Design and synthesis of uracil urea derivatives as potent and selective fatty acid amide hydrolase inhibitors

Article abstract of DOI:10.1039/c7ra02237a

Fatty acid amide hydrolase (FAAH) is one of the key enzymes involved in the biological degradation of endocannabinoids, especially anandamide. Pharmacological blockage of FAAH restores the levels of endocannabinoids, providing therapeutic benefits in the management of inflammation, depression and multiple sclerosis. In this study, a series of uracil urea derivatives as FAAH inhibitors were designed and synthesized. Structural modifications at the C5 position and side chain of N-hexyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-carboxamide (1a) led to FAAH inhibitors with improved potency and selectivity. Structure-activity relationship (SAR) studies indicated that C5 electron-withdrawing substituents were preferred for optimal potency but not for selectivity, whereas replacement of the alkyl chain with phenylalkyl moieties or biphenyl groups significantly improved both inhibitory potency and selectivity towards FAAH. Two highly potent picomolar FAAH inhibitors (4c, IC₅₀ = 0.3 ± 0.05 nM; 4d, IC₅₀ = 0.8 ± 0.1 nM) were developed. Compound 4c inhibited FAAH in a rapid, selective, noncompetitive, and irreversible pattern. This study provides several highly potent and selective FAAH inhibitors and an optimized chemical scaffold for the development of FAAH inhibitors. We anticipate that these FAAH inhibitors will enable new possibilities in understanding FAAH functions and development of therapeutics for pain and inflammatory diseases.

Full text of DOI:10.1039/c7ra02237a

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PAPER
Design and synthesis of uracil urea derivatives as
potent and selective fatty acid amide hydrolase
inhibitors†
Cite this: RSC Adv., 2017, 7, 22699
Yan Qiu,‡a Jie Ren,‡a Hongwei Ke,bc Yang Zhang,a Qi Gao,a Longhe Yang,d
b
ab
*
Canzhong Lu
and Yuhang Li
Fatty acid amide hydrolase (FAAH) is one of the key enzymes involved in the biological degradation of
endocannabinoids, especially anandamide. Pharmacological blockage of FAAH restores the levels of
endocannabinoids, providing therapeutic benefits in the management of inflammation, depression and
multiple sclerosis. In this study, a series of uracil urea derivatives as FAAH inhibitors were designed and
synthesized. Structural modifications at the C5 position and side chain of N-hexyl-2,4-dioxo-3,4-
dihydropyrimidine-1(2H)-carboxamide (1a) led to FAAH inhibitors with improved potency and selectivity.
Structure–activity relationship (SAR) studies indicated that C5 electron-withdrawing substituents were
preferred for optimal potency but not for selectivity, whereas replacement of the alkyl chain with
phenylalkyl moieties or biphenyl groups significantly improved both inhibitory potency and selectivity
towards FAAH. Two highly potent picomolar FAAH inhibitors (4c, IC50 ¼ 0.3 ꢀ 0.05 nM; 4d, IC50 ¼ 0.8 ꢀ
0.1 nM) were developed. Compound 4c inhibited FAAH in a rapid, selective, noncompetitive, and
irreversible pattern. This study provides several highly potent and selective FAAH inhibitors and an
optimized chemical scaffold for the development of FAAH inhibitors. We anticipate that these FAAH
inhibitors will enable new possibilities in understanding FAAH functions and development of therapeutics
for pain and inflammatory diseases.
Received 23rd February 2017
Accepted 17th April 2017
DOI: 10.1039/c7ra02237a
rsc.li/rsc-advances
So far, there are two main categories of FAAH inhibitors that
have been developed.12–14 First, reversible inhibitors, such as the
1. Introduction
a-ketoheterocycle-based inhibitors, bind to the active serine
residue of FAAH with a reversible hemiketal bond. OL-135 is the
most well-studied potent and reversible FAAH inhibitor.4,15,16
Second, irreversible inhibitors, including aryl carbamates,17–20
ureas21–25 and sulfonyl uoride analogues,26 which form cova-
lent binding with the catalytic site of FAAH, e.g., URB597.27
Administration of URB597 to rats elevated endogenous AEA
levels and exhibited anxiolytic effects in animal models.5,28
Additionally, benzothiazole,29 b-lactam,30 (thio)hydantoins31
and some nonsteroidal anti-inammatory drugs (NSAIDs)32–34
also have been reported to inhibit FAAH.
