Synthesis and anti-HIV evaluation of novel 1,3-disubstituted thieno[3,2-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxides(TTDDs)

Article abstract of DOI:10.1016/j.bmc.2007.10.013

A series of novel 1,3-disubstituted thieno[3,2-c] [1,2,6]thiadiazin-4(3H)-one 2,2-dioxides (TTDDs), designed as non-nucleoside reverse transcriptase inhibitors (NNRTIs), was synthesized, structurally confirmed by spectral analysis and evaluated for their

Full text of DOI:10.1016/j.bmc.2007.10.013

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 157–163
Synthesis and anti-HIV evaluation of novel 1,3-disubstituted
thieno[3,2-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxides(TTDDs)
Yongqiang Lin,a Xinyong Liu,a,* Renzhang Yan,a Jin Li,a Christophe Pannecouque,b
Myriam Witvrouwb and Erik De Clercqb,*
aInstitute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University,
No. 44 Wenhuaxi Road, Jinan 250012, China
bRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 6 August 2007; revised 24 September 2007; accepted 2 October 2007
Available online 10 October 2007
Abstract—A series of novel 1,3-disubstituted thieno[3,2-c] [1,2,6]thiadiazin-4(3H)-one 2,2-dioxides (TTDDs), designed as non-nucle-
oside reverse transcriptase inhibitors (NNRTIs), was synthesized, structurally confirmed by spectral analysis and evaluated for their
anti-HIV-1 activities by inhibition of HIV-1(IIIB)-induced cytopathogenicity in MT-4 cell culture. The results showed that TTDD
analogues exhibited marked potency as anti-HIV-1 agents. The most active and selective compound was 1-(3-cyano)benzyl-3-ben-
zyl-thieno[3,2-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (5f) with a 50% effective concentration (EC50) of 4.0 lM and a selectivity
index (SI) of >76. The structure–activity relationship (SAR) is discussed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
from the polymerase active site, causing a distortion of
the catalytic aspartate triad.2,3 The efficacy of NNRTIs,
and of the other anti-AIDS agents, has been limited by
the emergence of drug-resistant viral strains and possible
side effects.4–6 Therefore, new NNRTIs with more potent
activity and lesser toxicity are still needed.
The introduction of highly active anti-retroviral therapy
(HAART) has dramatically decreased the morbidity
and mortality from the infection by HIV, the causative
agent of acquired immunodeficiency syndrome (AIDS).
However, the AIDS prevalence remainsone of the world’s
most serious health problems, causing millions of deaths
each year.1 The principal chemotherapeutic agents that
have been used in the clinic to block the replication of
HIV are the reverse transcriptase inhibitors (RTIs), prote-
ase inhibitors (PIs) and a fusion inhibitor. The HIV-1 re-
verse transcriptase (HIV-1 RT)-catalyzed step in the life
cycle of HIV is an attractive target for anti-AIDS drug
development. Three classes of HIV-1 RTIs are currently
available: nucleoside (and nucleotide) reverse transcrip-
tase inhibitors (NRTIs, NtRTIs) and non-nucleoside re-
verse transcriptase inhibitors (NNRTIs). NNRTIs are
structurally diverse compounds specifically targeting at
In recent studies aimed at the discovery of new NNR-
TIs, Dr. S. Vega and his colleagues reported that a series
of
2,4-disubstituted-1,1,3-trioxo-2H,4H-thieno[3,4-e]
[1,2,4]thiadiazines (TTDs) effectively inhibited, at the re-
verse transcription step, the replication of a variety of
HIV-1 strains, including strains that are resistant to
AZT (azidothymidine, zidovudine), but not HIV-2
(ROD).7,8 The prototype compounds QM96521,
QM96539 and QM96639 (Fig. 1), containing a benzyl
or 2-halogenated benzyl moiety at the N2 position and
a cyanomethyl chain linked to the N4 position, were
found to selectively inhibit HIV-1 (III-B) replication in
MT and CEM cell cultures. The cross-resistance pattern
of these compounds against other NNRTI-resistant mu-
tant HIV-1 strains and molecular modelling of the HIV-
1 RT binding site were both found to be similar to that
of nevirapine. Furthermore, molecular modification by
replacement of the N4 cyanomethyl with a substituted
benzyl group led to the discovery of a new precursor
˚
an allosteric site of HIV-1 RT, approximately 10–15 A
Keywords: HIV-1 NNRTIs; Thieno[3,2-c][1,2,6]thiadiazine; Synthesis.
*
Corresponding authors. Tel.: +86 531 838 2005; fax: +86 531 838
2264 (X.L.); tel.: +32 16 337341; fax: +32 16 337340 (E.D.C.); e-mail
addresses: xinyongl@sdu.edu.cn; erik.declercq@rega.kuleuven.ac.be
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.013

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Y. Lin et al. / Bioorg. Med. Chem. 16 (2008) 157–163
O O
S
O
N
O O
S
S
N
N
N
S
S
S O
O
N
O
N
O
X
R
CN
Cl
QM 96521 X=H
QM 96639 X= F
QM 96539 X=Cl
1,3-Disubstituted-thieno[3,2-c][1,2,6]
thiadiazin-4(3 )-one 2,2-dioxides(TTDDs)
QM 96625
H
Figure 1. Structures of lead compounds TTDs and newly designed TTDDs.
