Synthesis and biological evaluation of novel 2-(substituted phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones as potent HIV-1 NNRTIs

Article abstract of DOI:10.1016/j.bmc.2009.09.035

A series of novel 2-(phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones have been designed and synthesized. All of the new compounds were evaluated for their anti-HIV activities in MT-4 cells. Most of these new compounds showed mod

Full text of DOI:10.1016/j.bmc.2009.09.035

Bioorganic & Medicinal Chemistry 17 (2009) 7749–7754
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel 2-(substituted phenylamino-
carbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones
as potent HIV-1 NNRTIs
a
a
b
b
Mingyan Yu a, Xinyong Liu a, , Zhenyu Li , Shuai Liu , Christophe Pannecouque , Erik De Clercq
*
a Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, No. 44 Wenhuaxi Road, Jinan 250012, PR China
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2009
Revised 17 September 2009
Accepted 18 September 2009
Available online 24 September 2009
A series of novel 2-(phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones have
been designed and synthesized. All of the new compounds were evaluated for their 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 ranging from 4.48
lM to 0.18 lM. Among them, 2-[(4-bromophenylamino)carbonylmethyl-
thio]-6-(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one 4b3 was identified as the most promising
compound (EC50 = 0.18 0.06
(SAR) of these new congeners is discussed.
l
M, CC50 >243.56
l
M, SI >1326). The structure–activity relationship
Keywords:
HIV-1
AIDS
Ó 2009 Elsevier Ltd. All rights reserved.
NNRTIs
S-DABOs
Anti-HIV-1 activity
1. Introduction
determinant for exhibition of anti-HIV-1 activity. 6-(2,6-Dichlorob-
enzyl) substituent of S-DABOs have been suggested to exert their
favorable effect on anti-HIV-1 activity by enhancement of the
The current therapy against the human immunodeficiency virus
type 1 (HIV-1), which is the etiological agent of acquired immuno-
deficiency syndrome (AIDS), is based on six of categories drugs:
nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtR-
TIs); non-nucleoside reverse transcriptase inhibitors (NNRTIs);
protease inhibitors (PIs); cell entry inhibitors [fusion inhibitors
(FIs) and co-receptor inhibitors (CRIs)]; and integrase inhibitors
(INIs).1 Among them, the HIV-1 NNRTIs serve as a representative
of most frontline AIDS combination therapies which are highly
effective with less side effects.
NNRTIs are highly specific for HIV-1 and include more than 50
structurally different classes of molecules. Among NNRTIs,
dihydroalkyloxybenzyloxopyrimidines (DABOs) are an interesting
class of compounds active at nanomolar concentrations.2,3 They
were first disclosed in 19924,5 and further developed into three gen-
erations during the following years: dihydroalkyloxybenzyl-oxo-
pyrimidines (O-DABOs); dihydroalkylthiobenzyloxopyrimidines
(S-DABOs); dihydroalkylaminodifluorobenzyloxopyrimidines (N-
DABOs)6–9 (Fig. 1).
putative charge-transfer interactions between the p-stacking aro-
matic rings of the inhibitor and Y188 and Y181 in HIV-1 RT.10–15
On the basis of these results, we kept the 6-(2,6-dichlorobenzyl)
substituent fixed and systematically modified the C-2 position by
increasing the length of the linker connecting the S atom to the
phenyl ring. A new series of S-DABOs (Fig. 1) was prepared, by
the insertion of a phenylaminocarbonylmethylthio chain at C-2,
and hydrogen or methyl substituents at the C-5 position of the
pyrimidine ring. We proposed that the introduction of the C-2 side
chain of S-DABOs might enhance the interaction between the
inhibitors and the RT, and further exploration of the structure–
activity relationships of S-DABOs may yield the discovery of new
more potent HIV-1 inhibitors. Herein, the detailed synthesis,
anti-HIV activity and preliminary SAR studies of these new congen-
ers are described.
