Design, synthesis and biological evaluation of N2,N 4-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6] thiatriazine derivatives as HIV-1 NNRTIs

Article abstract of DOI:10.1016/j.bmc.2013.09.009

A series of N₂,N₄-disubstituted-1,1,3-trioxo-2H,4H- pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives (PTTDs) was designed and synthesized by a facile route. The biological assay results showed that five most potent compounds displayed inhibitory activity against HIV-1 at low micromolar concentrations (EC₅₀ = 5.1-8.9 μM). Structure-activity relationship analysis indicated that N₂-(3-halogenated-benzyl) analogues were more potent than N₂-(unsubstituted-benzyl) analogues. The N₄-substitutions contributed to the antiviral activity in the following order: 2-/3-cyano substituted benzyl > 2-/3-halogenated benzyl > non-substituted benzyl > 4-halogenated benzyl. Docking studies of the representative compound revealed the binding conformation of these compounds and provided critical insights for the further development of PTTD analogues.

Full text of DOI:10.1016/j.bmc.2013.09.009

Bioorganic & Medicinal Chemistry 21 (2013) 7091–7100
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of
N2,N4-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]
thiatriazine derivatives as HIV-1 NNRTIs
Wenmin Chen a, Peng Zhan a, Erik De Clercq b, Christophe Pannecouque b, Jan Balzarini b, Xin Jiang a,
Xinyong Liu a,
⇑
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University,
No. 44, West Culture Road, 250012 Jinan, Shandong, PR China
b Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 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:
A series of N2,N4-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives (PTTDs)
was designed and synthesized by a facile route. The biological assay results showed that five most potent
compounds displayed inhibitory activity against HIV-1 at low micromolar concentrations (EC50 = 5.1–
Received 10 August 2013
Revised 3 September 2013
Accepted 4 September 2013
Available online 14 September 2013
8.9 lM). Structure–activity relationship analysis indicated that N2-(3-halogenated-benzyl) analogues
were more potent than N2-(unsubstituted-benzyl) analogues. The N4-substitutions contributed to the
antiviral activity in the following order: 2-/3-cyano substituted benzyl > 2-/3-halogenated benzyl >
non-substituted benzyl > 4-halogenated benzyl. Docking studies of the representative compound
revealed the binding conformation of these compounds and provided critical insights for the further
development of PTTD analogues.
Keywords:
HIV
Reverse transcriptase
NNRTIs
Thienothiadiazine
Pyrrolothiatriazine
Heterocycle
Drug design
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
wild-type HIV-1 strains as well as a variety of mutant HIV-1
strains. Prototype compounds QM96521, QM96539, QM96639,
Non-nucleoside reverse transcriptase inhibitors (NNRTIs),
which target an allosteric pocket of reverse transcriptase (RT), rep-
resent an important component of drug combination therapy for
the treatment of HIV-infected patients.1–3 Up to date, five NNRTIs
have been approved for clinical application by the FDA: nevirapine
(NVP),4 delavirdine (DLV),5 efavirenz (EFV),6 etravirine (ETR),7 and
rilpivirine (RPV).8 First generation of NNRTIs, NVP and EFV suffer
from severe side-effects during long term use, as well as an inevi-
table emergence of HIV-1-resistant strains.3 Although the second
generation of NNRTIs, ETR, and RPV perform better against a vari-
ety of variants bearing clinically prevalent mutations,9–11 the
development of novel NNRTI families are still demanded to address
issues of side effects, poor solubility, cross-resistance, and virologic
failure.12,13
and QM96652 were endowed with EC50 values in the low micro-
molar range in HIV-1 infected MT-4 cell cultures (Fig. 1).14–16 Ini-
tial structure–activity relationship (SAR) analysis revealed that
dual substitutions containing
p-electrons at the N2 and N4 sites
of TTDs were necessary for antiviral activity. Particularly, com-
pounds with a benzyl or 3-halogenated benzyl moieties connected
at the N2 position combined with a cyanomethyl group or a halo-
genated benzyl moiety substituted at the N4 position showed opti-
mal activities. With the aim to develop novel heterobicyclic
compounds structurally related to TTDs, our team has synthesized
a collection of novel five-membered heterocycle-fused thiadia-
zines, viz. pyrazolo[4,5-e][2,1,3]thiadiazines (PTDDs) and 7-meth-
ylpyrazolo[4,5-e][1,2,4]thiadiazines (PTDs),17–21 generally with
diminished or even annihilated anti-HIV activity.
In 1998, a class of 2,4-disubstituted-1,1,3-trioxo-2H,4H-thie-
no[3,4-e][1,2,4]thiadiazine derivatives (TTDs) was discovered as
potent NNRTIs, which can effectively inhibit the replication of
Compared with TTDs, the physicochemical properties (calcu-
lated by molinspiration software)22–24 of PTDDs and PTDs display
no significant difference with the exception of the lower lipophil-
icity (expressed by log P), which affects permeability, hydrophobic
ligand–receptor interactions, and toxicity of bioactive compounds
(Fig. 1).25 This results indicated a preference for hydrophobicity
over hydrophilicity at the central heterobicyclic moiety in term
⇑
Corresponding author. Tel.: +86 531 88382005; fax: +86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.009

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W. Chen et al. / Bioorg. Med. Chem. 21 (2013) 7091–7100
Lead Compounds of TTDs
PTDDs
O
N
O
O
N
2
The Newly Designed PTTDs
S
N
N
S
O
N
N
S
O
O
O
N
N
O
X
S
N
R
N
4
X
R
PTDs
O
QM 96521: R=CN, X=H
O
X
O
QM
QM
96639: R=CN, X= F
R
S
R=CN, X=Cl
96539:
N
N
QM 96652: R=2-Cl-benzyl,
X=H
N
O
X
R
EC50: for the anti-HIV-1 activity; miLog P: molinspiration predicted Log P; natoms: no. of atoms; MW:
molecular weight; nON: no. of hydrogen bond acceptors; nOHNH: no.of hydrogen bond donors; nViol:
number of violations; nrotb: no. of rotatable bonds.
Figure 1. Structures and representative physicochemical properties of TTDs, PTDs, PTDDs, and PTTDs (physicochemical properties was predicted by a computational
molinspiration method).
of anti-HIV-1 inhibition. Based on these findings, we further de-
signed series of 2,4-disubstituted-1,1,3-trioxo-2H,4H-pyrrol-
the subsequent hydrolysis reaction in a one-pot procedure at room
temperature to give intermediate 1-(N-benzylsulfamoyl)-1H-pyr-
role-2-carboxylic acids (3a–d). Next, the carboxylic acids (3a–d)
were converted to corresponding acyl azides using diphenylphos-
phoryl azide (DPPA)32 in the presence of triethylamine, which were
subjected to thermally Curtius rearrangement at 70 °C to form
N2-substitued pyrrolo[1,2-b][1,2,4,6]thiatriazines (4a–d). Further
alkylation of intermediates (4a–d) at their N4 positions with differ-
ent benzylhalides or chloroacetonitrile in the presence of sodium
hydride afforded the desired N2,N4-disubstituted pyrrolo[1,2-
b][1,2,4,6]thiatriazine derivatives (5a–d). Spectral data of all com-
pounds are in full agreement with the proposed structures. To the
best of our knowledge, this is the first report about the synthesis of
this unique pyrrolo[1,2-b][1,2,4,6]thiatriazine ring system.
a
o[1,2-b][1,2,4,6]thiatriazine derivatives (PTTDs) based on the
general principle of bioisosteric replacement (Fig. 1). These PTTDs
are demonstrated to possess similar lipophilicity with TTDs by a
computational Molinspiration method, thus to maximally simulate
molecular properties of TTDs. Additionally, the optimal substitu-
tions at the N2 and N4 position of TTDs elucidated by SAR studies
were retained in the new compounds.26,27 To further exploit the
nature and the pattern of substitutions on the N4-benzyl ring, the
nitrile group, which is ubiquitous in many highly potent NNRTIs,28
such as UK-453061,29 MK-4965,30 IDX-899,31 and TMC-125,10 was
also introduced to the N4-benzyl moiety of PTTD to investigate its
influence on the antiviral activity. Herein, we report the synthesis
and anti-HIV activity of these PTTD analogues, as well as the
molecular modeling analysis of the representative compound
within the RT binding pocket.
2.2. Biological activity
The newly synthesized N2,N4-disubstituted pyrrolo[1,2-
b][1,2,4,6]thiatriazine analogues were evaluated for their anti-
HIV activity and cytotoxicity in MT-4 cell cultures infected with
wild-type HIV-1 strain (IIIB) or HIV-2 strain (ROD), respectively.
