Discovery of memantyl urea derivatives as potent soluble epoxide hydrolase inhibitors against lipopolysaccharide-induced sepsis

Article abstract of DOI:10.1016/j.ejmech.2021.113678

Sepsis, a systemic inflammatory response, caused by pathogenic factors including microorganisms, has high mortality and limited therapeutic approaches. Herein, a new soluble epoxide hydrolase (sEH) inhibitor series comprising a phenyl ring connected to a

Full text of DOI:10.1016/j.ejmech.2021.113678

European Journal of Medicinal Chemistry 223 (2021) 113678
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of memantyl urea derivatives as potent soluble epoxide
hydrolase inhibitors against lipopolysaccharide-induced sepsis
,
c
Fangyu Du a, Wenjiao Sun a, Christophe Morisseau b, Bruce D. Hammock b, Xuefei Bao a
,
,
**
Qiu Liu c, Chao Wang c, Tan Zhang c, Hao Yang c, Jun Zhou c, Wei Xiao c
,
,
, *
Zhongbo Liu d ***, Guoliang Chen a
a Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenyang, Liaoning, 110016, China
b Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California Davis, One Shields Avenue, Davis, CA,
95616, USA
c Jiangsu Kanion Pharmaceutical Co., Ltd., Jiangning Industrial City, Economic and Technological Development Zone, Lianyungang, Jiangsu, 222001, China
d School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning, 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Sepsis, a systemic inflammatory response, caused by pathogenic factors including microorganisms, has
high mortality and limited therapeutic approaches. Herein, a new soluble epoxide hydrolase (sEH) in-
hibitor series comprising a phenyl ring connected to a memantyl moiety via a urea or amide linkage has
been designed. A preferential urea pharmacophore that improved the binding properties of the com-
pounds was identified for those series via biochemical assay in vitro and in vivo studies. Molecular
docking displayed that 3,5-dimethyl on the adamantyl group in B401 could make van der Waals in-
teractions with residues at a hydrophobic pocket of sEH active site, which might indirectly explain the
subnanomolar level activities of memantyl urea derivatives in vitro better than AR-9281. Among them,
compound B401 significantly improved the inhibition potency with human and murine sEH IC50 values
as 0.4 nM and 0.5 nM, respectively. Although the median survival time of C57BL/6 mice in LPS-induced
sepsis model was slightly increased, the survival rate did not reach significant efficacy. Based on safety
profile, metabolic stability, pharmacokinetic and in vivo efficacy, B401 demonstrated the proof of po-
tential for this class of memantyl urea-based sEH inhibitors as therapeutic agents in sepsis.
© 2021 Elsevier Masson SAS. All rights reserved.
Received 29 January 2021
Received in revised form
18 June 2021
Accepted 25 June 2021
Available online 29 June 2021
Keywords:
Soluble epoxide hydrolase
Sepsis
Memantine derivatives
Cytokine storm
1. Introduction
was around $20 billion, which caused serious economic and psy-
chological burden to patients [3]. The emerging of numerous pro-
Sepsis, also called cytokine storm or inflammation storm, a
systemic inflammatory response caused by pathogenic factors
including microorganisms, has high mortality and limited thera-
peutic approaches [1]. Epidemiological survey shows that global
estimates of 31.5 million sepsis patients and 19.4 million severe
sepsis cases and with potentially 5.3 million deaths every year [2].
Furthermore, the annual cost of hospital healthcare for severe
sepsis patients in the U.S. is the highest among other diseases and
inflammatory cytokines in the progression of sepsis, has been
identified [4]. Emerging evidence demonstrates that modulation of
the innate immune response is an effective strategy to fight against
infection caused by pathogenic microorganisms [5].
Coronavirus disease 2019 (COVID-19), caused by severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), characterized
by systemic hyperinflammation associated with a life-threatening
“cytokine storm” [6]. The pro-inflammatory cytokines levels
including TNF-
a, IL-6, IL-1b, IL-1, IL-8, G-CSF, MCP-1, IP-10, and MIP-
1
a
in serum for patients with COVID-19 was significantly increased
[7,8]. sEH inhibitors are effective not only in resolving pulmonary
inflammation, but also in preventing pulmonary fibrosis [9e11],
key hallmarks of COVID-19 that contribute to life-threatening
complications such as acute respiratory distress syndrome
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: xw_kanion@163.com (W. Xiao), 546265581@qq.com (Z. Liu),
chenguoliang@syphu.edu.cn (G. Chen).
https://doi.org/10.1016/j.ejmech.2021.113678
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
(ARDS). It is reported that widely available corticosteroid such as
dexamethasone can be applying for the treatment of cytokine
storm, but corticosteroids only can be administered in lower dosage
and short time because of their serious adverse effects [12,13].
Hence, there is an urgent need for new medicines for the treatment
of sepsis in clinic.
potential for this class of memantyl urea-based sEH inhibitors as
therapeutic agents in sepsis.
2. Results and discussion
2.1. Design compounds
Arachidonic acid (ARA), a 20-carbon polyunsaturated fatty acid
(PUFA) derived from cell membrane phospholipid, which is largely
biosynthesized into proinflammatory mediators by cyclooxygenase
(COX) and lipoxygenase (LOX) pathways [14]. Both pathways have
been pharmaceutically targeted, affording several pharmaceutical
compounds (e.g., nonsteroidal anti-inflammatory drugs aspirin and
blocking production with leukotriene inhibitor zileuton). Moreover,
the ARA converted by CYP450 enzymes into epoxide metabolites
epoxy-fatty acids (EpFAs) such as epoxyeicosatrienoic acids (EETs)
which include regioisomers 5,6-EET, 8,9-EET, 11,12-EET and 14,15-
EET [15]. Among them, 11,12-EET prevents the tumor necrosis fac-
Our internal sEH inhibitor research program sought to identify
small molecules that block the enzyme's catalytic function and
therefore inhibit the formation of inactive or even proinflammatory
diols. AR-9281 demonstrated impressive clinical response and has
progressed to phase II trials. From the perspective of structure, it
contained an amantadine group, leading to rapid cytochrome P450
based metabolism, and thus it has a short in vivo half-life [22].
Previous research revealed that piperine and piperlonguminine
from Scutellaria baicalensis Georgi displayed significant inhibitory
activity against sEH in vitro, with the IC50 values at micromolar
concentration [28,29]. On the other hand, comparing with aman-
tadine (antiviral drugs), memantine (anti-Alzheimer disease), has
good pharmacokinetic properties, with half-life (t1/2) as 60 h [30].
Therefore, we supposed that introduction of memantyl group could
increase half-life of target compounds. Meanwhile, we hypothe-
sized that the introduction of 3,5-dimethyl at adamantly group
should increase hydrophobic interactions with sEH and afford some
memantine derivatives. Taking into account for chemical character
of memantine, and excellent inhibitory effects of memantyl urea
derivatives, we replaced memantyl group with hydroxyl
substituted adamantyl to obtain some hydroxyl substituted ada-
mantyl urea derivatives as potent sEH inhibitors (Scheme 1).
Although the adamantly moiety of reported SAR of sEH inhibitors
should be lipophilicity, molecular docking displayed OH-
substituted adamantly could make extra hydrogen bond with
Gln384 of sEH protein (Fig. S1), which maybe further increase
inhibitory effect on sEH.
tor (TNF)-a induced activation of NF-kB and the subsequent in-
creases in vascular cell adhesion molecule (VCAM)-1 expression in
mice [16]. 14,15-EET also shows strongly anti-inflammatory effect
[17]. EETs turn down production of inflammatory eicosanoids and
cytokines through activating endoplasmic reticulum (ER) stress
pathways, moving the ER stress response back toward maintaining
homeostasis and inflammatory resolution [18]. However, the anti-
inflammatory EETs are rapidly metabolized by the soluble
epoxide hydrolase (sEH) to inactive or even proinflammatory diols
[19]. Inhibition of sEH could stabilize and elevate EETs level, which
have anti-inflammatory, anti-hypertensive, and analgesic proper-
ties, and also stimulates the production of pro-resolving mediators
[20,21].
To date, three sEH inhibitors were moved into human clinical
trials (Fig. 1). AR-9281 by Arete Therapeutics was in phase II clinical
trial for the treatment of hypertension and insulin resistance [22].
GSK-2256294 by GlaxoSmithKline for the treatment of patients
with chronic obstructive pulmonary disease (COPD) was investi-
gated in several phase I clinical trials [23]. EC-5026 by EicOsis for
the management of neuropathic and inflammatory chronic pain
was finished phase Ia clinical trial [24]. These data mean that the
safety, efficacy and toxicological studies of sEH inhibitors have been
substantially demonstrated in early preclinical investigation in vivo
and in vitro as a class and the value of sEH as a target. Based on the
significantly benefits of EETs or sEH inhibitors, we speculated that
sEH is a potential target for the treatment of cytokine storm caused
by SARS-CoV-2 or sepsis [25].
Structure-activity relationship (SAR) studies of sEH inhibitors
revealed that ureas, amides, or carbamates group act as central
primary pharmacophore, and attach to a relatively small group
such as a phenyl, cyclohexyl and adamantyl on one side. On the
other side could accommodate a large polar group termed the
secondary pharmacophore, such as amide, carboxyl, ester, etc.
[26,27] We report the discovery of a series of memantyl urea de-
rivatives as potent sEH inhibitor and the identification of candidate
B401 for in vivo studies in lipopolysaccharide-induced sepsis
model. The safety profile, metabolic stability, pharmacokinetic and
in vivo efficacy associated with B401 demonstrated the proof of
2.2. Chemistry
The compounds in Table 1 were synthesized, as shown in
Schemes 2 to 5. First, compounds A101 to A103 were synthesized in
two steps from commercially available amines through succinic
anhydride (1) acylation reaction. Then, commercially available
memantine was then coupled to the acid intermediates 2e4 under
standard amide coupling conditions (EDCI, HOBt, Et3N, DCM) to
give A101 ꢀ A103 as shown in Scheme 2. Acids 6 to 8 were obtained
using cis-butenedioic anhydride and amines as starting material.
Interestingly, acid 6 was a cis-isomer, and trans-isomer product was
not observed. The final compounds A201 ꢀ A203 were obtained
through an amide coupling condition reaction. The synthesis of
A301 ꢀ A303 began with the use of glutaric anhydride to transform
the acid compounds 10e12, then amide coupling to provide the
tittle compounds.
As described in Scheme 3, the synthesis of compounds
A401
ꢀ
A403 began with the halogenation reaction of 4-
(methoxycarbonyl)benzoic acid (13) with thionyl chloride at 80 ꢁC.