2. Results and discussion
2.1. Chemistry
Compared to O-DABOs, the S-DABOs were more potent and
selective, with the C-2 alkylthio chain being the most peculiar
The target compounds 4a1–4a9, 4b1–4b8 were prepared as de-
picted in Scheme 1. The b-ketoesters (2a, 2b) were prepared by a
simple and high-yielding method of Clay et al.16 by the reaction of
* Corresponding author. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.035

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M. Yu et al. / Bioorg. Med. Chem. 17 (2009) 7749–7754
O
N
O
O
R
R
HN
HN
R1
HN
R2
R2
R1
O
N
N
F
R2
S
N
X
H
F
Ar
R1
DABOs
R = H, Me, Et
R1 = H, 3-Me, 3,5-Me2
R2 = alkyl, cycloalkyl
S-DABOs
2, 6-F2-NH-DABOs
R1 = H, Me, Et
R2 = alkyl, cycloalkyl, ArCOCH2
X = CH2, S, CHCN, C=O
R = R1 = H, Me
R2 = alkyl, cycloalkyl
Ar = 1-naphthyl, 2, 6-Cl2-Ph, 2, 6-F2-Ph
O
O
N
R
R1
HN
HN
R3
R1
O
N
N
F
S
R2
F
NH
Cl
Cl
R2
Newly designed S-DABO derivatives:
2, 6-F2-N, N-DABOs
2-(Substituted phenylaminocarbonylmethylthio)-6-
(2, 6-dichlorobenzyl)-pyrimidin-4(3H)-ones
R = R1 = H, Me
R2, R3 = alkyl, (hetero)cycloalkyl
Figure 1. The DABOs family.
O
N
O
N
R1
R1
Cl
HN
HN
HS
O
O
O
O
O
ii
iii
i
S
EtO
OK
EtO
NH
Cl
Cl
Cl
Cl
R1
1a, 1b
R1
Cl
R2
2a, 2b
1a: R1 = H
1b: R1 = CH3
4a1 - 4a9
4b1 - 4b8
3a, 3b
Scheme 1. Reagents and conditions: (i) (a) MgCl2, Et3N, CH3CN, rt, 2 h; (b) 2-(2,6-dichlorophenyl)acetic acid, N,N0-carbonyldiimidazole, rt, overnight then reflux, 2 h; (ii)
thiourea, EtONa, reflux, 6–12 h; (iii) appropriate N-phenylacetamide halides, K2CO3, DMF, rt, 12 h.
2-(2,6-dichlorophenyl)acetic acid with N,N0-carbonyldiimidazole
(CDI) followed by treatment with potassium ethyl malonate or 2-
methylmalonate in the presence of anhydrous magnesium chloride
and triethylamine, which were sufficiently pure to be used for sub-
sequent reactions without further purification. Subsequent con-
densation of b-ketoesters (2a, 2b) with thiourea in the presence
of EtONa in refluxing ethanol gave the key intermediates 5-alkyl-
6-substituted thiouracil 3a, 3b.17 Next, selective S-alkylation of
3a, 3b with the appropriate N-phenylacetamide halides (1:1.1)
in the presence of K2CO3 in anhydrous DMF afforded the desirable
target compounds 4a1–4a9, 4b1–4b8. Both analytical and spectral
data of all the compounds are in full agreement with the proposed
structures.
The majority of these compounds exhibited moderate to good
activities against HIV-1 with EC50 values in the range of 4.48–
0.18
compound 4b3 with an EC50 value of 0.18 0.06
lue of >243.56 M. The SI value was greater than 1326, which is
lM. The most active and selectivity S-DABO derivative was
lM, and a CC50 va-
l
much better than for the reference drugs of NVP and DLV. Some
compounds, 4b2 and 4b6, also had high anti-HIV-1 potency
(EC50 = 0.32 and 0.69 lM, respectively) and good selectivity indices
(SI >836 and >387), while compounds4a7, 4a8, 4a9, 4b7, 4b8 were
totally inactive.
The SARs analysis showed that the nature of the substituents on
the C-2 side chain phenyl ring influenced the antiviral activity of
these S-DABOs. The monosubstituted anilide analogues (4a1–4a5,
4b1–4b4) were more active than the disubstituted ones ( 4a6–
4a9, 4b5–4b8). Disubstitutions at the meta-position and para-posi-
tion of the anilide (4a7, 4a8, 4a9, 4b7, 4b8) led to compounds de-
void of anti-HIV-1 activity, which might reflect a spatial restriction
in the target site of the HIV-1 enzyme. This feature can be used in
the design of new S-DABOs. Moreover, it was found that the nature
of the substituents at the C-5 position of the pyrimidine ring also
influenced the anti-HIV-1 activity of these new congeners. When
the C-5 was substituted by a methyl group (series 4b), a marked in-
crease of anti-HIV-1 activity was observed for all the title com-
pounds except for 6a1 and 6b1. The active sequence of the
2.2. Anti-HIV evaluation
The activity and cytotoxicity of the newly designed and syn-
thesized S-DABO analogues (compounds 4a1–4a9, 4b1–4b8)
were tested in MT-4 cells for their ability to inhibit HIV-1- and
HIV-2-induced cytopathogenicities. The results, expressed as
EC50, CC50 and SI (selectivity index), are illustrated in Table 1.
