7.17 (m,
1H, H4-phenyl), 7.27 (m, 4H, H2, H3, H5, H6-phenyl), 7.5
(dd, J=8.26, 3.15 Hz, 2H, H2, H6-aniline), 9.9 (s, 1H, NH);
1 3

Molecular Diversity
p.p.m.: 33.745, 37.059, 40.977, 119.469, 119.547, 125.778,
127.336, 127.534, 128.115, 128.253, 129.130, 129.657,
142.918, 144.181, 145.902, 171.214, 173.605; LC–MS: m/z
329 (M+1), 351 (M+23); Anal. Calcd. For C17H16N2O5: C,
62.19; H, 4.91; N, 8.53; Found: C, 62.33; H, 4.82; N, 8.45;
IR: (KBr) ν max (cm−1): 3583 (N–H), 2596–3325 (O–H),
1674, 1706 (C=O).
41.131, 42.778, 127.192, 127.379, 127.724, 128.402,
128.976, 129.037, 140.225, 144.368, 171.139, 173.741;
LC–MS: m/z 298 (M+1), 320 (M +23); Anal. Calcd. For
C18H19NO3: C, 72.71; H, 6.44; N, 4.71; N, 4.68; Found:
C, 72.58; H, 6.51; N, 4.78; IR: (KBr) ν max (cm−1): 3314
(N–H), 2351–3228 (O–H), 1685, 1718 (C=O).
5‑(4‑Fluorobenzyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6 k) 6 k as white powder (0.72 g, 87% yield) was synthe-
sized by general procedure using a mixture of intermedi-
ate 5 (0.5 g, 2.6 mmol) and 4-fuorobenzyl amine (0.3 mL,
2.6 mmol); mp. 136–138 °C; 1H NMR (400 MHz, DMSO-
d6) δ p.p.m.: 2.54 (m, 4H, CH2CH2), 3.51 (quin, 1H, CH-
benzyl), 4.05 (dd, J = 20.27, 10.21 Hz, 1H, CH2-benzyl),
4.23 (dd, J = 21.27, 9.21 Hz, 1H, CH2-benzylamine),
6.94 (t, J=8.21 Hz, 2H, H3, H5-benzyl), 7 (t, J=8.67 Hz,
1H, H4-phenyl), 7.25 (m, 6H, H2, H6-benzyl, H2, H3,
H5, H6-phenyl), 8.27 (t, J = 5.56 Hz, 1H, NH), 12 (br s,
1H, COOH); 13C NMR (125 MHz, DMSO-d6) δ p.p.m.:
39.355, 40.571, 41.068, 42.758, 115.545, 115.713, 127.247,
128.408, 129.056, 129.579, 129.644, 136.378, 136.399,
144.244, 160.875, 171.210, 173.670; LC–MS: m/z 316
(M + 1), 338 (M + 23); Anal. Calcd. For C18H18FNO3: C,
68.56; H, 5.75; N, 4.44; Found: C, 68.73; H, 5.67; N, 4.36;
IR: (KBr) ν max (cm−1): 3367 (N–H), 2512–3064 (O–H),
1623, 1705 (C=O).
5‑(Para‑tolyl amino)‑5‑oxo‑3‑phenyl pentanoic acid (6 h) 6h
as light yellow powder (0.70 g, 90% yield) was synthe-
sized by general procedure using a mixture of intermediate
5 (0.5 g, 2.6 mmol) and para-toluidin (0.28 g, 2.6 mmol);
mp. 145–146 °C; 1H NMR (400 MHz, DMSO-d6) δ p.p.m.:
2.21 (s, 3H, CH3), 2.61 (m, 4H, CH2CH2), 3.55 (quin, 1H,
CH-benzyl), 7.05 (d, J = 8.14, 2H, H3, H5-aniline), 7.16
(quin, 1H, H4-phenyl), 7.27 (m, 4H, H2, H3, H5, H6-phenyl),
7.37 (d, J=7.02 Hz, 2H, H2, H6-aniline), 9.74 (s, 1H, NH);
13C NMR (125 MHz, DMSO-d6) δ p.p.m.: 21.26, 39.054,
40.314, 43.542, 119.943, 120.041, 127.229, 128.262,
129.077, 129.844, 132.846, 137.300, 137.399, 144.441,
170, 173.672; LC–MS: m/z 298 (M+1), 320 (M+23); Anal.
