Inhibitory effects of 5-benzylidene barbiturate derivatives on mushroom tyrosinase and their antibacterial activities

Article abstract of DOI:10.1016/j.ejmech.2009.05.023

A series of novel 5-benzylidene barbiturate and thiobarbiturate derivatives were synthesized and evaluated as tyrosinase inhibitors and antibacterial agents. The results demonstrated that some compounds had more potent inhibitory activities than the parent compound 4-hydroxybenzaldehyde (IC₅₀ = 1.22 mM). Particularly, compounds 1a and 2a were found to be the most potent inhibitors with IC₅₀ value of 13.98 μM and 14.49 μM, respectively. The inhibition mechanism study revealed that these compounds were irreversible inhibitors. The circular dichroism spectra indicated that these compounds induced conformational changes of mushroom tyrosinase upon binding. In addition, these compounds exhibited selectively antibacterial activity against Staphylococcus aureus. All these results suggested that further development of such compounds may be of interest.

Full text of DOI:10.1016/j.ejmech.2009.05.023

European Journal of Medicinal Chemistry 44 (2009) 4235–4243
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibitory effects of 5-benzylidene barbiturate derivatives on mushroom
tyrosinase and their antibacterial activities
*
*
Qin Yan, Rihui Cao , Wei Yi, Zhiyong Chen, Huan Wen, Lin Ma, Huacan Song
School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 5-benzylidene barbiturate and thiobarbiturate derivatives were synthesized and
evaluated as tyrosinase inhibitors and antibacterial agents. The results demonstrated that some
compounds had more potent inhibitory activities than the parent compound 4-hydroxybenzaldehyde
(IC50 ¼ 1.22 mM). Particularly, compounds 1a and 2a were found to be the most potent inhibitors with
Received 29 December 2008
Received in revised form
15 May 2009
Accepted 20 May 2009
Available online 28 May 2009
IC50 value of 13.98 mM and 14.49 mM, respectively. The inhibition mechanism study revealed that these
compounds were irreversible inhibitors. The circular dichroism spectra indicated that these compounds
induced conformational changes of mushroom tyrosinase upon binding. In addition, these compounds
exhibited selectively antibacterial activity against Staphylococcus aureus. All these results suggested that
further development of such compounds may be of interest.
Keywords:
5-Benzylidene barbiturate
Thiobarbiturate
Ó 2009 Elsevier Masson SAS. All rights reserved.
Tyrosinase inhibitor
Circular dichroism spectra
Antibacterial activity
1. Introduction
In the last decades, benzaldehyde derivatives especially
hydroxylated analogues have been extensively investigated [4] and
Tyrosinase (EC1.14.18.1), a multifunctional copper-containing
oxygenase, is widely distributed in nature. It catalyzes the
hydroxylation of a monophenol and the conversion of o-diphenols
to the corresponding o-quinones [1]. It is responsible for melani-
zation in animals and browning in plants and fungi. The unfavor-
able darkening from enzymatic oxidation of phenols generally
results in a loss of nutritional value and has been of great concern. It
has recently been discovered that various dermatological disorders,
such as age spots and freckle, were also caused by the accumulation
of an excessive level of epidermal pigmentation. Therefore, tyros-
inase inhibitors have become increasingly important in food
industry as well as in the medicinal and cosmetic products [2,3].
Many efforts have been spent in the search for effective and safe
tyrosinase inhibitors, and a large number of naturally occurring and
synthetic tyrosinase inhibitors have already been reported. But
some of their individual activities are either not potent enough to
be considered of practical use or not compatible with safety regu-
lations for food and cosmetic additives. Therefore, it is still neces-
sary to search and develop novel tyrosinase inhibitors with potent
activities and lower side effects.
structure–activity relationship (SAR) analysis indicated that
electron-donating substituents at 4-position of benzaldehyde are
necessary for potent inhibitory activities [5]. Recently, N-hydroxy-
N0-phenylthiourea,
N-hydroxy-N0-phenylurea
and
thio-
semicarbazide derivatives were reported to exhibit potential
inhibitory activity for tyrosinase and oxygen, nitrogen and sulfur
atom of those compounds were able to complex the two copper ions
in the active site of tyrosinase [6].
More recently, barbiturate and thiobarbiturate derivatives
bearing urea and thiourea moiety, respectively, attracted consid-
erable attention owing to their various biological effects such as
inhibiting collagenase-3 (MMP-3) [7], matrix metalloproteinases
[8], recombinant cytochrome P450 enzymes [9], methionine
aminopeptidase-1 (MetAP-1) [10], and their broad spectrum
pharmacological properties including hypnotic [11], sedative [12]
and antibacterial [10]. In addition, it was well known that tyrosi-
nase contained two copper ions in the active center and a lipophilic
long-narrow gorge near to the active center, so we hypothesized
that barbiturates or thiobarbiturates could complex the binuclear
copper active site of tyrosinase, and interact with the hydrophobic
enzyme pocket, leading to increasing inhibitor–enzyme binding
affinity and improving inhibitory effects on mushroom tyrosinase.
Based on the above information, in the present investigation,
a series of novel 5-benzylidene barbiturate and thiobarbiturate
derivatives bearing hydrophobic alkyl substituents were designed
* Corresponding authors. Tel.: þ86 20 84110918; fax: þ86 20 84112245.
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), yjhxhc@mail.sysu.edu.cn
(H. Song).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.023

4236
Q. Yan et al. / European Journal of Medicinal Chemistry 44 (2009) 4235–4243
and synthesized by the condensation of 4-substituted benzalde-
hydes with barbituric acid or thiobarbituric acid. Their inhibitory
effects on the diphenolase activity of mushroom tyrosinase and
their antibacterial activities against Staphylococcus aureus
(S. aureus), Staphylococcus epidermidis (S. epidermidis), Staphylo-
coccus albus (S. albus), Bacillus cereus (B. cereus), Escherichia coli
(E. coli) and Pseudomonas aeruginosa (P. aeruginosa) were also
investigated. To the best of our knowledge, this is the first time to
report the inhibitory effects on the diphenolase activity of mush-
room tyrosinase of 5-benzylidene barbiturate and thiobarbiturate
derivatives.
10
80
60
40
2. Chemistry
The synthesis of 5-benzylidene barbiturate and thiobarbiturate
derivatives is summarized in Scheme 1. Mono-tosylates were
prepared by an excess of tosyl chloride and the corresponding
alcohols in catalytic amounts of NaOH as described previously
[13–15], and subsequently de-protected and simultaneously reacted
with 4-hydroxybenzaldehyde in dimethylformamide (DMF) with
potassium carbonate at 80ꢀC for 16 h [16,17] to afford alkylated
benzylaldehydes c–f. The condensation of 4-hydroxybenzaldehyde
and alkylation benzaldehydes b–f with barbituric acid (or thio-
barbituric acid) via well-known Knoevenagel condensation in
ethanol [18,19] gave the 5-benzylidene barbiturate and thio-
barbiturate derivatives 1 and 2 in good yield, and then followed by
reduction with NaBH4 in methanol to afford compounds 3 and 4,
respectively [20]. All the synthesized 5-benzylidene barbiturate and
thiobarbiturate derivatives were characterized by spectroscopic
data.
0
2
4
6
8
10
12
14
16
[I]( M)
Fig. 1. Effect of compound 1a on the diphenolase activity of mushroom tyrosinase for
the catalysis of L-DOPA at 25 ꢀC.
barbituric acid and thiobarbituric acid almost showed no activities
at concentration of 200 mM, and 4-hydroxybenzaldehyde exhibited
weak inhibitory activities with IC50 value of 1.22 mM. As predicted,
most of the 5-benzylidene barbiturate and thiobarbiturate deriva-
tives displayed more potent tyrosinase inhibitory activities than
parent compounds barbituric acid, thiobarbituric acid and
4-hydroxybenzylaldehyde.