Nevirapine (NVP), delaviridine (DLV), efavirenz (EFV) and
zidovudine (azidothymidine, AZT) were used as the reference
drugs.

M. Yu et al. / Bioorg. Med. Chem. 17 (2009) 7749–7754
7751
Table 1
Anti-HIV activities, cytotoxicities and selectivity indices of 2-(substituted phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones (series 4a1–4a9, 4b1–
4b8)
O
R1
HN
O
S
N
NH
Cl
Cl
R2
SIc (IIIB)
a
b
Compound
R1
R2
EC50
(lM)
CC50 (lM)
HIV-1 IIIB
HIV-2 ROD
4a1
4a2
4a3
4a4
4a5
4a6
4a7
4a8
4a9
4b1
4b2
4b3
4b4
4b5
4b6
4b7
4b8
NVPd
DLVd
EFVd
AZTd
H
H
H
H
H
H
H
H
4-OCH3
4-Cl
4-Br
4-CH3
4-NO2
3-OCH3, 4-OCH3
3-F, 4-F
3-Cl, 4-Cl
3-Cl, 4-F
4-OCH3
4-Cl
4-Br
4-NO2
3-OCH3, 4-OCH3
3-F, 4-F
3-Cl, 4-Cl
3-Cl, 4-F
0.92 0.16
0.95 0.11
0.74 0.08
0.88 0.28
2.07 1.29
4.48 1.62
>273.95
>25.21
>24.80
>22.84
>47.43
25.52 2.75
24.80 2.35
22.84 1.96
>or = 47.43
>268.64
165.67 11.22
>273.95
19.59 1.23
>264.41
10.84 7.00
>266.65
>243.56
>261.49
>252.84
>265.78
>248.40
>256.79
>15.02
>3.83
>6.34
>93.55
28
23
31
>or = 54
>130
28
>268.64
>165.67
>273.95
>19.58
>264.41
>10.84
>266.65
>243.56
P45.80
>252.84
>265.78
>248.40
>256.79
X1
>or = 84.63
>264.41
H
<1
4
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
2.74 3.00
0.32 0.11
0.18 0.06
0.99 0.27
1.45 1.13
0.69 0.02
P2.82
P41.09
0.21
0.32
0.0044
>836
>1326
>264
>175
>387
>or X88
X1
>72
>12
>1434
>6192
0.015
a
b
c
EC50: concentration of compound required to achieve 50% protection of MT-4 cells against HIV-induced cytotoxicity, as determined by the MTT method.
CC50: concentration required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.
SI: selectivity index (CC50/ EC50). The SI values: X1 stand for P1 or <1.
d
The antiviral properties of these compounds were previously described.18
substituents at the anilide moiety was as follows: 4-Br > 4-Cl > 3-F,
4-F > 4-NO2 > 3-OCH3, 4-OCH3 > 4-OCH3.
Lys103 backbone. Lengthening the C-2 side chain led the C-2 side
chain to go beyond the opening defined by Pro225 and Pro236.
Thus, the end of C-2 side chain was exposed to the solvent, causing
the lack of its hydrophobic interactions with the NNIBP. The 2,6-
In addition, all the title compounds were also evaluated for their
capability to inhibit the HIV-2 (strain ROD) replication in MT-4
cells, but none was found effective (Table 1). These findings
showed that this new series of S-DABOs was specific for HIV-1
and belonged to typical NNRTIs.
2.3. Molecular modeling analysis
With the aim to investigate the binding mode of our newly syn-
thesized compounds, molecular modeling study was performed by
means of Autodock Vina for docking. Compound 4b3 was chosen to
be docked into the NNRTIs binding pocket (NNIBP) of HIV-1 RT.
Three-dimensional coordinates of the HIV-1 RT/6-benzyl-1-
(benzyloxymethyl)-5-isopropylpyrimidine-2,4(1H,3H)-dione (TNK
-651) complex (Brookhaven Protein Data Bank entry 1RT2) were
used as the input structure for docking calculations because of
the high degree of similarity between TNK-651 and DABOs.19 De-
fault parameters were used as described in the Autodock Vina
manual unless otherwise specified. The theoretical binding mode
of 4b3 to the NNIBP is shown in Figure 2.
