Development of multitarget inhibitors for the treatment of pain: Design, synthesis, biological evaluation and molecular modeling studies

Article abstract of DOI:10.1016/j.bioorg.2020.104165

Multitarget-directed ligands are a promising class of drugs for discovering innovative new therapies for difficult to treat diseases. In this study, we designed dual inhibitors targeting the human fatty acid amide hydrolase (FAAH) enzyme and human soluble epoxide hydrolase (sEH) enzyme. Targeting both of these enzymes concurrently with single target inhibitors synergistically reduces inflammatory and neuropathic pain; thus, dual FAAH/sEH inhibitors are likely to be powerful analgesics. Here, we identified the piperidinyl-sulfonamide moiety as a common pharmacophore and optimized several inhibitors to have excellent inhibition profiles on both targeted enzymes simultaneously. In addition, several inhibitors show good predicted pharmacokinetic properties. These results suggest that this series of inhibitors has the potential to be further developed as new lead candidates and therapeutics in pain management.

Full text of DOI:10.1016/j.bioorg.2020.104165

BioorganicChemistry103(2020)104165
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Development of multitarget inhibitors for the treatment of pain: Design,
synthesis, biological evaluation and molecular modeling studies
Stephanie Wilta, Sean Kodanib, Thanh N.H. Lea, Lato Nguyena, Nghi Voa, Tanya Lya,
⁎
Mark Rodrigueza, Paula K. Hudsona, Christophe Morisseaub, Bruce D. Hammockb, Stevan Pecica,
a Department of Chemistry and Biochemistry, California State University Fullerton, Fullerton, CA 92831, United States
b Department of Entomology and Nematology, and UCD Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Multitarget-directed ligands are a promising class of drugs for discovering innovative new therapies for difficult
to treat diseases. In this study, we designed dual inhibitors targeting the human fatty acid amide hydrolase
(FAAH) enzyme and human soluble epoxide hydrolase (sEH) enzyme. Targeting both of these enzymes con-
currently with single target inhibitors synergistically reduces inflammatory and neuropathic pain; thus, dual
FAAH/sEH inhibitors are likely to be powerful analgesics. Here, we identified the piperidinyl-sulfonamide
moiety as a common pharmacophore and optimized several inhibitors to have excellent inhibition profiles on
both targeted enzymes simultaneously. In addition, several inhibitors show good predicted pharmacokinetic
properties. These results suggest that this series of inhibitors has the potential to be further developed as new
lead candidates and therapeutics in pain management.
Structure-Activity Relationship (SAR) study
Enzyme inhibition
Designed multiple ligands
Molecular modeling
Docking experiments
ADMET predictions
1. Introduction
therapeutics to treat acute and chronic pain, and only a few new drugs
have been introduced to the market in the past years. One approach
Pain results from a wide variety of conditions and illnesses.
Managing pain represents a unique challenge to health professionals,
which requires multidisciplinary strategies [1,2]. The most effective
analgesic drugs currently used to treat moderate-to-severe pain are
opioid agonists (e.g., oxycodone) [3]. The endogenous opioid system is
a part of the body’s natural defense network in the brain, and opioid-
based drugs (opioids) interact with opioid receptors, which leads to
pain relief [4]. Prolonged use of opioids will eventually lead to toler-
ance, physical dependence, and addiction [5]. Currently, the most
commonly used non-opioid drugs in pain management are nonsteroidal
anti-inflammatory drugs (NSAIDs) [6]. The anti-inflammatory action of
NSAIDs is a result of their inhibitory effect on the cyclooxygenase
(COX) enzymes, that are involved in the metabolism of arachidonic acid
into pro-inflammatory prostaglandins [7]. During inflammation, ara-
chidonic acid (AA) is released from the membrane phospholipids by
enzyme phospholipase A2 (PLA2) and converted to different in-
flammatory mediators (Fig. 1) [8,9]. NSAIDs can treat only mild and
moderate pain and, in addition, there are a number of adverse effects
associated with their use, but most common are dyspepsia, an increased
risk of gastric ulcer, and an increased risk of myocardial infarction
toward designing novel analgesics with improved efficacy and reduced
adverse effects is the poly-pharmacological approach, i.e., to design a
new, single drug that is able to modulate multiple molecular pathways
that are involved in pain regulation [12].
Soluble epoxide hydrolase (sEH) is a ubiquitous enzyme widely
distributed throughout the body with the most concentrated expression
in the liver, kidneys, lungs, and vascular tissues [13]. This enzyme is
selective for aliphatic epoxides of fatty acids, such as epoxyeicosa-
trienoic acids (EETs) [14] (Fig. 1). The EETs are one of the metabolic
derivatives of AA [15]. EETs exhibit vasodilatory effects in various
arteries and have also been shown to possess analgesic and anti-in-
flammatory properties [16]. AA is metabolized through three major
enzymatic pathways: cyclooxygenase (COX), lipoxygenase (LOX), and
cytochrome P450 (CYP) pathways [14]. The enzyme sEH mediates the
addition of water to EETs, leading to the corresponding diols, dihy-
droxyeicosatrienoic acids (DHETs), which show diminished biological
activity [17]. Therefore, inhibition of the enzyme sEH causes an in-
crease in EET concentration, which has beneficial therapeutic effects on
pain and inflammation [8]. Thus far, a large body of work has focused
on a class of urea-based inhibitors for sEH (e.g. AUDA, TPPU) shown in
Fig. 2 [18]. TPPU demonstrated to be active against both acute
[10,11]. Currently there is
a great demand for more effective
⁎ Corresponding author.
E-mail address: specic@fullerton.edu (S. Pecic).
https://doi.org/10.1016/j.bioorg.2020.104165
Received 9 July 2020; Received in revised form 5 August 2020; Accepted 12 August 2020
Availableonline28August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

S. Wilt, et al.
BioorganicChemistry103(2020)104165
we were able to successfully co-crystallize one of the non-urea in-
hibitors with human sEH [33]. Our docking experiments revealed that
an amide functional group binds in the proximity of key amino acids
needed for catalytic activity, two tyrosines (Y383 and Y466), and one
aspartic acid (D335) [34]. The orientation of this amide moiety is si-
milar to the orientation of the urea groups on urea sEH inhibitors (e.g.,
AUDA, Fig. 2); thus, the amide group likely satisfies the same hydrogen
bonding interactions with tyrosine and aspartic acid residues that
contribute to highly potent urea inhibitors. Using information obtained
from SAR studies in combination with molecular modeling and crys-
tallography data, we were able to determine a particular pharmaco-
phore for this series of sEH inhibitors (e.g., analog 2, shown in red,
Fig. 3) required to inhibit the sEH enzyme.
Phospholipids
PLA2
FAAH
Anandamide
Arachidonic acid
COX
CYP
EETs
sEH
LOX
Wang et al. (2010) performed an HTS and identified the ben-
zothiazole analog 3 (Fig. 3) as a potent rat FAAH inhibitor having an
IC50 of 18 nM [26]. Their SAR studies indicated that the sulfonamide
group, the piperidine ring, and benzothiazole on the left-hand side of
the molecule were key components to their activity. The sulfonamide
group likely forms hydrogen bonding interactions with the catalytic
serine group, similar to the hydrogen-bonded network between the
FAAH enzyme and the pyridyl nitrogen and oxazoyl oxygen of the α-
keto-oxazole FAAH inhibitors [35]. In addition, the modeling study
performed by Wang et al. (2010) also indicated that the benzothiazole
ring satisfies hydrophobic interactions within the hydrophobic binding
pocket of the rat FAAH enzyme that confers extraordinary potency
[26]. We also identified several potent 4-phenylthiazole FAAH in-
hibitors that possess the piperidine moiety connected to the phenyl ring
via a sulfonamide bond [36]. These studies have shown that all three
components, sulfonamide bond, piperidine ring and benzothiazole/4-
phenylthiazole moieties, are important for the FAAH inhibition, but
more work has to be done in order to access the particular relationship
of each of these moieties with the inhibition potencies.
Prostaglandins
Leukotrienes
Fig. 1. Metabolic pathways of Arachidonic acid and the role of enzymes fatty
acid amide hydrolase and soluble epoxide hydrolase.
inflammation and chronic pain conditions in several rodent and pri-
mate preclinical models [19–22].
FAAH is an integral membrane protein that hydrolyzes en-
docannabinoids [23]. Endocannabinoids are endogenous lipid ligands
that activate the cannabinoid receptors, CB1 and CB2 [24]. Two en-
docannabinoids have been identified in mammals, anandamide (AEA)
and 2-arachidonoylglycerol (2-AG). FAAH terminates AEA signaling by
hydrolyzing AEA to arachidonic acid and ethanolamine, two metabo-
lites that do not activate cannabinoid receptors (Fig. 1). The pharma-
cological inactivation of FAAH produces analgesic, anti-inflammatory,
anxiolytic and mild antidepressant effects, without showing the un-
desirable side effects of direct cannabinoid receptor agonists, indicating
that FAAH is a promising therapeutic target for pain management [25].
Several classes of FAAH inhibitors have been reported, including car-
bamates (e.g., URB597, URB937 – Fig. 2), ureas, and substrate-derived
inhibitors [26,27].
Overall, these extensive SAR studies indicate that modifications to
the aromatic ring on the left- and right-hand sides of the pharmaco-
phore (shown in red in Fig. 3) should allow for improved sEH and FAAH
inhibition. This data guided our design for the preparation of the dual
inhibitors wherein modifications to the right side of the aromatic ring
were carried out.
A recent study by Sasso et al. (2015) showed that the combination of
sEH inhibitor TPPU and FAAH inhibitor URB937 causes a significant
synergistic reduction in pain behavior in two rodent models of pain
[28]. We believe that this synergy may be exploited therapeutically to
achieve better pain control at lower drug dosages or to develop dual
agents that simultaneously inhibit both FAAH and sEH activities. Pre-
viously we have described dual sEH/FAAH inhibitors that are potent on
the human form of both enzymes but have poor metabolic stability and
water solubility [29,30].
Since inhibition of each enzyme showed analgesic effect in-
dividually [37,38], and co-administration of sEH and FAAH inhibitors
resulted in a significant synergistic reduction in pain behavior in animal
models of pain [28], we hypothesized that dual inhibitors will treat
pain at a lower dose, and consequently with fewer side-effects. We
decided to employ a Designed Multiple Ligands (DMLs) strategy
[12,39] wherein inhibitors used the core pharmacophore (a phenyl ring
connected to a piperidine moiety, which is connected to the sulfona-
mide bond) common to the sEH inhibitor 2 and the FAAH inhibitor 3
with modifications on the aromatic rings on either side. Fig. 3 shows
representative structures with the key pharmacophoric regions boxed.
Given that the benzothiazole ring contributes to high FAAH inhibitory
potency and bulky hydrophobic groups are well-tolerated in that po-
sition for sEH inhibitory potency [32,33], we kept this structure con-
stant (general structure 4) while modifying aromatic groups bound to
the sulfonamide group.
Here, we sought to take alternative approaches to design a new
series of dual sEH/FAAH inhibitors that may have a more favorable
physio-chemical profile to be used as a possible therapeutic. These in-
hibitors were designed by integrating a piperidine-sulfonamide-phenyl
pharmacophore common to the medicinal chemistry of both selective
sEH and FAAH inhibitors.
2. Results and discussion
Following the established synthetic procedure [34] shown in
Scheme 1, we started from the readily available 2-(4-aminophenyl)
benzothiazole and Boc-isonipecotic acid. EDC coupling yielded the
amide 5, which was subjected to Boc-deprotection with trifluoroacetic
acid (TFA) which provided the key amine intermediate 6. Coupling
with different R-sulfonylchlorides furnished final compounds 4–1 to
4–30 in moderate yields (16–84%).
2.1. Design and synthesis
Previously, we identified potent non-urea sEH inhibitors using high-
throughput screening (HTS) in combination with structure–activity
relationship (SAR) studies. These molecules were derivatives of iso-
nipecotic acid (e.g., inhibitor 1, Fig. 2). Our SAR studies showed that
the pharmacophore for the sEH inhibitors should include a central
sulfonamide moiety next to the piperidine ring. We also observed that
the bulky, hydrophobic groups on the left-hand side of the molecules
are positively correlated with inhibitory potency [31,32]. In addition,
2.2. Biological evaluation and Structure-activity relationship studies
All synthesized analogs 4–1 to 4–30 were tested in vitro in both
human sEH and human FAAH inhibition assays. The inhibition
2