As a whole, 5-benzylidene thiobarbiturates exhibited more
potent inhibitory effects than 5-benzylidene barbiturates.
Compounds 1c, 1e and 1f showed inhibitory activities with IC50
3. Biology
values of 75.42, 137.29 and >200
thiobarbiturate analogues 2c, 2e and 2f displayed inhibitory
activities with IC50 values of 28.43, 34.20 and 77.80 M, respec-
tively. Similarly, compounds 3a–e had no inhibitory activities at the
concentration of 200 M, whereas their thiobarbiturate analogues
4a–e demonstrated potent inhibitory activities with IC50 values in
the range of 70.02–179.31 M, respectively. These results suggested
that thiobarbiturate moiety was more favorable than barbiturate
moiety for their inhibitory effects on the diphenolase activity of
mushroom tyrosinase.
mM, respectively, while their
3.1. Inhibitory effects on the diphenolase activity
of mushroom tyrosinase
m
m
The inhibition activities of our synthetic 5-benzylidene barbi-
turate and thiobarbiturate derivatives on mushroom tyrosinase
were investigated by usual procedure [21] and compared with
those of 4-hydroxybenzaldehyde [22] and 4-methoxycinnamic acid
[23]. Figs. 1 and 2 showed that the remaining enzyme activities
rapidly decreased with the increasing concentration of compounds
1a and 2a. The IC50 values of all compounds investigated are
summarized in Table 1. As shown in Table 1, parent compounds
m
Among all the investigated compounds, compounds 1a and 2a
bearing a hydroxyl group at position-4 of phenyl ring were found to
OH
OR
X
DMF
TsCl
HN
O
NH
HOR
TsOR
+
+
THF
O
CHO
CHO
a
b~f
X =O or S
O
O
NaBH4
EtOH
EtOH
Reflux
NH
NH
X
RO
RO
O
N
H
X
O
N
H
1a~1f X=O
2a~2f X=S
3a~3f X=O
4a~4f X=S
a R= H; b R= CH2CH2OH; c R= CH2CH2OCH3; d R= CH2CH2OC4H9
e R= CH2CH2OCH2CH2OCH3; f R= (CH2)4OCH3
Scheme 1. Synthesis of 5-benzylidene barbiturate and thiobarbiturate derivatives.

Q. Yan et al. / European Journal of Medicinal Chemistry 44 (2009) 4235–4243
4237
Unlike compounds 1a–f and 2a–f having an arylidene substit-
uent at position-5, their reduced congeners 3a–f and 4a–f (except
4b), bearing a benzyl substituent at position-5, exhibited compar-
atively weaker tyrosinase inhibitory activities. This might be due to
the breaking of the conjugation of the barbituric moiety to the
aromatic phenyl ring, implying that the increase of molecular
flexibility might be detrimental to their inhibitory activities.
100
80
3.2. Inhibitory mechanism of compounds 1a and 2a
on mushroom tyrosinase
60
The inhibition mechanism of the most potent compounds 1a and
2a on mushroom tyrosinase for the oxidation of L-DOPA was first
determined. Figs. 3 and 4 showed the relationship of enzyme
activity with its concentration in the presence of different concen-
trations of compounds 1a and 2a. The results displayed that the
plots of the remaining tyrosinase activity versus the concentrations
of tyrosinase gave a family of parallel lines with the same slopes.
These results demonstrated that the inhibition of compounds 1a
and 2a on the tyrosinase enzyme were irreversible, suggesting that
5-benzylidene (thio)barbiturate derivatives might exhibit such
tyrosinase inhibitory effects by the mode of making the enzyme
inactivity forever.
40
0
2
4
6
8
10
12
14
16
[I]( M)
Fig. 2. Effect of compound 2a on the diphenolase activity of mushroom tyrosinase for
the catalysis of L-DOPA at 25 ꢀC.
be the most potent inhibitors with IC50 values of 13.98
mM and
14.49 M, respectively. Replacement of the hydroxyl of compounds
m
1a and 2a at position-4 of phenyl ring with 2-hydroxyethoxy
(compounds 1b and 2b), 2-methoxyethoxy (1c and 2c), 2-butox-
yethoxy (1d and 2d), 2-(2-methoxyethoxy) ethoxy (1e and 2e) and
4-methoxybutoxy (1f and 2f), respectively, led to a dramatic
decline in inhibitory activities, indicating that the increase of the
length of alkoxyl chain at position-4 of phenyl ring might cause
stereo-hindrance for inhibitors approaching the active site, and the
hydroxyl group was more favorable.
3.3. The influence of compounds 1a and 2a on the secondary
structure of mushroom tyrosinase
The influence of 5-benzylidene barbiturate and thiobarbiturate
derivatives 1a and 2a on the secondary structure of mushroom
tyrosinase was investigated by circular dichroism spectroscopy
[24,25]. The results are summarized in Table 2. As shown in Table 2,
the a-helix content of tyrosinase was 59.7% and the b-sheet was not
Table 1
Inhibitory effects on mushroom tyrosinase and antibiotic activities of 5-arylidene barbiturate (thiobarbiturate) derivatives.
Compound
X
R
Tyrosinase
MIC (mg/mL)
c
IC50
(m
M)
Inhibition rate (%) at 200
mM
S. aureus
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
O
O
O
O
O
O
S
S
S
S
S
H
13.98
>200
75.42
45.45
100
22.41
100
>25
>25
>25
>25
>25
>25
3.1
>25
>12.5
3.1
>12.5
>25
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>25
>25
>12.5
6.25
>12.5
>12.5
–
CH2CH2OH
CH2CH2OCH3
CH2CH2OCH2CH2CH2CH3
CH2CH2OCH2CH2OCH3
CH2CH2CH2CH2OCH3
H
CH2CH2OH
CH2CH2OCH3
CH2CH2OCH2CH2CH2CH3
CH2CH2OCH2CH2OCH3
CH2CH2CH2CH2OCH3
H
CH2CH2OH
CH2CH2OCH3
CH2CH2OCH2CH2CH2CH3
CH2CH2OCH2CH2OCH3
CH2CH2CH2CH2OCH3
H
CH2CH2OH
CH2CH2OCH3
CH2CH2OCH2CH2CH2CH3
CH2CH2OCH2CH2OCH3
CH2CH2CH2CH2OCH3
100
137.29
>200
14.49
>200
28.43
107.43
34.20
77.80
>200
>200
>200
>200
>200
>200
70.02
155.93
112.76
179.31
132.46
>200
>200
>200
1220a
410b
88.67
12.20
100
16.54
100
85.88
100
100
47.50
5.27
23.12
Nid
14.30
1.15
100
95.86
100
78.67
97.49
9.85
5.95
8.21
16.40
46.62
S
O
O
O
O
O
O
S
S
S
S
S
S
Barbituric acid
Thiobarbituric acid
4-Hydroxybenzaldehyde
4-Methoxycinnamic acid
Levofloxacin
–
–
–
0.4
a
Value in the literature [22] is 1.2 mM.
Value in the literature [23] is 420 mM.
Assay performed using mushroom tyrosinase. Values are means of three different experiments.
Not active against tyrosinase.
b
c
d

4238
Q. Yan et al. / European Journal of Medicinal Chemistry 44 (2009) 4235–4243
0.20
0.15
0.10
0.05
0.00
-0.05
0.20
0.15
1
2
3
0.10
0.05
0.00
-0.05
1
2
3
0
2
4
6
8
10
0
2
4
6
8
10
[E] (µM)
[E] (µM)
Fig. 3. The effect of concentrations of tyrosinase on its activity for the catalysis of L-
DOPA at different concentration of compound 1a. The concentrations of compound 1a
for curves 1–3 are 0, 0.5 and 2.0 mM, respectively.