QM96625 (N2-benzyl, N4-2-chlorobenzyl, EC50
0.1 lM, SI >1190. Fig. 1).7
=
In order to avoid producing a N1,N3- and N1,O4-disub-
stituted TTDDs mixture, an unambiguous synthetic
pathway was planned for the preparation of the newly
designed TTDDs 5a–m. Thus, we chose 3-substituted
TTDD 4 as the starting material, which was prepared
from methyl 3-amino-2-thiophene carboxylate 2 accord-
ing to the Cohen and Klarberg procedure,14 but with an
improvement by one-pot reaction. In brief, to an anhy-
drous toluene solution of the compound 2, N-ben-
zylsulfamoyl chloride 1 dissolved in toluene was added
dropwise at room temperature. The obtained solution
mixture was stirred for 4 h at 60 ꢁC and cooled to room
temperature, then neutralized with 5% aq KOH to ad-
just pH at 11, forming the solution mixture of thiophene
sulfamide derivative 3. The ring-closure reaction was
performed by continuing stirring of the two-phase solu-
tion at room temperature for 16 h without separation of
compound 3. The key intermediate of 3-benzyl thie-
no[3,2-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (4) was
precipitated after the water layer was separated and
treated with conc. HCl to adjust pH at 1, and purified
by recrystallization from ethanol in 45% of total yield.
Initial structure–activity relationship analysis together
with the molecular modelling disclosed that double sub-
stitutions containing p-electrons at the N2 and N4 sites
of TTDs were necessary for preserving anti-HIV-1 activ-
ity. Their structure is in perfect accord with the ‘butter-
fly-like’ three-dimensional (3D) model proposed by
Schafer et al.10
In continuing research of the new HIV-1 NNRTIs and
study of the structure–activity relationships of TTD ana-
logues, we designed a series of novel 1,3-disubstituted thi-
eno[3,2-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxides (TTDDs)
based on the general principle of bioisosteric replacement
in medicinal chemistry, since TTDDs can be considered as
the bioisosteres of TTDs. In the newly designed TTDD
analogues, the heterocyclic system of thieno[3,2-c]
[1,2,6]thiadiazin-4(3H)-one 2,2-dioxide served as a scaf-
fold. The benzyl group at N3 position, benzyl, substituted
benzyl, cyanomethyl and other groups containing p-elec-
trons at N1 position, which proved essential for anti-HIV
activity in TTD analogues, were respectively introduced
in the TTDD nuclear ring, so as to arrange molecules in
‘butterfly-like’ orientation, which is thought to be the
determinant for potent anti-HIV-1 activity of the NNR-
TIs.9 Here, we report the synthesis of novel TTDD ana-
logues and their anti-HIV activity.
Alkylation of 4 with cyanomethyl, benzyl or substituted
benzyl halides, in the presence of equal molar of sodium
hydride (NaH) and N, N-dimethylformamide (DMF)
solvent, achieved the target 1,3-disubstituted TTDDs
5a–m (Scheme 1).
The starting material N-benzylsulfamoyl chloride 1 was
prepared by the modified Kloek and Leschinsky proce-
dure.15 To a cooled (0 ꢁC) solution of benzylamine in
CH2Cl2 was added chlorosulfonic acid cautiously with
vigorous stirring. The resulting suspension was stirred
for 0.5 h at room temperature and then filtered. The col-
lected solids of N-benzylsulfamate were dissolved in tol-
uene and treated with phosphorus pentachloride. The
solution was refluxed for 1 h and the solid was filtered
off. The filtrate was concentrated in vacuo, and the syr-
upy residue (N-benzylsulfamoyl chloride 1) thus ob-
tained was used in the next synthetic step without
further purification.
2. Results and discussion
2.1. Chemistry
TTDD acyclonucleosides and two TTDD 1,3-dibenzyl
substituted analogues had been synthesized previously
and reported as potent human cytomegalovirus
(HCMV) inhibitors,11–13 in which thieno[3,2-c][1,2,6]thi-
adiazin-4(3H)-one 2,2-dioxide heterocycle was prepared
starting from methyl 3-amino-2-thiophene carboxylate 2
with sulfamoyl (ClSO2NH2) followed by two alkylations
at N1 and N3 position, respectively.11 Two weaknesses
were found in this process: the first-step alkylation at
N1 position usually produced a mixture of N1- and N1,
N3-disubstituted products; the second-step alkylation
at N3 position was found not only difficult to achieve
probably due to the steric hindrance, but also the side
product of O4-substituted compound was formed con-
comitantly with the TTDDs.13
2.2. Anti-HIV evaluation
The activity and cytotoxicity of the newly designed and
synthesized TTDDs 4 and 5 were tested in MT-4 cells
for inhibition of HIV-1-induced cytopathogenicity.
