Synthesis and anti-HIV-1 activity of S-dihydro(alkyloxy)benzyloxypyrimidine derivatives

Article abstract of DOI:10.1007/s00706-007-0834-8

Several 2-heteroaryl-, 2-heteroarylcarbonylmethyl-, 2-arylcarbonylmethyl, and 2-arylethyl derivatives of S-dihydro(alkyloxy)benzyloxypyrimidines have been synthesized and the anti-HIV activities of these compounds were tested in C8166 cell and against RT

Full text of DOI:10.1007/s00706-007-0834-8

Monatsh Chem 139, 967–974 (2008)
DOI 10.1007/s00706-007-0834-8
Printed in The Netherlands
Synthesis and anti-HIV-1 activity of S-dihydro(alkyloxy)benzyloxypyrimidine
derivatives
Zhi-Kun Rao1;y, Jing Long2;y, Cong Li1, Sui-Shuan Zhang1, Mei He1, Ling-Cheng Ou1,
Yong-Tang Zheng2;ꢀ, Yan-Ping He1;ꢀ
1
Key Laboratory of Medicinal Chemistry for Natural Resource Ministry of Education, School of Chemical
Science and Technology, Yunnan University, Kunming, People’s Republic of China
2
Laboratory of Molecular Immunopharmacology, Key Laboratory of Animal Models and Human Disease
Mechanisms, Chinese Academy of Sciences, Kunming Institute of Zoology, Kunming, People’s Republic of China
Received 10 August 2007; Accepted 23 October 2007; Published online 9 June 2008
# Springer-Verlag 2008
Abstract Several 2-heteroaryl-, 2-heteroarylcarbonyl-
methyl-, 2-arylcarbonylmethyl, and 2-arylethyl deri-
vatives of S-dihydro(alkyloxy)benzyloxypyrimidines
have been synthesized and the anti-HIV activities
of these compounds were tested in C8166 cell and
against RT enzyme. It was found that some of these
compounds showed good activity against HIV-1
(EC50 ¼ 0.014–0.8ꢀM) with low toxicity (CC50 val-
ue of 222–564 ꢀM) and high selectivity (SI value
of 278–37743). The structure-activity relationships
(SAR) of these compounds have also been discussed.
vention (CDC) in June 1981. Since then, it has been
widely spread in the world and seriously affect
healthy conditions of human being, HIV-1 reverse
transcriptase (RT) [1] played a critical role in the
process of HIV replication and has been identified
as one of the main targets for anti-HIV drug discov-
ery [2]. Two classifications of RT inhibitors have
been studied: nucleoside reverse transcriptase inhi-
bitors (NRTIs) and non-nucleoside transcriptase
inhibitors (NNRTIs). NNRTIs, being less toxic than
NRTIs, had received great attention [3–5]. Three
NNRTIs nevirapine [6], delavirdine [7], and efavir-
enz [8] have been approved by FDA. Not only being
effective RT inhibitors, these three NNRTIs also
acted as the key components of the combination
therapy [9–11]. However, NNRTIs can easily induce
drug-resistant variants of HIV-1, which have also
been seen in the treatment with other chemothera-
peutic agents. New anti-HIVagents with novel struc-
tures or mechanism(s) of action are still in demand
[12, 13].
Keywords NNRTIs; S-DABOs; Antiviral activity; HIV-1.
Introduction
Acquired immunodeficiency syndrome (AIDS) was
identified by Centers for Disease Control and Pre-
y
These authors contributed equally to this work
Correspondence: Yan-Ping He, Key Laboratory of Medicinal
Chemistry for Natural Resource Ministry of Education,
School of Chemical Science and Technology, Yunnan Univer-
sity, Kunming 650091, People’s Republic of China. E-mail:
yphe@ynu.edu.cn; Yong-Tang Zheng, Laboratory of Molec-
ular Immunopharmacology, Key Laboratory of Animal Mod-
els and Human Disease Mechanisms, Chinese Academy of
Sciences, Kunming Institute of Zoology, Kunming, Yunnan
650223, People’s Republic of China. E-mail: zhengyt@
mail.kiz.ac.cn
We have previously developed a series of 6-
naphthylmethyl substituted 2-(alkylthio)-5-alkyl-6-
arylmethyl-3,4-dihydro-4-oxopyrimidines (S-dihydro-
(alkyloxy)benzyloxypyrimidines) [14], characterized
by the presence of a ꢁ-carbonyl group on the C-2
side chain (1, Fig. 1) [15, 16]. These compounds
exhibited significant anti-HIV activity and more in-

968
Z. Rao et al.
Fig. 1 Structures of S-DABOs
teresting, they might interfere with a target that dif-
fered from HIV RT, or act on RT in a way that is
different from typical NNRTIs. To further optimize
the structure of S-DABO derivatives and improve
the anti-HIV functionality, we designed two series
of oxopyrimidine derivatives 2 based on previously
investigated scaffold 1 (Fig. 1). These novel chemi-
cal entities were characterized by (i) introduction of
a heteroaromatic ring at the end of C-2 side chain
(2a–2n); (ii) the presence of an arylalkylthio substit-
uent at position 2 (2m–2v) to investigate the impor-
tance of the ꢁ-carbonyl group; (iii) replacement of
the fused naphthylmethyl at the C-6 position present
in 1 with the phenylmethyl moiety, which might fa-
vor the entrance of intact molecular structure to the
binding pocket of the target due to the increasing
flexibility. Herein, we report the synthesis, antiviral
activity evaluation, and the structure activity rela-
tionships of these compounds.
uracil 5a–5f, which were subjected to S-alkylation
in anhydrous DMF with appropriate halide(R1X)
in the presence of K2CO3 to afford the desirable S-
DABO analogues 2a–2v (Scheme 1). Structures of
these compounds were determined by mass spectral,
1H NMR and 13C NMR data. A single crystal
X-ray structure analysis was also performed for 2j
(Fig. 2).
