Synthesis, molecular docking and antiviral screening of novel N'-substitutedbenzylidene-2-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4, 3-c][1,2]thiazin-1(4H)-yl)acetohydrazides

Article abstract of DOI:10.1007/s00044-013-0879-7

N'-Substitutedbenzylidene-2-(4-methyl-5,5-dioxido-3-phenylbenzo[e] pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)acetohydrazides (7a-u) were synthesized through a multistep reaction. The final compounds were screened for anti-HIV-1 and cytotoxic activities. Among

Full text of DOI:10.1007/s00044-013-0879-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0879-7
ORIGINAL RESEARCH
Synthesis, molecular docking and antiviral screening of novel
N0-substitutedbenzylidene-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)
acetohydrazides
•
Sana Aslam Matloob Ahmad Muhammad Makshoof Athar
•
•
•
•
•
Usman Ali Ashfaq John M. Gardiner Catherine Montero Mervi Detorio
•
•
Masood Parvez Raymond F. Schinazi
Received: 9 June 2013 / Accepted: 13 November 2013
Ó Springer Science+Business Media New York 2013
Abstract N0-Substitutedbenzylidene-2-(4-methyl-5,5-di-
oxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazides (7a–u) were synthesized through a
multistep reaction. The final compounds were screened for
anti-HIV-1 and cytotoxic activities. Among these com-
pounds, 7l and 7m exhibited the most significant anti-HIV-1
activity with EC50 values of 5.4 and 3.3 lM, respectively,
while 7j showed moderate anti-HIV activity with an EC50
value 17.1 lM. Molecular docking into HIV-1 Reverse
Transcriptase also showed the best fit for 7l and 7m followed
by 7j. In addition, compounds 7b, 7f, 7h, 7k, 7o, and 7s
exhibited no toxicity in all the three cell lines used i.e., pri-
mary human PBM, CEM, and Vero cells, while 7e, 7g, 7i, 7n,
7p, 7q, and 7t exhibited no cytotoxic activity in primary
human PBM cells. Moreover, it was found through molec-
ular docking that compounds 7l, 7m, and 7j bound efficiently
in the NTP-binding pocket of HIV-1 Reverse Transcriptase.
The structure–activity relationship established on these
results would facilitate the further development of new HIV
inhibitors based on this skeleton.
Keywords Anti-HIV-1 activity ꢀ 1,2-Benzothiazine ꢀ
Cytotoxic activity ꢀ Hydrazides ꢀ
Pyrazolobenzothiazine dioxides
Introduction
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0879-7) contains supplementary
material, which is available to authorized users.
The 1,2-benzothiazines ring system is a heterocyclic scaf-
fold of paramount importance in drug chemistry. The
potent anti-inflammatory drugs based on this pharmaco-
phore, e.g., piroxicam (Lomabardino et al., 1971),
This research article is dedicated to Dr. Hamid Latif Siddiqui
(Institute of Chemistry, University of the Punjab, Lahore-Pakistan),
our honorable teacher (SA and MA), our friend and colleague (MMA,
JMG, MP, and RFS) who passed away on July 26, 2012.
J. M. Gardiner
School of Chemistry and Manchester Institute of Biotechnology,
University of Manchester, Manchester M1 7DN, UK
S. Aslam
Department of Chemistry, Government College Women
University, Faisalabad, Pakistan
C. Montero ꢀ M. Detorio ꢀ R. F. Schinazi
S. Aslam ꢀ M. M. Athar
Laboratory of Biochemical Pharmacology, Emory University
School of Medicine/Veterans Affairs Medical Center, 1760
Haygood Drive, Atlanta, GA 30322, USA
Institute of Chemistry, University of the Punjab, Lahore 54590,
Pakistan
M. Ahmad (&)
Department of Chemistry, Government College University,
Faisalabad 38000, Pakistan
e-mail: Matloob_123@yahoo.com
M. Parvez
Department of Chemistry, The University of Calgary, 2500
University Drive NW, Calgary, AB T2N 1N4, Canada
U. A. Ashfaq
Department of Bioinformatics and Biotechnology, Government
College University, Faisalabad 38000, Pakistan
123

Med Chem Res
tenoxicam, meloxicam (Turck et al., 1995; Engelhardt
et al., 1995), ampiroxicam (Lombardino et al., 1973;
Myung and Soon, 2000; Marfat, 1985), lornoxicam (Caruso
et al., 1994), and droxicam (Soler, 1986), are classified as
oxicams within the broad class of NSAIDs. Various
derivatives of this class have been established as antimi-
crobial (Giuseppe et al., 1987), antiallergic (Ikeda et al.,
1992), anti-inflammatory (Suh et al., 1987), antianxiety,
antidepressant (Krapcho and Turk, 1975; Krapcho, 1973),
and antithrombotic agents (Constantine, 1967). Recently,
pyrazolobenzothiazine derivatives are reported as potent
HCV replication inhibitors (Barreca et al., 2013).
Results and discussion
Chemistry
N0-substitutedbenzylidene-2-(4-methyl-5,5-dioxido-3-phenyl
benzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)acetohydraz-
ides (7a–u) were synthesized via the reaction of
2-(4-methyl-3-phenyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzo-
thiazin-1(4H)-yl)acetohydrazide (6) with a range of substi-
tuted benzaldehydes (Scheme 2).
The starting material (N-phenacylsaccharine, 1) was
synthesized according to our previously reported thermal
method (Ahmad et al., 2010c), as well as using ultrasound
and microwave irradiation techniques (Table 1). The cor-
responding 1,2-benzothiazine derivative was synthesized by
the ring expansion reaction of 2-(2-oxo-2-phenylethyl)-1,2-
benzisothiazol-3(2H)-one-1,1-dioxide (1) using NaOMe in
MeOH (Ahmad et al., 2010d). N-Methylation of 3-benzoyl-
4-hydroxy-2H-1,2-benzothiazine-1,1-dioxide (2) was also
attempted using ultrasound (Table 2), which reduced reac-
tion times to 10–15 min with 85 % yield. The formation of
alkylated product was confirmed by the appearance of
Human Immunodeficiency Virus (HIV) is responsible
for Acquired Immune Deficiency Syndrome (AIDS). It is a
disease characterized by extensive immunosuppression that
predisposes patients to life-threatening opportunistic
infections and unusual forms of neoplasms (Gottlieb et al.,
1981; Masur et al., 1981; Siegal et al., 1981; Francis et al.,
1983; Broder and Gallo, 1984; Shaw et al., 1985; Feriono
et al., 1985). AIDS is thought to result from infection of T
cells by a pathogenic human retrovirus, human T-lym-
photropic virus type III (HTLV-III) or lymphadenopathy-
associated virus (LAV) (Barrd-Sinoussi et al., 1983;
Klatzman et al., 1984). This virus preferentially infects and
destroys OKT4? (helper/inducer) T-cells. Among the most
potent anti-HIV compounds, ribavirin (Fig. 1) (McCor-
mick et al., 1984), antimoniotungstate (HPA-23) (Rosen-
baum et al., 1985), ansamycin and phosphonoformic acid
(Sandstrom et al., 1985), and Crixivan (Fig. 1) are of sig-
nificant importance.
1
N–CH3 peak at 2.89 ppm in the H NMR spectrum and by
HR-MS spectrum. Treatment of compound 3 with hydrazine
monohydrate in refluxing ethanol resulted in the cyclization
of pyrazole ring to give 4-methyl-3-phenyl-2,4-dihydro-
benzo[e]pyrazole[4,3-c][1,2]thiazine (4)
(Scheme 1). In FTIR, NH and C=N functionalities were
5,5-dioxide
Table 1 Comparison of thermal and ultrasonic reaction conditions
for N-alkylation of 3-benzoyl-4-hydroxy-2H-1,2-benzothiazine-1,
1-dioxide (3)
To date, many drugs are known to inhibit HIV at
various stages of its life cycle. Yet, there remains an
urgent need for the development of new anti-HIV drugs
and therapeutic strategies. Our endeavors on the versatile
synthesis of a range of 1,2-benzothiazines led us to report
the synthesis of a number of libraries of biologically
active benzothiazine derivatives (Ahmad et al., 2010a, b,
2012, 2013a, b; Bukhari et al., 2011). Recently, we have
reported pyrazolobenzothiazine derivatives as the first
example of HIV inhibitors from this family of heterocy-
clic compounds (Aslam et al., 2013). Noting the
involvement of heterocyclic templates in a number of
antiviral agents (Fig. 1), we herein describe the potential
of various new derivatives of pyrazolobenzothiazine ring
system as anti-HIV-1 agents.
Entry
Technique used
Reaction time (min)
Yield (%)
1
2
Thermal method
30–40
10–15
80
85
Ultrasonic irradiation
Table 2 Synthesis of 2-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyr-
azolo[4,3-c][1,2]thiazin-1(4H)-yl)acetohydrazide (6) under different
reaction conditions
Entry Technique used
Reaction time (min) Yield (%)
1
2
3
Thermal method
240
80
71
75
Ultrasonic irradiation
30–40
Microwave irradiation 10–20
Fig. 1 The structures of anti-
HIV drugs, Ribavirin and
Crixivan (Indinavir)
O
N
N
Ph
O
N
N
OH
OH
O
NH2
H
N
N
O
N
HO
t-Bu
OH
N
HO
H
Ribavirin
Crixivan
123

Med Chem Res
Scheme 1 Microwave-assisted
synthesis of 4-methyl-3-phenyl-
2,4-dihydrobenzo[e]pyrazole
[4,3-c][1,2]thiazine 5,5-dioxide
(4)
O
O
O
O
Br
i
( )
NNa
N
+
S
S
Microwave, 30W
O
O
O
O
(1)
(ii)
OH O
N
OH O
(iii)
Ultrasonic bath
NH
S
S
CH3
O
O
(3)
O
O
(2)
3
M
0
i
W
c
r
o
(iv)
w
a
v
e
,
HN
N
N
S
CH3
O
O
(4)
Chemicals and reagents: (i) dimethylformamide (ii) NaOMe/MeOH, reflux
(iii) (CH3)2SO4/NaOH, acetone (iv) hydrazine monohydrate
observed at 3,368 and 1,655 cm-1, respectively. N-Alkyl-
ation of compound (4) with various alkyl chloroacetates was
affected using K2CO3 as a base. The structure was confirmed
from the O–CH3 peak at 3.27 ppm and N–CH2 at 5.77 ppm
in 1H NMR spectrum and a carbonyl stretch at 1,685 cm-1 in
the FTIR spectrum. For 5b, observation of a quartet at
3.96 ppm due to the O–CH2 and a triplet at 1.09 ppm for the
C–CH3 group supported structural assignment. Molecular
ion peaks were observed at 383.06 and 397.96 in the MS for
compounds 5a and 5b, respectively.
and it was observed that although the obtained yields were
almost same for all three methods, there was a significant
reduction in reaction times using microwave irradiation com-
pared to the thermal reaction times.
X-ray crystallographic studies
The molecular structure of 7b is shown in the ORTEP
(Fig. 2). The heterocyclic thiazine ring adopts a twist chair
conformation with atoms S1 and C6 displaced by 0.732(2)
˚
and -0.245(3) A, respectively, from the mean plane
Compound (5) was refluxed with hydrazine monohydrate
in absolute ethanol to provide 2-(4-methyl-5,5-dioxido-
3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)aceto-
hydrazide (6). Microwave irradiation resulted in 75 % yield
after a much reduced reaction time of only 10 min, compared
to the 4 h required for the thermal reaction. Absorption peaks
for C=N and NH groups appeared at 1,675 and 3,468 cm-1 in
formed by the remaining ring atoms (N1//C1/C7/C8). The
mean planes of benzene rings C1—C6 and C20—C25
make dihedral angles 22.94(8)° and 9.90(10)°, respec-
tively, with the mean plane of the pyrazolyl ring (N2/N3/
C7/C8/C10). The acetamide chain (O3/C12/N4/N5/C13) is
fully extended. The crystal data and structure refinement
parameters are described in Table 4.
1
the FTIR spectrum, respectively. In the H NMR spectrum,
NH2 and NH gave rise to resonances at 4.39 and 11.94 ppm,
respectively. N0-Substitutedbenzylidene-2-(4-methyl-5,5-di-
oxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)
acetohydrazides (7a–u) (Scheme 2) were obtained by the
reaction of compound 6 with a range of substituted benzalde-
hydes in refluxing ethanol using the conventional thermal
method, the microwave irradiation, and also using an ultra-
sonic bath. The reaction parameters are discussed in Table 3,
Virology
Anti-HIV-1 activity
The ability of these N0-substitutedbenzylidene-2-(4-methyl-
5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-
123