The resulting methyl 4-(chlorocarbonyl)benzoate (14) was reacted
Fig. 1. Chemical structures of sEH inhibitors that moved into clinical trials.
2

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
Scheme 1. Schematic diagram depicting the procedure for the design of the target compounds.
with memantine to yield the ester 15. Cleavage of the methyl ester
moiety with LiOH at rt gave the free acid 16, which coupled to the
corresponding amines under standard amide coupling conditions
to give A401 ꢀ A403.
in a similar mode as B401, as shown in Fig. 2D. Both compounds
bind to sEH's active site primarily through hydrogen bond in-
teractions by the urea moiety of the compounds (Fig. 2C).
Commercially available rimantadine was respectively coupled to
the acid intermediates 4 or 12 under standard amide coupling
conditions (EDCI, HOBt, Et3N, DCM) to give C103 or C303 as shown
in Scheme 4. Compounds D303 and E303 were prepared via amide
coupling reaction using 5-(isobutylamino)-5-oxopentanoic acid
2.4. sEH inhibition and StructureꢀActivity relationship
As the first step for the biological characterization of the amides,
their potency as human sEH (HsEH) and murine sEH (MsEH) in-
hibitors was tested, as shown in Table 1. Compound A302 was
potent inhibitor of the MsEH (IC50 ¼ 8.3 nM), while the compound
was poor inhibitor of the human enzyme (IC50 ¼ 1588.5 nM).
Compounds C103 and E303 showed IC50 values on the human and
murine sEH at nanomolar concentration (41.6 ꢀ 106.4 nM). Com-
pounds featured by an amide moiety and attaching to memantyl
were moderate inhibitors of the HsEH and MsEH at micromolar
range. Different spacers (two-, three-carbon, and phenyl group)
were used to evaluate the appropriate distance between the pri-
mary pharmacophore and the secondary pharmacophore. Results
showed that the length of the spacer seem not to affect the
inhibitory activity. For this reason, this set of derivatives was not
further evaluated.
(12),
2-aminoadamantane
(18)
and
3-amino-1-
hydroxyadamantane (19) as starting material.
Scheme 5 shows the synthetic pathways toward urea-based
inhibitors. Acylation of amines with 4-nitrobenzoyl chloride 20
followed by the hydrogenation reduction of the nitro group in
compounds 21e23 yielded the amino intermediates 24e26. Then,
the treatment with phenyl chloroformate in DCM yielded the key
intermediates of carbamate 27e29 that was sufficiently stable to be
isolated. Aminolysis of carbamates 27e29 with various ada-
mantane derivatives (for example, memantine, rimantadine, 2-
adamantanamine, trans-4-aminoadamantan-1-ol, 3-amino-1-
adamantanol) afforded the urea-based compounds, as shown in
Scheme 5.
Taking into consideration of the availability of inhibitors with a
urea as primary pharmacophore [24], then we have replaced the
aliphatic amide with selected aniline scaffold, affording three
memantyl urea-based inhibitors. Encouragingly, the in vitro
screening process shown in Table 2 revealed three compounds
(B401 ꢀ B403) with adequate potency on HsEH and MsEH. The
replacement of the terminal piperidine group with isobutylamine
did not significantly impact the activity (B401 vs B403). While the
replacement of the terminal piperidine with a cyclohexylamine led
to an obvious reduction (almost 6-fold) of the potency on human
sEH (B401 vs B402). Interestingly, compound B402 did not reduce
inhibitory effect on murine sEH with IC50 value of 0.4 nM. Molec-
ular modeling showed that the surrounding of memantyl group has
extra space volume which could accommodate the small sub-
stituents. Therefore, we decided to take rimantadyl moiety, con-
necting to urea group, since they would probe a very different
chemical space compared with the memantyl group. However, the
replacement of the memantyl moiety (B401 ꢀ B403) with riman-
tadyl (C401 ꢀ C403) gave different degrees of decrease in potency
on both targets, suggesting that the position of adamantyl directly
attached to urea group is important (Table 2). Among them, C401
displayed moderate inhibitory potency with IC50 values of 3.9 nM
and 2.4 nM on HsEH and MsEH.
2.3. Molecular docking
We used molecular modeling to predict possible binding mode
of representative inhibitors (B401 and B402) in the binding site of
sEH (PDB code: 3WKE) using BIOVIA Discovery Studio 2016, as
shown in Fig. 2. Generally, urea-group of compounds bound to the
catalytic triad formed by Tyr383, Asp335 and Tyr466. Hydrophobic
memantyl group bounds to the long branch formed by Trp336 and
Gln384. Terminal benzamide moiety bounds to the short branch
formed by Phe267, His524, Asp496 and Phe497. The carbonyl group
of the urea makes a hydrogen bond to Tyr383, and the NH groups of
the urea make two hydrogen bonds to Asp335 (Fig. 2B). The phenyl
ring of B401 binds in a hydrophobic pocket and makes
pꢀcation
interaction with His524. The methyl group at the 3-position of the
memantyl sits in a hydrophobic cavity made up of Leu499 and
Phe381. The methyl group at the 5-position of the memantyl sits in
a hydrophobic pocket formed from the side chains of Met469 and
phenyl ring of Tyr466. The terminal piperidinyl ring is deeply
buried in a hydrophobic pocket and the body of the ring makes van
der Waals contacts to the aliphatic portion of Val498 and His524.
The compound B402 contains a cyclohexylamino moiety and binds
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F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
Table 1
Human (HsEH) and Murine (MsEH) sEH IC50 of memantyl amide-based inhibitors a
.
Comp.
Chemical Structure
HsEH IC50 (nM)
2473.5
MsEH IC50 (nM)
21686.6
cLogPb
2.48
LLEc
3.13
A101
A102
7891.7
179.7
3.21
1.89
A103
A201
18915.9
21514.4
4129.3
2.54
2.87
2.18
1.79
81198.4
A202
A203
A301
24440.6
9624.6
5930.8
5516.1
21120.3
5839.1
3.61
2.94
2.93
1.00
2.07
2.29
A302
A303
1588.5
6225.8
8.3
3.67
3.00
2.13
2.21
224.8
A401
A402
26388.3
6287.8
60713.1
1021.8
3.92
4.66
0.65
0.54
A403
C303
C103
>100000
7322.2
50.8
6538.0
693.7
43.5
3.99
2.84
3.30
e
4.45
1.84
D303
E303
3581.8
41.6
2273.5
106.4
2.70
1.49
2.74
5.89
AR-9281
13.8
1.7
1.30
6.57
a
IC50 values are recorded on recombinant human sEH protein (HsEH) and murine sEH (MsEH) protein using PHOME as substrate at 50
The cLogP values were predicted by BIOVIA Discovery Studio 2016.
Ligand lipophilicity efficiency (LLE) ¼ pIC50-cLogP [32].
mM concentration [31]..
b
c
Meanwhile, the substitution of 1-adamantyl group with 2-
in vitro activity. This change resulted in less active compounds in
terms of the submicromolar potency. This effect might be attrib-
uted to an increased hydrophilic rather than an increased steric
hindrance. Indeed, hydroxyl group at C3 position of adamantyl
group (F401 ꢀ F403) showed similar inhibitory activity. Although
molecular docking displayed OH-substituted adamantly could
make extra hydrogen bond with Gln384 of sEH protein, regrettably,
adamantyl group did not alter the inhibition properties
(D401 ꢀ D403) with the IC50 values of subnanomolar levels, sug-
gesting that the position is not critical for interaction with the sEH
protein. We next introduced a hydroxyl group at C5 position
(E401 ꢀ E403) on the 2-adamantyl group with the expectation that
lowering the compound lipophilicity should favorably impact the
4

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
Scheme 2. Syntheses compounds A101 ꢀ A303a.
aReactions and conditions: i) corresponding amines, DCM, 0 ꢁC to room temperature (rt); ii) memantine hydrochloride, EDCI, HOBt, Et3N, DCM, rt; iii) piperidine, DCM, 0 ꢁC to rt; iv)
memantine hydrochloride, EDCI, HOBt, Et3N, DCM, rt.
Scheme 3. Syntheses compounds A401 ꢀ A403a.
aReactions and conditions: i) DMF, SOCl2, reflux; ii) memantine hydrochloride, DMAP, Et3N, DCM, 0 ꢁC to rt; iii) (1) LiOH H2O, THF, H2O, rt; (2) HCl aqueous; iv) corresponding
▪
amines, EDCI, HOBt, Et3N, DCM, rt.
Scheme 4. Syntheses compounds C103, C303, D303 and E303.
the inhibitory effect of these compounds didn't improve, and only
retain some potency.
2.5. In vivo safety
In addition, the cLogP values of all compounds were predicted
by BIOVIA Discovery Studio 2016. In particular, a further calculation
was conducted, affording ligand lipophilicity efficiency (LLE). Data
in Table 2 showed that adding the hydroxyl group function at
adamantane group not only significantly reduced inhibitory activity
on both sEH protein but also reduced cLogP and LLE. By attaching
memantyl or adamantyl group, cLogP values for the derivatives
(especially B and C series compounds) are higher than memantine
(cLogP ¼ 3.82). The BBB penetration property of the representative
compound B401 was predicted by SwissADME [33]. The results
revealed that those compounds showed favorable BBB perme-
ability; therefore, the application of these compounds may be
expanded to sepsis-associated encephalopathy which also could be
related with sEH reactivity. Taken together, compounds B401, B402,
B403, D401, D402 and D403 showed most promise as they dis-
played the highest LLE [34], which were selected for further studies.
In order to determine the potential toxicological effect, acute
toxicity tests were carried out in ICR (Institute of Cancer Research)
mice (4 males, 4 females). The compounds were administered by
intraperitoneal injection at single dose of 100 mg/kg, and advent
events and symptoms were continuously observed for 14 days. No
adverse effects were observed based on body weight as well as the
absence of apparent behavior problems in compounds B402 and
B403 groups, indicating that the two compounds are well tolerated
(Supporting information Table S1, S2 and S3). Compared with the
vehicle group, the average body weight gain of D401and D403
groups were reduced. In addition, abnormal performance was
observed based on eye secretion, indicating compounds B401,
D402, D403 and AR-9281 displayed adverse effects, and fortunately
abnormal performance of eye secretion is reversible (Supporting
information Table S4). However, phase I study demonstrated that
AR-9281 was safe (dosage of 1200 mg/d) and well tolerated in
5

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
Scheme 5. Syntheses of urea-based compoundsa.
aReactions and conditions: i) corresponding amines, Et3N, THF, 0 ꢁC to rt, 6 h; ii) H2, 5%PdeC, EtOH, 50 ꢁC, 12 h; iii) K2CO3, phenyl chloroformate, DCM, 0 ꢁC to rt, 7 h; iv) memantine,
Et3N, THF, reflux, 8 h; v) rimantadine, Et3N, THF, reflux, 8 h; vi) 2-adamantanamine, Et3N, THF, reflux, 8 h; vii) trans-4-aminoadamantan-1-ol, Et3N, THF, reflux, 8 h; viii) 3-amino-1-
adamantanol, Et3N, THF, reflux, 8 h.
