Design, synthesis, and biological evaluation of oxazolidone derivatives as highly potent N-acylethanolamine acid amidase (NAAA) inhibitors

Article abstract of DOI:10.1039/c6ra28734d

N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that catalyzes the hydrolysis of endogenous fatty acid ethanolamides (FAEs), such as N-palmitoylethanolamide (PEA). PEA exhibits anti-inflammatory and analgesic activities by engaging peroxisome proliferator-activated receptor α (PPAR-α). Preventing PEA degradation by inhibition of NAAA has been proposed as a novel strategy for the treatment of inflammation and pain. In the present study, we reported the discovery of the oxazolidone derivative as a novel scaffold for NAAA inhibitors, and studied the structure-activity relationship (SAR) by modification of the side chain and terminal lipophilic substituents. The results showed that the link chain length of C5, straight and saturated linkages were the preferred shape patterns for NAAA inhibition. Several nanomolar NAAA inhibitors were described, including 2f, 3h, 3i and 3j with IC₅₀ values of 270 nM, 150 nM, 100 nM and 190 nM, respectively. Enzymatic degradation studies suggested that 2f inhibited NAAA in a selective, noncompetitive and reversible pattern. Moreover, 2f showed high anti-inflammatory and analgesic activities after systemic and oral administration.

Full text of DOI:10.1039/c6ra28734d

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Design, synthesis, and biological evaluation of
oxazolidone derivatives as highly potent N-
acylethanolamine acid amidase (NAAA) inhibitors†
Cite this: RSC Adv., 2017, 7, 12455
a
ab
*
Jie Ren, Yuhang Li,
Hongwei Ke,c Yanting Li,a Longhe Yang,d Helin Yu,a
a
b
a
*
Rui Huang, Canzhong Lu and Yan Qiu
N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that catalyzes the hydrolysis
of endogenous fatty acid ethanolamides (FAEs), such as N-palmitoylethanolamide (PEA). PEA exhibits anti-
inflammatory and analgesic activities by engaging peroxisome proliferator-activated receptor a (PPAR-a).
Preventing PEA degradation by inhibition of NAAA has been proposed as a novel strategy for the
treatment of inflammation and pain. In the present study, we reported the discovery of the oxazolidone
derivative as a novel scaffold for NAAA inhibitors, and studied the structure–activity relationship (SAR) by
modification of the side chain and terminal lipophilic substituents. The results showed that the link chain
length of C5, straight and saturated linkages were the preferred shape patterns for NAAA inhibition.
Several nanomolar NAAA inhibitors were described, including 2f, 3h, 3i and 3j with IC50 values of 270 nM,
150 nM, 100 nM and 190 nM, respectively. Enzymatic degradation studies suggested that 2f inhibited
NAAA in a selective, noncompetitive and reversible pattern. Moreover, 2f showed high anti-inflammatory
and analgesic activities after systemic and oral administration.
Received 27th December 2016
Accepted 16th February 2017
DOI: 10.1039/c6ra28734d
rsc.li/rsc-advances
potential therapeutic effects in inammation and pain. Prevent-
ing PEA degradation by inhibition of NAAA10 may provide a novel
strategy for the treatment of inammatory and pain.4,11 For this
1 Introduction
N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lyso-
somal cysteine hydrolase widely expressed in many tissues,
especially those associated with immune responses, such as the
lungs, spleen, and small intestine. NAAA participates in the
control of inammatory and pain1 via involving in the hydrolysis
of endogenous palmitoylethanolamide (PEA). PEA is a multifunc-
tional lipid mediator that has been shown to inhibit peripheral
inammation2–4 and cell degranulation, and exhibits anti-
nociceptive activities in rat and mouse models of acute and
chronic pain.5–7 Moreover, PEA also could alleviate skin inam-
mation8 and neuropathic pain in humans.9 Those effects are
mainly attribute to the ability of PEA to engage nuclear receptor
peroxisome proliferator-activated receptor-alpha (PPAR-a). In
recent years, PEA has attracted increasing attention because of its
reason, NAAA has been pursued as a potential drug target.
Despite the increasing interest in the design of NAAA inhibi-
tors as anti-inammatory and analgesic agents, only few potent
NAAA inhibitors have been identied so far.12,13 There are two
main categories of NAAA inhibitors reported, irreversible cova-
lent inhibitors, such as b-lactone derivatives14–18 and non-
covalent reversible inhibitors, such as benzothiazole-piperazine
derivatives.19 Although covalent inhibitors, such as (S)-OOPP,4
ARN077,11 ARN726 (ref. 20) (Fig. 1) demonstrated profound anti-
inammatory effects in several animal models, their application
is limited due to the poor chemical and plasma stability. These
compounds degraded in phosphate buffered saline (PBS) within
120 min and were rapidly cleaved in plasma.16 Non-covalent
reversible inhibitors, such as, benzothiazole-piperazine deriva-
tives (Fig. 1),19 are selective, reversible, and competitive NAAA
inhibitors. However, only few potent reversible NAAA inhibitors
with drug-like properties have been identied so far. Highly
potent, stable and reversible NAAA inhibitors are still highly
desired for the treatment of inammation and pain.
aMedical College, Xiamen University, Xiamen, Fujian 361102, P. R. China. E-mail:
yuhangli@irsm.ac.cn; yanqiu@xmu.edu.cn
bXiamen Institute of Rare-earth Materials, Haixi Institutes, Chinese Academy of
Sciences, Fujian 361005, P. R. China
cCollege of Ocean and Earth Science, Xiamen University, Xiamen, Fujian, 361005, P. R.