Med Chem Res
Scheme 2 Layout for
microwave-assisted synthesis of
the titled compounds (7a–u)
RO
O
HN
S
N
N
S
N
(i)
N
N
CH3
CH3
O
O
O
O
(4)
5a; R = CH3
5b; R = C2H5
R'
(ii)
N
HN
H
N
O
H2N
N
S
N
N
S
N
O
(iii)
Microwave, 30W
N
N
O
CH3
CH3
O
O
O
(6)
(7a-u)
Chemical and Reagents: (i) alkyl chloroacetates, K2CO3, CH3CN (ii) hydrazine monohydrate, EtOH
(iii) RCHO, EtOH, 0.2 mL acetic acid
Table 3 Synthesis of N0-substitutedbenzylidene-2-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)acetohydrazide
(7a–u) under microwave irradiation, ultrasonic irradiation, and thermal reaction
Entry
Products
R0
Conventional thermal method
Microwaves irradiation
Ultrasonic bath
Reaction
time (min)
Yield (%)
Reaction
time (min)
Yield (%)
Reaction
time (min)
Yield (%)
1
7a
7b
7c
7d
7e
7f
4-Bromophenyl
26
25
20
22
17
20
21
24
21
22
23
30
28
20
18
17
24
20
21
19
20
81
82
81
80
79
80
80
81
80
82
85
80
77
79
82
82
80
80
79
80
79
3
83
84
87
82
85
86
85
85
90
90
87
81
80
82
84
85
86
84
81
84
83
4
4
4
5
4
6
5
4
4
3
3
7
7
8
8
6
5
6
5
6
4
79
79
84
82
81
83
85
83
89
87
81
79
78
80
80
83
81
80
80
81
82
2
2,6-Dichlorophenyl
4-Chlorophenyl
2
3
2.5
2
4
4-Dimethyl aminophenyl
2-Nitrophenyl
5
2.5
2
6
3-Nitrophenyl
7
7g
7h
7i
4-Diethyl aminophenyl
2,4-Dichlorophenyl
2,3,4-Trimethoxyphenyl
4-Methoxyphenyl
2,4-Dimethoxyphenyl
3-Ethoxy-4-hydroxyphenyl
2,4-Dihydroxyphenyl
2-Fluorophenyl
2.5
3
8
9
2
10
11
12
13
14
15
16
17
18
19
20
21
7j
3.5
4.5
3.5
2.5
3.5
3.5
2.5
3
7k
7l
7m
7n
7o
7p
7q
7r
7s
3,4,5-Trimethoxyphenyl
2-Bromophenyl
4-Acetamaidophenyl
3,4-Dichlorophenyl
4-Nitrophenyl
3.5
3
7t
3-Chlorophenyl
2.5
2.5
7u
3-Bromophenyl
123

Med Chem Res
Fig. 2 ORTEP diagram for N0-
(2,6-dichlorobenzylidene)-2-(4-
methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-
c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7b)
C18
C19
C17
C16
Cl2
C14
C15
C13
Cl1
N5
N4
C12
C11
O3
N3
N2
C5
C21
C10
C7
C4
C3
C22
C6
C20
C8
O2
C25
C1
C23
N1
C9
C2
S1
C24
O1
Cytotoxic activity
1(4H)-yl)acetohydrazides (7a–u) to inhibit viral growth in
activated primary human peripheral blood mononuclear
(PBM) cells was determined by screening against HIV-1
according to previously reported methodology (Schinazi
et al. 1990). The results obtained for these compounds are
summarized in Table 5. The data were compared with the
antiviral activity of 30-azido-30-deoxythymidine (AZT), a
potent clinical anti-HIV nucleoside reverse transcriptase
inhibitor (NRTI). From the results of the anti-HIV assay, it
is difficult to establish an obvious structure activity rela-
tionship (SAR) for the final compounds. Two compounds,
7l and 7m, exhibited the most significant anti-HIV-1
activity with EC50 values 5.4 and 3.3 lM, respectively,
while 7j showed moderate anti-HIV-1 activity with an EC50
value 17.1 lM. The SAR was established with the help of
molecular docking.
The final compounds were also tested for their cytotoxicity in
primary human PBM, CEM, and Vero cells as described by
Stuyver et al., 2002. The results of cell-based assays are
expressed as IC50 (median inhibitory concentration) (Table 5).
Out of the 21 N0-substitutedbenzylidene-2-(4-methyl-5,5-di-
oxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)
acetohydrazides evaluated (7a–u), six (7b, 7f, 7h, 7k, 7o, and
7s) showed significant nontoxic behavior in all three cell lines
usedi.e., primaryhumanPBM, CEM, andVerocells, while7e,
7g, and 7i were noncytotoxic to primary human PBM and
CEM cells. Moreover, compounds 7n, 7p, 7q, and 7t were
nontoxic in primary human PBM and Vero cells. Compound
7j, which is moderately active for anti-HIV-1 activity, is
nontoxic to human PBM cells. The nontoxic behavior in
123

Med Chem Res
Table 4 Crystal data and structure refinement for N0-(2,6-dichlorobenzylidene)-2-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-
c][1,2]thiazin-1(4H)-yl)acetohydrazide (7b)
Parameters
Values
Parameters
Values
CCDC number
Empirical formula
Formula weight
Temperature
915138
C25 H19 Cl2 N5 O3 S
540.41
Theta range for data collection 1.96°–27.67°
Index ranges
-10 B h B 10, -14 B k B 14, -19 B l B 19
173(2) K
Reflections collected
Independent reflections
9869
˚
0.71073 A
Wavelength
5385 [R(int) = 0.029]
Crystal system
Space group
Triclinic
Completeness to theta = 27.67° 98.2 %
Absorption correction multiscan
P-1
˚
Unit cell dimensions a = 7.8666(3) A, a = 89.500(2)° Max. and min. transmission
0.980 and 0.916
˚
b = 10.8263(5) A, b = 75.029(2)°
˚
c = 14.7797(6) A, c = 74.588(2)°
˚
1169.85(8) A
3
Volume
Refinement method
Full-matrix least-squares on F2
Z
2
Data/restraints/parameters
5385/0/329
Density (calculated)
1.534 Mg/m3
Goodness-of-fit on F2
1.10
Absorption coefficient 0.407 mm-1
Final R indices [I [ 2sigma(I)] R1 = 0.050, wR2 = 0.103
F(000)
556
0.22 9 0.22 9 0.05 mm3
R indices (all data)
R1 = 0.060, wR2 = 0.110
-3
˚
Crystal size
Largest diff. peak and hole
0.35 and -0.37 eꢀA
Table 5 Anti-HIV-1 and cytotoxicity screening of N0-substituted benzylidene-2-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-
c][1,2]thiazin-1(4H)-yl)acetohydrazides (7a–u)
Anti- HIV-1 activity in primary human PBM cells (lM)
Cytotoxicity (IC50, lM)
Compound Codes
R0
EC50
EC90
PBM
CEM
Vero
7a
7b
7c
7d
7e
7f
4-Bromophenyl
[100
[100
[100
[100
[100
[100
[100
[100
[100
17.1
[100
[100
[100
[100
[100
[100
[100
[100
[100
53.9
97.0
46.9
78.6
44.9
[100
88.9
99.6
[100
[100
[100
20.7
[100
\1
36.4
[100
20.5
2,6-Dichlorophenyl
4-Chlorophenyl
[100
[100
14.4
4-Dimethylaminophenyl
2-Nitrophenyl
[100
45.8
[100
[100
[100
[100
[100
[100
[100
8.7
3-Nitrophenyl
[100
23.5
7g
7h
7i
4-Diethylaminophenyl
2,4-Dichlorophenyl
2,3,4-Trimethoxyphenyl
4-Methoxyphenyl
2,4-Dimethoxyphenyl
3-Ethoxy-4-hydroxyphenyl
2,4-Dihydroxyphenyl
2-Fluorophenyl
88.9
19.6
7j
15.4
7k
7l
99.8
[100
15.5
[100
9.2
5.4
7m
7n
7o
7p
7q
7r
7s
3.3
5.9
7.8
\1
4.3
[100
[100
[100
[100
[100
[100
[100
[100
0.00037
[100
[100
[100
[100
[100
[100
[100
[100
0.014
[100
[100
[100
[100
38.0
23.4
75.4
36.6
44.6
19.3
[100
12.1
17.6
14.3
[100
[100
[100
C100
[100
[100
78.5
3,4,5-Trimethoxyphenyl
2-Bromophenyl
4-Acetamaidophenyl
3,4-Dichlorophenyl
4-Nitrophenyl
[100
51.6
7t
3-Chlorophenyl
7u
AZT
3-Bromophenyl
31.8
[100
56.0
[100
Where EC50 and IC50 are the median effective (antiviral) and inhibitory (cytotoxic) concentrations, respectively. All experiments were conducted
in replicate
123