Fatty acid amide hydrolase (FAAH) is a member of the amidase
signature family, responsible for the inactivation of endogenous
cannabinoids, especially anandamide (AEA).1–3 AEA modulates
a wide range of physiological processes through the interaction
with G protein-coupled CB1 and CB2 cannabinoid receptors.
Genetic and pharmacological blocking of FAAH leads to
increased endogenous AEA levels without the undesirable
“cannabinoid effects” caused by cannabinoid receptor
agonists.4–6 Therefore, restoring the endogenous AEA by FAAH
inhibition has become an alternative therapeutic strategy for
inammation, cancer, and neuropathic diseases.5,7–11
Although the overall physiological role of FAAH has been
reported, it is difficult to distinguish the function of FAAH in
each specic tissue and organ. The reason is the wide distri-
bution of FAAH in the whole body and lack of suitable topical
FAAH inhibitors. Most of current FAAH inhibitors have low
blood clearance and readily distributed and inhibit FAAH
throughout the body aer treatment. They are suitable for the
systemic inhibition of FAAH, but it's difficult to obtain targeted
FAAH inhibition in the specic tissues without inhibiting FAAH
in other non-targeted sites. Given the critical pharmacological
properties of FAAH, understanding the function of FAAH in
different tissues is of great signicance. FAAH inhibitors
aMedical College, Xiamen University, Xiamen, Fujian 361102, P. R. China. E-mail:
yuhangli@irsm.ac.cn
bXiamen Institute of Rare-earth Materials, Haixi Institutes, Chinese Academy of
Sciences, Fujian 361024, P. R. China
cCollege of Ocean and Earth Science, Xiamen University, Xiamen, Fujian, 361005, P. R.
China
dEngineering Research Centre of Marine Biological Resource Comprehensive
Utilization, Third Institute of Oceanography, State Oceanic Administration, Xiamen
361102, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra02237a
‡ Equal contribution.
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Scheme 1 The synthesis of compounds 1a, 1c, 2a, 2d, 2i–2k, 3a–3u,
4b–4d. Reagents and conditions: (a) (COCl)2, cat. DMF, CH2Cl2, then
NaN3 in H2O, 0 ꢁC; (b) toluene, reflux; (c) DMAP, pyridine, 60 ꢁC.
Fig. 1 The design strategies of uracil urea derivatives as highly potent
and selective FAAH inhibitors. The selectivity represents the selectivity
of uracil urea derivatives towards FAAH over NAAA.
(Scheme 2). 1e was synthesized by reaction of uracil with tri-
phosgene followed by treatment with N-methylhexanamine
(Scheme 3). 2b and 2c were prepared with 3-step procedure
starting from 2b-1 and 2c-1. Compound 2b-1 and 2c-1 were
protected by t-butyl di-methylsilyl chloride (TBSCl) and directly
used in the carbamoylation reaction, followed by deprotection
suitable for the topical administration is highly desired to
explore functions of FAAH in different tissues and organs.
In our previous study,35 we found that uracil urea 1a (Fig. 1),
an acid ceramidase (AC) inhibitor (IC50 ¼ 426 ꢀ 104 nM),36 was
also
a
potent FAAH (IC50
¼
492
ꢀ
53 nM) and N-
acylethanolamine-hydrolysing acid amidase (NAAA) inhibitor
(IC50 ¼ 990 ꢀ 170 nM). To develop FAAH inhibitors with
properties suitable for topical FAAH inhibition and improve the
selectivity of 1a towards FAAH, we modied the uracil electronic
properties and the side chain conformation of 1a. A series of
uracil urea derivatives designed as selective FAAH inhibitors
were synthesized and characterized. The results showed that
modication of 1a targeting the uracil ring improved the inhi-
bition potency towards FAAH, and the side chain replacement
with phenylalkyl moieties or biphenyl groups enhanced both
potency and selectivity (Fig. 1). Several highly potent and
selective FAAH inhibitors were reported, including two pico-
molar inhibitor, 4c (IC50 ¼ 0.3 ꢀ 0.05 nM) and 4d (IC50 ¼ 0.8 ꢀ
0.1 nM), a series of nanomolar inhibitors, e.g., 3f (IC50 ¼ 4 ꢀ 0.9
nM), 3u (IC50 ¼ 3 ꢀ 0.4 nM), 4a (IC50 ¼ 1.1 ꢀ 0.2 nM) and 4b
(IC50 ¼ 8.3 ꢀ 1.2 nM).