Calcd. For C18H19NO3: C, 72.71; H, 6.44; N, 4.71; Found:
C, 72.86; H, 6.37; N, 4.61; IR: (KBr) ν max (cm−1): 3336
(N–H), 2503–3124 (O–H), 1658, 1717 (C=O).
5‑(4‑Hydroxyphenyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6i) 6i as green crystalline powder (0.47 g, 60% yield) was
synthesized by general procedure using a mixture of inter-
mediate 5 (0.5 g, 2.6 mmol) and para-aminophenol (0.29 g,
5‑(4‑Methylbenzyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6 l) 6l as light yellow powder (0.62 g, 76% yield) was syn-
thesized by general procedure using a mixture of intermedi-
ate 5 (0.5 g, 2.6 mmol) and 4-methylbenzyl amine (0.3 mL,
2.6 mmol); mp. 109–111 °C; 1H NMR (400 MHz, DMSO-
d6) δ p.p.m.: 2.24 (s, 3H, CH3), 2.51 (m, 4H, CH2CH2),
3.52 (quin, 1H, CH-benzyl), 4.04 (d, J = 15.09 Hz, 1H,
CH2-benzyl), 4.19 (d, J=14.97 Hz, 1H, CH2-benzylamine),
6.84 (d, J = 7.33 Hz, 2H, H3, H5-benzyl), 7.01 (d,
J=7.36 Hz, 2H, H2, H6-benzylamine), 7.25 (m, 5H, H2, H3,
H4, H5, H6-phenyl), 8.21 (s, 1H, NH); 13C NMR (125 MHz,
DMSO-d6) δ p.p.m.: 21.497, 39.350, 40.6, 41.160, 42.801,
127.174, 127.731, 128.382, 129.033, 129.533, 136.405,
137.164, 144.416, 171.117, 173.766; LC–MS: m/z 312
(M+1), 334 (M+23), 645 (2 M+23), 972 (3 M+39); Anal.
Calcd. For C19H21NO3: C, 73.29; H, 6.80; N, 4.50; Found:
C, 73.42; H, 6.73; N, 4.44; IR: (KBr) ν max (cm−1): 3277
(N–H), 2609–3090 (O–H), 1636, 1700 (C=O).
1
2.6 mmol); mp. 180.2–182.2 °C; H NMR (400 MHz,
DMSO-d6) δ p.p.m.: 2.61 (m, 4H, CH2CH2), 3.56 (quin,
1H, CH-benzyl), 6.65 (d, J = 8.7 Hz, 2H, H3, H5-phenol),
7.22 (m, 7H, 5H -phenyl, H2, H6-phenol), 9.12 (s, 1H, OH-
phenol), 9.57 (s, 1H, NH), 12.06 (s, 1H, COOH); 13C NMR
(125 MHz, DMSO-d6) δ p.p.m.: 39.132, 40.375, 43.508,
115.813, 121.838, 127.192, 128.261, 129.058, 131.601,
144.535, 154.072, 169.486, 173.723; LC–MS: m/z 300
(M + 1), 322 (M + 23), 338 (M + 39); Anal. Calcd. For
C17H17NO4: C, 68.21; H, 5.72; N, 4.68; Found: C, 68.04;
H, 5.81; N, 4.76; IR: (KBr) ν max (cm−1): 3389 (N–H),
2493–3153 (O–H), 1653, 1677 (C=O).
5‑(Benzyl amino)‑5‑oxo‑3‑phenyl pentanoic acid (6j) 6j as
white powder (0.47 g, 60% yield) was synthesized by gen-
eral procedure using a mixture of intermediate 5 (0.5 g,
2.6 mmol) and benzylamine (0.28 mL, 2.6 mmol); mp.
