Synthesis and biological evaluation of novel 6-substituted 5-alkyl-2-(arylcarbonylmethylthio)pyrimidin-4(3H)-ones as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.01.039

A novel series of 2-arylcarbonylmethylthio-6-arylmethylpyrimidin-4(3H)-ones have been synthesized and evaluated for in vitro anti-HIV activities in MT-4 cells. Most of these new compounds showed moderate to potent activities against wild-type HIV-1 with a

Full text of DOI:10.1016/j.bmc.2008.01.039

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3887–3894
Synthesis and biological evaluation of novel 6-substituted
5-alkyl-2-(arylcarbonylmethylthio)pyrimidin-4(3H)-ones
as potent non-nucleoside HIV-1 reverse transcriptase inhibitors
Yue-Ping Wang,a Fen-Er Chen,a,* Erik De Clercq,b
Jan Balzarinib and Christophe Pannecouqueb
aDepartment of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
bRega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
Received 25 November 2007; revised 21 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract—A novel series of 2-arylcarbonylmethylthio-6-arylmethylpyrimidin-4(3H)-ones have been synthesized and evaluated for
in vitro anti-HIV activities in MT-4 cells. Most of these new compounds showed moderate to potent activities against wild-type
HIV-1 with an EC50 range from 8.97 lM to 0.010 lM. Among them, the 6-(3,5-dimethylbenzyl) analogue 5p was identified as
the most promising compound (EC50 = 0.010 lM, SI > 31,800) associated with moderate activity against the HIV-1 double mutant
RT strain K103N + Y181C. The structure–activity relationships of these new congeners were further discussed.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
detailed synthesis, anti-HIV-1 activity and preliminary
SAR studies of these new congeners are described.
Over the past decade, 2-alkylthio-6-benzylpyrimidin-
4(3H)-ones (S-DABOs)1 have been the subject of great
interest and have led in recent years to the identification
of several new structures (2–4, Fig. 1)2–11 displaying
excellent activities as non-nucleoside inhibitors of
HIV-1 reverse transcriptase (RT). Our recent work on
S-DABOs disclosed that the assembly of an arylcarbon-
ylmethyl moiety at the C-2 sulfur atom bound to the
pyrimidine nucleus has resulted in the synthesis of many
new analogues (4, Fig. 1)8 with potent antiviral activities
against HIV-1. These results indicated that this subclass
of S-DABO analogues raises the appealing possibility
for further structural modifications using 4 as the start-
ing point. In the course of our search for new anti-HIV-
1 agents, we became interested in investigating the effect
of the substituent at position 6 of the 2-arylcarbonylme-
thythio substituted S-DABOs (5, Fig. 1) on anti-HIV
activity with the aim to delineate the structure–activity
relationships (SAR) of S-DABOs and to further
improve their HIV-1 inhibitory potency. Herein, the
2. Chemistry
The synthesis of the target compounds 5a–q was
straightforward and is depicted in Scheme 1. The substi-
tuted b-ketoesters 7a–p were easily prepared using the
O
O
O
R2
R1
R2
HN34
HN
S
HN
5
6
2
N
1
R
Alkyl
1 S
N
F
S
n = 0-3
N
R
n
F
R
R1
R3
S
-DABOs
1
F2-S-DABOs
R = F, Cl
2
3
O
N
R2
HN
O
R1
R3
R2
R1
Ar
S
X
HN
S
O
N
O
X = CH2, S, CHCN, C=O
Keywords: HIV; NNRTIs; SAR; S-DABOs.
*
5
4
Corresponding author. Tel./fax: +86 21 65643811; e-mail:
rfchen@fudan.edu.cn
Figure 1. Chemical structures of S-DABOs.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.039

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Y.-P. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3887–3894
Scheme 1. Synthesis of compounds 5a–q. Reagents and conditions: (a) i—CDI, CH3CN, rt, 30 min; ii— R2CH(CO2Et)(CO2K), Et3N, anhydrous
MgCl2, rt, overnight, then reflux, 2 h. (b) Thiourea, EtONa, EtOH, reflux, 10 h. (c) R3COCH2X (X = Br or Cl), K2CO3, DMF, rt, 24 h.
method of Clay12 by exposure of substituted arylacetic
acids 6a–i to 1,10-carbonyldiimidazole (CDI) followed
by treatment with different ethyl potassium malonate
in the presence of anhydrous MgCl2 and Et3N, in which
the requisite starting materials 6a–i were commercially
available or could be readily prepared. Subsequent con-
densation of 7a–p with thiourea in the presence of
EtONa in refluxing EtOH led to the key intermediates
8a–p according to our previously reported procedure.13
Next, selective S-alkylation of 8a–p with the appropriate
arylcarbonylmethyl halides (1:1.1) in the presence of
K2CO3 in anhydrous DMF afforded the desirable target
compounds 5a–q. It is worth noting that in this reaction,
some by-products such as N-substituted or O-substi-
tuted or di-substituted compounds were also formed.
However, the S-alkylated compounds 5a–q were the
main products, which could be easily purified by column
chromatography or by crystallization.
3. Results and discussion
All of the newly synthesized S-DABO analogues (5a–q)
were first evaluated for their cytotoxicity and anti-HIV
activity in MT-4 cells infected with wild-type HIV-1
strain IIIB and HIV-2 strain ROD. The results, expressed
as CC50, EC50 and SI, are summarized in Table 1
together with those of efavirenz, delavirdine, AZT, and
DDI as reference drugs.
As can be seen from Table 1, most of the test com-
pounds inhibited HIV-1 replication in a lower micromo-
lar concentration range. It is worth noting that a
strikingly low cytotoxicity of these new S-DABOs was
observed; and the majority of them were non-cytotoxic
for MT-4 cells at doses as high as 236 lM. Among the
highly active compounds, 6-(3,5-dimethylbenzyl)-
5-ethyl-2-(phenylcarbonylmethylthio)pyrimidin-4(3H)-
one (5p) turned out to be the most potent inhibitor with
an EC50 of 0.010 lM against HIV-1 being about 7.2-
times higher than that of delavirdine. More importantly,
it also exhibited a high selectivity index (SI > 31,800).
Some other compounds, 5e, 5g, 5j, and 5n, also showed
high anti-HIV-1 potency (EC50 = 0.054, 0.044, 0.040,
and 0.018 lM, respectively) and excellent selectivity
indices (SI = 5704, 6318, 4675, and 13278, respectively).
All the target compounds 5a–q were characterized by
NMR, MS, IR and elemental analysis. Both analytical
and spectral data of all the compounds are in full agree-
ment with the proposed structures. Moreover, compari-
son of the spectroscopic data of the new compounds
with those of the previously reported analogues8 further
confirmed the above structures.