The biological evaluation results, expressed as 50% inhibitory con-
centration (EC50), 50% cytotoxic concentration (CC50), and SI (selec-
tivity, given by the CC50/EC50 ratio) values, are listed in Table 1. The
FDA-approved drug nevirapine (NVP) and lamivudine (3TC), and
zalcitabine (ddC) were used as reference drugs. For comparison,
previously reported antiviral data of QM96521 and QM96652 were
also included in Table 1.16
2. Results and discussion
2.1. Chemistry
The synthetic route for PTTDs is outlined in Scheme 1. The com-
mercially available pyrrole-2-carboxylic acid (1) was firstly con-
verted to its methyl ester (2) using SOCl2 in methanol. Then, the
methyl ester (2) was treated with N-benzylsulfamoyl chlorides5
in the presence of sodium hydride at 0 °C to afford methyl 1-(N-
benzylsulfamoyl)-1H-pyrrole-2-carboxylates, which underwent
O
O
O O
a
b
S
S
c
NH
NH
N
N
N
N
H
H
NHSO2Cl
COOH
COOH
CON3
COOCH3
X
X
X
3a-d
1
2
O
O
O
O
S
S
a X=H
N
N
heat
N
N
d
b X=F
c X=Cl
d X=Br
N
R
O
N
H
O
X
X
4a-d
5a1-5a13, 5b1-5b7,
5c1-5c2, 5d1-5d2
Scheme 1. Synthetic route of PTTDs. Reagents and conditions: (a) SOCl2, MeOH, 30 °C, rt; (b) NaH, THF, 0 °C then aq NaOH, room temperature, rt; (c) DPPA, Et3N, dioxane,
70 °C; (d) DMF, NaH, RCl or RBr, 50 °C.

W. Chen et al. / Bioorg. Med. Chem. 21 (2013) 7091–7100
7093
Table 1
Anti-HIV activity and cytotoxicity of N2,N4-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives (PTTDs) (5a–d)a
O
O
S
X
N
N
N
R
O
Compd
X
R
HIV-1 (IIIB)
HIV-2 (ROD)
a
b
a
b
EC50
(lM)
CC50
(
lM)
SIc
EC50
(lM)
CC50
(
lM)
SIc
5a1
5a2
5a3
5a4
5a5
5a6
5a7
5a8
H
H
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
F
F
F
Cl
Cl
Br
Br
Benzyl
43
5.1
6.5
23
30
40
>218
>190
>241
>187
>318
>802
25
52
5.6
11
29
7.2
>249
34
8.9
>251
18
196
>318
>318
291
228
196
218
190
241
187
>318
>802
226
288
>305
231
>269
208
249
172
>293
251
>260
>243
>119
503
5
>267
>318
>318
>311
>226
>239
>273
>150
>241
>180
>318
>802
>226
>324
>305
>262
>269
>212
>275
>187
>293
>265
>260
>243
267
<1
ꢀ1
ꢀ1
ꢀ1
<1
<1
<1
<1
<1
<1
ꢀ1
2-CN-benzyl
3-CN-benzyl
2-Cl-benzyl
2-Br-benzyl
3-Cl-benzyl
2,4-Di-Cl-benzyl
4-F-benzyl
4-Me-benzyl
CH2CN
Cinnamyl
>63
>49
12
8
5
<1
<1
<1
<1
ꢀ1
> or ꢀ1
9
6
>55
21
>9
29
<1
5
>318
>318
>311
226
239
273
150
241
180
>318
>802
226
>324
>305
262
>269
212
>275
187
5a9
5a10
5a11
5a12
5a13
5b1
5b2
5b3
5b4
5b5
5b6
5b7
5c1
5c2
5d1
5d2
Allyl
< or ꢀ1
<1
3-Methylbut-2-en-1-yl
Benzyl
ꢀ1
ꢀ1
<1
2-CN-benzyl
2-Cl-benzyl
2-Br-benzyl
3-Cl-benzyl
2,4-Di-Cl-benzyl
CH2CN
2-CN-benzyl
2,4-Di-Cl-benzyl
2-Cl-benzyl
2,4-Di-Cl-benzyl
ꢀ1
<1
ꢀ1
<1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
>33
<1
>293
>265
>260
>243
>14
ꢀ1
>119
559
>133
>47
>68
>243
0.1
0.9
0.11 0.09
1.8 0.6
1.4 0.05
QM96625d
QM96521d
NVPe
3TCe
ddCe
>15
87
>95
5.9 5.2
>87
>15
a
b
c
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-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).
d
e
The synthesis and antiviral properties of these lead compounds were previously described.16
These data were obtained from the same laboratory (Rega Institute for Medical Research, KU Leuven, Belgium).
According to the biological results, 15 PTTDs analogues were
found to be active against HIV-1 (IIIB) with EC50 values in the range
of 5.1–52 M, but none of the compounds was active against HIV-2
(ROD). The optimal activity against HIV-1 (IIIB) was observed with
compound 5a2 and 5b2, with EC50 values of 5.1 M and 5.6 M,
and SI values of >63 and >55, respectively, inferior to those of
the lead NNRTI compounds QM96625, QM96521 and NVP, but in
the same order of magnitude as that of the NNRTI reference drugs
ddC and 3TC. These results indicated that the pyrrolo[1,2-
b][1,2,4,6]thiatriazine ring (PTTD) is a promising bioisosteric moi-
ety of thieno[3,4-e][1,2,4]thiadiazine ring in the NNRTI lead
compounds.
As shown in Table 1, the substitution pattern of the N4 benzyl
moiety of PTTDs played a determinant role in their HIV-1 inhibi-
tory potency. Analysis of the anti-HIV-1 activity of PTTDs featuring
the same N2-benzyl group (5a1–5a13) revealed that the presence
of a CN group at the 2- or 3-position of the benzene ring of the
N4-benzyl group furnished two most potent compounds 5a2 and
N4-benzyl ring abolished their anti-HIV-1 activity, which was
exemplified by N4-(2,4-di-Cl-benzyl) analogue 5a7, N4-(4-F-ben-
zyl) compound 5a8, and N4-(4-Me-benzyl) analogue 5a9. These re-
sults indicated that the activities of PTTDs were significantly
affected by the electronic nature or steric hindrance of the para-
substitution at the N4-benzyl ring. In summary, the substitutions
at the N4 position contributed to the antiviral activity of PTTDs in
the following order: 2-/3-cyano substituted benzyl >2-/3-haloge-
nated benzyl > non-substituted benzyl > 4-substituted benzyl.
The same conclusion was also drawn from the SAR analysis of
the analogues featuring the same N2-(3-F-benzyl) group (5b1–
5b7): N4-(2-CN-benzyl) analogue 5b2 showed the best activity;
N4-(2-/3-halogenated benzyl) compounds (5b3–5b5) exhibited
better activity than non-substituted compound 5b1; N4-(2,4-di-
Cl-benzyl) compound 5b7 was completely inactive. In conclusion,
the SAR pattern of N4-benzyl substitutions of PTTDs was roughly
consistent with that of TTDs.26,33
l
l
l
In addition to different benzyl groups, other groups containing
5a3 (EC50 = 5.1
lM, SI >63; EC50 = 6.5
l
M, SI >55, respectively).
p-electrons, including cyanomethyl, cinnamyl, allyl, and a 3-meth-
Compounds (5a4–5a6) bearing a halogen atom (Cl or Br) at the
2- or 3-position of the N4-benzyl ring exhibited higher activities
compared with corresponding non-substituted compound 5a1.
However, the presence of a substitution at the 4-position of the
ylbut-2-en-1-yl group, were also introduced to the N4 position of
PTTDs to further explore the diversity of the N4-susbtitution.
Unfortunately, only 5a13 and 5b7 bearing a 3-methylbut-2-en-1-
yl group and a cyanomethyl group respectively at the N4 position

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W. Chen et al. / Bioorg. Med. Chem. 21 (2013) 7091–7100
exhibited moderate potency (EC50 = 25
respectively).
l
M
and 34 lM,
From the SAR point of view, introduction of halogens to the
3-position of the N2-benzyl ring generally brought a marked
improvement on the anti-HIV-1 activity and selectivity. For in-
stance, all the active N2-(3-F-benzyl) compounds (5b1–5b5)
showed improved anti-HIV-1 activity and higher SI values than
corresponding N2-benzyl compounds. However, all analogues
(5a6, 5b6, 5c6) bearing a cyano group at the N4 benzyl ring exhib-
ited similar antiviral activities (EC50 = 5.1, 5.6, 8.9 lM, respec-
tively), regardless of the substitution patterns of the N2-benzyl
groups. To some extent, these results further confirmed that the
cyano group, as the ‘privileged fragment’ of NNRTIs, could often re-
tain optimal biological activity compared with other counterparts.
2.3. Inhibition of HIV-1 RT
To confirm the drug target of PTTDs, the representative com-
pounds (5a2 and 5b2) endowed with the highest anti-HIV-1 po-
tency in MT-4 cell cultures were further evaluated for their
in vitro HIV-1 RT inhibitory activities using a RT assay Kit. As illus-
trated in Table 2, compounds 5a2 and 5b2 exhibited moderate
anti-RT activity at micromolar concentrations. Although the anti-
RT activity of PTTD analogues 5a2 and 5b2 was less potent than
the lead compound NVP, these biological results proved that the
newly PTTD derivatives exerted the anti-HIV-1 activity by inhibit-
ing RT, to some extent.
Figure 2. Superposition of the TTD lead compound Q96652 (purple), PTTD
analogue 5b2 (green) and efavirenz (pink) in the binding pocket of RT. (Docking
result was shown by PyMOL. Proposed hydrogen bonds and halogen bonds are
indicated with dashed lines in red.)