Scheme 2 The synthesis of compound 1b. Reagents and conditions:
(a) MeI, Cs2CO3, THF, room temperature; (b) HCl, CH3OH, room
temperature; (c) hexyl isocyanate, DMAP, pyridine, 60 ꢁC.
Scheme 3 The synthesis of compound 1e. Reagents and conditions:
(a) NaH, triphosgene, THF, room temperature; (b) HCl, CH3OH, room
temperature; (c) DMAP, pyridine, N-methylhexylamine, room
temperature.
2. Chemistry
The uracil ureas 1a, 1c, 1d, 2a, 2d, 2i–2k, 3a–3u, 4a–4d were
prepared by the reaction of isocyanates with 5-substituted
uracils following a reported procedures with minor modica-
tion.36 Isocyanates were synthesized via Curtius rearrangement
of appropriate acylazides prepared from successive reactions of
the corresponding acid with oxalyl chloride ((COCl)2) and
sodium azide (NaN3) (Scheme 1).20 1b and 1e were synthesized
following a literature procedure.36 1b was prepared by the
reaction of hexyl isocyanate with 3-methyl substituted uracil 1b-
1. 1b-1 was obtained by reaction of N-Boc uracil and iodo-
Scheme 4 The synthesis of compounds 2b and 2c. Reagents and
conditions: (a) pyridine, TBSCl, room temperature; (b) hexyl isocya-
methane (MeI), and then deprotection with HCl in MeOH nate, DMAP, pyridine, 60 ꢁC; (c) TBAF, THF, room temperature.
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4. Results and discussion
4.1. SAR study
It has been reported that carbamate and urea FAAH inhibitors
interact with the nucleophilic Ser241 of FAAH through the
electrophilic carbonyl group, and most of these inhibitors
contain O-aryl or N-aryl moiety as leaving group. We speculated
that the activity of uracil urea inhibitors may dependent on the
presence of N1-carboxamide as the electrophilic carbonyl group
and uracil as leaving group. Accordingly, two different chemical
modications were used to optimize the potency and selectivity
of uracil urea derivatives. First, inspired by existed urea FAAH
inhibitors, we replaced the uracil ring with chemical groups
with different electronic properties to increase the leaving
group ability of uracil. Second, we modied the side chain with
phenyl-containing groups that can mimic the conformation and
p-unsaturation of double bonds of anandamide, and provided
better drug-like characteristics and the opportunity for struc-
ture exploration (Fig. 1).
In preliminary SAR study, analogues (1a–1e, Table 1) bearing
a methyl were prepared to dene positions available for
substitution without adversely affecting the inhibitor potency.
The results showed that substitutions of position N3 (1b), C6
(1d) and N0 (1e) of 1a resulted in a greatly diminished or
complete loss in inhibition toward FAAH and NAAA, while
substitution of position C5 (1c) slightly decreased the inhibition
against both FAAH and NAAA. Therefore, we focused on the
modication of C5 in the following studies.
Scheme 5 The synthesis of compound 2e. Reagents and conditions:
(a) pyridine, CbzCl, room temperature; (b) hexyl isocyanate, DMAP,
pyridine, 60 ꢁC; (c) TMSI, THF, room temperature.
Scheme 6 The synthesis of compounds 2f, 2g and 2h. Reagents and
conditions: (a) pyridine, Ac2O, room temperature; (b) hexyl isocyanate,
DMAP, pyridine, 60 ꢁC.
Scheme 7 The synthesis of compound 2l. Reagents and conditions:
Next, we did systematic substitution of 1a at C5 (1c, 2a–2k) to
investigate the effect of electronic properties of ring substitu-
tion on the inhibitory activity of uracil urea derivatives toward
FAAH. Some analogues (2a, 2d, 2i, 2k) have been described by
other groups were also synthesized and tested as control
compounds.36,37 The results suggested that the electronic
properties of C5 substituents were of great signicance for the
inhibition towards both FAAH and NAAA (Table 2). Compared
to parent inhibitor 1a, the electron-withdrawing substituents
(2i–2k) exhibited 2–5 folds enhanced inhibition against FAAH.
(a) 10% Pd/C, H2, CH3OH, room temperature.