The docking simulation showed the binding mode of the 4b3
into the NNIBP. Results showed that the pyrimidine NH moiety at
position 3 was engaged in a hydrogen bond with the C@O moiety
of Lys101. The 2-phenylaminocarbonylmethylthio substituent
was well accommodated in the large pocket mainly defined by
Val106, Pro225, Pro236, and Phe227. A hydrogen bond is formed
between the C@O group of the C-2 side chain and NH group of
Figure 2. Model of 4b3 docked into the RT non-nucleoside binding site (PDB code:
1RT2) using Autodock Vina [http://vina.scripps.edu]. The docking result of 4b3 is
showed by PyMOL [http://pymol.sourceforge.net].

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M. Yu et al. / Bioorg. Med. Chem. 17 (2009) 7749–7754
dichlorobenzyl substituent at position 6 of the pyrimidinone ring
accommodated in a hydrophobic pocket mainly defined by the aro-
matic side chains of Tyr181, Tyr188, Phe227, and Trp229 as well as
by Leu234. In particular, the phenyl ring interacts favorably with
25 °C, and the resulting clear mixtures were stirred for a further
20 min. The organic layer was separated and concentrated, and
the residue was treated with EtOAc (200 mL). The aqueous layer
was extracted with EtOAc (3 ꢀ 150 mL), and the combined organic
layers were washed with saturated NaHCO3 (3 ꢀ 350 mL) and
brine (3 ꢀ 350 mL), dried (Mg2SO4), filtered and concentrated to
give the crude products 2a and 2b, respectively, which were di-
rectly used in the following step without further purification.
the Tyr188 side chain, giving rise to a positive p-stacking interac-
tion. Moreover, the methyl group at C-5 position was positioned in
the hydrophobic cavity formed by the Val179 side chains.
In summary, the results of the AutoDocking analysis supported
our newly designed and synthesized 2-(phenylaminocarbonylm-
ethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones. Further
optimization of 4b3 will consider these aspects in further design
attempts.
4.1.2. General procedure for the preparation of 5-alkyl-6-
substituted thiouracil 3a, 3b
Sodium metal (8.2 g, 356 mmol) was dissolved in 50 mL of
absolute ethanol, and thiourea (19 g, 249 mmol) and b-ketoesters
2a, 2b (178 mmol) were added to the clear solution at room tem-
perature. The reaction mixture was refluxed for 10–12 h (checked
by TLC) under a nitrogen atmosphere. The reaction mixture was
cooled to room temperature. Then, solvent was evaporated and
the residues were dissolved in H2O and were precipitated by addi-
tion of concd aq HCl and subsequent acidification to pH 4 with gla-
cial AcOH. The resulting precipitate was filtered under reduced
pressure. The solid was washed sequentially with H2O, EtOH, and
Et2O, then dried to give 3a, 3b, which is directly used in the next
step without further purification.
3. Conclusions
In summary, we designed and synthesized a series of novel 2-
(substituted phenylaminocarbonylmethylthio)-6-(2,6-dichlorob-
enzyl)-pyrimidin-4(3H)-ones (S-DABOs), which were structurally
confirmed by IR, 1H NMR and MS spectral analysis and evaluated
for their anti-HIV (HIV-1 IIIB and HIV-2 ROD) activities by inhibi-
tion of HIV-induced cytopathogenicity in MT-4 cell cultures.
Some of the new compounds displayed anti-HIV-1 activity in
the submicromolar range along with low cytotoxicity. Among
them, the most potent HIV-1 inhibitor was 4b3 (EC50 = 0.18
4.1.3. General procedure for the preparation of target
compounds 4a1–4a9, 4b1–4b8
0.06 lM), which was more effective than the reference drugs
nevirapine and efavirenz. The preliminary SAR among the newly
synthesized congeners and docking studies provided useful indica-
tions for guiding the further rational design of new S-DABO ana-
logues as more active and selective HIV-1 inhibitors.
Compounds 3a, 3b (5 mmol) and appropriate N-phenylacet-
amide halides (5.5 mmol) were suspended in anhydrous DMF
(25 mL) in the presence of anhydrous potassium carbonate
(0.759 g, 5.5 mmol) at room temperature. The mixtures were irra-
diated at room temperature for 12 h. The reaction mixture was
poured into cold H2O (200 mL), the resulting precipitate was col-
lected by filtration under reduced pressure and washed sequen-
tially with H2O, EtOH and Et2O and then dried to give the
corresponding crude product, which was purified by crystallization
to give the pure target compounds 4a1–4a9, 4b1–4b8.