Antiviral activity
The novel S-DABO derivatives (2a–2v) were tested
for their cytotoxicities and anti-HIV-1 activities in
C8166 cells infected by the HIV-1IIIB and compared
with AZT. The capability of these S-DABO deriva-
tives to inhibit HIV-1 RT was also measured by using
homopolymeric template primers. The activity data
were interpreted in CC50 values (cytotoxicities),
EC50 (anti-HIV-1 activities), SI (selectivity, given
by the CC50=EC50 ratio) and IC50 (RT inhibitory
activity) values (Table 1). In addition, the previously
reported 7–9 [15] were included in our assays for
comparison.
Results and discussion
Chemistry
The results of cellular assays showed (Table 1) that
the majority of the S-DABO derivatives, except for
2o, 2t, 2u, and 2v, exhibited anti-HIV-1 activities in the
low micromolar to submicromolar ranges. The cyto-
toxicity of most of these compounds against C8166
cells was at the dose of 194 ꢀM. In comparision, 2g,
2o, and 2t displayed slightly higher toxicity with
the CC50 value ranging from 88 to 112 ꢀM.
The synthetic route of novel S-DABO derivatives
2a–2v is outlined in Scheme 1. Following the pro-
cedure described previously [17, 18], the 3-oxo ester
4a–4f was prepared by reacting 1-naphthylacetoni-
trile or phenylacetonitrile with ethyl-2-bromoalkano-
ates 3a–3c in the presence of activated zinc dust in
refluxing THF for 8 h. Condensation of 4a–4f with
thiourea in the presence of sodium ethoxide in re-
fluxing ethanol afforded the key intermediate 2-thio-
In the series, 6-benzyl-5-isopropyl-2-[(phenyl-
carbonylmethyl)thio]pyrimidin-4(3H)-one (2c) was

Synthesis and anti-HIV-1 activity of S-DABOs
969
Scheme 1
the most promising compound. It exhibited ex-
tremely potent inhibitory activity against HIV-1 rep-
lication (EC50 ¼ 0.014 ꢀM) and highest selectivity
(SI >37743), which is somewhat better than those
found for AZT. Besides 2c, 2b, 2e, and 2j also
prevented the cytopathic effect of HIV-1IIIB at low
concentrations (EC50 ¼ 0.08, 0.048, and 0.095 ꢀM,
respectively) and were minimally toxic to C8166
cells (CC50>549, 507, and 543 ꢀM) resulting in a
remarkably high selectivity indices (SI >6859, 10560,
and 5714, respectively).
Using poly(rC) ligo(dG) as the template primer,
many of these analogues exhibited inhibitory activity
against HIV-1RT. Compounds with high activities in
cell assay also endowed with favorable activity in
enzyme analysis. For example, 2c, 2b, 2i, and 2j
displayed anti-HIV-1 activity in the range of micro-
molar concentration (IC50 ¼ 0.35–0.66 ꢀM). Howev-
er, as showed in Table 1, the IC50 values in enzyme
assay were 1–2 orders higher than the EC50 values in
cell assay. This might because these compounds also
interfered with another target except for HIV-1 RT.

970
Z. Rao et al.
The biological activity data (Table 1) indicated that
the anti-HIV-1 activity was strongly dependent on the
nature of the substituent at C-2, C-5, and C-6 of the
pyrimidin ring. In general, the 6-benzyl substituted
derivatives (2c, 2b, 2e, and 2i) were 10–20 fold more
active than the 6-napthylmethyl counterparts (8, 9,
7, and 2n) with the only exception of 2l, which was
about 9-fold more active than the corresponding 6-
benzyl derivative 2g.
It was also observed that the activities of these
novel S-DABO derivatives were closely related to
the length of the C-2 side chain. These C-2 substituent
groups having suitable length, such as PhCOCH2,
(40-OCH3)PhCOCH2, and 2-furanyl-COCH2, were
beneficial to the anti-HIV-1 activity. In contrast,
the 2-thiophen derivatives were almost inactive (2t,
2u, and 2v). Concerning the SAR of C-2 side chain
was concerned, the PhCOCH2 derivatives (2b, 2c,
and 9) were more potent than their 2-furanyl-
COCH2 counterparts (2i, 2j, and 2n). In the
6-benzyl series, the 2-furanyl-COCH2ꢁ derivative
2i was 3.3 folds more potent than its 2-thienyl-
COCH2 counterpart 2g. Otherwise, in the 6-nap-
thylmethyl series, the 2-furanyl-COCH2 derivative
2k and its 2-thienyl-COCH2ꢁ counterpart 2m ex-
hibited similar activities. When the C-2-ꢁ-carbonyl
was replaced with a methylene, the activity decreased
significantly (2q was 3-fold less active than 9 and 2r
was 25-fold less active than 8). These results showed
that the C-2-ꢁ–carbonyl is a crucial factor to improve
the activity. Based on a molecular modeling study, the
C-2 side chain was adjacent to amino acid residue
Try318 of HIV RT enzyme. We speculated that the
enhanced activity might be related to the formation of
hydrogen bond between C-2-ꢁ-carbonyl and the am-
ide of the main chain of Try318. The detailed SAR
analysis will be reported elsewhere.
