Synthesis and anti-HIV activity evaluation of novel 2,4-disubstituted 7-methyl-1,1,3-trioxo-2,4-dihydro-pyrazolo-[4,5-e][1,2]thiadiazines

Article abstract of DOI:10.1002/ardp.200700216

A series of novel 2,4-disubstituted 7-methyl-1,1,3-trioxo-2,4-dihydro- pyrazolo[4,5-e] [1,2,4]thiadiazines (PTDs) was synthesized, structurally confirmed by spectral analysis, and evaluated for their anti-HIV activities by inhibition of HIV-induced cytopathogenicity in MT-4 cell culture. The results showed that some compounds exhibited inhibitory activity specifically against HIV-2 replication. The most active HIV-2 inhibitor was compound 7i (R ₁ = benzyl, R₂ = 4-t-butyl-benzyl) with an EC₅₀ value of 18.7 μM and SI=15, which may provide a useful lead for further molecular optimization.

Full text of DOI:10.1002/ardp.200700216

216
Arch. Pharm. Chem. Life Sci. 2008, 341, 216–222
Full Paper
Synthesis and Anti-HIV Activity Evaluation of Novel 2,4-
Disubstituted 7-Methyl-1,1,3-trioxo-2,4-dihydro-pyrazolo-
[4,5-e][1,2]thiadiazines
Xin-yong Liu1, Ren-zhang Yan1, Yan Wang1, Peng Zhan1, Erik De Clercq2, Christophe
Pannecouque2, Myriam Witvrouw2, Maria Teresa Molina3, and Salvador Vega3
1 Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan, P. R.
China
2 Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
3
Instituto de Quimica Medica (CSIC), Madrid, Spain
A series of novel 2,4-disubstituted 7-methyl-1,1,3-trioxo-2,4-dihydro-pyrazolo[4,5-e] [1,2,4]thiadia-
zines (PTDs) was synthesized, structurally confirmed by spectral analysis, and evaluated for their
anti-HIV activities by inhibition of HIV-induced cytopathogenicity in MT-4 cell culture. The
results showed that some compounds exhibited inhibitory activity specifically against HIV-2 rep-
lication. The most active HIV-2 inhibitor was compound 7i (R1 = benzyl, R2 = 4-t-butyl-benzyl)
with an EC50 value of 18.7 lM and SI=15, which may provide a useful lead for further molecular
optimization.
Keywords: Phosphoinositide 3-kinase / phospholipases / HIV-1 / HIV-2 / NNRTIs / Pyrazolo[4,5-e][1,2,4]thiadiazines /
Received: October 21, 2007; accepted: December 12, 2007
DOI 10.1002/ardp.200700216
HIV-1 reverse transcriptase (HIV-1 RT) that catalyzed
Introduction
several steps in the replication of HIV is an attractive tar-
get for the development of anti-AIDS drugs. Three classes
of HIV-1 RTIs are currently available: nucleoside (and
nucleotide) reverse transcriptase inhibitors (NRTIs,
NtRTIs) and non-nucleoside reverse transcriptase inhibi-
tors (NNRTIs). NNRTIs are structurally diverse com-
pounds by specifically targeting an allosteric site of HIV-1
RT, approximately 10–15 ꢀ from the polymerase active
site, causing a distortion of the catalytic aspartate triad
[2]. The efficacy of NNRTIs like that of other types of anti-
AIDS agents has been limited by emergence of drug-
resistant viral strains and overwhelming side effects
[3, 4]. Therefore, discovery and development of new
NNRTIs with more potent, less toxic, and broad-spectrum
activity are urgently needed in medicinal research.
In recent years, studies aimed at the discovery of new
NNRTIs; Vega and his colleagues reported that a series of
2,4-disubstituted-1,1,3-trioxo-2,4-dihydro-thieno[3,4-e]-
[1,2,4]thiadiazines (TTDs) effectively inhibited the replica-
tion of a variety of HIV-1 strains at the reverse-transcrip-
tion steps, including strains that are resistant to AZT, but
not against HIV-2 (ROD) [5, 6]. The prototypes QM96521,
Although the highly active, anti-retroviral therapy
(HAART) combination regimen has dramatically
decreased the morbidity and mortality from infection by
HIV, the causative agent of acquired immunodeficiency
syndrome (AIDS), remains one of the world's most serious
health problems, causing millions of deaths each year [1].
Currently, three different classes of chemotherapeutic
agents have been used in HAART to block the replication
of HIV-1, namely reverse transcriptase inhibitors (RTIs),
protease inhibitors (PIs), and one-fusion inhibitor (enfu-
virtide).
Correspondence: Xinyong Liu, Institute of Medicinal Chemistry, School
of Pharmaceutical Sciences, Shandong University, No.44 Wenhuaxi
Road, Jinan 250012, P. R. China.