4. Experimental section
4.1. Chemistry
All melting points were determined on a micromelting point
apparatus and are uncorrected. Infrared spectra (IR) were recorded
with a Nexus 470FT-IR Spectrometer. NMR spectra were obtained
on a Brucker Avance-600 NMR-spectrometer in the indicated sol-
vents. Chemical shifts are expressed in d units and TMS as internal
reference. Mass spectra were taken on a LC Autosampler Device:
Standard G1313A instrument. TLC was performed on silica gel
GF254 for TLC (Merck) and spots were visualized by iodine vapors
or by irradiation with UV light (254 nm). Solvents were reagent
grade and, when necessary, were purified and dried by standard
methods. Concentration of the reaction solutions involved the
use of rotary evaporator at reduced pressure.
4.1.3.1. 2-[(4-Methoxyphenylamino)carbonylmethylthio]-6-
(2,6-dichlorobenzyl)-pyrimidin-4(3H)-one (4a1). Recrystallized
from EtOH/DMF as a white crystal, Yield: 25.4%, mp: 238–240 °C
(dec). 1H NMR (DMSO-d6, ppm) d: 12.80 (s, 1H, NH), 10.08 (s, 1H,
NH), 7.45–7.29 (m, 7H), 5.41 (s, 1H, CH@C@O), 4.05 (s, 2H, S-
CH2), 3.99 (s, 2H, CH2), 3.69 (s, OCH3, 3H); IR (KBr, cmꢁ1): 3276
(mNH), 3075 (mNH), 1661 (mC@O), 1247 (mC–N). ESI-MS: m/z 450.6
(M+1), 472.7 (M+Na). C20H17Cl2N3O3S (449.04).
4.1.3.2. 2-[(4-Chlorophenylamino)carbonylmethylthio]-6-(2,6-
dichlorobenzyl)-pyrimidin-4(3H)-one (4a2). Recrystallized from
EtOH–DMF as a white crystal, Yield: 23.6%, mp: 223–225 °C
(dec). 1H NMR (DMSO-d6, ppm) d: 12.77 (s, 1H, NH), 10.37 (s, 1H,
NH), 7.57–7.24 (m, 7H), 5.44 (s, 1H, CH@C@O), 4.00 (s, 2H, S-
4.1.1. General procedure for the preparation of b-ketoesters 2a
and 2b
To a well stirred solution of substituted diethyl malonate
(500 mmol) in anhydrous EtOH (400 mL) was added dropwise a
solution of KOH (28 g, 500 mmol) in EtOH (400 mL) at room tem-
perature over 4 h. Then the resulting mixture was allowed to stand
overnight until the pH of the final mixture had a value between 7
and 8. After removing the solvent, the residue was washed with a
small amount of ether and suspended in anhydrous CH3CN
(800 mL), Et3N (106 mL, 761 mmol) and MgCl2 (57 g, 595 mmol)
were added and the mixtures were stirred at room temperature
for 2 h. Then were added the solutions of arylacetyl imidazolide,
prepared 15 min before by reaction between 2-(2,6-dichloro-
phenyl)acetic acid (49 g, 238 mmol) and N,N0-carbonyldiimidazole
(CDI, 46 g, 286 mmol) in CH3CN (400 mL). The reaction mixtures
were stirred overnight at room temperature and then refluxed
for 2 h. After the mixture was cooled, a solution of 13% HCl
(800 mL) was added slowly while the temperature was kept below
CH2), 3.95 (s, 2H, CH2). IR (KBr, cmꢁ1): 3317 (
1657 ( C@O), 1240 ( C–N), 1206 (
mNH), 3052 (mNH),
m
m
mC–N). ESI-MS: m/z 456.3 (M+1),
478.3 (M+Na). C19H14Cl3N2S2O (454.96).
4.1.3.3. 2-[(4-Bromophenylamino)carbonylmethylthio]-6-(2,6-
dichlorobenzyl)-pyrimidin-4(3H)-one (4a3). Recrystallized from
EtOH–DMF as a white crystal, Yield: 23.1%, mp: 232–233 °C
(dec). 1H NMR (DMSO-d6, ppm) d: 12.82 (s, 1H, NH), 10.34 (s, 1H,
NH), 7.54 (d, J = 9.0 Hz, 2H), 7.49 (d, J = 9.0 Hz, 2H), 7.44 (d,
J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, H), 5.44 (s, 1H, CH@C@O), 4.03
(s, 2H, S-CH2), 3.97 (s, 2H, CH2); IR (KBr, cmꢁ1): 3320 (
NH), 1656 ( C@O), 1239 ( C–N), 1206 ( C–N). ESI-MS: m/z 498.3
(M+1), 520.2 (M+Na). C19H14BrCl2N3O2S (496.94).