Y.-P. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3887–3894
3889
Table 1. Biological activities of compounds 5a–q against HIV-1 and HIV-2 in MT-4 cellsa
SId (HIV-1 IIIB)
b
c
Compound
Ar
R1
R2
R3
EC50 (lM)
CC50 (lM)
HIV-1 IIIB
HIV-2 ROD
5a
5b
2-Br–Ph
3-Br–Ph
H
H
H
H
H
H
H
Ph
H
H
H
H
H
H
H
H
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
H
0.63 0.57
0.89 0.01
4.44 0.38
8.97 1.65
0.054 0.008
46.06 39.64
0.044 0.006
1.38 0.27
0.22 0.04
0.040 0.001
0.16 0.01
7.94 2.41
>266
>272
203
>59
>272
>432
P220
16
P196
70 22
>272
5c
5d
3-OH–Ph
4-Br–Ph
P226
60.62 29.97
>242
>30
>5704
>5
5e
3,5-Me2–Ph
3,5-(CF3)2–Ph
2,6-Cl2–Ph
Ph
>308
>242
5f
5g
>278
>274
>278
>274
>6318
>199
P1386
4675
887
5h
5i
1-Naphthyl
2-Br–Ph
3-Br–Ph
P42.20
>187
>142
P305
187 19
142 13
>264
5j
5k
MeO
MeO
MeO
MeO
MeO
MeO
H
Et
Et
5l
5m
4-Br–Ph
>264
>266
>33
nae
4-Ph–Ph
3,5-Me2–Ph
3,5-(CF3)2–Ph
3,5-Me2–Ph
Ph
Et
Et
>266
5n
5o
0.018 0.013
0.79 0.008
0.010 0.002
3.49 3.10
0.003
52.78 6.04
>236
P239
>236
>318
P13,278
299
Et
Et
5p
5q
154 135
>266
>31,800
76
Et
MeO
>266
Efavirenz
Delavirdine
AZT
>6.34
>3.62
>93.55
P529
2113
>50
0.072
0.005
5.37 0.1
>17,990
P98
DDI
2.71 0.25
a All data represent mean values for at least two separate experiments.
b EC50, effective concentration of compound required to protect the cell against viral cytopathogenicity by 50% in MT-4 cells.
c CC50, cytotoxic concentration of compound that reduces the normal uninfected MT-4 cell viability by 50%.
d SI, selectivity index; ratio CC50/EC50
e na, not active.
.
These promising results further highlighted that the
phenylcarbonylmethythio moiety was an optimal substi-
tuent at the C-2 position of the pyrimidine ring, and that
highly potent HIV-1 inhibitors could be obtained within
S-DABOs sharing these common structural features.
Also, the results shown in Table 1 revealed some impor-
tant SAR information on the role of different substitu-
tions in the aromatic ring of the C-6 benzyl moiety.
attributes on the substitution pattern of the phenyl ring
when more effective nanomolar HIV-1 inhibitors 5e, 5n
and 5p with exceptionally high SI index were considered.
Clearly, their high activity and selectivity toward HIV-1
could be attributed to the introduction of two meta-
methyl groups into the phenyl ring, whereas in other ser-
ies of S-DABOs this kind of structural variations had
negligible effects on HIV-1 inhibitory activity.3,5 The
underlying factors responsible for the significantly im-
proved antiviral potency of these compounds remain
to be elucidated. One explanation may be that the addi-
tional meta-dimethyl substituents on the aromatic ring
are in an appropriate spatial orientation to make exten-
sive favorable van der Waals contacts with the highly
conserved residue W229 located at the hydrophobic
binding pocket of RT.
In the case of monosubstituted analogues (5a–5d, and
5j–5m), a clear positional preference for substitution
on the phenyl ring was observed by direct comparison:
the inhibitory potency was higher for the ortho-substi-
tuted analogues (5a and 5j) than for the meta-substi-
tuted ones (5b and 5k) which, in turn, was more active
than the para-substituted congeners (5d and 5l). This
conclusion is in agreement with the previous relative
structure–activity relationship studies.4 Furthermore,
disubstitutions with two chlorine atoms at the ortho-po-
sition of the phenyl ring led to compound 5g capable of
inhibiting HIV-1 replication at nanomolar concentra-
tion, suggesting that this type of substitution pattern is
highly favored with regard to the HIV-1 inhibitory
activity. The above finding is also in agreement with
the earlier SAR studies4,9 concerning the 2,6-dichloro-
phenyl moiety of S-DABOs, which have been suggested
to exert their favorable effect on anti-HIV-1 activity by
enhancement of the putative charge-transfer interactions
between the p-stacking aromatic rings of the inhibitor
and Y188, and Y181 in RT.
As for C-5 modifications it has been demonstrated that
variation at this position plays an important role in
determining the activity of S-DABOs.8,14 Consistent
with the SAR for our lead compounds 2-arylcarbonylm-
ethyl-6-naphthylmethyl-substituted S-DABOs (4), a sig-
nificant increase (1.1- to 58-fold) of anti-HIV-1 potency
was found for all the newly synthesized analogues except
5h and 5q whose C-5 methyl group was replaced with an
ethyl group. It should be pointed out that the above
observations contrasted sharply with the conclusion
drawn by Artico et al. based on 2-alkyl-substituted
S-DABOs (2), in which the steric bulkiness of C-5 sub-
stituent is detrimental to HIV-1 inhibitory activity
(H P Me > Et > i-Pr).5
Although the SAR discussed above appeared to be very
similar to those found in other series of S-DABOs (i.e., 2
and 3), there emerged some strikingly different SAR
Overall, the discrepancy of SAR at position 5, together
with 3,5-dimethylsubstitution on the C-6 phenyl ring

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Y.-P. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3887–3894
Table 2. Activity of selected derivatives 5e, 5n and 5p against (K103N+Y181C) HIV-1 mutant strain in MT-4 cellsa
SId (RES056)
b
c
Compound
Ar
R1
R2
R3
EC50 (lM)
HIV-1 IIIB
CC50 (lM)
RES056f
5e
5n
3,5-Me2–Ph
3,5-Me2–Ph
3,5-Me2–Ph
H
H
H
Me
Et
OMe
OMe
H
>308
>239
19.82
0.38
0.054 0.008
0.018 0.013
0.010 0.002
0.003
>308
P239
>318
nae
nae
5p
Et
>16
17
nae
Efavirenz
Delavirdine
AZT
>6.34
>3.62
>93.55
>3.62
0.006
0.072
0.005
>15,592
a All data represent mean values for at least two separate experiments.
b–e See legend to Table 1.
f RES056, HIV-1 mutated strain bearing both K103N andY181C mutations.
being the most favorable structural variation, further
corroborates the rationale behind this study: 2-arylcar-
bonylmethyl-substituted S-DABOs might interact with
RT in a different way compared to closely related ana-
logues (e.g., 2); and more potent HIV-1 inhibitors with
improved antiviral profile, especially against HIV-1
resistant mutants, might be achieved by appropriate
chemical modifications on these unique S-DABOs.15
5. Experimental
5.1. Chemistry
5.1.1. General. Melting points were measured on a
WRS-1 digital melting point apparatus and are uncor-
rected. Infrared (IR) spectra (KBr) were recorded on a
Jasco FT/IR-4200 instrument. 1H NMR (400 MHz)
and 13C NMR (100 MHz) on a Brucker AV 400 MHz
spectrometer were recorded in DMSO-d6 or CDCl3.