China
dEngineering Research Center of Marine Biological Resource Comprehensive
Utilization, Third Institute of Oceanography, State Oceanic Administration, Xiamen
361005, P. R. China
In our previous study, we designed pyrrolidine-based deriv-
atives 1a and 1b (Fig. 1)21 as NAAA inhibitors. Substitution of the
saturated alkyl chain of 1a with an aryl-containing lipophilic
moiety leaded to a potent NAAA inhibitor 1b (IC50 ¼ 2.1 mM). In
vivo pharmacological studies indicated that 1b reduced the
mRNA expression levels of inducible nitric oxide synthase and
† Electronic supplementary information (ESI) available: Full experimental details,
characterization, and 1H-NMR and 13C-NMR spectra of the intermediate and nal
compounds. See DOI: 10.1039/c6ra28734d
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Compounds 1a, 1j–1o, and 1q–1t were prepared by the amida-
tion of palmitoyl chloride with the corresponding amine in the
presence of triethylamine (Scheme 1).21 1l and 1m were
prepared by the methylation of 1q and 1r with iodomethane in
the presence of sodium hydride, respectively (Scheme 2).23 1p
was prepared by Dess–Martin periodinane oxidation of 1j or 1k
(Scheme 3).24 1c and 1d were prepared by the acidic depro-
tection of pyrrolidine derivatives 1c-1 and 1d-1, which were
obtained by the amidation of (R)- or (S)-3-(tert-butox-
ycarbonylamino)pyrrolidine with palmitoyl chloride, respec-
tively (Scheme 4).25,26 1e and 1f were synthesized by a two-step
reaction starting from 1q and 1r, which were converted to
iodide derivatives by I2 and PPh3, respectively, followed by
hydrogenation with H2 (Scheme 5).27,28 1g, 2a–2h, 3a–3d and 3k–
3s were synthesized by the imidate reaction, employing the
corresponding acyl chloride with 2-pyrrolidone, 2-imidazoli-
done, or 2-oxazolidone in the presence of n-BuLi, respectively
(Scheme 6).29 3e–3g were synthesized by the amidation of 2-
oxooxazolidine-3-carbonyl chloride, generated by reaction of 2-
oxazolidone and bis(trichloromethyl) carbonate, with the cor-
responding substituted piperazine or piperidine (Scheme 7).30
HPLC analysis indicated >99% purity for compound 1g, 1e–1i,
2b, 2d, 2f–2h, 3c, 3e, and 3g, >98% purity for 1c–1d, 1f, 2a, 2e,
3a, 3b and 3g–3i, and >97% purity for 2c and 3d (Table S3†).
Fig. 1 The chemical structures of classic NAAA inhibitors.
2.2 NAAA inhibition by oxazolidone derivatives
Fig. 2 Design strategy for oxazolidone derivatives as NAAA inhibitors.
The NAAA inhibition capability of these newly synthesized
compounds were tested using heptadecenoylethanolamide as
a substrate. Since many NAAA inhibitors also block fatty acid
amide hydrolase (FAAH), the FAAH inhibition of these
compounds also was examined to obtain NAAA selective
inhibitors. (S)-OOPP and well known FAAH inhibitor URB597
were used as standard compounds in the enzymatic assay. The
results showed that substitution at 3-position of pyrrolidine
with an amino group (1c, 1d) slightly increased NAAA and FAAH
interleukin-6, as well as increased the intracellular PEA levels in
mouse macrophages with lipopolysaccharide-induced inam-
mation.21 Moreover, 1b exhibited excellent chemical stability
1
and plasma stability (t > 16 h).
2
In the present study, we further modied 1a to obtain more
potent and biologically stable NAAA inhibitors. A nanomolar
NAAA inhibitor 2f was discovered (IC50 ¼ 270 nM), which
exerted profound anti-inammatory and anti-nociceptive
effects in rats.22 We then carried out structure–activity rela-
tionship (SAR) studies on 2f by modication of the pyrrolidine
ring and the lipophilic chain, which led to the identication of
several more potent nanomolar NAAA inhibitors, such as 3h, 3i
and 3j (Fig. 2). Compound 2f was then selected as a model
molecule to study the NAAA inhibition mechanisms and in vivo
anti-inammatory and analgesic activities of oxazolidone
derivatives. The results showed that 2f inhibited NAAA in
a selective and highly reversible pattern, and efficiently reduced
inammatory and pain aer systemic and oral administration.
Oral administration of 2f (30 mg kgꢀ1) exhibited the same
efficacy with commercial nonsteroidal anti-inammatory drug
ibuprofen at a 6.7-fold higher dose (200 mg kgꢀ1).
