Discovery of uracil derivatives as potent inhibitors of Fatty Acid Amide Hydrolase

Article abstract of DOI:10.3390/molecules21020229

Fatty Acid Amide Hydrolase (FAAH) is an intracellular serine enzyme involved in the biological degradation of the fatty acid ethanolamide family of signaling lipids, which exerts neuroprotective, anti-inflammatory, and analgesic properties. In the present study, a conjugated 2,4-dioxo-pyrimidine-1-carboxamide scaffold was confirmed as a novel template for FAAH inhibitors, based on which, a series of analogues had been prepared for an initial structure-activity relationship (SAR) study. Most of the synthesized compounds displayed moderate to significant FAAH inhibitory potency. Among them, compounds 11 and 14 showed better activity than others, with IC₅₀ values of 21 and 53 nM. SAR analysis indicated that 2,4-dioxopyrimidine-1-carboxamides represented a novel class of potent inhibitors of FAAH, and substitution at the uracil ring or replacement of the N-terminal group might favor the inhibitory potency. Selected compounds of this class may be used as useful parent molecules for further investigation.

Full text of DOI:10.3390/molecules21020229

molecules
Communication
Discovery of Uracil Derivatives as Potent Inhibitors
of Fatty Acid Amide Hydrolase
Yan Qiu 1,*, Yang Zhang 1,2, Yuhang Li 1 and Jie Ren 1
1
Department of Basic Medical Sciences, Medical College, Xiamen University, Xiamen 361102, China;
zhyang880105@163.com (Y.Z.); mfaoxmu@126.com (Y.L.); renjie_7912@xmu.edu.cn (J.R.)
Quality Responsibility Pharmaceuticals Co., Ltd., Wuhan Research & Development Center,
2
Wuhan 430075, China
*
Correspondence: yanqiu@xmu.edu.cn; Tel.: +86-591-218-8681
Academic Editor: Derek J. McPhee
Received: 22 December 2015 ; Accepted: 14 February 2016 ; Published: 18 February 2016
Abstract: Fatty Acid Amide Hydrolase (FAAH) is an intracellular serine enzyme involved in
the biological degradation of the fatty acid ethanolamide family of signaling lipids, which exerts
neuroprotective, anti-inflammatory, and analgesic properties. In the present study, a conjugated
2,4-dioxo-pyrimidine-1-carboxamide scaffold was confirmed as a novel template for FAAH inhibitors,
based on which, a series of analogues had been prepared for an initial structure-activity relationship
(SAR) study. Most of the synthesized compounds displayed moderate to significant FAAH inhibitory
potency. Among them, compounds 11 and 14 showed better activity than others, with IC50 values
of 21 and 53 nM. SAR analysis indicated that 2,4-dioxopyrimidine-1-carboxamides represented a
novel class of potent inhibitors of FAAH, and substitution at the uracil ring or replacement of the
N-terminal group might favor the inhibitory potency. Selected compounds of this class may be used
as useful parent molecules for further investigation.
Keywords: fatty acid amide hydrolase (FAAH); uracil derivatives; FAAH inhibitor; amidation
1. Introduction
Anandamide (AEA) belongs to the endocannabinoid family, a series of endogenous fatty
acid ethanolamides (FAEs), which participates in the control of pain [
and cognition [4,5], through engaging cannabinoid receptors 1 and 2 (CB1, CB2) [
function of AEA is terminated by enzymatic hydrolysis in processes principally mediated by FAAH,
a distinct serine hydrolase [ ]. Selective FAAH inhibitors have been identified to elevate endogenous
1,
2
], inflammation [
3],
6]. The signaling
7,8
AEA levels, which display a more restricted spectrum of pharmacological effects than CB1 agonists.
Pharmacologic or genetic disruption of FAAH showed antihyperalgesia, antianxiety and antidepression
without exhibiting deficits in motility, cognition or body temperature [9,10], which suggests that FAAH
is an attractive therapeutic target for treatment of pain, inflammation, anxiety, depression and other
CNS disorders.
FAAH is a membrane-bound protein belonging to the amidase signature family of serine
hydrolases, and is located on the outer face of the endoplasmic reticulum and mitochondria [11].
Its bioactivity is optimal at pH 8.0–9.0. Mutagenesis studies revealed that FAAH possessed a distinct
serine-serine-lysine catalytic triad. Among which, Ser241 was served as the nucleophile and Lys142
as a key acid and base of the catalytic cycle [12]. The first-generation FAAH inhibitors such as the
substrate analogues
I [13] were proved invaluable for biochemical and structural studies of FAAH.
While, due to the high hydrophobicity and low selectivity, these compounds were not suitable to
Molecules 2016, 21, 229; doi:10.3390/molecules21020229
www.mdpi.com/journal/molecules

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serve as the templates for new drug development. Several classes of FAAH inhibitors with improved
selectivity and in vivo activity have been reported in recent years (Figure 1).
O
O
N
CF3
O
II, OL-135
N
I
CONH2
O
H
F3C
N
N
O
N
N
H
O
N
O
R
III, URB597, R = H
IV, URB937, R = OH
V, PF-3845
Figure 1. Structures of FAAH inhibitors.