Med Chem Res
Ser105, Va106, Asp192, Ile195, Pro236, Asp237, and
Asp320 (Fig. 3c). 3D images of 7m, 7l, and 7j docked in
the NTRI-binding site are shown in Fig. 4. Docking of
AZT with HIV-1 reverse transcriptase showed that it had
the interactions with Glu28, Lys32, Pro140, and Lys103
while also having contact with Ile31, Lys101, Lys102,
Ile132, Pro133, Ser134, Thr139, Gly141, Asp177, Val179,
Asp192, and Pro321 (Fig. 5).
Table 6 The interactions of compounds with HIV-1 reverse
transcriptase
Compound
EC50 (lM)
S score
Interacting residues
7l
5.4
-10.41616
-10.16434
-9.33224
-16.21676
Ser 105
Lys 102
Thr 107
7m
7j
3.3
17.1 lM
AZT
Glu28, Lys32,
Pro140, Lys103
Conclusion
human cell lines supports the potential of these compounds for
their medicinal use.
In short, we report an efficient synthesis of new N0-substituted
benzylidene-2-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyraz-
olo[4,3-c][1,2]thiazin-1(4H)-yl)acetohydrazides (7a–u). Fur-
Molecular Docking
thermore,
4-methyl-3-phenyl-2,4-dihydrobenzo[e]pyrazol-
The mechanism of anti-HIV activity of these compounds
was attempted by in silico investigation of active com-
pounds. The compounds (7m, 7l, and 7j) which showed the
most significant anti-HIV activity were docked into the
HIV-1 reverse transcriptase crystal structure. The HIV-1
reverse transcriptase structure was downloaded from the
PDB database using ID: 1C1B and was docked with active
compounds. After docking, the top conformations were
selected on the basis of the minimum S-score and were
analyzed to find potential interactions among compounds
and HIV-1 to understand the mechanism of inhibition.
Compound 7l was ranked as the top conformation, followed
sequentially by 7m and 7j. Compounds 7l and 7m exhibited
the most significant anti-HIV-1 activity, while 7j exhibited
lower anti-HIV-1 activity with EC50 value 17.1 lM. Com-
pound 7l, which had significant anti-HIV-1 activity
(EC50 = 5.4 lM), was also ranked as the top conformation
in docking results, followed in order by 7m and 7j. The
residue interactions of these compounds with HIV-1 Reverse
Transcriptase are listed in Table 6. AZT, which is a potent
anti-HIV drug, was also docked with this binding pocket of
HIV-1 reverse transcriptase to validate the docking proce-
dure. These three compounds and AZT bound efficiently
with the NTP-binding pocket of HIV-1 reverse transcriptase.
o[4,3-c][1,2]thiazine 5,5-dioxide (4) was efficiently synthe-
sized in high yield from low cost starting materials using
microwave irradiation. Two compounds 7l and 7m emerged as
potent anti-HIV-1 agents with EC50 values of 5.4 and 3.3 lM,
respectively, while 7j showed moderate activity with an EC50
value of 17.1 lM. Notably, six compounds (7b, 7f, 7h, 7k, 7o,
and 7s) showed no toxicity in the three cell lines used. Com-
pound 7j showed no toxicity in human PBM cells and thus
could have medicinal use. The mechanism of HIV inhibition
was studied through molecular docking, and thus, a rationale
for the efficacy of three lead compounds can be provided. This
provides a valuable basis for further SAR-driven optimization.
Experimental
All the chemicals were purchased from E.Merck, Sigma/
Aldrich or Fluka and used without purification. General
melting points were obtained on Gallenkamp melting point
apparatus and were uncorrected. IR spectra were recorded
in KBr pellets on Perkin Elmer infrared spectrophotometer.
1H NMR spectra were recorded in DMSO-d6 on Brucker
NMR (400 MHz), and TMS was used as internal standard
(chemical shifts, d in ppm). Mass spectra were recorded on
a Jeol MS Route instrument. Ultrasonic-mediated reactions
were carried out in Clifton Ultrasonic Bath (2 9 T2A,
300W, DU-4) made by Nickel Electro Ltd.
Interaction analysis
After postdocking analysis, it was found that the compound
7l had strong interactions with Ser 105 and also had close
contact with other pocket residues Lys102, Lys 103,
Lys104, Val 106, Asp 192, Ile195, Pro236, Tyr318,
Tyr319, Asp 320, Pro321, Lys 323, and Glu344 of the HIV
reverse transcriptase. Interactions are shown in Fig. 3a.
Compound 7m had strong interaction with Lys 102 and
also had contact with residues Ly103, Lys104, Tyr319,
Val317, Tyr318, Asp 320, Pro321, Lys323, and Glu344
(Fig. 3b). Compound 7j had the strong interactions with
Thr107 and also had contact with Ly102, Lys103, Lys104,
General procedure for the synthesis of 2-(2-oxo-2-
phenylethyl)-1,2-benzisothiazol-3(2H)-one-1,1-dioxide
(1)
Phenacyl bromide (0.222 mol) was added to the oven-dried
sodium saccharine (45.55 g, 0.222 mol) using DMF
(20 mL) as reaction medium. The reaction was completed
in 25 min under microwave irradiation of 30 W. Then, the
reaction mixture was cooled to ambient temperature and
poured onto crushed ice. The resulting white precipitates
123

Med Chem Res
Fig. 3 The binding interactions of compounds 7l, 7m, and 7j, respectively, with HIV-1 reverse transcriptase
Fig. 4 3D binding interaction of 7l, 7m, and 7j with HIV-1 reverse transcriptase
were filtered, washed with water, and recrystallized from
ethanol affording pure 1 (87 %). FT-IR (KBr) vmax:1715;
1345; 1165 cm-1; 1H NMR: (DMSO-d6, 400 MHz) d: 5.15
(2H, s, N–CH2), 7.47 (2H, t, J = 7.4 Hz, Ar–H), 7.59 (1H,
t, J = 7.4 Hz, Ar–H), 7.81–7.90 (2H, m, Ar–H), 7.92 (1H,
d, J = 6.6 Hz, Ar–H), 7.94 (2H, d, J = 6.8 Hz, Ar–H),
8.07 (1H, d, J = 7.8 Hz, Ar–H); 13C NMR (DMSO-d6):
51.3 (CH2), 123.2 (Ar–C), 124.1 (Ar–C), 126.3 (Ar–C),
129.2 (2C, Ar), 129.4 (2C, Ar), 133.5 (Ar–C), 134.8
(Ar–C), 135.3 (Ar–C), 137.0 (Ar–C), 141.1 (Ar–C), 169.5
(C=O), 191.3 (Ar–CO); MS m/z: ES-; 301.1 (M?), ?ES;
324.04 (M??Na?).
General procedure for the synthesis of 3-benzoyl-4-
hydroxy-2H-1,2-benzothiazine-1,1-dioxide (2)
Fresh sodium methoxide was first prepared by refluxing
sodium metal (5.34 g, 0.232 mol) in 60 mL of freshly
dried methanol under inert atmosphere maintained by N2
Fig. 5 Interactions of AZT bound to HIV-1 reverse transcriptase
123

Med Chem Res
gas. Subsequently, N-phenacylsaccharin (1) (10.0 g,
0.232 mol) was added to the sodium methoxide in reflux-
ing methanol. The reaction mixture immediately turned an
orange–red color. The mixture was further refluxed for 20
min and then cooled to room temperature and poured into
ice-cold 10 % HCl solution. The resulting white precipi-
tates were filtered and washed with excess water and dried
to afford 2 as a white crystalline solid (82 %). m.p. 156 °C
FT-IR (KBr)cm-1 3353; 3133; 1625; 1358; 1157; 1H
NMR: (DMSO-d6) (400 MHz) d: 5.79 (1H, s, SO2NH),
7.61 (1H, t, J = 7.8 Hz, Ar–H), 7.71 (1H, d, J = 7.8 Hz,
Ar–H), 7.95–7.99 (5H, m, Ar–H), 8.15 (2H, t, J = 4.6 Hz,
Ar–H), 14.67 (1H, s, O–H); 13C NMR (DMSO-d6): 110.1
(C-3, thiazine), 122.9 (Ar–C), 127.1 (Ar–C), 128.1 (Ar–C),
128.3 (Ar–C), 129.1 (Ar–C), 129.4 (2C, Ar), 132.1 (Ar–C),
132.3 (Ar–C), 134.0 (Ar–C), 137.1 (2C, Ar), 151.6 (C-4,
thiazine), 191.1 (C=O); MS m/z: 301.07 [M?].
refluxed in microwave oven at 30 W for 20 min. The unreacted
hydrazine monohydrate and ethanol were removed under
vacuum. The crude product was then dissolved in ice-cold
water. The resulting precipitateswere filtered and recrystallized
from ethanol to afford pure 4 as an off-white crystalline solid
(87 %). FT-IR (KBr) cm-1: 3368; 2965; 1655; 1337; 1165; 1H
NMR: (DMSO-d6) (400 MHz) d: 2.91 (3H, s, N–CH3), 7.49
(1H, t, J = 7.8 Hz, Ar–H), 7.61 (2H, d, J = 8.4 Hz, Ar–H),
7.64–7.71 (1H, dd, J1 = 1.2 Hz, J2 = 7.6 Hz, J3 = 11.2 Hz,
J4 = 16.8 Hz, Ar–H), 7.73–7.79 (1H, dd, J1 = 1.5 Hz, J2 =
8.4 Hz, Ar–H), 7.94–8.01 (3H, m, Ar–H), 8.17 (1H, d,
J = 7.6 Hz, Ar–H), 13.78 (1H, s, N–H); 13C NMR (DMSO-
d6): 39.9 (N–CH3), 122.2 (Ar–C), 124.2 (Ar–C), 124.7 (Ar–C),
124.9 (Ar–C), 125.8 (Ar–C), 125.9 (Ar–C), 127.3 (Ar–C),
128.6 (Ar–C), 130.1 (Ar–C), 130.3 (Ar–C), 130.4 (Ar–C),
134.2 (Ar–C), 134.4 (Ar–C), 138.2 (Ar–C), 140.4 (N=C, Ar–
C); MS m/z: 312.36 (ES?), HR-MS (?ES): 312.0801
(C16H13N3O2S).
General procedure for the synthesis of 3-benzoyl-4-
hydroxy-2-methyl-2H-1,2-benzothiazine-1,1-dioxide (3)
General procedure for the synthesis of alkyl 2-(4-
methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-
c][1,2]thiazin-1(4H)-yl)acetate (5)
3-Benzoyl-4-hydroxy-2H-1,2-benzothiazine-1,1-dioxide (2)
(10.0 g, 33.2 mmol) and aqueous sodium hydroxide (2.66 g in
5 mL water) were mixed in 50 mL of acetone. The mixture was
allowed to be stirred at ambient temperature for 10 min.
Dimethyl sulfate (8.06 mL, 64 mol) was added into the reaction
mixture drop wise. The mixture was placed in ultrasonic media
for 15 min at ambient temperature. Light yellow precipitates
were formed on addition of cold dilute HCl (20 mL; 5 %), were
filtered, washed with excess water, and then dried to afford 3 as a
light yellow crystalline solid (85 %). m.p. 147–148 °C. IR
4-Methyl-3-phenyl-2,4-dihydrobenzo[e]pyrazolo[4,3-c][1,2]
thiazine 5,5-dioxide (4) (6.2 g, 0.020 mol) and anhydrous
potassium carbonate (3.31 g, 0.024 mol) were mixed in ace-
tonitrile (30 mL). Then, methyl chloroacetate (2.60 g,
0.024 mol)/ethyl chloroacetate (0.024 mol) was added and
allowed to reflux for 8 h. The completion of reaction was
checked by TLC. The solvent was removed under reduced
pressure. The resulting residues were dissolved in cold water,
and neutral pH was maintained by addition of dilute HCl to
provide the dirty yellow crystalline product 5.
1
(KBr) cm-1: 3470; 2970; 1610; 1586; 1357; 1184; H-NMR
(CDCl3, 400 MHz) d: 2.89 (3H, s, N–CH3), 7.59 (2H, t,
J = 7.6 Hz, Ar–H), 7.69 (1H,d, J = 7.8 Hz, Ar–H), 7.73–7.77
(2H, dd, J1 = 2.8 Hz, J2 = 8.4 Hz, Ar–H), 7.94–7.97 (3H, m,
Ar–H), 8.17 (1H, t, J = 4.6 Hz, Ar–H), 14.69 (1H, s, O–H); 13C
NMR (DMSO-d6): 39.8 (N–CH3), 121.9 (C-3, thiazine), 123.2
(Ar–C), 124.2 (Ar–C), 124.7 (Ar–C), 127.2 (Ar–C), 128.6 (Ar–
C), 129.2 (Ar–C), 130.1 (Ar–C), 131.0 (Ar–C), 133.0 (Ar–C),
134.2 (Ar–C), 136.5 (Ar–C), 138.2 (Ar–C), 167.2 (C-4, thia-
zine), 190.0 (C=O); MS m/z: 315.34 [M?]. HR-MS (?ES):
315.0571 (M?H?) (C16H13NO4S).
Methyl 2-(4-methyl-5,5-dioxido-3-phenylbenzo[e]
pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)acetate (5a)
Yield: 89 %. FT-IR (KBr) cm-1: 3078; 2965; 1685; 1339;
1275; 1168; 1H NMR (DMSO-d6) (400 MHz): d: 2.91 (3H, s,
N–CH3), 3.27 (3H, s, O–CH3), 5.77 (2H, s, N–CH2), 7.44
(3H, t, J = 7.6 Hz, Ar–H), 7.63–7.67 (1H, dd, J1 = 2.4 Hz,
J2 = 7.8 Hz, Ar–H), 7.75–7.79 (1H, dd, J1 = 3.2 Hz,
J2 = 8.0 Hz, Ar–H), 7.94–8.01 (2H, dt, J1 = 2.8 Hz,
J2 = 7.8 Hz, J3 = 14.8 Hz, Ar–H), 8.2 (2H, d, J = 7.6 Hz,
Ar–H); 13C NMR (DMSO-d6): 39.8 (N–CH3), 51.4
(O–CH3), 53.9 (N–CH2), 122.3 (Ar–C), 124.2 (Ar–C), 124.7
(Ar–C), 124.9 (Ar–C), 125.9 (Ar–C), 126.0 (Ar–C), 127.2
(Ar–C), 128.6 (Ar–C), 130.2 (Ar–C), 130.4 (Ar–C), 130.6
(Ar–C), 132.4 (Ar–C), 134.29 (Ar–C), 134.41 (Ar–C), 139.0
(N=C, Ar–C), 167.8 (C=O); MS m/z: 383.06 (M?).
General procedure for the synthesis of 4-methyl-3-
phenyl-2,4-dihydrobenzo[e]pyrazolo [4,3-
c][1,2]thiazine 5,5-dioxide (4)
A mixture of 3-benzoyl-4-hydroxy-2-methyl-2H-1,2-benzo-
thiazine-1,1-dioxide (3) (5.0 g, 13.08 mmoles) and hydrazine
monohydrate (4.9 mL, 99 mmol) in 50 mL ethanol was
123