Scheme 1 The synthesis of compounds 1a, 1j, 1k, 1n, 1o, and 1q–1t.
Reagents and conditions: (a) Et3N, CH2Cl2, room temperature.
2 Results and discussion
2.1 Synthesis of oxazolidone derivatives
First, we synthesized a series of oxazolidone derivatives with
Scheme 2 The synthesis of compounds 1l and 1m. Reagents and
different side chains and terminal lipophilic substituents. conditions: (a) NaH, MeI, tetrahydrofuran (THF), 0 ꢁC.
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Scheme 3 The synthesis of compound 1p. Reagents and conditions:
(a) Dess–Martin periodinane, CH2Cl2, room temperature.
Scheme 6 The synthesis of compounds 1i, 2a–2h, 3a–3d and 3h–3s.
Reagents and conditions: (a) n-BuLi, THF, ꢀ78 ꢁC.
Scheme 4 The synthesis of compounds 1c and 1d. Reagents and
conditions: (a) Et3N, CH2Cl2, room temperature; (b) HCl, dioxane, 0 ꢁC.
¼ 3.4 ꢂ 0.42 mM), while 2f with the chain length of C5 exhibited
excellent potency (IC50 ¼ 0.27 ꢂ 0.04 mM), 3-fold more potent
than (S)-OOPP tested under the same experimental conditions.
Interestingly, sharply reduced or even abolished activity was
observed as the chain length decreased from C5 to C1 or
increased from C5 to C7. We are not quite sure why their
potency dramatically changed when we varied the link chain
length, probably because these oxazolidone imides with
different chain length are activated to different extent when
contacting the NAAA.
inhibition, and substitutions at the 2-position with (S)-methyl
(1f) or carbonyl (1g) slightly or moderately enhanced inhibition
towards NAAA without changing inhibition against FAAH.
However, (R)-methyl (1e), hydroxy (1j, 1k), methoxy (1l, 1m),
cyano (1n), formamide (1o), hydroxymethyl (1q, 1r) and
methoxymethyl (1s, 1t) substituents diminished the NAAA and
FAAH inhibitory effects (Table S1†). 1g exhibited low-
micromolar inhibition on NAAA activity (IC50 ¼ 4.6 ꢂ 0.48
mM), and it was over 5-fold more potent towards NAAA than
FAAH (Table 1). To further optimize the hydrophilic moieties,
a series of 1g isosteres were synthesized and tested (1h, 1i)
(Table 2). Enzymatic assay results showed that 1i with oxazoli-
done ring had similar effects on NAAA inhibition as 1g, but 1i
exhibited dramatically enhanced selectivity towards NAAA. Less
than 10% FAAH activity was inhibited by 1i even at a concen-
tration of 100 mM. Therefore, 2-oxazolidone ring was selected as
the template for further optimization.
To further optimize the NAAA inhibition activity of 2f, we
replaced the rotatable bond of the acyl chain of 2f with an
additional phenyl group. As shown in Table 4 and S2,† a series of
analogs corresponding to biphenyl (3a, 3k–3m), phenoxyphenyl
It has been reported that substitutions of the saturated alkyl
chain with aryl-containing lipophilic moieties could enhance
the inhibitory potency on NAAA and improved the drug-like
properties.14,21 Therefore, a series of oxazolidone derivatives
incorporating a phenyl ring at the terminus were synthesized
and tested. As shown in Table 3, 2a with the chain length of C0
exhibited low-micromolar inhibitory potency toward NAAA (IC50
Scheme
7 The synthesis of compounds 3e–3g. Reagents and
conditions: (a) bis(trichloromethyl) carbonate, Et3N, CH2Cl2, room
Scheme 5 The synthesis of compounds 1e and 1f. Reagents and temperature; (b) 4-benzylidenepiperidine, CH2Cl2, room temperature;
conditions: (a) PPH3, I2, THF, room temperature; (b) H2, Pd/C, CH3OH, (c) 4-benzylpiperidine, CH2Cl2, room temperature; (d) 1-benzylpi-
room temperature.
perazine, CH2Cl2, room temperature.
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Table
1 Inhibitory effects of compounds 1a–1g, (S)-OOPP and Table 3 Inhibitory effects of compounds 2a–2h on NAAA and FAAH
URB597 on NAAA and FAAH activities
activities
IC50 of
FAAH (mM)
IC50 of NAAA
(mM)
IC50 of FAAH
(mM)
Compound
n
IC50 of NAAA (mM)
Compound
R2
R3
2a
2b
2c
2d
2e
2f
0
1
2
3
4
5
6
7
3.4 ꢂ 0.42
>20
>20
12.4 ꢂ 2.0
8.3 ꢂ 1.8
0.27 ꢂ 0.04
6.3 ꢂ 1.2
>20
>20
>20
>20
>20
>20
>20
>20
>20
1a
1c
1d
1e
1f
1g
(S)-OOPP
URB597
–H
–H
–H
(R)-Me
(S)-Me
]O
–H
25 ꢂ 5.7
10 ꢂ 0.95
11 ꢂ 1.2
>100
15 ꢂ 2.3
4.6 ꢂ 0.48
0.83 ꢂ 0.1
>100
21 ꢂ 4.5
3.5 ꢂ 0.65
4.0 ꢂ 0.81
28 ꢂ 2.7
18 ꢂ 3.2
25 ꢂ 5.3
>20
(R)-NH2
(S)-NH2
–H
–H
–H
2g
2h
0.026 ꢂ 0.005
methylene unit to the aliphatic linker of 3c signicantly
decreased activity (3d). These results highlighted the impor-
tance of the optimal length and connecting pattern of the aryl-
containing lipophilic moieties.