Scheme 8 The synthesis of compound 2m. Reagents and conditions:
(a) K2CO3, DMF, room temperature.
with tetrabutylammonium uoride (TBAF) (Scheme 4). 5-Amino
uracil (2e-1) protected with benzyl carbonochloridate (CbzCl) in
pyridine was directly used in the carbamoylation reaction, and
followed by deprotection with trimethylsilyl iodide (TMSI) to
afford 2e (Scheme 5). 2f–2h were synthesized by treatment of the
uracil derivatives with acetic anhydride (Ac2O) followed by
reaction with isocyanates (Scheme 6). 2l was prepared by cata-
lytic hydrogenation of 1a (Scheme 7). 2m was obtained by
reaction of 1-bromooctane with uracil (Scheme 8).
Table 1 Inhibitory effects of compounds 1a–1e on rFAAH and rNAAA
activitiesa
R3
R5
R6
RN
IC50 of FAAH (nM) IC50 of NAAA (nM)
1a –H
1b –Me –H
1c –H
1d –H
1e –H
–H
–H
–H
–H
–H
–H
492 ꢀ 53
990 ꢀ 170
>10 000
3. Enzyme assay
7811 ꢀ 1563
1026 ꢀ 205
>10 000
–Me –H
–H
–H
3650 ꢀ 840
The inhibition activity of new compounds against rat FAAH
(rFAAH) and rat NAAA (rNAAA) was tested, using [3H]-AEA and
d4-PEA, respectively, as substrates. The residual hydrolysis
products of the substrates were determined by liquid
chromatography-mass spectrometer (LC-MS).
–Me –H
–H
>10 000
–Me >10 000
>10 000
a
Data are presented as IC50 ꢀ standard error of the mean. All
experiments were performed in triplicate.
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Table 2 Inhibitory effects of compounds 1a, 1c, 2a–2m on rFAAH and
Except from enhancing inhibition potency towards FAAH,
electron-withdrawing C5 substitution also increased the inhi-
bition potency of uracil urea derivatives toward NAAA. For
instance, acetyl substituents 2f–2h enhanced NAAA inhibition
by 12–27 folds compared to the unsubstituted derivatives (2b, 2c
and 2e). Electron-withdrawing C5 substituents exhibited weak
selectivity towards FAAH over NAAA. The electron-withdrawing
substituents failed to improve the selectivity towards FAAH
over acid ceramidase. The IC50 values of electron-withdrawing
substituents towards acid ceramidase (2i–2k) was an order of
magnitude lower than 1a.36 These results suggested that
electron-withdrawing substituents of uracil ring exhibited
enhanced the inhibition potency, but their selectivity towards
FAAH over acid ceramidase and NAAA was still not ideal.
Further modication was required to enable high selectivity for
FAAH over other competitive enzymes.
Finally, we further modied the side chain to improve the
selectivity towards FAAH. FAAH hydrolyzes unsaturated
substrates (e.g., anandamide) faster than saturated FAEs, while
NAAA and acid ceramidase strongly prefer saturated substrates
(e.g., PEA or ceramide).38 Replacement of the hexyl chain with
an unsaturated group might mimic the p-characteristics and
conformation of the anandamide, leading to binding affinity
preference towards FAAH. Therefore, we optimized the chain
terminus of C5 uracil substituents (–H, –F, –OAc) with a phenyl
ring, a chemical moiety that can mimic the conformation and
p-unsaturation of the D8,9/D11,12 double bonds of anandamide.
Three representative series where R5 ¼ H, F, OAc were designed
and synthesized. As shown in Table 3, most of the analogous
with an alkylphenyl group exhibited dramatically increased
inhibitory potency against FAAH. The inhibitory potency was
progressively improved by prolonging the linker length of the
alkylphenyl. Interestingly, the inhibition activity of these
substituents was independent of electron effect of C5 group,
which was previously expected to signicantly modulate the
rNAAA activitiesa
R5
IC50 of FAAH (nM) IC50 of NAAA (nM) Selectivity
1a
1c
2a
2b
2c
2d
2e
2f
2g
2h
2i
–H
–Me
–Et
492 ꢀ 53
1026 ꢀ 205
543 ꢀ 94
693 ꢀ 117
990 ꢀ 170
3650 ꢀ 840
1837 ꢀ 428
5049 ꢀ 1103
3265 ꢀ 519
1048 ꢀ 168
5265 ꢀ 864
190 ꢀ 20
2
3.6
3.4
7.3
7.6
1.5
3.5
1.7
2.2
1.9
6.6
11.5
22.4
–OH
–CH2OH 425 ꢀ 75
–OEt
–NH2
–OAc
703 ꢀ 136
1520 ꢀ 280
110 ꢀ 10
–CH2OAc 120 ꢀ 31
271 ꢀ 25
–NHAc
–F
221 ꢀ 44
38 ꢀ 9
423 ꢀ 78
250 ꢀ 50
2j
2k
URB597
–Ac
–CN
42 ꢀ 11
25 ꢀ 5
486 ꢀ 89
560 ꢀ 42
26 ꢀ 5
>10 000
a
Data are presented as IC50 ꢀ standard error of the mean. All
experiments were performed in triplicate.