One class of FAAH inhibitors is reported as electrophilic ketone compounds, which contains
α
-ketoheterocycles like II (OL-135) to form a reversible hemiacetal bond with the catalytic serine [14].
These compounds displayed excellent in vivo potency and selectivity, while, reversible action led
to elevated levels of several N-acylethanolamine (NAE) substrates, and only transient elevations
in AEA. URB597 (III) is a representative of carbamate inhibitors of FAAH [9]. It was shown that
carbamate inhibitors formed a covalent bond with active site Ser241 nucleophile by carbamylation in
an irreversible mechanism [15]. An additional hydroxy substitution to the proximal phenyl ring of
URB597 restricted the compound (IV, URB937) to cross the blood-brain barrier, avoiding the potential
CNS effects [16]. Urea inhibitors were reported since 2006 by some outstanding pharmaceutical
companies as Pfizer, Takeda, Johnson & Johnson, Lilly and Astellas [17]. As the most potent inhibitor,
PF-3845 (V) gains its potency from a more extended set of van der Waals interactions between the
biaryl ether piperidine moiety and the hydorphobic acyl chain-binding pocket (ACBP) of FAAH [18].
A computer-aided virtual screening combined with enzyme activity assay on in-house compounds
indicated that a known uracil derivative (
(IC50 = 0.11 0.03 M). Although in 2013, uracil derivatives were reported as a novel class of potent
inhibitors of Acid ceramidase (AC) [19 20], substituted 2,4-dioxopyrimidine-1-carboxamides were not
1) exhibited moderate enzyme inhibitory activity
˘
µ
,
applied as a template in design and synthesis of FAAH inhibitors. Considering that all of the reported
AC inhibitors have a retained C6 aliphatic chain [20], in the present work, we turned to replace it with
the more rigid aromatic group class to improve druggable properties. A series of uracil derivatives
were then prepared based on the parent molecule
of the role in FAAH inhibition was performed.
1 as potent enzyme inhibitors, and initial elucidation
2. Results
2.1. Chemistry
Compounds 1–15 were prepared by the amidation of uracil derivatives (or their analogues)
with the corresponding isocyanates in the presence of DMAP/triethylamine (Scheme 1). The above
isocyanates were produced by acyl azides reaction and Curtius rearrangement of the corresponding
carboxylic acids (Scheme 1).
The active substituents at position 5 of uracil ring, such as amino or hydroxy, should be
protected before amidation. Here, the carboxybenzyl group was used to protect amines, while the
t-butyl-dimethylsilyl group was used for active hydroxy protection. Compounds 16–18 were then

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synthesized by means of the amidation of protected uracil derivatives with phenylethyl isocyanates
in the presence of DMAP/pyridine (Scheme 2), and the protecting groups were removed after
amidation reaction.
Method A
R1
R1H
c
N3
a
b
HOOC
O C N
O
N
H
O
O
O
NH
NH
NH
1: R1=
2: R1=
3: R1=
N
O
O
N
O
N
O
O
N
O
N
N
NH
NH
4: R1=
5: R1=
6: R1=
N
O
O
O
O
Method B
R2
O
N
NH
R2
NH
O
N
H
c
O
a
b
R1 COOH
R1
N C O
O
R1
N3
R1
O
N
H
7: R1=
8: R1=
R2= CH3
9: R1=
R2= OCH3
10: R1=
R2= F
11: R1=
R2= CF3
R2= H
Ph
Ph
12: R1=
13: R1=
R2= F
14: R1=
15: R1=
R2= H
R2= H
R2= F
a: Oxalyl chloride, cat. DMF, DCM, rt; NaN3, acetone/water, 0 ℃; b: Toluene, 110 ℃, reflux, 2 h;
c: cat. DMAP, Et3N, Pyridine, 90 ℃, 3 h.
Scheme 1. Preparation of compounds 1–15.
Method A
O
NCO
Cbz O
HN
Cbz
HN
O
N
O
N
H2N
H2N
NH
NH
NH
NH
a
c
b
N
H
O
N
O
O
O
H
O
N
O
N
H
H
16
17
a: Cbz-Cl, NaOH, H2O; b: cat. DMAP, Pyridine, 90 ℃, 3 h; c: TMSI, CH2Cl2, 0 ℃.
Method B
O
N
O
O
N
HO
TBSO
NCO
TBSO
NH
b
NH
NH
a
O
N
H
O
O
O
N
O
N
H
H
18
a: cat. DMAP, Pyridine, 90 ℃, 3 h; b: TBAF, THF.
Scheme 2. Preparation of compounds 16–18.

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Compounds 19
–21 were prepared by N-alkylation reaction of uracil derivatives with the
corresponding alkyl iodide in the presence of NaH/THF (Scheme 3).
O
O
R1
R1
NaH, THF
NH
NH
R2I
N
O
N
O
H
R2
20: R1= F
R2=
19: R1= H
21: R1= F
H
R2=
R2=
N
O
Scheme 3. Preparation of compounds 19–21.