The results are listed in Table 1. The precursors
QM96521 and QM96625 were used as reference

Y. Lin et al. / Bioorg. Med. Chem. 16 (2008) 157–163
159
CH2NHSO2Cl
CH2NHSO3
CH2NH2
1
O
O
N
O
S
S
S
OCH3
NH2
OCH3
SO2NHCH2Ph
ii, iii
i
S O
N
N
H
O
H
2
3
4
O
N
S
N
iv
S O
O
R
5
Scheme 1. Reagents: (i) toluene/N-benzylsulfamoyl chloride; (ii) H2O/5%KOH; (iii) H2O/HCl; (iv) DMF/NaH/RCH2Cl.
Table 1. Structure, anti-HIV-1 activity and cytotoxicity of 1,3-disubstituted thieno[3,2-c][1,2,6] thiadiazin-4(3H)-one 2,2-dioxide (TTDD) analogues
4 and 5a–m
O
S
N
S O
N
O
R
Compound
CH2R
HIV-1 (IIIB)
EC50a ( lM)
CC50b ( lM)
SIc
4
5a
H
Benzyl
>373.9
>37.5
4.8
373.9
37.5
<1
<1
5b
5c
2-Cl-Benzyl
2-Br-Benzyl
2-CN-Benzyl
3-Cl-Benzyl
3-CN-Benzyl
2,4-Cl2-benzyl
4-Cl-Benzyl
4-Br-Benzyl
4-CN-Benzyl
4-NO2-Benzyl
CH2CN
>298.6
>270.0
>305.5
>298.6
>305.5
247.1
8.2
>62
>37
>59
>51
>76
5
7.3
5.1
5d
5e
5.9
4.0
5f
5g
52.1
>8.2
>6.6
>8.5
>268.0
>5.7
71.0
0.9
5h
5i
<1
<1
6.6
8.5
5j
5k
<1
<1
268.0
5.7
5l
<1
3
5m
CH3
CH2CN
2-Cl-Benzyl
202.4
502.7
>119.0
683
QM96521d
QM96625d
Nevirapined
AZTd
559
>1190
22,767
50,587
0.1
0.03
0.0007
35.6
a EC50: 50% effective concentration, or concentration of compound required to achieve 50% protection of MT-4 cell from HIV-1 induced cyto-
toxicity, as determined by the MTT method.
b CC50: 50% cytotoxic concentration, or concentration required to reduce the viability of mock-infected cells by 50%, as determined by the MTT
method.
c SI: selectivity index (CC50/EC50).
d Values for anti-HIV-1 activities reported in Refs.7–9. All data represent mean values for at least two separate experiments.
compounds for comparative purposes; AZT (Zidovu-
dine) and nevirapine were used as the reference drugs.
The new TTDDs derivatives were confirmed to be both
potent and selective HIV-1 inhibitors.
and the selective indexes (SI, ratio of CC50 for cell
growth to EC50 for virus replication) in the range be-
tween 37 and 76. The most active and selectivity TTDD
derivative was compound 5f (N1- 3-Cl-benzyl, N3-ben-
zyl) with an EC50 value of 4.0 lM and selective index
SI >76. Compounds 5g and 5m proved slightly active
against HIV-1, while compounds 4 and 5k were totally
Compounds 5b–f emerged as the most active HIV-1
inhibitors with EC50 values in the range of 4–7 lM

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Y. Lin et al. / Bioorg. Med. Chem. 16 (2008) 157–163
inactive. Other compounds displayed no selectivities be-
cause of the high cytotoxicity, compounds 5h–j and 5l
being cytotoxic for MT-4 cells at concentrations lower
than 10 lM. In addition, all synthesized compounds
were screened for activity against HIV-2 (strain ROD),
but none of the compounds was found to inhibit HIV-
2 replication.
(HIV-1 IIIB and HIV-2 ROD) activities by inhibition
of HIV-induced cytopathogenicity in MT-4 cell cultures.
The results showed that TTDD analogues exhibited
high potency as anti-HIV-1 agents. The most active
and selective compound was 5f with an EC50 value of
4.0 lM and a SI of > 76. TTDD analogues may seem
promising for further activity optimization studies.
These biological data led us to take into consideration
the structure–activity relationship (SAR) analysis of
these compounds. The active anti-HIV-1 agents were
the N1, N3- disubstituted TTDDs, N3-monosubstituted
derivative 4 was inactive, which is accordant with the re-
sults obtained for the SAR in the TTD series. Appar-
ently, the molecule that lacked the N1-substituent did
not meet the structural requirements of the ‘butterfly-
like’ conformation, which seems particularly important
in other known NNRTIs, i.e. TIBO, a-APA, nevirapine
and thiazolidones.16 The essential substituents are aryl-
methyl groups such as benzyl, or substituted benzyl.
Other groups having no p-electron character, such as al-
kyl (5m), lost or decreased the potency and selectivity
against HIV-1 replication.
4. Experimental
4.1. Chemistry
Melting points were determined on a Gallenkamp capil-
lary apparatus and are uncorrected.1H NMR
(600 MHz) and 13C NMR (150 MHz) spectra were ob-
tained on a Bruker Avance-600 instrument in the indi-
cated solvent. Chemical shifts are expressed in d units
with tetramethylsilane (TMS) as internal reference.