2.4. Molecular modeling analysis
ety of the K101 residue, which may contribute substantially to the
free energy of binding for the inhibitor. The N4-2-CN-benzyl groups
of 5b2 which acted as the cyclopropyl–propynyl group of efavirenz
and were positioned in the top sub-pocket, apparently developed
extensive van der Waals and p–p stacking interactions with adja-
cent aromatic residues of Tyr181, Tyr188, and Trp229. It was well
Molecular modeling of the lead compound Q96652 and the rep-
resentative PTTD compound 5b2 by docking into the NNRTI bind-
ing pocket (NNIBP) of HIV-1 RT was performed using Surflex-Dock
module of SYBYL-X 1.1 to predict the binding mode and rationalize
the SAR conclusion. Coordinates of the NNIBP were taken from the
crystal structure of the RT complex with efavirenz (PDB code:
IFK9),34 which also contains a fused heterocyclic scaffold.
known that introduction of a cyano group at the phenyl ring lo-
cated in this Tyr181–Tyr188–Trp229 p-rich region furnished many
highly potent compounds, such as UK-453061,29 MK-4965,30 and
IDX-899.31 This is also true for PTTD analogues, which was vali-
dated by the most potent compounds 5a2 and 5b2 that were char-
acterized by a cyano group at the N4-benzyl ring. This observation
further proved the cyano group to be a ‘privileged fragment’ in
NNRTIs.28 It seems possible through introduction of the 3,5-diCN
group at the N4-benzyl ring which is known to be able to make
compensatory contacts with the conserved residue Trp229, to im-
prove the resistance profiles of PTTDs. This drug design strategy
should be considered in the further optimization of PTTD com-
pounds. In addition, it was noticed that the 4-position of the
N4-benzyl group pointed to the indole ring of Trp229, with a min-
imal distance of 2.86 Å. Therefore, introduction of substitutions to
the 4-position of the N4-benzyl ring was likely to cause a steric
hindrance, thus to destroy the favorable binding mode, which
may explain the disappointing activity observed with all the
N4-(4-substitued benzyl) analogues.
The docking results suggested that PTTD 5b2 was fairly super-
posable to the TTD lead compound Q966525, and both of them
have similar binding orientations and conformation with that of
efavirenz. As illustrated in Figure 2, the pyrrolothiatriazine ring
of 5b2, which overlapped with the thienothiadiazine ring of
Q966525, was sandwiched between the side chains of Val106
and Try318, as expected, and involved in a series of hydrophobic
contacts. In this case, pyrrolothiatriazine had been shown to act
as essential pharmacophore elements, favorably contributing to li-
gand binding. The observation supports our initial presumption
that the lipophilic properties of the central heterobicyclic ring
has significant influence on the biological activity, indicating that
pyrrolothiatriazine could serve as a suitable surrogate for the thie-
nothiadiazine ring of TTDs.
Additionally, pyrrolothiatriazine serving as the core building
block, oriented its N2 and N4 substituents in the appropriate con-
formation. As the extension of pyrrolothiatriazine, the N2-(3-F-
benzyl) group was tolerated by the entrance channel defined by
the residues of Lys103, Glu138 and Val179. The fluoro atom was
found to be engaged in a halogen bond with the backbone NH moi-
Thus, the docking simulation helped to interpret the binding
conformation and to identify the potential key pharmacophore ele-
ments of PTTDs, which are in agreement with the SAR and provide
guide for the design of more potent NNRTIs with this scaffold.
3. Conclusion
Table 2
In vitro inhibitory activity of representative PTTD compounds against HIV-1 RT
In summary, based on the analysis of SAR and predicted physi-
cochemical properties of TTD derivatives, a novel series of 2,4-
disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine
derivatives (PTTDs) was designed as potential NNRTIs based on the
principle of bioisosteric replacement. A facile methodology for the
Compound
aIC50
M)
5a2
5b2
NVP
(
l
56.9
58.3
6.2
a
IC50: inhibitory concentration required to inhibit biotin deoxyuridine triphos-
phate (biotin-dUTP) incorporation into the HIV-1 RT by 50% (RT kit, Roche).

W. Chen et al. / Bioorg. Med. Chem. 21 (2013) 7091–7100
7095
synthesis of 1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine
as an original heterobicyclic ring system was reported for the first
time. Subsequent biological evaluation for their anti-HIV-1 activi-
ties revealed that most of the synthesized compounds were effec-
tive to inhibit the replication of HIV-1 at micromolar
concentrations. The preliminary SAR and molecular modeling stud-
ies of these unique inhibitors provide valuable information for ra-
tional structural optimization. Further structural modifications are
warranted to develop novel PTTD analogues with higher potency
against mutant variants and lower cytotoxicity.
8.31 (s, 1H, NH), 12.84 (bs, 1H, OH); 13C NMR (75 MHz, DMSO-
d6) d: 47.5 (CH2), 109.4, 122.6, 122.4, 127.9, 128.1, 128.7, 129.7,
136.7, 161.2 (C@O); ESI-MS: 281.5 [M+H]+.
4.1.2.2. 1-(N-(3-Fluorobenzyl)sulfamoyl)-1H-pyrrole-2-carbox-
ylic acid (3b).
Reagents: methyl 2-pyrrolecarboxylate (2)
(1.88 g, 0.015 mol) and N-(3-fluororobenzyl)sulfamoyl chloride
(6.71 g, 0.030 mol). Product as a white solid, yield 56%. Mp: 120–
121 °C. ESI-MS: 295.5 [M+H]+.
4.1.2.3. 1-(N-(3-Chlorobenzyl)sulfamoyl)-1H-pyrrole-2-carbox-
ylic acid (3c).
Reagents: methyl 2-pyrrolecarboxylate (2)
4. Experimental
4.1. Chemistry
(1.88 g, 0.015 mol) and N-(3-chlorobenzyl)sulfamoyl chloride
(7.20 g, 0.030 mol). Product as a white solid, yield 59%. Mp: 115–
117 °C. ESI-MS: 315.4 [M+H]+, 317.3 [M+H]+.
The key reactants including pyrrole-2-carboxylic acid, benzyl-
amines, benzyl halides, alkyl halides, and DPPA were purchased
from Adamas-beta Co. Ltd. and used without further purification.
Thin layer chromatography was performed on pre-coated HUANG-
HAIÒ HSGF254, 0.15–0.2 mm TLC-plates. Flash column chroma-
tography was performed on columns packed with silica gel 60
(200–300 mesh). NMR spectra were recorded on INOVA-600
(600 MHz) or Bruker AVANCE-300 (300 MHz) NMR-spectrometer,
using solvents as indicated (CDCl3 or DMSO-d6) with tetramethyl-
silane (TMS) as internal standard. Chemical shifts were reported in
d values (ppm), signals are expressed as s (singlet), d (doublet), t
(triplet), q (quartet) or m (multiplet), and J values were reported
in hertz (Hz). Mass spectrometry was performed on a LC Autosam-
pler Device: Standard G1313A instrument. Melting points were
determined on a micromelting point apparatus RY-1 (produced
by Tian Jin Analytical Instrument Factory) and are uncorrected.
4.1.2.4. 1-(N-(3-Bromobenzyl)sulfamoyl)-1H-pyrrole-2-carbox-
ylic acid (3d).
Reagents: methyl 2-pyrrolecarboxylate (2)
(1.88 g, 0.015 mol) and N-(3-bromobenzyl) sulfamoyl chloride
(8.54 g, 0.030 mol). Product as a white solid, yield 59%. Mp: 110–
112 °C. ESI-MS: 361.3 [M+H]+, 359.4 [M+H]+.
4.1.3. General procedure for the preparation of N2-benzyl-1,1,3-
trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazines (4a–d)
To a solution of intermediates 3a–d (0.010 mol) and diphenyl-
phosphoryl azide (DPPA) (3.3 g, 0.012 mol) in anhydrous 1,4-diox-
ane was added triethylamine (1.2 g, 0.012 mol). The reaction
mixture was stirred at 70 °C for 12 h. Then, the solvent was re-
moved under reduced pressure. Further, the residue was purified
by column chromatography using ethyl acetate/petroleum ether
as eluent to afford N2-benzyl-1,1,3-trioxo-2H,4H-pyrrolo[1,2-
b][1,2,4,6]thiatriazines (4a–d).
4.1.1. The preparation of methyl 1H-pyrrole-2-carboxylate (2)
To a solution of pyrrole-2-carboxylic acid (1) (16.65 g, 0.15 mol)
in 10 mL of MeOH was added 50 mL of SOCl2 dropwise within
30 min at 0 °C. Subsequently, the reaction mixture was stirred at
35 °C for 24 h. Then, the solvent was evaporated in vacuo, and
the residue was purified by column chromatography using ethyl
acetate/petroleum ether as eluent, giving intermediate 2 as a white
solid, yield 85%, mp: 72–73 °C.
4.1.3.1. N2-Benzyl-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thi-
atriazine (4a).
Reagents: 3a (2.77 g, 0.01 mol) and DPPA
(3.30 g, 0.012 mol). Product as a white solid, yield 58%. Mp: 128–
130 °C; 1H NMR (300 MHz, DMSO-d6) d: 5.02 (s, 2H, CH2), 5.66
(dd, J = 3.60 Hz, 1.50 Hz, 1H, pyrrole-H), 6.42 (t, J = 3.40 Hz, 1H,
pyrrole-H), 7.13 (dd, J = 3.30 Hz, 1.50 Hz, 1H, pyrrole-H), 7.27–
7.38 (m, 5H, Ar-H), 11.92 (s, 1H, NH); 13C NMR (75 MHz, DMSO-
d6) d: 45.9 (CH2), 93.3, 110.8, 114.4, 136.4, 128.3, 128.9, 128.4,
129.0, 147.9 (C@O); ESI-MS: 278.3 [M+H]+.