E-mail: xinyongl@sdu.edu.cn
Fax: +86 531 8838-2264
Abbreviations: reverse transcriptase inhibitor (RTI); nucleoside (and nu-
cleotide) reverse transcriptase inhibitor (NRTI, NtRTI); non-nucleoside re-
verse transcriptase inhibitor (NNRTI); 2,4-disubstituted 1,1,3-trioxo-2,4-
dihydro-thieno[3,4-e][1,2,4]thiadiazine (TTD); pyrazolo[4,5-e][1,2,4]thia-
diazine (PTD)
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Arch. Pharm. Chem. Life Sci. 2008, 341, 216–222
Novel PTD Analogues
217
QM96539 and QM96639 (Fig. 1), containing a benzyl or 2-
halogenated benzyl moiety at the N2 position and a cya-
nomethyl chain linked to the N4 position, were found to
selectively inhibit HIV-1 (III-B) replication in MT and CEM
cell cultures [6, 7]. The cross-resistance pattern of these
compounds against other NNRTI-resistant, mutant HIV-1
strains and molecular modelling of the HIV-1 RT binding
site were found both to be similar to those on nevirapine
[6]. Furthermore, molecular modification by replace-
ment of the N4 cyanomethyl with a substituted benzyl
group led to the discovery of a new precursor QM96625
(N2-benzyl, N4-2-chlorobenzyl, EC50 = 0.1 lM, SI A 1190;
Fig. 1) [7].
Figure 1. Lead compounds of 2,4-disubstituted-1,1,3-trioxo-
2,4-dihydro-thieno[3,4-e][1,2,4]thiadiazines (TTDs) and newly
designed 2,4-disubstituted-7-methylpyrazolo[4,5-e][1,2,4]thia-
diazines (PTDs).
The initial structure-activity relationship analysis
together with the molecular modelling disclosed that
double substitutions containing p-electrons groups at
the N2 and N4 sites of TTDs were necessary for preserving
anti-HIV-1 activity, which perfectly match the “butterfly-
like” three-dimensional (3D) model proposed by Schafer
et al. [8] for filling the space of the NNRTI binding pocket.
As a continuation of the study on the structure-activity
relationships of TTDs, we undertook a study of the substi-
tuted pyrazolo[4,5-e][1,2,4]thiadiazines (PTDs) series,
because of the known bio-isosterism of TTDs-PTDs [9]. In
recent publications, we reported the regioselective syn-
thesis of novel N2- or N4-monosubstituted 7-methylpyra-
zolo[4,5-e][1,2,4]thiadiazines, as well as the synthesis of
N2, N4-disubstituted 7-methylpyrazolo[4,5-e][1,2,4]thiadia-
zines by an one-pot reaction [10, 11]. In this paper, in
order to find potent HIV replication inhibitors, a new ser-
ies of 2,4-disubstituted PTDs was designed, in which, the
PTD nuclear ring was alkylated at the N2 and N4 positions
by those substituents active against HIV-1 activity in the
TTD series. Herein, we reported the synthesis of the novel
PTD analogues and their anti-HIV activities in cell cul-
ture.
Reagents: (a) N2H4 N H2O/EtOH; (b) 2N HCl, NaNO2, H2O; (c) D/toluene
Scheme 1. Synthesis route of compounds 1–5.
Reagents: (i) NaH/R1CH2X (1 : 1), DMF; (ii) NaH/R2CH2X (1 : 1), DMF
Scheme 2. Synthesis route of compounds 6 and 7a–l.
cautiously dried without further purification and then
subjected to a Curtius rearrangement by heating in the
refluxing dry toluene. The intermediate isocyanate 4
spontaneously cyclized to the desired 7-methyl-1,1,3-tri-
oxo-2,4-dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine (PTD, 5)
(Scheme 1) [5–7].
Compound 5 is a key intermediate for the preparation
of N2-monosubstituted derivatives 6, from which differ-
ent substituents can be introduced at the N4 position by
alkylation under the same conditions to obtain the target
compounds of N2, N4-disubstituted 7-methyl-1,1,3-trioxo-
2,4-dihydro-pyrazolo[4,5-e][1,2,4]thiadiazines (PTDs, 7a–l,
Scheme 2).
The N2,N4-disubstituted PTDs containing the same sub-
stituents were prepared starting from the nuclear ring 5
with two equivalents of sodium hydride in DMF solvent
at a temperature a108C, followed by addition of two
equivalents of alkyl halides at 40–808C for 2–8 h, achiev-
ing the N2, N4-disubstituted products 7m–r in good yields
(Scheme 3).
Results and discussion
Chemistry
Similar to the synthesis of 1,1,3-trioxo-2,4-dihydro-
thieno[3,4-e][1,2,4]thiadiazine (TTD), 1,1,3-trioxo-pyra-
zolo[4,5-e][1,2,4]thiadiazine (PTD, 5) was synthesized
starting from ethyl 1-methyl-5-sulfamoyl-1H-pyrazole-4-
carboxylate 1 through hydrazinolysis, with excess hydra-
zine hydrate in the refluxed ethanolic solution to gave
the 5-sulfamoylthiophene-4-carbohydrazide 2, which was
converted to the carbonyl azide 3 in almost quantitative
yield by reaction with sodium nitrite in diluted hydro-
chloric acid at a temperature a108C. Because of the
potential explosion hazard of acyl azides, crude 3 was
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Arch. Pharm. Chem. Life Sci. 2008, 341, 216–222
Table 1. Anti-HIV activities, cytotoxicities, and selectivity indices of 2,4-disubstituted 7-methyl-1,1,3-trioxo-2,4-dihydro-pyrazolo[4,5-
e][1,2,4]thiadiazines (TTDs) 7a–r.