mNH), 3051
(m
m
m
m

M. Yu et al. / Bioorg. Med. Chem. 17 (2009) 7749–7754
7753
4.1.3.4. 2-[(4-Methylphenylamino)carbonylmethylthio]-6-(2,6-
dichlorobenzyl)-pyrimidin-4(3H)-one (4a4). Recrystallized from
EtOH–DMF as a white crystal, Yield: 26.7%, mp: 235–237 °C
(dec). 1H NMR (DMSO-d6, ppm) d: 12.84 (s, 1H, NH), 10.11 (s, 1H,
NH), 7.45–7.12 (m, 7H), 5.45 (s, 1H, CH@C@O), 4.05 (s, 2H, S-
CH2), 3.95 (s, 2H, CH2), 2.25 (S, 3H, CH3); IR (KBr, cmꢁ1): 3287
4.1.3.11. 2-[(4-Chlorophenylamino)carbonylmethylthio]-6-(2,6-
dichlorobenzyl)-5-methylpyrimidin-4(3H)-one (4b2). Recrystal-
lized from EtOH–DMF as a white crystal, Yield: 27.6%, mp: 256–
258 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.71 (s, 1H, NH), 10.00
(s, 1H, NH), 7.53 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.24
(d, J = 7.8 Hz, 2H), 7.04 (d, J = 7.8 Hz, H), 4.11 (s, 2H, S-CH2), 3.72
(m
NH), 3034 (
m
NH), 1663 (
m
C@O), 1241 (
m
C–N). ESI-MS: m/z 434.7
(s, 2H, CH2), 2.04 (s, CH3, 3H); IR (KBr, cmꢁ1) 3327 (
NH), 1660 ( C@O), 1260 ( C–N), 1243 ( C–N). ESI-MS: m/z 468.5
(M+1). C20H16Cl3N3O2S (467).
mNH), 3053
(M+1), 456.5 (M+Na). C20H17Cl2N3O2S (433.34).
(m
m
m
m
4.1.3.5. 2-[(4-Nitrophenylamino)carbonylmethylthio]-6-(2,6-
dichlorobenzyl)-pyrimidin-4(3H)-one (4a5). Recrystallized from
EtOH–DMF as a white crystal, Yield: 29.6%, mp: 227–229 °C.
1H NMR (DMSO-d6, ppm) d: 12.78 (s, 1H, NH), 10.82 (s, 1H, NH),
7.56 (dd, J1 = 9.6 Hz, J2 = 2.4 Hz, 2H), 7.8 (dd, J1 = 9.6 Hz, J2 = 2.4 Hz,
2H), 7.36 (d, J = 7.8 Hz, 2H), 7.23 (d, J = 7.8 Hz, H), 5.52 (s, 1H,
4.1.3.12. 2-[(4-Bromophenylamino)carbonylmethylthio]-6-(2,6-
dichlorobenzyl)-5-methylpyrimidin-4(3H)-one (4b3). R. ecrystal-
lized from EtOH–DMF as a white crystal, Yield: 29.2%, mp: 259–
261 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.70 (s, 1H, NH), 9.99
(s, 1H, NH), 7.49–7.04 (m, 7H), 4.11 (s, 2H, S-CH2), 3.72 (s, 2H,
CH@C@O), 4.08 (s, 2H, S-CH2), 4.02 (s, 2H, CH2). IR (KBr, cmꢁ1
3323 ( NH), 3077 ( NH), 1655 ( C@O), 1241 ( C–N), 1205 ( C–N). ESI-
MS: m/z 465.5 (M+1). C19H14Cl2N4O4S (464.01).
)
CH2), 2.04 (s, CH3, 3H); IR (KBr, cmꢁ1): 3307 (
1656 ( C@O), 1256 ( C–N), 1242 (
C20H16BrCl2N3O2S (510.95).
mNH), 3051 (mNH),
m
m
m
m
m
m
m
mC–N). ESI-MS: m/z 514.5 (M+3).