4.1.2. General procedure for the preparation of 1-(N-
benzylsulfamoyl)-1H-pyrrole-2-carboxylic acids (3a–d)
To an anhydrous THF suspension of sodium hydride (60% dis-
persion in mineral oil, 1.80 g, 0.045 mol) was added methyl 2-pyr-
rolecarboxylate (2) (1.88 g, 0.015 mol) by portions at 0 °C. The
resulting mixture was stirred for 30 min and then corresponding
N-benzylsulfamoyl chlorides (0.03 mol) dissolved in anhydrous
THF was added dropwise at 0 °C. The mixture was stirred for
12 h at room temperature. Then, water was cautiously added to
quench the reaction, followed by addition of 30 mL of 5 N aqueous
NaOH solution. This mixture was stirred for another 12 h. Then, the
organic layer was evaporated in vacuo and the remaining aqueous
layer was acidified with concentrated hydrochloric acid to adjust
to pH 9. The resulting precipitate was filtered-off and purified by
column chromatography using ethyl acetate/petroleum ether as
eluent to give intermediates 3a–d.
4.1.3.2.
b][1,2,4,6]thiatriazine (4b).
N2-(3-Fluorobenzyl)-1,1,3-trioxo-2H,4H-pyrrolo[1,2-
Reagents: 3b (2.98 g, 0.01 mol)
and DPPA (3.30 g, 0.012 mol). Product as a white solid, yield 25%.
Mp: 122–124 °C. ESI-MS: 296.3 [M+H]+.
4.1.3.3.
b][1,2,4,6] thiatriazine (4c).
N2-(3-Chlorobenzyl)-1,1,3-trioxo-2H,4H-pyrrolo[1,2-
Reagents: 3c (3.15 g, 0.01 mol)
and DPPA (3.30 g, 0.012 mol). Product as a white solid, yield 29%.
Mp: 117–119 °C. ESI-MS: 312.3 [M+H]+, 314.2 [M+H]+.
4.1.3.4.
b][1,2,4,6] thiatriazine (4d).
N2-(3-Bromobenzyl)-1,1,3-trioxo-2H,4H-pyrrolo[1,2-
Reagents: 3d (3.59 g, 0.01 mol)
and DPPA (3.30 g, 0.012 mol). Product as a white solid, yield 30%.
Mp: 115–117 °C. ESI-MS: 358.2 [M+H]+, 356.3 [M+H]+.
4.1.4. General procedure for the preparation of
4.1.2.1. 1-(N-Benzylsulfamoyl)-1H-pyrrole-2-carboxylic acid
N2,N4-disubstitued-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]
thiatriazines (5a1–5a13, 5b1–5b7, 5c1–5c2, 5d1–5d2)
(3a).
Reagents: methyl 2-pyrrolecarboxylate (2) (1.88 g,
0.015 mol) and N-benzylsulfamoyl chloride (6.17 g, 0.030 mol).
Product as a white solid, yield 82%, mp: 124–126 °C. 1H NMR
(300 MHz, DMSO-d6) d: 4.21 (s, 2H, CH2), 6.18 (t, J = 3.30 Hz, 1H,
pyrrole-H), 6.92 (dd, J = 3.60 Hz, J = 1.80 Hz, 1H, pyrrole-H), 7.17–
7.25 (m, 5H, Ar-H), 7.32 (dd, J = 3.00 Hz, 1.8 Hz, 1H, pyrrole-H),
To a solution of the 3a–d (2 mmol) in dry DMF (10 mL) was
added sodium hydride (60% dispersion in mineral oil, 96 mg,
2.4 mmol) at 0 °C. After stirring for 15 min, the alkyl halide
(2.4 mmol) was added at 0 °C. The resulting mixture was stirred
at 75 °C for 12 h. Thereafter, the solvent was evaporated off in

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W. Chen et al. / Bioorg. Med. Chem. 21 (2013) 7091–7100
vacuo. The residue was purified by flash column chromatography
using dichloromethane/petroleum ether as eluent to give target
compounds.
was obtained from reaction of compound 4a (0.55 g, 2 mmol) with
2-bromobenzyl chloride (0.60 g, 2.4 mmol), and purified by flash
column chromatography using dichloromethane/petroleum ether
as eluent. Product as a white solid: yield 20%, mp: 115–117 °C.
1H NMR (600 MHz, DMSO-d6) d: 5.11 (s, 2H, CH2), 5.13 (s, 2H,
CH2), 5.9 (dd, J = 3.60 Hz, 1.20 Hz, 1H, pyrrole-H), 6.5 (t,
J = 3.60 Hz, 1H, pyrrole-H), 7.06 (d, J = 7.80 Hz, 1H, Ar-H), 7.25 (t,
J = 7.20 Hz, 1H, Ar-H), 7.30 (dd, J = 3.60 Hz, 1.20 Hz, 1H, pyrrole-
H), 7.32 (t, J = 7.20 Hz, 2H, Ar-H), 7.36–7.40 (m, 4H, Ar-H), 7.68
(d, J = 7.20 Hz, 1H, Ar-H); 13C NMR (75 MHz, CDCl3) d: 47.3 (CH2),
49.6 (CH2), 94.8, 111.3, 113.3, 122.5, 126.8, 128.0, 128.5, 128.7,
128.9, 129.2, 129.6, 133.0, 133.5, 135.0, 148.6 (C@O); ESI-MS:
446.2 [M+H]+, 448.2 [M+H]+.
4.1.4.1.
N2-Benzyl-N4-benzyl-1,1,3-trioxo-2H,4H-pyrrolo[1,2-
This compound was obtained
b][1,2,4,6] thiatriazine (5a1).
from reaction of compound 4a (0.55 g, 2 mmol) with benzyl bro-
mide (0.41 g, 2.4 mmol), and purified by flash column chromatog-
raphy using dichloromethane/petroleum ether as eluent. Product
as a white solid, yield 34%, mp: 113–117 °C. 1H NMR (600 MHz,
DMSO-d6) d: 5.10 (s, 2H, CH2), 5.18 (s, 2H, CH2), 6.00 (d,
J = 3.00 Hz, 1H, pyrrole-H), 6.45 (t, J = 3.00 Hz, 1H, pyrrole-H),
7.15 (t, J = 7.50 Hz, 1H, Ar-H), 7.22–7.27 (m, 4H, Ar-H and pyr-
role-H), 7.32–7.38 (m, 6H, Ar-H); 13C NMR (100 MHz, CDCl3) d:
47.2 (CH2), 49.7 (CH2), 94.9, 111.0, 113.3, 126.8, 127.9, 128.4,
128.7, 128.8, 128.9, 134.9, 135.2, 148.7 (C@O); ESI-MS: 368.2
[M+H]+.
4.1.4.6. N2-Benzyl-N4-(m-chlorobenzyl)-1,1,3-trioxo-2H,4H-pyr-
rolo[1,2-b][1,2,4,6]thiatriazine (5a6).
This compound was
obtained from reaction of compound 4a (0.55 g, 2 mmol) with
3-chlorobenzyl chloride (0.39 g, 2.4 mmol), and purified by flash
column chromatography using dichloromethane/petroleum ether
as eluent. Product as a white solid: yield 26%, mp: 155–158 °C.
1H NMR (600 MHz, DMSO-d6) d: 5.12 (s, 2H, CH2), 5.14 (s, 2H,
CH2), 6.01 (dd, J = 3.60 Hz, 1.80 Hz, 1H, pyrrole-H), 6.46 (t,
J = 3.60 Hz, 1H, pyrrole-H), 7.23 (d, J = 7.20 Hz, 1H, Ar-H), 7.27
(dd, J = 3.60 Hz, 1.80 Hz, 1H, pyrrole-H), 7.32–7.39 (m, 7H, Ar-H);
13C NMR (75 MHz, CDCl3) d: 47.3 (CH2), 49.2 (CH2), 94.7, 111.2,
113.2, 124.8, 127.0, 128.2, 128.5, 128.8, 129.6, 130.2, 134.8,
135.0, 137.0, 148.7 (C@O); ESI-MS: 402.3 [M+H]+, 404.3 [M+H]+.
4.1.4.2. N2-Benzyl-N4-(o-cyanobenzyl)-1,1,3-trioxo-2H,4H-pyr-
rolo[1,2-b][1,2,4,6]thiatriazine (5a2).
This compound was
obtained from reaction of compound 4a (0.55 g, 2 mmol) with
2-cyanobenzylchloride (0.37 g, 2.4 mmol), and purified by flash
column chromatography using dichloromethane/petroleum ether
as eluent. Product as a white solid: yield 27%, mp: 149–153 °C.
1H NMR (600 MHz, DMSO-d6) d: 5.10 (s, 2H, CH2), 5.31 (s, 2H,
CH2), 6.02 (dd, J = 3.60 Hz, 1.80 Hz, 1H, pyrrole-H), 6.48 (t,
J = 1.80 Hz, 1H, pyrrole-H), 7.28 (d, J = 8.40 Hz, 1H, Ar-H), 7.30 (d,
J = 3.60 Hz, 1.80 Hz, 1H, pyrrole-H), 7.32 (d, J = 7.20 Hz, 1H, Ar-H),
7.36–7.40 (m, 4H, Ar-H), 7.50 (t, J = 7.20 Hz, 1H, Ar-H), 7.67 (t,
J = 7.20 Hz, 1H, Ar-H), 7.90 (d, J = 7.80 Hz, 1H, Ar-H); 13C NMR
(75 MHz, CDCl3) d: 47.3 (CH2), 47.5 (CH2), 94.8, 111.3, 111.5,
113.3, 116.9, 126.2, 128.4, 128.5, 128.7, 128.8, 129.2, 133.1,
133.6, 134.9, 138.6, 148.8 (C@O); ESI-MS: 393.2 [M+H]+.