Fig. 2. Molecular modeling of sEH inhibitor bound to sEH (PDB code: 3WKE). A: Surface map of B401 in blue and B402 in pink bound to sEH in wheat; B: sEH in wheat bound to
compound B401 in blue. The carbonyl group of the urea makes a hydrogen bond to Tyr383, and the NH groups of the urea make two hydrogen bonds to Asp335. The phenyl ring of
B401 binds in a hydrophobic pocket and makes
(in white) presenting the surface of sEH binding pocket. Three compounds bind to sEH's active site primarily through hydrogen bond interactions by the urea moiety of the
p
ꢀcation interaction with His524. C: Docked poses of B401 (in blue) and B402 (in pink) overlayed over X-ray pose of native ligand
compounds. D: sEH in wheat bound to compound B402 in pink. The carbonyl group of the urea makes a hydrogen bond to Tyr383, and the NH groups of the urea make two
hydrogen bonds with Asp335. The phenyl ring of B401 binds in a hydrophobic pocket and makes
pꢀcation interaction with His524.
healthy volunteers [22]. Noteworthy, this adverse effect presented
gender differences, among male mouse appearing to this perfor-
mance. The observed efficacy and safety profile associated with
these compounds demonstrated the proof of concept for this class
of memantyl urea-based sEH inhibitors as therapeutic agents in
related diseases.
2.6. Microsomal stability
The microsomal stability of B401 was evaluated in human and
rat liver microsomes, which are widely used to determine the likely
degree of primary metabolic clearance in the liver. As shown in
Table 3, the in vitro metabolism of B401 remained in both human
6

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
Table 2
Human (HsEH) and Murine (MsEH) sEH IC50 of urea-based inhibitorsa.
Comp.
Structure
HsEH IC50 (nM)
0.4
MsEH IC50 (nM)
0.5
cLogPb
3.83
LLEc
5.57
B401
B402
2.3
0.4
4.57
4.07
B403
C401
0.7
3.9
0.4
2.4
3.90
4.13
5.25
4.28
C402
C403
217.2
59.8
13.7
28.8
4.86
4.20
1.80
3.03
D401
D402
0.4
1.4
0.4
0.4
3.53
4.27
5.86
4.58
D403
E401
E402
0.9
0.4
3.60
2.32
3.05
5.44
4.35
3.25
216.8
493.0
496.6
462.6
E403
F401
248.9
120.4
2348.6
88.8
2.39
2.08
4.22
4.84
F402
795.6
94.2
2.82
3.28
F403
949.1
13.8
290.6
1.7
2.15
1.30
3.87
6.57
AR-9281
a
IC50 values are recorded on recombinant human sEH protein (HsEH) and murine sEH (MsEH) protein using PHOME as substrate at 50
The cLogP values were predicted by BIOVIA Discovery Studio 2016.
m
M concentration.31
.
b
c
Ligand lipophilicity efficiency (LLE) ¼ pIC50-cLogP.32
.
and rat liver microsomes, with half-lives of 25 and 31 min,
respectively.
Table 3
Mean of concentrations of B401 in microsomal buffer at different time (ng/mL).
2.7. Pharmacokinetic study of B401
Time (min)
0
5
10
20
30
60
t1/2 (min)
Human
Rat
100.0
100.0
58.91
17.70
42.97
15.48
26.38
9.33
17.15
6.04
12.42
5.27
25.37
30.94
To identify the optimal candidate for evaluation in vivo, the
pharmacokinetics profile of B401 was determined with a single
7

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
dose of 30 mg/kg by oral (po) and a single dose of 3 mg/kg by
intravenous (iv) administration in the Sprague Dawley (SD) rats.
Pharmacokinetic parameters are shown in Table 4. After oral
administration blood levels were detected up to 24 h. The
level, and increased the median survival time of C57BL/6 mice in
LPS-induced sepsis model. Despite survival rate did not reach sig-
nificant efficacy vs vehicle, based on safety profile, metabolic sta-
bility, pharmacokinetic and in vivo efficacy, compound B401
demonstrated the proof of potential for this class of memantyl
urea-based sEH inhibitors as therapeutic agents in sepsis. Consid-
ering the drug efficacy in vivo is determined by the concentration of
free drug, rather than the total concentration in plasma; therefore,
this moderate efficacy could be partly attributed to the high %PPB
(98.6%). Further optimization of the pharmaceutical properties of
this class of compounds and evaluation on combination with
dexamethasone to decrease the dosage of dexamethasone will be
reported in due course.
maximum total concentration of B401 (0.33
1.36 h; total exposure area under the curve AUC0ꢀ24 was
1.304 M▪h. Poor exposures of compound B401 were obtained,
mM) was reached after
m
indicating bioavailability of 7.1%. The pharmacokinetic properties
revealed compound B401 has a plasma half-life (t1/2) of 2.24 h.
According to pharmacokinetic parameters, compound B401
displayed large apparent volume of distribution (Vd), which indi-
cated that compound B401 could be highly bound to plasma pro-
tein. Therefore, the percentage plasma protein binding (%PPB) of
mice was determined using equilibrium dialysis method. The result
of this screen demonstrated that compound B401 exhibited high %
PPB (98.6%) value. The high %PPB can be partly attributed to the
presence of the memantyl urea and benzamide moieties, as plasma
protein binding interactions are hydrophobic in nature.
4. Experimental section
4.1. General information
Unless otherwise stated, solvents and all commercially available
reagents were used directly without further purification. Melting
points were measured with an X-4 melting point apparatus and
were uncorrected. 1H NMR spectra were recorded on an Ascent
400 MHz spectrometer using Me4Si (TMS) as the internal standard.
Electrospray ionization (ESI) mass spectra was recorded using an
1100 Series MSD Trap SL. High-resolution mass spectrometry
(HRMS) results were recorded using an Agilent Technologies 6530
Accurate-Mass Q-TOF MS. The reactions were monitored by thin-
layer chromatography (TLC, HG/T2354e92, GF254), and termi-
nated as judged by the consumption of starting material.
2.8. In Vivo Efficacy
To determine if the administration of compound B401 to C57BL/
6 mice would produce a survival benefit (an increase in the median
survival time), in vivo efficacy was further evaluated in lipopoly-
saccharide (LPS) induced sepsis model, and compound B401 was
then designed with three doses of 1, 5 and 20 mg/kg, as shown in
Table 5. The numbers in parentheses indicated the number of sur-
viving mice.
The cumulative deaths of vehicle group at 24 h was 4, and the
survival rate was 71.4%. The survival rate within 72 h was 28.6%, and
the median survival time was 31.2 h. Adrenocortical steroid dexa-
methasone significantly improved survival rate (100%) vs vehicle
(28.6%). Mice treated with B401 at doses of 1 mg/kg and 5 mg/kg
couldn't improve survival rate and median survival time. The both
cumulative deaths at 24 h were 6. The median survival was 31.7 and
25.8, respectively. Mice treated with higher dose B401 (20 mg/kg)
showed increased survival, with a median survival rate of 53.8 h
compared with 31.2 h for the vehicle mice. The cumulative deaths
at 24 h were 3, survival rate of 24 h and 72 h were 78.6 and 35.7,
respectively. (Fig. 3). Considering the compound B401 is highly
bound to mouse plasma proteins, the amount of drug available to
reach the target is reduced. Subsequently, the efficacy of compound
B401 may be significantly reduced. Despite not reaching significant
efficacy, the ability to provide a survival advantage in this LPS-
induced sepsis model illustrates the potential of B401 as a lead
compound for the further optimization.
4.2. Chemistry
General procedure for acylation of anhydrides with amines.
Amines (50.0 mmol) was added dropwise to a solution of anhy-
drides (50.0 mmol) in dichloromethane (30 mL) at 0 ꢁC. The reac-
tion mixture was allowed to warm to rt and stirred for 2 h. Then
solvent was evaporated, and the crude product was purified by
recrystallization in EtOAc to obtain the title compound as white
solid.
General procedure for acylation of carboxylic acids with
amines. To a solution of EDCI (0.78 g, 4.05 mmol), HOBt (0.36 g,
4.05 mmol), Et3N (1.36 g, 13.50 mmol) in dry DCM (20 mL) was
added carboxylic acids derivatives (2.70 mmol) at rt and stirred for
30 min. Then, corresponding amines or amines hydrochloride
(2.70 mmol) was added. The mixture was stirred at rt for 4 h. Then,
the reaction mixture was poured into water (20 mL). The resulting
mixture was extracted with DCM (20 mL ꢂ 3). The combined
organic layers were washed twice with saturated NaHCO3 aqueous
(20 mL), once with water (20 mL) and once with brine (20 mL). The
separated organic layer was dried with MgSO4, filtered, and
concentrated under vacuum produce crude product, which was
purified by recrystallization in acetone to obtain the title com-
pound as a white solid.
3. Conclusion
A novel series of memantyl based sEH inhibitors was created
from an initial AR-9281, memantine, piperine and piperlongumi-
nine via molecular hybridization, bioisosterism and computer aided
drug design. The ligand lipophilicity efficiency (LLE) and potency of
the initial compounds were improved considerably during the
course of these optimization activities. These efforts culminated
with the identification of an optimized molecule, compound B401,
which exhibited potent in vitro enzyme activity in subnanomolar
Compounds A101 ꢀ A303 were synthesized via two steps ac-
cording to general procedure for acylation of anhydrides with
amines and carboxylic acids with amines.
N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-oxo-4-
Table 4
Pharmacokinetics of B401 in rats following intravenous and oral administration (n ¼ 6).
Route
Tmax (h)
Cmax
(m
M)
t1/2 (h)
CL (L/h/kg)
Vz (L/kg)
AUC
(mM$h)
AUC
(mM$h)
Bioavailability (%)
(0-24)
(0-∞)
iv (3 mg/kg)
po (30 mg/kg)
0.64
1.36
3.64
0.33
1.23
2.24
4.30
269.79
7.83
1097.80
1.834
1.304
1.835
1.305
7.1
8

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
Table 5
Survival rate of 24 h and 72 h (n ¼ 14).
