Identification of highly potent N-acylethanolamine acid amidase (NAAA) inhibitors: Optimization of the terminal phenyl moiety of oxazolidone derivatives

Article abstract of DOI:10.1016/j.ejmech.2017.08.004

N-acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that participates in the deactivation of fatty acid ethanolamides, such as palmitoylethanolamide (PEA). NAAA inhibition may provide a potential therapeutic strategy for the treatment of diseases in which higher PEA level is desired. In the present study, we reported the structure-activity relationship (SAR) studies for oxazolidone derivatives as NAAA inhibitors. A series of substituents or alkyl replacements for the terminal phenyl ring of oxazolidone derivatives were examined. The results showed that the inhibition potency of these oxazolidone derivatives towards NAAA depends on the sizes, flexibility, and lipophilicity of the terminal groups. SAR results suggested that small lipophilic 3-phenyl substituents or hydroxy-containing 4-phenyl substituents were preferable for optimal potency. Furthermore, the distal aliphatic replacement is also preferred for high inhibitory potency. Rapid dilution and kinetic analysis suggested that oxazolidone derivatives with different terminal phenyl moieties inhibited NAAA via different mechanisms. This study identified several highly potent NAAA inhibitors, including 1a (F215, IC₅₀ = 0.009 μM), 1o (IC₅₀ = 0.061 μM) and 2e (IC₅₀ = 0.092 μM), and also determined structural requirements of oxazolidone derivatives for potent inhibition against NAAA.

Full text of DOI:10.1016/j.ejmech.2017.08.004

Accepted Manuscript
Identification of highly potent N-acylethanolamine acid amidase (NAAA) inhibitors:
Optimization of the terminal phenyl moiety of oxazolidone derivatives
Yuhang Li, Qi Chen, Longhe Yang, Yanting Li, Yang Zhang, Yan Qiu, Jie Ren,
Canzhong Lu
PII:
S0223-5234(17)30607-4
10.1016/j.ejmech.2017.08.004
EJMECH 9648
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 June 2017
Revised Date: 24 July 2017
Accepted Date: 2 August 2017
Please cite this article as: Y. Li, Q. Chen, L. Yang, Y. Li, Y. Zhang, Y. Qiu, J. Ren, C. Lu, Identification
of highly potent N-acylethanolamine acid amidase (NAAA) inhibitors: Optimization of the terminal phenyl
moiety of oxazolidone derivatives, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.08.004.
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ACCEPTED MANUSCRIPT
Identification of highly potent N-acylethanolamine acid amidase (NAAA) inhibitors:
Optimization of the terminal phenyl moiety of oxazolidone derivatives
Yuhang Lia,b, Qi Chenc, Longhe Yangc, Yanting Lic, Yang Zhangc, Yan Qiuc*, Jie
Renc, and Canzhong Lua,b*
a CAS Key Laboratory of Design and Assembly of Functional Nanostructures, and
Fujian Provincial Key Laboratory of Nanomaterials, Fujian Institute of Research on
the Structure of Matter, Chinese Academy of Sciences;
b Xiamen Institute of Rare-earth Materials, Haixi Institutes, Chinese Academy of
Sciences, Fujian 361005, P. R. China;
c Medical College, Xiamen University, Xiamen, Fujian 361102, P. R. China;
* Corresponding author:
Canzhong Lu, czlu@fjirsm.ac.cn (CL.)
Yan Qiu, yanqiu@xmu.edu.cn (YQ)
1

ACCEPTED MANUSCRIPT
Graphical abstract
Highlights
Structure-activity relationship studies on oxazolidone derivatives to identify the
structural requirements for inhibition of N-acylethanolamine acid amidase (NAAA) .
Selectivity and inhibition mechanism studies on the newly synthesized NAAA
inhibitors based on oxazolidone derivatives.
Discovery of 1a (F215) as a highly selective and nanomolar potent NAAA inhibitor.
2