Fig. 4. The effect of concentrations of tyrosinase on its activity for the catalysis of
L-DOPA at different concentration of compound 2a. The concentrations of compound
2a for curves 1–3 are 0, 0.5 and 2.0 mM, respectively.
found in the absence of inhibitor. While the b-sheet was observed
in the presence of inhibitors without obvious regularity. On the
other hand, it was observed that the content of rigidity structure
Gram-positive S. aureus with the MIC value of 3.1 mg/mL, suggesting
that these molecules might be served as lead compounds for
further designing and developing more potent compounds.
(
a
-helix þ
structure (
panying the increasing of concentration of compound 1a and 2a
from 0 to 150 M. These results suggested that the enhanced
b
b
-sheet) of tyrosinase was decreased while that of loop
-turn þ random) of tyrosinase was increased accom-
4. Conclusion
m
flexibility of enzyme completely destructed the hydrogen bonds of
the protein structure and produced irreversible damage on the
structure of tyrosinase leading to the enzyme inactivation. Inter-
In the present investigation, for the first time, we synthesized
a series of novel 5-benzylidene barbiturate and thiobarbiturate
derivatives and determined their inhibitory effects on the diphe-
nolase activity of mushroom tyrosinase and their antibacterial
activities against Gram-positive and Gram-negative bacteria. The
results showed that most of compounds had potential tyrosinase
inhibitory activities. Particularly, compounds 1a and 2a were found
estingly, the content of rigidity structure (
tyrosinase was dramatically increased while the content of loop
structure
-turn þ random) of tyrosinase was significantly
decreased accompanying the increasing of concentration of
compound 1a from 150 to 200 M. These results implied that the
a-helix þ
b-sheet) of
(
b
m
to be the most potent inhibitors with IC50 value of 13.98
mM and
enhanced rigidity of enzyme might increase the ratio of misfolded
tyrosinase and further prevent the formation of natural confor-
mation of tyrosinase.
14.49 M, respectively. Preliminary SARs analysis suggested that:
m
(1) the inhibitory effects of 5-benzylidene thiobarbiturates on
tyrosinase were more potent than 5-benzylidene barbiturates; (2)
the increase of molecular flexibility might be detrimental to their
inhibitory activities; (3) the increase of the length of alkyl chain at
hydroxyl group located in position-4 might cause stereo-hindrance
for the inhibitors approaching the active site. The inhibition
mechanism study revealed that these compounds were irreversible
tyrosinase inhibitors. Circular dichroism spectra indicated that
these compounds induced conformational changes of mushroom
tyrosinase upon binding. Interestingly, thiobarbiturates 2a, 2d and
4d exhibited potent and selective antibacterial effects on Gram-
positive bacteria S. aureus. These results suggested that further
development of such compounds may be of interest.
3.4. Antibacterial activities in vitro
In the present study, antibiotic activities of 5-benzylidene
(2-thio)barbiturate derivatives were screened on S. aureus,
S. epidermidis, S. albus, B. cereus, E. coli and P. aeruginosa in vitro by
broth diffusion method [26]. All the synthesized compounds
showed no antibacterial activities in vitro against S. epidermidis,
S. albus, B. cereus, E. coli and P. aeruginosa at the concentration of
1 mg/mL (data not shown), whereas some thiobarbiturates
exhibited potent antibacterial activities against S. aureus. The
results are summarized in Table 1.
Previous report described that barbiturate and thiobarbiturate
derivatives displayed potent antibacterial activity against E. coli
[10]. Unfortunately, our synthesized 5-benzylidene (2-thio)-
barbiturate derivatives were inactive against E. coli at the concen-
tration of 1 mg/mL. Interestingly, thiobarbiturates 2a, 2d and 4d
exhibited selective antibacterial activities against S. aureus with the
minimum inhibitory concentration (MIC) value of 3.1, 3.1 and
5. Experimental protocols
5.1. Reagents and general procedures
Melting points were determined on a WRS-1B digital instru-
ment without correction. NMR spectra were recorded on a Varian
Mercury-Plus 300 spectrometers in CDCl3 or DMSO-d6 at 25 ꢀC. All
6.25
derivatives 1a, 1d and 3d had no antibacterial activities at the
concentration of 25, 25 and 12.5 g/mL, respectively. These results
mg/mL, respectively, whereas their corresponding barbiturate
chemical shifts (d) are quoted in parts per million downfield from
m
TMS and coupling constants (J) are given in Hertz. Mass spectra
were obtained from VG ZAB-HS, LCMS-2010A or LCQ DECA XP
spectrometer. Elemental analyses were performed on a Vario EI
instrument and were with in ꢁ0.4% of the theoretical values.
suggested that sulfur atom was more favorable than oxygen atom
for their antibacterial activities. Moreover, compounds 2a and 2d
were found to be the most potent antibacterial agents against

Q. Yan et al. / European Journal of Medicinal Chemistry 44 (2009) 4235–4243
4239
Table 2
The influence of compounds 1a and 2a on the secondary structure of tyrosinase.
Compound
Concentration (
m
M)
a
-Helix (%)
b
-Sheet (%)
a
-Helix þ
b
-sheet (%)
b-Turn (%)
Random (%)
b
-Turn þ random (%)
1a
0
50
100
150
200
59.7
37.7
42.3
36.2
83.3
0.0
8.9
3.4
7.2
0.0
59.7
46.6
45.7
43.4
83.0
16.2
20.2
21.4
19.4
0.0
24.1
33.3
32.8
37.2
16.7
40.3
53.5
54.2
56.6
16.7
2a
50
100
150
200
48.7
39.3
45.1
79.4
0.0
5.2
0.0
0.0
48.7
44.5
45.1
79.4
20.3
22.4
20.5
0.0
31.1
33.0
34.5
20.6
51.4
55.4
55.0
20.6
Infrared (IR) spectra were measured on VECTOR 22 spectrometer
using a potassium bromide (KBr) disk, scanning from 400 to
4000 cmꢂ1. All reactions were monitored by TLC (Merck Kieselgel
60 F254) and spots were visualized with UV light or iodine. All
commercially available reagents and solvents were used without
further purification. Mushroom tyrosinase (specific activity of the
(d, J ¼ 8.4 Hz, 2H, ArH), 4.19 (t, J ¼ 5.1 Hz, 2H, CH2), 3.79 (t, J ¼ 4.5 Hz,
2H, CH2), 3.47 (s, 3H, CH3); ESI-MS m/z (%): 180 (50) (Mþ).
5.2.4. 4-(2-Butoxyethoxy)benzaldehyde (d)
Compound d was obtained as yellow liquid (1.64 g, 74%). IR
(KBr, cmꢂ1 : 2958, 1693, 1601, 1509, 1259, 1161, 1126, 833; 1H NMR
) n
enzyme is 6680 U/mg) and
were purchased from Sigma Chemical Co.
L
-DOPA (
L
-3,4-dihydroxyphenylalanine)
(CDCl3, 300 MHz)
d
: 9.88 (s, 1H, CHO), 7.82 (d, J ¼ 8.3 Hz, 2H, ArH),
7.02 (d, J ¼ 8.4 Hz, 2H, ArH), 4.20 (t, 2H, CH2), 3.80 (t, 2H, CH2), 3.54
(t, 2H, CH2), 1.63–1.55 (m, 2H, CH2), 1.43–1.32 (m, 2H, CH2), 0.93
(t, 3H, CH3); ESI-MS m/z (%): 222 (53) (Mþ).