No
R1CH2
R2CH2
EC50 (lM)a)
HIV-2 ROD
CC50 (lM)b)
SIc)
HIV-2 ROD
HIV-1 IIIB
HIV-1 IIIB
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
4-CH3-benzyl
2-Br-benzyl
4-(t-Bu)benzyl
2,4-Cl2-benzyl
4-Cl-benzyl
4-Br-benzyl
CH2CN
benzyl
A251.4
A319.4
A271.1
A6.7
A209.4
A327.2
A271.1
A112.7
A300.1
44.7
A56.2
A277.2
18.7
A198.0
A307.1
A23.8
A304.9
A231.5
A5.3
38.1
A5.8
A151.2
F209.4
F319.4
A271.1
54.7
A300.1
A300.1
53.9
A277.2
A285.4
F198.0
A307.1
24.2
F170.5
A231.5
7.1
61
61
61
a1
61
61
61
a1
2-Br-benzyl
4-Br-benzyl
2-Cl-benzyl
3-Cl-benzyl
4-Cl-benzyl
2,4-Cl2-benzyl
4-(t-Bu)-benzyl
2-CN-benzyl
3-CN-benzyl
4-CN-benzyl
4-CH3-benzyl
2-Br-benzyl
4-(t-Bu)benzyl
2,4-Cl2-benzyl
4-Cl-benzyl
4-Br-benzyl
A300.1
A300.1
A47.5
A277.2
A285.4
A265.4
A307.1
A20.4
A170.5
A231.5
A7.5
61
61
a1
61
>7
a1
61
61
61
61
a1
61
F15
61
61
a1
61
61
a1
61
61
a1
>7
a1
a1
A253.0
A6.7
A151.2
0.9
A253.0
61
a1
7q
7r
6.7
161.6
502.7
A119.0
683
a1
QM96521d)
QM96625d)
Nevirapined)
AZTd)
559
A1190
22767
50587
0.1
0.03
0.0007
35.6
a)
Dose of compound required to achieve 50% protection of MT-4 cell from HIV (HIV-1 IIIB, HIV-2 ROD)-induced cytotoxicity, as deter-
mined by the MTT method.
CC50 : dose required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.
SI: selectivity index (CC50/EC50); the SI values: 6 1 stand for =1 or a1. All data represent mean values for at least two separate
experiments.
b)
c)
d)
The synthesis and antiviral properties of these compounds were previously described [5, 7].
tive purposes; AZT and nevirapine were used as the refer-
ence drugs.
As illustrated in Table 1, the new PTD derivatives, pre-
pared by pyrazole substitution for the thiophene of the
TTD derivatives, had completely lost their anti-HIV-1
activity, although the active substituents of TTDs were
retained in the PTD molecules. Putatively, an increased
hydrophilicity of the pyrazole-ring moiety, in compari-
son with the thiophene in the corresponding TTD ana-
logues, did not fit for the requirement of a “butterfly
Reagents:NaH/RCH2X (2 : 2), DMF
Scheme 3. Synthesis route of compounds 7m–r.
Anti-HIV evaluation
The activity and cytotoxicity of the newly designed and like” model with its lipophilic center binding through
synthesized PTDs 7a-r were tested for inhibition of HIV van–der–Waals and p-p interactions with aromatic
(HIV-1 IIIB, HIV-2 ROD)-induced cytopathogenicity in MT-4 amino acid residues in the RT binding pocket [6]. This fea-
cells culture. The results are listed in Table 1. EC50 and ture should be taken into account in the future for
CC50 for QM96521 and QM96625 are given for compara- design of novel TTDs.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 216–222
Novel PTD Analogues
219
USA). Flash column chromatography was performed on column
packed with silica gel 60 (230–400 mesh). Solvents were reagent
grade and, when necessary, were purified and dried by standard
methods. Concentration of the reaction solutions involved the
use of rotary evaporator at reduced pressure.
To our surprise, some PTD compounds, 7f, 7i, and 7p,
exhibited inhibitory activities specifically against HIV-2
(ROD) replication in MT-4 cell culture. The most active
one was compound 7i (R1 = benzyl, R2 = 4-t-butyl-benzyl)
with an EC50 value of 18.7 lM and SI F 15. Compounds 7f
and 7p emerged as moderately active HIV-2 inhibitors
with EC50 values in a similar range (44.7 lM and 38.1 lM,
respectively).
General procedure for the preparation of compounds N2-
benzyl, N4-substituted 7-methyl-1,1,3-trioxo-2,4-dihydro-
pyrazolo[4,5-e][1,2,4]thiadiazines 7a–l
HIV-2 infection had appeared predominantly in West
Africa, but gradually spread to other parts of the world in
recent years. In-vitro (laboratory) studies suggest that
non-nucleoside reverse transcriptase for inhibitors
(NNRTIs) are usually specific for HIV-1 and inactive
against HIV-2. Therapeutic options for HIV-2 infection are
therefore limited to nucleoside reverse transcriptase
inhibitors (NRTIs) and protease inhibitors (PIs) [12]. There
is insufficient data to recommend any specific drug or
drug combinations for the treatment of HIV-2 infection
[13]. PTD analogues could act as useful leads for further
optimization, which will provide a stimulus for struc-
ture-based design of more potent anti-HIV-2 agents.