ACCEPTED MANUSCRIPT
Abstract
N-acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that participates in
the deactivation of fatty acid ethanolamides, such as palmitoylethanolamide (PEA).
NAAA inhibition may provide a potential therapeutic strategy for the treatment of
diseases in which higher PEA level is desired. In the present study, we reported the
structure-activity relationship (SAR) studies for oxazolidone derivatives as NAAA
inhibitors. A series of substituents or alkyl replacements for the terminal phenyl ring
of oxazolidone derivatives were examined. The results showed that the inhibition
potency of these oxazolidone derivatives towards NAAA depends on the sizes,
flexibility, and lipophilicity of the terminal groups. SAR results suggested that small
lipophilic 3-phenyl substituents or hydroxy-containing 4-phenyl substituents were
preferable for optimal potency. Furthermore, the distal aliphatic replacement is also
preferred for high inhibitory potency. Rapid dilution and kinetic analysis suggested
that oxazolidone derivatives with different terminal phenyl moieties inhibited NAAA
via different mechanisms. This study identified several highly potent NAAA
inhibitors, including 1a (F215, IC50 = 0.009 µM), 1o (IC50 = 0.061 µM) and 2e (IC50 =
0.092 µM), and also determined structural requirements of oxazolidone derivatives for
potent inhibition against NAAA.
Keywords
N-acylethanolamine acid amidase (NAAA), palmitoylethanolamide (PEA), NAAA
inhibitor, oxazolidone derivatives, structure-activity relationship (SAR)
3

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1. Introduction
N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase enzyme widely
expressed in immune cells, e.g., macrophages and B-lymphocytes, is one of the key
enzymes in the degradation of fatty acid ethanolamides, especially for
palmitoylethanolamide (PEA) [1-3]. PEA involves in various physiological processes,
such as the regulation of pain, inflammation and cell degranulation, through the
activation of nuclear peroxisome proliferator-activated receptor α (PPAR-α) [4-7].
Pharmacological blocking of NAAA increases endogenous PEA levels in rodent
models, inducing analgesic and anti-inflammatory effects [8, 9]. Inhibition of NAAA
showed no undesirable cardiovascular effects and gastrointestinal haemorrhaging as
seen with Cyclo-oxygenase-2 (COX-2) inhibitors [10, 11]. NAAA inhibitors,
therefore, provide a potential therapeutic strategy for the treatment of pain,
inflammatory and other related diseases [12].
Although development of potent NAAA inhibitors has attracted increasing interest
recently, only a few potent NAAA inhibitors have been identified so far [12-14]. Most
of the recently reported potent inhibitors are the reactive β-lactone/β-lactam
derivatives, including ARN077 and ARN726, which can form a covalent bond with
the cysteine nucleophile of NAAA, thereby inactivating NAAA [8, 15-19]. However,
inhibitors with such structure show low chemical and plasma stability, thus limiting
their therapeutic applications [18]. Another category of NAAA inhibitors are
oxazolidone derivatives, such as 1 (F96) and 2 [10, 11]. Inhibitors with such structure
are potent and selective towards NAAA inhibition, and are also systemically active
and exhibited low toxicity in animal models [10, 11]. In vivo studies showed that
compound 1 effectively normalized PEA levels in inflamed tissues. The profound
analgesic and anti-inflammatory effects was seemed in several animal models,
including acetic acid-induced visceral pain, formalin-induced inflammatory pain,
sciatic
nerve
injury
(SNI)-induced
neuropathic
pain,
and
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema models [10, 11].
We recently reported a structure-activity relationship (SAR) study targeting the
oxazolidone ring and the link chain of 1, and disclosed several selective and potent
NAAA inhibitors [11]. In the present study (Figure 1), we further modified the
terminal phenyl group of 1 and 2 to develop more potent NAAA inhibitors and to
better understand the structure-activity relationship of oxazolidone derivatives. We
explored the sizes, flexibility, and lipophilic requirements of the terminal moiety for
potent NAAA inhibition, and also evaluated the impact of these structural features on
the inhibition mechanism. These studies allowed us to identify a series of highly
potent NAAA inhibitors, including 1a (IC50 = 0.009 µM), 1o (IC50 = 0.061 µM) and
2e (IC50 = 0.092 µM), and disclosed the structural requirements of the terminal moiety
of oxazolidone derivatives for potent NAAA inhibition.
4