Med Chem Res
Ethyl 2-(4-methyl-5,5-dioxido-3-phenylbenzo
benzaldehyde were dissolved in equimolar quantity in
ethanol followed by the addition of 0.2–0.5 mL of glacial
acetic acid. The mixture was allowed to reflux for half to
1 h depending on the reactivity of aromatic aldehydes.
The reaction was also carried out under microwave irra-
diation of 30 W or alternatively using an ultrasonic bath.
The reaction parameters are described in Table 3. The
resulting precipitates were filtered and washed with hot
methanol and dried.
[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)acetate (5b)
Dirty yellow crystalline; FT-IR (KBr) cm-1: 3082;2959; 2901;
1681; 1344; 1272; 1161; 1H NMR (DMSO-d6) (400 MHz) d:
1.09 (3H, t, J = 8.4 Hz,C–CH3), 2.91 (3H, s, N–CH3), 3.96
(2H, q, J = 8.4 Hz, O–CH2), 5.75 (2H, s, N–CH2), 7.41 (3H, t,
J = 7.6 Hz, Ar–H), 7.61–7.65 (1H, dd, J1 = 2.8 Hz,
J2 = 8.0 Hz, Ar–H), 7.74–7.78 (1H, dd, J1 = 3.2 Hz,
J2 = 8.4 Hz, Ar–H), 7.95–8.02 (2H, dt, J1 = 2.8 Hz,
J2 = 7.8 Hz, J3 = 14.8 Hz, Ar–H), 8.25 (2H, d, J = 7.6 Hz,
Ar–H); 13C NMR (DMSO-d6): 13.4 (CH3, ester), 39.8
(N–CH3), 51.1 (O–CH2), 53.9 (N–CH2), 122.2 (Ar–C), 124.3
(Ar–C), 124.8 (Ar–C), 124.9 (Ar–C), 125.8 (Ar–C), 126.0 (Ar–
C), 127.2 (Ar–C), 128.62 (Ar–C), 130.2 (Ar–C), 130.4 (Ar–C),
130.8 (Ar–C), 132.6 (Ar–C), 134.2 (Ar–C), 134.4 (Ar–C),
139.0 (N=C, Ar–C), 167.8 (C=O); MS m/z: 397.96 (M?).
N0-(4-Bromobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7a)
Off-white crystals; Yield: 83 %; m.p. 260 °C; FT-IR (KBr)
cm-1: 3469; 3060; 2948; 1694; 1604; 1486; 1348; 1279; 1189;
1021; 754; 1H NMR (DMSO-d6) (400 MHz) d: 2.88 (3H, s, N–
CH3), 5.87 (2H, s, N–CH2), 7.43 (1H, t, J = 7.6 Hz, Ar–H),
7.53 (1H, t, J = 7.6 Hz, Ar–H), 7.62 (1H, d, J = 8.8 Hz, Ar–
H), 7.67 (1H, d, J = 4 Hz, Ar–H), 7.71–7.73 (3H, dd,
J1 = 2.8 Hz, J2 = 8.4 Hz, Ar–H), 7.83(2H, t, J = 8.4 Hz,
Ar–H), 7.96 (2H, t, J = 7.2 Hz, Ar–H), 8.03 (2H, d,
General procedure for the synthesis of 2-(4-methyl-5,5-
dioxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-
1(4H)-yl)acetohydrazide (6)
A
mixture of methyl 2-(4-methyl-5,5-dioxido-3-phen-
J = 8.0 Hz, Ar–H), 8.69 (1H, s, CH), 11.94 (1H, s, NH); 13
C
ylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-yl)acetate (5a)
(5.0 g, 13.04 mmol) and hydrazine monohydrate (4.9 mL,
99 mmol) in 50 mL absolute ethanol was refluxed for 4 h. The
unreacted hydrazine monohydrate and ethanol were then
removed under vacuum. The crude product was dissolved in
ice-cold water, and the pH was maintained at about 5.0–5.5 by
addition of dil. HCl. The resulting precipitates were filtered and
finally dried to provide pure 6 (80 %). Reaction in an ultrasonic
bath was complete in 30 min with 71 % yield, and reaction in a
microwave oven was complete in 15 min with 75 % yield. FT-
NMR (DMSO-d6): 38.9 (N–CH3), 53.65 (N–CH2), 122.3 (Ar–
C), 123.80 (Ar–C), 124.6 (Ar–C), 125.0 (Ar–C), 125.4 (Ar–C),
125.77 (Ar–C), 128.6 (Ar–C), 129.0 (Ar–C), 129.2 (Ar–C),
129.8 (Ar–C), 130.0 (Ar–C), 130.2 (Ar–C), 130.7 (Ar–C),
130.8 (Ar–C), 131.4 (Ar–C), 131.5 (Ar–C), 131.8 (Ar–C),
132.0 (Ar–C), 133.5 (Ar–C), 135.1 (Ar–C), 142.5 (N=C, Ar–
C), 146.9 (N=CH), 167.9 (C=O); MS m/z: 549.0 (M?), 551.0
(M??2); Anal. Calcd. for C25H20BrN5O3S: C, 54.55; H, 3.66;
N, 12.72. Found: C, 54.63; H, 3.60; N, 12.68.
IR (KBr) cm-1: 3468; 3010; 1675; 1337, 1165; H NMR:
1
N0-(2,6-Dichlorobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7b)
(DMSO-d6) (400 MHz) d: 2.90 (3H, s, N–CH3), 4.39 (2H, s,
NH2), 5.78 (2H, s, N–CH2), 7.49 (3H, t, J = 7.6 Hz, Ar–H),
7.59–7.61 (1H, dd, J1 = 2.8 Hz, J2 = 8.4 Hz, Ar–H),
7.71–7.73 (1H, dd, J1 = 2.8 Hz, J2 = 8.4 Hz, Ar–H), 7.83
(2H, t, J = 8.4 Hz, Ar–H), 8.11 (2H, d, J = 7.8 Hz, Ar–H)
11.94 (1H, br–s, NH); 13C NMR (DMSO-d6): 39.8 (N–CH3),
53.9 (N–CH2), 122.2 (Ar–C), 124.2 (Ar–C), 124.7 (Ar–C),
124.9 (Ar–C), 125.9 (Ar–C), 126.0 (Ar–C), 127.2 (Ar–C),
128.6 (Ar–C), 130.2 (Ar–C), 130.4 (Ar–C), 130.6 (Ar–C),
132.4 (Ar–C), 134.2 (Ar–C), 134.4 (Ar–C), 138.9 (N=C, Ar–
C), 165.6 (C=O); MS m/z: 383.07 (M?).
Off-white crystals; Yield: 84 %; m.p. 240 °C; FT-IR (KBr)
cm-1:3457; 3069; 2964; 1685; 1598; 1466; 1345; 1291; 1168;
1
1065; 747; H NMR (DMSO-d6) (400 MHz)d: 2.88 (3H, s,
N–CH3), 5.79 (2H, s, N–CH2), 7.44 (2H, q, J = 7.2 Hz,
Ar–H), 7.53 (1H, t, J = 7.6 Hz, Ar–H), 7.58 (2H, d,
J = 8.0 Hz, Ar–H), 7.75(1H,t,J = 7.2 Hz, Ar–H), 7.80(1H,
d, J = 7.6 Hz, Ar–H), 7.85 (2H, d, J = 7.2 Hz, Ar–H),
7.96–8.02 (3H, dd, J1 = 7.2 Hz, J2 = 8.0 Hz, Ar–H),
8.38(1H, s, CH), 12.17 (1H, s, NH); 13C NMR (DMSO-d6):
38.9 (N–CH3), 53.5 (N–CH2), 122.3 (Ar–C), 123.8 (Ar–C),
124.5 (Ar–C), 125.1 (Ar–C), 125.4 (Ar–C), 125.8 (Ar–C), 128.6
(Ar–C), 129.0 (Ar–C), 129.2 (Ar–C), 129.5 (Ar–C), 129.8 (Ar–
C), 130.1 (Ar–C), 130.2 (Ar–C), 130.8 (Ar–C), 130.8 (Ar–C),
131.3 (Ar–C), 131.5 (Ar–C), 131.7 (Ar–C), 132.0 (Ar–C), 133.5
(Ar–C), 142.2 (N=C, Ar–C), 145.9 (N=CH), 168.8 (C=O);
General procedure for the synthesis of N0-
substitutedbenzylidene-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7a–u)
2-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c]
[1,2]thiazin-1(4H)-yl)acetohydrazide (6) and substituted
123