S. Wilt, et al.
BioorganicChemistry103(2020)104165
O
O
F
F
O
O
O
N
N
H
N
F
H
N
N
OH
H
H
O
AUDA
TPPU
IC50 = 3.7 nM
IC50 = 2.6 nM
NH2
H
H2N
O
N
H
N
O
O
O
O
HO
URB597
IC50 = 4.6 nM
URB937
IC50 = 26.8 nM
Cl
O
N
H
N
Cl
S
O
O
1
IC50 = 20 nM
Fig. 2. Known FAAH and sEH inhibitors.
Pharmacophore
O
Hydrophobic
left-side
fragment
O
Pharmacophore
S
N
S
N
O
S
NH
H
N
N
O
Benzothiazole-phenyl
left-side fragment
O
O
S
HOOC
Aromatic
right-side
fragment
FAAH Inhibitor 3
IC50 = 18 nM
sEH Inhibitor 2
IC50 = 23 nM
Aromatic
right-side
fragment
Pharmacophore
S
N
O
NH
N
S
R
O
O
Dual Inhibitors 4-1 to 4-30
Fig. 3. Design strategy for synthesis of dual sEH/FAAH inhibitors.
potencies of analyzed analogs against both enzymes are summarized in
Table 1. Our initial SAR investigation started with the synthesis of the
thiophene-2-yl analog, 4–1. This analog showed inhibition potency in
the low nanomolar range for human FAAH enzyme (IC50 = 16 nM), but
only moderate inhibition potency at the human sEH enzyme
(IC50 = 420 nM). The introduction of bromine and chlorine atoms on
the thiophene ring (analog 4–2) led to significantly diminished in-
hibition potencies at both enzymes. We decided to replace the thio-
phene ring with a phenyl ring, which allowed us to access many
sterically and electronically diverse chemical groups, which could in
turn improve inhibition profiles at both enzymes. The phenyl analog
4–3, showed excellent inhibition potency with the human FAAH en-
zyme, having an IC50 of 8.6 nM, but only low micromolar inhibition for
human sEH enzyme (IC50 = 1100 nM). Fluoro-, chloro-, bromo- and
methyl- groups placed at the ortho position (4–4, 4–5, 4–6, and 4–7,
respectively) were all well tolerated in the human FAAH binding pocket
and led to low nanomolar inhibition potency on human FAAH enzyme.
The bulkier, electron-donating methoxy- group (4–8) was less potent
for human FAAH (IC50 = 80 nM). Placement of chloro-, bromo-, or
methyl- groups in the ortho position improved potency at the sEH en-
zyme relative to the unsubstituted inhibitor (4–3). This led to the best
dual sEH/FAAH inhibitor of the series (4–5) with equally high potency
for sEH (IC50 = 9.6 nM) and FAAH (IC50 = 7 nM). Next, we introduced
the same replacements into the meta-position on the phenyl ring. We
noticed that adding fluoro-, chloro-, bromo-, methyl- or methoxy-
groups at the meta position (4–9, 4–10, 4–11, 4–12 and 4–13, respec-
tively) had comparable potencies relative to the unsubstituted inhibitor
(4–3). These inhibitors retain low nanomolar inhibition potencies for
3

S. Wilt, et al.
BioorganicChemistry103(2020)104165
COOH
O
S
N
a
N
Boc
S
N
NH2
NH
N
Boc
5
2-(4-aminophenyl)
benzothiazole
Boc-isonipecotic acid
b
O
O
O
NH
4-1 to 4-30
c
NH
S
N
N
S
R
O
S
N
NH
6
Scheme 1. Reagents and conditions: (a) EDC, DCM, rt, 24 h, 48%; (b) TFA, DCM, rt, 24 h, 87%; (c) R-sulfonyl chloride,
Et3N, DCM, rt, 24 h, 16-84%.
Scheme 1. Reagents and conditions: (a) EDC, DCM, rt, 24 h, 48%; (b) TFA, DCM, rt, 24 h, 87%; (c) R-sulfonyl chloride, Et3N, DCM, rt, 24 h, 16–84%.
human FAAH enzyme with low potency at the human sEH enzyme
(IC50s ranged from 620 to 2400 nM). Modification of the para position
with fluoro-, chloro, bromo-, methyl- and methoxy- substitutions (4–14,
4–15, 4–16, 4–17 and 4–18, respectively) led to a loss of potency to-
wards FAAH relative to the unsubstituted inhibitor (4–3). Surprisingly,
the introduction of the 2,4-disubstitutions for fluoro-, chloro-, bromo-
and methoxy- groups (4–19, 4–20, 4–21, 4–22 and 4–23, respectively)
led to inhibitors with potency at both enzymes comparable to the same
single ortho- substitutions and improved potency relative to para- sub-
stitutions. Thus, the benefit from ortho- substitutions is greater than the
loss from para- substitutions. The fluoro- and methyl- 3,5-disubstitu-
tions (4–24 and 4–25) had low potency at both enzymes
(IC50s > 10,000 nM on FAAH and 1,000 nM on sEH). Additionally, the
fluoro-, chloro-, methyl- and isopropyl- tri-substitutions (4–26, 4–27,
4–28 and 4–29, respectively) and the pentafluoro- substitution (4–30)
had lower potency than the 2,4-disubstituted molecules. This suggests
compounds that have high bulk are unfavored in the active sites of
FAAH and sEH.
define the most important residues within the binding pocket of the
FAAH enzyme and tried to explain the increased in vitro inhibition
potencies by observing and analyzing the type of contacts of the most
active FAAH inhibitor identified in this study, 4–4, with the inhibitory
potency of 1.3 nM, and other analogs with low nanomolar inhibition
potencies, e.g., 4–5, 4–6, 4–10 and 4–22. The aromatic part of the in-
hibitor 4–4 is found to be embedded between several hydrophobic
amino acid residues (F192, S193, Y194, I238, G239, G240, S241, F244,
F381, L404, I407, V422, L429, F432, L433, M436, T488, V491, I530,
W531) and forms other important non-covalent interactions (M191,
G216, S217, L380, L401, G485, M495), which we believe all contribute
to high inhibitory potency of this analog (Table 2A). The best dual in-
hibitor identified herein, 4–5 has a very similar defined binding pocket,
showing the importance of selected hydrophobic and other non-cova-
lent interactions for the low nanomolar inhibition potency (Fig. 5A,
Fig. 5B, Table 2A). The poor correlation of docking energies with in
vitro experiments were observed with only three analogs, 4–24, 4–25
and 4–30, but visual inspection of these compounds within the binding
pocket of the human FAAH homology model reveals the absence of
several important contacts with residues, S241 and S217, that are
present with the biologically active analogs, which could explain the
lower inhibition potencies of these three analogs (Table 2A).
2.3. Molecular modeling studies
Our design and evaluation of synthesized analogs were com-
plemented with in silico experiments. Since the crystal structure of the
human FAAH enzyme has not been reported, we built and evaluated a
homology model for the human FAAH enzyme [36]. We docked all
synthesized analogs, 4–1 to 4–30, in both the human FAAH enzyme
homology model and the human sEH enzyme crystal structure derived
from PDB: 4HAI. Docking scores obtained in these experiments are
shown in Table 1 and all non-covalent interactions are shown in
Table 2A and Table 2B for FAAH and sEH enzymes, respectively. The
ICM docking score represents unitless approximations of the binding
free energy between the inhibitor and the enzyme where a lower
docking score suggests a higher chance the inhibitor is bound to the
enzyme [40]. For the human FAAH enzyme, the low values for the
potential energy (docking scores) were obtained for all the analogs that
also show in vitro low nanomolar inhibition potencies (e.g., 4–7, 4–11,
4–12, 4–21, 4–23, etc.). Analysis of these values show that most of the
obtained docking energies are correlated with inhibitory potency
(R2 = 0.3255, p = 0.0023) with the exception of compounds with very
poor potency on FAAH (IC50 > 10,000) (Fig. 4A). This suggests that
the potency of these inhibitors is primarily based on Van der Waals
interactions between the enzyme active site and inhibitors. After visual
inspection of the top binding modes of the most active inhibitors, we
noticed that all docked compounds are located in the proximity of S241
and S217, both residues of the catalytic triad, S241-S217-K142, which
is responsible for the hydrolytic cleavage of the amide bond of the
substrate anandamide by the FAAH enzyme [25]. We were able to
The docking experiments of all synthesized inhibitors in the human
sEH enzyme model revealed that most of the analogs are located in the
proximity of key amino acids within the catalytic pocket that are in-
volved in the hydrolysis of EETs. Compared with docking scores for the
FAAH enzyme, the docking scores of sEH poorly correlated with sEH
potency (Fig. 4B). All potent sEH inhibitors (e.g. 4–5, 4–6, 4–7, 4–8,
4–20, 4–21 and 4–22) have good docking scores (Table 1), however,
the analogs that were inactive in the in vitro inhibition assay, also have
good docking scores in our docking experiments. This possibly indicates
that sEH potency is primarily determined based on hydrogen bonding
interactions with the catalytic residues rather than hydrophobic inter-
actions with the binding pocket which probably is responsible for the
good docking scores in this series of analogs. Our previous molecular
modeling studies and X-ray crystallographic structure showed that two
tyrosine residues (Y383 and Y466) and one aspartic acid residue
(D335), located in the hydrolase catalytic pocket of sEH, are involved in
hydrogen bonding with the inhibitors. One of the most potent sEH in-
hibitors identified in this study, and the best dual inhibitor, 4–5, is in
the close proximity of these three amino acid residues (Fig. 6A, Fig. 6B,
Table 2B). This inhibitor forms a hydrogen bond with D335 via amide
bond, and has several important hydrophobic interactions (F267,
W336, M339, P371, I375, F381, Y383, F387, L408, L417, M419, L428,
M469, N472, V498, L499, H524), that, combined with other non-
covalent interactions (P268, Q384, Y466, W525), contribute to the high
inhibition potency of this compound. We also noticed that all low
4