2.2. FAAH Inhibition
The substituted 2,4-dioxopyrimidine-1-carboxamides and the related analogues described
above were subjected to in vitro biological evaluation toward recombinant rat FAAH derived from
˝
HEK293-rFAAH cell. Enzyme assays were performed at 37 C by incubating 30
µg of rFAAH
protein with 25 M anandamide as substrate in Tris-HCl buffer containing fatty acid-free BSA (0.05%).
µ
When the reactions were terminated, 1 nmol heptadecanoic acid (17:0 FFA) was added as an internal
standard. URB597 was used as a positive control. The hydrolyzate of AEA (arachidonic acid), together
with heptadecanoic acid was detected in HPLC/MS/MS to estimate the potency of rFAAH inhibition
according to the methods previously described [21]. IC50 values of the above compounds were
expressed in Table 1.
Table 1. FAAH inhibitory activity of compounds 1–21 and URB597.
Compound
IC50 of FAAH Inhibition (µM)
Compound
IC50 of FAAH Inhibition (µM)
1
2
3
4
5
6
7
8
9
0.11 ˘ 0.03
12
13
14
15
16
17
18
19
20
0.81 ˘ 0.10
1.21 ˘ 0.22
0.053 ˘ 0.006
0.10 ˘ 0.01
0.12 ˘ 0.03
1.33 ˘ 0.13
0.54 ˘ 0.07
>100
>100
>100
0.035 ˘ 0.008
>10
>10
0.86 ˘ 0.08
0.25 ˘ 0.03
1.27 ˘ 0.21
0.30 ˘ 0.07
1.07 ˘ 0.14
0.531 ˘ 0.08
0.098 ˘ 0.01
0.021 ˘ 0.004
10
11
21
URB597
3. Discussion
The purpose of the present work was to investigate substituted 2,4-dioxo-pyrimidine-1
-carboxamides as a potent class of FAAH inhibitors. We firstly examined the role of the uracil
scaffold. Compound
which highlighted the important function of the C5–C6 double bond. Replacement of the uracil ring
with tetrahydro-2-one-pyrimidine (compound ) also led to the loss of FAAH inhibitory activities.
We next evaluated the role of the urea linkage. Compared with , when the carboxamide was reduced
2, the dihydro derivative of 1, showed no inhibitory activity toward FAAH,
3
1
to methylene as in 19 and 20, FAAH activity was not inhibited at all. Even when the carboxamide
group was retained, destroying of urea linkage by inserting a methylene into the urea bond could lead
to a total loss of the FAAH inhibitory activity (21, IC50 > 100 µM). It was suggested that a complete

Molecules 2016, 21, 229
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conjugated 2,4-dioxo-pyrimidine-1-carboxamide system was necessary for enzyme inhibition, which
represents a novel template for FAAH inhibitor development and research.
As the flexible aliphatic chain may weaken the prospects of pharmaceutical development of
FAAH inhibitor, we turned to using the more rigid aromatic group class to replace it. Replacement
of the hexyl chain of with a naphthylmethyl group led to a 8-fold decrease in inhibitory activity
12, IC50 = 0.81 0.10 M), and substitution with a 2-phenylethyl side chain in the same position caused
a slight 3-fold decrease in potency (compound vs. ). Notably, changing the steric configuration of the
1 as a
1
(
˘
µ
7
1
lipophilic chain by substitution of 4-biphenylmethyl group resulted in a new double digit nanomolar
inhibitor 14 (IC50 = 0.053 ˘ 0.006 µM).
We finally focused our attention on uracil ring substitutions to further investigate the
structure-activity relationships. Methylation on unsubstituted positions of the uracil ring of
then explored, which was not beneficial for the activity. The introduction of a methyl group at 3-,
or 6-position of the uracil ring led to 8- to 10- fold decrease in potency ( vs. ). Considering that the
1 was
4
,
6
1
5-methyl derivatives were only 2- to 3- fold less potent than the related parent compounds (5 vs. 1, 8
vs. 7), additional substitutions were then kept at position 5 for further SAR study.
As illustrated in Table 1, introduction of a methoxy, amino and hydroxy group at 5-position of
declined the inhibitory activity toward FAAH, as compound 17 and 18 yielded IC50 values
of 0.53, 1.33 and 0.54 M, respectively. Substitution of the amine group of compound 17 by
7
9,
µ
N-benzyloxycarbonyl amino group (16) increased the inhibitory potency toward FAAH for about
10 times, which suggested that a free amine group in position 5 of the uracil ring is unfavorable to
the activity. Replacing the methyl group of
and –CF3, resulted in two novel double digit nanomolar FAAH inhibitors 10 (IC50 = 0.098
and 11 (IC50 = 0.021 0.004 M), which had a more favorable impact on the inhibitory potency than
8
with electron-withdrawing substituents, such as –F
˘
0.01 M)
µ
˘
µ
substitution of electron-donating groups. Meanwhile, fluorine substitution at the uracil ring did not
always cause an increase in potency. Replacement of a hydrogen atom with fluorine at 5-position of 12
led to a slightly decrease in potency (13 vs. 12), and compound 15 showed a 2-fold less potent than 14
,
which might be affected by additional features that reduced the binding site affinity.