Chemical shifts are reported in d (ppm) units relative
to the internal standard tetramethylsilane (TMS). Mass
spectra were obtained on an Agilent MS/5975 mass
spectrometer. Elemental analyses were performed on a
CARLOERBA 1106 instrument and the results of ele-
mental analyses for C, H, N, and S were within 0.4%
of the theoretical values. All chemicals and solvents used
were of reagent grade and were purified and dried by
standard methods before use. All air-sensitive reactions
were run under a nitrogen atmosphere. All the reactions
were monitored by thin-layer chromatography (TLC)
on pre-coated silica gel G plates at 254 nm under a
UV lamp using ethyl acetate/hexane as eluents. Flash
chromatography separations were obtained on silica
gel (300–400 mesh). Compounds 6a–6d and 6f–6i were
obtained commercially; 3,5-dimethylphenyl acetic acid
(6e) was prepared according to literature procedure.16,17
In the light of these, we further evaluated the capability
of compounds 5e, 5n and 5p to inhibit the replication of
HIV-1 strain (RES056) bearing both NNRTI-character-
istic K103N and Y181C mutations in cell culture (Table 2).
It was found that compound 5p exhibited some activity
against the mutant strain at the lower micromolar
concentrations.
In addition, all the title compounds were also tested for
their antiviral activity against HIV-2 in MT-4 cells, but
none was found effective except for compounds 5e, 5i,
and 5n displaying moderate potency at micromolar con-
centrations that were close to their toxicity/solubility
threshold (Table 1).
4. Conclusions
In summary, a good agreement between our newly syn-
thesized analogues and other series of S-DABOs was
observed when the phenyl ring of the C-6 benzyl moiety
was substituted with halogens. In particular, compound
5g containing two chlorine atoms at the ortho-position
exhibited potent anti-HIV-1 activity (EC50 = 0.044 lM,
SI > 6318). However, the most favorable structural var-
iation in this new series of S-DABOs turned out to be
the introduction of two methyl groups into the meta-po-
sition of the phenyl ring leading to more potent and
selective HIV-1 inhibitors 5e, 5n and 5p (EC50 = 0.054,
0.018, and 0.010 lM, SI > 5704, 13278, and 31,800,
respectively), while such variations had little effects in
other series of S-DABOs. These peculiar and promising
results imply that this new series of S-DABOs may inter-
act with RT in a different way compared to the closely
related analogues (2). Thus, 2-arylcarbonylmethylthio-
6-(3,5-dimethylbenzyl)substituted-S-DABOs, especially
compound 5p, show great potential as candidates for
further development of new HIV-1 specific inhibitors
with an improved antiviral profile.
5.1.2. General procedure for the preparation of
b-ketoesters 7a–p. To a well stirred solution of substi-
tuted diethyl malonate (517 mmol) in anhydrous EtOH
(345 mL) was added dropwise a solution of KOH
(29 g, 517 mmol) in EtOH (345 mL) at room tempera-
ture over 4 h. Then the resulting mixture was allowed
to stand at this temperature for four to twelve hours un-
til the pH of the final mixture had a value between 7 and
8. After removing the solvent, the residue was rinsed
with Et2O and suspended in anhydrous CH3CN
(800 mL), Et3N (100 mL, 717 mmol) and MgCl2 (54 g,
567 mmol) were added and the mixture continued being
stirred at room temperature for 2 h. Then a solution of
arylacetyl imidazolide, which was prepared from aryl-
acetic acid (214 mmol) and N,N0-carbonyldiimidazole
(CDI, 45 g, 259 mmol) in CH3CN (400 mL) 30 min be-
fore, was added and the reaction mixture was stirred
overnight at room temperature. After refluxing for 2 h
and then cooled to 0 ꢁC, a solution of 13 % HCl
(800 mL) was added slowly and the resulting clear

Y.-P. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3887–3894
3891
mixture was stirred for further 15 min. The organic
phase was separated and concentrated, and the aqueous
phase was extracted with EtOAc (3 · 300 mL). The
combined organic layers were washed with saturated
NaHCO3 (3 · 350 mL) and brine (3 · 350 mL), dried
(Mg2SO4), filtered and concentrated in vacuo to afford
the crude products 7a–p to be used directly in the next
step without further purification.
CH2), 1666 (C@O), 1638 (C@O), 1595, 1570, 1548,
1
1476 (aryl); H NMR (DMSO-d6) d 1.91 (s, 3H, CH3),
3.66 (s, 2H, ArCH2), 3.86 (s, 3H, OCH3), 4.64 (s, 2H,
SCH2), 7.02–7.11 (m, 4H, ArH5,6 and Ar0H3,5), 7.26 (s,
1H, ArH2), 7.32 (d, 1H, J = 7.6 Hz ArH4), 7.96 (d,
2H, J = 8.8 Hz, Ar0H2,6]), 12.73 (br s, 1H, CONH);
13C NMR (DMSO-d6) d 10.47 (CH3), 37.36 (ArCH2),
39.60 (SCH2), 55.75 (OCH3), 114.09 (2C), 116.25 (C-
5), 121.63, 128.00, 128.86, 129.24, 130.38, 130.74 (2C),
131.42, 140.85, 155.82 (C-6), 159.27 (C-2), 162.97 (C-
4), 163.52 (Ar0-C4), 191.53 (C@O); MS (EI) m/z 458
(M+); Anal. calcd for C21H19BrN2O3S: C, 54.91; H,
4.17; N, 6.10; S, 6.98. Found: C, 54.95; H, 4.14; N,
6.12; S, 7.01.
5.1.3. General procedure for the preparation of 5-alkyl-6-
substituted thiouracil 8a–p. To a stirred solution of
EtONa (250 mmol) in EtOH (30 mL) was added thio-
urea (15.3 g, 200 mmol) at room temperature. After stir-
ring for 30 min, the corresponding b-ketoesters 7a–p
(160 mmol) was added and the reaction mixture was re-
fluxed for 16 h under a nitrogen atmosphere. The reac-
tion mixture was cooled to room temperature and
concentrated under reduced pressure, and the remaining
residue was dissolved in H2O and neutralized with 13%
HCl to precipitate. The solid was collected, washed
sequentially with H2O, EtOH, and Et2O, then dried to
give 8a–p to be used in the next step without further
purification.