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Table 1
Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) inhibitory activities and docking scores of analogs.
Compound
R
FAAH IC50 (nM)
sEH IC50 (nM)
Docking Score FAAH
Docking Score sEH
URB 597
AUDA
4–1
–
–
38
–
–
−31.45
−22.18
−21.04
−22.16
−25.83
−22.35
2.6
420
16
4–2
2700
5900
−12.3
−21.02
4–3
8.6
1.3
7
1100
150
9.6
−23.77
−28.05
−28.93
−26.88
−34.41
−29.93
−35.20
−32.98
−32.17
−36.5
−30.32
−32.42
−33.03
−30.93
−32.44
−32.98
−34.19
−29.41
−29.25
−30.08
−32.16
−28.91
4–4
4–5
4–6
6.1
9.6
80
24
4–7
35
4–8
26
4–9
13.4
7.2
51.6
7
1700
2400
1100
940
620
1300
4–10
4–11
4–12
4–13
4–14
14.6
77
−26.88
−19.99
4–15
260
710
−22.49
−27.61
(continued on next page)
5

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Table 1 (continued)
Compound
R
FAAH IC50 (nM)
185
sEH IC50 (nM)
580
Docking Score FAAH
Docking Score sEH
−31.23
4–16
−23.64
4–17
4–18
> 10000
> 10000
370
84
−29.02
−18.66
−32.60
−31.59
4–19
4–20
4–21
11.4
31.3
10
195
13.1
36
−27.22
−23.64
−37.38
−33.84
−34.11
−34.20
4–22
4–23
5.5
29.2
180
−33.14
−33.22
−32.08
−34.47
36.2
4–24
> 10000
1100
−36.69
−32.39
4–25
4–26
> 10000
250
3600
6600
−41.62
−26.88
−33.18
−30.58
4–27
270
1300
−33.37
−29.05
(continued on next page)
6

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Table 1 (continued)
Compound
R
FAAH IC50 (nM)
84
sEH IC50 (nM)
790
Docking Score FAAH
Docking Score sEH
−31.33
4–28
−28.97
4–29
4–30
2233
840
−18.61
−24.48
−27.90
> 10000
2670
−37.58
nanomolar sEH inhibitors identified in this study are docked in the
proximity of the key amino acid residues, Y383, Y466, and D335, lo-
cated in the catalytic site of the sEH enzyme (Table 2B), while inactive
compounds lack this interaction.
rotatable bonds. The logS for aqueous solubility of a molecule, ex-
pressed in mol/L, helps to predict oral absorption. The acceptable range
for logS is between −6.5 and −0.5 mol/liter. In this study, all syn-
thesized analogs showed values close to, but below, −6.5 mol/L, sug-
gesting a moderate aqueous solubility. Improving solubility of these
potential drug candidates, if needed, can be addressed in the future
guided design of follow-up inhibitors or in the drug formulation pro-
cess. The next important parameter to consider for the drug design and
development is permeability, and we assessed it using the Caco-2 pre-
diction tool [48]. The ICM-Chemist-Pro Caco-2 prediction scores higher
than −5 suggest a highly permeable drug candidate, while scores of
below −6 represent a poorly permeable compound. As shown in
Table 3, all compounds synthesized in this study have predicted scores
for Caco-2 between −5 and −6, suggesting moderately permeable drug
candidates. We also wanted to predict the plasma half-life of synthe-
sized dual inhibitors. Our analysis showed that inhibitors 4–26, 4–27
and 4–28 have the longest predicted half-life in hours (10.10 h)
whereas the inactive compound 4–2 had the shortest predicted half-life
of 1.2 h. The most potent inhibitors identified in this study, 4–5, 4–6,
4–20, 4–21, and 4–22 showed a moderate predicted half-life of 3.2 to
5.7 h. As part of our Tox studies, we analyzed several important tox-
icology descriptors, LD50, hERG inhibition, and the Tox score. Predic-
tion of the hERG inhibition is performed because pharmacological
blockade of the hERG channel results in a severe life-threatening car-
diac side effect possibly causing sudden death, leading to the with-
drawal of many drugs from the development process [49,50]. The ICM-
Chemist-Pro tool predicts that scores above or equal to 0.5 will prob-
ably exhibit some hERG inhibition at 100 µM or less, while compounds
with predicted values below 0.5 will likely not be hERG inhibitors. No
compounds synthesized in this study exceeded a value of 0.5. As part of
the toxicity study predictions, we calculated the Tox score for each
compound synthesized. This value represents the identification of po-
tentially toxic parts/bi-products (during metabolism) of the molecule.
The only compound that showed a toxicity score was analog 4–30.
Finally, we calculated a “drug-like” properties for each synthesized
compound. This is a purely empirical value and is based on several
factors calculated above. The scores are on a scale of −1 to 1 and
should not exceed 1. Only two compounds, 4–15 and 4–29, slightly
exceed a value of 1. In addition, these two analogs show moderate and
low inhibition profiles against both enzymes, respectively, and will not
be considered for future follow up studies.
Finally, we looked at the binding poses of the potent dual inhibitors,
4–5, 4–6, 4–20, 4–21, and 4–22, within both, human FAAH and sEH
enzyme binding pockets. We observed that the benzothiazole moiety of
all aforementioned ligands is located in the proximity of the two hy-
drophobic residues, V422 and L433 in the human FAAH binding
pockets, while this group probably interacts with one of the methionine
residues (M419 or M469) within human sEH binding pocket.
2.4. In silico ADMET studies
Based on the encouraging biological results described above, we
performed some selected absorption, distribution, metabolism, excre-
tion, and toxicology (ADMET) predictions to assess their drug-like
properties. Unfavorable ADMET properties have been identified as a
major cause of drug candidate failure in the pharmaceutical industry
[41,42]. In silico ADMET prediction represents the use of computer
modeling software to understand structure–property relationships and
predicts the in vivo behavior of potential drug candidates in the human
body [43].
Using the ICM-Chemist-Pro tool, we started our investigations (see
Table 3) by calculating simple physicochemical descriptors, such as
cLogP and aqueous solubility (cLogS). It has been shown previously that
these parameters are good predictors for drug candidate permeability
[44,45]. High logP values usually mean poor absorption/barrier pene-
tration and is also an integral part of the well-known Lipinski Rule of 5
prediction [46]. All synthesized analogs in this study, 4–1 to 4–30, are
in the agreement with Lipinski Rule of 5 in terms of cLogP, the number
of hydrogen bond acceptors and the number of hydrogen bond donors.
Several inhibitors have molecular weights that exceed 500 g/mol, but
these numbers are very close to 500 Da and as the prediction rule states,
potential orally active drugs should not violate more than one of the
four criteria. Therefore, we believe that these inhibitors represent ex-
cellent candidates for future follow-up SAR studies and drug develop-
ment. In addition, another important rule that predicts oral bioavail-
ability, Veber’s Rule, states that drug candidates will have good oral
bioavailability if the number of rotatable bonds present in a molecule is
less than 10 [47]. All compounds synthesized in this study has 6 to 9
7

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Table 2A
The list of hydrogen bonds, hydrophobic interactions and other non-covalent interactions of analogs docked in human FAAH enzyme.
Compound
H-bonds
Hydrophobic interactions
Other non-covalent interactions
URB-597
AUDA
4–1
G485
G272
N/A
F192, S193, Y194, G239, F244, L401, L404, I407, M436, T488, V491, W531
M191, F192, S193, Y194, I238, G239, F244, V270, Q273, L278, V491
F192, S193, Y194, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433, M436,
T488, V491, I530, W531
M191, G240, S241, F381, D403, R486, I530
S190, S217, S241, C269, Y271, E274
M191, G216, S217, L380, L401, G485
4–2
G485
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
G485
G485
G485
N/A
N/A
F192, S193, Y194, I238, G239, G240, S241, F244, L404, I407, V422, L429, L433, T488, V491,
I530, W531
M191, G216, S217, F381, L401, P484
G216, S217, M191, I238, G240
4–3
F192, S193, Y194, G239, S241, F244, L380, F381, L404, I407, V422, L429, F432, L433, M436,
T488, V491, I530, W531
4–4
F192, S193, Y194, I238, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433,
M436, T488, V491 I530, W531
M191, G216, S217, L380, L401, G485, M495
M191, G216, S217, L380, G485, M495
M191, S241, F244, F381, G485
4–5
F192, S193, Y194, I238, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433,
M436, T488, V491 I530, W531
4–6
F192, S193, Y194, I238, G239, L380, L404, I407, V422, L429, L433, M436, T488, V491, M495,
I530, W531
4–7
F192, S193, Y194, I238, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433,
M436, T488, V491, I530, W531
M191, G216, S217, L380, L401, G485, M495
M191, G216, S217, L380, G485, M495
M191, G216, S217, L380, L401, G485
M191, G216, S217, L380, L401, G485
M191, G216, F381, L401, P484
4–8
F192, S193, Y194, I238, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433,
M436, T488, V491, I530, W531
4–9
F192, S193, Y194, I238, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433,
M436, T488, V491, I530, W531
4–10
4–11
4–12
4–13
4–14
4–15
F192, S193, Y194, I238, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433,
M436, T488, V491, I530, W531
F192, S193, Y194, I238, G239, G240, S241, F244, L404, I407, V422, L429, L433, T488, V491,
I530, W531
F192, S193, Y194, L380, F381, L404, I407, V422, L429, F432, L433, T488, V491, M495, I530,
W531
M191, T377, L401, D403, P484
F192, S193, Y194, I238, G239, S241, F244, L404, I407, V422, L429, L433, T488, V491, I530,
W531
M191, S217, F381, L401, D403, P484
M191 G216, S217, L380
F192, S193, Y194, G239, G240, S241, F244, F381, L404, I407, V422, L429, F432, L433, M436,
T488, V491, I530, W531
M191, F192, S193, Y194, I238, G239, F381, L404, I407, V422, L429, F432, L433, M436, T488,
V491, I530, W531
S241, F244, M495
4–16
4–17
4–18
N/A
N/A
N/A
M191, F192, S193, G239, L404, I407, V422, L429, F432, L433, M436, T488, I530, W531
M191, F192, S193, I238, G239, L404, I407, V422, L429, F432, L433, M436, T488, I530, W531
M191, F192, S193, I238, G239, L380, F381, L404, I407, V422, L429, F432, L433, M436, T488,
I530, W531
Y194, S217, S241, F244, G485, V491
Y194, S217, S241, F244, G485, V491
Y194, S241, F244, V491
4–19
N/A
M191, F192, S193, I238, G239, L380, F381, L404, I407, V422, L429, F432, L433, M436, T488,
V491, I530, W531
Y194, S241, F244, M495
4–20
4–21
N/A
N/A
M191, F192, S193, Y194, I238, G239, L404, I407, V422, L429, L433, M436, T488, I530, W531
M191, F192, S193, Y194, I238, G239, L380, L404, I407, L429, L433, M436, T488, V491, I530,
W531
G216, S217, S241, F244, L401, G485, V491
G216, S241, F244, G485
4–22
4–23
G485
N/A
F192, S193, Y194, G239, G240, S241, F244, L404, I407, V422, L429, L433, T488, V491, M495,
I530, W531
M191, G216, F381, P484
M191, F192, S193, Y194, I238, G239, L380, F381, L404, I407, L429, L433, T488, V491, I530,
W531
S217, S241, F244, L401, P484, G485
4–24
4–25
G485
G485
Y194, G239, F381, F432, L404, I407, V422, L429, F432, L433, T488, V491, M495, I530, W531
F192, S193, Y194, L380, F381, L404, I407, V422, L429, F432, L433, T488, V491, M495, I530,
W531
F192, T377, L401, D403, P484
M191, T377, L401, D403, P484
4–26
4–27
N/A
N/A
M191, F192, S193, Y194, I238, G239, L380, L404, I407, V422, L429, F432, L433, M436, T488,
I530, W531
G216, S217, S241, F244, G485, V491, M495
S190, S217, T236, G240, S241, K267, V270
M191, F192, S193, Y194, I238, G239, L266, G268, Y271, T377, F381, L404, F432, M436,
T488, V491, M495
4–28
4–29
N/A
I238
M191, F192, S193, Y194, I238, G239, L380, L404, I407, V422, L429, L433, M436
M191, F192, S193, Y194, G239, F244, L266, G268, Y271, L278, T377, L380, F381, L404,
F432, M436, T488, V491, M495
G216, S217, S241, F244, P484, G485
S190, S217, T236, D237, G240, S241, K267, C269,
V270, L401, P484
4–30
G485
S193, Y194, G239, S376, T377, L380, F381, D403, L404, I407, V422, F432, L433, T488, V491,
M495, I530, W531
F192, L401, P484
3. Conclusion
investigated as potential therapeutics in many diseases, i.e., cardio-
vascular [51], pulmonary [52,53], and Parkinson’s disease [54,55]. In
this study, we observed several novel compounds that have inhibitor
activity for one of these two enzymes. Compounds that were found to
inhibit only one particular enzyme, sEH (e.g., 4–18) or FAAH (e.g.,
4–4), will be very useful to further develop as novel therapeutics for the
aforementioned diseases and other disorders beyond pain.
We successfully designed, synthesized, and identified several potent
dual FAAH/sEH inhibitors. Inhibitors 4–5, 4–6, 4–20, 4–21, and 4–22
were identified as the most potent compounds in the benzothiazole
series against both enzymes. Molecular modeling studies of these
compounds revealed important residues within the catalytic sites of
both enzymes that are responsible for the low nanomolar potencies of
these inhibitors. This knowledge will be used in future design and
follow-up SAR studies. In addition, these selected dual inhibitors
showed acceptable predicted ADMET properties, and further study is
currently underway to test their stability in liver microsome assays. In
addition, the inhibitors of both enzymes have been individually
4. Material and methods
All solvents and reagents were obtained from Sigma–Aldrich, Matrix
Scientific, TCI, and Acrōs Organic and used without further purification.
Analytical thin-layer chromatography (TLC) was performed on
8