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Previous studies
Explored herein
O
O
O
O
N
O
Cl
N
O
1a, IC50 = 0.009 µM
1, IC50 = 0.27 µM
O
O
O
Explored herein
O
N
N
O
O
O
O
2e, IC50 = 0.092 µM
2, IC50 = 0.68 µM
Figure 1. Design strategy for oxazolidone derivatives as NAAA inhibitors.
2. Chemistry
Oxazolidone imides 1a-1d, 1f, 1k-1n, 1p, 1aa, 1bb, 2a, 2c, 2d-2i and 2k were
prepared by imidation of the appropriate acyl chloride with 2-oxazolidone. The acyl
chlorides were obtained by reaction of corresponding acids with oxalyl dichloride
(Scheme 1) [11]. Acids 1a-1~1d-1, 1f-1, 1k-1~1n-1, 1p-1, 1aa-1, 1bb-1 were
synthesized by a catalytic hydrogenation of the respective intermediates, prepared by
the Wittig reaction in the presence of lithium hexamethyldisylazane (LHMDS)
(Scheme 2) [20]. Acids 2a-1, 2c-1 and 2d-1 were prepared by copper-mediated
cross-coupling reactions with methyl 4-hydroxybenzoate and phenylboronic acids,
and subsequent hydrolysis of corresponding esters (Scheme 3) [21]. Acids 2e-1~2i-1
and 2k-1 were obtained by reactions of methyl 4-hydroxybenzoate with alcohols and
subsequent hydrolysis of esters (Scheme 4) [21]. Oxazolidone imides 1e, 1g, 1h, 1o,
1q and 1r were prepared by catalytic hydrogenation of the O-protected intermediates,
obtained by the reactions of acids 1e-1, 1g-1, 1h-2, 1o-1, 1q-1, 1r-2 with
2-oxazolidone (Scheme 5). Acids 1e-1, 1g-1, 1o-1, 1q-1 were synthesized by Wittig
reaction of the phosphonium salt and corresponding O-protected aldehyde (Scheme 6)
[20]. Acids 1h-2 and 1r-2 were synthesized by the Wittig reactions of commercially
unavailable aldehyde 1h-1 and 1r-1, generated by a palladium catalyzed arylation of
allylic alcohol with iodobenzene (Scheme 7) [22]. Compounds 1i and 1s were
obtained by reaction of 1h and 1r with oxalyl dichloride, respectively, followed by
treatment with ammonium bicarbonate (NH4HCO3) in acetonitrile (CH3CN) (Scheme
8). Compounds 1j and 1t were obtained by the Curtius rearrangement of appropriate
acylazide, prepared by the 1h and 1r with diphenylphosphorylazide (DPPA),
respectively (Scheme 9) [20].
3. Enzyme assay
The NAAA inhibition activity of newly synthesized compounds was tested using
heptadecenoylethanolamide (C17:1 FAE) as substrate, and the residual hydrolysis
products of the substrate were determined by liquid chromatography-mass
spectrometer (LC-MS). NAAA inhibitor (S)-OOPP (10 µM) was used as the positive
5