5.1.4.3. 6-(3-Hydroxybenzyl)-2-(4-methoxyphenylcar-
bonylmethylthio)-5-methylpyrimidin-4(3H)-one (5c).
Yield 64%; recrystallized from AcOEt as an off-white
powder; mp 185.0–185.4 ꢁC; FT-IR (KBr) m 3447 (NH,
OH), 2965 (CH3), 2919, 2840 (CH2), 1644 (C@O),
1
1598, 1544, 1509, 1456 (aryl); H NMR (DMSO-d6) d
1.82 (s, 3H, CH3), 3.57 (s, 2H, ArCH2), 3.83 (s, 3H,
OCH3), 4.65 (s, 2H, SCH2), 6.45 (d, 1H, J = 7.6 Hz,
ArH2), 6.56 (m, 2H, ArH5,6), 6.95 (t, 1H, J1 = 7.6 Hz,
J2 = 7.6 Hz, ArH4), 7.03 (d, 2H, J = 8.8 Hz, Ar0H3,5),
7.96 (d, 2H, J = 8.8 Hz, Ar0H2,6), 9.24 (br s, 1H, ArOH),
12.54 (br s, 1H, CONH); 13C NMR (DMSO-d6) d10.51
(CH3), 37.27 (ArCH2), 55.70 (OCH3), 113.29, 114.08
(2C), 115.14 (C-5), 115.55, 119.55, 128.88, 129.24,
130.75 (2C), 139.35, 156.53 (C-6), 157.40 (Ar-C3),
160.99 (C-2), 163.51 (2C, C-4 and Ar0-C4), 191.87
(C@O); MS (EI) m/z 396 (M+); Anal. calcd for
C21H20N2O4S: C, 63.62; H, 5.08; N, 7.07; S, 8.09.
Found: C, 63.65; H, 5.04; N, 7.05; S, 8.06.
5.1.4. General procedure for the preparation of target
compounds 5a–q. To a stirred solution of 8a–p (3 mmol)
in anhydrous DMF (18 mL) was added K2CO3 (0.46 g,
3.3 mmol) at room temperature. After stirring for
20 min,
appropriate
arylcarbonylmethyl
halide
(3.3 mmol) was added, and stirring was continued at this
temperature for another 18 h. The reaction mixture was
poured into cold H2O (150 mL), the resulting precipitate
was collected by filtration and washed sequentially with
small portions of H2O, MeOH and Et2O and then dried
in vacuo at 40 ꢁC to afford the corresponding crude
product, which was purified by flash chromatography
or by crystallization to give the pure target compounds
5a–q.
5.1.4.4. 6-(4-Bromobenzyl)-2-(4-methoxyphenylcar-
bonylmethylthio)-5-methylpyrimidin-4(3H)-one (5d).
Yield 17%; recrystallized from THF as a white solid;
mp 199.2–199.6 ꢁC; FT-IR (KBr) m 3433 (NH), 2954
(CH3), 2919, 2835 (CH2), 1667 (C@O), 1641 (C@O),
5.1.4.1. 6-(2-Bromobenzyl)-2-(4-methoxyphenylcar-
bonylmethylthio)-5-methylpyrimidin-4(3H)-one (5a).
Yield 21%; recrystallized from MEK as a pale yellow
crystal; mp 204.3–205.2 ꢁC; FT-IR (KBr) m 3407 (NH),
2958 (CH3), 2917, 2841 (CH2), 1671 (C@O), 1658
1
1595, 1573, 1547, 1457 (aryl); H NMR (DMSO-d6) d
1.98 (s, 3H, CH3), 3.68 (s, 2H, ArCH2), 3.92 (s, 3H,
OCH3), 4.71 (s, 2H, SCH2), 7.04 (d, 2H, J = 8.4 Hz,
ArH2,6), 7.12 (d, 2H, J = 8.8 Hz, Ar0H3,5), 7.33 (d, 2H,
J = 8.4 Hz, ArH3,5), 8.03 (d, 2H, J = 8.8 Hz, Ar0H2,6);
13C NMR (DMSO-d6) d 10.43 (CH3), 37.38 (ArCH2),
39.36 (SCH2), 55.71 (OCH3), 114.09 (2C), 115.25 (C-
5), 119.39, 128.84, 130.68 (2C), 131.06 (4C), 137.49,
156.70 (C-6), 160.09 (C-2), 163.45 (C-4), 163.52 (Ar0-
C4), 191.43 (C@O); MS (EI) m/z 458 (M+); Anal. calcd
for C21H19BrN2O3S: C, 54.91; H, 4.17; N, 6.10; S,
6.98. Found: C, 54.95; H, 4.18; N, 6.04; S, 6.96.
1
(C@O), 1597, 1513, 1458 (aryl); H NMR (DMSO-d6)
d 1.94 (s, 3H, CH3), 3.82 (s, 2H, ArCH2), 3.87 (s, 3H,
OCH3), 4.50 (s, 2H, SCH2), 7.02 (d, 2H, J = 8.8 Hz,
Ar0H3,5), 7.04–7.12 (m, 3H, ArH4,5,6), 7.44 (d, 1H,
ArH3), 7.80 (d, 2H, J = 8.8 Hz, Ar0H2,6), 12.73 (br s,
1H, CONH); 13C NMR (DMSO-d6) d 10.45 (CH3),
37.61 (ArCH2), 40.04 (SCH2), 55.72 (OCH3), 113.97
(2C), 115.61 (C-5), 124.30, 127.44, 128.35, 128.56,
130.60 (2C), 131.40, 132.22, 137.51, 156.48 (C-6),
159.15 (C-2), 163.36 (C-4), 163.53 (Ar0-C4), 191.23
(C@O); MS (EI) m/z 458 (M+); Anal. calcd for
C21H19BrN2O3S: C, 54.91; H, 4.17; N, 6.10; S, 6.98.
Found: C, 54.89; H, 4.18; N, 6.11; S, 6.96.
5.1.4.5. 6-(3,5-Dimethylbenzyl)-2-(4-methoxyphenyl-
carbonylmethylthio)-5-methylpyrimidin-4(3H)-one (5e).
Yield 38%; column chromatography (eluent CH2Cl2/
AcOE 100:20), off-white plate crystal; mp 197.3–
197.7 ꢁC; FT-IR (KBr) m 3433 (NH), 2950 (CH3),
2914, 2848 (CH2), 1673 (C@O), 1655 (C@O), 1599,
1573, 1554, 1509, 1457 (aryl); 1H NMR (CDCl3) d
1.90 (s, 3H, CH3), 2.13 (s, 6H, Ar(CH3)2), 3.56 (s, 2H,
ArCH2), 3.84 (s, 3H, OCH3), 4.67 (s, 2H, SCH2), 6.63
(s, 2H, ArH2,6), 6.74 (s, 1H, ArH4), 7.04 (d, 2H,
5.1.4.2. 6-(3-Bromobenzyl)-2-(4-methoxyphenylcar-
bonylmethylthio)-5-methylpyrimidin-4(3H)-one (5b).