To a solution of 7-methyl-1,1,3-trioxo-2,4-dihydro-pyrazolo[4,5-
e][1,2,4]thiadiazine (5, 1 eq) in dry DMF (1 mmol, 4 mL) was
added sodium hydride (60% dispersion in mineral oil, 1 eq) in
portions, under inert atmosphere (N2) and keeping the tempera-
ture at 108C. After 30 minutes stirring, the alkyl halide (1 eq)
was added dropwise. The mixture was stirred at room tempera-
ture for 20 min and 30–508C for 12–20 h (checked by TLC). After
the solvent was evaporated off under reduced pressure, the
crude product was purified by recrystallization from ethanol.
2-Benzyl-4-cyanomethyl-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7a
Compound 5 reacted with chloroacetonitrile at 508C for 20 h to
give compound 7a, which was purified by recrystallization from
ethanol as brown solid (51%, mp. 176–1788C). IR (KBr, cm– 1):
1686 (C=O), 1333, 1197 (SO2). 1H-NMR (CDCl3, 600 MHz) d: 7.28 (s,
1H, PyH), 7.33–7.51 (m, 5H, PhH), 5.08 (s, 2H, NCH2), 4.77 (s, 2H,
NCH2), 4.19 (s, 3H, CH3). 13C-NMR (CDCl3, 150 MHz) d: 148.4 (C=O),
134.5 (C-19), 128.8, 128.4, 128.2, 123.9 (C-5), 123.6 (C-4a), 123.0 (C-
7a), 113.0 (CN), 45.1 (N2-CH2), 39.2 (N4-CH2), 33.1 (CH3). MS
(C14H13N5O3S 331.35): m/z (ESI) 332.4 [M + 1].
Conclusion
In conclusion, we designed and synthesized a series of
novel 2,4-disubstituted-7-methyl-1,1,3-trioxo-2,4-dihydro-
pyrazolo[4,5-e][1,2,4]thiadiazines (PTDs), which were
structurally confirmed by IR, 1H-NMR, 13C-NMR, and MS
spectral analysis and evaluated for their inhibition of
HIV (HIV-1 IIIB and HIV-2 ROD)-induced cytopathogenicity
in MT-4 cell culture. The results indicated that PTD ana-
logues derived from the TTDs by pyrazole-ring substitu-
tion for thiophene exhibited decreased anti-HIV-1 activ-
ity. Unexpected was the finding that PTD analogues were
potential and specific HIV-2 inhibitors. The most active
compound was compound 7i (R1 = benzyl, R2 = 4-t-butyl-
benzyl) with an EC50 value of 18.7 lM and SI=15, which
provides a useful lead for further molecular optimiza-
tion.
2,4-Dibenzyl-7-methyl-1,1,3-trioxo-2,4-dihydro-
pyrazolo[4,5-e][1,2,4]thiadiazine 7b
Compound 5 reacted with benzyl bromide at 308C for 12 h to
give compound 7b, which was purified by recrystallization from
ethanol as white solid (72%, mp. 128–1308C). IR (KBr, cm– 1):
1668 (C=O), 1331, 1193 (SO2). 1H-NMR (CDCl3, 600 MHz) d: 7.16 (s,
1H, PyH), 7.51 (d, 2H, J = 8.50 Hz, PhH), 7.21 (d, 2H, J = 8.03 Hz,
PhH), 7.29–7.37 (m, 6H, PhH), 5.13 (s, 2H, NCH2), 5.07 (s, 2H,
NCH2), 4.12 (s, 3H, CH3). 13C-NMR (CDCl3, 150 MHz) d: 149.8 (C=O),
135.1, 135.9, 129.2, 128.9, 128.7, 127.1, 128.3, 128.2, 126.0 (C-5),
125.4 (C-4a), 123.2 (C-7a), 49.7 (N2-CH2), 44.9 (N4-CH2), 39.2 (CH3).
MS (C19H18N4O3 S 382.44): m/z (ESI) 383.4 [M + 1].
2-Benzyl-4-(o-bromobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7c
Experimental
Compound 5 reacted with 2-bromobenzyl bromide at 308C for
12 h to give compound 7c, which was purified by recrystalliza-
tion from ethanol as white solid (71%, mp. 106–1088C). IR (KBr,
cm– 1): 1692 (C=O), 1324, 1192 (SO2). 1H-NMR (CDCl3, 600 MHz) d:
7.11 (s, 1H, PyH), 7.60 (d, 1H, J = 7.87 Hz, PhH), 7.51 (d, 2H, J =
7.46 Hz, PhH), 6.91 (d, 1H, J = 7.57 Hz, PhH), 6.90–7.52 (m, 5H,
PhH), 5.17 (s, 2H, NCH2), 5.14 (s, 2H, NCH2), 4.15 (s, 3H, CH3). 13C-
NMR (CDCl3, 150 MHz) d: 148.3 (C=O), 135.3, 133.3, 129.2, 128.3,
132.9, 129.2, 127.9, 127.8, 126.9, 125.3 (C-5), 125.1 (C-4a), 122.9
(C-7a), 122.2, 49.1 (N2-CH2), 44.6 (N4-CH2), 38.9 (CH3). MS
(C19H17BrN4O3S 461.33): m/z (ESI) 461.3 [M+], 463.3 [M + 2].