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control. All statistical analysis was performed using the Graphpad Prism 5 software
package, and IC50 values were calculated from concentration response curves using
nonlinear regression analysis. All experiments were performed in triplicate (n = 3).
Scheme 1. Synthesis of compounds 1a-1d, 1f, 1k-1n, 1p, 1aa, 1bb, 2a, 2c-2k. Reagents
and conditions: (a) oxalyl dichloride, cat. DMF, CH2Cl2, 0°C-rt, 1 h; (b) n-BuLi,
2-oxazolidone, THF, −78°C-rt, 4 h.
Scheme 2. Synthesis of compounds 1a-1~1d-1, 1f-1, 1k-1~1n-1, 1p-1, 1aa-1, 1bb-1.
Reagents and conditions: (a) LHMDS, THF, −78°C-room temperature (rt), overnight; (b)
H2, Pd/C, CH3OH, rt, 2 h.
O
O
R4
R3
a, b
OH
R4
R3
HO
O
+
(HO)2B
O
2a-1, 2c-1, 2d-1
R3 = Cl, R4 = H, 2a-1
R3 = Me, R4 = H, 2c-1
R3 = H, R4 = Me, 2d-1
6

ACCEPTED MANUSCRIPT
Scheme 3. Synthesis of compounds 2a-1, 2c-1 and 2d-1. Reagents and conditions: (a)
Cu(OAc)2, pyridine, 4 Å molecular sieves; (b) NaOH, EtOH/H2O, then 3N HCl
O
O
O
a
b
R OH
O
+
O
HO
OH
OR
OR
3
2e-1~2i-1, 2k-1
2g-1
2h-1
2i-1
R =
2e-1
2f-1
O
2k-1
Scheme 4. Synthesis of compounds 2e-1~2i-1 and 2k-1. Reagents and conditions: (a)
DIAD, PPh3, THF, 0°C-rt, 4 h; (b) NaOH, EtOH/H2O, then 3N HCl.
R4
R3
1e-1
O
HO
O
O
R4 = H, R3 = OBn
R4 = H, R3 = CH2OBn 1g-1
R4 = OBn, R3 = H 1o-1
R4 = CH2OBn, R3 = H 1q-1
R3
R4
N
4
O
a-c
1e, 1g, 1h, 1o, 1q, 1r
R4 = H, R3 = OH
1e
R4 = H, R3 = CH2OH 1g
R4 = H, R3 = COOH 1h
R4
R3
O
1o
1q
1r
R4 = OH,
R3 = H
R4 = CH2OH, R3 = H
R4 = COOH, R3 = H
HO
R3 = COOBn, R4 = H, 1h-2
R3 = H, R4 = COOBn, 1r-2
Scheme 5. Synthesis of compounds 1e, 1g, 1h, 1o, 1q and 1r. Reagents and conditions: (a)
oxalyl dichloride, cat.DMF, CH2Cl2, 0°C-rt, 1 h; (b) n-BuLi, 2-oxazolidone, THF,
−78°C-rt, 4 h; (c) H2, Pd/C, CH3OH, rt, 2 h
O
R4
O
a
OHC
R3
R4
PPh3Br
HO
+
4
HO
R3
3
1e-1, 1g-1, 1o-1, 1q-1
R4 = H, R3 = OBn
1e-1
R4 = H, R3 = CH2OBn 1g-1
R4 = OBn,
R3 = H 1o-1
R4 = CH2OBn, R3 = H 1q-1
Scheme 6. Synthesis of compounds 1e-1, 1g-1, 1o-1 and 1q-1. Reagents and conditions:
(a) LHMDS, THF, −78°C-rt, overnight;
7