5.2. General procedures for the synthesis of 5-benzylidene
barbiturate and thiobarbiturate derivatives
5.2.5. 4-(2-(2-Methoxyethoxy)ethoxy)benzaldehyde (e)
5.2.1. 4-(2-Hydroxyethoxy)benzaldehyde (b)
Compound e wasꢂo1btained as yellow solid (1.63 g, 73%). Mp
A solution of 2-chloroethanol (0.03 mol) in 10 mL of n-BuOH
was added over a period of 1 h to a boiling solution of 4-hydrox-
ybenzaldehyde (0.01 mol) and NaOH (0.011 mol) in a mixture of
100 mL of n-BuOH and 15 mL of water. The reaction mixture was
refluxed for an additional 12 h, the solvent was evaporated in
vacuo, and the residue was purified by silica gel column chroma-
tography (PE/EtOAc 2:1) to afford compound b (1.35 g, 81%) as
44–45 ꢀC; IR (KBr, cm
) n: 2891, 1689, 1601, 1509, 1129, 1104, 1034,
836; 1H NMR (CDCl3, 300 MHz)
d: 9.89 (s, 1H, CHO), 7.81
(d, J ¼ 8.7 Hz, 2H, ArH), 7.02 (d, J ¼ 8.6 Hz, 2H, ArH), 4.23
(t, J ¼ 5.0 Hz, 2H, CH2), 3.90 (t, J ¼ 4.7 Hz, 2H, CH2), 3.73 (dd,
J ¼ 2.0 Hz, 4.8 Hz, 2H, CH2), 3.59 (dd, 2H, J ¼ 2.0 Hz, 4.8 Hz, CH2),
3.34 (s, 3H, CH3); ESI-MS m/z (%): 225 (82) (M þ 1)þ.
yellow liquid. IR (KBr, cmꢂ1
1112, 1034, 839; 1H NMR (CDCl3, 300 MHz)
)
n
3370, 1684, 1600, 1509, 1309, 1164,
9.89 (s, 1H, CHO), 7.79
5.2.6. 4-(4-Methoxybutoxy)benzaldehyde (f)
d
Compound f was obtained as yellow liquid (1.44 g, 69%). IR
(d, J ¼ 8.3 Hz, 2H, ArH), 7.32 (d, J ¼ 8.4 Hz, 2H, ArH), 4.75 (t, 2H,
(KBr, cmꢂ1 : 2950,1691,1600, 1509,1253,1160, 1125, 834; 1H NMR
) n
CH2), 4.42 (t, 2H, CH2); ESI-MS m/z (%): 167 (68) (M þ 1)þ.
(CDCl3, 300 MHz)
d
: 9.90 (s, 1H, CHO), 7.76 (d, J ¼ 8.4 Hz, 2H, ArH),
7.01 (d, J ¼ 8.4 Hz, 2H, ArH), 4.15 (t, J ¼ 4.5 Hz, 2H, CH2), 3.76
(t, J ¼ 6.3 Hz, CH2, 2H,), 3.37 (s, 3H, OCH3), 1.75–1.72 (m, 2H, CH2),
1.66–1.64 (m, 2H, CH2); ESI-MS m/z (%): 209 (35) (M þ 1)þ.
5.2.2. General procedure for the synthesis of c–f
The various alcohols (3 mmol) were dissolved in 15 mL of THF,
and a solution of NaOH (360 mg, 9 mmol) in H2O (10 mL) was
added. The mixture was cooled to 0 ꢀC, and then a solution of
4-toluenesulfonyl chloride (1.2 g, 6.3 mmol) in THF (15 mL) was
added dropwise for a period of 2 h. The mixture was stirred for 5 h
at 20 ꢀC. The white precipitate, which was formed in organic layer,
was dissolved by adding 20 mL of 2 M NaOH solution. The organic
phase was separated, and the residue was extracted with CH2Cl2
(30 mL ꢃ 2). The combined organic phase was washed with water
(150 mL ꢃ 3), and dried over Na2SO4. The solvent was removed, and
the residue was purified on silica column to afford the mono-
tosylate product.
5.2.7. General procedure for the synthesis of 1a–f and 2a–f
To a solution of barbituric acid or thiobarbituric acid (1 mmol) in
hot ethanol (20 mL), aromatic aldehyde (1 mmol) was added. The
reaction mixture was heated 2 h at 80 ꢀC and cooled to room
temperature. The precipitated solid was filtered, washed with
ethanol and dried under vacuum. The crude product was recrys-
tallized from N,N-dimethylformamide and water.
5.2.8. 5-(4-Hydroxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-
trione (1a)
Solid 4-hydroxybenzaldehyde (1.22 g, 10 mmol) was added to
a mixture of K2CO3 (4.14 g, 30 mmol) in dry DMF (20 mL) at 80 ꢀC.
The mixture was stirred at this temperature for 1 h before addition
of the mono-tosylate product. The reaction mixture was refluxed
for 16 h and cooled to room temperature, then pulled into 100 mL
cool water, and extracted with CH2Cl2 (30 mL ꢃ 3). The organic
phase was washed generously with water to remove DMF and dried
over MgSO4. The solvent was removed and the crude product was
purified on a silica column using petroleum ether/ethyl acetate.
Compound 1a was obtained as yellow powder (0.20 g, 87%),
mp >250 ꢀC (mp: 299–301, lit. [18]); IR (KBr, cmꢂ1
)
n
: 3479, 3124,
3049, 2907, 1708, 1563, 1490, 1318, 1205, 897; 1H NMR (DMSO-d6,
300 MHz) : 11.21 (s, 1H, NH), 11.09 (s, 1H, NH), 10.77 (s, 1H, OH),
8.32 (d, J ¼ 8.7 Hz, 2H, ArH), 8.18 (s, 1H, CH), 6.87 (d, J ¼ 8.7 Hz, 2H,
ArH); 13C NMR (DMSO-d6, 75 MHz)
: 163.89,162.43,158.67,156.05,
d
d
151.66, 137.42, 133.24 (2C), 119.49, 115.52 (2C); FAB-MS m/z (%): 233
(100) (M þ 1)þ; Anal. Calcd for C11H8N2O4: C, 56.90; H, 3.47;
N, 12.06. Found: C, 56.77; H, 3.53; N, 11.95.
5.2.3. 4-(2-Methoxyethoxy)benzaldehyde (c)
5.2.9. 5-(4-(2-Hydroxyethoxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (1b)
Compound
IR(KBr, cmꢂ1
(CDCl3, 300 MHz)
c
was obtained as yellow liquid (1.47 g, 82%).
:2925,1690,1600,1508,1258,1159,1124,833;1H NMR
:9.90(s,1H, CHO), 7.79(d, J ¼ 8.4 Hz,2H, ArH), 7.02
)
n
Compound 1b was obtained as a yellow powder (0.22 g, 79%),
d
mp >250 ꢀC; IR (KBr, cmꢂ1
) n: 3485, 3171, 3056, 2841, 1689, 1546,

4240
Q. Yan et al. / European Journal of Medicinal Chemistry 44 (2009) 4235–4243
1410, 1245, 1174, 1060, 908, 529; 1H NMR (DMSO-d6, 300 MHz)
: 11.26 (s, 1H, NH), 11.13 (s, 1H, NH), 8.33 (d, J ¼ 8.7 Hz, 2H, ArH),
7.92 (s, 1H, CH), 7.04 (d, J ¼ 8.8 Hz, 2H, ArH), 4.13–4.10 (m, 2H, CH2),
3.75–3.72 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
: 164.41,
162.50, 160.13, 156.01, 152.76, 133.66, 129.96 (2C), 120.84, 115.58
(2C), 70.81, 59.96; EI-MS m/z (%): 276 (45) (Mþ), 231 (23), 215 (38),
188 (42), 145 (59), 118 (100), 89 (71), 45 (64).