Chemistry
All melting points were determined on a micro melting-point
apparatus and are uncorrected. The 1H- and 13C-NMR spectra
were obtained on a Brucker Avance-600 (600 MHz; Bruker Bio-
science, Billerica, MA, USA). Chemical shifts are expressed in d
units and TMS as internal reference. Infrared spectra (IR) were
recorded with a Nexus 470FT-IR Spectrometer (Thermo Nicolet,
Madison, WI, USA). Mass spectra were taken on a LC Autosampler
Device: Standard G1313A instrument (Agilent, Palo Alto, CA,
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X. Liu et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 216–222
PhH), 6.87 (d, 1H, J = 8.39 Hz, PhH), 7.32–7.37 (m, 3H, PhH), 5.13
(s, 2H, NCH2), 5.12 (s, 2H, NCH2), 4.15 (s, 3H, CH3).13C-NMR (CDCl3,
150 MHz) d: 149.2 (C=O), 134.2, 135.2, 133.1, 129.4, 128.5, 128.3,
127.9, 127.8, 127.5, 125.1 (C-5), 124.7 (C-4a), 122.9 (C-7a), 46.1 (N2-
CH2), 44.5 (N4-CH2), 38.9 (CH3). MS (C19H16Cl2N4O3S 451.33): m/z
(ESI) 451.3 [M+].
2-Benzyl-4-(p-bromobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7d
Compound 5 and 4-bromobenzyl bromide at 308C for 12 h gave
compound 7d, which purified by recrystallization from ethanol
as white solid (73%, mp. 168–1708C). IR (KBr, cm– 1): 1694 (C=O),
1330, 1149 (SO2). 1H-NMR (DMSO-d6, 600 MHz) d: 7.83 (s, 1H, PyH),
7.31 (d, 2H, J = 8.43 Hz, PhH), 7.21 (d, 2H, J = 8.42 Hz, PhH), 7.53–
7.55 (m, 5H, PhH), 5.07 (s, 2H, NCH2), 5.01 (s, 2H, NCH2), 4.05 (s,
3H, CH3). 13C-NMR (DMSO-d6, 150 MHz) d: 148.9 (C=O), 135.6,
135.4, 131.7 131.4 130.0, 129.4, 126.2 (C-5), 125.5 (C-4a), 122.1 (C-
7a), 121.0, 120.9, 48.2 (N2-CH2), 43.5 (N4-CH2), 38.1 (CH3). MS
(C19H17BrN4O3S 461.33): m/z (ESI) 461.3 [M+], 463.3 [M + 2].
2-Benzyl-4-(p-(t-butyl)chlorobenzyl)-7-methyl-1,1,3-
trioxo-2,4-dihydro-pyrazolo[4,5-e][1,4]thiadiazine 7i
Compound 5 reacted with p-(t-butyl)chlorobenzyl chloride at
508C for 20 h to give compound 7i, which was purified by recrys-
tallization from ethanol as white solid (55%, mp. 76–788C). IR
(KBr, cm– 1): 1690 (C=O), 1327, 1190 (SO2). 1H-NMR (CDCl3,
600 MHz) d: 7.20 (s, 1H, PyH), 7.15 (d, 2H, J = 8.26 Hz, PhH), 7.32–
7.52 (m, 7H, PhH), 5.12 (s, 2H, NCH2), 5.03 (s, 2H, NCH2), 4.12 (s,
3H, CH3), 1.30 (s, 9H, CH3). 13C-NMR (CDCl3, 150 MHz) 150.8 (C=O),
131.5, 135.4, 149.2, 128.4, 128.2, 127.7, 126.5, 125.6, 125.5, 125.0
(C-4a), 122.5 (C-7a), 48.9 (N2-CH2), 44.3 (N4-CH2), 38.8 (CH3), 34.3
(C), 31.0 (3C, CH3). MS (C23H26N4O3S 438.54): m/z (ESI) 439.5
[M + 1].
2-Benzyl-4-(o-chlorobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7e
Compound 5 reacted with 2-chlorobenzyl chloride at 458C for
20 h to give compound 7e, which was purified by recrystalliza-
tion from ethanol as white solid (65%, mp. 104–1068C). IR (KBr,
cm– 1): 1691 (C=O), 1326, 1193 (SO2). 1H-NMR (CDCl3, 600 MHz) d:
6.95 (s, 1H, PyH), 7.13–7.51 (m, 9H, PhH), 5.19 (s, 2H, NCH2), 5.14
(s, 2H, NCH2), 4.15 (s, 3H, CH3). 13C-NMR (CDCl3, 150 MHz) d: 149.3
(C=O), 132.5, 135.3, 128.6, 128.3, 131.8, 129.7, 129.0, 127.9,
127.2, 170.0, 125.3 (C-5), 124.9 (C-4a), 120.0 (C-7a), 46.6 (N2-CH2),
44.5 (N4-CH2), 38.9 (CH3). MS (C19H17ClN4O3S 416.88): m/z (ESI)
417.4 [M + 1].
2-Benzyl-4-(o-cyanobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7j
Compound 5 reacted with 2-cyanobenzyl chloride at 508C for
20 h to give compound 7j, which was purified by recrystalliza-
tion from ethanol as white solid (45%, mp. 136–1388C). IR (KBr,
cm– 1): 2224 (CN), 1690 (C=O), 1328, 1196 (SO2).1H-NMR (DMSO-d6,
600 MHz) d: 7.84 (s, 1H, PyH), 7.15 (d, 1H, J = 7.88 Hz, PhH), 7.26–
7.89 (m, 8H, PhH), 5.29 (s, 2H, NCH2), 5.02 (s, 2H, NCH2), 4.09 (s,
3H, CH3). MS (C20H17N5O3S 407.45): m/z (ESI) 408.5 [M + 1].