4.1.4.7. N2-Benzyl-N4-(2,4-dichlorobenzyl)-1,1,3-trioxo-2H,4H-
pyrrolo[1,2-b][1,2,4,6]thiatriazine (5a7).
This compound
was obtained from reaction of compound 4a (0.55 g, 2 mmol) with
2,4-dichlorobenzyl chloride (0.47 g, 2.4 mmol), and then purified
by flash column chromatography using dichloromethane/petro-
leum ether as eluent. Product as a white solid: yield 25%, mp:
122–124 °C. 1H NMR (600 MHz, DMSO-d6) d: 5.10 (s, 2H, CH2),
5.15 (s, 2H, CH2), 5.95 (dd, J = 3.60 Hz, 1.20 Hz, 1H, pyrrole-H),
6.45 (t, J = 3.60 Hz, 1H, pyrrole-H), 7.17 (d, J = 8.40 Hz, 1H, Ar-H),
7.29 (dd, J = 3.60 Hz, 1.20 Hz, 1H, pyrrole-H), 7.32–7.34 (m, 1H,
Ar-H), 7.36–7.38 (m, 4H, Ar-H), 7.41 (dd, J = 7.80 Hz, 2.10 Hz, 1H,
Ar-H), 7.70 (d, J = 1.80 Hz, 1H, Ar-H); 13C NMR (75 MHz, CDCl3) d:
46.6 (CH2), 47.4 (CH2), 94.7, 111.4, 113.3, 127.7, 127.9, 128.5,
128.7, 128.8, 129.4, 129.6, 130.7, 133.4, 134.2, 134.9, 148.6
(C@O); ESI-MS: 436.3 [M+H]+, 438.2 [M+H]+, 437.2 [M+H]+.
4.1.4.3. N2-Benzyl-N4-(m-cyanobenzyl)-1,1,3-trioxo-2H,4H-pyr-
rolo[1,2-b][1,2,4,6]thiatriazine (5a3).
This compound was
obtained from reaction of compound 4a (0.55 g, 2 mmol) with
3-cyanobenzyl bromide (0.47 g, 2.4 mmol), and purified by flash
column chromatography using dichloromethane/petroleum ether
as eluent. Product as a white solid: yield 32%, mp: 122–124 °C.
1H NMR (600 MHz, DMSO-d6) d: 5.12 (s, 2H, CH2), 5.18 (s, 2H,
CH2), 6.00 (dd, J = 3.60 Hz, 1.20 Hz, 1H, pyrrole-H), 6.46 (t,
J = 3.60 Hz, 1H, pyrrole-H), 7.28 (dd, J = 3.00 Hz, 1.20 Hz, 1H, pyr-
role-H), 7.30 (t, J = 6.60 Hz, 1H, Ar-H), 7.37–7.41 (m, 4H, Ar-H),
7.56–7.61 (m, 2H, Ar-H), 7.77–7.78 (m, 2H, 2H, Ar-H); 13C NMR
(75 Hz, CDCl3) d: 47.5 (CH2), 48.9 (CH2), 94.7, 111.5, 113.1, 113.2,
118.3, 128.6, 128.7, 128.8, 129.4, 129.8, 130.4, 131.1, 131.8,
134.8, 136.6, 148.7 (C@O); ESI-MS: 393.5 [M+H]+.
4.1.4.8. N2-Benzyl-N4-(p-fluorobenzyl)-1,1,3-trioxo-2H,4H-pyr-
rolo[1,2-b][1,2,4,6]thiatriazine (5a8).
This compound was
obtained from reaction of compound 4a (0.55 g, 2 mmol) with
4-fluorobenzyl chloride (0.35 g, 2.4 mmol), and purified by flash
column chromatography using dichloromethane/petroleum ether
as eluent. Product as a white solid: yield 22%, mp: 120–122 °C.
1H NMR (600 MHz, DMSO-d6) d: 5.01 (s, 2H, CH2), 5.11 (s, 2H,
CH2), 6.02 (dd, J = 3.60 Hz, 1.80 Hz, 1H, pyrrole-H), 6.5 (t,
J = 3.60 Hz, 1H, pyrrole-H), 7.18 (t, J = 9.00 Hz, 2H, Ar-H), 7.26 (d,
J = 1.20 Hz, 1H, pyrrole-H), 7.32–7.40 (m, 6H, Ar-H); 13C NMR
(100 MHz, CDCl3) d: 47.3 (CH2), 49.1 (CH2), 94.8, 111.2, 113.2,
115.8 (2JC–F = 22 Hz), 128.4, 128.6, 128.7, 128.8 (3JC–F = 6 Hz),
4.1.4.4. N2-Benzyl-N4-(o-chlorobenzyl)-1,1,3-trioxo-2H,4H-pyr-
rolo[1,2-b][1,2,4,6]thiatriazine (5a4).
This compound was
obtained from reaction of compound 4a (0.55 g, 2 mmol) with
2-chlorobenzyl chloride (0.39 g, 2.4 mmol) and purified by flash
column chromatography using dichloromethane/petroleum ether
as eluent. Product as a white solid: yield 27%, mp: 122–124 °C.
1H NMR (600 MHz, DMSO-d6) d: 5.11 (s, 2H, CH2), 5.18 (s, 2H,
CH2), 5.93 (dd, J = 3.60 Hz, 1.20 Hz, 1H, pyrrole-H), 6.45 (t,
J = 3.60 Hz, 1H, pyrrole-H), 7.12 (d, J = 7.20 Hz, 1H, pyrrole-H),
7.28–7.31 (m, 2H, Ar-H), 7.32–7.34 (m, 2H, Ar-H), 7.36–7.40 (m,
4H, Ar-H), 7.51 (d, J = 7.80 Hz, 1H, Ar-H); 13C NMR (75 MHz, CDCl3)
d: 47.0 (CH2), 49.3 (CH2), 94.7, 111.2, 113.2, 126.8, 127.4, 128.4,
128.7, 128.8, 128.9, 129.6, 129.7, 132.0, 132.7, 135.1, 148.6
(C@O); ESI-MS: 402.4 [M+H]+, 404.4 [M+H]+.
129.7, 130.
7
(4JC–F = 3 Hz), 135.1, 148.64 (C@O), 162.4
(1JC–F = 245 Hz); ESI-MS: 386.4 [M+H]+.
4.1.4.9. N2-Benzyl-N4-(p-methylbenzyl)-1,1,3-trioxo-2H,4H-pyr-
rolo[1,2-b][1,2,4,6]thiatriazine (5a9).
This compound was
obtained from reaction of compound 4a (0.55 g, 2 mmol) with
4-methylbenzyl chloride (0.34 g, 2.4 mmol), and purified by flash
column chromatography using dichloromethane/petroleum ether
as eluent. Product as a white solid, yield 75%, mp: 86–88 °C. 1H
NMR (400 MHz, CDCl3) d ppm: 2.31 (s, CH3, 3H), 5.00 (s, 2H,
4.1.4.5. N2-Benzyl-N4-(o-bromobenzyl)-benzyl-1,1,3-trioxo-2H,
4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine (5a5).
This compound

W. Chen et al. / Bioorg. Med. Chem. 21 (2013) 7091–7100
7097
CH2), 5.11 (s, 2H, CH2), 5.55 (dd, J = 3.48 Hz, 1.52 Hz, 1H, pyrrole-
H), 6.25 (t, J = 3.48 Hz, 1H, pyrrole-H), 6.89 (dd, J = 3.40 Hz,
1.52 Hz, 1 H, pyrrole-H), 7.09–7.14 (m, 4H, Ar-H), 7.30–7.36 (m,
3H, Ar-H), 7.47 (d, J = 2.60 Hz, 2H, Ar-H); 13C NMR (100 MHz,
CDCl3) d ppm: 21.1 (CH3), 47.2 (CH2), 49.6 (CH2), 94.8, 110.9,
113.2, 126.8, 128.3, 128.7, 128.8, 129.5, 129.9, 131.9, 135.2, 148.6
(C@O); ESI-MS: 382.5 [M+H]+.
was obtained from reaction of compound 4b (0.59 g, 2 mmol) with
benzyl bromide (0.41 g, 2.4 mmol), and purified by flash column
chromatography using dichloromethane/petroleum ether as elu-
ent. Product as a white solid: yield 32%, mp: 101–106 °C. 1H NMR
(300 MHz, DMSO) d: 5.10 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.00 (d,
J = 3.00 Hz, 1H, pyrrole-H), 6.46 (t, J = 3.00 Hz, 1H, pyrrole-H), 7.15
(t, J = 7.20 Hz, 1H, Ar-H), 7.23 (t, J = 8.70 Hz, 2H, Ar-H), 7.26 (d,
J = 2.40 Hz, 1H, pyrrole-H), 7.32–7.38 (m, 6H, Ar-H); 13C NMR
(75 MHz, CDCl3) d: 46.5 (CH2), 49.8 (CH2), 95.1, 111.1, 113.4, 115.4
4.1.4.10. N2-Benzyl-N4-cyanolmethyl-1,1,3-trioxo-2H,4H-pyrrol-
o[1,2-b][1,2,4,6]thiatriazine (5a10).