Med Chem Res
MS m/z: 539.0 (M?); Anal. Calcd. for C25H19Cl2N5O3S C,
2-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c]
[1,2]thiazin-1(4H)-yl)-N0-(2-nitrobenzylidene)
acetohydrazide (7e)
55.56; H, 3.54; N, 12.96. Found: C, 55.50; H, 3.58; N, 12.90.
N0-(4-Chlorobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7c)
Dirty yellow powder; Yield: 85 %; m.p. 227 °C; FT-IR (KBr)
cm-1: 3427; 3082; 2970; 1655; 1569; 1519; 1443; 1343; 1206;
1184; 953; 747; 1H NMR (DMSO-d6) (400 MHz) d: 2.88 (3H,
s, N–CH3), 5.89 (2H, s, N–CH2), 7.08–7.14 (2H, dd,
J1 = 8.4 Hz, J2 = 18.4 Hz, Ar–H), 7.32–7.45 (3H, m, Ar–H),
7.50 (2H, t, J = 7.8 Hz, Ar–H), 7.58 (1H, d, J = 7.2 Hz, Ar–
H), 7.68 (2H, t, J = 7.8 Hz, Ar–H), 7.96 (2H, d, J = 9.2 Hz,
Ar–H), 8.03(1H, d, J = 7.6 Hz, Ar–H), 8.13 (1H, s, CH), 11.69
(1H, s, NH); 13C NMR (DMSO-d6): 39.9 (N–CH3), 54.8 (CH2),
121.4 (Ar–C), 122.3 (Ar–C), 122.7 (Ar–C), 123.3 (Ar–C),
123.8 (Ar–C), 124.0 (Ar–C), 124.7 (Ar–C), 125.1 (Ar–C),
125.6 (Ar–C), 125.8 (Ar–C), 127.8 (Ar–C), 128.6 (Ar–C),
129.0 (Ar–C), 129.6 (Ar–C), 130.8 (Ar–C), 131.6 (Ar–C),
132.1 (Ar–C), 133.9 (Ar–C), 134.4 (Ar–C), 142.6 (N=C, Ar–
C), 146.8 (N=CH), 148.8 (Ar–C), 169.7 (C=O); MS m/z: 516.0
(M?); Anal. Calcd. for C25H20N6O5S C, 58.13; H, 3.90; N,
16.27. Found: C, 58.17; H, 3.97; N, 16.19.
Off-white powder; Yield: 87 %; m.p. 256 °C; FT-IR
(KBr) cm-1: 3467; 3061; 2949; 1684; 1614; 1486; 1345;
1274; 1182; 1021; 1H NMR (DMSO-d6) (400 MHz) d:
2.88 (3H, s, N–CH3), 5.88 (2H, s, N–CH2), 7.44 (1H, d,
J = 7.2 Hz, Ar–H), 7.51 (1H, q, J = 7.8 Hz, Ar–H),
7.57 (3H, d, J = 8.8 Hz, Ar–H), 7.74–7.80 (2H, dd,
J1 = 8.4 Hz, J2 = 16.4 Hz, Ar–H), 7.89 (3H, d,
J=8.4 Hz, Ar–H), 7.97 (2H, d, J = 7.2 Hz, Ar–H), 8.00
(1H, d, J = 7.4 Hz, Ar–H), 8.70 (1H, s, CH), 11.88 (1H,
s, NH); 13C NMR (DMSO-d6): 38.9 (N–CH3), 53.6
(CH2), 122.4 (Ar–C), 123.9 (Ar–C), 124.8 (Ar–C), 125.3
(Ar–C), 126.0 (Ar–C), 126.7 (Ar–C), 128.6 (Ar–C),
128.8 (Ar–C), 129.2 (Ar–C), 129.8 (Ar–C), 130.1 (Ar–
C), 130.7 (Ar–C), 130.8 (Ar–C), 131.3 (Ar–C), 131.5
(Ar–C), 131.8 (Ar–C), 132.5 (Ar–C), 135.9 (Ar–C),
137.4 (Ar–C), 137.8 (Ar–C), 142.5 (N=C, Ar–C), 146.9
(N=CH), 161.1 (C=O); MS m/z: 505.0 (M?); Anal.
Calcd. for C25H20Cl N5O3S C, 59.34; H, 3.98; N, 13.84.
Found: C, 59.30; H, 3.86; N, 13.77.
2-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c]
[1,2]thiazin-1(4H)-yl)-N0-(3-nitrobenzylidene)
acetohydrazide (7f)
Off-white crystalline; Yield: 86 %; m.p. 204 °C; FT-IR
(KBr) cm-1 : 3457; 3085; 2956; 1687; 1598; 1528; 1466;
1348; 1267; 1183; 1022; 734; 1H NMR (DMSO-d6)
(400 MHz) d: 2.88(3H, s, N–CH3), 5.94 (2H, s, N–CH2),
7.43 (1H, d, J = 9.2 Hz, Ar–H), 7.53 (2H, t, J = 7.6 Hz,
Ar–H), 7.72 (2H, t, J = 7.8 Hz, Ar–H), 7.82 (2H, t,
J = 8.0 Hz, Ar–H) 7.97 (2H, d, J = 7.2 Hz, Ar–H), 8.03
(1H, t, J = 7.6 Hz, Ar–H) 8.24 (1H, t, J = 8.4 Hz, Ar–H),
8.30 (1H, d, J = 7.6 Hz, Ar–H), 8.36–8.38 (1H, dd,
J1 = 2.4 Hz, J2 = 7.2 Hz, Ar–H), 8.92 (1H, s, CH), 12.19
(1H,s,NH); 13CNMR (DMSO-d6): 39.7 (N–CH3), 54.8
(CH2), 121.4 (Ar–C), 122.3 (Ar–C), 122.6 (Ar–C), 123.3
(Ar–C), 123.8 (Ar–C), 124.3 (Ar–C), 124.5 (Ar–C), 125.0
(Ar–C), 125.4 (Ar–C), 125.8 (Ar–C), 128.6 (Ar–C), 129.0
(Ar–C), 129.8 (Ar–C), 130.0 (Ar–C), 130.6 (Ar–C), 133.2
(Ar–C), 133.5 (Ar–C), 134.2 (Ar–C), 135.5 (Ar–C), 142.5
(N=C, Ar–C), 145.7 (N=CH), 148.2 (Ar–C), 168.6 (C=O);
MS m/z: 516.0 (M?); Anal. Calcd. for C25H20N6O5S C,
58.13; H, 3.90; N, 16.27. Found: C, 58.11; H, 3.99; N, 16.19.
N0-(4-(Dimethylamino)benzylidene)-2-(4-methyl-5,5-
dioxido-3-phenylbenzo[e]pyrazolo [4,3-c][1,2]thiazin-
1(4H)-yl)acetohydrazide (7d)
Dirty off-white powder; Yield: 82 %; m.p. 234 °C; FT-IR
(KBr) cm-1: 3422; 3082; 2968; 1685;1531; 1467; 1344;
1
1287; 1184; 1021; 749; H NMR (DMSO-d6) (400 MHz) d:
2.88 (3H, s, N–CH3), 2.95 (6H, s, N–CH3), 5.81 (2H, s, N–
CH2), 6.68–6.74 (2H, dd, J1 = 11.6 Hz, J2 = 18.4 Hz, Ar–
H), 7.44 (1H, q, J = 9.2 Hz, Ar–H), 7.53 (2H, t,
J = 8.0 Hz, Ar–H), 7.58 (2H,t, J = 6.8 Hz, Ar–H), 7.84
(3H, t, J = 7.8 Hz, Ar–H), 7.96–7.99 (2H, dd, J1 = 2.4 Hz,
J2 = 10.0 Hz, Ar–H), 8.02 (1H, d, J = 8.0 Hz, Ar–H), 8.14
(1H, s, CH), 11.60 (1H, s, NH); 13C NMR (DMSO-d6): 38.7
(N–CH3), 40.2 (2C, N–CH3), 53.8 (CH2), 111.6 (Ar–C),
111.8 (Ar–C), 121.1 (Ar–C), 122.2 (Ar–C), 122.9 (Ar–C),
123.9 (Ar–C), 124.3 (Ar–C), 124.6 (Ar–C), 125.9 (Ar–C),
126.7 (Ar–C), 128.6 (Ar–C), 128.8 (Ar–C), 128.9 (Ar–C),
129.1 (Ar–C), 129.2 (Ar–C), 129.4 (Ar–C), 131.2 (Ar–C),
132.9 (Ar–C), 133.5 (Ar–C), 142.0 (N=C, Ar–C), 146.7
(N=CH), 151.5 (Ar–C), 168.1 (C=O); MS m/z: 514.0 (M?);
Anal. Calcd. for C27H26N6O3S C, 63.02; H, 5.09; N, 16.33.
Found: C, 63.08; H, 5.17; N, 16.30.
N0-(4-(Diethylamino)benzylidene)-2-(4-methyl-5,5-
dioxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-
1(4H)-yl)acetohydrazide (7g)
Bright yellow powder; Yield: 85 %; m.p. 245 °C; FT-IR
(KBr) cm-1: 3445; 3069; 2976; 1679; 1595; 1468; 1347;
123