IC50 of
IC50 of
Structure
FAAH (nM)
NAAA (nM)
2l
>10 000
>10 000
>10 000
>10 000
2m
Among compounds 2a–2k, 2i–2k were the three most potent binding affinity of these derivatives. The results suggested that
inhibitors, being equipotent with URB597 tested under the van der Waals interactions, strongly related to the closeness
same conditions. On the contrary, the electron-donating between inhibitors and protein surfaces, might be the most
substituents (1c, 2a–2e) showed slightly decreased inhibitory signicant contribution to binding affinity.
activity against FAAH. We then tested whether further modu-
These uracil urea derivatives exhibited different inhibition
lation of the electron-donating C5 substituents with electron- pattern against NAAA (Table 3). Most compounds with alkyl-
withdrawing group can restore the potency. The results phenyl replacement were less active than the parent hexyl
showed that the addition of acetyl to the compounds 2f–2h derivatives. The optimal potency was observed with the linker
resulted in a modest (3–7 folds) increase of inhibitory potency length of C5 (for 5-F series) or C4 (for 5-H and 5-OAc series)
compared to corresponding derivatives (2b, 2c and 2e). These methylene spacer, and the inhibition potency progressively
results suggested that the electron-withdrawing C5 substitution decreased with lengthened or shortened linker. The tight
was critical for potent FAAH inhibition. The reason might be binding pocket of NAAA, may explain the loss of NAAA inhibi-
such modulation increases the leaving group ability of uracil, tory and gain in FAAH selectivity by bulkier structures. Most of
and makes molecules more sensitive to nucleophilic attack of the alkylphenyl derivatives were more selective for FAAH
Ser241 of FAAH. To examine this hypothesis, two 1a derivative compared to parent inhibitors (1a, 2f and 2i). Among these
was synthesized and tested. Derivative 2l is the saturated alkylphenyl derivatives 3g, 3t and 3u were the most promising
derivative of 1a without the leaving group uracil. Derivative 2m inhibitors with high potency and selectivity towards FAAH. We
without electrophilic carbonyl group was obtained by replace- further substituted the phenylalkyl chain of 3t and 3u with
ment of N1-carboxamide with methylene. The data showed that biphenyl group and tested whether biphenyl modication will
inhibitory activity of 2l and 2m against FAAH was completely further enhance the potency. As showed in Table 4, the biphenyl
eliminated. These results conrmed that uracil leaving group derivatives (4a, 4c and 4d) were 3–16 folds more potent or
and N1-carboxamide group are critical for FAAH inhibition.
similar potent (4b) compared to the parent inhibitors (3t and
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Table 3 Inhibitory effects of compounds 3a–3u on rFAAH and rNAAA
for the uracil urea derivatives. Representative inhibitors (2f, 2i,
3u and 4c) were selected and their selectivity towards FAAH was
examined. As shown in Table 5, the inhibitors were highly
selective for FAAH over MAGL. Hexyl derivatives 2f and 2i
exhibited inhibitory effects against all the three enzymes.
Phenyl ring substitutions at the terminus of the hexyl side chain
enhanced the binding affinity of the inhibitors to FAAH over
NAAA (2f, 1.7 folds; 2i, 6.5 folds; 3u and 4c, >3000 folds), but it
did not decrease their inhibition potency towards AC. Notably,
biphenyl group substitution dramatically increased both
potency and selectivity of the derivatives towards FAAH. The
most potent inhibitor 4c was more than 3000 folds selective for
FAAH over NAAA, and modestly selective for FAAH over AC
(Table 5).