1
169–171 °C; H NMR (400 MHz, DMSO-d6) δ p.p.m.:
5‑(4‑Methoxybenzyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6 m) 6 m as white powder (0.58 g, 67% yield) was synthe-
sized by general procedure using a mixture of intermediate
5 (0.5 g, 2.6 mmol) and 4-methoxybenzyl amine (0.34 mL,
2.56 (m, 4H, CH2CH2), 3.53 (quin, 1H, CH-benzyl), 4.1 (dd,
J = 33.78, 4.91 Hz, 1H, CH2-benzyl), 4.27 (dd, J = 33.82,
5.87 Hz, 1H, CH2-benzylamine), 6.95 (d, J=6.76 Hz, 2H,
H2, H6-benzyl), 7.25 (m, 8H, H3, H4, H5-benzyl, H2, H3, H4,
H5, H6-phenyl), 8.26 (s, 1H, NH), 12 (br s, 1H, COOH);
13C NMR (125 MHz, DMSO-d6) δ p.p.m.: 39.352, 40.551,
1
2.6 mmol); mp. 129.4–130.4 °C; H NMR (400 MHz,
DMSO-d6) δ p.p.m.: 2.51 (m, 4H, CH2CH2), 3.63 (m, 1H,
CH-benzyl), 3.9 (s, 5H, CH2-benzylamine, CH3-methoxy),
1 3

Molecular Diversity
6.75 (br s, 2H, H3, H5-benzyl), 6.78 (br s, 2H, H2,
H6-benzyl), 7.19 (br s, 5H, H2, H3, H4, H5, H6-phenyl), 8.21
(br s, 1H, NH); LC–MS: m/z 328 (M+1), 350 (M+23), 677
(2 M+23), 1020 (3 M+39); Anal. Calcd. For C19H21NO4:
C, 69.71; H, 6.47; N, 4.28; Found: C, 69.56; H, 6.54; N,
4.35; IR: (KBr) ν max (cm−1): 2538–3252 (O–H), 3355
(N–H), 1685, 1697 (C=O).
the reference inhibitor for assay. The enzyme and inhibi-
tors were incubated for 5 min at 30 °C. Activities were
determined by monitoring the appearance of 6-methoxy-
2-naphthaldehyde by fuorescence detection with an exci-
tation wavelength of 330 nm and an emission wavelength
of 465 nm. All test samples and AUDA were dissolved in
DMSO and each compound was assayed at 5 concentra-
tions in triplicate.
5‑(4‑Acetylphenyl amino)‑5‑oxo‑3‑phenyl pentanoic acid
(6n) 6n as yellow powder (0.65 g, 76% yield) was synthe-
sized by general procedure using a mixture of intermediate
5 (0.5 g, 2.6 mmol) and para-aminoacetophenone amine
(0.36 g, 2.6 mmol); mp. 162–164 °C; 1H NMR (400 MHz,
DMSO-d6) δ p.p.m.: 2.58 (m, 7H, CH3, CH2CH2), 3.49
(quin, 1H, CH-benzyl), 7.14 (br s, 1H, H4-phenyl), 7.26
(br s, 4H, H2, H3, H5, H6-phenyl), 7.64 (d, J = 7.74 Hz,
2H, H2, H6-acetophenone), 7.87 (d, J = 8.09 Hz, 2H,
H3, H5-acetophenone), 10.4 (s, 1H, COOH); 13C NMR
(125 MHz, DMSO-d6) δ p.p.m.: 27.225, 39.841, 40.342,
43.738, 119.056, 119.139, 127.081, 128.271, 129.030,
130.243, 132.368, 144.330, 144.897, 171.169, 197.332;
LC–MS: m/z 326 (M+1), 348 (M+23), 364 (M+39); Anal.
Calcd. For C19H19NO4: C, 70.14; H, 5.89; N, 4.31; Found:
C, 70.32; H, 5.77; N, 4.23; IR: (KBr) ν max (cm−1): 3352
(N–H), 2609–3285 (O–H), 1676, 1706 (C=O).
Modeling study
Molecular modeling was performed using AutoDock
Tools version 1.5.6rc3 (http://mgltools.scripps.edu). The
atomic coordinates of the crystal structure of human sEH
(PDB code: 3ANS) with its complex ligand (4-cyano-N-
[(1S,2R)-2-phenylcyclopropyl]benzamide) was derived
from the RCSB Protein Data Bank. The resolution of sEH
structure was 1.98 Å. The original ligand and water mol-
ecules were omitted, and polar hydrogen atoms and Kollman
united atom partial charges were added onto the protein.
The enzyme and ligands were considered as rigid and fex-
ible, respectively. The structures of the compounds were
optimized by MM+force feld using HyperChem8 (http://
www.hyper.com) and converted to pdbqt format fle by
AutoDock Tools. Each docking system was performed by
100 runs of the AutoDock using the Lamarckian genetic
algorithm (LGA). The lowest docking-energy conformation
of the highest populated cluster was considered as the most
stable orientation and could be selected for analysis. Finally,
a cluster analysis was performed on the docking results using
a root mean square deviation (RMSD) of 0.5 Å. The image
was produced via Pymol software version 1.5.0.1 (http://
pymol.fndmysoft.com).