Infrared spectra (IR) were recorded with a Nexus
470FT-IR Spectrometer. Mass spectra were recorded
on a LC Autosampler Device: Standard G1313A instru-
ment. All compounds were routinely checked by TLC
on pre-coated silica gel G plates with fluorescent indica-
tor at 254 nm, which were prepared in our laboratory.
Developed plates were visualized by UV light. Solvents
were of reagent grade and, when necessary, were puri-
fied and dried by standard methods. Concentration of
the reaction solutions involved the use of rotary evapo-
rator under reduced pressure.
In the N1, N3-disubstituted benzyl series (5a–k), the
activities of the substituents at N1-benzyl group were
found in the following declining trends: the ortho and
meta substituted benzyl (5b–f) >2,4-Cl2-benzyl (5g) >
para substituted benzyl (5h–k). The results demon-
strated that the N1-o-substituted benzyl groups were still
effective substituents, as previously reported for the
SAR of the TTDs,7 and further indicated that the intro-
duction of m-substituted benzyl group in the N1 position
could also result in the congeners endowed with the
equivalent potency and selectivity. Whereas the N1para
(p) substituted benzyls, such as 2,4-dichlorobenzyl 5g, p-
halogenatedbenzyl 5h–i, p-cyanobenzyl 5j and p-nitro-
benzyl 5k, had reduced or abolished activities in contrast
to the ortho and meta monosubstituted benzyl ano-
logues, 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 TTDDs.
4.1.1. N-Benzylsulfamoyl chloride (1). To a 0 ꢁC cooled
and stirred solution of benzylamine (10.7 g, 0.1 mol) in
CH2Cl2 (100 mL) was added chlorosulfonic acid
(3.49 g, 0.03 mol) cautiously. The resulting suspension
was stirred for 0.5 h at room temperature and then fil-
tered. The collected solids of N-benzylsulfamate were
dissolved in toluene (50 mL) and treated with phospho-
rus pentachloride (6.24 g, 0.03 mol). A mild exothermic
reaction took place. The solution was refluxed for 1 h
and the solid was filtered off. The filtrate was concen-
trated in vacuo and N-benzylsulfamoyl chloride 1 was
obtained as syrupy residue.
The newly designed TTDD compounds presented de-
creased activities, leading us to hypothesize that the
changed positions of the heteroatoms N and S in the
TTDD heterocycle, giving rise to the changed direction
of ‘butterfly wing’, affected the binding orientation in
the aromatic-rich non-nucleoside binding site of HIV-1
RT, surrounded by the aromatic side chains, such as
Tyr181, Tyr188, Phe227 and Trp 229. In particular, it
affected p–p interaction of the phenyl ring with the
Tyr181 side chain.5,7 Molecular modelling of TTDDs
by AutoDocking analysis is underway.
4.1.2. 3-Benzyl-1H-thieno[3,2-c][1,2,6]thiadiazin-4(3H)-
one 2,2-dioxide (4). To an anhydrous toluene solution
of the 3-amino-2-thiophene carboxylate (2) (2.30 g,
0.015 mol), N-benzylsulfamoyl chloride (1) dissolved in
toluene was added dropwise at room temperature. The
obtained mixture solution was stirred for 4 h at 60 ꢁC
and cooled to room temperature, then neutralized with
5% aq KOH to adjust pH at 11, forming the mixture
solution of thiophene sulfamide derivative 3. The
ring-closure reaction was performed without separation
of compound 3 by continuing stirring of the two-phase
solution at room temperature for 16 h. The intermediate
4 was precipitated after the water layer was separated
and treated with conc. HCl (pH 1), and purified by
recrystallization from ethanol in 45% of total yield.
mp 180–182 ꢁC.1H NMR (DMSO-d6) d: 8.02 (d, 1H,
J = 5.2 Hz, thiophene), 7.35–7.26 (m, 5H, benzene),
7.25 (m, 1H, NH), 6.93 (d, 1H, J = 5.1 Hz, thiophene),
4.97 (s, 2H, CH2); 13C NMR (DMSO-d6) d: 158.67
3. Conclusions
In summary, we designed and synthesized a series of
novel 1,3-disubstituted thieno[3,2-c][1,2,6]thiadiazin-
4(3H)-one 2,2-dioxides (TTDDs), which were structur-
1
ally confirmed by IR, H NMR, 13C NMR and MS
spectral analysis and evaluated for their anti-HIV

Y. Lin et al. / Bioorg. Med. Chem. 16 (2008) 157–163
161
(C@O), 137.22(C-1), 135.28, 128.52(3C), 127.97(3C),
127.55, 121.41(C-7), 44.30 (CH2–N3); IR (KBr, cmÀ1):
3274 (N1-H), 3094 (Ar–H), 1645 (C@O), 1490 (Ar, C–
C), 1373, 1179 (SO2); ESI-MS: m/z 295.4 [M + 1].