4. Experimental Section
4.1. General
In the present study, all chemicals were purchased from Sigma-Aldrich (Shanghai, China),
seeking the highest grade commercially available unless otherwise indicated. Some solvents were
distilled prior to use, such as methylene chloride (CH2Cl2), distilled from phosphorus pentoxide,
dimethylformamide (DMF), distilled from calcium hydride and tetrahydrofuran (THF), distilled from
sodium benzophenone ketyl. Silica gel (300–400 mesh) from Yantai Athy Chemical Technology Co. Ltd.
(Zhifu, China) was used for column chromatography. Mixtures of increasing polarity of ethyl acetate
1
and petroleum ether (PE) (60–90 ˝C) were used as eluents unless otherwise stated. H-NMR spectra
were recorded on a Bruker 400 (400 MHz) spectrometer (Bruker, Fallanden, Switzerland) using CDCl3,
DMSO-d6, or CD3OD as the solvent with Me4Si as the internal standard. 13C-NMR spectra were
recorded on a Bruker 400 (100 MHz) spectrometer. IR spectra were recorded on a Nicolet AVATAR
360 RT-IR spectrophotometer (Thermo Electron Inc., San Jose, CA, USA). Mass spectra were recorded
on an MDS SCIEX 3200Q TRAP mass spectrometry (MS) system (Applied Biosystems/MDS Sciex,
Concard, ON, Canada) with electrospray ionization (ESI) and direct injection. Elemental analyses
were performed on an Elementar Vario EL analyzer (Vario EL, Elementar Analyser systeme GmbH,
Hanau, Germany). HPLC analyses were run on a 1200 series HPLC (Agilent, Shanghai, China) system
equipped with a photodiode array detector. PDA range was 210–600 nm. Analyses were performed
on a Hypersil Gold C18 column (dimensions 250
ˆ
4.6 mm, particle size 5 µm, Thermo Scientific,
Waltham, MA, USA). A constant mobile phase was 85% methanol in H2O, and the flow rate was set at
1 mL¨ min´1. All final compounds showed ě95% purity by HPLC and NMR analysis.

Molecules 2016, 21, 229
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4.2. General Procedure for the Synthesis of Uracil Derivatives 1–15
Compounds 1–15 were synthesized according to Scheme 1. The appropriately substituted
carboxylic acid (0.5 mmol) was dissolved in dry CH2Cl2 (5 mL). Oxalyl chloride (0.6 mmol, 0.05 mL)
and DMF (0.01 mL) were added, and the reaction mixture was stirred at 0 ˝C for 1 h. The solvent
was evaporated under reduced pressure, keeping the temperature below 30 ˝C. The crude residue
was redissolved with anhydrous acetone (5 mL) and then dropwise added into a stirred and ic˝e-cold
solution of NaN3 (1.0 mmol, 33 mg) in H2O (1 mL). The resulting mixture was stirred at 0 C for
30 min, and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The residue was then dissolved in toluene (15 mL),
and refluxed for 2 h. The reaction solution was cooled to room temperature, and dropwise added to
a solution of˝the corresponding uracil derivatives (0.5 mmol) in dry pyridine (0.5 mL). After being
stirred at 90 C for 3 h, the mixture was cooled and evaporated. The crude was purified by flash
chromatography on silica gel to afford compounds 1–15.
N-Hexyl-2,4-dioxo-pyrimidine-1-carboxamide (
1). This compound was obtained in 84% yield as a white
amorphous powder. Detailed MS, 1H- and 13C-NMR results were according to the literature data [19].
N-Hexyl-2,4-dioxotetrahydropyrimidine-1-carboxamide (
2). This compound was obtained in 67% yield as a
white amorphous powder, Detailed MS, 1H- and 13C-NMR results matched the literature values [20].
N-Hexyl-2-oxotetrahydropyrimidine-1-carboxamide (3). This compound was obtained as a white
amorphous powder in 74% yield 1H-NMR (D2O-CDCl3)
δ 0.86 (t, J = 6.8 Hz, 3H), 1.23–1.28 (m, 6H),
1.47–1.52 (m, 2H), 1.95–2.00 (m, 2H) 3.24–3.29 (m, 2H), 3.35 (m, 2H), 3.78–3.84 (m, 2H); 13C-NMR
(CDCl3) δ 14.3, 21.5, 22.5, 26.3, 29.1, 31.3, 32.5, 40.8, 40.9, 152.0, 161.0.
N-Hexyl-3-methyl-2,4-dioxopyrimidine-1-carboxamide (4). This compound was obtained as a white
amorphous powder in 49% yield, Detailed MS, 1H- and 13C-NMR results were in accord with the
literature [20].
N-Hexyl-5-methyl-2,4-dioxopyrimidine-1-carboxamide (5). This compound was obtained in 55% yield as
a white amorphous powder. Detailed MS, 1H- and 13C-NMR results were in good accord with the
literature [20].