Yield 70%; column chromatography (eluent CH2Cl2/
AcOEt 100:20), white plate crystal; mp 180.8–181.3 ꢁC;
FT-IR (KBr) m 3428 (NH), 2935, 2910, 2839 (CH3,

3892
Y.-P. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3887–3894
J = 8.8 Hz, Ar0H3,5), 8.00 (d, 2H, J = 8.8 Hz, Ar0H2,6),
12.66 (br s, 1H, CONH); 13C NMR (DMSO-d6) d
10.50 (CH3), 20.93 (2C, Ar(CH3)2), 37.22 (ArCH2),
55.68 (OCH3), 114.04 (2C), 115.01 (C-5), 126.45 (2C),
127.73, 128.99, 130.66 (2C), 137.21 (2C), 137.83,
156.48 (C-6), 160.84 (C-2), 163.47 (2C, C-4 and Ar0-
C4), 191.69 (C@O); MS (EI) m/z 408 (M+); Anal. calcd
for C23H24N2O3S: C, 67.62; H, 5.92; N, 6.86; S, 7.85.
Found: C, 67.60; H, 5.95; N, 6.88; S, 7.81.
C27H24N2O3S: C, 71.03; H, 5.30; N, 6.14; S, 7.02.
Found: C, 71.08; H, 5.28; N, 6.12; S, 7.05.
5.1.4.9. 5-Methyl-6-(1-naphthylmethyl)-2-(phenylcar-
bonylmethylthio)pyrimidin-4(3H)-one (5i). Yield 21%;
recrystallized from dioxone as a white solid; mp 194.3–
195.3 ꢁC; FT-IR (KBr) m 3432 (NH), 2928, 2840 (CH3,
CH2), 1690 (C@O), 1633 (C@O), 1595, 1579, 1553,
1
1509, 1480, 1448 (aryl); H NMR (DMSO-d6) d 1.07
(s, 3H, CH3), 4.14 (s, 2H, CH2-naphthyl), 4.48 (s, 2H,
SCH2), 7.10–7.94 (m, 12H, aryl), 12.72 (br s, 1H,
CONH); 13C NMR (DMSO-d6) d 10.57 (CH3), 37.45
(CH2-naphthyl), 37.75 (SCH2), 115.66 (C-5), 124.13–
135.81 (16C, aryl), 156.50 (C-6), 160.63 (C-2), 163.54
(C-4), 193.18 (C@O); MS (EI) m/z 400 (M+); Anal. calcd
for C24H20N2O2S: C, 71.98; H, 5.03; N, 6.99; S, 8.01.
Found: C, 71.95; H, 5.07; N, 7.03; S, 7.99.
5.1.4.6. 6-(3,5-Bis(trifluoromethyl)benzyl)-2-(4-meth-
oxyphenylcarbonylmethylthio)-5-methylpyrimid in-
4(3H)-one (5f). Yield 35%; recrystallized from AcOEt
as a white needle crystal; mp 202.8–203.1 ꢁC; FT-IR
(KBr) m 3397 (NH), 2932, 2894, 2848 (CH3, CH2),
1
1653 (C@O), 1602 (C@O), 1572, 1552, 1509 (aryl); H
NMR (DMSO-d6) d 1.93 (s, 3H, CH3), 3.81 (s, 3H,
OCH3), 3.94 (s, 2H, ArCH2), 4.51 (s, 2H, SCH2), 6.94
(d, 2H, J = 8.8 Hz, Ar0H3,5), 7.74–7.77 (m, 3H, ArH),
7.78 (d, 2H, J = 8.8 Hz, Ar0H2,6), 12.76 (br s, 1H,
CONH); 13C NMR (DMSO-d6) d 10.37 (CH3), 37.11
(ArCH2), 38.99 (SCH2), 55.65 (OCH3), 113.88 (2C),
115.09 (C-5), 120.00 (sept, J = 4 Hz, Ar-C4), 123.45 (q,
2C, J = 271.1 Hz, CF3), 128.52, 129.83 (2C), 129.99 (q,
2C, J = 32.5 Hz, Ar-C3,5), 130.42 (2C), 141.60, 155.06
(C-6), 158.20 (C-2), 162.77 (C-4), 163.45 (Ar0-C4),
191.12 (C@O); MS (EI) m/z 516 (M+); Anal. calcd for
C23H18F6N2O3S: C, 53.49; H, 3.51; N, 5.42; S, 6.21.
Found: C, 53.53; H, 3.46; N, 5.40; S, 6.22.
5.1.4.10. 6-(2-Bromobenzyl)-5-ethyl-2-(4-methoxyphe-
nylcarbonylmethylthio)pyrimidin-4(3H)-one (5j). Yield
62%; recrystallized from THF as white solid; mp
213.2–213.8 ꢁC; FT-IR (KBr) m 3420 (NH), 2962
(CH3), 2926, 2845 (CH2), 1649 (C@O), 1601 (C@O),
1
1570, 1546, 1509, 1422 (aryl); H NMR (DMSO-d6) d
0.97 (t, 3H, J = 7.2 Hz, CH2CH3), 2.42 (q, 2H,
J = 7.2 Hz, CH2CH3), 3.84 (s, 2H, ArCH2), 3.87 (s,
3H, OCH3), 4.49 (s, 2H, SCH2), 7.01 (d, 2H,
J = 8.8 Hz, Ar0H2,6), 7.05–7.13 (m, 3H, ArH4,5,6), 7.43
(d, 1H,, J = 7.6 Hz, ArH3), 7.80 (d, 2H, J = 8.8 Hz,
Ar0H2,6), 12.70 (br s, 1H, CONH); 13C NMR (DMSO-
d6) d 12.30 (CH2CH3), 18.25 (CH2CH3), 37.55 (ArCH2),
55.76 (OCH3), 113.99 (2C), 121.86 (C-5), 124.34, 127.47,
128.39, 128.56, 130.64 (2C), 131.39, 132.23, 137.73,
155.87 (C-6), 158.39 (C-2), 162.59 (C-4), 163.53 (Ar0-
C4), 191.26 (C@O); MS (EI) m/z 472 (M+); Anal. calcd
for C22H21BrN2O3S: C, 55.82; H, 4.47; N, 5.92; S,
6.77. Found: C, 55.86; H, 4.45; N, 5.90; S, 6.78.
5.1.4.7. 6-(2,6-Dichlororbenzyl)-2-(4-methoxyphenyl-
carbonylmethylthio)-5-methylpyrimidin-4(3H)-one (5g).