4.1.3.6. 2-[(3,4-Dimethoxyphenylamino)carbonylmethylthio]-
6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-one (4a6). Recrystal-
4.1.3.13. 2-[(4-Nitrophenylamino)carbonylmethylthio]-6-(2,6-
dichlorobenzyl)-5-methylpyrimidin-4(3H)-one (4b4). Recrystal-
lized from EtOH–DMF as a white crystal, Yield: 24.8%, mp: 250–
252 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.76 (s, 1H, NH), 10.47
(s, 1H, NH), 8.25–6.97 (m, 7H), 4.10 (s, 2H, S-CH2), 3.79 (s, 2H,
lized from EtOH–DMF as a white crystal, Yield: 22.6%, mp: 221–
223 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.80 (s, 1H, NH), 10.03
(s, 1H, NH), 7.51–6.86 (m, 6H), 5.47 (s, 1H, CH@C@O), 4.08 (s, 2H,
S-CH2), 4.02 (s, 2H, CH2), 3.72 (2 ꢀ OCH3, 6H); IR (KBr, cmꢁ1
)
CH2), 2.03 (s, CH3, 3H); IR (KBr, cmꢁ1): 3412 (
1649 ( C@O), 1620 ( C@O), 1331 ( C–N), 1307 (
m
NH), 3085 (mNH),
3292 (mNH), 3136 (mNH), 1664 (mC@O), 1231.65 (mC–N), 1218 (mC–N).
m
m
m
mC–N). ESI-MS: m/z
ESI-MS: m/z 482.4 (M+3). C21H19Cl2N3O4S (479.05).
479.2 (M+1), 501.2 (M+Na). C20H16Cl2N4O4S (478.03).
4.1.3.7. 2-[(3,4-Difluorophenylamino)carbonylmethylthio]-6-
(2,6-dichlorobenzyl)-pyrimidin-4(3H)-one (4a7). Recrystal-
4.1.3.14. 2-[(3,4-Dimethoxyphenylamino)carbonylmethylthio]-
6-(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one
(4b5).
lized from EtOH–DMF as a white crystal, Yield: 24.2%, mp: 228–
230 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.78 (s, 1H, NH), 10.45
(s, 1H, NH), 7.74–7.28 (m, 6H), 5.52 (s, 1H, CH@C@O), 4.05 (s, 2H,
Recrystallized from EtOH–DMF as a white crystal, Yield: 52.3%,
mp: 246–249 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.68 (s, 1H,
NH), 9.75 (s, 1H, NH), 7.29–6.89 (m, 6H), 4.13 (s, 2H, S-CH2),
3.73–3.72 (2 ꢀ OCH3, 6H), 3.67 (s, 2H, CH2), 2.04 (s, CH3, 3H); IR
S-CH2), 3.93 (s, 2H, CH2); IR (KBr, cmꢁ1): 3297 (
m
NH), 3085 (mNH),
1659 (mC@O), 1241 (mC–N), 1210 (mC–N). ESI-MS: m/z 456.5 (M+1),
478.5 (M+Na). C19H13Cl2F2N3O2S (455.01).
(KBr, cmꢁ1): 3272 (
1262 ( C–N), 1234 (mC–N). ESI-MS: m/z 494.3 (M+1), 516.3 (M+Na).
mNH), 3050 (mNH), 1671 (mC@O), 1651 (mC@O),
m
C22H21Cl2N3O4S (493.06).
4.1.3.8. 2-[(3,4-Dichlorophenylamino)carbonylmethylthio]-6-
(2,6-dichlorobenzyl)-pyrimidin-4(3H)-one (4a8). Recrystallized
from EtOH–DMF as a white crystal, Yield: 26.8%, mp: 236–238 °C
(dec) 1H NMR (DMSO-d6, ppm) d: 12.87 (s, 1H, NH), 10.50 (s, 1H,
NH), 7.95 (d, J = 2.4 Hz, H), 7.58 (d, J = 8.4 Hz, H), 7.45 (dd,
J1 = 8.4 Hz, J2 = 2.4 Hz, H), 7.42 (d, J = 7.8 Hz, 2H), 7.25 (d,
J = 7.8 Hz, 1H), 5.44 (s, 1H, CH@C@O), 4.04 (s, 2H, S-CH2), 3.95 (s,
4.1.3.15. 2-[(3,4-Difluorophenylamino)carbonylmethylthio]-6-
(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one (4b6) . Recrys-
tallized from EtOH–DMF as a white crystal, Yield: 29.6%, mp:
247–250 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.71 (s, 1H, NH),
10.10 (s, 1H, NH), 7.68–7.03 (m, 6H), 4.11 (s, 2H, S-CH2), 3.72 (s,
2H, CH2), 2.04 (s, CH3, 3H); IR (KBr, cmꢁ1): 3313 (
1675 ( C@O), 1647 ( C@O), 1257 ( C–N), 1206 (
m
NH), 3060 (
mNH),
2H, CH2); IR (KBr, cmꢁ1): 3291 (
1234 ( C–N), 1206 ( C–N). ESI-MS: m/z 488.3 (M+1), 510.1 (M+Na).
mNH), 3091 (mNH), 1660 (mC@O),
m
m
m
mC–N). ESI-MS: m/z
m
m
470.3 (M+1), 492.1 (M+Na). C20H15 Cl2F2N3O2S (469.02).