Finally, the activity of S-DABO derivatives in-
creased proportionally to the modication of the C-5
substitution in the order of Me ! Et ! i-Pr, which
was also shown in previous studies [15]. One excep-
tion here was the pairs 2e, 2f, and 2q, 2r, wherein the
5-ethyl derivatives were 3–4 fold more active.
In conclusion, we discovered a series of HIV inhi-
bitors with high activities. The EC50 values to HIV-1 of
four compounds in the series, 2b, 2c, 2e, and 2j, were
at the range of submicromolar and all showed low
toxicity and high selectivity exponents. To explore
the use of these compounds as anti-HIV-1 candidates,
further biological studies are still in progress.
Fig. 2 X-Ray crystal structure of 2j
Table 1 Cytotoxicity and anti-HIV-1 activity of target com-
poundsa
Comp.
EC50=ꢀMb
CC50=ꢀMc
SId
IC50=ꢀMe
2a
2b
2c
2d
2e
2f
2g
2h
2i
0.80
0.08
0.014
1.47
0.048
0.144
5.39
0.28
0.16
0.095
1.92
0.62
1.82
1.43
20.9
5.40
1.79
7.11
1.04
43.3
14.1
24.1
0.427
0.280
0.579
0.017
222
278
1.85
0.35
0.66
1.10
1.06
1.46
17.8
37.8
0.45
0.43
1.86
9.67
5.99
17.5
449
517
499
482
549
509
548
528
>549
>528
>526
>507
>489
95.7
>6859
>37743
>357
>10560
>3399
17.7
194
679
>564
>543
>492
227
>512
201
112
>517
321
>482
>549
88.3
280
230
>450
>467
>482
4413
>3399
>5714
>256
367
>281
140
5.37
>95
179
>68
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
2u
2v
7
8
9
AZT
>5268
2.04
19.9
9.55
>1052
>1666
>833
260243
a
All data represent mean values for three separate
experiments
Concentration required to protect the cell against viral
cytopathogenicity by 50% in C8166 cells
Concentration that reduces the C8166 cell viability by 50%
Selectivity index ratio CC50=EC50
Concentration required to inhibit the HIV-1 rRT activity by
b
c
d
e
50%

Synthesis and anti-HIV-1 activity of S-DABOs
971
DMSO-d6): ꢂ ¼ 19.9 (2CH3), 27.4 (CH), 37.4 (CH2Ph), 37.9
(SCH2), 123.7 (C-5), 126.4–138.8 (12C, 2Ph), 156.1 (C-6),
159.2 (C-2), 163.0 (C-4), 193.60 (C¼O) ppm; MS (EI):
m=z ¼ 378 (Mþ).
Experimental
Melting points were determined on a XT-4 binocular micro-
scope melting point instrument. Infrared (IR) spectra were
recorded on an AVATAR FT-IR spectrometer as KBr pellets.
1H NMR and 13C NMR spectra were obtained on a Bruker
AM-500MHz spectrometer using DMSO-d6 as solvent.
Chemical shifts are reported in ꢂ (ppm) units relative to the
internal reference tetramethylsilane (TMS). Mass spectra were
obtained on an Agilent LC=MSD TOF mass spectrometer.
Regents and solvents were all analytical grade and were puri-
fied and dried by stands methods before use. All air-sensitive
reactions were run under an atmosphere of N2.
6-Benzyl-5-methyl-2-[(40-methoxyphenylcarbonylmethyl)-
thio]pyrimidin-4(3H)-one (2d, C21H20N2O3S)
Yield 45%; mp 162–163ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2925,
1
1674, 1643 cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.89
(s, 3H, CH3), 3.61 (s, 2H, SCH2), 3.83 (s, 3H, OCH3), 4.65 (s,
2H, CH2Ph), 7.01–7.99 (m, 9H, Harmo), 12.73 (s, br, s, 1H,
NH) ppm; 13C NMR (500 MHz, DMSO-d6): ꢂ ¼ 10.6 (CH3),
37.6 (CH2Ph), 38.6 (SCH2), 55.9 (OCH3), 110.2 (C-5), 114.3–
138.3 (12C, 2Ph), 59.8 (C-6), 161.3 (C-4), 163.8 (C-2), 191.8
(C¼O) ppm; MS (EI): m=z ¼ 380 (Mþ).
General procedure for preparation of derivatives 2a–2v
To a solution of 2-thiouracil 5 [17, 18] (2mmol) in anhydrous
DMF (8cm3) were added K2CO3 (2.2mmol) and halide (R1X)
(2.2mmol). The mixture was stirred at room temperature for
8–24 h. After TLC (EtOAc:PE) revealed the disappearance of
the starting material, the reaction mixture was filtered. The
filtrate was concentrated in vacuo, and CH2Cl2 (60–80cm3)
was added to the residue. The organic phase was washed with
H2O (2ꢂ50 cm3), dried over anhydrous Na2SO4 and evapo-
rated in vacuo to give the crude products 2a–2v. The crude
products were purified by column chromatography (eluent:
CH2Cl2:EtOAc:hexane). Selected analytical data for com-
pounds 2a–2v are presented below.