Vehicle
B401 (1 mg/kg)
31.7
B401 (5 mg/kg)
B401 (20 mg/kg)
Dexamethasone (5 mg/kg)
Median survival/h
31.2
25.8
53.8
Undefined
Survival rate
71.4 (10)
57.1 (8)
57.1 (8)
78.6 (11)
100 (14)
% (24 h)
Survival rate % (72 h)
28.6 (4)
21.4 (3)
7.1 (1)
35.7 (5)
100 (14)
234e236 ꢁC. 1H NMR (400 Hz, CDCl3)
d
(ppm) 6.84 (d, J ¼ 14.8 Hz,
1H), 6.76 (d, J ¼ 14.8 Hz, 1H), 5.81 (d, J ¼ 7.6 Hz, 1H), 5.72 (br, 1H),
3.89-3.81 (m, 1H), 2.18e2.14 (m, 1H), 1.96e1.92 (m, 2H), 1.87 (d,
J ¼ 2.7 Hz, 2H), 1.76e1.69 (m, 6H), 1.65e1.60 (m, 1H), 1.43e1.29 (m,
6H), 1.23e1.13 (m, 5H), 0.86 (s, 6H). 13C NMR (100 Hz, CDCl3)
d
(ppm) 163.5, 134.7, 132.6, 54.1, 50.6, 48.6, 47.3, 42.6, 40.1, 33.0,
32.4, 30.1, 30.0, 25.5, 24.9. ESI MS m/z: 359.3 [MþH]þ.
N
[1]-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-N [4]-iso-
butylfumaramide (A203). White solid (0.33 g, 36%). m.p. 97e99 ꢁC.
1H NMR (400 Hz, CDCl3)
d
(ppm) 7.36 (br, 1H), 6.03 (d, J ¼ 14.0 Hz,
1H), 6.01 (q, J ¼ 14.0 Hz, 1H), 3.15 (t, J ¼ 5.7 Hz, 2H), 2.7e2.15 (m,
1H), 1.89 (br, 2H), 1.73e1.68 (m, 2H), 1.65e1.63 (m, 1H), 1.42e1.39
(m, 2H), 1.32e1.29 (m, 2H), 1.28e1.24 (m, 2H), 1.21e1.13 (m, 2H),
0.96 (s, 3H), 0.95 (s, 3H), 0.86 (s, 6H). 13C NMR (100 Hz, CDCl3)
Fig. 3. Increased survival of C57BL/6 mice in LPS-induced sepsis following B401
treatment. Mice treated with 20 mg/kg B401 by intraperitoneal injection (ip) (wheat,
n ¼ 14) exhibited a survival advantage compared to mice treated with soybean oil
vehicle (black, n ¼ 14).
d
(ppm) 165.1, 164.3, 133.5, 132.3, 54.1, 50.6, 47.2, 47.1, 42.7, 39.7,
32.4, 30.1, 29.7, 28.3, 20.2. ESI MS m/z: 333.3 [MþH]þ.
N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-5-oxo-5-(piper-
idin-1-yl)pentanamide (A301). White solid (0.34 g, 35%). m.p.
98e100 ꢁC. 1H NMR (400 Hz, CDCl3)
d (ppm) 5.65 (br, 1H), 3.54 (t,
(piperidin-1-yl)butanamide (A101). White solid (0.50 g, 53%). m.p.
J ¼ 5.4 Hz, 2H), 3.40 (t, J ¼ 5.4 Hz, 2H), 2.40 (t, J ¼ 7.0 Hz, 2H), 2.18 (t,
J ¼ 7.1 Hz, 2H), 2.14e2.11 (m, 1H), 1.67e1.62 (m, 6H), 1.58e1.52 (m,
4H), 1.40e1.35 (m, 2H),1.29e1.26 (m, 2H), 1.20e1.10 (m, 2H), 0.84 (s,
102e103 ꢁC. 1H NMR (400 Hz, CDCl3)
d
(ppm) 3.55 (t, J ¼ 5.2 Hz, 2H),
3.42 (t, J ¼ 4.9 Hz, 2H), 2.65 (t, J ¼ 6.6 Hz, 2H), 2.53 (br, 2H),
2.14e2.11 (m, 1H), 1.83 (d, J ¼ 2.4 Hz, 2H), 1.68e1.62 (m, 6H),
1.58e1.51 (m, 4H), 1.39e1.35 (m, 2H), 1.29e1.26 (m, 2H), 1.20e1.15
(m, 1H), 1.13e1.10 (m, 1H), 0.84 (s, 6H). 13C NMR (100 Hz, CDCl3)
6H). 13C NMR (100 Hz, CDCl3)
d (ppm) 171.9, 170.9, 53.4, 50.6, 47.6,
46.7, 42.7, 42.6, 40.2, 36.7, 32.4, 32.1, 30.1, 30.0, 26.6, 25.6, 24.5, 21.6.
ESI MS m/z: 361.3 [MþH]þ.
d
(ppm) 171.7, 170.1, 53.3, 50.6, 47.5, 46.5, 42.9, 42.7, 40.1, 32.7, 32.3,
N [1]-cyclohexyl-N [5]-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-
yl)glutaramide (A302). White solid (0.35 g, 34%). m.p.
30.1, 30.0, 28.8, 26.4, 25.6, 24.5. ESI-MS m/z: 347.3 [MþH]þ.
N [1]-cyclohexyl-N [4]-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-
yl)succinimide (A102). White solid (0.38 g, 39%). m.p. 128e130 ꢁC.
155e157 ꢁC.1H NMR (400 Hz, CDCl3)
d (ppm) 5.72 (s, 1H), 5.51 (br,
1H), 3.80e3.71 (m, 1H), 2.23 (t, J ¼ 7.1 Hz, 2H), 2.17 (t, J ¼ 7.0 Hz, 2H),
2.14e2.12 (m, 1H), 1.94e1.87 (m, 4H), 1.82 (d, J ¼ 2.5 Hz, 2H),
1.73e1.67 (m, 2H), 1.64 (s, 4H), 1.61e1.60 (m, 1H), 1.41e1.34 (m, 4H),
1.32e1.27 (m, 2H), 1.22e1.09 (m, 5H), 0.85 (s, 6H). 13C NMR (100 Hz,
1H NMR (400 Hz, CDCl3)
d (ppm) 5.88 (br, 1H), 5.67 (br, 1H),
3.78e3.68 (m, 1H), 2.46 (br, 4H), 2.15e2.12 (m, 1H), 1.90e1.86 (m,
2H), 1.82 (d, J ¼ 2.3 Hz, 2H), 1.72e1.66 (m, 2H), 1.63 (br, 4H),
1.60e1.58 (m, 1H), 1.39e1.33 (m, 4H), 1.30e1.26 (m, 3H), 1.21e1.20
(m, 1H), 1.19e1.10 (m, 4H), 1.84 (s, 6H). 13C NMR (100 Hz, CDCl3)
CDCl3)
d (ppm) 171.9, 171.7, 53.5, 50.6, 48.1, 47.6, 42.7, 40.2, 36.3,
35.6, 33.2, 32.4, 33.2, 32.4, 30.1, 30.0, 25.5, 24.9, 22.3. ESI-MS m/z:
375.4 [MþH]þ.
d
(ppm) 171.4, 171.3, 53.5, 50.6, 48.2, 47.5, 42.7, 40.1, 33.2, 33.0, 32.3,
32.2, 30.1, 30.0, 25.5, 24.8. ESI-MS m/z: 361.3 [MþH]þ.
N
[1]-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-N [5]-iso-
N
[1]-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-N [4]-iso-
butylglutaramide (A303). White solid (0.39 g, 41%). m.p.
130e132 ꢁC.1H NMR (400 Hz, CDCl3)
d
(ppm) 5.86 (br, 1H), 5.45 (br,
butylsuccinamide (A103). White solid (0.40 g, 44%). m.p.
123e125 ꢁC. 1H NMR (400 Hz, CDCl3)
d
(ppm) 6.16 (br 1H), 5.60 (br,
1H), 3.07 (dd, J ¼ 6.7 Hz, 6.1 Hz, 2H), 2.27 (t, J ¼ 7.1 Hz, 2H), 2.17 (t,
J ¼ 7.4 Hz, 2H), 2.14e2.12 (m, 1H), 1.95e1.88 (m, 2H), 1.82 (d,
J ¼ 2.9 Hz, 2H), 1.80e1.73 (m, 1H), 1.64 (s, 4H), 1.39e1.36 (m, 2H),
1.30e1.27 (m, 2H), 1.20e1.11 (m, 2H), 0.92 (s, 3H), 0.91 (s, 3H), 0.85
1H), 3.06 (q, J ¼ 6.7 Hz, 2H), 2.50e2.44 (m, 4H), 2.14e2.11 (m, 1H),
1.80 (d, J ¼ 2.7 Hz, 2H), 1.79e1.72 (m, 1H), 1.62 (s, 4H), 1.38e1.35 (m,
2H), 1.29e1.26 (m, 2H), 1.19e1.14 (m, 2H), 0.91 (s, 3H), 0.90 (s, 3H),
0.84 (s, 6H). 13C NMR (100 Hz, CDCl3)
d (ppm) 172.4, 171.4, 53.5,
(s, 6H). 13C NMR (100 Hz, CDCl3)
d (ppm) 172.7, 171.9, 53.5, 50.6,
50.6, 47.5, 46.9, 42.7, 40.1, 33.1, 32.3, 32.1, 30.1, 30.0, 28.5, 20.1. ESI-
47.6, 46.9, 42.7, 40.2, 36.3, 35.4, 32.4, 30.1, 30.0, 28.5, 22.3, 20.1. ESI
MS m/z: 355.3 [MþNa]þ.
MS m/z: 349.3 [MþH]þ.
(Z)-N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-oxo-4-
(piperidin-1-yl)but-2-enamide (A201). White solid (0.10 g, 11%). m.p.
Methyl 4-(chlorocarbonyl)benzoate (14).
A mixture of 4-
(methoxycarbonyl)benzoic acid 13 (18.0 g, 100.0 mmol) and DMF
(0.5 mL) in SOCl2 (75 mL) was heated to reflux for 2 h. After cooling
to r.t., the reaction mixture was concentrated in vacuo to obtain.
After cooling to rt, the reaction mixture was concentrated in vacuo
to obtain methyl 4-(chlorocarbonyl)benzoate 14 as a light yellow
solid. It was used in the following reaction without any further
purification.
162e164 ꢁC.1H NMR (400 Hz, CDCl3)
d (ppm) 7.35 (br, 1H), 6.26 (d,
J ¼ 12.9 Hz, 1H), 5.97 (d, J ¼ 12.5 Hz,1H), 3.61 (t, J ¼ 5.6 Hz, 2H), 3.42
(t, J ¼ 5.5 Hz, 2H), 2.15e2.12 (m, 1H), 1.85 (d, J ¼ 2.0 Hz, 2H),
1.70e1.54 (m, 10H), 1.40e1.37 (m, 2H), 1.29e1.26 (m, 2H), 1.20e1.10
(m, 2H), 0.85 (s, 6H). 13C NMR (100 Hz, CDCl3)
d (ppm) 165.7, 164.2,
132.1, 128.6, 53.7, 50.7, 47.7, 47.3, 42.7, 42.5, 39.9, 32.3, 30.1, 26.4,
25.5, 24.4. ESI-MS m/z: 345.3 [MþH]þ.