(3b, 3n–3p), and benzyloxyphenyl (3c, 3q–3s) were obtained and
examined. Compared to 2a, 1,4-substituted phenoxyphenyl (3b,
IC50 ¼ 0.68 ꢂ 0.11 mM) and benzyloxyphenyl (3c, IC50 ¼ 2.3 ꢂ
0.36 mM) analogs were approximately 10-fold and 2-fold more
potent. However, 1,4-substituted bipheny analog 3a (IC50 ¼ 9.4 ꢂ
1.2 mM) was 2-fold less potent (Table 4), and 1,3-substituted
isomers (3k, 3l, 3n, 3o, 3q, 3r) were completely inert for NAAA
inhibition (Table S2†). The results indicated that straight
structures are preferred shape for the hydrophobic channel of
NAAA, which is in agreement with previous reports.14,17 The
binding pocket of NAAA proximal to the carbonyl moieties
seemed to be narrow, as such, only a phenyl moiety is tolerated
at this region. Therefore, incorporation of an extra methylene
group between the biaryl and carbonyl groups (3m, 3p and 3s)
signicantly decreased NAAA inhibition.
It's worth noting that the position of the terminal phenyl
groups in biaryl series compounds (3a–3d) is corresponding to
the location of phenyl moieties in the phenylalkyl series
compounds (2e–2h). Increasing the length of the terminal aryl
group of 3a by insertion of an oxygen atom or a methyleneoxy
unit between the phenyl rings led to 14-fold (3b) or 4-fold (3c)
higher potency, whereas the insertion of an additional
It has been reported that NAAA prefer saturated substrates
e.g., N-myristoylethanolamine and PEA, rather than unsatu-
rated substrates, e.g., AEA.1 Therefore, we studied whether the
replacement of a phenyl ring with a 6-member ring can improve
the NAAA inhibition. A series of additional derivatives of 2f were
then synthesized and examined (Table 5). The results showed
that introducing hexane ring further enhanced the NAAA
inhibitory potency. We found two highly potent NAAA inhibitors
3h and 3i with a IC50 value of 150 nM and 100 nM, respectively,
around 2-fold more potent than 2f. Moreover, saturated link-
ages were found to be more benecial for the activity than
unsaturated benzene ring (3h, 3i vs. 3a–3c; 3j vs. 2f). However,
nitrogen atoms substitution of the saturated hexane signi-
cantly reduced the activity (3e–3g).
2.3 NAAA inhibition pattern
To further understand the NAAA inhibition pattern of these
oxazolidone derivatives, compound 2f was selected as a model
Table 4 Inhibitory effects of compounds 3a–3d on NAAA and FAAH
activities
Table 2 Inhibitory effects of compounds 1h–1j on NAAA and FAAH
activities
IC50 of
IC50 of
Compound
n
R
IC50 of NAAA (mM)
FAAH (mM)
Compound
X
IC50 of NAAA (mM)
FAAH (mM)
3a
3b
3c
3d
0
0
0
0
4-Ph
4-OPh
4-OBn
4-OCH2CH2Ph
9.4 ꢂ 1.2
0.68 ꢂ 0.11
2.4 ꢂ 0.36
>20
>20
>20
>20
>20
1g
1h
1i
C
NH
O
4.6 ꢂ 0.48
>100
4.8 ꢂ 0.63
25 ꢂ 5.3
27 ꢂ 4.9
>100
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Table 5 Inhibitory effects of compounds 3e–3j on NAAA and FAAH
activities
IC50 of NAAA
IC50 of FAAH
Compound
R
(mM)
(mM)
3e
>20
>20
>20
3f
15.3 ꢂ 2.4
3g
3h
>20
21.2 ꢂ 4.2
>20
0.15 ꢂ 0.03
0.10 ꢂ 0.03
Fig. 3 Characterization of 2f as a reversible and noncompetitive NAAA
inhibitor. (A) Concentration-dependent inhibitory effect of 2f on the
rat NAAA. (B) NAAA activity in the presence of vehicle (open columns)
or 2f (closed columns) before and 12 h after dialysis. ** P < 0.01 vs.
vehicle, n ¼ 3; (C) rapid dilution NAAA assay in the presence of vehicle
(1% DMSO, open circles) or 2f (closed circles). (D) Michaelis–Menten
analysis of the NAAA reaction in the presence of vehicle (1% DMSO,
closed circles) and 2f (0.5 mM, triangles).