Yield 52%; recrystallized from dioxone as a white block
crystal; mp 230.5–235.6 ꢁC; FT-IR (KBr) m 3430 (NH),
2962 (CH3), 2927, 2842 (CH2), 1657 (C@O), 1596,
1
1569, 1509, 1434 (aryl); H NMR (DMSO-d6) d 2.03
(s, 3H, CH3), 3.90 (s, 3H, OCH3), 4.08 (s, 2H, ArCH2),
4.39 (s, 2H, SCH2), 7.02–7.15 (m, 5H, ArH3,4,5 and
ArH03,5), 7.73 (d, 2H, J = 8.4 Hz, ArH02,6), 12.69 (br s,
1H, CONH); 13C NMR (DMSO-d6) d 10.16 (CH3),
35.25 (ArCH2), 38.02 (SCH2), 55.81 (OCH3), 114.02
(2C), 115.19 (C-5), 127.82 (2C), 128.10, 128.83, 130.60
(2C), 134.48, 135.40 (2C), 156.38 (C-6), 157.88 (C-2),
163.17 (C-4), 163.69 (Ar0-C4), 190.28 (C@O); MS (EI)
m/z 448 (M+); Anal. calcd for C21H18Cl2N2O3S: C,
56.13; H, 4.04; N, 6.23; S, 7.14. Found: C, 56.10; H,
4.08; N, 6.21; S, 7.14.
5.1.4.11. 6-(3-Bromobenzyl)-5-ethyl-2-(4-methoxyphe-
nylcarbonylmethylthio)pyrimidin-4(3H)-one (5k). Yield
47%; column chromatography (eluent CH2Cl2/AcOE
100:20), white needle crystal; mp 201.9–203.0 ꢁC; FT-
IR (KBr) m 3431 (NH), 2967 (CH3), 2924, 2840 (CH2),
1652 (C@O), 1600 (C@O), 1570, 1550, 1510, 1423 (aryl);
1H NMR (DMSO-d6) d 0.91 (t, 3H, J = 7.2 Hz,
CH2CH3), 2.41 (q, 2H, J = 7.2 Hz, CH2CH3), 3.67 (s,
2H, ArCH2), 3.85 (s, 3H, OCH3), 4.64 (s, 2H, SCH2),
7.03–7.09 (m, 4H, ArH5,6 and Ar0H3,5), 7.28 (s, 1H,
ArH2), 7.32 (d, 1H, ArH4), 7.96 (d, 2H, J = 8.8 Hz,
Ar0H2,6), 12.72 (br s, 1H, CONH); 13C NMR (DMSO-
d6) d 13.21 (CH2CH3), 18.25 (CH2CH3), 37.38 (ArCH2),
55.74 (OCH3), 114.08 (2C), 121.59, 121.93 (C-5), 127.99,
128.84, 129.21, 130.34, 130.73 (2C), 131.45, 141.21,
155.97 (C-6), 159.19 (C-2), 162.67 (C-4), 163.51 (Ar0-
C4), 191.50 (C@O); MS (EI) m/z 472 (M+); Anal. calcd
for C22H21BrN2O3S: C, 55.82; H, 4.47; N, 5.92; S,
6.77. Found: C, 55.80; H, 4.51; N, 5.89; S, 6.81.
5.1.4.8. 6-(a-Phenylbenzyl)-2-(4-methoxyphenylcar-
bonylmethylthio)-5-methylpyrimidin-4(3H)-one
(5h).
Yield 26%; recrystallized from THF as white solid; mp
220.5–221.6 ꢁC; FT-IR (KBr) m 3419 (NH), 2912, 2841
(CH3, CH2), 1671 (C@O), 1636 (C@O), 1601, 1574,
1
1542, 1510, 1493, 1423 (aryl); H NMR (DMSO-d6) d
1.93 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.52 (s, 2H,
SCH2), 5.49 (s, 1H, ArCH), 7.04–7.12 (m, 12H,
2 · ArH and Ar0H3,5), 7.87 (d, 2H, J = 9.2 Hz Ar0H2,6),
12.77 (br s, 1H, CONH); 13C NMR (DMSO-d6) d 10.16
(CH3), 38.03 (SCH2), 53.17 (ArCH), 55.74 (OCH3),
114.05 (2C), 115.64 (C-5), 126.41 (2C), 128.04 (4C),
128.47, 129.19 (4C), 130.73 (2C), 141.49 (2C), 156.31
(C-6), 161.86 (C-2), 163.60 (2C, C-4 and Ar0-C4),
190.75 (C@O); MS (EI) m/z 456 (M+); Anal. calcd for
5.1.4.12. 6-(4-Bromobenzyl)-5-ethyl-2-(4-methoxyphe-
nylcarbonylmethylthio)pyrimidin-4(3H)-one (5l). Yield
58%; column chromatography (eluent CH2Cl2/AcOEt
100:20), white solid; mp 205–206.7 ꢁC; FT-IR (KBr) m
3434 (NH), 2963 (CH3), 2928 (CH2), 2870 (CH3), 2836

Y.-P. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3887–3894
3893
(CH2), 1666 (C@O), 1638 (C@O), 1593, 1571, 1543,
1457 (aryl); 1H NMR (DMSO-d6) d 0.98 (t, 3H,
J = 7.2 Hz, CH2CH3), 2.46 (q, 2H, J = 7.2 Hz,
CH2CH3), 3.69 (s, 2H, ArCH2), 3.91 (s, 3H, OCH3),
4.69 (s, 2H, SCH2), 7.04 (d, 2H, J = 8.0 Hz, ArH2,6),
7.11 (d, 2H, J = 8.8 Hz, Ar0H3,5), 7.31 (d, 2H,
J = 8.0 Hz, ArH3,5), 8.00 (d, 2H, J = 8.8 Hz, Ar0H2,6),
12.73 (br s, 1H, CONH); 13C NMR (DMSO-d6) d
13.35 (CH2CH3), 18.26 (CH2CH3), 37.45 (ArCH2),
38.83 (SCH2), 55.79 (OCH3), 114.14 (2C), 119.42,
121.79 (C-5), 128.86, 130.76 (2C), 131.07 (2C), 131.17
(2C), 137.84, 156.10 (C-6), 159.12 (C-2), 162.56 (C-4),
163.56 (Ar0-C4), 191.42 (C@O); MS (EI) m/z 472 (M+);
Anal. calcd for C22H21BrN2O3S: C, 55.82; H, 4.47; N,
5.92; S, 6.77. Found: C, 55.85; H, 4.43; N, 5.91; S, 6.75.