4.1.3.4. 1-(2-Cyano)benzyl-3-benzyl-thieno[3,2-c][1,2,6]
thiadiazin-4(3H)-one 2,2-dioxide (5d). Reagents: Com-
pound
4 (1.2 g, 4 mmol), 2-cyanobenzyl chloride
(0.91 g, 6 mmol). Conditions: 80 ꢁC, 18 h. Purification:
recrystallization. Yield 0.9 g (55%) as a white solid: mp
1
4.1.3. General procedure for the preparation of 1,3-disub-
stituted TTDDs (5a–m). To a solution of the 3-benzyl-1H-
thieno[3,2-c] [1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (4)
(1.2 g, 4 mmol) in dry DMF (15 mL), under N2, was
added sodium hydride (60% dispersion in mineral oil,
0.24 g, 6 mmol) by portions, maintaining the temperature
below 10 ꢁC. After 15 min, the alkyl halide (6 mmol) was
added and the reaction mixture was stirred at 50–80 ꢁC
for 16–24 h. The solvent was evaporated in vacuo to dry-
ness and the crude oil washed with 5% aq KOH, extracted
with CH2Cl2, evaporated in vacuo and the crude product
was recrystallized from the appropriate solvent.
102–103 ꢁC (EtOH); H NMR (DMSO-d6) d: 8.22 (d,
1H, J = 5.4 Hz, thiophene), 7.89 (d, 1H, J = 7.8 Hz ben-
zene H-3), 7.65–7.30 (m, 8H, benzene), 7.22 (d, 1H,
J = 5.3 Hz, thiophene), 5.30 (s, 2H, CH2), 5.01 (s, 2H,
CH2); 13C NMR (DMSO-d6) d: 157.57 (C@O),
143.63(C-1), 138.47(C-1), 136.61, 136.06, 133.79,
129.31, 128.71 (3C), 128.11 (3C), 127.98, 121.27,
119.01 (CN), 117.11(C-7), 111.05(C-2), 52.77 (CH2–
N1), 46.06 (CH2–N3); IR (KBr, cmÀ1): 3110 (Ar–H),
2231 (CN), 1656 (C@O), 1535 (Ar, C–C), 1382, 1176
(SO2); ESI-MS: m/z 410.5 [M + 1].
4.1.3.5. 1-(3-Chloro)benzyl-3-benzyl-thieno[3,2-c][1,2,6]-
thiadiazin-4(3H)-one 2,2-dioxide (5e). Reagents: Com-
pound 4 (1.2 g, 4 mmol), 3-chlorobenzyl chloride (1.0 g,
6 mmol). Conditions: 80 ꢁC, 18 h. Purification: recrystalli-
zation. Yield 0.75 g (45%) as a white solid: mp 108–
4.1.3.1. 1,3-Dibenzyl-thieno[3,2-c][1,2,6]thiadiazin-4(3H)-
one 2,2-dioxide (5a). Reagents: Compound 4 (1.2 g,
4 mmol), benzyl bromide (1.0 g, 6 mmol). Conditions:
60 ꢁC, 10 h. Purification: recrystallization. Yield 0.65 g
(42.3%) as a white solid: mp 96–98 ꢁC (EtOH); 1H
NMR (DMSO-d6) d: 8.21 (d, 1H, J = 5.4 Hz, thiophene),
7.43–7.13 (m, 11H, benzene and thiophene), 5.16 (s, 2H,
CH2), 4.97 (s, 2H, CH2); 13C NMR (DMSO-d6) d:
157.59 (C@O), 143.43(C-1), 136.54(C-1), 136.12, 134.57,
128.83(2C), 128.75(2C), 128.55,128.23(4C), 128.03,
121.32, 118.72(C-7), 54.02 (CH2–N1), 45.80 (CH2–N3);
IR (KBr, cmÀ1): 3104 (Ar–H), 1666 (C@O), 1539 (Ar,
C–C), 1371, 1173 (SO2); ESI-MS: m/z 385.2 [M + 1].
1
109 ꢁC (EtOH); H NMR (DMSO-d6) d: 8.23 (d, 1H,
J = 5.3 Hz, thiophene), 7.41–7.28 (m, 9H, benzene), 7.10
(d, 1H, J = 5.3 Hz, thiophene), 5.18 (s, 2H, CH2), 4.99
(s, 2H, CH2); 13C NMR (DMSO-d6) d: 157.53 (C@O),
143.39(C-1), 137.28(C-1), 136.78, 136.08, 133.42, 130.80,
128.75(2C), 128.52, 128.12(2C), 128.03(2C), 126.65,
121.05, 118.50(C-7), 53.13 (CH2–N1), 45.86 (CH2–N3);
IR (KBr, cmÀ1): 3112 (Ar–H), 1660 (C@O), 1535 (Ar,
C–C), 1375, 1174 (SO2); ESI-MS: m/z 419.3 [M + 1].