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R4
R3
R4
R3
R4
b
a
O
HO
I
OHC
R3
1h-2, 1r-2
1h-1, 1r-1
R3 = COOBn, R4 = H, 1h-2
R3 = H, R4 = COOBn, 1r-2
R3 = COOBn, R4 = H, 1h-1
R3 = H, R4 = COOBn, 1r-1
Scheme 7. Synthesis of compounds 1h-2 and 1r-2. Reagents and conditions: (a) allyl
alcohol, Pd(OAC)2, TBACl, NaHCO3, DMF, 50°C; (b) BrPh3PCH2CH2COOH, LHMDS,
THF, −78°C-rt, overnight
O
O
O
O
a, b
R3
R4
R3
R4
N
N
4
4
O
O
1h, 1r
1i, 1s
R4 = H, R3 = COOH 1h
R4 = COOH, R3 = H 1r
R4 = H, R3 = CONH2 1i
R4 = CONH2, R3 = H 1s
Scheme 8. Synthesis of compounds 1i and 1s. Reagents and conditions: (a) oxalyl
dichloride, CH2Cl2, rt, 1 h; (b) NH4HCO3, rt, 2 h.
O
O
O
O
a, b
R3
R4
R3
R4
N
N
4
4
O
O
1h, 1r
1j, 1t
R4 = H, R3 = COOH 1h
R4 = COOH, R3 = H 1r
R4 = H, R3 = NH2 1j
R4 = NH2, R3 = H 1t
Scheme 9. Synthesis of compounds 1j and 1t. Reagents and conditions: (a) DPPA, Et3N,
toluene, rt, 30 min; (b) reflux, 30 min.
4. Results and discussion
4.1 Substitution of the terminal phenyl ring of 1
In preliminary SAR study, a series of phenyl substitutions for the terminal phenyl ring
of 1 were evaluated. (Table 1, Table S1). Substitution at the position 2 with either
lipophilic or polar groups (2-CH3, 3; 2-Cl, 4; or 2-OH, 5) reduced inhibitory potency
compared to the unsubstituted phenyl derivative 1, indicating that position 2 is
unsuitable for further modification. Substitution at the position 3 with lipophilic
groups afforded effective inhibitors (1a-1d), while polar substituents greatly
diminished the potency (1e-1j). The potency of these derivatives was dependent on
the size of the 3-substituent. 3-Cl (1a) and 3-CF3 (1u) significantly enhanced
inhibitory activity, while 3-Br (1b) and 3-CH3 (1v) matched the activity of 1. Bulky
substituents (3-I, 1c; 3-SCH3, 1d) dramatically decreased NAAA inhibition potency.
In contrast, the 4-substituents showed different patterns of potency changes.
Lipophilic functionality (1k-1n, 1p, 1w) was poorly tolerated in position 4, while
polar hydroxyl-containing substituents (4-OH, 1o; 4-CH2OH, 1q) significantly
enhanced the potency. However, much more polar (4-COOH, 1r; 4-CONH2, 1s) and
8

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basic (4-NH2, 1t) groups substantially decreased the potency. 4-CH3 substituted 1x
(IC50 = 0.44 µM) was less potent than hydroxyl-containing analogs (1o, IC50 = 0.061
µM and 1q, IC50 = 0.19 µM). We speculated that 4- substitution sits in a small
hydrophilic niche in the protein, which prefers polar hydroxyl (1o and 1q) to
lipophilic methyl (1x). Previous studies suggested that the acyl chain of oxazolidone
derivatives occupies a hydrophobic pocket of NAAA that binds the fatty acyl chain of
substrates, and incorporation of polar moieties into the acyl side chain substantially
decreased the binding affinity [11, 23]. We are surprised to find that the polar
hydroxyl can be well tolerated in the hydrophobic pocket. The preliminary SAR study
showed that introduction of small lipophilic groups in the position 3 or
hydroxyl-containing substituents in the position 4 significantly improved inhibitory
potency. Several oxazolidone derivatives were identified as nanomolar potent NAAA
inhibitors, e.g., 1a (IC50 = 0.009 µM) and 1o (IC50 = 0.061 µM).
Table 1. Inhibitory effects of compounds 3-5, 1a-1x, 1aa-1dd on rat NAAA activities. Data
are presented as mean ± standard deviation (SD) of three independent experiments mean. All
experiments were performed in triplicate (n = 3).
R2
-CH3
-Cl
IC50 ± SD (µM)
R2
IC50 ± SD (µM)
> 20
> 20
-OH
> 20
3
4
5
R4
R3
O
O
N
O
R3
R4
IC50 ± SD (µM)
0.009 ± 0.001
0.43 ± 0.02
3.12 ± 0.98
12.8 ± 4.01
8.64 ± 1.25
16.30 ± 5.80
> 20
R3
R4
IC50 ± SD (µM)
4.60 ± 0.83
8.51 ± 2.90
> 20
-Cl
-Br
-Cl
-Br
1a
1b
1c
1d
1e
1f
1k
1l
-I
-I
1m
1n
1o
1p
1q
1r
1s
-SCH3
-OH
-SCH3
-OH
> 20
0.061 ± 0.015
> 20
-OCH3
-CH2OH
-COOH
-CONH2
-NH2
-OCH3
-CH2OH
-COOH
-CONH2
-NH2
-H
-H
0.19 ± 0.04
> 20
1g
1h
1i
> 20
> 20
> 20
> 20
15.70 ± 6.20
1.04 ± 0.23
1j
1t
-CF3
0.058 ± 0.012
-CF3
1u
1w
9