Med Chem Res
1
1266; 1182; 1021; 766; H NMR (DMSO-d6) (400 MHz)
J1 = 5.2 Hz, J2 = 6.4 Hz, Ar–H), 7.98 (2H, t, J = 7.2 Hz,
Ar–H), 8.01 (1H, d, J = 8.0 Hz, Ar–H),8.28 (1H, s, CH),
11.76 (1H, s, NH); 13C NMR (DMSO-d6): 38.9 (N–CH3),
55.4 (CH2), 58.9 (O–CH3), 59.9 (O–CH3), 60.3 (O–CH3),
106.5 (Ar–C), 120.1 (Ar–C), 120.7 (Ar–C), 121.2 (Ar–C),
122.1 (Ar–C), 122.8 (Ar–C), 123.9 (Ar–C), 124.2 (Ar–C),
124.9 (Ar–C), 125.6 (Ar–C), 125.9 (Ar–C), 127.2 (Ar–C),
128.7 (Ar–C), 129.4 (Ar–C), 129.9 (Ar–C), 130.9 (Ar–C),
132.5 (Ar–C), 139.5 (Ar–C), 141.9 (N=C, Ar–C), 146.6
(N=CH), 149.9 (Ar–C), 150.2 (Ar–C), 167.9 (C=O); MS
m/z: 561.4(M?); Anal. Calcd. for C28H27N5O6S 59.88; H,
4.85; N, 12.47. Found: 59.80; H, 4.81; N, 12.59.
d: 1.14 (6H, t, J = 6.4 Hz, C–CH3), 2.88 (3H, s, N–CH3),
3.44 (4H, q, J = 6.8 Hz, C–CH2), 5.80 (2H, s, N–CH2),
6.69–6.74 (2H, dd, J1 = 2.4 Hz, J2 = 7.2 Hz, Ar–H), 7.44
(2H, d, J = 7.6 Hz, Ar–H), 7.42–7.51 (2H, dd, J1 =
2.0 Hz, J2 = 9.8 Hz, Ar–H), 7.59 (2H, d, J = 8.8 Hz, Ar–
H), 7.84 (1H, t, J = 8.4 Hz, Ar–H), 7.93–7.99 (4H, m, Ar–
H), 8.44 (1H, s, CH), 11.49 (1H, s, NH); 13C NMR
(DMSO-d6): 12.4 (2C, C–CH3), 38.8 (N–CH3), 43.7 (2C,
N–CH2), 53.8 (CH2), 110.9 (Ar–C), 120.2 (Ar–C), 120.7
(Ar–C), 122.1 (Ar–C), 122.8 (Ar–C), 123.9 (Ar–C), 124.3
(Ar–C), 124.6 (Ar–C), 125.8 (Ar–C), 126.6 (Ar–C), 128.6
(Ar–C), 128.9 (Ar–C), 129.0 (Ar–C), 129.2 (Ar–C), 129.4
(Ar–C), 129.8 (Ar–C), 130.3 (Ar–C), 130.8 (Ar–C), 133.5
(Ar–C), 142.8 (N=C, Ar–C), 146.3 (N=CH), 151.1 (Ar–C),
169.5 (C=O); MS m/z: 542.1 (M?); Anal. Calcd. for
C29H30N6O3S C, 64.19; H, 5.57; N, 15.49. Found: C,
64.27; H, 5.44; N, 15.51.
N0-(4-Methoxybenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7j)
Off-white powder; 90 % Yield; m.p. 208 °C; FT-IR (KBr)
cm-1 : 3440; 3056; 2940; 1677; 1595; 1470; 1344; 1269;
1167; 1069; 746; 1H NMR (DMSO-d6) (400 MHz) d: 2.88
(3H, s, N–CH3), 3.79 (3H, s, O–CH3), 5.85 (2H, s, N–CH2),
6.99 (2H, d, J = 9.2 Hz, Ar–H), 7.44 (1H, d, J = 7.6 Hz,
Ar–H), 7.53(3H, t, J = 7.6 Hz, Ar–H), 7.69 (2H, q,
J = 8.8 Hz, Ar–H), 7.84(1H, t, J = 10.2 Hz, Ar–H), 7.98
(3H, t, J = 7.2 Hz, Ar–H), 8.03 (1H, d, J = 8.0 Hz, Ar–
H), 8.23(1H, s, CH), 11.69 (1H, s, NH); 13C NMR (DMSO-
d6): 38.9 (N–CH3), 55.6 (N–CH2), 58.9 (O–CH3), 110.4
(Ar–C), 120.4 (Ar–C), 120.8 (Ar–C), 121.4 (Ar–C), 122.1
(Ar–C), 122.8 (Ar–C), 123.3 (Ar–C), 124.3 (Ar–C), 125.4
(Ar–C), 125.9 (Ar–C), 127.2 (Ar–C), 128.5 (Ar–C), 128.9
(Ar–C), 129.6 (Ar–C), 130.1 (Ar–C), 130.6 (Ar–C), 133.2
(Ar–C), 133.5 (Ar–C), 134.2 (Ar–C), 141.8 (N=C, Ar–C),
145.8 (N=CH), 159.8 (Ar–C), 168.1 (C=O); MS m/z:
501.4(M?); Anal. Calcd. for C26H23N5O4S C, 62.26; H,
4.62; N, 13.96. Found: C, 62.30; H, 4.67; N, 13.89.
N0-(2,4-Dichlorobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7h)
Light yellow powder; Yield: 85 %; m.p. 212 °C; FT-IR
(KBr) cm-1: 3447; 3078; 2960; 1682; 1599; 1467; 1343;
1274; 1155; 1050; 747; 1HNMR (DMSO-d6) (400 MHz) d:
2.76 (3H, s, N–CH3), 5.45 (2H, s, N–CH2), 7.04 (1H, s, Ar–
H), 7.45–7.47 (1H, dd, J1 = 2.8 Hz, J2 = 8.4 Hz, Ar–H),
7.58 (3H, d, J = 4.4 Hz, Ar–H), 7.69 (2H, t, J = 3.2 Hz,
Ar–H), 7.83 (2H, t, J = 7.6 Hz, Ar–H), 7.92 (1H, d,
J = 7.6 Hz, Ar–H), 7.97 (1H, d, J = 8.8 Hz, Ar–H),
8.03(1H, d, J = 7.6 Hz, Ar–H), 8.31 (1H, s, CH), 11.94 (1H, s,
NH); 13C NMR (DMSO-d6): 38.9 (N–CH3), 53.5 (N–CH2),
121.5 (Ar–C), 122.3 (Ar–C), 122.7 (Ar–C), 124.5 (Ar–C),
125.1 (Ar–C), 125.4 (Ar–C), 125.9 (Ar–C), 128.6
(Ar–C), 129.0 (Ar–C), 129.2 (Ar–C), 129.5 (Ar–C), 129.8
(Ar–C), 130.1 (Ar–C), 130.2 (Ar–C), 130.8 (Ar–C), 131.3
(Ar–C), 131.5 (Ar–C), 131.7 (Ar–C), 132.0 (Ar–C), 133.5
(Ar–C), 142.2 (N=C, Ar–C), 145.9 (N=CH), 168.8 (C=O); MS
m/z: 538.9 (M?); Anal. Calcd. for C25H19Cl2N5O3S C, 55.56;
H, 3.54; N, 12.96. Found: C, 55.51; H, 3.65; N, 12.99.
N0-(2,4-Dimethoxybenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7k)
Light yellow powder; Yield: 87 %; m.p. 178 °C; FT-IR
(KBr) cm-1: 3454; 3065; 2965; 1685; 1610; 1460; 1341;
1
1274; 1155; 1067; 746; H NMR (DMSO-d6) (400 MHz)
2-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo
[4,3-c][1,2]thiazin-1(4H)-yl)-N0-(2,3,4-trimethoxy
benzylidene)acetohydrazide (7i)
d: 2.88 (3H, s, N–CH3), 3.79 (3H, s, O–CH3), 3.86 (3H, s,
O–CH3), 5.82 (2H, s, N–CH2), 6.64 (1H, s, Ar–H), 7.34
(1H, d, J = 7.4 Hz, Ar–H), 7.53 (3H, t, J = 8 Hz, Ar–H),
7.72 (2H, t, J = 8.0 Hz, Ar–H), 7.83(2H, t, J = 8.8 Hz,
Ar–H), 7.96–8.01 (3H, dd, J1 = 7.2 Hz, J2 = 12.4 Hz,
Ar–H), 8.34 (1H, s, CH), 11.69 (1H, s, NH); 13C NMR
(DMSO-d6): 38.9 (N–CH3), 55.4 (CH2), 59.4 (O–CH3),
59.9 (O–CH3), 106.9 (Ar–C), 120.1 (Ar–C), 120.7 (Ar–C),
121.2 (Ar–C), 122.1 (Ar–C), 122.8 (Ar–C), 123.8 (Ar–C),
124.2 (Ar–C), 124.9 (Ar–C), 125.7 (Ar–C), 127.2 (Ar–C),
Off-white powder; Yield: 90 %; m.p. 241 °C; FT-IR (KBr)
cm-1: 3449; 3067; 2940; 1673; 1593; 1462; 1343; 1266;
1168; 1066; 741; 1H NMR (DMSO-d6) (400 MHz) d: 2.88
(3H, s, N–CH3), 3.88 (9H, s, O–CH3), 5.83 (2H, s, N–CH2),
6.86–6.93 (1H, dd, J1 = 8.8 Hz, J2 = 18.4 Hz, Ar–H),
7.53 (3H, t, J = Hz, Ar–H), 7.58 (1H, d, J = 4.8 Hz, Ar–
H), 7.65 (1H, d, J = 9.2 Hz, Ar–H), 7.83–7.87 (2H, dd,
123