6-Benzyl-5-ethyl-2-[(40-methoxyphenylcarbonylmethyl)-
thio]pyrimidin-4(3H)-one (2e, C22H22N2O3S)
Yield 29%; mp 190–191ꢃC; FT-IR (KBr): ꢃꢀ¼ 3424, 2924,
1
1683, 1644 cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 0.88
(t, 3H, J ¼ 7.15Hz, CH3), 2.40 (q, J ¼ 7.25Hz, 2H, CH2), 3.62
(s, 2H, SCH2), 3.83 (s, 3H, OCH3), 4.65 (s, 2H, CH2Ph),
7.04–7.99 (m, 9H, Harmo), 12.68 (s, br, s, 1H, NH) ppm; 13C
NMR (500 MHz, DMSO-d6): ꢂ ¼ 13.4 (CH3), 18.5 (CH2), 37.6
(PhCH2), 39.4 (SCH2), 56.0 (OCH3), 116.2 (C-5), 114.3–
138.6 (12C, 2Ph), 156.5 (C-6), 160.8 (C-2), 163.8 (C-4),
193.5 (C¼O) ppm; HRMS: m=z ¼ 394.1366, calcd. 394.1351.
6-Benzyl-5-methyl-2-[(phenylcarbonylmethyl)thio]-
pyrimidin-4(3H)-one (2a, C20H18N2O2S)
6-Benzyl-5-isopropyl-2-[(40-methoxyphenylcarbonylmethyl)-
thio]pyrimidin-4(3H)-one (2f, C23H24N2O3S)
Yield 27%; mp 166–167ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2970,
Yield 51%; mp 185.5–186.5ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2924,
1
1673, 1654cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.89
1
1696, 1641 cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.11
(s, 3H, CH3), 3.57 (s, 2H, SCH2), 4.78 (s, 2H, CH2Ph), 6.96–
8.03 (m, 10H, Harmo), 12.75 (s, br, s, 1H, NH) ppm; 13C NMR
(500 MHz, DMSO-d6): ꢂ ¼ 10.7 (CH3), 37.9 (CH2Ph), 39.4
(SCH2), 116.2 (C-5), 126.5–138.3 (12C, 2Ph), 159.8 (C-6),
160.3 (C-4), 161.5 (C-2), 193.5 (C¼O) ppm; MS (EI): m=z ¼
350 (Mþ).
(d, 6H, J ¼ 6.60Hz, 2CH3), 3.02 (m, 1H, J ¼ 6.60Hz, CH),
3.64 (s, 2H, SCH2), 3.83 (s, 3H, OCH3), 4.65 (s, 2H, CH2Ph),
6.99–8.00 (m, 9H, Harmo), 12.59 (s, br, s, 1H, NH) ppm; 13C
NMR (500 MHz, DMSO-d6): ꢂ ¼ 19.9 (2CH3), 27.4 (CH),
37.5 (PhCH2), 39.5 (SCH2), 55.9 (OCH3), 110.7 (C-5),
114.3–138.9 (12C, 2Ph), 156.1 (C-6), 159.2 (C-2), 163.7
(C-4), 193.6 (C¼O) ppm; MS (EI): m=z ¼ 408 (Mþ).
6-Benzyl-5-ethyl-2-[(phenylcarbonylmethyl)thio]pyrimidin-
4(3H)-one (2b, C21H20N2O2S)
Yield 21%; mp 158–159ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2924,
6-Benzyl-5-ethyl-2-[(thiophen-2-ylcarbonylmethyl)thio]py-
rimidin-4(3H)-one (2g, C19H18N2O2S2)
1
1679, 1628cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 0.88
Yield 57%; mp 195–196ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2972,
(t, 3H, J ¼ 7.05Hz, CH3), 2.40 (q, J ¼ 7.20 Hz, 2H, CH2), 3.59
(s, 2H, SCH2), 4.70 (s, 2H, CH2Ph), 6.99–7.99 (m, 10H,
Harmo), 12.64 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 13.4 (CH3), 18.5 (CH2), 37.9 (CH2Ph), 39.4
(SCH2), 116.2 (C-5), 126.4–134.4 (12C, 2Ph), 156.5 (C-6),
160.8 (C-2), 163.6 (C-4), 193.5 (C¼O) ppm; MS (EI): m=z ¼
364 (Mþ).
1
1679, 1663 cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 0.89
(t, 3H, J ¼ 7.20Hz, CH3), 1.17 (q, J ¼ 7.10Hz, 2H, CH2), 3.19
(s, 2H, SCH2), 4.02 (s, 2H, CH2, phenyl), 7.02–8.26 (m, 8H,
Harmo), 12.65 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 13.4 (CH3), 18.8 (CH2), 37.2 (CH2Ph), 37.8
(SCH2), 116.2 (C-5), 124.4–144.4 (6C, Ph, 4C, thienyl), 156.5
(C-6), 160.8 (C-2) ppm; MS (EI): m=z ¼ 370 (Mþ).