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-CH3
-Me
-Cl
0.35 ± 0.05
> 20
-CH3
-OH
-OH
0.44 ± 0.06
> 20
1v
1x
1cc
1dd
-Me
-F
-Cl
-F
1aa
1bb
0.026 ± 0.004
0.16 ± 0.04
Next, we tested whether di-substitution of the terminal phenyl group of 1 by highly
preferable 3- and 4-substituents can enhance the potency (Table 1). Unfortunately, the
NAAA inhibition potency of di-substituted compounds 1aa (3-CH3, 4-CH3) and 1cc
(3-Cl, 4-OH) was almost completely diminished compared to their mono-substituted
analogues. However, additional -F can be tolerated in 3-Cl and 4-OH substituted
oxazolidone derivatives (1bb, 1dd), but -F modification produced 2.9- and 2.6-fold
decreases in potency with respect to the mono-substituted 1a and 1o.
4.2 Modification of the terminal phenyl ring of 2
Except from the distal moieties, the flexibility of the link chain of oxazolidone
derivatives also has critical influence on the NAAA inhibition potency [11].
Analogues of 1 bearing conformationally flexible hexanoyl link chain, which may
favour the correct positioning of different distal moieties, thus showed high potency.
We then tested whether the identified preferable distal moieties can increase the
potency of oxazolidone derivatives containing conformationally constrained link
chain. We then did a SAR study on compound 2, a low micromolar NAAA inhibitor
(IC50 = 0.68 µM) with restricted phenoxyl link chain. By modification of the terminal
phenyl ring, a series of phenyl substitutions for the distal phenyl group of 2 were
synthesized and tested (Table 2). Unfortunately, all 3- or 4-substituted phenyl
analogues (2a-2d) were less effective than unsubstituted 2, indicating the restricted
link chain may restrict the movements of distal phenyl ring of inhibitor towards the
binding site. Next, oxazolidone derivatives bearing flexible aliphatic replacement for
2 were evaluated. Compared to unsubstituted 2, the cycloalkyl substitutions (2e, 2f)
were 6-fold more effective, while 3-pentenyl (2g) and hexyl derivatives (2i) were
comparable potent. The results are in agreement with previous observation that
replacement of the phenyl ring of 1 with aliphatic group exceeded the potency [11].
Incorporation of polar hydroxyl (2j) or oxygen (2k) into the aliphatic ring didn’t
affect the potency. However, butyl derivative (2h) resulted in nearly 2-fold decrease
in inhibitory potency. It has been reported that sp2 hybridized atoms can’t be well
tolerated at the end of hydrophobic pocket of NAAA, thus NAAA inhibitors with
such groups exhibited weak potency [24]. Replacement of the phenyl ring with the
aliphatic groups may avoid the unfavourable binding occupancy of the hydrophobic
pocket by the sp2 hybridized carbon, therefore enhanced the NAAA inhibition. These
results confirmed the beneficial effects of the terminal aliphatic ring of oxazolidone
derivatives towards NAAA inhibition, and several potent NAAA inhibitors, e.g., 2e
(IC50 = 0.092 µM) were identified.
10