Med Chem Res
N0-(2-Fluorobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7n)
128.6 (Ar–C), 129.0 (Ar–C), 129.4 (Ar–C), 130.2 (Ar–C),
130.9 (Ar–C), 132.5 (Ar–C), 133.5 (Ar–C), 142.0 (N=C,
Ar–C), 146.4 (N=CH), 159.9 (Ar–C), 160.7 (Ar–C), 168.3
(C=O); MS m/z: 531.4 (M?); Anal. Calcd. for
C27H25N5O5S C, 61.00; H, 4.74; N, 13.17. Found: C,
61.09; H, 4.82; N, 13.24.
Off-white powder; Yield: 82 %; m.p. 213 °C; FT-IR (KBr)
cm-1: 3447; 3078; 2960; 1682; 1599; 1467; 1343; 1274;
1155; 1050; 747; 1H NMR (DMSO-d6) (400 MHz) d: 2.89
(3H, s, N–CH3), 5.88 (2H, s, N–CH2), 7.26–7.30 (2H, m,
Ar–H), 7.53 (2H, t, J = 7.6 Hz, Ar–H), 7.58 (1H, d,
J = 4.4 Hz, Ar–H), 7.74 (3H, t, J = 7.8 Hz, Ar–H), 7.85
(2H, d, J = 9.2 Hz, Ar–H), 7.92 (1H, d, J = 7.4 Hz, Ar–
H), 7.97–8.02 (2H, dd, J1 = 7.2 Hz, J2 = 13.2 Hz, Ar–H),
8.31(1H, s, CH), 11.99 (1H, s, NH); 13C NMR (DMSO-d6):
38.9 (N–CH3), 53.5 (N–CH2), 122.4 (Ar–C), 123.9 (Ar–C),
124.8 (Ar–C), 125.5 (Ar–C), 125.9 (Ar–C), 126.8 (Ar–C),
128.7 (Ar–C), 128.9 (Ar–C), 129.3 (Ar–C), 129.8 (Ar–C),
130.2 (Ar–C), 130.8 (Ar–C), 130.9 (Ar–C), 131.3 (Ar–C),
131.5 (Ar–C), 131.8 (Ar–C), 132.2 (Ar–C), 133.0 (Ar–C),
137.4 (Ar–C), 142.3 (N=C, Ar–C), 143.5 (Ar–C), 146.9
(N=CH), 167.9 (C=O); MS m/z: 489.4(M?); Anal. Calcd.
for C25H20FN5O3S C, 61.34; H, 4.12; N, 14.31. Found: C,
61.39; H, 4.17; N, 14.27.
N0-(3-Ethoxy-4-hydroxybenzylidene)-2-(4-methyl-5,5-
dioxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-
1(4H)-yl)acetohydrazide (7l)
Off-white powder; Yield: 81 %; m.p. 196 °C; FT-IR (KBr)
cm-1: 3449; 3367; 3077; 2941; 1693; 1599; 1482; 1349;
1
1267; 1178; 1056; 743; H NMR (DMSO-d6) (400 MHz)
d:1.48 (3H, t, C–CH3), 2.89 (3H, s, N–CH3), 4.89 (2H, q,
O–CH2), 5.85 (2H, s, N–CH2), 7.38 (2H, t, J = 7.2 Hz, Ar–H),
7.49 (3H, d, J = 7.6 Hz, Ar–H), 7.53 (2H, t, J = 8.4 Hz,
Ar–H), 7.60 (1H, s, Ar–H), 7.67–7.85 (2H, dd, J1 = 5.2 Hz,
J2 = 6.4 Hz, Ar–H), 7.96 (2H, d, J = 10.8 Hz, Ar–H), 8.21
(1H, s, CH), 9.87 (1H, s, OH), 11.91 (1H, s, NH); 13C NMR
(DMSO-d6): 12.6 (CH3), 38.9 (N–CH3), 55.5 (N–CH2), 59.9
(O–CH2), 111.4 (Ar–C), 120.5 (Ar–C), 120.9 (Ar–C), 121.4
(Ar–C), 122.2 (Ar–C), 122.9 (Ar–C), 123.8 (Ar–C), 124.3
(Ar–C), 125.8 (Ar–C), 127.4 (Ar–C), 128.5 (Ar–C), 128.8
(Ar–C), 129.6 (Ar–C), 130.1 (Ar–C), 130.6 (Ar–C), 133.7
(Ar–C), 134.4 (Ar–C), 135.2 (Ar–C), 142.8 (N=C, Ar–C),
145.6 (N=CH), 147.8 (Ar–C), 150.3 (Ar–C), 168.8 (C=O); MS
m/z: 531.1(M?); Anal. Calcd. for C27H25N5O5S C, 61.00; H,
4.74; N, 13.17. Found: C, 61.09; H, 4.72; N, 13.22.
2-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c]
[1,2]thiazin-1(4H)-yl)-N0-(3,4,5-trimethoxybenzylidene)
acetohydrazide (7o)
Off-white powder; Yield: 84 %; m.p. 238 °C; FT-IR (KBr)
cm-1: 3445; 3055; 2954; 1687; 1590; 1461; 1344; 1264;
1
1155; 1027; 746; H NMR (DMSO-d6) (400 MHz)d: 2.89
N0-(2,4-Dihydroxybenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7m)
(3H, s, N–CH3), 3.69 (3H, s, O–CH3), 3.81 (6H, s, O–CH3),
5.88 (2H, s, N–CH2), 7.04 (1H, s, Ar–H), 7.09 (1H, s, Ar–
H), 7.54 (2H, t, J = 8.0 Hz, Ar–H), 7.75 (2H, t,
J = 8.0 Hz, Ar–H), 7.86(1H, d, J = 7.6 Hz, Ar–H), 7.97
(2H, d, J = 7.2 Hz, Ar–H), 8.02 (2H, t, J = 6.4 Hz, Ar–
H), 8.21 (1H, s, CH), 11.94 (1H, s, NH); 13C NMR
(DMSO-d6): 38.9 (N–CH3), 55.4 (CH2), 59.2 (O–CH3),
59.8 (O–CH3), 60.1 (O–CH3), 105.7 (Ar–C), 106.5 (Ar–C),
120.7 (Ar–C), 121.2 (Ar–C), 122.8 (Ar–C), 124.2 (Ar–C),
125.6 (Ar–C), 125.9 (Ar–C), 127.2 (Ar–C), 128.7 (Ar–C),
129.0 (Ar–C), 129.4 (Ar–C), 130.0 (Ar–C), 130.2 (Ar–C),
130.9 (Ar–C), 132.6 (Ar–C), 133.5 (Ar–C), 142.1 (N=C,
Ar–C), 146.5 (N=CH), 158.0 (Ar–C), 159.7 (Ar–C), 160.1
(Ar–C), 168.0 (C=O); MS m/z: 561.5 (M?); Anal. Calcd.
for C28H27N5O6S 59.88; H, 4.85; N, 12.47. Found: 59.80;
H, 4.80; N, 12.40.
Off-white powder; Yield: 80 %; m.p. 201 °C; FT-IR (KBr)
cm-1: 3529; 3396; 3071; 2961; 1695; 1595; 1489; 1345;
1
1267; 1173; 1057; 740; H NMR (DMSO-d6) (400 MHz)
d: 2.88 (3H, s, N–CH3), 5.84 (2H, s, N–CH2), 7.34 (2H, d,
J = 7.6 Hz, Ar–H), 7.51 (1H, s, Ar–H), 7.53 (2H, t,
J = 7.8 Hz, Ar–H), 7.75 (2H, t, J = 8.4 Hz, Ar–H),
7.82–7.87 (2H, dd, J1 = 7.6 Hz, J2 = 14.6 Hz, Ar–H),
7.90 (1H, t, J = 8.8 Hz, Ar–H), 7.96–8.03 (2H, dd,
J1 = 7.6 Hz, J2 = 12.6 Hz, Ar–H), 8.43 (1H, s, CH), 9.89
(2H, s, OH), 11.89 (1H, s, NH); 13C NMR (DMSO-d6):
38.9 (N–CH3), 53.5 (N–CH2), 120.2 (Ar–C), 121.2 (Ar–C),
122.3 (Ar–C), 123.7 (Ar–C), 124.5 (Ar–C), 125.4 (Ar–C),
125.8 (Ar–C), 127.2 (Ar–C), 128.6 (Ar–C), 128.9 (Ar–C),
129.5 (Ar–C), 129.8 (Ar–C), 130.0 (Ar–C), 130.7 (Ar–C),
131.3 (Ar–C), 132.5 (Ar–C), 133.5 (Ar–C), 134.1 (Ar–C),
142.2 (N=C, Ar–C), 146.6 (N=CH), 149.7 (Ar–C), 150.2
(Ar–C), 168.1 (C=O); MS m/z: 503.4 (M?); Anal. Calcd.
for C25H20N6O5S C, 59.63; H, 4.20; N, 13.91. Found: C,
59.54; H, 4.12; N, 13.97.
N0-(2-Bromobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7p)
Off-white powder; Yield: 85 %; m.p. 256 °C; FT-IR (KBr)
cm-1: 3465; 3069; 2946; 1695; 1599; 1486; 1348; 1275;
123