5.2.14. 5-(4-Hydroxybenzylidene)-2-thioxo-dihydropyrimidine-
4,6(1H,5H)-dione (2a)
d
Compound 2a was obtained as yellow powder (0.24 g, 97%),
d
mp >250 ꢀC,(mp >300 ꢀC, lit. [19]); IR (KBr, cmꢂ1
)
n
: 3485, 3118,
2973, 1644, 1561, 1430, 1208, 1205, 847; 1H NMR (DMSO-d6,
300 MHz) : 12.30 (s, 1H, NH), 12.20 (s, 1H, NH), 10.94 (s, 1H,
d
OH), 8.37 (d, J ¼ 8.7 Hz, 2H, ArH), 8.20 (s, 1H, CH), 6.89
(d, J ¼ 8.7 Hz, 2H, ArH); 13C NMR (DMSO-d6, 75 MHz)
d: 178.66,
162.23, 160.09, 156.79, 149.08, 132.22, 125.47 (2C), 118.77, 115.50
(2C); FAB-MS m/z (%): 249 (100) (M þ 1)þ; Anal. Calcd for
C11H8N2O3S: C, 53.22; H, 3.25; N, 11.28. Found: C, 53.07; H, 3.33;
N, 11.11.
5.2.10. 5-(4-(2-Methoxyethoxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (1c)
Compound 1c was obtained as yellow powder (0.27 g, 92%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3195, 3055, 2948, 2846, 1735,
1671, 1603, 1439, 1395, 1268, 1186, 1113, 1037, 839, 509; 1H NMR
(DMSO-d6, 300 MHz) : 11.27 (s, 1H, NH), 11.14 (s, 1H, NH), 8.33
5.2.15. 5-(4-(2-Hydroxyethoxy)benzylidene)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (2b)
d
(d, J ¼ 8.8 Hz, 2H, ArH), 8.22 (s, 1H, CH), 7.04 (d, J ¼ 8.0 Hz, 2H, ArH),
Compound 2b was obtained as yellow powder (0.21 g, 73%),
4.12 (t, J ¼ 4.2 Hz, 2H, CH2), 3.69 (t, J ¼ 4.5 Hz, 2H, CH2), 3.31 (s, 3H,
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3393, 3120, 2972, 1632, 1552, 1417,
OCH3); 13C NMR (DMSO-d6, 75 MHz)
d: 163.87, 162.76, 160.09,
1195, 1073, 830, 543; 1H NMR (DMSO-d6, 300 MHz)
d: 12.34 (s, 1H,
155.98, 152.85, 134.16, 128.72 (2C), 120.28, 114.57 (2C), 71.81, 68.32,
58.26; FAB-MS m/z (%): 291 (85) (M þ 1)þ; Anal. Calcd for
C14H14N2O5: C, 57.93; H, 4.86; N, 9.65. Found: C, 57.51; H, 5. 09;
N, 8.92.
NH), 12.25 (s, 1H, NH), 8.39 (d, J ¼ 8.1 Hz, 2H, ArH), 8.24 (s, 1H, CH),
7.06 (d, J ¼ 8.1 Hz, 2H, ArH), 4.13 (t, J ¼ 2.7 Hz, 2H, CH2),
3.74 (t, J ¼ 3.0 Hz, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 178.71,
162.29, 160.13, 156.34, 154.44, 132.39, 125.39 (2C), 118.66, 114.42
(2C), 70.29, 59.32; EI-MS m/z (%): 292 (90) (Mþ), 247 (100), 231
(85), 188 (30), 150 (45), 118 (25), 89 (60), 45 (73).
5.2.11. 5-(4-(2-Butoxyethoxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (1d)
Compound 1d was obtained as yellow powder (0.30 g, 89%), mp
5.2.16. 5-(4-(2-Methoxyethoxy)benzylidene)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (2c)
>250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3206, 3082, 2945, 2855, 1742, 1680, 1578,
1440, 1248, 1200, 1124, 815, 526; 1H NMR (DMSO-d6, 300 MHz)
d:
Compound 2c was obtained as yellow powder (0.27 g, 88%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3120, 2933, 1656, 1535, 1425, 1258,
11.26 (s, 1H, NH), 11.13 (s, 1H, NH), 8.33 (d, J ¼ 8.3 Hz, 2H, ArH), 8.22
(s,1H, CH), 7.05 (d, J ¼ 8.0 Hz, 2H, ArH), 4.23–4.20 (m, 2H, CH2), 3.74–
3.70 (m, 2H, CH2), 3.45 (t, 2H, CH2),1.52–1.45 (m, 2H, CH2),1.36–1.28
(m, 2H, CH2), 0.88 (t, J ¼ 6.0 Hz, 3H, CH3); 13C NMR (DMSO-d6,
1184, 815; 525; 1H NMR (DMSO-d6, 300 MHz)
d: 12.34 (s, 1H, NH),
12.25 (s, 1H, NH), 8.38 (d, J ¼ 8.8 Hz, 2H, ArH), 8.24 (s, 1H, CH), 7.06
(d, J ¼ 8.9 Hz, 2H, ArH), 4.12 (t, J ¼ 4.2 Hz, 2H, CH2), 3.38
(t, J ¼ 3.9 Hz, 2H, CH2), 3.23 (s, 3H, OCH3); 13C NMR (DMSO-d6,
75 MHz) d: 164.12,162.44,159.29,155.93,153.02,132.31,129.27 (2C),
118.92, 115.47 (2C), 70.56, 69.87, 68.32, 31.69, 19.25, 14.20; FAB-MS
m/z (%): 333 (65) (M þ 1)þ; Anal. Calcd for C17H20N2O5: C, 61.44; H,
6.07; N, 8.43. Found: C, 59.99; H, 6.14; N, 8.76.
75 MHz) d: 178.57, 162.24, 160.09, 156.37, 154.71, 132.28, 125.48
(2C), 118.78, 115.41 (2C), 70.12, 68.10, 58.29; FAB-MS m/z (%): 307
(100) (M þ 1)þ.
5.2.17. 5-(4-(2-Butoxyethoxy)benzylidene)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (2d)
5.2.12. 5-(4-(2-(2- Methoxyethoxy)ethoxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (1e)
Compound 2d was obtained as yellow powder (0.32 g, 93%),
Compound 1e was obtained as yellow powder (0.27 g, 80%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3122, 2933, 1674, 1531, 1445, 1395,
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3201, 3075, 2943, 2862, 1684, 1551,
1281, 1187, 1052, 815; 523; 1H NMR (DMSO-d6, 300 MHz)
d
: 12.29
1418, 1257, 1184, 1037, 808, 513; 1H NMR (DMSO-d6, 300 MHz)
(s, 1H, NH), 12.20 (s, 1H, NH), 8.33 (d, J ¼ 8.9 Hz, 2H, ArH), 8.19
(s, 1H, CH), 7.02 (d, J ¼ 8.9 Hz, 2H, ArH), 4.20–4.17 (m, 2H, CH2),
3.68–3.65 (m, 2H, CH2), 3.40 (t, 2H, CH2), 1.47–1.40 (m, 2H, CH2),
1.30–1.23 (m, 2H, CH2); 0.83 (t, J ¼ 5.8 Hz, 3H, CH3); 13C NMR
d
: 11.27 (s, 1H, NH), 11.13 (s, 1H, NH), 8.34 (d, J ¼ 8.7 Hz, 2H, ArH),
8.22 (s, 1H, CH), 7.03 (d, J ¼ 8.7 Hz, 2H, ArH), 4.21 (t, J ¼ 5.4 Hz, 2H,
CH2), 3.69 (t, J ¼ 5.4 Hz, 2H, CH2), 3.57–3.54 (m, 2H, CH2), 3.45–3.42
(m, 2H, CH2), 3.21 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75 MHz)
(DMSO-d6, 75 MHz)
d: 178.74, 163.80, 162.65, 160.39, 156.48,
d: 164.40, 162.58, 156.03, 153.74, 152.35, 131.08, 126.17 (2C), 120.84,
138.42, 125.77 (2C), 116.02, 115.01 (2C), 70.60, 69.80, 68.28, 31.67,
19.25, 14.18; FAB-MS m/z (%): 349 (70) (M þ 1)þ; Anal. Calcd for
C17H20N2O4S: C, 58.60; H, 5.79; N, 8.04. Found: C, 58.33; H, 5.83;
N, 8.20.