The potency and selectivity of inhibitors were depended on
the compound reactivity as well as the molecular shape and
exibility. The inhibition prole of uracil urea derivatives was
modulated by electron properties of the C5 substituents and the
conformation of the side chain. Electron-withdrawing substit-
uent enhances both potency and selectivity for FAAH over
NAAA, but failed to improve the selectivity towards FAAH over
AC.36 Electron-withdrawing substitution turning 1a to a dual
inhibitor of FAAH–AC. On the other hand, the side chain is the
major factor contributing to the selectivity of uracil urea deriv-
atives. Incorporation of phenyl-containing group into the side
chain signicantly enhanced their inhibition potency and
selectivity towards FAAH over NAAA and AC, turning the uracil
urea scaffold from a multi-target inhibitor to a FAAH-selective
inhibitor.
activitiesa
R5
n
IC50 of FAAH (nM) IC50 of NAAA (nM) Selectivity
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
–H
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
155 ꢀ 23
333 ꢀ 105
19 ꢀ 7
13 ꢀ 4
11 ꢀ 1
4 ꢀ 0.9
3 ꢀ 0.8
122 ꢀ 10
98 ꢀ 12
21 ꢀ 3
16 ꢀ 2
13 ꢀ 3
4 ꢀ 1
8020 ꢀ 1290
6340 ꢀ 1080
1572 ꢀ 326
305 ꢀ 57
52
19
83
23
435 ꢀ 63
40
2490 ꢀ 372
4768 ꢀ 541
4655 ꢀ 482
4510 ꢀ 371
440 ꢀ 96
623
1589
38
9.4
20
30
8
73
274
115
57
25
12
355
1648
3127
–F
481 ꢀ 35
105 ꢀ 11
290 ꢀ 30
3 ꢀ 0.8
51 ꢀ 10
96 ꢀ 12
32 ꢀ 6
18 ꢀ 3
10 ꢀ 1
5 ꢀ 1.1
3 ꢀ 0.4
822 ꢀ 85
–OAc
5880 ꢀ 647
5440 ꢀ 1023
810 ꢀ 100
220 ꢀ 25
3s
3t
3u
3545 ꢀ 641
7908 ꢀ 855
9382 ꢀ 1425
a
Data are presented as IC50 ꢀ standard error of the mean. All
experiments were performed in triplicate.
4.3. Investigations of the inhibition mechanism
Table 4 Inhibitory effects of compounds 4a–4d on rFAAH and rNAAA
To explore the pharmacological properties of uracil urea deriv-
atives, we analysed their interaction pattern with FAAH.
Representative inhibitor 4c was selected in these studies. As
well as its signicant inhibition towards recombinant rat FAAH
protein (IC50 ¼ 0.3 ꢀ 0.05 nM, Table 4), 4c displayed a profound
inhibition toward recombinant human FAAH (IC50 ¼ 0.6 ꢀ 0.1
nM) (Fig. 2A). Kinetic analysis revealed that 4c rapidly inhibited
rFAAH (t1/2 < 1 minute) (Fig. 2B), and changed the maximal
catalytic velocity (Vmax) of rFAAH (Vmax in pmol per min per mg
protein, vehicle: 2056 ꢀ 107; and 4c, 50 nM: 698 ꢀ 84), but it did
not affect Michaelis–Menten constant Km (Km in mM, vehicle: 9.8
ꢀ 0.3; and 4c, 50 nM: 10.4 ꢀ 0.6) (Fig. 2C). No recovery of the
rFAAH activity was observed by dialysis of the 4c–rFAAH
activitiesa
n
IC50 of FAAH (nM)
IC50 of NAAA (nM)
4a
4b
4c
4d
1
2
3
4
1.1 ꢀ 0.2
8.3 ꢀ 1.2
0.3 ꢀ 0.05
0.8 ꢀ 0.1
>10 000
>10 000
>10 000
>10 000
a
Data are presented as IC50 ꢀ standard error of the mean. All
experiments were performed in triplicate.
Table 5 Selectivity screening of compounds 2f, 2i, 3u and 4ca
IC50 of rFAAH
(nM)
IC50 of rNAAA
(nM)
IC50 of rAC
(nM)
IC50 of
rMAGL (nM)
3u). Moreover, the selectivity of biphenyl substituents towards
FAAH was signicantly enhanced. 4a–4d exhibited profound
selectivity (>3000 folds) towards FAAH over NAAA.