3i
3j
>20
0.19 ꢂ 0.02
>20
a antagonist MK886 (2 mg kgꢀ1, i.p.), which indicated that 2f
relieve pain in a PPAR-a dependent pathway.
molecule and its interaction mode with NAAA protein in cells
was studied. The results showed that 2f displayed a profound
inhibition toward recombinant human NAAA (IC50 ¼ 0.27 ꢂ
0.04 mM, Fig. S1A†) and recombinant rat NAAA in NAAA-
transfected HEK293 cells (IC50 ¼ 0.42 ꢂ 0.04 mM, Fig. S1B†),
which were similar to the data obtained with the NAAA protein
extract. Dialysis (Fig. 3B) and rapid dilution (Fig. 3C) of the 2f–
NAAA interaction complex almost completely restored the NAAA
activity. Moreover, Michaelis–Menten kinetic analysis revealed
The anti-inammatory activity of 2f was tested in 12-o-
tetradecanoylphorbol-13-acetate (TPA) induced mice ear edema
model. A commercial nonsteroidal anti-inammatory drug,
ibuprofen (200 mg kgꢀ1) was intragastrically administrated as
a positive control. The results showed that intragastrical
administration of 2f (30, 100 mg kgꢀ1) signicantly reduce ears
edema. Oral administration of compound 2f (30 mg kgꢀ1) pre-
sented the same efficacy with ibuprofen (200 mg kgꢀ1). More-
over, 2f signicantly prevent the production of proinammatory
cytokines, including TNF-a, IL-6, COX-2 and NF-kB in mRNA
level (Fig. 5).
For many nonsteroidal anti-inammatory drugs, such as
COX-2 inhibitors, adverse gastrointestinal effects and increased
cardiovascular risks are two main limitations for their clinical
application. We tested the toxicity of 2f by monitoring its
inuence on food intake, body weight and ether-a-go-go-related-
gene (hERG) channel activity. Long-term studies suggested that
intragastrical administration of 2f (30 mg kgꢀ1 for 30 days,
100 mg kgꢀ1 for 10 days) had no signicant effects on food
intake and body weight in mice (Fig. S2†). Mice stomach
anatomy indicated that 2f did not cause gastrointestinal
hemorrhages, while indomethacin (20 mg kgꢀ1 for 3 days, ig)
resulted in obvious hemorrhages (Fig. S3†). The low gastroin-
testinal side effects of 2f may be because it induces analgesia
and anti-inammatory via enhancement of PEA rather than
reducing prostaglandins (PGs) in the gut. Moreover, the activity
of hERG channels in CHO cells was only inhibited by 38.3% by
that 2f induced changes in the maximal catalytic velocity (Vmax
)
of NAAA (Vmax in nmol per min per mg protein, vehicle: 6.0 ꢂ
0.51; and 2f, 0.3 mM: 1.9 ꢂ 0.15, Fig. 3D) but did not affect
Michaelis–Menten constant Km (Km in mM, vehicle: 174 ꢂ 26;
and 2f, 0.3 mM: 212 ꢂ 24). These results indicated that 2f is
a reversible and noncompetitive NAAA inhibitor.
2.4 In vivo anti-inammatory and analgesic activities of 2f
Finally, the in vivo anti-inammation and analgesic activities of
2f was studied in different pain and inammation mice models.
Experimental mice were treated with 2f via intraperitoneal
injection or intragastrical administration. The behavioural
response of Mice to formalin stimulation was divided to the
rst-phase of acute pain (0–10 min) and the second-phase of
chronic pain (15–35 min). Single treatment of 2f (10 mg kgꢀ1
,
i.p.) decreased second-phase but not rst-phase (Fig. 4), sug-
gesting a central mechanism of action of 2f. The reduced licking
time by 2f at the second-phase was reversed by PPAR-
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3 Conclusion
In conclusion, we report the discovery of oxazolidone deriva-
tives as a novel scaffold of NAAA inhibitors. The NAAA inhibi-
tion activity of oxazolidone derivatives was modulated by the
side chain and terminal lipophilic substituents. The results
showed that the link chain length of C5, straight and saturated
linkages were the preferred shape patterns. Several nanomolar
NAAA inhibitors (2f, 3b, 3h, 3i and 3j) were developed.
Compound 2f exhibited highly potent and selective inhibitory
activity towards NAAA via a competitive and reversible pattern.
Moreover, in vivo study showed that 2f had signicant anti-
inammatory and analgesic effects with low side effects. Our
study demonstrated that oxazolidone derivatives is a class of
potent NAAA inhibitors holds great promise for clinical trans-
lation. We anticipate that these oxazolidone derivatives will
help better understand the function of NAAA and facilitate the
development of improved therapies for inammation and pain.
Fig. 4 2f attenuated formalin-induced pain behaviors. (A) 2f (10 mg
kgꢀ1, i.p., open squares), injected 45 min prior to formalin treatment,
produced time-dependent changes in composite pain score relative
to vehicle (5% PEG/5% Tween-80 in saline, 10 mL kgꢀ1, i.p., open
cycles), or a combination of 2f and PPAR-a antagonist MK886 (2 mg
kgꢀ1, i.p., close squares). * P < 0.1 vs. vehicle; (B) 2f (close bar) reduced
the licking time during phase 2 of the formalin response, ** P < 0.1 vs.
vehicle (open bar). MK886 (2 mg kgꢀ1, i.p., close bar) prevented the
anti-nociceptive effects of F96, # P < 0.1 vs. 2f. V represents vehicle
(5% PEG/5% Tween-80 in saline), and MK represents PPAR-a antago-
nist MK 886.