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Table 2B
The list of hydrogen bonds, hydrophobic interactions and other non-covalent interactions of analogs docked in human sEH enzyme.
Compound
H-bonds
Hydrophobic interactions
Other non-covalent interactions
URB-597
AUDA
L417, Y466
Y466, H524,
W525
F267, D335, W336, T360, Y383, M419, V498, L499, H524, W525
D335, W336, M339, T360, F381, Y383, S412, M419, F497, V498, L499
Q384, L408, S415
F267, Q384, L408, R410, A411, K495,
G523
4–1
4–2
4–3
4–4
4–5
4–6
4–7
4–8
D335
F267, W336, M339, P371, I375, F381, Y383, L408, L417, M419, L428, M469, N472, V498, L499,
Q384, Y466, W525
H524
Y466
D335
Y466
D335
D335
N/A
F267, W336, M339, P371, I375, F381, Y383, L408, L417, M419, L428, M469, N472, V498, L499,
P268, D335, Q384, F387, S418
Q384, Y466, W525
H524, W525
F267, W336, M339, P371, I375, F381, Y383, L408, L417, M419, L428, M469, N472, V498, L499,
H524
D335, W336, M339, T360, P361, I363, F381, Y383, L407, R410, S415, L417, M419, V498, L499,
Q384, S407, A411, M469, D496
P268, Q384, Y466, W525
M503, H524, W525
F267, W336, M339, P371, I375, F381, Y383, F387, L408, L417, M419, L428, M469, N472, V498,
L499, H524
F267, W336, M339, P371, I375, F381, Y383, F387, L408, L417, M419, L428, M469, N472, V498,
P268, Q384, Y466, W525
L499, H524
D335, W336, M339, T360, P361, I363, F381, Y383, L408, R410, S415, L417, M419, Y466, V498,
F267, Q384, S407, A411, M469
D335, Q384, S407, A411, M469, D496
L499, M503, H524, W525
Y466
W336, M339, T360, P361, I363, I375, F381, Y383, L408, R410, S415, L417, M419, V498, L499,
M503, H524, W525
4–9
Y466
D335
Y466
F267, W336, M339, P371, I375, F381, Y383, L408, L417, M419, L428, M469, V498, L499, H524
D335, Q384, F387
4–10
4–11
W336, M339, P361, I363, F381, Y383, L408, R410, S415, L417, M419, Y466, V498, L499, M503
P268, Q384, Y466, W525
D335, Q384, S407, A411, M469, D496
W336, M339, T360, P361, I363, F381, Y383, L408, R410, S415, L417, M419, V498, L499, M503,
H524, W525
4–12
4–13
4–14
4–15
4–16
4–17
4–18
4–19
4–20
4–21
4–22
4–23
4–24
4–25
4–26
4–27
4–28
4–29
4–30
Y466
N/A
F267, D335, W336, M339, P371, I375, F381, Y383, Q384, L408, L417, M419, L428, Y466, M469,
D335, Q384, F387
V498, L499, H524
D335, W336, M339, T360, P361, I363, F381, Y383, L408, R410, S415, L417, M419, Y466, V498,
F267, Q384, S407, A411, M469
P268, D335, Q384, S418, Y466, W525
P268, D335, Q384, S418, W525
D335, Q384, F387, S415, L428
P268, D335, Q384, W473, W525
L499, M503, H524, W525
N/A
F267, W336, M339, P371, I375, F381, Y383, L408, L417, M419, L428, M469, N472, V498, L499,
H524
N/A
F267, W336, M339, P371, I375, F381, Y383, L408, L417, M419, L428, Y466, M469, N472, V498,
L499, H524
Y466
N/A
F267, D335, W336, M339, P371, I375, F381, Y383, L408, L417, M419, M469, V498, L499, H524,
W525
F267, W336, M339, P371, I375, F381, Y383, L408, L417, M419, L428, Y466, M469, N472, V498,
L499, H524
Y466
D335
N/A
W336, M339, I363, I375, F381, Y383, L408, R410, S415, L417, M419, V498, L499, M503, H524,
F267, D335, T360, Q384, S407, A411,
D496
W525
F267, W336, M339, P371, I375, F381, F387, Y383, L408, L417, M419, L428, M469, N472, L499,
P268, Q384, S418, Y466, W525
V498, H524
F267, W336, M339, P371, I375, F381, Y383, F387, L408, L417, M419, L428, M469, N472, V498,
P268, D335, Q384, S418, Y466, W525
D335, Q384, F387, S415
L499, H524
Y466
N/A
F267, W336, M339, P371, I375, F381, Y383, L408, L417, M419, L428, M469, N472, V498, L499,
H524, W525
F267, W336, M339, P371, I375, F381, Y383, F387, L408, L417, M419, L428, M469, N472, V498,
P268, D335, Q384, Y466, W473, W525
L499, H524
Y466
Y466
N/A
W336, M339, P361, I363, I375, F381, Y383, L408, R410, S415, L417, M419, V498, L499, M503,
H524, W525
F267, D335, T360, Q384, S407, A411,
D496
W336, M339, I375, F381, Y383, L408, R410, S415, L417, M419, M469, L499, V498, H524, W525
D335, T360, Q384, S407, A411, N472,
D496
W336, M339, I375, F381, Y383, L408, R410, S415, L417, M419, Y466, M469, V498, L499, H524,
F267, D335, T360, Q384, S407, A411,
D496
W525
Y466
N/A
W336, M339, T360, P361, I363, F381, Y383, L408, R410, S415, L417, M419, M469, V498, L499,
M503, H524, W525
F267, D335, Q384, S407, A411, D496
F267, W336, M339, P371, I375, F381, Y383, F387, L408, L417, M419, L428, M469, N472, V498,
L499, H524, W525
P268, D335, Q384, S418, Y466
P268, Q384, S418, Y466
D335
Y466
Y466
F267, W336, M339, P371, I375, F381, Y383, F387, L408, L417, M419, L428, M469, N472, V498,
L499, H524
W336, M339, T360, P361, I363, P371, I375, F381, Y383, L408, R410, L417, M419, Y466, M469,
V498, L499, M503, H524, W525
D335, S374, Q384, S407, D496
F267, D335, Q384, S407, A411, D496
W336, M339, T360, P361, I363, I375, F381, Y383, L408, R410, S415, L417, M419, M469, V498,
L499, M503, H524, W525
aluminum plates precoated with silica gel, also obtained from
Sigma–Aldrich. Column chromatography was carried out on Merck 938S
silica gel. Proton and carbon NMR spectra were recorded with a Bruker
400 MHz NMR spectrometer. Spectra were referenced to the residual
solvent peak: proton chemical shifts are reported relative to the residual
reported relative to the residual deuterated solvent signals (chloro-
form = 77.2 ppm, or dimethyl sulfoxide = 39.5 ppm). All compounds
described were of > 95% purity. Purity was confirmed by high-re-
solution liquid chromatography mass spectrometer (ThermoFisher
Scientific system). Elution was isocratic with water (30%, +0.1% formic
acid) and acetonitrile (70%, +0.1% formic acid) at a flow rate of
0.4 mL/min. Melting points were measured with a MEL-TEMP II melting
point apparatus and are reported uncorrected. Human recombinant
FAAH enzyme (Item No. 100101183, Batch No. 0523867) and human
recombinant sEH enzyme (Item No. 10011669) were obtained from
solvent peak (chloroform
=
7.26 ppm or dimethyl sulf-
oxide = 2.50 ppm) as follows: chemical shift (δ), proton ID, multiplicity
(s = singlet, bs = broad singlet, d = doublet, bd = broad doublet,
dd = doublet of doublets, t = triplet, q = quartet, m = multiplet, in-
tegration, coupling constant(s) in Hz). Carbon chemical shifts are
9