N-Hexyl-6-methyl-2,4-dioxopyrimidine-1-carboxamide (
amorphous powder (63% yield). H-NMR (CDCl3/pyridine-d5)
6
). This compound was obtained as a white
1
δ
0.90 (t, J = 6.8 Hz, 3H), 1.29–1.38
(m, 6H), 1.55–1.65 (m, 2H), 1.76 (s, 3H), 3.34–3.44 (m, 2H), 6.25 (br, 1H), 8.24 (br, 1H), 9.25 (br s, 1H);
13C-NMR (CDCl3/pyridine-d5)
(ESI, m/z): 252 (M ´ H) .
δ 9.4, 14.0, 22.5, 26.5, 29.2, 31.4, 41.2, 102.5, 145.3, 150.0, 155.1, 163.1; MS
´
N-(2-Phenylethyl)-2,4-dioxopyrimidine-1-carboxamide (
7). This compound was obtained as a white
amorphous powder (75% yield). IR (
δ 2.84 (t, J = 7.0 Hz, 2H), 3.50–3.55 (m, 2H), 5.80 (d, J = 8.4 Hz, 1H), 7.22–7.31
(m, 5H), 8.21 (d, J = 8.4 Hz, 1H), 9.20 (br, 1H), 11.72 (br, 1H); 13C-NMR (DMSO-d6)
ν
max, cm´1): 2950, 2918, 2849, 1725, 1580, 1547, 1402, 1383;
1H-NMR (DMSO-d6)
δ
35.2, 42.4, 104.1,
´
126.8, 128.9, 129.2, 139.1, 139.3, 150.4, 152.0, 163.2; MS (ESI, m/z): 258 (M ´ H) .
5-Methyl-N-(2-phenylethyl)-2,4-dioxopyrimidine-1-carboxamide (
8). This compound was obtained as white
amorphous powder in 70% yield. IR (
ν
max, cm´1): 3456, 2950, 2917, 2849, 1580, 1068; 1H-NMR (CDCl3)
δ
1.98 (s, 3H), 2.91 (t, J = 7.1 Hz, 2H), 3.62–3.65 (m, 2H), 7.21–7.31 (m, 5H), 8.22 (s, 1H), 9.16 (br, 1H),
9.41 (br, 1H); 13C-NMR (CDCl3)
163.6; MS (ESI, m/z): 272 (M ´ H) .
δ 12.4, 35.5, 42.5, 112.4, 126.7, 128.7, 128.8, 134.5, 138.3, 150.1, 151.5,
´

Molecules 2016, 21, 229
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5-Methoxy-N-(2-phenylethyl)-2,4-dioxopyrimidine-1-carboxamide (
9
). This compound was obtained in 65%
1
yield as a white amorphous powder. H-NMR (DMSO-d6)
δ
2.84 (t, J = 7.0 Hz, 2H), 3.50–3.55 (m, 2H),
35.2, 42.4,
3.68 (s, 3H), 7.22–7.31 (m, 5H), 7.70 (s, 1H), 9.25 (m, 1H), 12.0 (s, 1H); 13C-NMR (DMSO-d6)
δ
´
56.5, 116.2, 126.8, 128.3, 128.7, 130.2, 136.5, 150.1, 150.3, 158.8; MS (ESI, m/z): 288 (M ´ H) .
5-Fluoro-N-(2-phenylethyl)-2,4-dioxopyrimidine-1-carboxamide (10). This compound was obtained as a
white amorphous powder in 39% yield. Detailed MS and 1H-NMR results were in agreement with the
literature [22].
N-(2-Phenylethyl)-5-trifluoromethyl-2,4-dioxopyrimidine-1-carboxamide (11). This compound was obtained
1
in 23% as a white amorphous powder. H-NMR (CDCl3)
δ
2.76 (t, J = 6.9 Hz, 2H), 3.41–3.45 (m, 2H),
7.25–7.35 (m, 5H), 8.93–9.01 (m, 3H); 13C-NMR (CDCl3)
128.9, 129.2, 139.1, 139.7, 149.0, 150.7, 156.8; MS (ESI, m/z): 326 (M ´ H) .
δ
35.4, 42.4, 114´.3, 121.2 (q, J = 268 Hz), 126.8,
N-(1-Naphthylmethyl)-2,4-dioxopyrimidine-1-carboxamide (12). This compound was obtained as white
˝
crystals (20% yield). Mp: 84.0–84.6 C; IR (
1H-NMR (DMSO-d6)
4.66 (d, J = 5.3 Hz, 2H), 5.81(d, J = 8.4 Hz, 1H), 7.50–7.51 (br, 3H), 7.85 (s, 1H),
7.89–7.90 (br, 3H), 8.23 (d, J = 8.4 Hz, 1H), 9.67 (br, 1H), 11.77 (br, 1H); 13C-NMR (DMSO-d6)
44.6,
ν
max, cm´1): 3426, 2955, 2917, 2849, 1726, 1578, 1380;
δ
δ
104.1, 126.0, 126.3, 126.4, 126.7, 128.0, 128.1, 128.5, 132.7, 133.3, 136.3, 139.3, 150.9, 152.0, 163.3; MS
´
(ESI, m/z): 294 (M ´ H) .