Med Chem Res
1188; 1024; 754; 1H NMR (DMSO-d6) (400 MHz) d: 2.88
(3H, s, N–CH3), 5.89 (2H, s, N–CH2), 7.36–7.43 (2H, m,
Ar–H), 7.54 (2H, t, J = 7.4 Hz, Ar–H), 7.59 (3H, d,
J = 10.8 Hz, Ar–H),7.69 (1H, d, J = 7.6 Hz, Ar–H),
7.83–7.85 (2H, m, Ar–H), 7.97 (1H, d, J = 7.6 Hz, Ar–H),
8.01(1H, t, J = 8.0 Hz, Ar–H), 8.06–8.08 (1H, dd,
J1 = 1.6 Hz, J2 = 7.6 Hz, Ar–H), 8.32 (1H, s, CH), 11.92
(1H, s, NH); 13C NMR (DMSO-d6): 38.9 (N–CH3), 53.6
(N–CH2), 122.3 (Ar–C), 123.8 (Ar–C), 124.6 (Ar–C),
125.1 (Ar–C), 125.4 (Ar–C), 125.8 (Ar–C), 128.6 (Ar–C),
129.1 (Ar–C), 129.2 (Ar–C), 129.8 (Ar–C), 130.0 (Ar–C),
130.2 (Ar–C), 130.5 (Ar–C), 130.8 (Ar–C), 131.3 (Ar–C),
131.5 (Ar–C), 131.8 (Ar–C), 132.0 (Ar–C), 133.6 (Ar–C),
142.2 (N=C, Ar–C), 143.5 (Ar–C), 146.9 (N=CH), 167.9
(C=O); MS m/z: 549.4 (M?), 551.4(M??2); Anal. Calcd.
for C25H20BrN5O3S C, 54.55; H, 3.66; N, 12.72. Found: C,
54.52; H, 3.68; N, 12.85.
8.06 (1H, s, Ar–H), 8.08(1H, d, J = 5.8 Hz, Ar–H), 8.28
(1H, s, CH), 12.04 (1H, s, NH); 13C NMR (DMSO-d6):
38.9 (N–CH3), 53.6 (CH2), 122.4 (Ar–C), 123.7 (Ar–C),
124.5 (Ar–C), 125.1 (Ar–C), 125.5 (Ar–C), 125.7 (Ar–C),
127.3 (Ar–C), 128.6 (Ar–C), 128.9 (Ar–C), 129.0 (Ar–C),
129.2 (Ar–C), 129.8 (Ar–C), 130.0 (Ar–C), 130.2 (Ar–C),
130.9 (Ar–C), 131.4 (Ar–C), 131.6 (Ar–C), 131.8 (Ar–C),
132.0 (Ar–C), 133.6 (Ar–C), 142.1 (N=C, Ar–C), 145.9
(N=CH), 168.8 (C=O); MS m/z: 539.5 (M?); Anal. Calcd.
for C25H19Cl2N5O3S C, 55.56; H, 3.54; N, 12.96. Found:
C, 55.63; H, 3.58; N, 12.88.
2-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-
c][1,2]thiazin-1(4H)-yl)-N0-(4-nitrobenzylidene)
acetohydrazide (7s)
Off-white powder; Yield: 81 %; m.p. 243 °C; FT-IR (KBr)
cm-1: 3452; 3085; 2952; 1689; 1597; 1525; 1460; 1349; 1267;
1
1186; 1026; 738; HNMR (DMSO-d6)(400 MHz) d: 2.89
4-((2-(2-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo
[4,3-c][1,2]thiazin-1(4H)-yl)acetyl)hydrazono)methyl)
benzamide (7q)
(3H, s, N–CH3), 5.93(2H, s, N–CH2), 7.43(1H, t, J = 7.2 Hz,
Ar–H), 7.54 (2H, t, J = 7.6 Hz, Ar–H), 7.58 (1H, d,
J = 4.8 Hz, Ar–H), 7.85(2H,t,J = 7.8 Hz, Ar–H), 7.97(2H,
d, J = 7.2 Hz, Ar–H), 8.03 (2H, t, J = 7.6 Hz, Ar–H), 8.19
(1H, s, CH), 8.24 (3H, t, J = 7.8 Hz, Ar–H), 12.17 (1H, s,
NH);13CNMR(DMSO-d6):39.9(N–CH3), 54.9(CH2), 121.4
(Ar–C), 122.7 (Ar–C), 123.3 (Ar–C), 123.8 (Ar–C), 124.0
(Ar–C), 124.8 (Ar–C), 125.1 (Ar–C), 125.6 (Ar–C), 125.8
(Ar–C), 127.8 (Ar–C), 128.6 (Ar–C), 129.0 (Ar–C), 129.4
(Ar–C), 129.6 (Ar–C), 130.8 (Ar–C), 131.6 (Ar–C), 132.2
(Ar–C), 133.9 (Ar–C), 134.4 (Ar–C), 142.2 (N=C, Ar–C),
144.6 (Ar–C), 148.8 (N=CH), 169.8 (C=O); MS m/z:
516.4(M?); Anal. Calcd. for C25H20N6O5S C, 58.13; H, 3.90;
N, 16.27. Found: C, 58.17; H, 3.97; N, 16.35.
Off-white powder; Yield: 86 %; m.p. 145 °C; FT-IR (KBr)
cm-1 :3449; 3066; 2968; 1679; 1654; 1483; 1345; 1268;
1184; 1022; 747; 1H NMR (DMSO-d6) (400 MHz) d: 2.88
(3H, s, N–CH3), 5.84 (2H, s, N–CH2), 7.43 (2H, t,
J = 7.6 Hz, Ar–H), 7.53 (3H, t, J = 7.6 Hz, Ar–H),7.66
(4H, d, J = 9.8 Hz, Ar–H), 7.84 (2H, s, NH2), 7.98(3H, t,
J = 7.2 Hz, Ar–H), 8.04 (1H, d, J = 9.6 Hz, Ar–H), 8. 22
(1H, s, CH), 11.80 (1H, s, NH); 13C NMR (DMSO-d6):
39.7 (N–CH3), 55.6 (N–CH2), 118.8 (Ar–C), 120.4 (Ar–C),
120.8 (Ar–C), 121.4 (Ar–C), 122.1 (Ar–C), 122.8 (Ar–C),
123.4 (Ar–C), 123.8 (Ar–C), 124.3 (Ar–C), 125.7 (Ar–C),
125.9 (Ar–C), 127.8 (Ar–C), 128.5 (Ar–C), 128.9 (Ar–C),
129.0 (Ar–C), 129.8 (Ar–C), 130.6 (Ar–C), 133.2 (Ar–C),
133.5 (Ar–C), 134.2 (Ar–C), 141.8 (N=C, Ar–C), 145.7
(N=CH), 168.1 (C=O, hydrazide), 171.8 (C=O); MS m/z:
528.5 (M?); Anal. Calcd. for C26H22N6O4S C, 60.69; H,
4.31; N, 16.33. Found: C, 60.61; H, 4.38; N, 16.25.
N0-(3-Chlorobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7t)
Off-white powder; Yield: 84 %; m.p. 233 °C; FT-IR (KBr)
cm-1: 3469; 3067; 2949; 1684; 1609; 1486; 1345; 1272;
1
1180; 1021; 738; HNMR (DMSO-d6) (400 MHz) d: 2.88
N0-(3,4-Dichlorobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7r)
(3H, s, N–CH3), 5.91 (2H, s, N–CH2), 7.44 (2H, q,
J = 8.4 Hz, Ar–H), 7.53 (2H, t, J = 8.0 Hz, Ar–H),7.58
(2H,d, J = 4.8 Hz, Ar–H), 7.68–7.71 (2H, dd,, J1 = 3.6 Hz,
J2 = 7.6 Hz, Ar–H), 7.84 (1H, d, J = 4.8 Hz, Ar–H),
7.91(1H, t, J = 8.0 Hz, Ar–H), 7.98 (2H, t, J = 8.4 Hz, Ar–
H), 8.02 (1H, s, Ar–H), 8.08 (1H, s, CH), 11.99 (1H, s, NH);
13C NMR (DMSO-d6): 38.9 (N–CH3), 53.5 (CH2), 122.3
(Ar–C), 123.9 (Ar–C), 124.8 (Ar–C), 125.3 (Ar–C), 125.9
(Ar–C), 126.6 (Ar–C), 128.6 (Ar–C), 128.8 (Ar–C), 129.2
(Ar–C), 129.8 (Ar–C), 130.1 (Ar–C), 130.7 (Ar–C), 130.8
(Ar–C), 131.4 (Ar–C), 131.5 (Ar–C), 131.8 (Ar–C), 132.5
(Ar–C), 132.9 (Ar–C), 137.5 (Ar–C), 142.3 (N=C, Ar–C),
Off-white powder; Yield: 84 %; m.p. 245 °C; FT-IR (KBr)
cm-1: 3454; 3068; 2966; 1682; 1597; 1462; 1344; 1290;
1
1168; 1066; 746; HNMR (DMSO-d6) (400 MHz) d: 2.88
(3H, s, N–CH3), 5.91(2H, s, N–CH2), 7.43(1H, t,
J = 7.6 Hz, Ar–H), 7.53 (2H, t, J = 7.6 Hz, Ar–H),7.57
(1H, d, J = 4.4 Hz, Ar–H), 7.67 (1H, d, J = 3.2 Hz, Ar–
H), 7.72–7.76 (2H, dd, J1 = 7.8 Hz, J2 = 9.2 Hz, Ar–H),
7.83 (2H, d, J = 8.4 Hz, Ar–H), 7.95–8.02 (1H, m, Ar–H),
123

Med Chem Res
143.5 (Ar–C), 146.9 (N=CH), 167.1 (C=O); MS m/z:
505.4(M?); Anal. Calcd. for C25H20Cl N5O3S C, 59.34; H,
3.98; N, 13.84. Found: C, 59.38; H, 3.87; N, 13.88.
difference Fourier map was essentially featureless. The
Figure was plotted with the aid of ORTEP-3 (Farrugia,
1997).
N0-(3-Bromobenzylidene)-2-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-1(4H)-
yl)acetohydrazide (7u)
Anti-HIV-1 assay
The synthesized compounds were screened for their
in vitro antiviral effects in human PBM cells according to
the standardized assay (Schinazi et al., 1990). Cells
obtained from Life South Community Blood Centers
(Atlanta, GA) were isolated by Histopaque (Sigma-
Aldrich, St. Louis, MO) and discontinuous gradient cen-
trifugation from healthy seronegative donors. The median
effective concentration (EC50) was determined using a
reported method (Belen’kii and Schinazi, 1994). Assays
were conducted using at least two different donor cells in
duplicate or triplicate. The results are expressed in Table 5.
Off-white powder; Yield: 83 %; m.p. 251 °C; FT-IR (KBr)
cm-1 : 3465; 3069; 2946; 1695; 1599; 1486; 1348; 1275;
1
1188; 1024; 754; HNMR (DMSO-d6) (400 MHz) d: 2.88
(3H, s, N–CH3), 5.91 (2H, s, N–CH2), 7.39 (1H, t,
J = 8.0 Hz, Ar–H), 7.45 (1H, t, J = 7.6 Hz, Ar–H), 7.53
(3H, t, J = 7.6 Hz, Ar–H), 7.58 (2H, d, J = 5.8 Hz, Ar–
H), 7.75 (1H, dd, J1 = 4.8 Hz, J2 = 7.6 Hz, Ar–H), 7.90
(2H, d, J = 5.2 Hz, Ar–H), 7.95(2H, t, J = 8.4 Hz, Ar–H),
8.02 (1H, s, Ar–H), 8.07 (1H, s, CH), 11.99 (1H, s, NH);
13C NMR (DMSO-d6): 38.9 (N–CH3), 53.6 (N–CH2),
122.3 (Ar–C), 123.7 (Ar–C), 124.6 (Ar–C), 125.0 (Ar–C),
125.5 (Ar–C), 125.7 (Ar–C), 128.6 (Ar–C), 129.0 (Ar–C),
129.3 (Ar–C), 129.8 (Ar–C), 130.0 (Ar–C), 130.2 (Ar–C),
130.7 (Ar–C), 130.8 (Ar–C), 131.3 (Ar–C), 131.5 (Ar–C),
131.7 (Ar–C), 131.9 (Ar–C), 133.5 (Ar–C), 142.2 (Ar–C),
143.5 (N=C, Ar–C), 146.9 (N=CH), 167.8 (C=O); MS m/z:
549.3 (M?), 551.3 (M??2); Anal. Calcd. for
C25H20BrN5O3S C, 54.55; H, 3.66; N, 12.72. Found: C,
54.58; H, 3.60; N, 12.79.
Cytotoxicity assay
Primary human PBM, CEM, and Vero cells were cultured
in 96-well plates (5 9 104 cells per well) along with
increasing concentrations of the test compound. Cell via-
bility was measured after 5-day incubation period using the
Cell Titer 96 Aqueous One Solution cell proliferation assay
(Promega, Madison, WI) by incubating in an incubator at
37 °C with 5 % CO2 for human PBM cells (Stuyver et al.,
2002). The results are summarized in Table 5.
X-ray crystallographic studies
A colorless plate crystal of (7b) was coated with Paratone
8277 oil (Exxon) and mounted on a glass fiber. All mea-
surements were made on a Nonius KappaCCD diffractom-
eter with graphite monochromated Mo-Ka radiation. Details
of crystal data and structure refinement have been provided
in Table 4. The data were collected at a temperature of
173(2) K using x and u scans, corrected for Lorentz and
polarization effects and for absorption using multiscan
method (Hooft, 1998; Otwinowski and Minor, 1997).
Molecular docking
The high resolution structure of HIV-1 Reverse Transcriptase
was retrieved from the Protein data bank using accession no.
1C1B. The docking procedure was carried out using Molec-
ular operating environment (MOE). The 3D structure was
optimized by removing water molecules and then protonating
the structure. Energy of the structure was minimized using
parameters such as gradient: 0.05, Force Field:
MMFF94X ? Solvation, and Chiral Constraint: Current
Geometry. The structures of ligands were drawn using
ChemDraw software and were converted to 3D structures.
Energy was minimized to optimize the structures using
parameters such as gradient: 0.05, Force Field: MMFF94X,
Chiral Constraint, and Current Geometry. The docking algo-
rithm of MOE software was utilized to dock the compounds
with binding pocket of HIV-1 reverse transcriptase. After
docking,top-rankingconformationswereselectedonthebasis
of S-score for interaction analysis. Interaction analysis was
done using Chimera structure visualization software.
The structure was solved by the direct methods (Altomare
et al., 1993) and expanded using Fourier techniques
(Beurskens et al., 1994). The nonhydrogen atoms were
refined anisotropically. Hydrogen atoms were included at
geometrically idealized positions and were not refined
except for the amino H atom which was located from a dif-
ference map and was allowed to refine freely. The final cycle
of full-matrix least-squares refinement using SHELXL97
(Sheldrick, 2008) converged with unweighted and weighted
agreement factors, R and wR = 0.0436 (all data) and 0.0919
(all data) and the goodness of fit, S = 1.079. The weighting
scheme was based on counting statistics, and the final
123

Med Chem Res
Acknowledgments The authors (SA and MA) are grateful to Higher
Education Commission, Pakistan and Institute of Chemistry, University
of the Punjab, Lahore, for financial assistance. We are also thankful to
International Centre for Chemical and Biological Sciences, HEJ
Research Institute of Chemistry, University of Karachi, Karachi, for
spectral measurements. This work was also supported in part by NIH
grant 2P30-AI-050409 and the Department of Veterans Affairs (RFS),
USA. The X-ray coordinate of the compound has been deposited at
Cambridge Crystallographic Data Centre with CCDC number 915138.
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