C19H13Cl4N3O2S (486.95).
4.1.3.16. 2-[(3,4-Dichlorophenylamino)carbonylmethylthio]-6-
(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one (4b7). Recrys-
tallized from EtOH–DMF as a white crystal, Yield: 25.7%, mp:
256–258 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.74 (s, 1H, NH),
10.14 (s, 1H, NH), 7.87 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 9 Hz, 1H),
7.38 (dd, J1 = 9 Hz, J2 = 1.8 Hz, 1H), 7.21 (d, J = 7.8 Hz, 2H), 7.02 (d,
J = 7.8 Hz, 1H), 4.11 (s, 2H, S-CH2), 3.73 (s, 2H, CH2), 2.04 (s, CH3,
4.1.3.9. 2-[(3-Chloro-4-fluorophenylamino)carbonylmethylthio]-
6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-one
(4a9). Recrystal-
lized from EtOH–DMF as a white crystal, Yield: 21.8%, mp: 237–
239 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.75 (s, 1H, NH), 10.41
(s, 1H, NH), 7.87–7.25 (m, 6H), 5.50 (s, 1H, CH@C@O), 4.02 (s, 2H,
S-CH2), 3.93 (s, 2H, CH2); IR (KBr, cmꢁ1): 3290 (
m
NH), 3042 (mNH),
1658
(mC@O), 1221 (mC–N). ESI-MS: m/z 472.6 (M+1), 494.6
3H); IR (KBr, cmꢁ1): 3283 (
C@O), 1257 C–N), 1236
C20H15Cl4N3O2S (500.96).
m
NH), 3040 (mNH), 1673 (mC@O), 1646
(M+Na). C19H13Cl3FN3O2S (470.98).
(m
(m
(mC–N). ESI-MS: m/z 504.3 (M+3).
4.1.3.10. 2-[(4-Methoxyphenylamino)carbonylmethylthio]-6-
(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one (4b1). Recrys-
tallized from EtOH–DMF as a white crystal, Yield: 54.0%, mp: 248–
251 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.71 (s, 1H, NH), 9.72 (s,
1H, NH), 7.40–6.68 (m, 7H), 4.05 (s, 2H, S-CH2), 3.99 (s, 2H, CH2),
4.1.3.17. 2-[(3-Chloro-4-fluorophenylamino)carbonylmethyl-
thio]-6-(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one (4b8).
Recrystallized from EtOH–DMF as a white crystal, Yield: 26.6%, mp:
259–261 °C (dec). 1H NMR (DMSO-d6, ppm) d: 12.68 (s, 1H, NH),
10.06 (s, 1H, NH), 7.96–7.02 (m, 6H), 4.11 (s, 2H, S-CH2), 3.72 (s,
2.04 (s, CH3, 3H); IR (KBr, cmꢁ1): 3323 (
C@O), 1248
C21H19Cl2N3O3S (463.05).
m
NH), 3041 (mNH), 1654
(m
(mC–N). ESI-MS: m/z 464.5 (M+1), 502.6 (M+K).
2H, CH2), 2.04 (s, CH3, 3H); IR (KBr, cmꢁ1): 3294 (
mNH), 3052

7754
M. Yu et al. / Bioorg. Med. Chem. 17 (2009) 7749–7754
(
m
NH), 1673 (
m
C@O), 1648 (
m
C@O), 1260 (
m
C–N), 1221 (
m
C–N). ESI-MS:
Cooperation, Ministry of Science and Technology of China
(2003DF000033) and Research Fund for the Doctoral Program of
Higher Education of China (070422083). We are grateful to Kristien
Erven and Kris Uyttersprot for technical assistance with the antivi-
ral experiments.
m/z 488.2 (M+3). C20H15Cl3FN3O2S (484.99).
4.2. Anti-HIV activity assays
Evaluation of the antiviral activity of the compounds against
HIV-1 strain IIIB and HIV-2 strain (ROD) in MT-4 cells was per-
formed using the MTT assay as previously described.20,21 Stock
solutions (10 ꢀ final concentration) of test compounds were added
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Research work in the authors’ laboratory has been supported by
the National Natural Science Foundation of China (NSFC Nos.
30371686, 30772629, 30873133), Key Project of The International