Methyl
4-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)carba-
N [1]-cyclohexyl-N [4]-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-
yl)fumaramide (A202). White solid (0.33 g, 34%). m.p.
moyl)benzoate (15). To a solution of Et3N (12.6 g, 0.13 mol), DMAP
(0.56 g, 5.0 mmol) in dry DCM (30 mL) was added menantine
9

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
hydrochloride (5.40 g, 25.0 mmol). To the reaction mixture was
added dropwise solution of 14 (9.90 g, 50.0 mmol) in dry DCM
(30 mL) at 0 ꢁC. The resulting mixture was allowed to warm to rt
and stirred for 8 h. The reaction mixture was poured into water
(40 mL), extracted with DCM (30 mL ꢂ 3). The combined organic
layers were washed with saturated NaHCO3 aqueous (30 mL ꢂ 2),
once with water (30 mL) and once with brine (30 mL). The sepa-
rated organic layer was dried with MgSO4, filtered, and concen-
trated under vacuum produce crude 15 as a white solid (4.80 g,
80%). It was used in the following reaction without any further
purification.
4-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)carbamoyl)ben-
zoic acid (16).Crude 15 (1.0 g, 2.93 mmol) was dissolved in THF
(10 mL), followed by the addition of H2O (7 mL) and LiOH▪H2O
(0.37 g, 8.79 mmol) at rt. After 3 h, the reaction was filtered and
concentrated under vacuum. To the resides was added H2O (20 mL),
after cooling to 0 ꢁC, the pH was adjusted to 1 using 2 N HCl. The
resulting precipitation was filtered and washed with H2O to give a
white solid (0.90 g, 94%).
General procedure for the syntheses of A401 ꢀA403. To a so-
lution of EDCI (0.26 g, 1.38 mmol), HOBt (0.19 g, 1.38 mmol), Et3N
(0.46 g, 4.55 mmol) in dry DCM (20 mL) was added 16 (0.30 g,
0.92 mmol) at rt and stirred for 30 min. Then, corresponding
amines (1.10 mmol) was added. The mixture was stirred at rt for
24 h. Then, the reaction mixture was poured into water (20 mL).
The resulting mixture was extracted with DCM (20 mL ꢂ 3). The
combined organic layers were washed once with water (20 mL) and
once with brine (20 mL). The separated organic layer was dried
with MgSO4, filtered, and concentrated under vacuum to produce
crude product, which was purified by column chromatography on
silica gel to obtain a white solid.
2H), 2.60e2.49 (m, 4H), 1.97 (br, 3H), 1.80e1.74 (m, 1H), 1.72e1.60
(m, 6H), 1.54e1.45 (m, 6H), 1.00 (d, J ¼ 6.9 Hz, 3H), 0.90 (d,
J ¼ 6.7 Hz, 6H). 13C NMR (100 Hz, CDCl3)
d (ppm) 172.3, 171.7, 53.1,
47.0, 38.3, 37.0, 35.7, 32.4, 32.2, 28.5, 28.3, 20.1, 14.5.ESI-HRMS: m/z
[MþNa]þ calcd for C20H34N2O2Na: 357.2512; found: 357.2529.
N
[1]-(1-((3r,5r,7r)-adamantan-1-yl)ethyl)-N
[5]-iso-
butylglutaramide (C303). According to General procedure for acyl-
ation of carboxylic acids with amines. C303 was obtained as a white
solid as a white solid (0.44 g, 46%). m.p. 191e193 ꢁC. 1H NMR
(400 Hz, CDCl3)
d (ppm) 5.95 (br, 1H), 5.66 (br, 1H), 3.74e3.67 (m,
1H), 3.08 (t, J ¼ 6.6 Hz, 2H), 2.31e2.27 (m, 4H), 2.00e1.94 (m, 5H),
1.82e1.74 (m, 1H), 1.72e1.60 (m, 6H), 1.55e1.46 (m, 6H), 1.01 (d,
J ¼ 6.9 Hz, 3H), 0.92 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 Hz, CDCl3)
d
(ppm) 172.6,171.9, 52.9, 46.9, 38.4, 37.0, 35.7, 35.5, 28.5, 28.3, 22.4,
20.1, 14.6. ESI-HRMS: m/z [MþNa]þ calcd for C21H36N2O2Na:
371.2669; found: 371.2684.
N
[1]-((1S,2R,5R)-adamantan-2-yl)-N [5]-isobutylglutaramide
(D303). According to General procedure for acylation of carboxylic
acids with amines. D103 was obtained as a white solid (0.44 g, 51%).
m.p. 186e188 ꢁC. 1H NMR (400 Hz, DMSO‑d6)
d (ppm) 7.74 (t,
J ¼ 5.4 Hz, 1H), 7.64 (t, J ¼ 7.5 Hz, 2H), 3.83 (d, J ¼ 7.3 Hz, 1H), 2.85 (t,
J ¼ 6.2 Hz, 2H), 2.13 (t, J ¼ 7.4 Hz, 2H), 2.06 (t, J ¼ 7.4 Hz, 2H),
1.99e1.95 (m, 2H), 1.79e1.62 (m, 13H), 1.48e1.45 (m, 2H). 13C NMR
(100 Hz, DMSO‑d6)
d (ppm) 172.2, 171.7, 53.2, 46.4, 37.7, 37.4, 35.3,
35.2, 32.0, 31.4, 28.5, 27.3, 27.2, 22.5, 20.6. ESI-HRMS: m/z [MþNa]þ
calcd for C19H32N2O2Na: 343.2356; found: 343.2361.
N
[1]-((5s,7s)-5-hydroxyadamantan-2-yl)-N
[5]-iso-
butylglutaramide (E303). According to General procedure for acyl-
ation of carboxylic acids with amines. E303 was obtained as a white
solid (0.49 g, 49%). m.p. 181e183 ꢁC. 1H NMR (400 Hz, DMSO‑d6)
d
(ppm) 7.74 (t, J ¼ 5.5 Hz, 1H), 7.59 (d, J ¼ 7.4 Hz, 1H), 4.37 (s, 1H),
N-((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)-4-(piperidine-1-
carbonyl)benzamide (A401). White solid (0.20 g, 56%). m.p.
3.76e3.74 (m, 1H), 2.85 (t, J ¼ 6.1 Hz, 2H), 2.12 (t, J ¼ 7.4 Hz, 2H),
2.06 (t, J ¼ 7.4 Hz, 2H),1.97 (br,1H),1.88e1.84 (m, 4H),1.73e1.63 (m,
5H), 1.62e1.56 (m, 4H), 1.31e1.28 (m, 2H), 0.82 (d, J ¼ 6.7 Hz, 6H).
148e150 ꢁC.1H NMR (400 Hz, CDCl3)
7.42 (d, J ¼ 8.2 Hz, 2H), 5.83 (s, 1H), 3.68 (br, 2H), 3.31 (br, 2H),
2.21e2.18 (m, 1H), 1.96 (d, J ¼ 2.2 Hz, 2H), 1.81e1.73 (m, 4H), 1.67
(br, 6H), 1.45e1.32 (m, 4H), 1.26e1.16 (m, 2H), 0.59 (s, 6H). 13C NMR
d
(ppm) 7.73 (d, J ¼ 8.2 Hz, 2H),
13C NMR (100 Hz, DMSO‑d6)
d (ppm) 172.2, 171.9, 66.0, 52.6, 46.4,
45.8, 45.1, 35.3, 35.2, 33.8, 30.4, 29.6, 28.5, 22.4, 20.6. ESI-HRMS: m/
z [MþNa]þ calcd for C19H32N2O3Na: 359.2305; found: 359.2332.
General procedure for the syntheses of compounds 21 e 23. To a
solution of amines (80.8 mmol), Et3N (16.4 g, 0.16 mol) in dry THF
(25 mL) was added dropwise a solution of 4-nitrobenzoyl chloride
(10.0 g, 53.9 mmol) in THF (40 mL) at 0 ꢁC under ice bath. The re-
action mixture was allowed to warm to rt and stirred for 6 h. Then
solvent was evaporated, and the residues were dissolved in EtOAc
(50 mL) and treated with water (40 mL). The organic layer was
separated, and the water layer was extracted with EtOAc
(30 mL ꢂ 3). The combined organic layers were washed twice with
1 N hydrochloric acid (30 mL), twice with water (30 mL), once with
brine (30 mL), dried over MgSO4, filtered, and concentrated to
produce yellow oil. The crude product was triturated with diethyl
ether to afford the title compound as a white solid.
(100 Hz, CDCl3)
d (ppm) 169.4, 166.0, 139.0, 136.8, 126.93, 126.91,
54.1, 50.6, 47.6, 42.7, 40.2, 32.5, 30.2, 30.1, 24.5. ESI-HRMS: m/z
[MþNa]þ calcd for C25H34N2O2Na: 417.2512; found: 417.2543.
N [1]-cyclohexyl-N [4]-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-
yl)terephthalamide (A402). White solid (0.21 g, 57%). m.p.
257e259 ꢁC. 1H NMR (400 Hz, CDCl3)
d
(ppm) 7.76 (q, J ¼ 14.6,
8.7 Hz, 4H), 6.00 (d, J ¼ 7.9 Hz, 1H), 5.84 (s, 1H), 2.21e2.19 (m, 1H),
2.06e2.02 (m, 2H), 1.97 (d, J ¼ 2.0 Hz, 2H), 1.81e1.75 (m, 6H),
1.69e1.64 (m, 1H), 1.48e1.30 (m, 6H), 1.28e1.17 (m, 5H), 0.89 (s,
6H). 13C NMR (100 Hz, CDCl3)
d (ppm) 165.8, 138.4, 137.3, 127.1,
126.9, 54.2, 50.6, 48.9, 47.6, 42.7, 40.2, 33.2, 32.5, 30.2, 30.1, 25.6,
24.9. ESI-HRMS: m/z [MþNa]þ calcd for C26H36N2O2Na: 431.2669;
found: 431.2697.
N
[1]-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-N [4]-iso-
butylterephthalamide (A403). White solid (0.21 g, 60%). m.p.
234e236 ꢁC. 1H NMR (400 Hz, CDCl3)
General procedure for the syntheses of compounds 24 e 26.