2-Benzyl-4-(m-chlorobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7f
Compound 5 reacted with 3-chlorobenzyl chloride at 458C for
20 h to give compound 7f, which was purified by recrystalliza-
tion from ethanol as white solid (64%, mp. 106–1088C). IR (KBr,
cm– 1): 1665 (C=O), 1320, 1194 (SO2). 1H-NMR (CDCl3, 600 MHz) d:
7.15 (s, 1H, PyH), 7.19 (s, 1H, PhH), 7.07 (d, 1H, J = 6.97 Hz, PhH),
7.20–7.51 (m, 7H, PhH), 5.12 (s, 2H, NCH2), 5.03 (s, 2H, NCH2),
4.14 (s, 3H, CH3). 13C-NMR (CDCl3, 150 MHz) d: 149.2 (C=O), 135.2,
136.6, 134.7, 130.0, 128.4, 128.3, 128.3, 128.1, 127.8, 126.8, 125.2
(C-5), 124.7 (C-4a), 122.7 (C-7a), 48.6 (N2-CH2), 44.4 (N4-CH2), 38.8
(CH3). MS (C19H17ClN4O3S 416.88): m/z (ESI) 417.4 [M + 1].
2-Benzyl-4-(m-cyanobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7k
Compound 5 reacted with 3-cyanobenzyl chloride at 508C for
20 h to give compound 7k, which was purified by recrystalliza-
tion from ethanol as white solid (46%, mp. 208–8108C). IR (KBr,
1
cm– 1): 2229 (CN), 1689 (C=O), 1331, 1192 (SO2). H-NMR (CDCl3,
2-Benzyl-4-(p-chlorobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7g
600 MHz) d: 7.14 (s, 1H, PyH), 7.28–7.61 (m, 9H, PhH), 5.12 (s, 2H,
NCH2), 5.07 (s, 2H, NCH2), 4.16 (s, 3H, CH3). 13C-NMR (CDCl3,
600 MHz) d: 144.4 (C=O), 130.3, 131.5, 126.9, 126.2, 125.4, 124.9,
123.2, 123.7, 123.6, 108.3, 120.3, 119.6, 118.1, 113.1 (CN), 43.7
(N2-CH2), 39.8 (N4-CH2), 34.2 (CH3). MS (C20H17N5O3S 407.45): m/z
(ESI) 408.5 [M + 1].
Compound 5 reacted with 4-chlorobenzyl chloride at 458C for
20 h to give compound 7g, which was purified by recrystalliza-
tion from ethanol as white solid (65%, mp. 92–948C). IR (KBr,
cm– 1): 1671 (C=O), 1320, 1193 (SO2). 1H-NMR (CDCl3, 600 MHz) d:
7.28 (s, 1H, PyH), 7.13–7.50 (m, 9H, PhH), 5.11 (s, 2H, NCH2), 5.02
(s, 2H, NCH2), 4.13 (s, 3H, CH3). 13C-NMR (CDCl3, 150 MHz) d: 149.2
(C=O), 133.8, 135.2, 133.1, 127.8, 128.9, 128.5, 128.3, 128.1, 125.2
(C-5), 124.7 (C-4a), 122.7 (C-7a), 48.5 (N2-CH2), 44.4 (N4-CH2), 38.9
(CH3). MS (C19H17ClN4O3S 416.88): m/z (ESI) 417.4 [M + 1].
2-Benzyl-4-(p-cyanobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7l
Compound 5 reacted with 4-cyanobenzyl chloride at 508C for
20 h to give compound 7l, which was purified by recrystalliza-
tion from ethanol as white solid (47%, mp. 148–1508C). IR (KBr,
2-Benzyl-4-(2,4-dichlorobenzyl)-7-methyl-1,1,3-trioxo-
2,4-dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7h
1
cm– 1): 2229 (CN), 1693 (C=O), 1325, 1196 (SO2). H-NMR (CDCl3,
Compound 5 reacted with 2,4-dichlorobenzyl chloride at 508C
for 20 h to give compound 7h, which was purified by recrystalli-
zation from ethanol as white solid (63%, mp. 112–1148C). IR
(KBr, cm– 1): 1697 (C=O), 1324, 1190 (SO2). 1H-NMR (CDCl3,
600 MHz) d: 7.12 (s, 1H, PyH), 7.49 (d, 2H, J = 6.98 Hz, PhH), 7.43
(d, 1H, J = 2.68 Hz, PhH), 7.14 (dd, 1H, J = 8.38 Hz, J = 2.09 Hz,
600 MHz) d: 7.11 (s, 1H, PyH), 7.49 (d, 2H, J = 7.60 Hz, PhH), 7.28-
7.63 (m, 7H, PhH), 5.11 (s, 2H, NCH2), 5.10 (s, 2H, NCH2), 4.15 (s,
3H, CH3). 13C-NMR (CDCl3, 150 MHz) d: 149.2 (C=O), 135.1, 139.9,
132.6, 128.5, 128.3, 127.9, 127.2, 111.9, 125.1 (C-5), 124.4 (C-4a),
123.0 (C-7a), 118.0 (CN), 48.7 (N2-CH2), 44.6 (N4-CH2), 38.9 (CH3).