S. Wilt, et al.
BioorganicChemistry103(2020)104165
0
-0.5
-1
R² = 0.3255
-1.5
-2
-2.5
-3
-3.5
-4
-4.5
10
15
20
25
30
35
40
45
Fig. 4A. Correlation of inhibitory potency (-log(IC50)) vs. Docking Scores for FAAH enzyme.
Cayman Chemical. Molecular modeling studies and docking experiments
were performed using ICM Pro Molsoft software.
mp: 238–240 °C. 1H NMR (400 MHz, CDCl3) δ ppm 8.05 – 8.02 (m, 3H),
7.88 (d, J = 7.2 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.47 (t,
J = 7.2 Hz, 1H), 7.36 (t, J = 6.8 Hz, 1H), 4.17 (bs, 1H), 2.77 (t,
J = 11.2 Hz, 2H), 2.43 – 2.37 (m, 1H), 1.89 (d, J = 10.8 Hz, 2H), 1.79
– 1.70 (m, 3H), 1.47 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 172.9,
167.5, 154.8, 154.2, 140.4, 135.0, 129.6, 128.5, 126.4, 125.2, 123.1,
121.7, 119.9, 79.9, 44.5, 28.7, 28.5 ppm.
5. Experimental
5.1. Chemistry
The amide 5 (250 mg, 0.57 mmol) was dissolved in anhydrous di-
chloromethane, DCM (10 mL), and stirred in an ice bath at 0 °C.
Trifluoroacetic acid, TFA (1 mL, 11.4 mmol) was added dropwise into
the solution and the reaction mixture was stirred at room temperature
for 24 h under an argon atmosphere. Following concentration in vacuo,
the crude product was triturated with diethyl ether and filtered. The
product 6 was obtained as a TFA salt and used for next step without
further purification. A small amount was free-based and used for NMR
analysis: N-(4-(benzo[d]thiazol-2-yl)phenyl)piperidine-4-carboxamide,
6 pale green solid, yield 91% (0.57 mmol, 234 mg), mp: 242–244 °C. 1H
NMR (400 MHz, DMSO‑d6) δ ppm 10.31 (s, 1H), 8.11 (d, J = 8 Hz, 1H),
8.02 (t, J = 9.2 Hz, 3H), 7.81 (d, J = 8.8 Hz, 2H), 7.52 (t, J = 8.4 Hz,
1H), 7.43 (t, J = 8 Hz, 1H), 4.23 (bs, 1H), 3.14 (d, J = 12.4 Hz, 2H),
2.68 (t, J = 12.4 Hz, 2H), 2.59 – 2.55 (m, 1H), 1.82 (d, J = 11.6 Hz,
2H), 1.65 (q, J = 12, 11.6 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δ
173.5, 166.9, 153.6, 142.1, 134.2, 127.9, 127.4, 126.5, 125.2, 122.5,
122.2, 119.3, 44.1, 42.1, 27.5 ppm.
5.1.1. General procedure for the preparation of benzothiazole-phenyl
analogs
The mixture of N-Boc-4-piperidinecarboxylic acid (250 mg,
1.09 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC
(252 mg, 1.31 mmol) and a catalytic amount of 4-dimethylaminopyr-
idin, DMAP were dissolved in anhydrous tetrahydrofuran, THF (40 mL).
The reaction mixture was stirred at room temperature for 1.5 h under
an argon atmosphere. Then, 2-(4-aminophenyl) benzothiazole (197 mg,
0.87 mmol) was added to the stirring solution. The reaction mixture
was stirred at room temperature for 48 h under an argon atmosphere.
After removal of the solvent under reduced pressure, the residue was
dissolved in ethyl acetate (40 mL). The mixture was transferred to a
separatory funnel and the organic layer was washed with an aqueous
solution of 1 M hydrochloric acid, HCl (3x25 mL), followed by an
aqueous solution of saturated sodium bicarbonate, NaHCO3 (25 mL),
dried over anhydrous sodium sulfate, Na2SO4, filtered and con-
centrated. The crude product 5 was purified by flash chromatography
(1:4 ethyl acetate/hexane solvent system) and recrystallized from die-
thyl ether: tert-butyl 4-((4-(benzo[d]thiazol-2-yl)phenyl)carbamoyl)pi-
peridine-1-carboxylate, 5 white solid, yield 70% (0.87 mmol, 267 mg),
The amine 6, as a TFA salt (100 mg, 0.22 mmol), was dissolved in
anhydrous DCM (20 mL), and stirred in an ice bath at 0 °C.
Triethylamine (0.19 mL, 1.33 mmol) was added and the reaction
0
-0.5
-1
-1.5
-2
-2.5
-3
R² = 0.1614
-3.5
-4
-4.5
0
5
10
15
20
25
30
35
Fig. 4B. Correlation of inhibitory potency (-log(IC50)) vs. Docking Scores for sEH enzyme.
10

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Fig. 5A. A 3D docking pose of the inhibitor 4–5 in the catalytic site of the human fatty acid amide hydrolase (FAAH) enzyme.
mixture was warmed to room temperature. Corresponding benzene-
sulfonyl chloride (0.33 mmol) was added to the reaction mixture and
the reaction mixture was stirred for 48 h at room temperature under an
argon atmosphere. Next, the mixture was transferred to a separatory
funnel where the organic layer was washed with an aqueous solution of
saturated NaHCO3 (30 mL). The organic layer was dried over anhy-
drous sodium sulfate, filtered and concentrated. The crude product was
purified by flash chromatography (1:4 ethyl acetate/hexane solvent
system) and recrystallized from diethyl ether.
piperidine-4-carboxamide, 4–1 was obtained as an off-white solid in the
amount of 18 mg (17% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.19 (s,
1H), 8.12 (d, J = 7.6 Hz, 1H), 8.07–8.00 (m, 4H), 7.78 (d, J = 8.8 Hz,
2H), 7.67 (d, J = 2.4 Hz, 1H), 7.52 (t, J = 8 Hz, 1H), 7.44 (t,
J = 12 Hz, 1H), 7.31 (t, J = 5.2 Hz, 1H), 3.68 (d, J = 12 Hz, 2H),
2.47–2.38 (m, 3H), 1.95 (d, J = 14 Hz, 2H), 1.70 (q, J = 16, 8.8 Hz,
2H). 13C NMR (100 MHz, DMSO‑d6): δ 173.0, 167.0, 153.7, 142.0,
135.5, 134.3, 133.8, 133.0, 128.4, 128.0, 127.6, 126.6, 125.5, 122.6,
122.3, 119.4, 45.4, 41.4, 27.5 ppm. HRMS-ESI+: calculated for
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-ylsulfonyl)
C23H21N3O3S3 + H: 484.0823; Found: 484.0818.
Fig. 5B. 2D representation for the lowest energy conformation of inhibitor 4–5 in the binding pocket of human FAAH. Green shading represents hydrophobic region;
gray parabolas represent accessible surface for large areas; broken thick line around ligand shape indicates accessible surface; size of residue ellipse represents the
strength of the contact.
11

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Fig. 6A. A 3D docking pose of the inhibitor 4–5 in the catalytic site of the human soluble epoxide hydrolase (sEH) enzyme.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((4-bromo-5-chlorothiophen-2-
141.9, 135.9, 134.5, 134.2, 132.3, 131.5, 130.9, 127.9, 127.8, 127.5,
126.6, 125.2, 122.5, 122.2, 119.4, 99.5, 44.6, 41.7, 27.9 ppm. HRMS-
yl)sulfonyl)piperidine-4-carboxamide, 4–2 was obtained as an off-white
solid in the amount of 55 mg (42% yield): 1H NMR (400 MHz, DMSO‑d6)
δ 10.20 (s, 1H), 8.12 (d, J = 7.2 Hz, 1H), 8.03 (t, J = 8.8 Hz, 3H), 7.80
(t, J = 22.4 Hz, 2H), 7.53 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 6.8 Hz, 1H),
3.69 (d, J = 12 Hz, 2H), 2.62 (t, J = 12 Hz, 2H), 1.97 (d, J = 10.4 Hz,
2H), 1.74 (q, J = 11.6, 9.6 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6): δ
173.0, 167.0, 153.6, 141.6, 134.9, 133.9, 133.5, 132.2, 127.9, 127.1,
126.6, 125.3, 122.6, 122.2, 119.4, 112.1, 45.3, 41.4, 27.5 ppm. HRMS-
ESI+: calculated for C25H22ClN3O3S2
512.0858.
+ H: 512.0869; Found:
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2-bromophenyl)sulfonyl)pi-
peridine-4-carboxamide, 4–6 was obtained as an off-white solid in the
amount of 96 mg (78% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.26 (s,
1H), 8.11 (t, J = 6.8 Hz, 1H), 8.02 (t, J = 10.4 Hz, 4H), 7.91–7.89 (m,
1H), 7.79 (d, J = 7.6 Hz, 2H), 7.64–7.49 (m, 3H), 7.45–7.40 (m, 1H),
3.76 (d, J = 12.8 Hz, 2H), 2.86 (t, J = 12.0 Hz, 2H), 2.55 (d,
J = 11.6 Hz, 1H), 1.89 (d, J = 12.0 Hz, 2H), 1.63 (q, J = 11.2, 11.2 Hz,
2H). 13C NMR (100 MHz, DMSO‑d6): δ 173.0, 166.9, 153.6, 142.0,
137.5, 135.8, 134.4, 134.2, 131.6, 128.2, 127.9, 127.4, 126.5, 125.2,
122.5, 122.2, 119.5, 119.3, 44.6, 41.7, 27.9 ppm. HRMS-ESI+: calcu-
lated for C25H22BrN3O3S2 + H: 556.0364; Found: 556.0356.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-(o-tolylsulfonyl)piperidine-4-
carboxamide, 4–7 was obtained as an off-white solid in the amount of
91 mg (84% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.24 (s, 1H), 8.11
(d, J = 7.6 Hz, 1H), 8.02 (t, J = 8.8 Hz, 3H), 7.83 (d, J = 8 Hz, 1H),
7.79, (d, J = 8.8 Hz, 2H), 7.60–7.50 (m, 2H), 7.48–7.41 (m, 3H), 7.78
(d, J = 8.8 Hz, 2H), 7.67 (d, J = 2.4 Hz, 1H), 7.52 (t, J = 8 Hz, 1H),
3.66 (d, J = 12.4 Hz, 2H), 2.70 (t, J = 12 Hz, 2H), 2.58 (s, 3H), 1.90 (d,
J = 10.4 Hz, 2H), 1.68–1.59 (m, 2H). 13C NMR (100 MHz, DMSO‑d6): δ
177.0, 173.1, 167.0, 153.7, 142.1, 137.3, 135.9, 134.3, 133.1, 133.0,
132.9, 129.6, 128.0, 128.0, 127.6, 126.6, 126.5, 126.4, 126.4, 125.3,
125.2, 122.6, 122.3, 119.4, 114.7, 110.1, 44.4, 41.9, 27.9, 20.2 ppm.
HRMS-ESI+: calculated for C26H25N3O3S2 + H: 492.1416; Found:
492.1407.
ESI+: calculated for
595.9529.
C23H19BrClN3O3S3 + H: 595.9539; Found:
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-(phenylsulfonyl)piperidine-4-
carboxamide, 4–3 was obtained as an off-white solid in the amount of
40 mg (38% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.15 (s, 1H), 8.11
(d, J = 7.2 Hz, 1H), 8.01 (t, J = 9.2 Hz, 3H), 7.79–7.65 (m, 5H), 7.52
(t, J = 6.8 Hz, 1H), 7.43 (t, J = 8 Hz, 1H), 3.69 (d, J = 12 Hz, 2H),
2.36 (t, J = 9.6 Hz, 3H), 1.87 (d, J = 10.4 Hz, 2H), 1.69 (q, J = 11.6,
9.2 Hz, 2H).13C NMR (100 MHz, DMSO‑d6): δ 173.0, 167.0, 153.7,
142.0, 135.7, 134.3, 133.3, 129.5, 128.1, 127.5, 126.7, 125.3, 122.7,
122.3, 119.4, 45.4, 41.6, 27.4 ppm. HRMS-ESI+: calculated for
C25H23N3O3S2 + H: 478.1259; Found: 478.1252.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2-fluorophenyl)sulfonyl)pi-
peridine-4-carboxamide, 4–4 was obtained as an off-white solid in the
amount of 51 mg (46% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.22 (s,
1H), 8.11 (d, J = 7.2 Hz, 1H), 8.02 (t, J = 8.8 Hz, 3H), 7.84–7.77 (m,
4H), 7.53, (t, J = 8.4 Hz, 2H), 7.44 (q, J = 9.2, 7.2 Hz, 2H), 3.75 (d,
J = 12.8 Hz, 2H), 2.66 (t, J = 11.6 Hz, 2H), 2.48–2.45 (m, 1H), 1.92
(d, J = 10.4 Hz, 2H), 1.65 (q, J = 11.6, 8.8 Hz, 2H). 13C NMR
(100 MHz, DMSO‑d6): δ 172.9, 166.9, 159.3, 156.9, 153.6, 141.9,
135.9, 135.8, 134.2, 130.8, 127.9, 127.5, 126.5, 125.2, 124.8, 122.5,
122.2, 119.3, 117.6, 117.4, 54.8, 44.8, 41.4, 27.7 ppm. HRMS-ESI+:
calculated for C25H22FN3O3S2 + H: 496.1165; Found: 496.1154.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2-chlorophenyl)sulfonyl)pi-
peridine-4-carboxamide, 4–5 was obtained as an off-white solid in the
amount of 93 mg (83% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.25 (s,
1H), 8.11 (d, J = 7.2 Hz, 1H), 8.02 (t, J = 8.8 Hz, 4H), 7.79 (d,
J = 8.8 Hz, 2H), 7.73 (q, J = 8, 10 Hz, 2H), 7.59–7.50 (m, 2H), 7.43 (t,
J = 8 Hz, 1H), 3.78 (d, J = 12.8 Hz, 2H), 2.84 (t, J = 12.4 Hz, 2H),
2.57–2.52 (m, 1H), 1.89 (d, J = 13.2 Hz, 2H), 1.65 (q, J = 15.6, 12 Hz,
2H). 13C NMR (100 MHz, DMSO‑d6): δ 178.8, 173.0, 166.9, 153.6,
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2-methoxyphenyl)sulfonyl)pi-
peridine-4-carboxamide, 4–8 was obtained as a white solid in the amount
of 61 mg (54% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.23 (s, 1H), 8.11
(d, J = 7.6 Hz, 1H), 8.02 (t, J = 8.4 Hz, 3H), 7.78 (t, J = 8.8 Hz, 3H),
7.65 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.43 (t, J = 7.2 Hz,
1H), 7.27 (d, J = 7.6 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 3.91 (s, 3H), 3.74
(d, J = 10.4 Hz, 2H), 2.68 (t, J = 12 Hz, 2H), 1.87 (d, J = 13.6 Hz, 2H),
1.61 (t, J = 12.4 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 173.1, 166.9,
156.6, 153.6, 142.0, 134.8, 134.2, 130.6, 127.9, 126.5, 126.0, 124.8,
123.4, 122.5, 122.2, 120.2, 119.3, 113.1, 55.9, 44.9, 41.9, 28.1 ppm.
HRMS-ESI+: calculated for C26H25N3O4S2 + H: 508.1365; Found:
508.1354.
12