Nitro compounds (21e23) (42.7 mmol) was dissolved in EtOH
(100 mL), and the solution was vacuumed briefly then put under an
argon atmosphere. Then 5%Pd/C (10% w/w) was quickly added to
the solution that was vacuumed and put under argon atmosphere.
This was repeated three times. The solution was briefly vacuumed
to remove the argon atmosphere then put under a H2 atmosphere;
this was repeated three times. The mixture was heated to 50 ꢁC for
12 h. After the reaction mixture was cooled to rt, the reaction was
filtered through a celite pad and concentrated in vacuum to give a
white solid.
d
(ppm) 7.76 (q, J ¼ 16.7,
8.1 Hz, 4H), 6.22 (br, 1H), 5.85 (br, 1H), 3.30 (t, J ¼ 6.2 Hz, 2H),
2.21e2.19 (m, 1H), 1.97 (d, J ¼ 2.2 Hz, 2H), 1.95e1.88 (m, 1H),
1.82e1.74 (m, 4H),1.46e1.32 (m, 4H),1.26e1.17 (m, 2H), 0.99 (s, 3H),
0.98 (s, 3H), 0.89 (s, 6H). 13C NMR (100 Hz, CDCl3)
d (ppm) 166.8,
165.9, 138.4, 137.2, 127.1, 127.0, 54.2, 50.6, 47.6, 47.5, 42.7, 40.2, 32.5,
30.2, 30.1, 29.7, 28.6, 20.2. ESI-HRMS: m/z [MþNa]þ calcd for
C
24H34N2O2Na: 405.2512; found: 405.2533.
N
[1]-(1-((3r,5r,7r)-adamantan-1-yl)ethyl)-N
[4]-iso-
butylsuccinamide (C103). According to General procedure for acyl-
ation of carboxylic acids with amines. C103 was obtained as a white
solid (0.38 g, 42%). m.p. 186e188 ꢁC. 1H NMR (400 Hz, CDCl3)
General procedure for the syntheses of compounds 27 e 29. To
a solution of Compounds (24e26) (26.9 mmol), K2CO3 (40.4 mmol)
in DCM (20 mL) was added dropwise a solution of phenyl chlor-
oformate (35.3 mmol) in DCM (20 mL) at 0 ꢁC. The reaction mixture
d
(ppm) 6.24 (br,1H), 5.94 (br,1H), 3.71e3.63 (m, 1H), 3.13e3.00 (m,
10

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
was allowed to warm to rt and stirred for 7 h. The reaction mixture
was poured into water (40 mL), the resulting precipitation was
filtered and washed with H2O to give a crude product, which was
triturated with diethyl ether to afford the title compound as a white
solid.
General procedure for the syntheses of urea-based compounds.
To a solution of carbamate intermediates (compounds 27e29)
(1.54 mmol), Et3N (3.08 mmol) in THF (5 mL) was added corre-
sponding amines (1.70 mmol). The reaction mixture was heated to
reflux for 8 h. After cooling to the rt, the resulting precipitation was
filtered and washed with THF to give a tittle product as a white
solid.
DMSO‑d6)
d
(ppm) 165.4, 155.0, 143.7, 128.7, 127.4, 116.6, 53.2, 48.6,
38.5, 37.1, 36.0, 33.0, 28.3, 25.8, 25.5. ESI-HRMS: m/z [MþNa]þ calcd
for C26H37N3O2Na: 446.2778; found: 446.2800.
4-(3-(1-((1s,3s)-adamantan-1-yl)ethyl)ureido)-N-iso-
butylbenzamide (C403)¶. White solid (0.38 g, 60%). m.p. 207e209 ꢁC.
1H NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.59 (s, 1H), 8.21 (t, J ¼ 5.7 Hz,
1H), 7.73 (d, J ¼ 8.8 Hz, 2H), 7.42 (d, J ¼ 8.8 Hz, 2H), 6.08 (d,
J ¼ 9.3 Hz, 1H), 3.42e3.34 (m, 1H), 3.05 (t, J ¼ 6.1 Hz, 2H), 1.96 (br,
3H), 1.86e1.76 (m, 1H), 1.69e1.58 (m, 6H), 1.55e1.45 (m, 6H), 0.97
(d, J ¼ 6.8 Hz, 3H), 0.88 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 Hz,
DMSO‑d6)
d (ppm) 166.3, 155.1, 143.8, 128.5, 127.3, 116.7, 53.2, 47.1,
38.5, 37.1, 36.0, 28.6, 28.3. 20.7, 15.3. ESI-HRMS: m/z [MþNa]þ calcd
1-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-3-(4-(piperidine-
1-carbonyl)phenyl)urea (B401). White solid (0.41 g, 65%). m.p.
204e205 ꢁC. Compound B401 used for animal experiment exhibi-
ted >99.9% purity by HPLC (Shimadzu 2010A). Purity assessment of
for C24H35N3O2Na: 420.2621; found: 420.2647.
1-((1S,2R,5R)-adamantan-2-yl)-3-(4-(piperidine-1-carbonyl)
phenyl)urea (D401). White solid (0.40 g, 68%). m.p. 265e267 ꢁC. 1H
NMR (400 Hz, DMSO‑d6)
d (ppm) 8.58 (s, 1H), 7.42e7.39 (m, 2H),
B401 was performed by injecting 2
terest in 1:1 MeOH onto a reverse-phase Agilent ZORBA ꢂ 80A
m) column under the following
mobile-phase conditions: MeCN/H2O ¼ 75%/25%. The mobile-phase
flow rate was 0.8 mL/min, and the column temperature was set to
25 ꢁC. Analytical UV detection was performed at wavelength of 210
and 230 nm. The retention time of B401 was 4.949 min 1H NMR
m
L (2 mg/mL) of sample of in-
7.26e7.23 (m, 2H), 6.50 (d, J ¼ 8.0 Hz, 1H), 3.77 (d, J ¼ 7.8 Hz, 1H),
3.43 (br, 4H), 1.87e1.74 (m, 10 H), 1.70e1.49 (m, 10H). 13C NMR
Extend-C18 (4.6 ꢂ 150 mm, 5
m
(100 Hz, DMSO‑d6) d (ppm) 169.5, 154.7, 142.2, 128.9, 128.4, 117.0,
53.3, 37.6, 37.3, 32.5, 31.7, 27.2, 27.1, 26.2, 25.6, 24.6. ESI-HRMS: m/z
[MþNa]þ calcd for C23H31N3O2Na: 404.2308; found: 404.2335.
4-(3-((1S,2R,5R)-adamantan-2-yl)ureido)-N-cyclo-
hexylbenzamide (D402)¶. White solid (0.39 g, 66%). m.p. >300 ꢁC. 1H
(400 Hz, CDCl3)
d
(ppm) 8.41 (s, 1H), 7.37 (d, J ¼ 8.6 Hz, 2H), 7.22 (d,
NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.67 (s,1H), 7.94 (d, J ¼ 8.0 Hz,1H),
J ¼ 8.6 Hz, 2H), 5.95 (s, 1H), 3.42 (br, 4H), 2.10e2.08 (m, 1H),
7.73 (d, J ¼ 8.7 Hz, 2H), 7.41 (d, J ¼ 8.8 Hz, 2H), 6.54 (d, J ¼ 8.0 Hz,
1H), 3.78e3.72 (m, 2H), 1.87e1.71 (m, 16H), 1.62e1.55 (m, 3H),
1.34e1.24 (m, 4H), 1.15e1.10 (m, 1H). 13C NMR (100 Hz, DMSO‑d6)
1.76e1.75 (m, 2H), 1.61e1.55 (m, 6H), 1.49 (br, 4H), 1.35e1.24 (m,
4H), 1.12 (s, 2H), 0.83 (s, 6H). 13C NMR (100 Hz, DMSO‑d6)
d (ppm)
169.5, 154.3, 142.2, 128.8, 128.3, 117.1, 52.0, 50.8, 48.1, 42.8, 40.6,
d (ppm) 165.4, 154.6, 143.5, 128.7, 127.5, 116.6, 53.3, 48.6, 37.6, 37.3,
32.4, 30.5, 30.1, 26.1, 24.6. ESI-HRMS: m/z [MþNa]þ calcd for
33.0, 32.5, 31.7, 27.2, 27.1, 25.8, 25.5. ESI-HRMS: m/z [MþNa]þ calcd
C
25H35N3O2Na: 432.2621; found: 432.2646.
for C24H33N3O2Na: 418.2465; found: 418.2490.
N-cyclohexyl-4-(3-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)
4-(3-((1S,2R,5R)-adamantan-2-yl)ureido)-N-isobutylbenzamide
(D403). White solid (0.41 g, 70%). m.p. 270e272 ꢁC. 1H NMR
ureido)benzamide (B402). White solid (0.39 g, 62%). m.p. >300 ꢁC.
1H NMR (400 Hz, DMSO‑d6)
2H), 7.71 (d, J ¼ 8.7 Hz, 2H), 7.77 (d, J ¼ 8.7 Hz, 2H), 5.97 (s,1H), 3.73-
3.72 (m, 1H), 2.10e2.08 (m, 1H), 1.79e1.73 (m, 6H), 1.61e1.58 (m,
5H), 1.34e1.25 (m, 8H), 1.15e1.09 (m, 3H), 0.83 (s, 6H). 13C NMR
d
(ppm) 8.47 (s, 1H), 7.94 (d, J ¼ 8.0 Hz,
(400 Hz, DMSO‑d6)
d
(ppm) 8.88 (s, 1H), 8.22 (t, J ¼ 5.7 Hz, 1H), 7.73
(d, J ¼ 8.7 Hz, 2H), 7.44 (d, J ¼ 8.7 Hz, 2H), 6.69 (d, J ¼ 8.0 Hz, 1H),
3.78-3.77 (m, 2H), 3.05 (t, J ¼ 6.2 Hz, 2H), 1.91e1.70 (m, 13H),
1.57e1.54 (m, 2H), 0.88 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 Hz,
(100 Hz, DMSO‑d6)
d
(ppm) 165.3, 154.2, 143.6, 128.6, 127.4, 116.6,
DMSO‑d6) d (ppm) 166.3, 154.7, 143.7, 128.6, 127.3, 116.6, 53.3, 47.1,
52.1, 50.7, 48.6, 48.1, 42.8, 40.6, 33.0, 32.4, 30.6, 30.1, 25.8, 25.5. ESI-
HRMS: m/z [MþNa]þ calcd for C26H37N3O2Na: 446.2778; found:
446.2810.
37.6, 37.3, 32.6, 31.7, 28.6, 27.2, 27.1, 20.7. ESI-HRMS: m/z [MþNa]þ
calcd for C22H31N3O2Na: 392.2308; found: 392.2326.