J = 7.2 Hz, CH2CH3), 3.82 (s, 3H, OCH3), 3.97 (s, 2H,
ArCH2), 4.53 (s, 2H, SCH2), 6.95 (d, J = 8.8 Hz,
Ar0H3,5), 7.76 (s, 2H, ArH2,6), 7.78 (s, 1H, ArH4), 7.80
(d, J = 8.8 Hz, Ar0H2,6), 12.78 (br s, 1H, CONH); 13C
NMR (DMSO-d6) d 13.15 (CH2CH3), 18.15 (CH2CH3),
37.14 (ArCH2), 38.50 (SCH2), 55.67 (OCH3), 113.88
(2C), 120.04 (sept, J = 4 Hz, Ar-C4), 122.45 (C-5),
123.48 (q, 2C, J = 271.1 Hz, CF3), 128.55, 129.84 (2C),
130.00 (q, 2 C, J = 32.3 Hz, Ar-C3,5), 130.46 (2C),
141.93, 156.31 (C-6), 157.85 (C-2), 162.49 (C-4), 163.46
(Ar0-C4), 191.16 (C@O); MS (EI) m/z 530 (M+); Anal.
calcd for C24H20F6N2O3S: C, 54.34; H, 3.80; N, 5.28;
S, 6.04. Found: C, 54.31; H, 3.84; N, 5.30; S, 6.10.
5.1.4.16. 6-(3,5-Dimethylbenzyl)-5-ethyl-2-(phenylcar-
bonylmethylthio)pyrimidin-4(3H)-one (5p). Yield 38%;
column chromatography (eluent CH2Cl2/AcOEt
100:20), yellow solid; mp 165.5–167.0 ꢁC; FT-IR (KBr)
m 3431 (NH), 2964 (CH3), 2929 (CH2), 2869 (CH3),
5.1.4.13. 6-(4-Phenylbenzyl)-5-ethyl-2-(4-methoxyphe-
nylcarbonylmethylthio)pyrimidin-4(3H)-one (5m). Yield
30%; column chromatography, (eluent CH2Cl2/AcOEt
100:20), white solid; mp 201.2–201.6 ꢁC; FT-IR (KBr)
m 3421 (NH), 2965 (CH3), 2920 (CH2), 2870 (CH3),
2837 (CH2), 1660 (C@O), 1601 (C@O), 1575, 1551,
1509 (aryl); 1H NMR (DMSO-d6) d 0.96 (t, 3H,
J = 7.2 Hz, CH2CH3), 2.46 (q, 2H, J = 7.2 Hz,
CH2CH3), 3.71 (s, 2H, ArCH2), 3.80 (s, 3H, OCH3),
4.69 (s, 2H, SCH2), 6.99–8.00 (m, 13H, aryl), 12.68 (br
1
1637 (C@O), 1600, 1570, 1542, 1450 (aryl); H NMR
(DMSO-d6) d 0.89 (t, 3H, J = 7.4 Hz, CH2CH3), 2.11
(s, 6H, Ar(CH3)2), 2.39 (q, 2H, J = 7.4 Hz, CH2CH3),
3.54 (s, 2H, ArCH2), 4.71 (s, 2H, SCH2), 6.61 (s, 2H,
ArH2,6), 6.71 (s, 1H, ArH4), 7.52 (t, 2H, Ar0H3,5), 7.65
(t, 1H, Ar0H4), 7.90 (d, 2H, Ar0H2,6), 12.69 (br s, 1H,
CONH); 13C NMR (DMSO-d6) d 13.14 (CH2CH3),
18.33 (CH2CH3), 20.95 (2C, Ar(CH3)2), 37.65 (ArCH2),
121.19 (C-5), 126.48 (2C), 127.72, 128.31 (2C), 128.86
(2C), 133.56, 136.24, 137.17 (2C), 138.17, 156.00 (C-6),
160.35 (C-2), 163.04 (C-4), 193.39 (C@O); MS (EI) 392
(M+); Anal. calcd for C23H24N2O2S: C, 70.38; H, 6.16;
N, 7.14; S, 8.17. Found: C, 70.33; H, 6.19; N, 7.13; S,
8.21.
s, 1H, CONH); 13C NMR (DMSO-d6)
d 13.21
(CH2CH3), 18.20 (CH2CH3), 37.39 (ArCH2), 55.60
(OCH3), 114.00–140.00 (18C, aryl and C-5), 155.60 (C-
6), 160.71 (C-2), 162.45 (C-4), 163.49 (Ar0-C4), 191.46
(C@O); MS (EI) m/z 470 (M+). Anal. calcd for
C28H26N2O3S: C, 71.46; H, 5.57; N, 5.95; S, 6.81.
Found: C, 71.43; H, 5.61; N, 5.93; S, 6.85.
5.1.4.14.
6-(3,5-Dimethylbenzyl)-5-ethyl-2-(4-meth-
5.1.4.17. 5-Ethyl-2-(4-methoxyphenylcarbonylmethyl-
thio)-6-(a-phenylbenzyl)pyrimidin-4(3H)-one (5q). Yield
12%; column chromatography (eluent CH2Cl2/AcOEt
100:10), white solid; mp 214.3–214.9 ꢁC; FT-IR (KBr)
m 3397 (NH), 2962 (CH3), 2916 (CH2), 2869 (CH3),
1672 (C@O), 1637 (C@O), 1600, 1574, 1543, 1494,
1457 (aryl); 1H NMR (DMSO-d6) d 0.94 (t, 3H,
J = 7.2 Hz, CH2CH3), 2.54 (q, 2H, J = 7.2 Hz,
CH2CH3), 3.91 (s, 3H, OCH3), 4.60 (s, 2 H, ArCH2),
5.56 (s, 1H, ArH), 7.11–7.20 (m, 12H, 2 · ArH and
Ar0H3,5), 7.95 (d, 2H, J = 8.8 Hz, Ar0H2,6), 12.82 (br s,
1H, CONH); 13C NMR (DMSO-d6) d 13.37 (CH2CH3),
18.01 (CH2CH3), 37.99 (SCH2), 52.58 (ArCH), 55.73
(OCH3), 114.04 (2C), 121.63 (C-5), 126.39 (2C), 128.02
(4C), 128.47, 129.15 (4C), 130.72 (2C), 141.84 (2C),
156.16 (C-6), 161.16 (C-2), 163.09 (C-4), 163.58 (Ar0-
C4), 190.68 (C@O); MS (EI) m/z 470 (M+); Anal. calcd
for C28H26N2O3S: C, 71.46; H, 5.57; N, 5.95; S, 6.81.
Found: C, 71.41; H, 5.59; N, 5.89; S, 6.83.
oxyphenylcarbonylmethylthio)pyrimidin-4(3H)-one (5n).