4 Materials and methods
4.1 Materials and instruments
All reagents used in the present study were purchased from
Sigma-Aldrich (Shanghai, China) unless otherwise indicated.
Tetrahydrofuran was distilled prior to use from sodium
benzophenone ketyl. Dichloromethane (CH2Cl2) was distilled
from phosphorus pentoxide. Dimethylformamide (DMF) was
distilled from calcium hydride. Silica gel (300–400 mesh) from
Yantai Athy Chemical Technology Co. Ltd. (Zhifu, China) was
used for column chromatography, and compounds were eluted
ꢁ
with an ethyl acetate/petroleum ether (PE) (60–90 C) mixture
(unless otherwise stated).
The 1H-NMR and 13C-NMR spectra were recorded on
1
a Bruker 400 spectrometer (400 MHz for H, 100 MHz for 13C),
using CDCl3, d6-DMSO, or CD3OD as solvent and Me4Si as
internal substance. IR spectra were recorded on a Nicolet Avatar
360 RT-IR spectrophotometer. Mass spectra were recorded on
an Applied Bio systems MDS SCIEX 3200Q-TRAP mass spec-
trometry (MS) system with electro spray ionization (ESI†) and
direct injection. The HRFABMS spectra were recorded on
a Bruker APEX-FTMS apparatus. Elemental analysis was per-
formed using a Vario RL analyzer. Melting points were deter-
mined on a Yanaco MP-500 melting point apparatus and are
uncorrected. HPLC analysis were run on Agilent-1200 series
HPLC system equipped with a photodiode array detector, using
a Hypersil Gold C18 column (dimensions 250 ꢃ 4.6 mm,
particle size 5 mm). Photodiode array (PDA) detector range was
set as 210–600 nm.
Fig. 5 Compound 2f alleviated TPA-induced inflammation. Effects of
oral administration of 2f and ibuprofen on mice ears edema (A) and
mRNA expression levels of TNF-a (B), IL-6 (C), COX-2 (D) and NF-kB
(E) in mice ear treated with vehicle (open bars) or TPA (closed bars).
Vehicle, 0.1% DMSO; TPA, 0.03% in acetone. ### P < 0.001 vs. vehicle;
* P < 0.1, ** P < 0.01, *** P < 0.001 vs. TPA control, n ¼ 8–10.
4.2 Synthesis of oxazolidone derivatives
40 mM 2f (Fig. S4†), a concentration far above its threshold for
NAAA inhibition. However, cisapride, a COX-2 inhibitor,
inhibited 50% hERG at a concentration of 0.1 mM. These results
suggested that with lower gastrointestinal and cardiovascular
side effects, oxazolidone derivatives are promising anti-
inammatory drug candidates in clinical translation.
4.2.1 Synthesis of amides 1a, 1j, 1k, 1n, 1o and 1q–1t.
Palmitoyl chloride (83 mg, 0.3 mmol) in CH2Cl2 (2 mL) was
added dropwise to a mixture of amine (0.3 mmol) and Et3N (0.08
mL, 0.6 mmol) in anhydrous CH2Cl2 (2 mL) under a nitrogen
atmosphere. The resulting solution was stirred at room
temperature for 4 h, then quenched with saturated aqueous
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ammonium chloride, and extracted with CH2Cl2 (3 ꢃ 10 mL). was slowly added. The reaction mixture was stirred at ꢀ78 ꢁC for
The combined organic phases were washed with brine, dried 0.5 h and allowed to warm slowly to room temperature over 5 h.