This compound was ob-
(2JC–F = 21 Hz), 115.7 (2JC–F = 17 Hz), 124.4 (4JC–F
= 3 Hz), 128.8,
tained from reaction of compound 4a (0.55 g, 2 mmol) with
chloroacetonitrile (0.18 g, 2.4 mmol), and purified by flash column
chromatography using dichloromethane/petroleum ether as elu-
ent. Product as a white solid: yield 63%, mp: 131–134 °C. 1H
NMR (600 MHz, DMSO-d6) d: 5.10 (s, 2H, CH2), 5.12 (s, 2H, CH2),
6.23 (dd, J = 3.60 Hz, 1.80 Hz, 1H, pyrrole-H), 6.57 (t, J = 3.60 Hz,
1H, pyrrole-H), 7.32–7.34 (m, 1H, Ar-H), 7.35 (dd, J = 3.60 Hz,
1.80 Hz, 1H, pyrrole-H), 7.37–7.41 (m, 4H, Ar-H); 13C NMR
(75 MHz, CDCl3) d: 33.6 (CH2), 48.0 (CH2), 94.4, 112.3, 113.1,
113.3, 128.0, 128.7, 128.8, 129.0, 134.3, 148.0 (C@O); ESI-MS:
ESI-MS: 317.2 [M+H]+.
128.0, 128.9, 129.8, 130.3 (3JC–F = 8 Hz), 134.8, 137.5 (3JC–F = 8 Hz),
148.6 (C@O), 162.9 (1JC–F = 245 Hz); EI-MS: 386.4 [M+H]+.
4.1.4.15. N2-(m-Fluorobenzyl)-N4-(o-cyanobenzyl)-1,1,3-trioxo-
2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine (5b2).
This com-
pound was obtained from reaction of compound 4b (0.59 g,
2 mmol) with 2-cyanobenzylchloride (0.37 g, 2.4 mmol), and puri-
fied by flash column chromatography using dichloromethane/
petroleum ether as eluent. Product as a white solid: yield 37%,
mp: 162–165 °C. 1H NMR (600 MHz, DMSO-d6) d: 5.09 (s, 2H,
CH2), 5.29 (s, 2H, CH2), 6.00 (dd, J = 3.60 Hz, 1.20 Hz, 1H, pyrrole-
H), 6.4 (t, J = 3.60 Hz, 1H, pyrrole-H), 7.13 (td, J = 8.40 Hz, 2.40 Hz,
1H, Ar-H), 7.17–7.21 (m, 2H, Ar-H), 7.28 (dd, J = 3.60 Hz, 1.80 Hz,
1H, pyrrole-H), 7.29 (d, J = 7.80 Hz, 1H, Ar-H), 7.37–7.41 (m, 1H,
Ar-H), 7.47 (t, J = 7.80 Hz, 1H, Ar-H), 7.65 (td, J = 7.80 Hz, 1.20 Hz,
1H, Ar-H), 7.87 (d, J = 7.80 Hz, 1H, Ar-H); 13C NMR (75 Hz, CDCl3)
4.1.4.11.
N2-Benzyl-N4-cinnamyl-1,1,3-trioxo-2H,4H-pyrrol-
This compound was ob-
o[1,2-b][1,2,4,6]thiatriazine (5a11).
tained from reaction of compound 4a (0.55 g, 2 mmol) with
cinnamyl chloride (0.37 g, 2.4 mmol), and purified by flash column
chromatography using dichloromethane/petroleum ether as elu-
ent. Product as a colorless oil, yield 78%. 1H NMR (400 MHz, CDCl3)
d ppm: 4.61 (dd, J = 6.00 Hz, 1.40 Hz, 2H, CH2), 5.09 (s, 2H, CH2),
5.72 (dd, J = 3.56 Hz, 1.56 Hz, 1H, pyrrole-H), 6.18-6.23 (m, 1H,
–CH@), 6.33 (t, J = 3.44 Hz, 1H, pyrrole-H), 6.61 (d, J = 15.96 Hz,
1H, – CH@), 6.93 (dd, J = 3.40 Hz, 1.56 Hz, 1H, pyrrole-H), 7.25–
7.34 (m, 8H, Ar-H), 7.34 (d, J = 1.40 Hz, 2H, Ar-H); 13C NMR
(100 MHz, CDCl3) d ppm: 47.1 (CH2), 48.5 (CH2), 94.3, 111.0,
113.3, 121.7, 126.6, 128.2, 128.4, 128.6, 128.7, 128.9, 129.9,
133.9, 135.1, 135.9, 148.1 (C@O). ESI-MS: 394.5 [M+H]+.
d: 46.8 (CH2), 47.4 (CH2), 95.0, 111.3, 111.6, 113.4, 115.6 (2JC–F
=
23 Hz), 115.7 (2JC–F = 21 Hz), 116.9, 124.4 (4JC–F = 3 Hz), 126.6,
128.5, 129.1, 130.4 (3JC–F = 8 Hz), 133.1, 133.6, 137.2 (3JC–F = 8 Hz),
138.5, 148.7 (C@O), 161.30 (1JC–F = 202 Hz); ESI-MS: 411.4 [M+H]+.
4.1.4.16. N2-(m-Fluorobenzyl)-N4-(o-chlorobenzyl)-1,1,3-trioxo-
2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine (5b3).
This com-
pound was obtained from reaction of compound 4b (0.59 g,
2 mmol) with 2-chlorobenzyl chloride (0.39 g, 2.4 mmol), and
purified by flash column chromatography using dichlorometh-
ane/petroleum ether as eluent. Product as a white solid: yield
30%, mp: 113–114 °C. 1H NMR (600 MHz, DMSO-d6) d: 4.46 (s, 2H,
CH2), 4.50 (s, 2H, CH2), 5.95 (d, J = 1.80 Hz, 1H, pyrrole-H), 6.46 (t,
J = 2.40 Hz, 1H, pyrrole-H), 7.16–7.24 (m, 4H, Ar-H), 7.29–7.35 (m,
3H, Ar-H and pyrrole-H), 7.42–7.45 (m, 1H, Ar-H), 7.51 (d,
J = 7.80 Hz, 1H, Ar-H); 13C NMR (75 Hz, CDCl3) d: 46.6 (CH2), 47.1
4.1.4.12. N2-Benzyl N4-allyl-1,1,3-trioxo-2H,4H-pyrrolo[1,2-
b][1,2,4,6]thiatriazine (5a12).
This compound was obtained
from reaction of compound 4a (0.55 g, 2 mmol) with allyl chloride
(0.18 g, 2.4 mmol), and purified by flash column chromatography
using dichloromethane/petroleum ether as eluent. Product as a
colorless oil, yield 65%. 1H NMR (400 MHz, CDCl3) d ppm: 4.45 (d,
J = 5.36 Hz, 1H, CH2), 5.07 (s, 2H, CH2), 5.24–5.28 (m, 2H, CH2@),
5.65 (dd, J = 3.44 Hz, 1.44 Hz, 1H, pyrrole-H), 5.79–5.89 (m, 1H,
– CH@), 6.33 (t, J = 3.48 Hz, 1H, pyrrole-H), 6.92 (dd, J = 3.36 Hz,
1.48 Hz, 1H, pyrrole-H), 7.28–7.34 (m, 3H, Ar-H), 7.36 (d,
J = 2.04 Hz, 2H, Ar-H); 13C NMR (100 MHz, CDCl3) d ppm: 47.1
(CH2), 48.7 (CH2), 94.2, 111.0, 113.2, 118.4, 128.3, 128. 7, 128.8,
129.9, 130.5, 135.1, 148.1 (C@O); ESI-MS: 318.5 [M+H]+.
2
2
(CH2), 95.0, 111.4, 113.5, 115.5 (d, JC–F = 18 Hz), 115.6 (d, JC–
4
F = 18 Hz), 124.4 (d, JC–F = 3 Hz), 126.8, 127.4, 129.0, 129.5, 129.8,
3
3
130.3 (d, JC–F = 8 Hz), 131.9, 132.7, 137.4 (d, JC–F = 8 Hz), 148.5
(C@O), 162.8 (d, 1JC–F = 245 Hz); ESI-MS: 420.3 [M+H]+, 422.3 [M+H]+.
4.1.4.17. N2-(m-Fluorobenzyl)-N4-(o-bromobenzyl)-1,1,3-trioxo-
2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine (5b4).
This com-
pound was obtained from reaction of compound 4b (0.59 g,
2 mmol) with 2-bromobenzyl chloride (0.60 g, 2.4 mmol), and
purified by flash column chromatography using dichlorometh-
ane/petroleum ether as eluent. Product as a white solid: yield
29%, mp: 113–116 °C. 1H NMR (300 MHz, CDCl3) d: 5.11 (s, 2H,
CH2), 5.16 (s, 2H, CH2), 5.52 (dd, J = 3.60 Hz, 1.50 Hz, 1H, pyrrole-
H), 6.29 (t, J = 3.60 Hz, 1H, pyrrole-H), 6.93 (dd, J = 3.30 Hz,
1.50 Hz, 1H, pyrrole-H), 7.00–7.06 (m, 2H, Ar-H), 7.11–7.37 (m,
6H, Ar-H); 13C NMR (75 MHz, CDCl3) d: 46.6 (CH2), 49.6 (CH2),
4.1.4.13. N2-Benzyl-N4-(4-(3-methylbut-2-en-1-yl)-1,1,3-trioxo-
2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine (5a13).