6-Benzyl-5-isopropyl-2-[(phenylcarbonylmethyl)thio]-
pyrimidin-4(3H)-one (2c, C22H22N2O2S)
6-Benzyl-5-isopropyl-2-[(thiophen-2-ylcarbonylmethyl)thio]-
pyrimidin-4(3H)-one (2h, C22H20N2O2S2)
Yield 50%; mp 187.1–187.8ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2924,
1
Yield 20%; mp 187.1–187.8ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2953,
1696, 1641cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.09
1
1675, 1641 cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.19
(d, 6H, J ¼ 6.75Hz, 2CH3), 3.01 (m, 1H, J ¼ 6.75Hz, CH),
3.61 (s, 2H, SCH2), 4.69 (s, 2H, CH2Ph), 6.95–8.01 (m, 10H,
Harmo), 12.62 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
(d, 6H, J ¼ 7.35Hz, 2CH3), 3.11 (m, 1H, J ¼ 6.85Hz, CH),
3.28 (s, 2H, SCH2), 3.95 (s, 2H, CH2Ph), 7.14–7.33 (m, 8H,

972
Z. Rao et al.
Harmo), 12.69 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 19.4 (2CH3), 27.0 (CH), 37.0 (CH2Ph), 37.2
(SCH2), 116.2 (C-5), 123.6–134.3 (6C, Ph, 4C, thienyl), 156.2
(C-6), 160.4 (C-2), 162.8 (C-4), 192.7 (C¼O) ppm; MS (EI):
m=z ¼ 384 (Mþ).
(s, 3H, CH3), 4.15 (s, 2H, SCH2), 4.32 (s, 2H, CH2, naphthyl),
6.58–7.99 (m, 10H, Harmo), 12.78 (s, brs, 1H, NH) ppm;
13C NMR (500MHz, DMSO-d6): ꢂ ¼ 12.59 (CH3), 36.6
(CH2, naphthyl), 37.8 (SCH2), 110.2 (C-5), 113.0–151.5
(10C, naphthyl, 4C, furanyl), 159.9 (C-6), 160.3 (C-4),
161.6 (C-2), 182.0 (C¼O) ppm; MS (EI): m=z ¼ 390 (Mþ).
6-Benzyl-5-ethyl-2-[(furan-2-ylcarbonylmethyl)thio]pyrim-
idin-4(3H)-one (2i, C19H18N2O3S)
5-Ethyl-2-[(furan-2-ylcarbonylmethyl)thio]-6-(1-naphthyl-
methyl)pyrimidin-4(3H)-one (2n, C23H20N2O3S)
Yield 56%; mp 165–166ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2924,
1
1675, 1642cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 0.87
Yield 64%; mp 164.5–165.5ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2953,
1683, 1639cmꢁ1; 1H NMR (500MHz, DMSO-d6): ꢂ ¼ 0.85 (t,
3H, J ¼ 7.35Hz, CH3), 2.42 (q, J ¼ 7.35 Hz, 2H, CH2), 4.15
(s, 2H, SCH2), 4.28 (s, 2H, CH2, naphthyl), 6.51–7.98 (m, 10H,
Harmo), 12.72 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 13.3 (CH3), 18.5 (CH2), 36.6 (CH2, naphthyl),
37.8 (SCH2), 110.2 (C-5), 113.0–151.5 (10C, naphthyl, 4C,
furanyl), 159.3 (C-6), 160.1 (C-4), 160.9 (C-2), 182.2 (C¼O)
ppm; MS (EI): m=z ¼ 404 (Mþ).
(t, 3H, J ¼ 7.40Hz, CH3), 2.40 (q, J ¼ 6.95 Hz, 2H, CH2), 3.65
(s, 2H, SCH2), 4.49 (s, 2H, CH2Ph), 6.72–8.01 (m, 8H,
Harmo), 12.70 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 13.3 (CH3), 18.5 (CH2), 36.6 (CH2Ph), 37.8
(SCH2), 110.2 (C-5), 113.0–151.5 (6C, Ph, 4C, furanyl),
159.9 (C-6), 160.39 (C-4), 161.6 (C-2), 182.0 (C¼O) ppm;
HRMS: m=z ¼ 354.1042, calcd. 354.1038.
6-Benzyl-5-isopropyl-2-[(furan-2-ylcarbonylmethyl)thio]-
pyrimidin-4(3H)-one (2j, C20H20N2O3S)
5-Isopropyl-2-[(40-methoxy-phenylethyl)thio]-6-(1-naphthyl-
methyl)pyrimidin-4(3H)-one (2o, C27H28N2O2S)
Yield 31%; mp 163–164.8ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2924,
1
1675, 1642cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.03
Yield 13%; mp 137–138ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2953,
1641cmꢁ1
;
1H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.15 (d,
(d, 6H, J ¼ 7.35Hz, 2CH3), 3.01 (m, 1H, J ¼ 6.85Hz, CH),
3.64 (s, 2H, SCH2), 4.49 (s, 2H, CH2Ph), 6.71–8.26 (m, 8H,
Harmo), 12.71 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 19.82 (2CH3), 27.38 (CH), 36.54 (CH2Ph),
39.42 (SCH2), 109.54 (C-5), 113.0–151.6 (6C, Ph, 4C, fura-
nyl), 156.1 (C-6), 159.2 (C-2), 163.0 (C-4), 182.1 (C¼O)
ppm; HRMS: m=z ¼ 368.1204, calcd. 368.1195.