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Fig. 6B. 2D representation for the lowest energy conformation of inhibitor 4–5 in the binding pocket of human sEH. Green shading represents hydrophobic region;
gray parabolas represent accessible surface for large areas; broken thick line around ligand shape indicates accessible surface; size of residue ellipse represents the
strength of the contact.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((3-fluorophenyl)sulfonyl)pi-
peridine-4-carboxamide, 4–9 was obtained as an off-white solid in the
amount of 79 mg (72% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.17 (s,
1H), 8.12 (d, J = 7.6 Hz, 1H), 8.02 (t, J = 8.8 Hz, 3H), 7.79–7.71 (m,
3H), 7.65–7.62 (m, 3H), 7.53 (t, J = 6.8 Hz, 1H), 7.44 (t, J = 8.0 Hz,
1H), 3.72 (d, J = 12 Hz, 2H), 2.42 (q, J = 12.0, 9.6 Hz, 3H), 1.91 (d,
J = 10.8 Hz, 2H), 1.66 (q, J = 11.6, 9.2 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6): δ 173.0, 167.0, 160.6, 153.6, 142.0, 137.5, 134.3, 131.9,
128.0, 127.5, 126.6, 125.3, 123.7, 122.6, 122.3, 120.5, 119.4, 114.3,
45.3, 41.4, 27.6 ppm. HRMS-ESI+: calculated for C25H22FN3O3S2 + H:
496.1165; Found: 496.1154.
amount of 75 mg (61% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.16 (s,
1H), 8.11 (d, J = 7.2 Hz, 1H), 8.04–7.95 (m, 5H), 7.91 (s, 1H), 7.78 (t,
J = 10.0 Hz, 3H), 7.64 (t, J = 8 Hz, 1H), 7.53 (t, J = 8 Hz, 1H), 7.44 (t,
J = 8 Hz, 1H), 3.71 (d, J = 11.6 Hz, 2H), 2.44 (t, J = 11.6 Hz, 3H),
1.91 (d, J = 11.6 Hz, 2H), 1.67 (t, J = 7.6 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6): δ 172.9, 166.9, 153.6, 136.0, 134.2, 131.6, 129.5, 127.9,
126.5, 122.5, 122.4, 122.2, 119.3, 45.2, 41.3, 27.5 ppm. HRMS-ESI+:
calculated for C25H22BrN3OS2 + H: 556.0364; Found: 556.0356.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-(m-tolylsulfonyl)piperidine-4-
carboxamide, 4–12 was obtained as an off-white solid in the amount of
53 mg (49% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.14 (s, 1H), 8.11
(d, J = 7.2 Hz, 1H), 8.01 (t, J = 8.8 Hz, 3H), 7.76 (d, J = 8.8 Hz, 2H),
7.58 (s, 1H), 7.57–7.50 (m, 4H), 7.43 (t, J = 8 Hz, 1H), 3.67 (d,
J = 12 Hz, 2H), 2.43 (s, 3H), 2.38 – 2.31 (m, 3H) 1.90 (d, J = 10.4 Hz,
2H) 1.70 – 1.62 (m, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 174.5,
172.9, 166.9, 159.0, 153.6, 152.5, 150.8, 150.6, 145.0, 143.3, 141.9,
139.1, 135.4, 134.2, 133.7, 129.1, 127.9, 127.5, 127.4, 126.5, 126.0,
125.2, 124.5,122.5, 122.2, 119.3, 116.7, 115.2, 110.4 ppm. HRMS-ESI
+: calculated for C26H25N3O3S2 + H: 492.1416; Found: 492.1407.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((3-methoxyphenyl)sulfonyl)
piperidine-4-carboxamide, 4–13 was obtained as a white solid in the
amount of 59 mg (47% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.14 (s,
1H), 8.11 (d, J = 8 Hz, 1H), 8.01 (t, J = 8.8 Hz, 3H), 7.76 (d,
J = 8.8 Hz, 2H), 7.58 (t, J = 8.4 Hz, 1H), 7.52 (t, J = 7.2 Hz, 1H),
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((3-chlorophenyl)sulfonyl)pi-
peridine-4-carboxamide, 4–10 was obtained as an off-white solid in the
amount of 81 mg (72% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.61 (s,
1H), 8.11 (d, J = 7.2 Hz, 1H), 8.04 (q, J = 2.4, 8.8 Hz, 2H), 7.84–7.69
(m, 4H), 7.52 (t, J = 7.2 Hz, 1H), 7.43 (t, J = 8 Hz, 1H), 3.71 (d,
J = 12 Hz, 2H), 2.46–2.38 (m, 3H), 1.91 (d, J = 10.8 Hz, 2H),
1.70–1.61 (m, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 172.9, 166.9,
153.6, 141.9, 137.8, 134.2, 134.1, 133.1, 131.5, 127.9, 127.5, 126.8,
126.5, 126.1, 125.2, 122.5, 122.2, 119.4, 99.5, 45.3, 41.4, 27.5 ppm.
HRMS-ESI+: calculated for C25H22ClN3O3S2 + H: 512.0869; Found:
512.0858.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((3-bromophenyl)sulfonyl)pi-
peridine-4-carboxamide, 4–11 was obtained as an off-white solid in the
13