MS (C20H17N5O3S 407.45): m/z (ESI) 408.5 [M + 1].
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Arch. Pharm. Chem. Life Sci. 2008, 341, 216–222
Novel PTD Analogues
221
General procedure for the preparation of compounds
N2,N4-disubstituted 7-methyl-1,1,3-trioxo-2,4-dihydro-
pyrazolo[4,5-e][1,2,4]thiadiazines 7m–r
2,4-Di-(2,4-dichlorobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7p
Compound 5 reacted with 2,4-dichlorobenzyl chloride at 60–
708C for 20 h, purification by recrystallization from ethanol
gave compound 7r as white solid (73%), mp. 176–1788C. IR (KBr,
cm– 1): 1698 (C=O), 1326, 1186 (SO2). 1H-NMR (DMSO-d6, 600 MHz)
d: 7.82 (s, 1H, PyH), 7.69 (d, 1H, J = 2.12 Hz, PhH), 7.66 (d, 1H, J =
2.14 Hz, PhH), 7.31 (d, 1H, J = 8.47 Hz, PhH), 7.12 (d, 1H, J =
8.41 Hz, PhH), 7.38–7.43 (m, 2H, PhH), 5.13 (s, 2H, NCH2), 5.08 (s,
2H, NCH2), 4.08 (s, 3H, CH3).13C-NMR (DMSO-d6, 600 MHz) d: 148.6
(C=O), 133.0, 133.2, 133.1, 132.7, 132.6, 132.2, 129.5, 129.4,
129.1, 128.9, 127.8, 127.7, 126.4 (C-5), 125.8 (C-4a), 122.0 (C-7a),
47.1 (N2-CH2), 41.4 (N4-CH2), 39.1 (CH3). MS (C19H14Cl4N4O3S
520.22): m/z (ESI) 520.2 [M+].
To a solution of compound (5, 1 eq) in dry DMF (1 mmol, 4 mL)
was added sodium hydride (60% dispersion in mineral oil, 2 eq)
in portions, under inert atmosphere (N2) and keeping the tem-
perature at 108C. After 60 minutes stirring, the alkyl halide (RX,
2 eq) was added dropwise. The mixture was stirred at room tem-
perature for 20 min., and 30–808C for 12–20 h (checked by
TLC). After the solvent was evaporated off under reduced pres-
sure, the crude product was purified by recrystallization from
ethanol.
2,4-Di-(p-methylbenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7m
2,4-Di-(p-chlorobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7q
Compound 5 reacted with 4-methylbenzyl chloride at 60–708C
for 20 h, purification by recrystallization from ethanol gave
compound 7m as white solid (60%), mp. 116–1188C. IR (KBr, cm–
1): 1668 (C=O), 1331, 1192 (SO2). 1H-NMR (DMSO-d6, 600 MHz) d:
7.80 (s, 1H, PyH), 7.12–7.24 (m, 8H, PhH), 5.04 (s, 2H, NCH2), 4.98
(s, 2H, NCH2), 4.04 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.25 (s, 3H, CH3).
13C-NMR (DMSO-d6, 150 MHz) d: 148.6 (C=O), 137.1, 137.2, 133.2,
133.0, 129.4, 129.1, 128.0, 127.3, 126.3 (C-5), 125.5 (C-4a), 122.3
(C-7a), 48.4 (N2-CH2), 43.8 (N4-CH2), 39.0 (Py-CH3), 20.8, 20.9 (2C,
CH3). MS (C21H22N4O3S 410.49): m/z (ESI) 411.5 [M + 1].
Compound 5 reacted with 4-chlorobenzyl chloride at 50–608C
for 20 h, purification by recrystallization from ethanol gave
compound 7q as white solid (75%), mp. 146–1488C. IR (KBr,
cm–1): 1696 (C=O), 1332, 1190 (SO2). 1H-NMR (DMSO-d6, 600 MHz)
d: 7.84 (s, 1H, PyH), 7.28 (d, 2H, J = 8.50 Hz, PhH), 7.37-7.42 (m, 6H,
PhH), 5.09 (s, 2H, NCH2), 5.03 (s, 2H, NCH2), 4.05 (s, 3H, CH3). 13C-
NMR (DMSO-d6, 150 MHz) d: 149.0 (C=O), 135.0, 135.3, 132.5,
132.6, 129.8, 129.2, 128.9, 128.6, 126.3 (C-5), 125.5 (C-4a), 122.2
(C-7a), 48.2 (N2-CH2), 43.4 (N4-CH2), 39.1 (CH3). MS (C19H16Cl2N4O3S
451.33): m/z (ESI) 451.4 [M+].