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Table 2. Inhibitory effects of compounds 2a-2k on rat NAAA activities. Data are presented
as mean ± standard deviation (SD). All experiments were performed in triplicate (n = 3).
O
O
R4
R3
N
O
O
R3
-H
-Cl
-H
R4
-H
IC50 ± SD (µM)
0.68 ± 0.11
7.74 ± 1.84
> 20
R3
-Me
-H
R4
-H
IC50 ± SD (µM)
5.21 ± 1.33
2
2c
-H
-Me
6.40 ± 1.57
2a
2b
2d
-OH
O
O
N
O
R
O
R
IC50 ± SD (µM)
R
IC50 ± SD (µM)
0.092 ± 0.028
0.64 ± 0.18
2e
2f
2i
2j
OH
O
0.12 ± 0.03
0.21 ± 0.05
0.33 ± 0.08
0.48 ± 0.10
1.34 ± 0.30
2g
2h
2k
4.3 Selectivity evaluation
Acid ceramidase (AC) is another lysosomal cysteine amidase that showed 33-35%
amino acid identity with NAAA. To understand the selectivity of oxazolidone
derivatives towards NAAA against AC, the activity of the most active oxazolidone
derivatives (1a, 1o and 2e) against h-AC was tested. As shown in Table 3, 1a, 1o and
2e showed weak inhibition activity against AC. All the three representative
oxazolidone derivatives reducing no more than 20% at a concentration of 20 µM, at
which concentration NAAA was completely inhibited by these oxazolidone
derivatives. The results suggested that oxazolidone derivatives were highly selective
for NAAA over AC.
Table 3. Effects of compounds 1, 1a, 1o and 2e on rat-AC activity. Data are presented
as mean ± standard deviation (SD). All experiments were performed in triplicate (n = 3).
1 (20µM)
1a (20µM)
1o (20µM)
0.061 ± 0.015 0.092 ± 0.028
95.1 ± 17.4 81.7 ± 9.4
2e (20µM)
Vehicle
---
IC50 ± SD (µM)
0.27 ± 0.04 0.009 ± 0.001
AC activity (%) 100.0 ± 21.4 90.6 ± 18.2 85.3 ± 12.5
4.4 Mechanistic study
11