5‑(Phenethyl amino)‑5‑oxo‑3‑phenyl pentanoic acid (6o) 6o as
light yellow powder (0.66 g, 81% yield) was synthesized by
general procedure using a mixture of intermediate 5 (0.5 g,
2.6 mmol) and phenylethyl amine (0.34 mL, 2.6 mmol); mp.
154–156 °C; 1H NMR (400 MHz, DMSO-d6) δ p.p.m.: 2.51
(m, 6H, CH2-phenethyl, CH2CH2), 3.46 (quin, 1H, CH-ben-
zyl), 4.8 (t, J=7.17 Hz, 2H, CH2-phenethyl), 6.94 (d, 1H,
J=6.93 Hz, H4-phenethyl), 7.21 (m, 9H, H-phenethyl), 8.17
(d, J=7.96 Hz, 1H, NH); 13C NMR (125 MHz, DMSO-d6)
δ p.p.m.: 23.133, 39.316, 40.443, 42.783, 48.443, 126.504,
126.789, 127.135, 127.178, 127.395, 128.305, 128.386,
128.873, 128.980, 129.031, 144.407, 145.472, 170.301,
173.689; LC–MS: m/z 312 (M + 1), 334 (M + 23), 350
(M+39); Anal. Calcd. For C19H21NO3: C, 73.29; H, 6.80;
N, 4.50; Found: C, 73.38; H, 6.76; N, 4.43; IR: (KBr) ν max
(cm−1): 3319 (N–H), 2548–3076 (O–H), 1697, 1721 (C=O).
Conclusions
In this study, 15 novel amide derivatives of 3-phenylglutaric
acid were designed, synthesized and evaluated. Most of the
synthesized compounds provided nanomolar range inhibi-
tion against sEH enzyme. The docking study of compound
6o showed that this structure had afnity to the hydrolase
catalytic pocket of sEH and ft properly. Also, this compound
with phenethyl in the R position and IC50 value of 0.5 nM
showed the highest inhibitory activity due to proper lipo-
philicity and the appropriate distance of phenyl ring from
the amide group. Also, incorporating a carboxylic moiety
into these designed structures by forming carboxylate salts
would increase the solubility, and improving physicochemi-
cal properties. In summary, these structures will be a valu-
able lead scafold to develop new potent sEH inhibitors.
Pharmacological activity
A fuorescence-based assay was used to determine biologi-
cal activities by a Cayman human soluble epoxide hydro-
lase assay kit (item number 10011671). A stock solution
of the enzyme and inhibitors were prepared in 25 mM
Bis–Tris/HCl bufer (200 μL; pH 7.0). 3-Phenyl-cyanoIJ6-
methoxy-2-naphthalenyl)-methylester-2-oxiraneacetic acid
(PHOME) was used as the substrate for assay [26]. AUDA,
one of the most efective inhibitors of sEH, was used as
1 3

Molecular Diversity
library design and structure-based virtual screening. J Med Chem
54:1211–1222. https://doi.org/10.1021/jm101382t
Acknowledgements This work was supported by two grants from the
Research Council of Shahid Beheshti University of Medical Sciences
[Grant Nos. 7765 and 8457].
13. Tran KL, Aronov PA, Tanaka H, Newman JW, Hammock BD,
Morisseau C (2005) Lipid sulfates and sulfonates are allosteric
competitive inhibitors of the N-terminal phosphatase activity of the
mammalian soluble epoxide hydrolase. Biochemistry 44:12179–
12187. https://doi.org/10.1021/bi050842g
Compliance with ethical standards
Conflict of interest The authors confrm that this article content has no
conficts of interest.
14. Arand M, Wagner H, Oesch F (1996) Asp333, Asp495, and His523
form the catalytic triad of rat soluble epoxide hydrolase. J Biol Chem
271:4223–4229. https://doi.org/10.1074/jbc.271.8.4223
15. Langhlin LT, Tzeng HF, Lin S, Armstrong RN (1998) Mechanism of
microsomal epoxide hydrolase. Semifunctional sitespecifc mutants
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https://doi.org/10.1021/bi972737f
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1 3