Yield 33%; column chromatography (eluent CH2Cl2/
AcOEt 100:20), white needle crystal; mp 201.7–
202.6 ꢁC; FT-IR (KBr) m 3432 (NH), 2968, 2928, 2867
(CH3, CH2), 1653 (C@O), 1600 (C@O), 1571, 1542,
1
1509, 1458 (aryl); H NMR (DMSO-d6) d 0.90 (t, 3H,
J = 7.2 Hz, CH2CH3), 2.13 (s, 6H, Ar(CH3)2), 2.40 (q,
2H, J = 7.2 Hz, CH2CH3), 3.32 (s, 2H, ArCH2), 4.67(s,
2H, SCH2), 6.65 (s, 2H, ArH2,6), 6.74 (s, 1H, ArH4),
7.0 (d, 2H, J=8.8 Hz, Ar0H3,5), 7.99 (d, 2H,
J = 8.8 Hz, Ar0H2,6), 12.68 (br s, 1H, CONH); 13C
NMR (DMSO-d6) d 13.11 (CH2CH3), 18.31 (CH2CH3),
20.93 (2C, Ar(CH3)2), 37.25 (ArCH2), 55.70 (OCH3),
114.04 (2C), 121.09 (C-5), 126.49 (2C), 127.71, 128.98,
130.67 (2C), 137.17 (2C), 138.19, 156.27 (C-6), 160.28
(C-2), 163.13 (C-4), 163.48 (Ar0-C4), 191.66 (C@O);
MS (EI) m/z 422 (M+); Anal. calcd for C24H26N2O3S:
C, 68.22; H, 6.20; N, 6.63; S, 7.59. Found: C, 68.18;
H, 6.23; N, 6.64; S, 7.55.
5.2. Anti-HIV activity assays
5.1.4.15. 6-(3,5-Bis(trifluoromethyl)benzyl)-5-ethyl-2-
(4-methoxyphenylcarbonylmethylthio)pyrimid in-4(3H)-
one (5o). Yield 55%; column chromatography (eluent
Petroleum ether/acetone 100:25), white solid; mp
172.3–173.3 ꢁC; FT-IR (KBr) m 3422 (NH), 2966
(CH3), 2933, 2848 (CH2), 1671 (C@O), 1640 (C@O),
The activity of the compounds against wild-type HIV-1,
the double RT mutant virus strain (RES056) and HIV-2
(ROD) was based on the inhibition of a virus-induced
cytopathic effect in MT-4 cells using the 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method.18 Briefly, virus stocks were titrated in MT-4
cells and expressed as 50% cell culture infective dose
1
1597, 1576, 1557, 1513, 1458 (aryl); H NMR (DMSO-
d6) d 0.92 (t, 3H, J = 7.4 Hz, CH2CH3), 2.45 (q, 2H,

3894
Y.-P. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3887–3894
5. Artico, M.; Mai, A.; Sbardella, G.; Massa, S.; La Colla, P.
Med. Chem. Res. 2000, 10, 30.
6. Mai, A.; Sbardella, G.; Artico, M.; Ragno, R.; Massa, S.;
Novellino, E.; Greco, G.; Lavecchia, A.; Musiu, C.; La
Colla, M.; Murgioni, C.; La Colla, P.; Loddo, R. J. Med.
Chem. 2001, 44, 2544.
7. Artico, M. Drugs Future 2002, 27, 159.
8. He, Y. P.; Chen, F. E.; Yu, X. J.; Wang, Y. P.; De Clercq,
E.; Balzarini, J.; Pannecouque, C. Bioorg. Chem. 2004, 32,
536.
(CCID50). MT-4 cells were suspended in culture medium
at 1 · 105 cells/mL and infected with HIV at a multiplic-
ity of infection of 0.02. Immediately after virus infection,
100 lL of the cell suspension was brought into each well
of a flat-bottomed microtiter tray containing various
concentrations of the test compounds. The test com-
pounds were dissolved in DMSO at 50 mM. After a 4-
day incubation at 37 ꢁC, the number of viable cells
was determined using the MTT method. Compounds
were tested in parallel for cytotoxic effect in uninfected
MT-4 cells.
´
´
9. Manetti, F.; Este, J. A.; Clotet-Codina, I.; Armand-Ugon,
M.; Maga, G.; Crespan, E.; Cancio, R.; Mugnaini, C.;
Bernardini, C.; Togninelli, A.; Carmi, C.; Alongi, M.;
Petricci, E.; Massa, S.; Corelli, F.; Botta, M. J. Med.
Chem. 2005, 48, 8000.
Acknowledgments
10. Sun, G. F.; Kuang, Y. Y.; Chen, F. E.; De Clercq, E.;
Balzarini, J.; Pannecouque, C. Arch. Pham. Chem. Life
Sci. 2005, 338, 457.
11. Ji, L.; Chen, F. E.; De Clercq, E.; Balzarini, J.; Panne-
couque, C. J. Med. Chem. 2007, 50, 1778.
We are grateful to the National Natural Science Foun-
dation of China (No. 30672536) and the Geconcerteerde
Onderzoeksacties (GOA No. 05/15) for the financial
support of this research.
12. Clay, R. J.; Collom, T. A.; Karrick, G. L.; Wemple, J. A.
Synthesis 1993, 290.
13. Meng, G.; Chen, F. E.; De Clercq, E.; Balzarini,
J.; Pannecouque, C. Chem. Pharm. Bull. 2003, 51,
779.
References and notes
14. Sbardella, G.; Mai, A.; Artico, M.; Chimenti, P.; Maasa,
S.; Loddo, R.; Marongiu, M. E.; La Colla, P.; Pani, A.
Antiviral Chem. Chemother. 2001, 12, 37.
1. Mai, A.; Artico, M.; Sbardella, G.; Massa, S.; Loi, A. G.;
Tramontano, E.; Scano, P.; La Colla, P. J. Med. Chem.
1995, 38, 3258.
15. Hopkins, A. L.; Ren, J. S.; Milton, J.; Hazen, R. J.; Chan,
J. H.; Stuart, D. I.; Stammers, D. K. J. Med. Chem. 2004,
47, 5912.
2. Mai, A.; Artico, M.; Sbardella, G.; Quartarone, S.; Massa,
S.; Loi, A. G.; De Montis, A.; Scintu, F.; Putzolu, M.; La
Colla, P. J. Med. Chem. 1997, 40, 1447.
16. Cadogan, J. I. G.; Inward, P. W. J. Chem. Soc. 1962, 4170.
17. Glamkowski, E. J.; Chiang, Y. L. J. Heterocyclic Chem.
1987, 24, 1599.
3. Vig, R.; Mao, C.; Venkatachalam, T. K.; Tuel-Ahlgren,
L.; Sudbeck, E. A.; Uckun, F. M. Bioorg. Med. Chem.
Lett. 1998, 8, 1461.
18. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols,
D.; Herdewijn, P.; Desmyter, J.; De Clercq, E. J. Virol.
Methods 1988, 20, 309.
4. Mai, A.; Artico, M.; Sbardella, G.; Massa, S.; Novellino,
E.; Greco, G.; Loi, A. G.; Tramontano, E.; Marongiu, M.
E.; La Colla, P. J. Med. Chem. 1999, 42, 619.