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Oxazolidone derivatives share similar electrophilic structures with β-lactone
derivatives, one type of NAAA inhibitors that are known to interact covalently with
the active site of NAAA. Therefore, we postulated that oxazolidone derivatives may
inactivate NAAA via similar mechanisms as β-lactone. To demonstrate this
hypothesis, we investigated the interaction mechanism of 1a with NAAA by a
Yonetani-Theorell analysis, an approach widely used to discriminate whether two
inhibitors potentially binding to the same or overlapping site [25-26]. 1a was
subsequently tested in combination with ARN077 [16], a well known covalent
β-lactone NAAA inhibitor, or 3-biphenylpropaonoyl pyrrolidine [13], a competitive
inhibitor that binds non-covalently in the same active site of NAAA as ARN077.
Results showed a series of parallel lines, indicating that 1a bound to NAAA in a
mutually exclusive fashion with ARN077 (Figure. 2A) and 3-biphenylpropaonoyl
pyrrolidine (Figure. S1). These data suggested that 1a binds to the active site of
NAAA similar to β-lactone derivative. Further kinetic analysis showed that 1a
decreased the maximal catalytic velocity (Vmax) of NAAA, but did not affect
Michaelis-Menten constant Km (Figure. 2B), suggesting a non-competitive and
probably covalently inhibition mechanism. Moreover, rapid dilution revealed that 1a
inhibited NAAA via a slowly reversible mechanism (Figure. 2C). When combined,
these results suggested that oxazolidone derivatives may form a covalent yet
reversible bond with NAAA, thus inhibited the enzyme activity.
However, the enzyme kinetics data showed that 1k, the 4-regioisomer of 1a, was a
weak, competitive and rapidly reversible NAAA inhibitor (Figure 2B, 2C, Table 4).
There may be two reasons for the different inhibition mechanism and potency. First,
the 4-Cl decreases the binding affinity of the inhibitors, preventing the formation of
the NAAA-inhibitor complex, thus resulting in poor potency and rapidly reversible
binding. Second, the 4-Cl enhances the degradation of NAAA-inhibitor complex
compared to 3-Cl, and inhibitors containing 4-Cl are rapidly converted to other
non-active structures. Therefore, the degradation of 1a and 1k by NAAA was studied
to test the possible mechanisms. The results showed that compound 1a was slowly
cleaved by NAAA (Figure 2D), and there was 92.6% of 1a remaining after 6 h
incubation, suggesting that 1a was covalently attacked by NAAA, and slowly
converting to other compounds. However, for weak inhibitor 1k, no significant
degradation was observed when tested under the same conditions. The results
suggested that the different inhibition mechanisms of 1a and 1k wasn’t due to the
different conversion rate. Instead, it’s more possible that different group influenced
the binding affinity of inhibitors, thus affected the inhibition mechanism and potency.
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Figure 2. Characterization of the inhibition mechanisms of oxazolidone derivatives 1a and 1k.
(A) Yonetani-Theorell plots for the inhibition of rat-NAAA by various combinations of 1a
and ARN077 (open circle, vehicle; closed square, 50 nM; closed triangle, 250 nM); (B)
Kinetic analysis of rat NAAA inhibition by vehicle (open circle, 1% DMSO), 1a (black circle,
0.05 µM) and 1k (black triangle, 10 µM); (C) Rapid dilution assay of NAAA in the presence
of vehicle (open circle), 1a (black circle) and 1k (black triangle); (D) Degradation experiment
of 1a (circle, 10 µM) and 1k (triangle, 10 µM) in the present of vehicle (open plot, 1% DMSO)
or NAAA (close plot, 300 ng). Data are presented as mean ± standard deviation (SD) of the
mean. All experiments were performed in triplicate (n = 3).
Table 4. Michaelis–Menten analysis of compounds 1a and 1k
Vmax (nmol/min/mg protein)
9383 ± 937
Km (µM)
1395 ± 260
1278 ± 207
1671 ± 340
Vehicle
1a
2793 ± 236
9905 ± 1147
1k
5. Conclusion
Here, we further optimized the terminal phenyl moiety of oxazolidone derivatives 1
and 2 to develop more potent NAAA inhibitors and explored the structural
requirements for potent NAAA inhibition. SAR studies suggested that substitution of
the terminal phenyl ring of 1 with small lipophilic groups at position 3, or
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hydroxyl-containing substituents at position 4 significantly improved the inhibitory
potency. Replacement of the terminal phenyl group of 2 with aliphatic groups also
effectively enhanced the NAAA inhibition activity. Rapid dilution and kinetic
analysis suggested that oxazolidone derivatives with different structures inhibited
NAAA via different mechanisms. 1a (F215) inhibited NAAA through a
non-competitive and slowly reversible mechanism, while 1k inhibited NAAA in a
competitive and rapidly reversible pattern. In brief, current exploration of the sizes,
flexibility, and lipophilicity of the terminal groups allowed us to define the structural
requirements for potent NAAA inhibition. 1a represents a useful probe for
investigating the biological function of NAAA in inflammation and analgesia, and a
promising anti-inflammatory and analgesic drug candidates.
6. Author contributions
YL and YZ synthesised compounds; QC performed NMR and LC-MS assays; LY,
YtL and JR performed enzymatic assays; YL wrote the paper; YL, CL, and YQ
conceived and designed the experiments.
7. Acknowledgements
We thank Dr Xiang Gao for technical consultation. This work was supported by funds
from Key Research Program of Frontier Science, CAS (Grant No.
QYZDJ-SSW-SLH033), the Strategic Priority Research Program of the Chinese
Academy of Sciences (Grant No. XDB20000000), the National Natural Science
Foundation of China (Grant No. 21401192, 81602974), the Major State Basic
Research Development Program of China (Grant No. 2014CB965101) and the
Natural Science Foundation of Fujian Province (2006L2005).
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