S. Wilt, et al.
BioorganicChemistry103(2020)104165
Table 3
Predicted ADMET properties of synthesized analogs.
Compound Mol. Wt. cLogP cLogS
Drug Likeness Bad Groups # of Rot
# of H-B
Acceptors
# of H-B
Donors
Caco2
Half-Life
(hours)
hERG
Tox Score
Bonds
Inhibition
URB-597
AUDA
4–1
338.407 4.022 −6.168
392.584 6.107 −6.679
483.619 5.317 −6.918
596.957 6.757 −8.513
477.597 5.386 −7.327
495.587 5.535 −7.804
512.039 5.980 −7.900
556.493 6.117 −8.070
491.624 5.667 −7.664
507.623 5.356 −7.277
495.587 5.655 −7.506
512.039 6.100 −8.086
556.493 6.237 −8.273
491.624 5.787 −7.519
507.623 5.476 −7.339
495.587 5.655 −7.844
5112.039 6.100 −8.285
556.493 6.237 −8.476
491.624 5.787 −7.783
507.623 5.476 −7.587
513.577 5.805 −8.010
546.481 6.695 −8.826
635.389 6.967 −9.177
505.651 6.068 −7.953
537.649 5.447 −7.40
513.577 5.925 −7.285
505.651 6.188 −7.451
531.568 5.954 −8.057
580.923 7.289 −9.402
519.678 6.349 −7.788
603.840 8.500 −10.319
567.549 6.013 −9.372
1.008
−0.016
0.584
0.740
0.553
0.541
0.644
0.396
0.428
0.382
0.595
0.658
0.409
0.379
0.625
0.911
1.013
0.722
0.498
0.817
0.511
0.6212
0.362
−0.018
0.385
0.662
0.719
0.943
0.9648
0.772
1.060
0.738
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
15
6
6
6
6
6
6
6
7
6
6
6
6
7
6
6
6
6
7
6
6
6
6
8
6
6
6
6
6
9
6
5
5
8
8
8
8
8
8
8
9
8
8
8
8
9
8
8
8
8
9
8
8
8
8
10
8
8
8
8
8
8
8
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
−4.985
−4.760
−5.12
0.85
2.838
2.05
1.213
2.873
3.20
3.205
3.21
3.71
4.47
7.20
7.44
7.44
7.26
5.78
3.23
3.23
3.24
3.46
4.20
5.28
5.28
5.29
5.87
6.69
5.56
5.00
0.714
0.601
0.106
0.106
0.149
0.255
0.182
0.106
0.182
0.182
0.255
0.182
0.106
0.182
0.182
0.255
0.182
0.106
0.182
0.182
0.357
0.182
0.106
0.274
0.371
0.357
0.274
0.310
0.310
0.255
0.342
0.342
2
0.878
0
4–2
−5.348
−5.055
−5.252
−5.180
−5.276
−5.216
−5.217
−5.289
−5.231
−5.319
−5.289
−5.225
−5.298
−5.141
−5.270
−5.237
−5.195
−5.343
−5.346
−5.397
−5.347
−5.262
−5.370
−5.382
0
4–3
0
4–4
0
4–5
0
4–6
0
4–7
0
4–8
0
4–9
0
4–10
4–11
4–12
4–13
4–14
4–15
4–16
4–17
4–18
4–19
4–20
4–21
4–22
4–23
4–24
4–25
4–26
4–27
4–28
4–29
4–30
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
−5.391 10.10
−5.426 10.10
−5.361 10.10
0
0
0
−5.396
−5.263
3.90
4.45
0
2.408
7.429 (t, J = 7.2 Hz, 1H), 7.31 (q, J = 8 Hz, 6.8 Hz, 2H), 7.22 (s, 1H),
3.85 (s, 3H), 3.69 (d, J = 12 Hz, 2H), 2.38 (t, J = 11.6 Hz, 3H), 1.90 (d,
J = 11.6 Hz, 2H), 1.66 (q, J = 11.6 Hz, 9.6 Hz, 2H). 13C NMR
(100 MHz, DMSO‑d6): δ 172.6, 166.7, 159.9, 153.1, 142.5, 136.9,
134.1, 130.6, 127.9, 127.6, 126.1, 125.7, 122.5, 122.2, 119.3, 188.9,
112.2 55.6, 45.3, 41.4, 27.5 ppm. HRMS-ESI+: calculated for
7.52 (t, J = 8.4 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 3.67 (d, J = 12.0 Hz,
2H), 2.43–2.36 (m, 3H), 1.90 (d, J = 13.6 Hz, 2H), 1.66 (q, J = 15.2,
9.2 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 172.9, 166.9, 153.6,
141.9, 135.0, 134.2, 132.4, 129.3, 127.9, 127.5, 127.1, 126.5, 122.5,
122.2, 119.3, 45.1, 41.3, 27.5 ppm. HRMS-ESI+: calculated for
C25H22BrN3OS2 + H: 556.0364; Found: 556.0355.
C
26H25N3O4S2 + H: 508.1365; Found: 508.1354.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-tosylpiperidine-4-carbox-
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((4-fluorophenyl)sulfonyl)pi-
amide, 4–17 was obtained as an off-white solid in the amount of 73 mg
(68% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.14 (s, 1H), 8.11 (d,
J = 8 Hz, 1H), 8.01 (t, J = 8.8 Hz, 3H), 7.76 (d, J = 9.2 Hz, 2H), 7.65
(d, J = 8 Hz, 2H), 7.52 (t, J = 6.8 Hz, 1H), 7.44 (q, J = 8.2 Hz, 3H),
3.65 (d, J = 12 Hz, 2H), 2.42 (s, 3H), 2.31 (q, J = 11.6, 9.6 Hz, 3H),
1.89 (d, J = 10.4 Hz, 2H), 1.66 (q, J = 11.2, 9.6 Hz, 2H). 13C NMR
(100 MHz, DMSO‑d6: δ 173.0, 166.9, 153.6, 143.4, 141.9, 134.2, 132.6,
129.8, 127.9, 127.4, 127.4, 126.5, 125.2, 122.2, 119.3 ppm. HRMS-ESI
+: calculated for C26H25N3O3S2 + H: 492.1416; Found: 492.1409.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((4-methoxyphenyl)sulfonyl)
piperidine-4-carboxamide, 4–18 was obtained as a white solid in the
amount of 54 mg (48% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.17 (s,
1H), 8.11 (d, J = 8 Hz, 1H), 8.01 (t, J = 8.8 Hz, 3H), 7.7 (d,
J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.52 (t, J = 7.2 Hz, 1H),
7.43 (t, J = 7.2 Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H), 3.64 (d,
J = 12 Hz, 2H), 2.38–2.28 (m, 3H), 1.89 (d, J = 10.8 Hz, 2H), 1.66 (q,
J = 11.2 Hz, 9.6 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 173.0,
166.9, 162.6, 153.6, 141.9, 134.2, 129.6, 127.9, 122.5, 122.2, 119.3,
114.5, 55.7, 45.2, 41.4, 27.5 ppm. HRMS-ESI+: calculated for
peridine-4-carboxamide, 4–14 was obtained as an off-white solid in the
amount of 67 mg (61% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.17 (s,
1H), 8.12 (d, J = 7.2 Hz, 1H), 8.02 (t, J = 8.8 Hz, 3H), 7.86 (q,
J = 5.2, 4.0 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.51–7.49 (m, 3H), 7.44
(t, J = 8.0 Hz, 1H), 3.69 (d, J = 11.6 Hz, 2H), 2.39 (t, J = 9.6 Hz, 3H),
1.91 (d, J = 10.8 Hz, 2H), 1.67 (q, J = 11.6, 9.2 Hz, 2H). 13C NMR
(100 MHz, DMSO‑d6): δ 172.9, 166.9, 163.2, 153.6, 141.9, 134.2,
132.0, 130.5, 130.4, 127.9, 127.5, 126.5, 125.2, 122.5, 122.2, 119.3,
116.7, 116.4, 45.2, 41.4, 27.5 ppm. HRMS-ESI+: calculated for
C
25H22FN3O3S2 + H: 496.1165; Found: 496.1154.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((4-chlorophenyl)sulfonyl)pi-
peridine-4-carboxamide, 4–15 was obtained as an off-white solid in the
amount of 84 mg (75% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.19 (s,
1H), 8.12 (d, J = 7.6 Hz, 1H), 8.02 (t, J = 8.8 Hz, 3H), 7.79 (q,
J = 9.2, 12, Hz, 6H), 7.53 (t, J = 7.2 Hz, 1H), 7.43 (t, J = 8 Hz, 2H),
3.68 (d, J = 12 Hz, 2H), 2.42 (q, J = 12, 9.2 Hz, 3H), 1.91 (d,
J = 11.2 Hz, 2H), 1.69 (q, J = 11.6, 9.2 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6): δ 173.3, 167.5, 138.5, 133.9, 129.6, 129.3, 127.9, 122.6,
122.3, 119.4, 45.2, 41.8, 27.5 ppm. HRMS-ESI+: calculated for
C
26H25N3O4S2 + H: 508.1365; Found: 508.1356.
C
25H22ClN3O3S2 + H: 512.0869; Found: 512.0858.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2,4-difluorophenyl)sul-
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((4-bromophenyl)sulfonyl)pi-
fonyl)piperidine-4-carboxamide, 4–19 was obtained as an off-white
solid in the amount of 28 mg (25% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.22 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.03 (t, J = 8.8 Hz,
3H), 7.90 (q, J = 8.4, 6.4 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.64 (t,
J = 10.8 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H),
peridine-4-carboxamide, 4–16 was obtained as an off-white solid in the
amount of 38 mg (31% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.17 (s,
1H), 8.11 (d, J = 76 Hz, 1H), 8.01 (t, J = 8.8 Hz, 3H), 7.88 (d,
J = 8.8 Hz ,2H), 7.77 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H),
14