5-Fluoro-N-(1-naphthylmethyl)-2,4-dioxopyrimidine-1-carboxamide (13). This compound was obtained as
1
a white amorphous powder in 27% yield. H-NMR (DMSO-d6)
δ 4.67 (d, J = 5.6 Hz, 2H), 7.49–7.52
(m, 3H), 7.84–7.91 (m, 4H), 8.42 (d, J = 7.6 Hz, 1H), 9.67 (t, J = 6.0 Hz, 1H), 12.30 (d, J = 4.4 Hz, 1H);
13C-NMR (DMSO-d6)
142.3, 150.6, 157.6; MS (ESI, m/z): 312 (M ´ H) .
δ
44.7, 123.6, 125.4, 126.3, 126.5, 126.7, 127.9, 128.0, 128.5, 132.7, 133.3, 137.5, 140.0,
´
N-[2-(4-Biphenyl)methyl]-2,4-dioxopyrimidine-1-carboxamide (14). This compound was obtained in 30%
yield as a white amorphous powder, yield 30%. IR (ν
max, cm´1): 3440, 2956, 2917, 2849, 1723, 1578,
1464, 1380; 1H-NMR (CDCl3)
(br, 4H), 8.28 (br, 1H), 8.43 (d, J = 8.5 Hz, 1H), 9.47 (br, 1H); MS (ESI, m/z): 320 (M ´ H) .
δ
4.61 (d, J = 4.6 Hz, 2H), 5.90 (d, J = 8.5 Hz, 1H), 7.34–7.44 (m, 5H), 7.57
´
N-[2-(4-biphenyl)methyl]-5-fluoro-2,4-dioxo-pyrimidine-1-carboxamide (15). This compound was obtained
1
as a white amorphous powder (46% yield). H-NMR (DMSO-d6)
δ
4.54 (d, J = 5.6 Hz, 2H), 7.34–7.38
(m, 1H), 7.43–7.48 (m, 4H), 7.63–7.66 (m, 4H), 8.41 (br, 1H), 9.61 (t, J = 6.0 Hz, 1H), 12.29 (s, 1H);
13C-NMR (DMSO-d6)
157.6; MS (ESI, m/z): 338 (M ´ H) .
δ
44.3, 123.1, 123.5, 127.1, 127.2, 127.9, 128.5, 129.4, 137.9, 139.6, 140.4, 142.3, 150.6,
´
4.3. Synthesis of Uracil Derivatives 16–17
According to Method A, Scheme 2, 5-aminouracil (1270 mg, 10 mmol) and NaOH (600 mg,
˝
15 mmol) were dissolved in H2O (20 mL) and then stirred at 0 C for 10 min. Cbz-Cl (1.7 mL,
12 mmol) was added dropwise to the above mixture, and stirred overnight at room temperature.
The resulting mixture was adjusted to pH 3 with dilute hydrochloric acid, and extracted with EtOAc.
The organic layer was dried over anhydrous Na2SO4, and evaporated under reduced pressure to
afford 5-(N-benzyloxycarbonyl)aminouracil.
5-(N-benzyloxycarbonyl)aminouracil (261.2 mg, 1 mmol), DMAP (6.1 mg, 0.05 mmol) and dried
triethylamine (0.3 mL, 2 mmol) were dissolved in anhydrous pyridine (4.0 mL) and then stirred at
˝
90 C for 10 min under N2 protection. Phenylethyl isocyanate (220.8 mg, 1.5 mmol) were added
dropwise, and the reaction mixture were stirred at 90 ˝C for 3 h. After cooling to room temperature,
the reaction solution was concentrated under reduced pressure. The crude residue was purified by
flash chromatography on silica gel to afford compound 16 (121.4 mg).

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Compound 16 (98.2 mg, 0.24 mmol) was dissolved in anhydrous CH2Cl2 (4.0 mL), and stirred for
10 min at 0 ˝C. TMSI (0.1 mL, 0.48 mmol) was added dropwise in the above solution, and stirred for
2 h at the same temperature. CH3OH (1.0 mL) was added slowly to quench the reaction. After being
stirred for 10 min, the reaction mixture was cooled and concentrated to remove the solvent. The residue
was purified by flash chromatography on silica gel to afford compound 17 (21.6 mg).
Benzyl (2,4-dioxo-1-{[(2-phenylethyl)amino]carbonyl}-1,2,3,4-tetrahydro-5-pyrimidinyl)carbamate (2H)-carboxamide
(
16). This compound was obtained as a white amorphous powder in 30% yield. IR (
2917, 2849, 1580, 1383, 1258, 1207; 1H-NMR (CDCl3)
2.90 (t, J = 7.1 Hz, 2H), 3.62–3.66 (m, 2H), 5.18
(s, 2H), 7.11 (s, 1H), 7.20 (s, 1H), 7.23–7.25 (m, 1H), 7.28–7.37 (m, 8H), 9.07–9.12 (br, 2H), 9.79(s, 1H);
ν
max, cm´1): 2957,
δ
13C-NMR (CDCl3)
149.6, 149.8, 152.8, 159.4; MS (ESI, m/z): 407 (M ´ H) .