115.83 (2C), 71.35, 70.01, 68.82, 68.36, 58.28; FAB-MS m/z (%): 335
(28) (M þ 1)þ; Anal. Calcd for C16H18N2O6: C, 57.48; H, 5.43; N, 8.38.
Found: C, 57.69; H, 5.35; N, 8.45.
5.2.13. 5-(4-(4- Methoxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (1f)
5.2.18. 5-(4-(2-(2- Methoxyethoxy)ethoxy)benzylidene)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (2e)
Compound 1f was obtained as yellow powder (0.26 g, 82%),
Compound 2e was obtained as yellow powder (0.26 g, 75%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3072, 2927, 1649, 1518, 1409, 1258,
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3201, 2933, 2892, 1680, 1529, 1418,
1199, 1170, 1110, 952, 857, 510; 1H NMR (DMSO-d6, 300 MHz)
1257, 1184, 808, 515; 1H NMR (DMSO-d6, 300 MHz)
d
: 12.29 (s, 1H,
d
: 11.26 (s, 1H, NH), 11.13 (s, 1H, NH), 8.38 (d, J ¼ 8.7 Hz, 2H, ArH),
NH), 12.18 (s, 1H, NH), 8.37 (d, J ¼ 8.7 Hz, 2H, ArH), 8.21 (s, 1H, CH),
7.03 (d, J ¼ 8.7 Hz, 2H, ArH), 4.22 (t, J ¼ 5.4 Hz, 2H, CH2), 3.70
(t, J ¼ 5.4 Hz, 2H, CH2), 3.58–3.54 (m, 2H, CH2), 3.47–3.42 (m, 2H,
8.24 (s, 1H, CH), 7.04 (d, J ¼ 8.6 Hz, 2H, ArH), 4.12 (t, 2H, CH2),
3.36–3.39 (m, 2H, CH2), 3.23 (s, 3H, CH3), 1.75–1.80 (m, 2H, CH2),
1.62–1.67 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 163.98,
CH2), 3.23 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75 MHz)
d: 178.71,
162.31, 160.29, 155.51, 153.17, 134.42, 125.77 (2C), 119.89, 115.11
(2C), 71.95, 68.54, 58.24, 26.01, 25.96; EI-MS m/z (%): 318 (25)
(Mþ), 231 (40), 188 (87), 87 (100), 45 (60).
162.25, 160.10, 157.34, 156.40, 132.33, 125.51 (2C), 118.92, 115.42
(2C), 71.31, 69.85, 68.72, 68.40, 58.08; FAB-MS m/z (%): 351 (67)
(M þ 1)þ.

Q. Yan et al. / European Journal of Medicinal Chemistry 44 (2009) 4235–4243
4241
5.2.19. 5-(4-(4-Methoxyethoxy)benzylidene)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (2f)
(d, J ¼ 8.1 Hz, 2H, ArH), 4.00 (t, J ¼ 3.9 Hz, 2H, CH2), 3.81
(t, J ¼ 3.9 Hz, 1H, CH), 3.65 (t, J ¼ 3.9 Hz, 2H, CH2), 3.42 (t, J ¼ 6.6 Hz,
2H, CH2), 3.18 (t, J ¼ 3.6 Hz, 2H, CH2), 1.53–1.43 (m, 2H, CH2),
1.35–1.27 (m, 2H, CH2), 0.87 (t, J ¼ 7.5 Hz, 3H, CH3); 13C NMR
Compound 2f was obtained as yellow powder (0.27 g, 81%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3064, 2919, 1656, 1525, 1409, 1258,
1184, 844; 509; 1H NMR (DMSO-d6, 300 MHz)
d
: 12.34 (s, 1H, NH),
(DMSO-d6, 75 MHz) d: 170.53 (2C), 156.94, 150.42, 132.61, 129.87
12.25 (s, 1H, NH), 8.38 (d, J ¼ 8.8 Hz, 2H, ArH), 8.24 (s, 1H, CH),
7.06 (d, J ¼ 8.9 Hz, 2H, ArH), 4.15–4.10 (m, 2H, CH2), 3.39–3.36
(m, 2H, CH2), 3.23 (s, 3H, OCH3), 1.80–1.73 (m, 2H, CH2), 1.69–1.62
(2C),115.24 (2C), 70.86, 70.17, 68.52, 48.97, 33.45, 31.71, 19.30, 14.19;
EI-MS m/z (%): 334 (45) (Mþ), 87 (100); Anal. Calcd for C17H22N2O5:
C, 61.07; H, 6.63; N, 8.38. Found: C, 60.98; H, 6.29; N, 8.19.
(m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 178.71, 163.95,
162.66, 160.38, 156.56, 138.50, 125.63 (2C), 115.85, 114.95 (2C),
71.93, 68.44, 58.28, 25.99, 25.86; FAB-MS m/z (%): 335 (55)
(M þ 1)þ; Anal. Calcd for C16H18N2O4S: C, 57.47; H, 5.43; N, 8.38.
Found: C, 57.21; H, 5.63; N, 8.35.
5.2.25. 5-(4-(2-(2-Methoxyethoxy)ethoxy)benzyl)pyrimidine-
2,4,6(1H,3H,5H)trione (3e)
Compound 3e was obtained as yellow powder (0.28 g, 82%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3247, 2925, 1756, 1572, 1514, 1392,
1301, 1248, 1178, 1111, 1035, 842, 753, 694, 544; 1H NMR (DMSO-d6,
300 MHz)
5.2.20. General procedure for the synthesis of 3a–f, 4a–f
d
: 11.13 (s, 2H, NH), 6.96 (d, J ¼ 8.1 Hz, 2H, ArH), 6.80
5-Benzylidene barbituric acid or thiobarbituric acid (1 mmol)
was dissolved in 20 mL anhydrous ethanol, and NaBH4 (0.11 g,
3 mmol) was added three times. The mixture was stirred at room
temperature for 2 h, and ethanol was removed under reduced
pressure. To the residue was added 10 mL water and neutralized
(pH 7) with 1 M HCl. The precipitated solid was filtered and the
crude product was recrystallized from methanol.
(d, J ¼ 8.1 Hz, 2H, ArH), 4.01 (t, J ¼ 4.5 Hz, 2H, CH2), 3.81
(t, J ¼ 6.0 Hz, 1H, CH), 3.69 (t, 2H, CH2), 3.57–3.54 (m, 2H, CH2),
3.45–3.42 (m, 2H, CH2), 3.23 (s, 3H, CH3), 3.18 (d, J ¼ 5.7 Hz, 2H,
CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 170.40 (2C), 158.74, 150.30,
133.58, 130.87 (2C), 115.18 (2C), 71.75, 70.17, 69.91, 68.79, 58.27,
50.31, 33.57; FAB-MS m/z (%): 336 (100) (Mþ).