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BioorganicChemistry103(2020)104165
7.36 (t, J = 8.0 Hz, 1H), 3.74 (d, J = 12.0 Hz, 2H), 2.68 (t,
J = 12.0 Hz, 2H), 1.92 (d, J = 10.8 Hz, 2H), 1.65 (q, J = 11.6, 8.8 Hz,
2H). 13C NMR (100 MHz, DMSO‑d6): δ 172.9, 166.9, 153.6, 141.9,
134.2, 126.5, 125.2, 122.5, 122.2, 119.3, 54.8, 44.7, 41.5, 27.7 ppm.
HRMS-ESI+: calculated for C25H21F2N3O3S2 + H: 514.1071; Found:
514.1065.
sulfonyl)piperidine-4-carboxamide, 4–25 was obtained as an off-white
solid in the amount of 70 mg (63% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.13 (s, 1H), 8.12 (d, J = 7.2 Hz, 1H), 8.01 (t, J = 8.8 Hz,
3H), 7.76 (d, J = 9.2 Hz, 2H), 7.52 (t, J = 7.2 Hz, 1H), 7.43 (t,
J = 8 Hz, 1H), 7.36 (d, J = 6 Hz, 3H), 3.66 (d, J = 11.6 Hz, 2H), 2.38
(s, 6H), 2.33 (t, J = 10 Hz, 3H), 1.90 (d, J = 10.8 Hz, 2H), 1.66 (q,
J = 15.6, 9.2 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 173.0, 166.9,
153.6, 141.9, 138.9, 135.2, 134.4, 134.2, 127.9, 127.4, 126.5, 125.2,
124.8, 122.5, 122.2, 119.3, 45.3, 41.5, 27.5, 20.7 ppm. HRMS-ESI+:
calculated for C27H27N3O5S2 + H: 538.1470; Found: 538.1462.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2,4,6-trifluorophenyl)sul-
fonyl)piperidine-4-carboxamide, 4–26 was obtained as an off-white
solid in the amount of 19 mg (16% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.24 (s, 1H), 8.12 (d, J = 7.2 Hz, 1H), 8.03 (t, J = 8.8 Hz,
3H), 7.79 (d, J = 8.8 Hz, 2H), 7.54–7.50 (m, 3H), 7.43 (t, J = 8.0 Hz,
1H), 3.77 (d, J = 12.4 Hz, 2H), 2.77 (t, J = 11.2 Hz, 2H), 1.95 (d,
J = 10.4 Hz, 2H), 1.67 (q, J = 11.6, 9.2 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6): δ 172.9, 166.9, 153.6, 141.9, 134.2, 127.9, 127.5, 126.5,
125.2, 122.5, 122.2, 119.4, 44.5, 41.3, 27.6 ppm. HRMS-ESI+: calcu-
lated for C25H22F3N3O3S2 + H: 532.0976; Found: 532.0968.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2,4-dichlorophenyl)sul-
fonyl)piperidine-4-carboxamide, 4–20 was obtained as an off-white
solid in the amount of 58 mg (48% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.27 (s, 1H), 8.11 (d, J = 7.2 Hz, 1H), 8.05–7.94 (m, 4H),
7.81 (s, 1H), 7.79 (d, J = 9.2 Hz, 2H), 7.67 (d, J = 10.8 Hz, 1H), 7.52
(t, J = 7.2 Hz, 1H), 7.43 (t, J = 8 Hz, 1H), 3.77 (d, J = 12.8 Hz, 2H),
2.86 (t, J = 12.6 Hz, 2H), 2.54 (t, J = 11.6 Hz, 1H), 1.89 (d,
J = 10.8 Hz, 2H), 1.65 (q, J = 12, 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6): δ 173.0, 166.9, 153.6, 141.9, 138.4, 134.2, 132.9, 132.2,
131.7, 127.9, 127.5, 126.6, 125.2, 122.6, 122.2, 119.4, 44.7, 41.6,
27.9 ppm. HRMS-ESI+: calculated for
546.0480; Found: 546.0476.
C25H21Cl2N3O3S2 + H:
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2,4-dibromophenyl)sul-
fonyl)piperidine-4-carboxamide, 4–21 was obtained as an off-white
solid in the amount of 39 mg (28% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.26 (s, 1H), 8.19 (s,1H), 8.12 (d, J = 7.6 Hz, 1H), 8.04
(d, J = 7.6 Hz, 3H), 7.95 (d, J = 8.4 Hz, 1H), 7.85–7.79 (m, 3H), 7.53
(t, J = 7.2 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 3.75 (d, J = 11.2 Hz, 2H),
2.88 (t, J = 12.0 Hz, 2H), 2.58–2.55 (m, 1H), 1.89 (d, J = 13.2 Hz,
2H), 1.64 (q, J = 10.8, 12.0 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6): δ
173.0, 166.9, 153.6, 141.8, 137.6, 137.0, 134.2, 133.0, 131.3, 127.9,
127.1, 126.5, 125.0, 122.5, 122.2, 120.7, 119.3, 44.6, 41.6, 27.9 ppm.
HRMS-ESI+: calculated for C25H21Br2N3O3S2 + H: 633.9469; Found:
633.9461.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2,4,6-trichlorophenyl)sul-
fonyl)piperidine-4-carboxamide, 4–27 was obtained as an off-white solid
in the amount of 65 mg (51% yield): 1H NMR (400 MHz, DMSO‑d6) δ
10.28 (s, 1H), 8.11 (d, J = 8 Hz, 1H), 8.03 (t, J = 8.8 Hz, 3H), 7.93 (s,
2H), 7.79 (d, J = 8.8 Hz, 2H), 7.52 (t, J = 8.8 Hz, 1H), 7.45 (t, J = 8 Hz,
1H), 3.84 (d, J = 13.2 Hz, 2H), 2.98 (t, J = 12 Hz, 2H), 2.58 (t,
J = 11.2 Hz, 1H), 1.91 (d, J = 11.2 Hz, 2H), 1.61 (t, J = 9.2 Hz, 2H).
13C NMR (100 MHz, DMSO‑d6): δ 172.9, 166.9, 153.6, 141.9, 135.4,
134.1, 131.5, 129.6, 126.6, 122.5, 122.2, 119.4, 45.8, 44.6, 41.7,
27.9 ppm. HRMS-ESI+: calculated for C25H20Cl3N3O3S2 + H: 580.0090;
Found: 580.0081.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2,4-dimethylphenyl)sul-
fonyl)piperidine-4-carboxamide, 4–22 was obtained as an off-white
solid in the amount of 83 mg (75% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.22 (s, 1H), 8.11 (d, J = 7.2 Hz, 1H), 8.02 (t, J = 8.8 Hz,
3H), 7.78 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8 Hz, 1H), 7.52 (t,
J = 6.8 Hz, 1H) 7.43 (t, J = 8 Hz, 1H), 7.24 (t, J = 11.2 Hz, 1H), 3.63
(d, J = 12.4 Hz, 2H), 2.66 (t, J = 12 Hz, 2H), 2.53 (s, 3H), 2.35 (s, 3H),
1.89 (d, J = 10.4 Hz, 2H), 1.62 (q, J = 12, 9.2 Hz, 2H). 13C NMR
(100 MHz, DMSO‑d6): δ 173.0, 166.9, 153.6, 143.3, 141.9, 137.0,
134.2, 133.4, 132.7, 129.8, 127.9, 127.4, 126.8, 126.5, 125.2, 122.5,
122.2, 119.3, 44.2, 41.8, 27.8, 20.7, 20.0 ppm. HRMS-ESI+: calculated
for C27H27N3O3S2 + H: 506.1572; Found: 506.1566.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-(mesitylsulfonyl)piperidine-4-
carboxamide, 4–28 was obtained as an off-white solid in the amount of
72 mg (63% yield): 1H NMR (400 MHz, DMSO‑d6) δ 10.29 (s, 1H), 8.11
(d, J = 7.2 Hz, 1H), 8.02 (t, J = 8.8 Hz, 3H), 7.79 (d, J = 8.8 Hz, 2H),
7.52 (t, J = 6.8 Hz, 1H), 7.43 (t, J = 8.4 Hz, 1H), 7.09 (s, 2H), 3.52 (d,
J = 12.8 Hz, 2H), 2.77 (t, J = 12.4 Hz, 2H), 2.56 (s, 6H), 2.28 (s, 3H),
1.88 (d, J = 10.8 Hz, 2H), 1.58 (q, J = 16, 9.2 Hz, 2H). 13C NMR
(100 MHz, DMSO‑d6): δ 173.3, 167.1, 153.8, 142.6, 142.2, 139.8,
136.0, 134.4, 132.0, 131.8, 130.0, 128.1, 127.7, 126.7, 125.4, 122.7,
122.4, 119.5, 43.4, 42.3, 27.8, 22.9, 22.5, 20.6 ppm. HRMS-ESI+:
calculated for C28H29N3O3S2 + H: 520.1729; Found: 520.1721.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2,4,6-triisopropylphenyl)
sulfonyl)piperidine-4-carboxamide, 4–29 was obtained as an off-white
solid in the amount of 92 mg (69% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.30 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 8.02 (t, J = 8.8 Hz,
2H), 7.79 (d, J = 8.8 Hz, 2H), 7.52 (t, J = 6.8 Hz, 1H) 7.43 (t,
J = 7.2 Hz, 1H), 7.29 (s, 2H), 4.14–4.07 (m, 2H), 3.53 (d, J = 12 Hz,
2H), 2.97–2.90 (m, 1H), 2.79 (t, J = 10.4 Hz, 2H) 2.56 (t, J = 10.8 Hz,
1H), 1.93 (d, J = 10.8 Hz, 2H), 1.60 (q, J = 11.6, 9.6 Hz, 2H), 1.21 (d,
J = 6.8 Hz, 18H). 13C NMR (100 MHz, DMSO‑d6): 13C NMR (100 MHz,
DMSO‑d6): δ 173.0, 166.9, 154.1, 153.0. 151.0, 142.0, 134.2, 129.8,
127.9, 127.4, 126.5, 125.0, 123.8, 122.5, 122.2, 119.3, 43.1, 41.9,
33.3, 28.7, 27.6, 24.5, 23.3 ppm. HRMS-ESI+: calculated for
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((2,4-dimethoxyphenyl)sul-
fonyl)piperidine-4-carboxamide, 4–23 was obtained as an off-white
solid in the amount of 73 mg (62% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.22 (s, 1H), 8.11 (d, J = 8 Hz, 1H), 8.02 (t, J = 8.8 Hz,
3H), 7.79 (d, J = 8.8 Hz, 2H), 7.67, (d, J = 8.4 Hz, 1H), 7.52 (t,
J = 8 Hz, 1H), 7.43 (t, J = 8 Hz, 1H), 6.75 (s, 1H), 6.66 (d, J = 8.8 Hz,
1H), 3.87 (d, J = 11.6 Hz, 3H), 3.69 (d, J = 10 Hz, 2H), 2.62 (t,
J = 10.4, 2H), 2.44 (d, J = 11.2, 1H), 1.86 (d, J = 10.8, 2H), 1.60 (q,
J = 11.6 Hz, 9.2 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 173.4,
167.1, 164.5, 158.4, 153.8, 142.2, 134.4, 132.7, 128.1, 127.6, 126.7,
122.7, 122.4, 119.5, 118.2, 105.2, 99.7, 56.2, 55.9, 45.1, 42.2,
28.3 ppm. HRMS-ESI+: calculated for C27H27N3O5S2 + H: 538.1470;
Found: 538.1462.
C
34H41N3O3S2 + H: 604.2668; Found: 604.2658.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((3,5-difluorophenyl)sul-
fonyl)piperidine-4-carboxamide, 4–24 was obtained as an off-white
solid in the amount of 60 mg (53% yield): 1H NMR (400 MHz,
DMSO‑d6) δ 10.19 (s, 1H), 8.12 (d, J = 7.2 Hz, 1H), 8.03 (t, J = 8.8 Hz,
3H), 7.80–7.71 (m, 3H), 7.57–7.52 (m, 3H), 7.44 (t, J = 8.0 Hz, 1H),
3.75 (d, J = 12.4 Hz, 2H), 2.55–2.41 (m, 4H), 1.92 (d, J = 10.4 Hz,
2H), 1.67 (q, J = 11.6, 9.2 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6): δ
172.9, 166.9, 153.6, 141.9, 134.2, 127.9, 127.5, 126.5, 125.2, 122.5,
122.2, 119.3, 111.3, 111.0, 45.2, 41.3, 27.5 ppm. HRMS-ESI+: calcu-
lated for C25H21F2N3O3S2 + H: 514.1071; Found: 514.1064.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((3,5-dimethylphenyl)
N-(4-(benzo[d]thiazol-2-yl)phenyl)-1-((perfluorophenyl)sulfonyl)
piperidine-4-carboxamide, 4–30 was obtained as an off-white solid in
the amount of 95 mg (76% yield): 1H NMR (400 MHz, DMSO‑d6) δ
10.26 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.03 (t, J = 8.8 Hz, 3H), 7.80
(d, J = 8.8 Hz, 2H), 7.53 (t, J = 6.8 Hz, 1H), 7.44 (t, J = 6.8 Hz, 1H),
3.78 (d, J = 11.6 Hz, 2H), 2.85 (t, J = 11.2 Hz, 2H), 1.99 (d,
J = 10.8 Hz, 2H), 1.71 (q, J = 12.0, 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6): δ 172.8, 166.9, 153.6, 141.9, 134.2, 127.9, 127.5, 126.5,
122.5, 122.2, 119.4, 44.5, 41.2, 27.6 ppm. HRMS-ESI+: calculated for
C25H18F5N3O3S2 + H: 568.0788; Found: 568.0780.
15

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BioorganicChemistry103(2020)104165
5.2. sEH and FAAH IC50 assay conditions
tolerability and acceptability of oxycodone for cancer-related pain in adults: an
updated Cochrane systematic review, BMJ Support. Palliat. Care 8 (2) (2018)
117–128.
Measurement of sEH potency was performed using cyano(2-meth-
oxynaphthalen-6-yl)methyl trans-(3-phenyloxyran-2-yl) methyl carbo-
nate (CMNPC) as the fluorescent substrate [56]. Human sEH (1 nM) was
incubated with the inhibitor for 5 min in pH 7.0 Bis–Tris/HCl buffer
(25 mM) containing 0.1 mg/mL of bovine serum albumin (BSA) at 30 °C
prior to substrate introduction ([S] = 5 μM). Activity was determined
by monitoring the appearance of 6-methoxy-2-naphthaldehyde over
10 min by fluorescence detection with an excitation wavelength of
330 nm and an emission wavelength of 465 nm. Reported IC50 values
are the average of the three replicates with at least two data points
above and at least two below the IC50. Measurement of FAAH potency
was performed using the substrate N-(6-methoxypyridin-3-yl) octana-
mide (OMP) ([S]final = 50 μM) in sodium phosphate buffer (0.1 M,
pH = 8, 0.1 mg/mL BSA). Progress of the reaction was measured by
fluorescence detection of 6-methoxypyridin-3-amine at an excitation
wavelength of 303 nm and an emission wavelength of 394 nm at 37 °C
by the use of microplate reader (Molecular Devices., CA, USA). All
experiments were run in triplicate, and values reported as average +/-
SD. The substrate OMP was synthesized following a previously reported
synthetic procedure and reaction conditions [36].
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For the docking studies of the dual sEH/FAAH inhibitors, a crystal
structure of human soluble epoxide hydrolase complexed with N-cy-
cloheptyl-1-(mesitylsulfonyl)piperidine-4-carboxamide (PDBfile: 4HAI)
[33] and a homology model of human FAAH enzyme [36] were used.
PDB file 4HAI was first converted to an ICM file and the inhibitor N-
cycloheptyl-1-(mesitylsulfonyl) piperidine-4-carboxamide was re-
moved. Docking experiments were performed following the program
guidelines. ICM scores were obtained after this procedure. ADMET
properties for all synthesized target analogs were calculated using the
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Research reported in this publication was supported by the National
Institute of General Medical Sciences of the National Institutes of Health
under Award Number SC2GM135020 and, in part, by a grant from the
National Institute of Environmental Health Sciences (NIEHS) Grant
R35ES030443, and NIEHS Superfund Research Program P42 ES004699.
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of
Health. Instrumentation support was provided by the National Science
Foundation MRI (CHE1726903) for acquisition of an UPLC-MS.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
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