δ
35.4, 42.7, 67.6, 115.7, 123.6, 126.7, 127.1, 128.3, 128.5, 128.6, 128.7, 135.5, 138.3,
´
5-Amino-N-(2-phenylethyl)-2,4-dioxopyrimidine-1-carboxamide (17). This compound was obtained as a
white amorphous powder in 33% yield. IR (ν
max, cm´1): 3337, 2955, 2921, 2851, 1723, 1613, 1580, 1462,
1378; 1H-NMR (DMSO-d6)
δ 2.81–2.85 (m, 2H), 3.50–3.55 (m, 2H), 4.48 (s, 2H), 7.21–7.32 (m, 5H), 7.57
(s, 1H), 9.30–9.36 (m, 1H), 11.80 (br, 1H); 13C-NMR (DMSO-d6)
129.1, 139.4, 150.7, 151.0, 160.9; MS (ESI, m/z): 273 (M ´ H) .
δ
35.3, 42.2, 112.2, 124.5, 126.7, 128.9,
´
4.4. Synthesis of Compound 18
According to Method B, Scheme 2, 5-OTBS-uracil (96.9 mg, 0.4 mmol), DMAP (2.4 mg, 0.02 mmol)
and anhydrous triethylamine (0.3 mL, 2 mmol) were dissolved in dry pyridine (4.0 mL). The reaction
˝
mixture was stirred for 10 min at 90 C under N2 protection and then phenylethyl isocyanate (220.8 mg,
1.5 mmol) was added dropwise. After being stirred for 3 h at the same temperature, the reaction
solution was cooled and concentrated under reduced pressure. The residue was purified by flash
chromatography on silica gel to afford 5-OTBS-N-(2-phenylethyl)-2,4-dioxopyrimidine-1-carboxamide
(25.2 mg). This reaction product (24.6 mg, 0.06 mmol) and TBAF 3H2O (39.9 mg, 0.13 mmol) were
˝
dissolved in THF (3.0 mL), and stirred for 30 min at 0 C. After gradually warming to room temperature,
the reaction solution was stirred for another 2 h. Solvent was evaporated under reduced pressure,
and the crude residue was purified by flash chromatography on silica gel to afford compound
5-hydroxy-N-(2-phenylethyl)-2,4-dioxopyrimidine-1-carboxamide (18) in 10% yield as a white amorphous
powder. IR (
ν
max, cm´1): 2956, 2918, 2850, 1726, 1383, 1071; 1H-NMR (MeOD)
δ
2.90 (t, J = 7.2 Hz, 2H),
´
3.61 (t, J = 7.2 Hz, 2H), 7.22–7.32 (m, 5H), 7.88 (s, 1H); MS (ESI, m/z): 274 (M ´ H) .
4.5. General Procedure for the Synthesis of Uracil Derivatives 19–21
A solution of uracil derivative (0.5 mmol) in THF (3 mL) was added slowly to a suspension of NaH
(60% in mineral oil, 30 mg) in anhydrous THF (5 mL) under a nitrogen atmosphere at
´
20 ˝C. After
˝
stirring for 5 min at
´20 C, a solution of the appropriate iodide (0.6 mmol) in THF (2 mL) was slowly
added. The reaction mixture was stirred at 0 ˝C for 5 h and then quenched with saturated aqueous
ammonium chloride solution (5 mL) followed by extraction with EtOAc (3
organic layers were dried over anhydrous Na2SO4 and filtered. The concentrated residue was purified
ˆ
5 mL). The combined
by flash chromatography on silica gel to afford compounds 19–21.
1-Heptyl-2,4-dioxopyrimidine (19). This compound was obtained in 44% yield as a white amorphous
max, cm´1): 2920 (N-H), 2852 (N-H), 1667 (C=O), 1461; 1H-NMR (CDCl3)
powder. IR (ν δ 0.89
(t, J = 6.8 Hz, 3H), 1.28–1.32 (m, 8H), 1.67–1.71 (m, 2H), 3.73 (t, J = 7.6 Hz, 2H), 5.71 (d, J = 7.6 Hz,
1H), 7.16 (d, J = 7.6 Hz, 1H), 9.56 (s, 1H); 13C-NMR (CDCl3)
δ 14.0, 22.6, 26.4, 29.0, 29.1, 31.7, 48.9,
´
102.1, 144.5, 151.0, 164.0; MS (ESI, m/z): 209 (M
´
H) . Anal. Calcd. for C11H18N2O2: C 62.83; H 8.63;
N 13.32. Found: C 62.59; H 8.64; N 13.30. This is a known compound, although the detailed MS,
1H- and 13C-NMR data are not reported in the previous literature.

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5-Fluoro-1-heptyl-2,4-dioxopyrimidine (20). This compound was obtained as a white amorphous powder
1
in 31% yield. IR (νmax, cm´1): 2920 (N-H), 2852 (N-H), 1667 (C=O), 1461; H-NMR (CDCl3)
δ 0.88
(t, J = 6.8 Hz, 3H), 1.26–1.33 (m, 8H), 1.67–1.69 (m, 2H), 3.70 (t, J = 7.6 Hz, 2H), 7.23 (d, J = 5.2 Hz, 1H),
9.33 (s, 1H); 13C-NMR (CDCl3)
14.0, 22.6, 26.3, 28.9, 29.0, 31.7, 49.1, 128.7, 139.2, 149.3, 157.3; MS
H)´. Anal. Calcd for C11H17FN2O2: C 57.88; H 7.51; N 12.27. Found: C 58.05;
δ
(ESI, m/z): 227 (M
H 7.52; N 12.29.