5.2.26. 5-(4-(4-Methoxyethoxy)benzyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (3f)
5.2.21. 5-(4-Hydroxybenzyl)pyrimidine-2,4,6(1H,3H,5H)-trione
(3a)
Compound 3f was oꢂb1tained as white powder (0.28 g, 88%),
Compound 3a was obtained as yellow powder (0.18 g, 77%),
mp >250 ꢀC; IR (KBr, cm
) n: 3240, 2926, 2864, 2650, 1756, 1572,
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3387, 3120, 3055, 2917, 1708, 1653,
1514, 1395, 1301, 1177, 1124, 1036, 841, 694, 544; 1H NMR (DMSO-d6,
300 MHz)
1489, 1205, 867; 1H NMR (DMSO-d6, 300 MHz)
d: 11.10 (s, 1H, NH),
d
: 11.13 (s, 1H, NH), 6.97 (d, J ¼ 7.0 Hz, 2H, ArH), 6.79
11.09 (s, 1H, NH), 6.90 (d, J ¼ 8.4 Hz, 2H, ArH), 6.77 (d, J ¼ 8.4 Hz, 2H,
ArH), 3.80 (t, J ¼ 6.0 Hz, 1H, CH), 3.18 (t, J ¼ 6.0 Hz, 2H, CH2);
(d, J ¼ 6.8 Hz, 2H, ArH), 3.90–3.80 (m, 3H, CH2, CH), 3.35–3.31
(m, 2H, CH2), 3.31–3.28 (m, 2H, CH2), 3.21 (s, 3H, OCH3), 1.70–1.61
13C NMR (DMSO-d6, 75 MHz)
d: 170.39 (2C), 157.95, 150.96, 130.42,
(m, 4H, 2 ꢃ CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 170.47 (2C),
129.24 (2C), 115.62 (2C), 49.87, 33.38; FAB-MS m/z (%): 235 (25)
157.94, 150.97, 130.48, 129.29 (2C), 114.67 (2C), 72.01, 68.58, 58.24,
50.03, 33.36, 26.12, 26.05; EI-MS m/z (%): 320 (35) (Mþ), 233 (54),
188 (67), 87 (100), 45 (80); Anal. Calcd for C16H20N2O5: C, 59.99;
H, 6.29; N, 8.74. Found: C, 59.30; H, 6.28; N, 8.71.
(M þ 1)þ.
5.2.22. 5-(4-(2-Hydroxyethoxy)benzyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (3b)
Compound 3b was oꢂb1tained as yellow powder (0.17 g, 61%),
5.2.27. 5-(4-Hydroxybenzyl)-2-thioxo-dihydropyrimidine-
4,6(1H,5H)-dione (4a)
mp >250 ꢀC; IR (KBr, cm
) n: 3392, 3112, 2970, 2801, 1704, 1635,
1510, 1387, 1296, 1239, 1076, 902, 811, 769, 538; 1H NMR (DMSO-d6,
300 MHz)
Compound 4a was obtained as yellow powder (0.18 g, 71%),
d
: 11.12 (s, 2H, NH), 6.95 (d, J ¼ 7.8 Hz, 2H, ArH), 6.79
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3327, 3119, 2983, 1634, 1469, 1205,
(d, J ¼ 7.5 Hz, 2H, ArH), 3.90 (t, J ¼ 3.9 Hz, 2H, CH2), 3.80
(t, J ¼ 6.0 Hz, 1H, CH), 3.66 (t, J ¼ 3.9 Hz, 2H, CH2), 3.18 (t, J ¼ 6.0 Hz,
877; 1H NMR (DMSO-d6, 300 MHz)
d: 12.10 (s, 2H, NH), 7.11
(d, J ¼ 8.7 Hz, 2H, ArH), 7.00 (d, J ¼ 8.7 Hz, 2H, ArH), 3.89
2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 170.43 (2C), 158.15,
(t, J ¼ 4.5 Hz, 1H, CH), 3.23 (t, J ¼ 4.5 Hz, 2H, CH2); 13C NMR (DMSO-
150.87, 131.22, 130.24 (2C), 115.11 (2C), 49.95, 33.51; EI-MS m/z (%):
d6, 75 MHz) d: 178.15,173.31 (2C),157.85,132.43, 129.24 (2C), 115.81
278 (56) (Mþ), 231 (30), 188 (85) 108 (100).
(2C), 49.51, 33.38; FAB-MS m/z (%): 251 (63) (M þ 1)þ.
5.2.23. 5-(4-(2-Methoxyethoxy)benzyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (3c)
5.2.28. 5-(4-(2-Hydroxyethoxy)benzyl)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (4b)
Compound 3c was obtained as yellow powder (0.21 g, 71%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3192, 2938, 2846, 1744, 1615, 1580,
Compound 4b was obtained as pale powder (0.20 g, 69%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3303, 2972, 1628, 1579, 1450, 1237,
1387, 1296, 1186, 1076, 811, 749, 540; 1H NMR (DMSO-d6, 300 MHz)
1136, 816; 1H NMR (DMSO-d6, 300 MHz)
d: 12.05 (s, 2H, NH), 7.10
d
: 11.11 (s, 2H, NH), 6.97 (d, J ¼ 8.1 Hz, 2H, ArH), 6.71 (d, J ¼ 8.1 Hz,
(d, J ¼ 7.8 Hz, 2H, ArH), 6.96 (d, J ¼ 7.8 Hz, 2H, ArH), 4.01
(t, J ¼ 2.7 Hz, 2H, CH2), 3.91 (t, J ¼ 4.5 Hz, 1H, CH), 3.74 (t, J ¼ 3.0 Hz,
2H, ArH), 4.03 (t, J ¼ 4.2 Hz, 2H, CH2), 3.81 (t, J ¼ 5.4 Hz, 1H, CH),
3.66 (t, J ¼ 3.9 Hz, 2H, CH2), 3.31 (s, 3H, OCH3), 3.16 (t, J ¼ 5.1 Hz, 2H,
2H, CH2), 3.31 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 178.68,
CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 171.04 (2C), 158.18, 150.67,
172.09 (2C), 156.84, 132.35, 129.19 (2C), 114.92 (2C), 70.21, 59.72,
49.23, 33.40; FAB-MS m/z (%): 294 (67) (Mþ); Anal. Calcd for
C13H14N2O4S: C, 53.05; H, 4.79; N, 9.52. Found: C, 53.29; H, 5.16;
N, 9.05.
133.61,130.45 (2C),115.32 (2C), 71.68, 68.97, 58.30; FAB-MS m/z (%):
293 (100) (M þ 1)þ.
5.2.24. 5-(4-(2-Butoxyethoxy)benzyl)pyrimidine-2,4,6(1H,3H,5H)-
trione (3d)
Compound 3d was obtained as yellow powder (0.29 g, 88%),
5.2.29. 5-(4-(2-Methoxyethoxy)benzyl)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (4c)
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3239, 2928, 2839, 1758, 1573, 1515,
1394,1301,1249,1177,1125,1037, 838, 695, 546; 1H NMR (DMSO-d6,
300 MHz)
: 11.13 (s, 2H, NH), 6.96 (d, J ¼ 8.1 Hz, 2H, ArH), 6.80
Compound 4c waꢂs1obtained as white powder (0.22 g, 72%), mp
d
>250 ꢀC; IR (KBr, cm
) n: 3220, 2943, 2865, 1565, 1447, 1328, 1231,

4242
Q. Yan et al. / European Journal of Medicinal Chemistry 44 (2009) 4235–4243
1095, 810; 1H NMR (DMSO-d6, 300 MHz)
d
: 12.04 (s, 1H, NH), 7.08
dose–response curves. 4-Hydroxybenzaldehyde and 4-methox-
ycinnamic acid were used as reference compounds. Moreover, the
inhibition type of the selected compounds 1a and 2a were assayed
by a Lineweaver–Burk plot.