2f
2i
3u
4c
110 ꢀ 10
38 ꢀ 9
190 ꢀ 20
250 ꢀ 50
9382 ꢀ 1425
>10 000
102 ꢀ 23
45 ꢀ 9
>10 000
>10 000
>10 000
>10 000
3 ꢀ 0.4
68 ꢀ 10
360 ꢀ 74
0.3 ꢀ 0.05
4.2. Selectivity evaluation
a
Data are presented as IC50 ꢀ standard error of the mean. All
NAAA, acid ceramidase (AC) and endocannabinoid hydrolase
monoacylglycerol lipase (MAGL) are three competitive targets
experiments were performed in triplicate.
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distributed throughout the body, involved in a complex
anandamide-based signalling system. Most of current FAAH
inhibitors, e.g. PF-04457845, are distributed throughout all
tissues and organs aer administration. They are suitable for
the systemic application, but probably not optimal agents to
investigate the function of FAAH in specic organ or tissue. The
inhibitors we developed exert acute FAAH inhibitory in the
target site and rapidly inactivated to prevent unwanted systemic
effects. Our ndings may offer a new topically applied tool to
investigate the function of FAAH at a selected tissue or organ.
5. Conclusions
Here, we report the synthesis and characterization of a series of
uracil urea derivatives, designed as FAAH inhibitors by
progressive modication of 1a. SAR studies suggested that
electron-withdrawing substituents in the C5 position enhanced
the potency of FAAH inhibition and the replacement of the side
chain with phenylalkyl moieties or biphenyl groups signi-
cantly improved both the inhibitory potency and selectivity
towards FAAH. The two most potent inhibitors, 4c (IC50 ¼ 0.3 ꢀ
0.05 nM) and 4d (IC50 ¼ 0.8 ꢀ 0.1 nM) showed moderate to high
selectivity towards FAAH over other endocannabinoid-
metabolizing enzymes. Rapid dilution and kinetic analysis
indicate that 4c is a selective, irreversible and non-competitive
FAAH inhibitor. This study provides us some promising FAAH
inhibitors and an optimized scaffold for the development of
strategies to investigate tropical FAAH function, and to explore
new therapeutics for inammation, cancer, and neuropathic
diseases.
Fig. 2 Effect of 4c on FAAH activity in vitro. (A) Dose-dependent
inhibition of recombinant rat FAAH (rFAAH) and recombinant human
FAAH (hFAAH) activity by 4c. (B) Time course of rFAAH inhibition by 4c
(50 nM). (C) Michaelis–Menten analysis of the FAAH reaction in the
presence of vehicle (open circles, 1% DMSO) or 4c (closed triangles, 50
nM). (D) FAAH activity in the presence of vehicle (open bars 1% DMSO)
or 4c (closed bars, 50 nM) before dialysis and 12 hours after dialysis.
Results are expressed as mean ꢀ SEM (n ¼ 4–6). ***P < 0.001.
interaction complex even aer 12 hours (Fig. 2C). These results
indicated that 4c was an irreversible and non-competitive FAAH
inhibitor, thereby providing strong evidence for the formation
of covalent bonds between compound and enzyme.
Acknowledgements
We thank Dr Daniele Piomelli at the University of California,
Irvine for the kind gis of the HEK293–rNAAA cells and the
HEK293–rFAAH cells. This study was supported by grants from
National Natural Sciences Foundation of China (No. 81373273
to QY, No. 81602974 to YL), Key Research Program of Frontier
Science, CAS, Grant No. QYZDJ-SSW-SLH033, The Major State
Basic Research Development Program of China (grant number
2014CB965101), and China Postdoctoral Science Foundation
Funded Project (2016M592876XB) to YL.
4.4. Biological stability evaluation
The hydrolysis of compounds (2f, 2i and 4c) was studied in 80%
(v/v) rat plasma at 37 ꢁC. The results showed that more than
60% of compounds were hydrolyzed aer 10 min incubation
(Table 6). Analogues 2f and 2i containing less sterically
hindered hexyl chain were cleaved more rapidly than the bulkier
compound 4c. LC-MS analysis of degradation products of
inhibitors (2f, 2i and 4c) showed the formation of hexylamine
and 4-biphenylpropylamine, which resulted from the hydrolysis
of the N1-carboxamide (data not shown). FAAH is broadly
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