2,4-Di-(p-bromobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7r
2,4-Di-(o-bromobenzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7n
Compound 5 reacted with 4-bromobenzyl bromide at 40–508C
for 15 h, purification by recrystallization from ethanol gave
compound 7o as white solid (83%), mp. 182–1848C. IR (KBr,
cm–1): 1693 (C=O), 1329, 1189 (SO2). 1H-NMR (DMSO-d6, 600 MHz)
d: 7.83 (s, 1H, PyH), 7.31 (d, 2H, J = 8.41 Hz, PhH), 7.21 (d, 2H, J =
8.38 Hz, PhH), 7.53–7.55 (m, 4H, PhH), 5.07 (s, 2H, NCH2), 5.01 (s,
2H, NCH2), 4.05 (s, 3H, CH3). 13C-NMR (DMSO-d6, 150 MHz) 150.0
(C=O), 135.4, 135.6, 131.7 131.4 130.0, 129.4, 126.2 (C-5), 125.5 (C-
4a), 122.1 (C-7a), 121.0, 120.9, 48.2 (N2-CH2), 43.5 (N4-CH2), 39.0
(CH3). MS (C19H16Br2N4O3S 540.23): m/z (ESI) 541.2 [M + 1].
Compound 5 reacted with 2-bromobenzyl bromide at 40–508C
for 15 h, purification by recrystallization from ethanol gave
compound 7n as white solid (81%), mp. 120–1228C. IR (KBr, cm–
1): 1697 (C=O), 1322, 1193 (SO2). 1H-NMR (DMSO-d6, 600 MHz) d:
7.79 (s, 1H, PyH), 7.67 (dd, 1H, J = 7.95 Hz, J = 1.10 Hz, PhH), 7.40
(d, 1H, J = 7.52 Hz, PhH), 7.03 (dd, 1H, J = 7.70 Hz, J = 1.23 Hz,
PhH), 7.23–7.38 (m, 5H, PhH), 5.12 (s, 2H, NCH2), 5.06 (s, 2H,
NCH2), 4.09 (s, 3H, CH3). 13C-NMR (DMSO-d6, 150 MHz) d: 148.7
(C=O), 134.4, 134.6, 133.2, 132.7, 129.7, 129.6, 128.3, 128.1,
127.7, 127.2, 126.4, 125.9 (C-5), 122.1, 122.0, 121.6, 49.7 (N2-CH2),
44.2 (N4-CH2), 39.2 (CH3). MS (C19H16Br2N4O3S 540.23): m/z (ESI)
541.3 [M + 1].
Anti-HIV activity assays
The anti-HIV activity and cytotoxicity were evaluated against
wild type HIV-1 strain IIIB and HIV-2 (ROD) in MT-4 cells using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) method [14] MT-4 cells were suspended in culture
medium at 16105 cells/mL and infected with HIV at a multiplic-
ity of infection (MOI) of 0.02. Immediately after viral infection,
100 lL of the cell suspension were placed in each well of a flat-
bottomed microtiter tray containing various concentrations of
the test compounds. Stock solutions of the test compounds were
prepared in DMSO at a concentration of 10 mg/mL. After four
days of incubation at 378C, the number of viable cells was deter-
mined using the MTT method. Compounds were tested in paral-
lel for cytotoxic effects in uninfected MT-4 cells.
2,4-Di-(p-(t-butyl)benzyl)-7-methyl-1,1,3-trioxo-2,4-
dihydro-pyrazolo[4,5-e][1,2,4]thiadiazine 7o
Compound 5 reacted with 4-(t-butyl)benzyl chloride at 70–808C
for 20 h, purification by recrystallization from ethanol gave
compound 7p as white solid (65%), mp. 130–1328C. IR (KBr,
1
cm–1): 1695 (C=O), 1334, 1186 (SO2). H-NMR (CDCl3, 600 MHz) d:
7.19 (s, 1H, PyH), 7.44 (d, 2H, J = 8.33 Hz, PhH), 7.37 (d, 2H, J =
8.39 Hz, PhH), 7.32 (d, 2H, J = 8.33 Hz, PhH), 7.14 (d, 2H, J =
8.30 Hz, PhH), 5.09 (s, 2H, NCH2), 5.03 (s, 2H, NCH2), 4.12 (s, 3H,
CH3), 1.32 (s, 9H, CH3), 1.30 (s, 9H, CH3).13C-NMR (CDCl3, 150 MHz)
d: 150.7 (C=O), 131.5, 132.3, 150.6, 149.2, 128.3, 126.5, 125.6,
125.5, 125.2, 125.0, 122.5 (C-4a), 122.0 (C-7a), 48.8 (N2-CH2), 44.0
(N4-CH2), 38.8 (CH3), 34.3, 34.2 (2C), 31.0, 30.9 (6C, CH3). MS
(C27H34N4O3S 494.65): m/z (ESI) 495.5 [M + 1].
The 50% effective antiviral concentration (EC50) was defined as
the compound concentration required protecting 50% of the
virus-infected cells against viral cytopathicity. The 50% cytotoxic
concentration (CC50) was defined as the compound concentra-
tion required reducing the viability of mock-infected cells by
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

222
X. Liu et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 216–222
50%. The symbol ”A” is used to indicate the highest concentra-
tion at which the compounds were tested and still found to be
non-cytotoxic. Average EC50 and CC50 values for at least two sepa-
rate experiments are presented.#
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The financial support from the National Natural Science Foun-
dation of China (NSFC No.30772629 and 30371686), key project
of The International Cooperation, Ministry of Science and Tech-
nology of China (2003DF000033) and Natural Science Founda-
tion of Shandong Province (Y2003C11), is gratefully acknowl-
edged.
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The authors have declared no conflict of interest.
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i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com