This com-
pound was obtained from reaction of compound 4a (0.55 g,
2 mmol) with 1-chloro-3-methyl-2-butene (0.25 g, 2.4 mmol),
and purified by flash column chromatography using dichlorometh-
ane/petroleum ether as eluent. Product as a yellow oil, yield 62% 1H
NMR (400 MHz, CDCl3) d ppm: 1.77 (s, 3H, CH3), 1.72 (s, 3H, CH3),
4.44 (d, J = 6.44 Hz, 2H, CH2), 5.06 (s, 2H, CH2), 5.18 (m, 1H, – CH@),
5.60 (dd, J = 3.52 Hz, 1.60 Hz, 1H, pyrrole-H), 6.33 (t, J = 3.52 Hz,
1H, pyrrole-H), 6.92 (dd, J = 3.40 Hz, 1.56 Hz, 1H, pyrrole-H),
7.23–7.36 (m, 3H, Ar-H), 7.46–7.48 (m, 2H, Ar-H); ESI-MS: 346.3
[M+H]+.
2
2
95.1, 111.4, 113.5, 115.5 (d, JC–F = 18 Hz), 115.8 (d, JC–F = 18 Hz),
4
122.5, 124.4 (d, JC–F = 3 Hz), 126.7, 128.0, 129.3, 129.5, 130.3 (d,
3JC–F = 8 Hz), 133.0, 133.4, 137.4 (d, JC–F = 8 Hz), 148.5 (C@O),
3
162.8 (d, JC–F = 245 Hz). ESI-MS: 464.3 [M+H]+, 466.3 [M+H]+.
1
4.1.4.18.
oxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine
compound was obtained from reaction of compound 4b (0.59 g,
N2-(m-Fluorobenzyl)-N4-(m-chlorobenzyl)-1,1,3-tri-
4.1.4.14.
N2-(m-Fluorobenzyl)-N4-benzyl-1,1,3-trioxo-2H,4H-
This compound
(5b5). This
pyrrolo[1,2-b][1,2,4,6]thiatriazine (5b1).

7098
W. Chen et al. / Bioorg. Med. Chem. 21 (2013) 7091–7100
2 mmol) with 3-chlorobenzyl chloride (0.39 g, 2.4 mmol), and
purified by flash column chromatography using dichlorometh-
ane/petroleum ether as eluent. Product as a white solid: yield
31%, mp: 122–124 °C. 1H NMR (600 MHz, DMSO-d6) d: 5.13 (s,
2H, CH2), 5.18 (s, 2H, CH2), 5.95 (dd, J = 3.60 Hz, 1.80 Hz, 1H, pyr-
role-H), 6.46 (t, J = 3.60 Hz, 1H, pyrrole-H), 7.16–7.24 (m, 4H, Ar-
H), 7.29–7.35 (m, 3H, Ar-H and pyrrole-H), 7.42–7.46 (m, 1H, Ar-
H), 7.51 (dd, J = 7.20 Hz, 1.20 Hz, 1H); 13C NMR (75 Hz, CDCl3) d:
purified by flash column chromatography using dichlorometh-
ane/petroleum ether as eluent. Product as a white solid: yield
28%, mp: 164–168 °C. 1H NMR (600 MHz, DMSO-d6) d: 5.11 (s,
2H, CH2), 5.16 (s, 2H, CH2), 5.97 (dd, J = 3.60 Hz, 1.80 Hz, 1H, pyr-
role-H), 6.47 (t, J = 3.60 Hz, 1H, pyrrole-H), 7.21 (d, J = 7.80 Hz,
1H, Ar-H), 7.21 (dd, J = 3.60 Hz, 1.80 Hz, 1H, pyrrole-H), 7.35 (d,
J = 7.20 Hz, 1H, Ar-H), 7.40–7.44 (m, 4H, Ar-H), 7.70 (d,
J = 2.40 Hz, 1H, Ar-H); 13C NMR (75 MHz, CDCl3) d: 46.6 (CH2),
46.7 (CH2), 95.0, 111.5, 113.4, 127.0, 127.8, 127.9, 128.8, 128.9,
129.3, 129.6, 130.0, 130.6, 133.4, 134.3, 134.6, 136.9, 148.5
(C@O); ESI-MS: 470.2 [M+H]+, 472.2 [M+H]+, 473.2 [M+H]+.
2
46.6 (CH2), 47.1 (CH2), 95.0, 111.4, 113.5, 115.5 (d, JC–F = 18 Hz),
2
4
115.7 (d, JC–F = 19 Hz), 124.4 (d, JC–F = 2 Hz), 126.9, 127.4, 129.0,
3
3
129.5, 129.8, 130.3 (d, JC–F = 8 Hz), 131.9, 132.7, 137.4 (d, JC–F
=
1
8 Hz), 148.5 (C@O), 161.25 (d, JC–F = 245 Hz); ESI-MS: 420.3
[M+H]+, 422.3 [M+H]+.
4.1.4.23. N2-(m-Bromobenzyl)-N4-(o-chlorobenzyl)-1,1,3-trioxo-
2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine (5d1).
This com-
4.1.4.19. N2-(m-Fluorobenzyl)-N4-(2,4-dichlorobenzyl)-1,1,3-tri-
pound was obtained from reaction of compound 4d (0.71 g,
2 mmol) with 2-chlorobenzyl chloride (0.39 g, 2.4 mmol), and
purified by flash column chromatography using dichlorometh-
ane/petroleum ether as eluent. Product as a white solid: yield
31%, mp: 103–105 °C. 1H NMR (300 MHz, CDCl3) d: 5.08 (s, 2H,
CH2), 5.19 (s, 2H, CH2), 5.55 (dd, J = 3.60 Hz, 1.50 Hz, 1H, pyrrole-
H), 6.29 (t, J = 3.60 Hz, 1H, pyrrole-H), 6.94 (dd, J = 3.60 Hz,
1.50 Hz, 1H, pyrrole-H), 7.03 (dd, J = 9.30 Hz, 2.40 Hz, 1H, Ar-H),
7.17–7.26 (m, 3H, Ar-H), 7.38–7.48 (m, 3H, Ar-H), 7.63 (s, 1H, Ar-
H), 13C NMR (100 MHz, CDCl3) d: 46.4 (CH2), 47.1 (CH2), 95.0,
111.4, 113.5, 122.7, 126.8, 127.4, 129.0, 129.5, 129.8, 130.3,
131.7, 131.8, 131.9, 132.7, 137.3, 148.5 (C@O); ESI-MS: 480.1
[M+H]+, 482.2 [M+H]+.
oxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine
(5b6).
This
compound was obtained from reaction of compound 4b (0.59 g,
2 mmol) with 2,4-dichlorobenzyl chloride (0.47 g, 2.4 mmol), and
purified by flash column chromatography using dichlorometh-
ane/petroleum ether as eluent. Product as a white solid: yield
30%, mp: 109–116 °C. 1H NMR (600 MHz, DMSO-d6) d: 5.08 (s,
2H, CH2), 5.13 (s, 2H, CH2), 5.9 (dd, J = 3.00 Hz, 1.80 Hz, 1H, pyr-
role-H), 6.4 (t, J = 3.00 Hz, 1H, pyrrole-H), 7.13–7.20 (m, 4H, Ar-
H), 7.27 (dd, J = 3.00 Hz, 1.80 Hz, 1H, pyrrole-H), 7.35–7.42 (m,
2H, Ar-H), 7.47 (d, J = 1.80 Hz, 1H, Ar-H); 13C NMR (100 MHz,
CDCl3) d: 46.6 (CH2), 46.7 (CH2), 94.9, 111.5, 113.4, 115.5 (2JC–F
=
20 Hz), 115.7 (2JC–F = 21 Hz), 124.4 (4JC–F = 3 Hz), 127.8 (3JC–F = 10 Hz),
129.3, 129.6, 130.3, 130.6, 131.0, 133.4, 134.3, 137.3 (3JC–F = 10 Hz),
148.5 (C@O), 162.8 (1JC–F = 255 Hz); ESI-MS: 454.3 [M+H]+, 456.3 [M+H]+.
4.1.4.24. N2-(m-Bromobenzyl)-N4-(2,4-dichlorobenzyl)-1,1,3-tri-
oxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine
(5d2).
This
4.1.4.20.