4.1.3.2. 1-(2-Chloro)benzyl-3-benzyl-thieno[3,2-c][1,2,6]-
thiadiazin-4(3H)-one 2,2-dioxide (5b). Reagents: Com-
4.1.3.6. 1-(3-Cyano)benzyl-3-benzyl-thieno[3,2-c][1,2,6]-
thiadiazin-4(3H)-one 2,2-dioxide (5f). Reagents: Com-
pound 4 (1.2 g, 4 mmol), 3-cyanobenzyl chloride (0.91 g,
6 mmol). Conditions: 80 ꢁC, 24 h. Purification: recrystal-
lization. Yield 0.80 g (49%) as a white solid: mp 83–85 ꢁC
(EtOH); 1H NMR (DMSO-d6) d: 8.25 (d, 1H, J = 5.3 Hz,
thiophene), 7.80 (d, 1H, J = 7.3 Hz benzene H-6), 7.66 (s,
1H, benzene H-2), 7.48–7.28 (m, 8H, benzene and thio-
phene), 5.23 (s, 2H, CH2); 4.99 (s, 2H, CH2); 13C NMR
(DMSO-d6) d: 157.43 (C@O), 143.24(C-1), 136.89(C-1),
136.45, 136.00, 132.75, 132.33, 131.68, 130.15, 128.72
(3C), 128.05 (3C), 121.05 (CN), 118.52(C-7), 111.70(C-
3), 52.86 (CH2–N1), 45.76 (CH2–N3); IR (KBr, cmÀ1):
3107 (Ar–H), 2231(CN), 1658 (C@O), 1534 (Ar, C–C),
1375, 1172 (SO2); ESI-MS: m/z 410.5 [M + 1].
pound
4 (1.2 g, 4 mmol), 2-chlorobenzyl chloride
(1.0 g, 6 mmol). Conditions: 80 ꢁC, 18 h. Purification:
recrystallization. Yield 0.50 g (30%) as a white solid:
1
mp 83–84 ꢁC (EtOH); H NMR (DMSO-d6) d: 8.16 (d,
1H, J = 5.3 Hz, thiophene), 7.49 (d, 1H, J = 7.9 Hz ben-
zene H-3), 7.36–7.27 (m, 8H, benzene), 7.09 (d, 1H, J =
5.3 Hz, thiophene), 5.20 (s, 2H, CH2), 5.05 (s, 2H, CH2);
13C NMR (DMSO-d6) d: 157.64 (C@O), 143.60(C-1),
136.42(C-1), 136.11, 132.50, 132.40, 130.23, 129.93,
129.74, 128.7 (2C), 128.15(2C), 127.98, 127.72, 121.04,
118.50(C-7), 51.82 (CH2–N1), 45.98 (CH2–N3); IR
(KBr, cmÀ1): 3109 (Ar–H), 1668 (C@O), 1538 (Ar, C–
C), 1373, 1168 (SO2); ESI-MS: m/z 419.2 [M + 1].
4.1.3.3. 1-(2-Bromo)benzyl-3-benzyl-thieno[3,2-c][1,2,6]-
thiadiazin-4(3H)-one 2,2-dioxide (5c). Reagents: Com-
pound 4 (1.2 g, 4 mmol), 2-bromobenzyl bromide (1.5 g,
6 mmol). Conditions: 60 ꢁC, 8 h. Purification: recrystalli-
zation. Yield 0.6 g (32%) as a red solid: mp 103–105 ꢁC
(EtOH); 1H NMR (DMSO-d6) d: 8.16 (d, 1H, J = 5.3 Hz,
thiophene), 7.67 (d, 1H, J = 7.8 Hz benzene H-3), 7.36–
7.23 (m, 8H, benzene), 7.03 (d, 1H, J = 5.3 Hz, thiophene),
5.16(s, 2H, CH2);5.06(s, 2H, CH2);13C NMR(DMSO-d6)
d: 157.72 (C@O), 143.70(C-1), 136.49(C-1), 136.20, 134.16,
133.27, 130.45, 129.57, 128.75(2C), 128.43, 128.22(2C),
128.04, 122.50, 120.97, 118.53(C-7), 54.23 (CH2–N1),
46.09 (CH2–N3); IR (KBr, cmÀ1): 3108 (Ar–H), 1669
(C@O), 1538 (Ar, C–C), 1373, 1167 (SO2); ESI-MS: m/z
463.2 [M+], 465.2 [M + 2].
4.1.3.7. 1-(2,4-Dichloro)benzyl-3-benzyl-thieno[3,2-c]-
[1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (5g). Reagents:
Compound 4 (1.2 g, 4 mmol), 2,4-dichlorobenzyl chloride
(1.17 g, 6 mmol). Conditions: 80 ꢁC, 24 h. Purification:
recrystallization. Yield 0.95 g (52%) as a white solid:
1
mp 112–114 ꢁC (EtOH); H NMR (DMSO-d6) d: 8.18
(d, 1H, J = 5.3 Hz, thiophene), 7.67–7.28 (m, 8H, ben-
zene), 7.15 (d, 1H, J = 5.3 Hz, thiophene), 5.18 (s, 2H,
CH2); 5.03 (s, 2H, CH2); 13C NMR (DMSO-d6) d:
157.61 (C@O), 143.54(C-1), 136.48(C-1), 136.07, 133.91,
133.51, 131.79, 131.28, 129.43, 128.68(2C), 128.12 (2C),
127.98, 127.88, 121.22, 118.70(C-7), 51.51 (CH2–N1),
45.97 (CH2–N3); IR (KBr, cmÀ1): 3095 (Ar–H), 1652
(C@O), 1536 (Ar, C–C), 1375, 1185 (SO2); ESI-MS: m/z
453.3 [M+].