over anhydrous Na2SO4, ltered, and concentrated under The reaction was quenched with saturated aqueous ammonium
reduced pressure. The residue was puried by ash chroma- chloride (1.0 mL) and extracted with EtOAc (3 ꢃ 5 mL). The
tography on silica gel and 1a, 1j, 1k, 1n, 1o and 1q–1t were then combined organic layers were treated using the same procedure
obtained.21
4.2.2 Synthesis of amides 1l and 1m. A solution of 1-(3-
as 2.2.1 to afford 1i, 2a–2h, 3a–3d and 3h–3s.29
4.2.6 Synthesis of acids 3q-1 and 3t-1. Thionyl dichloride
hydroxypyrrolidin-1-yl)hexadecan-1-one (19 mg, 0.06 mmol) in (0.2 mL, 3 mmol) was slowly added to 10 mL CH3OH solution of
THF (1 mL) was added slowly to a suspension of NaH (60% in the corresponding carboxylic acid (2.0 mmol) under a nitrogen
mineral oil, 5 mg) in anhydrous THF (5 mL), under a nitrogen atmosphere at 0 ꢁC. The reaction mixture was stirred at 0 ꢁC for
ꢁ
atmosphere at 0 C. Aer stirring at the same temperature for 2 h and evaporated under reduced pressure. The product was
5 min, a solution of CH3I (0.15 mmol) in THF (0.5 mL) was dissolved in acetone (15 mL) and treated with K2CO3 (552 mg, 4
ꢁ
slowly added. The reaction mixture was stirred at 0 C for 3 h mmol) and alkyl bromide (2.5 mmol). The reaction mixture was
before quenched with saturated aqueous ammonium chloride ltered and concentrated under reduced pressure aer being
(5 mL) and extracted with EtOAc (3 ꢃ 5 mL). The combined reuxed for 8 h. The residue was dissolved in a 1 : 1 mixture of
organic layers were treated using the same procedure as 2.2.1 to aqueous 2 M NaOH and MeOH (16 mL), stirred at room
afford 1l and 1m.23
temperature for 4 h, then treated with 2 M HCl, and extracted
4.2.3 Synthesis of amides 1c and 1d. A solution of 4 M HCl with EtOAc (3 ꢃ 5 mL). The combined organic layers were dried
(1 mmol, 0.25 mL dioxane) was added slowly to 10 mL tert-butyl over anhydrous Na2SO4, ltered, and concentrated under
1-palmitoylpyrrolidin-3-ylcarbamate (60.0 mg, 0.14 mmol) reduced pressure. The residue was puried by ash chroma-
methanol solution at 0 ꢁC under a nitrogen atmosphere. The tography on silica gel to afford 3q-1 and 3t-1.14
resulting solution was stirred at 0 ꢁC for 2 h, quenched with
4.2.7 Synthesis of carbamates 3e–3g. A CH2Cl2 solution (2
a saturated aqueous solution of Na2CO3, and extracted with mL) of bis(trichloromethyl) carbonate (98 mg, 0.33 mmol) was
EtOAc (3 ꢃ 20 mL). The combined organic phases were washed added dropwise to a solution of 2-oxazolidone (44 mg, 0.5
with brine, dried over anhydrous Na2SO4, ltered, and mmol) and Et3N (0.16 mL, 1.2 mmol) in CH2Cl2 (5 mL) under
ꢁ
concentrated under reduced pressure. The residue was puried a nitrogen atmosphere. Aer stirring at 0 C for 1 h and room
by ash chromatography on silica gel (eluent : MeOH/CH2Cl2 ¼ temperature for 2 h, the amine (0.4 mmol) in CH2Cl2 (1 mL) was
1 : 15) to afford 1c and 1d.25,26
added dropwise to the mixture under a nitrogen atmosphere.
4.2.4 Synthesis of amides 1e and 1f. I2 solution (76 mg, 0.3 The resulting mixture was stirred at room temperature for 5 h,
mmol) in THF (2 mL) was added slowly to a mixture of imid- quenched with saturated aqueous ammonium chloride, and
azole (27 mg, 0.4 mmol) and PPh3 (79 mg, 0.3 mmol) in THF (5 extracted with CH2Cl2 (3 ꢃ 10 mL). The combined organic
ꢁ
mL), under a nitrogen atmosphere at 0 C. Aer stirring at the phases were washed with brine, dried over anhydrous Na2SO4,
same temperature for 1 h, a THF solution (2 mL) of 1-(2- ltered, and concentrated under reduced pressure. The residue
(hydroxymethyl) pyrrolidin-1-yl) hexadecan-1-one (71 mg, 0.2 was puried by ash chromatography on silica gel to afford 3e–
mmol) was slowly added. The reaction mixture was stirred at 3g.30
0 ꢁC for 0.5 h and allowed to warm slowly to room temperature
over 2 h. The reaction was quenched with a saturated solution of
4.3 In vitro biological evaluation of oxazolidone derivatives
Na2S2O3 and extracted with EtOAc (3 ꢃ 5 mL). The combined
organic layers were dried over anhydrous Na2SO4, ltered, and
4.3.1 HPLC-MS/MS analysis. A HPLC-MS/MS method was
concentrated under reduced pressure. The mixture in EtOAc (10 developed and validated for free fatty acids (FFAs) quantica-
mL) was treated with 10% Pd/C (100 mg) and purged with H2. tion. An ABI 3200 Q-Trap mass spectrometer equipped with
Aer stirring for 12 h at room temperature, the reaction mixture Agilent 1200-Hꢁ PLC system was used. FFAs were eluted at 1.0 mL
was ltered through celite and concentrated. The residue was minꢀ1 at 40 C with MeOH : water 95 : 5, v/v, each containing
puried by ash chromatography on silica gel (eluent : EtOAc/ 0.25% acetic acid and 5 mmol Lꢀ1 ammonium acetate, pH ¼
PE 1 : 5) to afford 1e and 1f.27,28
7.4. The molecular ions were monitored by ESI† negative mode
4.2.5 Synthesis of oxazolidone imides 1i, 2a–2h, 3a–3d and at m/z ¼ 267 for heptadecenoic acid, m/z 303 for arachidonic
3h–3s. A CH2Cl2 (5 mL) solution of oxalyl dichloride (0.06 mL, acid, and m/z 269 for C17:0 FFAs.