1-((5s,7s)-5-hydroxyadamantan-2-yl)-3-(4-(piperidine-1-
carbonyl)phenyl)urea (E401). White solid (0.42 g, 69%). m.p.
4-(3-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)ureido)-N-iso-
butylbenzamide (B403). White solid (0.33 g, 52%). m.p. >300 ꢁC. 1H
270e272 ꢁC. 1H NMR (400 Hz, DMSO‑d6)
d (ppm) 8.58 (s, 1H), 7.42
NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.68 (s, 1H), 8.22 (t, J ¼ 5.8 Hz, 1H),
(d, J ¼ 8.6 Hz, 2H), 7.25 (d, J ¼ 8.6 Hz, 2H), 6.44 (d, J ¼ 7.8 Hz, 1H),
4.41 (s, 1H), 3.71e3.69 (m, 1H), 3.43 (br, 4H), 2.03 (br, 1H), 1.93 (br,
2H), 1.76e1.62 (m, 10H), 1.49e1.34 (m, 6H). 13C NMR (100 Hz,
7.72 (d, J ¼ 8.8 Hz, 2H), 7.38 (J ¼ 7.1 Hz, 2H), 6.13 (s, 1H), 3.05 (t,
J ¼ 6.1 Hz, 2H), 2.10-2.08 (m, 1H), 1.86e1.79 (m, 1H), 1.76e1.75 (m,
2H), 1.62e1.55 (m, 4H), 1.35e1.24 (m, 4H), 1.12 (s, 2H), 0.88 (s, 3H),
DMSO‑d6)
d (ppm) 169.5, 154.7, 142.1, 129.0, 128.4, 117.0, 65.9, 52.7,
0.86 (s, 3H), 0.83 (s, 6H). 13C NMR (100 Hz, DMSO‑d6)
d
(ppm) 166.3,
45.8, 44.9, 34.3, 30.6, 29.6, 26.1, 24.6. ESI-HRMS: m/z [MþNa]þ calcd
154.3, 143.8, 128.5, 127.2, 116.6, 52.0, 50.8, 48.1, 47.1, 42.8, 40.6, 32.4,
for C23H31N3O3Na: 420.2258; found: 420.2281.
30.5, 30.1, 28.6, 20.7. ESI-HRMS: m/z [MþNa]þ calcd for
N-cyclohexyl-4-(3-((5s,7s)-5-hydroxyadamantan-2-yl)ureido)
benzamide (E402). White solid (0.46 g, 75%). m.p. >300 ꢁC. 1H NMR
C
24H35N3O2Na: 420.2661; found: 420.2655.
1-(1-((1s,3s)-adamantan-1-yl)ethyl)-3-(4-(piperidine-1-
(400 Hz, DMSO‑d6)
d
(ppm) 8.63 (s, 1H), 7.94 (d, J ¼ 7.9 Hz, 1H), 7.73
carbonyl)phenyl)urea (C401). White solid (0.39 g, 62%). m.p.
(d, J ¼ 8.7 Hz, 2H), 7.41 (d, J ¼ 8.7 Hz, 2H), 6.45 (d, J ¼ 7.8 Hz, 1H),
4.41 (s, 1H), 3.71e3.69 (m, 2H), 2.03 (br, 1H), 1.93 (br, 2H), 1.79e1.68
(m, 8H), 1.64e1.62 (m, 5H), 1.45e1.38 (m, 2H), 1.34e1.23 (m, 4H),
181e183 ꢁC. 1H NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.50 (s, 1H), 7.41
(d, J ¼ 8.6 Hz, 2H), 6.02 (d, J ¼ 9.4 Hz, 1H), 3.42e3.34 (m, 5H), 1.96
(br, 3H), 1.69e1.58 (m, 8H), 1.55e1.45 (m, 10 H), 0.97 (d, J ¼ 6.8 Hz,
1.17e1.10 (m, 1H). 13C NMR (100 Hz, DMSO‑d6)
d (ppm) 165.4, 154.6,
3H). 13C NMR (100 Hz, DMSO‑d6)
d
(ppm) 169.5, 155.1, 142.3, 128.8,
143.5, 128.7, 127.5, 116.6, 65.9, 52.7, 48.6, 45.8, 44.9, 34.3, 33.0, 30.6,
29.6, 25.8, 25.5. ESI-HRMS: m/z [MþNa]þ calcd for C24H33N3O3Na:
434.2414; found: 434.2437.
128.3, 117.0, 53.2, 38.5, 37.2, 36.0, 28.3, 26.1, 24.6, 15.3. ESI-HRMS:
m/z [MþNa]þ calcd for C25H35N3O2Na: 432.2621; found: 432.2645.
4-(3-(1-((1s,3s)-adamantan-1-yl)ethyl)ureido)-N-cyclo-
4-(3-((5s,7s)-5-hydroxyadamantan-2-yl)ureido)-N-iso-
hexylbenzamide (C402)¶. White solid (0.40 g, 64%). m.p.
butylbenzamide (E403). White solid (0.44 g, 72%). m.p. 270e272 ꢁC.
280e283 ꢁC. 1H NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.58 (s, 1H), 7.94
1H NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.65 (s, 1H), 8.22 (t, J ¼ 5.6 Hz,
(d, J ¼ 7.9 Hz, 1H), 7.72 (d, J ¼ 8.7 Hz, 2H), 7.41 (d, J ¼ 8.8 Hz, 2H),
6.06 (d, J ¼ 9.4 Hz, 1H), 3.74e3.72 (m, 1H), 3.42-3.34 (m, 1H), 2.00
(br, 3H), 1.79e1.72 (m, 4H), 1.69e1.58 (m, 7H), 1.55e1.45 (m, 6H),
1.35e1.24 (m, 4H), 0.97 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 Hz,
1H), 7.74 (d, J ¼ 8.6 Hz, 2H), 7.42 (d, J ¼ 8.6 Hz, 2H), 6.47 (d,
J ¼ 7.7 Hz, 1H), 4.42 (s, 1H), 3.72e3.70 (m, 1H), 3.05 (t, J ¼ 6.3 Hz,
2H), 2.03 (br,1H),1.94 (br, 2H),1.86e1.81 (m,1H),1.79e1.57 (m, 9H),
1.41e1.38 (m, 2H), 0.87 (d, J ¼ 7.4 Hz, 6H). 13C NMR (100 Hz,
11

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
DMSO‑d6)
d
(ppm) 166.3, 154.6, 143.5, 128.6, 127.5, 116.7, 65.9, 52.7,
(including general conditions, behavioral activities, gait and
posture, eyes, nose and mouth, gastrointestinal function, skin coat,
urogenital tract). After that, clinical symptoms were monitored for
14 days. The animals’ weight was monitored for 7 days. Statistical
analysis for the body weight was performed using GraphPad Prism
6 software.
47.1, 45.8, 44.9, 34.3, 30.6, 29.6, 28.6, 25.6, 20.7. ESI-HRMS: m/z
[MþNa]þ calcd for C22H31N4O3Na: 408.2258; found: 408.2282.
N
[1]-((1r,3s,5R,7S)-3-hydroxyadamantan-1-yl)-N
[5]-iso-
butylglutaramide (F401). White solid (0.45 g, 73%). m.p. 245e247 ꢁC.
1H NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.42 (s, 1H), 7.37 (d, J ¼ 8.5 Hz,
2H), 7.23 (d, J ¼ 8.6 Hz, 2H), 6.00 (s, 1H), 4.80 (s, 1H), 3.42 (br, 4H),
2.14 (br, 2H), 1.85e1.42 (m, 6H), 1.61e1.42 (m, 12H). 13C NMR
4.5. Microsomal stability
(100 Hz, DMSO‑d6)
d (ppm) 169.5, 154.2, 142.2, 128.8, 128.3, 117.1,
67.8, 53.0, 50.1, 44.7, 41.0, 35.3, 30.6, 26.2, 24.6. ESI-HRMS: m/z
[MþNa]þ calcd for C23H31N3O3Na: 420.2258; found: 420.2279.
N-cyclohexyl-4-(3-((1r,3s,5R,7S)-3-hydroxyadamantan-1-yl)ure-
ido)benzamide (F402). White solid (0.43 g, 71%). m.p. 263e265 ꢁC.
Human and SD rat hepatic microsomes were purchased from
Research Institute for Liver Diseases (Shanghai, China) Co., Ltd. The
incubation mixture consisted of microsomal protein in PBS buffer
(100 mM, pH ¼ 7.4) and 100 ng/mL B401 in a final volume of 100
mL.
1H NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.54 (s, 1H), 7.94 (d, J ¼ 7.9 Hz,
The concentration of human hepatic microsomal protein was
0.4 mg/mL (0.53 mg/mL of SD rat). A 0.83 mg/mL NADPH solution
was prepared and added to the PBS buffer. After the addition of the
NADPH-generating system, the resulting mixture was incubated at
37 ꢁC for 0,5,10,20,30 and 60 min. The reaction was terminated by
1H), 7.72 (d, J ¼ 8.8 Hz, 2H), 7.37 (d, J ¼ 8.8 Hz, 2H), 6.08 (s, 1H), 4.48
(s,1H), 3.74e3.72 (m,1H), 2.14 (br, 2H),1.85e1.77 (m, 8H),1.73e1.72
(m, 2H), 1.61e1.51 (m, 5H), 1.47e1.42 (m, 2H), 1.35e1.24 (m, 4H),
1.13e1.10 (m, 1H). 13C NMR (100 Hz, DMSO‑d6)
d (ppm) 165.4, 154.1,
143.6, 128.6, 127.4, 116.6, 67.8, 53.0, 50.1, 48.6, 44.7, 41.0, 35.3, 33.0,
30.6, 25.8, 25.5. ESI-HRMS: m/z [MþNa]þ calcd for C24H33N3O3Na:
434.2414; found: 434.2439.
the addition of methanol 300 mL containing diphenhydramine
(0.02 ng/mL). The mixture was vortexed for 1 min, centrifuged at
21,528ꢂg for 10 min at 4 ꢁC. After centrifugation, 200
m
L of the
4-(3-((1r,3s,5R,7S)-3-hydroxyadamantan-1-yl)ureido)-N-iso-
supernatant was transferred to 96-well plate, and 400 mL of purified
butylbenzamide (F403). White solid (0.46 g, 74%). m.p. 156e158 ꢁC.
water was added, and the solution was mixing with shaker at
500 rpm for 5 min. The concentrations of B401 in the reaction
mixture were measured by LC/MS/MS, using an AB SCIEX 4500MD
mass spectrometer (Rockwell Allen-Bradley, USA).4.6 In Vivo
Pharmacokinetics (PK) study.