(d, J ¼ 8.7 Hz, 2H, ArH), 6.96 (d, J ¼ 8.7 Hz, 2H, ArH), 3.90 (m, 3H,
CH2, CH), 3.36 (t, J ¼ 3.9 Hz, 2H, CH2), 3.25 (m, 2H, CH2), 3.21 (s, 3H,
CH3); 13C NMR (DMSO-d6, 75 MHz)
d: 178.59, 171.98 (2C), 157.38,
132.30, 125.43 (2C), 115.35 (2C), 70.21, 68.53, 48.91, 33.42; FAB-MS
Circular dichroism (CD) spectra were obtained at 25 ꢀC using
a Jasco J-810 spectropolarimeter. The quartz cells had path lengths
of 0.2 cm for measurements in the far-UV region (190–250 nm). For
the determination of the spectra, solutions of the enzymes
m/z (%): 309 (89) (M þ 1)þ.
5.2.30. 5-(4-(2-Butoxyethoxy)benzyl)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (4d)
(39
mg/mL) were equilibrated against a 50 mM phosphate buffer
Compound 4d was obtained as pale powder (0.26 g, 75%),
(pH 6.8) containing 20
m
L different concentrations of samples. The
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3130, 2893, 2750, 1576, 1440, 1269,
samples were dissolved in 1.0 mL methanol, and methanol had
a final concentration of 2%. Each spectrum was the average of six
repetitive scans and was corrected by subtracting the average
spectrum of the buffer and methanol.
1187, 1061, 820; 1H NMR (DMSO-d6, 300 MHz)
d: 12.00 (s, 2H, NH),
7.03 (d, J ¼ 8.7 Hz, 2H, ArH), 6.93 (d, J ¼ 8.7 Hz, 2H, ArH), 3.93–3.89
(m, 3H, CH2, CH), 3.61–3.58 (m, 2H, CH2), 3.37 (t, 2H, CH2), 3.24
(d, J ¼ 6.0 Hz, 2H, CH2), 1.47–1.40 (m, 2H, CH2), 1.30–1.23 (m, 2H,
CH2); 0.83 (t, J ¼ 5.8 Hz, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz)
5.4. In vitro antibacterial activity assays
d
: 178. 26, 172.35 (2C), 157.14, 132.58, 129.89 (2C), 115.27 (2C), 70.96,
70.07, 68.75, 48.97, 33.37, 31.71, 19.30, 14.19; FAB-MS m/z (%): 351
(100) (M þ 1)þ.
All the synthesized compounds were tested for their antibac-
terial activity against Gram-positive (S. aureus, B. cereus,
S. epidermidis and S. albus) and Gram-negative (E. coli and P. aer-
uginosa) bacteria strains. Antibacterial assays were performed by
the agar dilution method according to the Clinical and Laboratory
Standard Institute (CLSI) (formerly National Committee for Clinical
Laboratory Standards) [26]. Twofold serial dilutions of the
compounds and reference drugs were prepared in Mueller–Hinton
agar (MHA). The overnight cultures (after 16–18 h of incubation at
37 ꢀC) of all the bacteria were used for the assay and adjusted to
the turbidity of a 0.5 McFarland Standard. The stock solution
(1 mg/mL) of all the test chemicals was prepared by dissolving
1 mg of the test chemical in 1 mL of dimethylsulfoxide (DMSO),
and DMSO was used as control for all the test compounds. Twenty
milliliters of MHA and 500 mL of each test bacterial culture of
16–18 h incubation adjusted at 0.5 McFarland were mixed and
poured into sterilized and labeled plates. The wells of 6 mm
diameter were punched in the solidified agar plates. Test chem-
icals of 100 mL were added to individual wells. The loaded plates
were incubated at 37 ꢀC for 24 h. The diameter of zone of growth
inhibition around each well was measured after incubation using
a Vernier Caliper. The MIC value is the lowest concentration of the
antimicrobial agent that prevents the development of viable
5.2.31. 5-(4-(2-(2-Methoxyethoxy)ethoxy)benzyl)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (4e)
Compound 4e was obtained as white powder (0.24 g, 69%),
mp >250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3138, 2916, 2884, 1615, 1559, 1417,
1223, 1174, 809, 520; 1H NMR (DMSO-d6, 300 MHz)
d: 12.01 (s, 2H,
NH), 6.97 (d, J ¼ 7.8 Hz, 2H, ArH), 6.92 (d, J ¼ 7.8 Hz, 2H, ArH), 4.02
(t, J ¼ 5.1 Hz, 2H, CH2), 3.88 (t, J ¼ 6.3 Hz, 1H, CH), 3.67 (t, J ¼ 5.1 Hz,
2H, CH2), 3.55–3.51 (m, 2H, CH2), 3.43–3.38 (m, 2H, CH2), 3.23
(s, 3H, OCH3), 3.18 (d, J ¼ 6.0 Hz, 2H, CH2); 13C NMR (DMSO-d6,
75 MHz) d: 178.30, 172.29 (2C), 157.37, 132.30, 125.55 (2C), 115.44
(2C), 71.12, 69.67, 68.32, 67.94, 58.08, 49.19, 33.33; FAB-MS m/z (%):
353 (78) (M þ 1)þ.
5.2.32. 5-(4-(4-Methoxybutoxy)benzyl)-2-thioxo-
dihydropyrimidine-4,6(1H,5H)-dione (4f)
Compound 4f was obtained as white powder (0.24 g, 73%), mp
>250 ꢀC; IR (KBr, cmꢂ1
)
n
: 3217, 2922, 2743, 1570, 1432, 1246, 1178,
1120, 1079, 1051, 1001, 801; 1H NMR (DMSO-d6, 300 MHz)
d: 12.03
(s, 1H, NH), 7.03 (d, J ¼ 8.3 Hz, 2H, ArH), 7.02 (d, J ¼ 8.3 Hz, 2H, ArH),
3.89 (t, J ¼ 6.3 Hz, 3H, CH2, CH), 3.50 (m, 2H, CH2), 3.33 (t, J ¼ 6.3 Hz,
2H, CH2), 3.21 (s, 3H, OCH3), 1.66–1.72 (m, 2H, CH2), 1.57–1.65 (m,
growth after 16–18 h incubation. MIC values (mg/mL) were deter-
2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
d: 178.11, 173.05 (2C),
mined on Mueller–Hinton (MH) agar with medium containing
dilutions of antibacterial agents by the literature method [27].
Stock solution of 1 mg/mL was prepared in DMSO and was
appropriately diluted to get final concentrations of 50, 25, 12.5,
156.76, 133.32, 125.60 (2C), 115.45 (2C), 71.99, 68.67, 58.27, 49.31,
33.38, 26.01, 25.96; EI-MS m/z (%): 336 (57) (Mþ), 107 (88), 87 (100),
45 (60).
6.25, 3.1, 1.5, 0.8, 0.4 and 0.2 mg/mL. Levofloxacin (purchased from
5.3. Tyrosinase assay
Darui Chemical Co.) was used as positive control.
Tyrosinase inhibition assay was performed according to the
method reported by Liu et al. [21] with slight modifications. Briefly,
all the synthesized compounds were screened for the diphenolase
Acknowledgment
This work was supported by the Natural Science Foundation of
Guangdong Province, China (2004B30101007).
inhibitory effects of mushroom tyrosinase using
substrate. The reaction media (1 mL) for activity assay contained
0.5 mM -DOPA in 50 mM Na2HPO4–NaH2PO4 buffer (pH 6.8). The
L-DOPA as
L
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