162
Y. Lin et al. / Bioorg. Med. Chem. 16 (2008) 157–163
4.1.3.8. 1-(4-Chloro)benzyl-3-benzyl-thieno[3,2-c][1,2,6]-
4.1.3.12. 1-Cynomethyl-3-benzyl-thieno[3,2-c][1,2,6]-
thiadiazin-4(3H)-one 2,2-dioxides (5l). Reagents: Com-
pound 4 (1.2 g, 4 mmol), chloroacetonitrile (0.45 g,
6 mmol). Conditions: 80 ꢁC, 24 h. Purification: recrys-
tallization. Yield 0.80 g (60%) as a white solid: mp
thiadiazin-4(3H)-one 2,2-dioxide (5h). Reagents: Com-
pound 4 (1.2 g, 4 mmol), 4-chlorobenzyl chloride (1.0 g,
6 mmol). Conditions: 80 ꢁC, 18 h. Purification: recrystalli-
zation. Yield 0.60 g (36%) as a white solid: mp 77–79 ꢁC
(EtOH); 1H NMR (DMSO-d6) d: 8.22 (d, 1H, J = 5.3 Hz,
thiophene), 7.42–7.14 (m, 10H, benzene and thiophene),
5.15 (s, 2H, CH2); 4.98 (s, 2H, CH2); 13C NMR (DEPT)
(DMSO-d6) d: 157.48 (C@O), 143.22(C-1), 136.64(C-1),
136.02, 133.57, 133.16, 130.07(2C), 128.78(2C), 128.66
(2C), 128.17(2C), 127.98, 121.24, 118.75(C-7), 53.26
(CH2–N1), 45.71 (CH2–N3); IR (KBr, cmÀ1): 3112 (Ar–
H), 1672 (C@O), 1535 (Ar, C–C), 1376, 1173 (SO2); ESI-
MS: m/z 419.2 [M + 1].
1
148–150 ꢁC (EtOH); H NMR (DMSO-d6) d: 8.35 (d,
1H, J = 5.3 Hz, thiophene), 7.53–7.35 (m, 5H, benzene),
7.33 (d, 1H, J = 7.14 Hz, thiophene), 5.24 (s, 2H, CH2);
5.07 (s, 2H, CH2); 13C NMR (DMSO-d6) d: 157.30
(C@O), 142.11(C-1), 137.25, 135.75, 128.71(2C),
128.11(2C), 128.09, 122.01, 120.93(C-7), 114.69 (CN),
46.45 (CH2–N3), 39.24 (CH2–N1); IR (KBr, cmÀ1):
3091 (Ar–H), 1686 (C@O), 1527 (Ar, C–C), 1384, 1185
(SO2); ESI-MS: m/z 334.5 [M + 1].
4.1.3.9. 1-(4-Bromo)benzyl-3-benzyl-thieno[3,2-c][1,2,6]
thiadiazin-4(3H)-one 2,2-dioxide (5i). Reagents: Com-
pound 4 (1.2 g, 4 mmol), 4-bromobenzyl chloride
(1.5 g, 6 mmol). Conditions: 60 ꢁC, 8 h. Purification:
recrystallization. Yield 0.50 g (27%) as a white solid:
4.1.3.13. 1-Methyl-3-benzyl-thieno[3,2-c][1,2,6]thiadia-
zin-4(3H)-one 2,2-dioxides (5m). Reagents: Compound 4
(1.2 g, 4 mmol), (CH3)2SO4 (0.76 g, 6 mmol). Conditions:
60 ꢁC, 8 h. Purification: recrystallization. Yield 0.30 g
(24.3%) as a white solid: mp 94–96 ꢁC (EtOH); 1H
NMR (DMSO-d6) d: 8.24 (d, 1H, J = 5.3 Hz, thiophene),
7.41–7.36 (m, 5H, benzene), 7.35, 5.04 (s, 2H, CH2), 3.41
(3H); 13C NMR (DMSO-d6) d: 157.66 (C@O), 145.34(C-
1), 136.56, 136.27, 128.70, 127.94(2C), 127.92(2C),
121.06, 120.95, 117.24, 45.75 (CH2–N3), 37.06 (CH3-
N1); IR (KBr, cmÀ1): 3102 (Ar–H), 1666 (C@O), 1540
(Ar, C–C), 1368, 1175 (SO2); ESI-MS: m/z 309.5 [M + 1].