1H NMR (400 Hz, DMSO‑d6)
d
(ppm) 8.48 (s, 1H), 8.22 (t, J ¼ 5.7 Hz,
1H), 7.72 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 8.8 Hz, 2H), 6.03 (s, 1H), 4.49
(s, 1H), 3.05 (t, J ¼ 6.6 Hz, 2H), 2.14 (br, 2H), 1.85e1.76 (m, 7H),
1.57e1.42 (m, 6H), 0.87 (d, J ¼ 6.7 Hz, 6H). 13C NMR (100 Hz,
DMSO‑d6)
d
(ppm) 166.3, 154.1, 143.6, 128.5, 127.3, 116.7, 67.8, 53.0,
SD rats (6 females, 6 males, and 8 weeks old) were used in this
study. All animal procedures were performed in accordance with
the Guidelines for Care and Use of Laboratory Animals of Jiangsu
Kanion Pharmaceutical Co., Ltd. and experiments were approved by
the Animal Ethics Committee of Jiangsu Kanion Pharmaceutical Co.,
Ltd.12 SD rats were randomly divided into two groups (half females
and males). The 6 SD rats was administered iv with a single dose of
3 mg/kg of compound B401, and the other 6 SD rats were admin-
istered po with a single dose of 30 mg/kg of compound B401. An-
imals were weighed before each administration to adjust the
required volume. Blood samples (iv group) were collected at
different time: 0 min, 5 min, 15 min, 30 min, 1, 2, 4, 6, 8 and 12 h.
Blood samples (po group) were collected at different time: 0 min,
10 min, 30 min, 1, 2, 4, 6, 8, 12 and 24 h. Blood was taken from the
orbital vein. Blood was collected on Eppendorf tubes (1.5 mL)
containing of EDTA, then centrifuged at 4800 rpm for 10 min. Each
50.1, 47.1, 44.7, 41.0, 35.3, 30.6, 28.6, 25.6, 20.7. ESI-HRMS: m/z
[MþNa]þ calcd for C22H31N4O3Na: 408.2258; found: 408.2286.
4.3. In vitro biological activity assays
All HsEH and MsEH IC50 values is the concentration of a com-
pound that reduces the sEH activity by 50%. The IC50 values were
determined using a fluorescent-based assay [(3-phenyloxiranyl)
acetic
acid
cyano(6-methoxynaphthalen-2-yl)methyl
ester
(PHOME) as the substrate] [31]. The fluorescent assay was used
with purified recombinant human, or mouse sEH proteins. The
enzymes were incubated at 37 ꢁC with the inhibitors
([I]final ¼ 0.4 ꢀ 100,000 nM) for 10 min in 25 mM Tris HCl buffer
(pH ¼ 7.4) containing 0.1 mg/mL of BSA and 1% of DMSO. The
substrate (PHOME) was then added ([S]final ¼ 50
mM). Activity was
assessed by measuring the formation of the fluorescent 6-
methoxynaphthaldehyde product (lex ¼ 330 nm, lem ¼ 465 nm)
on a SpectraMax M2 (molecular devices). Results were obtained by
regression analysis from a linear region of the curve.
plasma sample (50
acetonitrile (5 ng/mL) and was centrifuged at 13,000 rpm for
10 min. After that, 50 L of supernatant was reconstituted with
200 L of acetonitrile, and resulting concentrations of compound
mL) was mixed with 150 mL of tolbutamide in
m
m
B401 analyzed by LC-MS.
4.4. In vivo safety
4.6. Percentage plasma protein binding (%PPB)
All animal experiments were performed under the guidelines of
the Jiangsu Kanion Pharmaceutical Co., Ltd. Committee for Use and
Care of Animals using an approved animal protocol. Mice used in
this study were ICR (half females and half males, 6 ꢀ 8 weeks old,
body weight 16e18 g) and purchased from Comparative Medicine
Center of Yangzhou University. The animals were housed under
sterile conditions at a constant temperature of 22e24 ꢁC under
daily cycles of light/darkness (12 h). After adaptive feeding three
days, mice were randomly divided into vehicle group, B401 group
and AR-9281 group (n ¼ 9 ꢀ 10). The vehicle was soybean oil, mice
were administered intraperitoneal injection (ip) with a single dose
of 100 mg/kg of test compounds. The volume of administration was
10 mL/kg. Animals were weighed before each administration to
adjust the required volume. After administration of 1, 3 and 6 h,
clinical symptoms and death/near death situation were observed
Compound B401 was dissolved in a 1: 19: 480 mixture of DMSO,
MeCN and 0.1 M PBS, and to achieve a concentration of 0.02 mM.
First, 96-well plate with 380
designated for mouse plasma respectively were preloaded. Then,
20 L of solution (0.02 mM) was spiked into the pre-loaded plasma
in the 96-well plate. The final concentration of B401 was 1 M,
containing 0.01% DMSO. Third, aliquots of 100 L of control dialysis
buffer were added to the receiver side of dialysis chambers. Ali-
quots of 100 L of the plasma spiking with B401 and warfarin (finial
concentration: 1 M) were added to the donor side of the dialysis
mL aliquots of plasma in the wells
m
m
m
m
m
chambers. Then, the dialysis block was covered with a plastic lid
and placed the entire apparatus in a shaker (120 rpm) for 5 h at
37 ꢁC. At end of incubation, each of post-dialysis samples was took
out from the buffer and plasma chambers. After mixing solution
12

F. Du, W. Sun, C. Morisseau et al.
European Journal of Medicinal Chemistry 223 (2021) 113678
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15 min. Then, 100
transferred into 96-well plate containing 100
the resulting mixture was further analyzed by LC-MS/MS. The
content of compound B401 was measured by LC-MS/MS-20 (TQ-
6500þ) with ACQUITY UPLC HSS T3 1.8 m (50 mm ꢂ 2.10 mm)
m
L of the supernatant from each well was
m
L of ultrapure water,
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m
column under the following mobile phase conditions:
0.01 ꢀ 0.3 min 90:10 to 5:95 A/B, 0.3 ꢀ 1 min 5:95 A/B,
1.01 ꢀ 1.5 min 90:10, where A ¼ 0.1% formic acid in water and
B ¼ 0.1% formic acid in MeCN. The mobile phase flow rate was
0.6 mL/min. The %PPB value of the compound was calculated by
following equation: % PPB ¼ 100 ꢂ ([Donor]5h -[Receiver]5h)/
[Donor]5h. The experimental system was calibrated using the
standard compound warfarin of known %PPB: mouse plasma
(94.6%).4.7 In Vivo Efficacy of B401.
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lipid mediator surge and ovarian cancer by a dual COX-2/sEH inhibitor, Proc.
Natl. Acad. Sci. U. S. A 116 (2019) 1698e1703.
All animal experiments were performed under the guidelines of
the Jiangsu Kanion Pharmaceutical Co., Ltd. Committee for Use and
Care of Animals using an approved animal protocol. Mice used in
this study were C57BL/6 (females, body weight 14 ꢀ 16 g) and
purchased from Comparative Medicine Center of Yangzhou Uni-
versity. The animals were housed under sterile conditions at a
constant temperature of 22e24 ꢁC under daily cycles of light/
darkness (12 h). After adaptive feeding seven days, 70C57BL/6 mice
were randomly divided into five groups (soybean oil vehicle, B401
three doses groups, dexamethasone group). The mice were fasted
for 6e8 h and freely drinking water before the experiment. Mice
were administered ip with LPS (30 mg/kg), then dosed by ip with
either vehicle or B401 at 1, 5, 20 mg/kg, administered at 0, 8 and
24 h intervals. Survival rate of mice was observed and record for
72 h. The survival curve was analyzed by log-rank (Mantel-Cox) test
of GraphPad Prism software.
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Funding sources
[17] C. Morin, M. Sirois, V. Echave, M.M. Gomes, E. Rousseau, 14,15-EET displays
anti-inflammatory effects in TNFa-stimulated human bronchi: putative role of
This work was funded by the Liaoning Revitalization Talents
Program (XLYC1908031), Basic Research Project of Department of
Education of Liaoning Province e natural sciences (2020LJC02), and
partial support was provided by the NIH e NIEHS (RIVER Award)
R35 ES030443-01, the NIEHS Superfund Research Program P42
ES004699.
CPI-17, Am. J. Respir. Cell Mol. Biol. 38 (2008) 192e201.
[18] K.M. Wagner, A. Gomes, C.B. McReynolds, B.D. Hammock, Soluble epoxide
hydrolase regulation of lipid mediators limits pain, Neurotherapeutics 17
(2020) 900e916.
[19] B. Inceoglu, A. Bettaieb, C.A. Trindade da Silva, K.S. Lee, F.G. Haj,
B.D. Hammock, Endoplasmic reticulum stress in the peripheral nervous sys-
tem is a significant driver of neuropathic pain, Proc. Natl. Acad. Sci. U.S.A. 112
(2015) 9082e9087.
€
[20] R. Blocher, K.M. Wagner, R.R. Gopireddy, T.R. Harris, H. Wu, B. Barnych,
Declaration of competing interest
S.H. Hwang, Y.K. Xiang, E. Proschak, C. Morisseau, B.D. Hammock, Orally
available soluble epoxide hydrolase/phosphodiesterase 4 dual inhibitor treats
inflammatory pain, J. Med. Chem. 61 (2018) 3541e3550.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[21] E. Ono, S. Dutile, S. Kazani, M.E. Wechsler, B.D. Yang, J. Hammock, D.N. Douda,
Y. Tabet, R. Khaddaj-Mallat, M. Sirois, C. Sirois, E. Rizcallah, E. Rousseau,
R. Martin, E.R. Sutherland, M. Castro, N.N. Jarjour, E. Israel, B.D. Levy, Lipoxin
generation is related to soluble epoxide hydrolase activity in severe asthma,
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S.K. Anandan, R. Gless, H.K. Webb, Pharmacokinetics and pharmacodynamics
of AR9281, an inhibitor of soluble epoxide hydrolase, in single- and multiple-
dose studies in healthy human subjects, J. Clin. Pharmacol. 52 (2012)
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D.E. Newby, I.B. Wilkinson, R. Tal-Singer, R.J. Mayer, J. Cheriyan, Pharmaco-
kinetics, pharmacodynamics and adverse event profile of GSK2256294, a
novel soluble epoxide hydrolase inhibitor, Br. J. Clin. Pharmacol. 81 (2016)
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[24] K.S.S. Lee, J.C. Ng, J. Yang, S.H. Hwang, C. Morisseau, K. Wagner, B.D. Hammock,
Preparation and evaluation of soluble epoxide hydrolase inhibitors with
improved physical properties and potencies for treating diabetic neuropathic
pain, Bioorg. Med. Chem. 28 (2020) 115735e115746.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113678.
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