0.66 mmol) was added slowly to a solution of the suitable acid
4.3.2 Protein preparation and enzymatic assay. HEK293
(0.55 mmol) and dimethylformamide (0.05 mL) under cells overexpressing rat NAAA (HEK293-rNAAA) or rat FAAH
a nitrogen atmosphere at 0 ꢁC. The reaction mixture was stirred (HEK293-rFAAH) were maintained in Dulbecco's modied Eagle
at 0 ꢁC for 1 h and evaporated under reduced pressure. The acid medium (DMEM, Hyclone, Beijing, China) supplemented with
chloride obtained was used as such for the next reaction due to 10% fetal bovine serum (FBS, Gibco®, Shanghai, China) con-
its high instability. A hexane solution (0.2 mL) of 2.5 Mn-BuLi taining 0.3 mg mLꢀ1 geneticin G418. HEK293-rNAAA or
(0.55 mmol, hexane) was added slowly to a THF solution (5 HEK293-rFAAH cells were harvested, washed with PBS, soni-
mL) of 2-oxazolidone (44 mg, 0.5 mmol), under a nitrogen cated in 20 mM Tris–HCl (pH 7.5) containing 0.32 M sucrose,
ꢁ
atmosphere at ꢀ78 C. Aer stirring at the same temperature and centrifuged at 800 ꢃ g for 15 min at 4 ꢁC. The supernatants
for 10 min, a THF solution (2 mL) of acid chloride (0.55 mmol) were collected, and the protein concentrations were measured
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by a BCA protein assay kit (Pierce, Shanghai, China). NAAA Compound 2f (30, 100 mg kgꢀ1) was then administered by
activity was measured by incubating 30 mg recombinant rNAAA intragastric gavage 30 min before TPA application. TPA (0.03%
protein with 25 mM heptadecenoylethanolamide at 37 ꢁC for in acetone, 10 mL) was applied evenly to both sides of the le ear
30 min in 0.2 mL of phosphate buffer (50 mM, pH 5.0) con- of each mouse to cause edema and inammation, while the
taining 0.1% Triton X-100 and 3 mM dithiothreitol with or same amount of acetone alone was smeared to the right ear.
without the tested compounds. FAAH activity was measured by Animals were sacriced 3 h aer treatment. Both ears were
incubating 30 mg of protein derived from the HEK293-rFAAH excised immediately and punched in the same position, with
cell extract at 37 ꢁC in Tris–HCl buffer (50 mM, pH 8.0) con- a diameter of 9 mm. The ear swelling was determined by
taining fatty acid-free bovine serum albumin (0.05%). Ananda- weighing pieces of the ears obtained. The mRNA expression
mide (25 mM) was used as the substrate for FAAH. The reactions levels of TNF-a, IL-6, COX-2 and NF-kB in mice ear were deter-
were terminated by adding 200 mL of methanol containing 1 mined following our previously reported method.21
nmol heptadecanoic acid as internal standard, and the
remaining substrates were analysed by HPLC-MS/MS. IC50 of with compound 2f (100 mg kgꢀ1 for 10 d, 30 mg kgꢀ1 for 30 d) or
each compound was analysed by GraphPad Prism 5.
indomethacin (20 mg kgꢀ1 for 3 d) via intragastrical adminis-
4.4.3 Long term toxicity of compound 2f. Mice were treat
4.3.3 Dialysis assay. The dialysis assay was performed tration with normal diet. Mice body weight and food
using Slide-A-Lyzer dialysis cassettes (Pierce, Shanghai, China). consumption were measured and recorded every day. The mice
Briey, 2 mg of NAAA protein was incubated with 2f or dimethyl were sacriced 6 h aer the last administration and stomachs
sulfoxide (DMSO) in 4 mL of Tris–HCl buffer (50 mM, pH 5.0) were harvested, rinsed with distilled water and photographed.
for 10 min at 37 ꢁC. The mixed reaction solution was loaded into
a dialysis cartridge using a syringe and incubated in Tris–HCl
buffer (50 mM, pH 5.0) at 4 ꢁC for 8 h. The samples in the
Acknowledgements
dialysis cassettes were collected for the NAAA enzymatic assay. We thank Dr Daniele Piomelli at the University of California,
4.3.4 Rapid dilution assay. The rapid dilution assay was Irvine for the kind gis of the HEK293-rNAAA cells and the
performed following a previous reported method.21 Briey, HEK293-rFAAH cells. YL also wants to thank William stung for
samples containing 100-fold concentrated rNAAA protein were English revision. This work was supported by funds from the
pre-incubated with 10-fold of the IC50-equivalent concentration National Natural Science Foundation of China (Grant No.
ꢁ
of compounds or vehicle (1% DMSO) for 10 min at 37 C. The 81373273 to QY and 81602974 to YL), Xiamen Southern Ocean
samples were then diluted 100-fold with assay buffer containing Research Center Project (No. 14GYY018NF18) to QY, the
substrate to initiate the reactions, and the time course of Fundamental Research Funds for the Central Universities (No.
product formation was determined by HPLC-MS analysis.
20720150054) to QY and China Postdoctoral Science Founda-
tion Funded Project (No. 2016M592876XB) to YL.
4.4 In vivo anti-inammatory and analgesic activity of
compound 2f
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