N2-(m-Fluorobenzyl)-N4-cyanomethyl-1,1,3-trioxo-
This com-
compound was obtained from reaction of compound 4d (0.71 g,
2 mmol) with 2,4-dichlorobenzyl chloride (0.47 g, 2.4 mmol), and
purified by flash column chromatography using dichlorometh-
ane/petroleum ether as eluent. Product as a white solid: yield
25%, mp: 113–117 °C. 1H NMR (300 MHz, CDCl3) d: 5.07 (s, 2H,
CH2), 5.14 (s, 2H, CH2), 5.53 (dd, J = 3.60 Hz, 1.80 Hz, 1H, pyrrole-
H), 6.30 (t, J = 3.60 Hz, 1H, pyrrole-H), 6.94 (J = 3.60 Hz, 1.80 Hz,
1H, pyrrole-H), 7.00 (s, 1H, Ar-H), 7.12–7.40 (m, 3H, Ar-H), 7.42
(d, J = 2.10 Hz, 1H, Ar-H), 7.45–7.48 (m, 1H, Ar-H), 7.62 (t,
J = 1.80 Hz, 1H, Ar-H); 13C NMR (75 MHz, CDCl3) d: 46.5 (CH2),
46.7 (CH2), 94.9, 111.6, 113.5, 122.7, 127.5, 127.8, 127.9, 129.2,
129.6, 130.3, 130.6, 131.7, 131.8, 133.4, 134.3, 137.1, 148.5
(C@O); ESI-MS: 516.1 [M+H]+, 514.1 [M+H]+, 518.2 [M+H]+.
2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine (5b7).
pound was obtained from reaction of compound 4b (0.59 g,
2 mmol) with chloroacetonitrile (0.18 g, 2.4 mmol), and purified
by flash column chromatography using dichloromethane/petro-
leum ether as eluent. Product as a white solid: yield 30%, mp:
122–127 °C. 1H NMR (300 MHz, CDCl3) d: 4.73 (s, 2H, CH2), 5.06
(s, 2H, CH2), 5.87 (dd, J = 3.60 Hz, 1.50 Hz, 1H, pyrrole-H), 6.43 (t,
J = 3.60 Hz, 1H, pyrrole-H), 7.00 (dd, J = 3.60 Hz, 1.50 Hz, 1H, pyr-
role-H), 7.06 (dd, J = 8.40 Hz, 2.40 Hz, 1H, Ar-H), 7.17–7.22 (m,
1H, Ar-H), 7.26 (s, 1H, Ar-H), 7.30–7.37 (m, 1H, Ar-H); 13C NMR
(75 MHz, CDCl3) d: 47.2 (CH2), 47.3 (CH2), 94.6, 112.4, 113.1,
113.5, 115.7 (2JC–F = 21 Hz), 115.9 (2JC–F = 23 Hz), 124.5 (4JC–F
=
3 Hz), 127.9, 130.4 (3JC–F = 8 Hz), 136.6 (3JC–F = 8 Hz), 147.9 (C@O),
162.8 (1JC–F = 245 Hz); ESI-MS: 335.4 [M+H]+.
4.2. Anti-HIV activity assays
4.1.4.21. N2-(m-Chlorobenzyl)-N4-(o-cyanobenzyl)-1,1,3-trioxo-
The target compounds were evaluated for their antiviral activi-
ties against wild-type HIV-1 strain (HIV-IIIB), double mutant HIV-1
IIIB strain RES056 (K103N+Y181C) and HIV-2 strain (ROD) in MT-4
cell cultures using the MTT method as previously described.35
Briefly, stock solutions (10 ꢀ final concentration) of test com-
2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine (5c1).
This com-
pound was obtained from reaction of compound 4c (0.62 g,
2 mmol) with 2-cyanobenzylchloride (0.37 g, 2.4 mmol), and puri-
fied by flash column chromatography using dichloromethane/
petroleum ether as eluent. Product as a white solid: yield 38%,
mp: 203–205 °C. 1H NMR (600 MHz, DMSO-d6) d: 5.11 (s, 2H,
CH2), 5.32 (s, 2H, CH2), 6.03 (dd, J = 3.60 Hz, 1.20 Hz, 1H, pyrrole-
H), 6.5 (t, J = 3.60 Hz, 1H, pyrrole-H), 7.31–7.32 (m, 2H, Ar-H and
pyrrole-H), 7.35–7.43 (m, 3H, Ar-H), 7.45 (s, 1H, Ar-H), 7.51 (t,
J = 7.80 Hz, 1H, Ar-H), 7.68 (t, J = 7.80 Hz, 1H, Ar-H), 7.90 (d,
J = 1.80 Hz, 1H, Ar-H); 13C NMR (75 MHz, CDCl3) d: 46.7 (CH2),
47.4 (CH2), 95.0, 111.3, 111.6, 113.5, 116.9, 126.6, 127.0, 128.5,
128.8, 128.9, 129.1, 130.1, 133.1, 133.6, 134.6, 136.8, 138.4, 148.7
(C@O); ESI-MS: 427.3 [M+H]+, 429.4 [M+H]+.
pounds were added in 25
lL volumes to two series of triplicate
wells to allow simultaneous evaluation of their effects on mock-
and HIV-infected cells at the beginning of each experiment. Serial
fivefold dilutions of test compounds were made directly in flat-
bottomed 96-well microtiter trays by adding 100
the 25 L stock solution and transferring 25 L of this solution
to another well that contained 100 L medium using a Biomek
lL medium to
l
l
l
3000 robot (Beckman instruments, Fullerton, CA). Untreated con-
trol HIV- and mock-infected cell samples were included for each
sample.
HIV-1(IIIB)36 or HIV-2 (ROD)37 stock (50
lL) at 100–300 CCID50
4.1.4.22. N2-(m-Chlorobenzyl)-N4-(2,4-dichlorobenzyl)-1,1,3-tri-
(50% cell culture infectious dose-50%) or culture medium was
added to either the infected or mock-infected wells of the microti-
ter plate. Mock-infected cells were used to evaluate the effect of
the test compounds on uninfected cells in order to assess their
oxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine
(5c2).
This
compound was obtained from reaction of compound 4c (0.62 g,
2 mmol) with 2,4-dichlorobenzyl chloride (0.47 g, 2.4 mmol), and

W. Chen et al. / Bioorg. Med. Chem. 21 (2013) 7091–7100
7099
cytotoxicity. Exponentially growing MT-4 cells were centrifuged
for 5 min at 1000 rpm and the supernatant was discarded. The
Waals, electrostatic, and torsional energy terms defined in the Tri-
pos force field. The best docking conformation of compounds 5b2
and Q96652 with the highest score was shown by PyMOL(http://
www.pymol.org/), in overlap with the ligand efavirine. The second-
ary structure of RT are shown in cartoons, and only the key resi-
dues for interactions with the inhibitors were shown in sticks
and labeled. The potential hydrogen-bondings were presented by
dashed lines.
MT-4 cells were resuspended at 6 ꢀ 105 cells/mL, and 50-
lL vol-
umes were transferred to the microtiter tray wells. Five days after
infection, the viability of mock- and HIV-infected cells was exam-
ined spectrophotometrically by the MTT assay. The 50% cytotoxic
concentration (CC50) was defined as the concentration of the test
compound that reduced the viability of the mock-infected MT-4
cell cultures by 50%. The concentration achieving 50% protection
from the cytopathic effect of the virus in infected cells was defined
as the 50% effective concentration (EC50).
Acknowledgments
We thank K. Erven, K. Uyttersprot and C. Heens for technical
assistance with the anti-HIV assays. The financial support from
the National Natural Science Foundation of China (NSFC Nos.
81273354, 81102320, 30873133, 30772629, 30371686), Key Pro-
ject of NSFC for International Cooperation (No. 30910103908), Re-
search Fund for the KU Leuven (GOA 10/014), Doctoral Program of
Higher Education of China (No. 20110131130005), and Shandong
Provincial Natural Science Foundation (No. ZR2009CM016) is
gratefully acknowledged.
4.3. Inhibition of HIV-1 RT
A RT assay kit produced by Roche was used for the RT inhibition
assay. All the reagents for performing the RT reaction are contained
in the kit and the ELSIA procedures for RT inhibition assay was per-
formed as described in the kit protocol.38,39
Briefly, the reaction mixture containing HIV-1 reverse transcrip-
tase (RT) enzyme, reconstituted template and viral nucleotides
(digoxigenin (DIG)-dUTP, biotin-dUTP and dTTP) in the incubation
buffer with or without inhibitors was incubated for 1 h at 37 °C.
Then, the reaction mixture was transferred to a streptavidine-
coated microtitre plate (MTP) and incubated for another 1 h at
37 °C. The biotin-labeled dNTPs that was incorporated in cDNA
chain in the presence of RT were bound to streptavidine. The un-
bound dNTPs were removed by rinsing using washing buffer fol-
lowed by the addition of anti-DIG-POD working solution into the
MTPs. After incubation for 1 h at 37 °C, the DIG-labeled dNTPs
incorporated in cDNA were bound to the anti-DIG-POD antibody.
The unbound anti-DIG-PODs were removed and the peroxide sub-
strate (ABST) solution was added to the MTPs. Incubation of the
reaction mixture at 25 °C until a green color was sufficiently devel-
oped for detection. The absorbance of the sample was determined
at O.D. 405 nm using microtiter plate ELISA reader. The percentage
inhibitory activity of RT inhibitors was calculated by formula as gi-
ven below:
References and notes
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Van Meervelt, L.; Vega, S.; Balzarini, J.; De Clercq, E. Antimicrob. Agents
Chemother. 1998, 42, 618.
%Inhibition ¼ ½O:D: value with RT but without inhibitors
ꢁ O:D: value with RT and inhibitorsꢂ=½O:D: value with RT and inhibitors
ꢁ O:D: value without RT and inhibitorsꢂ
The IC50 values corresponded to the concentrations of the inhibitors
required to inhibit biotin-dUTP incorporation by 50%.
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