6H, J ¼ 6.85Hz, 2CH3), 2.94 (m, 1H, J ¼ 6.85Hz, CH),
3.48 (t, 2H, J ¼ 7.45Hz, PhCH2), 3.83 (s, 3H, OCH3), 4.30
(s, 2H, CH2, naphthyl), 4.36 (t, 2H, J ¼ 7.45Hz, SCH2), 7.10–
8.15 (m, 11H, Harmo), 12.71 (s, br, s, 1H, NH) ppm; 13C NMR
(500MHz, DMSO-d6): ꢂ ¼ 19.9 (2CH3), 27.9 (CH), 31.3
(PhCH2), 37.7 (CH2, naphthyl), 39.9 (SCH2), 55.9 (OCH3,
123.70 (C-5), 124.0–134.8 (10C, naphthyl, 6C, Ph), 159.8
(C-6), 160.3 (C-2), 161.6 (C-4) ppm; MS (EI): m=z ¼ 444
(Mþ).
5-Methyl-2-[(thiophen-2-ylcarbonylmethyl)thio]-6-(1-naph-
thylmethyl)pyrimidin-4(3H)-one (2k, C22H18N2O2S2)
Yield 40%; mp 210–211ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2953,
1
1675, 1635cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.94
5-Methyl-2-[(phenylethyl)thio]-6-(1-naphthylmethyl)-pyrim-
idin-4(3H)-one (2p, C24H22N2OS)
(s, 3H, CH3), 4.14 (s, 2H, SCH2), 4.42 (s, 2H, CH2, naphthyl),
7.09–8.27 (m, 10H, Harmo), 12.55 (s, brs, 1H, NH) ppm; 13C
NMR (500MHz, DMSO-d6): ꢂ ¼ 10.7 (CH3), 37.3 (PhCH2),
37.7 (SCH2), 122.8 (C-5), 124.3–142.4 (10C, naphthyl, 4C,
thienyl), 159.9 (C-6), 160.3 (C-2), 161.6 (C-4), 186.4 (C¼O)
ppm; MS (EI): m=z ¼ 406 (Mþ).
Yield 21%; mp 175–176ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2953,
1654cmꢁ1
;
1H NMR (500MHz, DMSO-d6): ꢂ ¼ 2.05 (s,
3H, CH3), 2.56 (q, J ¼ 7.35 Hz, 2H, PhCH2), 2.99 (t, 2H,
J ¼ 7.35 Hz, SCH2), 4.37 (s, 2H, CH2, naphthyl), 6.92–8.16
(m, 12H, Harmo), 12.58 (s, br, s, 1H, NH) ppm; 13C NMR
(500MHz, DMSO-d6): ꢂ ¼ 10.8 (CH3), 31.0 (PhCH2), 34.87
(napthyl, CH2), 38.73 (SCH2), 122.7 (C-5), 124.6–140.1 (10C,
naphthyl, 6C, Ph), 159.9 (C-6), 160.39 (C-2), 161.56 (C-4)
ppm; MS (EI): m=z ¼ 386 (Mþ).
5-Ethyl-2-[(thiophen-2-ylcarbonylmethyl)thio]-6-(1-naphthyl-
methyl)pyrimidin-4(3H)-one (2l, C23H20N2O2S2)
Yield 38%; mp 191–192ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2953,
1
1675, 1641cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 0.85
(t, 3H, J ¼ 7.10Hz, CH3), 2.42 (q, J ¼ 6.90 Hz, 2H, CH2), 4.14
(s, 2H, SCH2), 4.41 (s, 2H, CH2, naphthyl), 7.04–8.30 (m, 10H,
Harmo), 12.75 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 13.1 (CH3), 18.6 (CH2), 36.4 (PhCH2), 37.3
(SCH2), 110.8 (C-5), 112.8–151.1 (10C, naphthyl, 4C, thie-
nyl), 159.9 (C-6), 160.3 (C-2), 161.6 (C-4), 181.8 (C¼O)
ppm; MS (EI): m=z ¼ 420 (Mþ).
5-Ethyl-2-[(phenylethyl)thio]-6-(1-naphthylmethyl)-pyrim-
idin-4(3H)-one (2q, C25H24N2OS)
Yield 27%; mp 123.5–124.5ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2965,
1
1649cmꢁ1; H NMR (500MHz, DMSO-d6): ꢂ ¼ 0.93 (t, 3H,
J ¼ 7.40 Hz, CH3), 2.08 (q, 2H, J ¼ 7.40 Hz, CH2), 2.56 (q,
2H, J ¼ 7.35Hz, PhCH2), 2.97 (q, 2H, J ¼ 7.35Hz, SCH2),
4.48 (s, 2H, CH2, naphthyl), 6.86–8.30 (m, 12H, Harmo),
12.56 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz, DMSO-
d6): ꢂ ¼ 13.4 (CH3), 18.6 (CH2), 30.9 (PhCH2), 34.9 (CH2,
naphthyl), 37.1 (SCH2), 122.8 (C-5), 124.6–140.1 (10C,
naphthyl, 6C, Ph), 159.9 (C-6), 160.3 (C-2), 161.6 (C-4)
ppm; MS (EI): m=z ¼ 400 (Mþ).