1
mp 91–93 ꢁC (EtOH); H NMR (DMSO-d6) d: 8.22 (d,
1H, J = 5.3 Hz, thiophene), 7.45–7.09 (m, 10H, benzene
and thiophene), 5.14 (s, 2H, CH2), 4.98 (s, 2H, CH2);
13C NMR (DMSO-d6) d: 157.48 (C@O), 143.22(C-1),
136.66(C-1), 136.01, 133.99, 131.71(2C), 130.36(2C),
128.67(2C), 128.17(2C), 127.97, 121.78, 121.21, 118.69
(C-7), 53.29 (CH2–N1), 45.71 (CH2–N3); IR (KBr,
cmÀ1): 3111(Ar–H), 1673 (C@O), 1536 (Ar, C–C),
1374, 1171 (SO2); ESI-MS: m/z 463.2 [M+], 465.2
[M + 2].
4.2. Anti-HIV activity assays
The anti-HIV activity and cytotoxicity were evaluated
against HIV-1 strain IIIB and HIV-2 (ROD) in MT-4
cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) method.17 MT-4 cells were
suspended in culture medium at 1 · 105 cells/ mL and in-
fected with HIV at a multiplicity of infection (MOI) of
0.02. Immediately after viral infection, 100 lL of the cell
suspension was placed in each well of a flat-bottomed
microtiter tray containing various concentrations of
the test compounds. Stock solutions of the test com-
pounds were prepared in DMSO at a concentration of
10 mg/ml. After 4 days of incubation at 37 ꢁC, the num-
ber of viable cells was determined using the MTT meth-
od. Compounds were tested in parallel for cytotoxic
effects in uninfected MT-4 cells.
4.1.3.10. 1-(4-Cyano)benzyl-3-benzyl-thieno[3,2-c][1,2,6]-
thiadiazin-4(3H)-one 2,2-dioxide (5j). Reagents: Com-
pound 4 (1.2 g, 4 mmol), 4-cyanobenzyl chloride (0.91 g,
6 mmol). Conditions: 80 ꢁC, 24 h. Purification: recrystalli-
zation. Yield 0.50 g (31%) as a white solid: mp 118–119 ꢁC
(EtOH); 1H NMR (DMSO-d6) d: 8.24 (d, 1H, J = 5.3 Hz,
thiophene), 7.74–7.73 (q, 2H, benzene H-4, H-5), 7.41–
7.28 (m, 8H, benzene and thiophene), 5.27 (s, 2H, CH2),
4.99 (s, 2H, CH2); 13C NMR (DMSO-d6) d: 157.45
(C@O), 143.29(C-1), 140.38(C-1), 136.90, 136.00, 132.74
(2C), 128.79(2C), 128.69(2C), 128.18(2C), 128.02, 120.92,
118.62 (CN), 118.35(C-7), 111.20(C-4), 53.21 (CH2–N1),
45.76 (CH2–N3); IR (KBr, cmÀ1): 3113 (Ar–H), 2226
(CN),1686 (C@O), 1533 (Ar, C–C), 1366, 1177 (SO2);
ESI-MS: m/z 410.5 [M + 1].
The 50% effective antiviral concentration (EC50) was de-
fined as the compound concentration required to protect
50% of the virus-infected cells against viral cytopathic-
ity. The 50% cytotoxic concentration (CC50) was defined
as the compound concentration required to reduce the
viability of mock-infected cells by 50%. The symbol
‘ > ’ is used to indicate the highest concentration at
which the compounds were tested and still found to be
non-cytotoxic. Average EC50 and CC50 values for at
least two separate experiments are presented.
4.1.3.11. 1-(4-Nitro)benzyl-3-benzyl-thieno[3,2-c][1,2,6]-
thiadiazin-4(3H)-one 2,2-dioxide (5k). Reagents: Com-
pound 4 (1.2 g, 4 mmol), 4-nitrobenzyl bromide (0.86 g,
6 mmol). Conditions: 60 ꢁC, 8 h. Purification: recrystalli-
zation. Yield 0.80 g (47%) as a yellow solid: mp
147–149 ꢁC (EtOH); 1H NMR (DMSO-d6) d: 8.24
(d, 1H, J = 5.3 Hz, thiophene), 8.12–8.10 (m, 2H, benzene
H-3, H-5), 7.43–7.29 (m, 8H, benzene and thiophene),
5.32 (s, 2H, CH2), 5.00 (s, 2H, CH2); 13C NMR
(DEPT) (DMSO-d6) d: 157.46 (C@O), 147.39(C-1),
143.28(C-1), 142.37(C-4), 136.95, 136.01, 129.15(2C),
128.66(2C), 128.25(2C), 128.00, 123.93 (2C), 120.95,
118.43(C-7), 52.98 (CH2–N1), 45.78 (CH2–N3); IR
(KBr, cmÀ1): 3098 (Ar–H), 1684 (C@O), 1520 (Ar, C–
C), 1351 (NO2), 1380, 1177 (SO2); ESI-MS: m/z 430.4
[M + 1].
Acknowledgments
The financial support of this work by National Natural
Science Foundation of China (NSFC No. 30772629,
No. 30371686), Key Project of The International Cooper-

Y. Lin et al. / Bioorg. Med. Chem. 16 (2008) 157–163
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