´
2-[5-Fluoro-2,4-dioxopyrimidinyl]-N-hexylacetamide (21). This compound was obtained in 46% yield as a
white amorphous powder. Detailed MS and 1H-NMR results matched the literature values [23].
4.6. Enzyme Activity Assay
The inhibition effects of uracil derivatives toward recombinant rat FAAH (rFAAH) were assessed
by enzyme activity assay [24]. HEK293-rFAAH cells were harvested, washed with PBS, sonicated in
20 mM Tris-HCl (pH 7.5) containing 0.32 M sucrose, and centrifuged at 800
The supernatants were collected and protein concentrations were measured by BCA protein assay kit
(Pierce, Shanghai, China). FAAH proteins (30
g) were incubated at 37 ˝C for 30 min in Tris buffer
(50 mM, pH = 8.0) containing fatty acid-free BSA (0.05%) and 25 M AEA (Sigma-Aldrich, Shanghai,
China) as substrate. The above reactions were stopped by adding 200 L methanol containing 1 nmol
heptadecanoic acid (17:0 FFA) as internal standard. The reaction mixture was extracted with 4 mL
chloroform and then vortex for 1 min. Lipid layer was separated by centrifugation at 3000 g for
ˆ
g for 15 min at 4 ˝C.
µ
µ
µ
ˆ
10 min and then transferred to a clean 10 mL V-bottom glass tube and dried under nitrogen (N2)
flow. After redissolved with 1 mL CHCl3, the lipid solution went through the solid-phase extraction,
and was eluted by methanol/chloroform (v/v, 1/9). The elution containing FAEs and FFAs was dried
under N2, and reconstituted in 100 µL methanol for HPLC-MS/MS analysis.
Samples were detected by an ABI 3200 Q-Trap mass spectrometer (Applied Biosystems/MDS
Sciex) equipped with 1100-LC system (Agilent, Shanghai, China). The mobile phase for FFAs detection
was consisted of MeOH and ultra-pure H2O (pH = 7.4, each containing 0.25% acetic acid and 5 mmol/L
ammonium acetate), and the elution condition was 95% methanol keeping for 3 min at a flow rate
of 1.0 mL
min´1. The column temperature was kept at 40 ˝C. Ion detection was monitored by ESI
¨
negative mode. The molecular ions were monitored at m/z 303 for AA, and m/z 269 for C17:0 FFA.
Inhibition curves and IC50 values of each compounds was calculated by GraphPad Prism (version 5.01,
GraphPad Software Inc., San Diego, CA, USA).
5. Conclusions
According to the crystal structure of protein rFAAH-MAFP (PDB: 1MT5), the catalytic core of
FAAH contains a series of channels and cavities including a cytosolic port (CP) which interacts with
the hydrophilic fragments and a acyl-chain binding pocket (ACBP) that may allow for the exit of the
acyl chain to support the catalytic reaction. Herein, we used the uracil ring as a novel hydrophilic
product scaffold, and different aromatic substituent groups to replace the acyl side chain. Urea bonds
were formed to connect these two fragments, and thus a series of structurally related substituted
2,4-dioxopyrimidine-1-carboxamides were designed and synthesized according to Schemes 1–3.
In the present study, we have discovered and identified uracil derivatives as a novel class of potent
FAAH inhibitors with IC50 values in the nanomolar or low-micromolar range. Initial SAR analysis
indicated that an entirely conjugated 2,4-dioxopyrimidine-1-carboxamide scaffold was necessary for
FAAH inhibition. Introduction of a 4-biphenylmetyhl moiety as the N-acyl chain was favorable for
activity, leading to a highly potent inhibitor. Although replacement of a 2-phenylethyl side chain
decreased the inhibitory potency, electron-withdrawing substituents in the 5-position of the uracil ring
afforded two novel double digit nanomolar FAAH inhibitors. An electron-withdrawing moiety rather
than an electron-donating group at position 5 of the 2,4-dioxopyrimidine is beneficial for approaching
potent FAAH inhibition. However, further investigation is necessary to elucidate the exact mechanism

Molecules 2016, 21, 229
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of action of this class of inhibitors, which should be useful to clarify the structure-activity relationships
to enhance FAAH affinity and selectivity.
Acknowledgments: This study was supported by grants from National Natural Sciences Foundation of China
(No. 81373273, 81273371), Xiamen Southern Ocean Research Center Project (No. 14GYY018NF18), and the
Fundamental Research Funds for the Central Universities (No. 20720150054).
Author Contributions: Y.Q. and Y.L. conceived and designed the experiments; Y.Q. and Y.Z. performed the
experiments; Y.Q., Y.L. and J.R. analyzed the data; Y.Q. wrote and revised the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–21 are available from the corresponding author.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).