5-Methyl-2-[(furan-2-ylcarbonylmethyl)thio]-6-(1-naph-
thylmethyl)pyrimidin-4(3H)-one (2m, C22H18N2O3S)
Yield 41%; mp 206–207ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2845,
1
1674, 1642cmꢁ1; H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.95

Synthesis and anti-HIV-1 activity of S-DABOs
973
Table 2 Crystal data and structure refinement for X-ray crys-
tal structures of compounds 2j
5-Isopropyl-2-[(phenylethyl)thio]-6-(1-naphthylmethyl)-
pyrimidin-4(3H)-one (2r, C26H26N2OS)
Yield 25%; mp 95–96ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2924,
Parameter
Compound 2j
1638 cmꢁ1
;
1H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.23 (d,
0.30ꢂ0.19ꢂ0.08 mm3
6H, J ¼ 6.85Hz, 2CH3), 2.58 (t, 2H, J ¼ 7.35 Hz, PhCH2),
3.01 (t, 2H, J ¼ 7.35 Hz, SCH2), 3.13 (m, 1H, J ¼ 6.80 Hz,
CH), 4.01 (s, 2H, CH2, naphthyl), 6.90–8.31 (m, 12H,
Harmo), 12.46 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 19.90 (2CH3), 27.4 (CH), 30.4 (PhCH2), 37.4
(CH2-naphthyl), 37.9 (SCH2), 123.7 (C-5), 126.4–138.8 (10C,
naphthyl, 6C, Ph), 156.1 (C-6), 159.2 (C-2), 163.0 (C-4) ppm;
MS (EI): m=z ¼ 414 (Mþ).
Crystal size
ꢃ
˚
Unit cell dimensions a ¼ 12.5785 (17) A ꢄ ¼ 90 ,
ꢃ
˚
b ¼ 9.6073 (13) A ꢁ ¼ 102.653 (2) ,
ꢃ
˚
c ¼ 15.676 (2) A ꢅ ¼ 90 .
F (000)
Space group
Cell volume
Z, Calculated density 4, 1.324 mg=m3
ꢆ range for data
collection
Limiting indices
776
Monoclinic, P2 (1)=c
3
˚
1848.4 (4)A
1.66 to 26.43ꢃ
6-Benzyl-5-isopropyl-2-[(phenylethyl)thio]-pyrimidin-4(3H)-
one (2s, C22H24N2OS)
ꢁ15 ꢄ h ꢄ 14,
Yield 29%; mp 130–131ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2953,
ꢁ10 ꢄ k ꢄ 12,
1642 cmꢁ1
;
1H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.18 (d,
ꢁ19 ꢄ l ꢄ 19.
Reflections
10773=3770 [R(int) ¼ 0.0348]
6H, J ¼ 6.80Hz, 2CH3), 2.86 (t, 2H, J ¼ 7.45 Hz, PhCH2),
3.11 (m, 1H, J ¼ 6.80Hz, CH), 3.27 (t, 2H, J ¼ 7.45Hz,
SCH2), 3.94 (s, 2H, CH2Ph), 7.15–7.33 (m, 10H, Harmo),
12.71 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz, DMSO-
d6): ꢂ ¼ 19.4 (2CH3), 27.0 (CH), 30.2 (PhCH2), 35.3 (CH2Ph),
37.2 (SCH2), 123.7 (C-5), 113.5–154.3 (12C, 2Ph), 156.1 (C-
6), 159.2 (C-2), 163.0 (C-4) ppm; MS (EI): m=z ¼ 364 (Mþ).
collected=unique
Data=restraints=
parameters
Goodness-of-fit on F2 1.071
3770=0=235
Final R indices
[I>2ꢇ(I)]
R indices (all data)
R1 ¼ 0.0910, wR2 ¼ 0.2691
R1 ¼ 0.1293, wR2 ¼ 0.3056
Absorption coefficient 0.197 mmꢁ1
5-Methyl-6-(1-naphthylmethyl)-2-[(thiophen-2-yl)thio]-
pyrimidin-4(3H)-one (2t, C20H16N2OS2)
ꢁ3
˚
Largest diff. peak
and hole
0.935 and ꢁ0.621 eA
Yield 46%; mp 218–219ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2953,
1669 cmꢁ1
;
1H NMR (500MHz, DMSO-d6): ꢂ ¼ 2.12 (s,
3H, CH3), 4.26 (s, 2H, CH2, naphthyl), 7.26–9.48 (m, 10H,
Harmo), 12.78 (s, br, s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): ꢂ ¼ 12.8 (CH3), 32.1 (CH2, naphthyl), 112.5 (C-
5), 118.7–133.6 (10C, naphthyl, 4C, thienyl), 149.30 (C-6),
161.85 (C-2), 174.66 (C-4) ppm; MS (EI): m=z ¼ 364 (Mþ).
X-Ray crystallography
Colorless cubic crystals of 2j were grown from ethyl acetate in
separate experiments by slow evaporation at room tempera-
ture. The crystals were mounted on glass fibers by using
epoxy, and X-ray diffraction data for a crystal (0.30ꢂ
0.19ꢂ0.08 mm) of 2j was collected at 22ꢃC by using a
SMART charge-coupled device X-ray detector (Bruker
Analytical X-Ray Systems, Madison, WI). Structure solution
and refinement were performed by using the SHELXTL suite
of programs (Bruker Analytical X-Ray Systems). All nonhy-
drogen atoms were refined using anisotropic displacement
parameters. Hydrogen atoms were placed at ideal positions
and refined as riding atoms with relative isotropic displace-
ment parameters (Table 2). Crystallographic data were depos-
ited at the Cambridge Crystallographic Data Centre with
CCDC No. 689793 and may be obtained free of charge from
the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK (fax: þ44-1223-336033, e-mail: deposit@ccdc.cam.ac.uk,
or http:==www.ccdc.cam.ac.uk).
5-Ethyl-6-(1-naphthylmethyl)-2-[(thiophen-2-yl)thio]-
pyrimidin-4(3H)-one (2u, C21H18N2OS2)
Yield 44%; mp 184–185ꢃC; FT-IR (KBr): ꢃꢀ¼ 3425, 2952,
1
1649 cmꢁ1; H NMR (500MHz, DMSO-d6): ꢂ ¼ 0.81 (t, 3H,
J ¼ 7.35Hz, CH3), 1.18 (q, J ¼ 7.35 Hz, 2H, CH2), 4.32 (s,
2H, CH2, naphthyl), 7.08–8.14 (m, 10H, Harmo), 12.49 (s, brs,
1H, NH) ppm; 13C NMR (500 MHz, DMSO-d6): ꢂ ¼ 13.2
(CH3), 18.1 (CH2), 32.2 (CH2, naphthyl), 112.8 (C-5),
118.7–133.6 (10C, naphthyl, 4C, thienyl), 149.3 (C-6), 161.9
(C-2), 174.7 (C-4) ppm; MS (EI): m=z ¼ 378 (Mþ).
5-Isopropyl-6-(1-naphthylmethyl)-2-[(thiophen-2-yl)thio]-
pyrimidin-4(3H)-one (2v, C22H20N2OS2)
Yield 56%; mp 219–220ꢃC; FT-IR (KBr): ꢃꢀ¼ 3541, 2965,
Antiviral assay procedures
1650 cmꢁ1
;
1H NMR (500 MHz, DMSO-d6): ꢂ ¼ 1.33 (d,
Cells and viruses
6H, J ¼ 7.35 Hz, 2CH3), 3.04 (m, 1H, J ¼ 6.85, CH), 4.30 (s,
2H, CH2, naphthyl), 6.84–8.92 (m, 10H, Harmo), 12.46 (s, br, s,
1H, NH) ppm; 13C NMR (500 MHz, DMSO-d6): ꢂ ¼ 19.8
(2CH3), 27.4 (CH), 32.2 (CH2, naphthyl), 112.8 (C-5),
118.7–133.6 (10C, naphthyl, 4C, thienyl), 149.31 (C-6),
161.88 (C-2), 174.67 (C-4) ppm; HRMS: m=z ¼ 392.1025,
calcd. 392.1017.
Human T-cell lines (C8166, H9) and HIV-1IIIB were kindly
donated by Medical Research Council, AIDS Regent Project.
The cells were maintained at 37ꢃC in 5% CO2 in RPMI-1640
medium supplemented with 10% heat-inactivating fetal calf
serum (Gibco). HIV-1IIIB was prepared from the supernatants
of H9=HIV-1IIIB cells. The 50% HIV-1 tissue culture in-

974
Z. Rao et al.
fectious dose (TCID50) in C8166 cells was determined and
calculated by Reed and Muench method. Virus stocks were
stored in small aliquots at ꢁ70ꢃC. The titer of virus stock was
6.0ꢂ105 TCID50 per cm.
(5Z0475C) to Dr. Y.P. He, the knowledge Innovation Program
of Chinese Academy of Sciences (KSCX1-YW-R-24), 973
Program (2006CB504200), Key Scientific and Technological
projects of Yunnan (2004NG12) to Dr. Y.T. Zheng. We would
like to acknowledge MRC AIDS Research Project (UK) for
providing cell lines and virus.
Quantification of the inhibitory effect of compounds
on reverse transcriptase
HIV-1 RT activity was measured by ELISA RT kit using a
commercially available kit (Roche) according to the instruc-
tions of the manufacturer. The compounds 2a–2v and 7–9
were dissolved in DMSO and stored at ꢁ4ꢃC. The compounds
were incubated with DIG-labeled reaction mixture at 37ꢃC for
2 h, then anti-DIG-POD solution was added, followed by sub-
strate ABTS. Foscarnet was used as a positive control. The
absorbency at 405 nm=490 nm (A405=490) was read on Bio-
Tek ELx 800 ELISA reader [19].
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Cytotoxicity assay
The cellular toxicity of compounds on C8166 cells was
assessed by MTT colorimetric assay as described previously
[20]. Briefly, 100 mm3 of 4ꢂ105 cells ꢅ cm3 were plated into
96-well microtiter plates, 100 mm3 of various concentrations
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ifed atmosphere of 5% CO2 for 72 h. Discard 100 mm3 super-
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Inhibition of syncytium formation
C8166 cells (4ꢂ105 cells ꢅ cm3) were treated with different
concentrations of the compounds and HIV-1IIIB (M.O.I. ¼
0.02), and incubated in a humidified incubator at 37ꢃC in final
volume of 200 mm3. AZT was used for positive drug control.
After 3 days incubation of culture, the cytopathic effect (CPE)
was measured by counting the number of syncytium (mul-
tinucleated giant cell) in each well under an inverted micro-
scope [21]. The Percentage inhibition of syncytial cell
formation (EC50) was calculated by the percentage of syncy-
tial cell number in compounds treated culture to that in
infected control culture.
19. Wang Q, Ding ZH, Liu JK, Zheng YT (2004) Antiviral
Res 64:189
20. Zheng YT, Zhang WF, Ben KL, Wang JH (1995) Immu-
nopharmacol Immunotoxicol 17:69
Acknowledgements
21. Wang YH, Tang JG, Wang RR, Yang LM, Dong ZJ, Du L,
Shen X, Liu JK, Zheng YT (2007) Biochem Biophys Res
Commun 355:1091
This research was supported by the National Natural Science
Foundation of China (Grant No. 30530179), the Natural
Science Foundation of education office of Yunnan province