Synthesis of novel pyrazolobenzothiazine 5,5-dioxide derivatives as potent anti-HIV-1 agents

Article abstract of DOI:10.1007/s00044-013-0718-x

A new series of pyrazolobenzothiazine-based carbohydrazides was prepared in a facile way, starting with commercially available sodium saccharine. The final products were sufficiently characterized by spectroscopic techniques. In addition, compound 5k was

Full text of DOI:10.1007/s00044-013-0718-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1309–1319
DOI 10.1007/s00044-013-0718-x
ORIGINAL RESEARCH
Synthesis of novel pyrazolobenzothiazine 5,5-dioxide derivatives
as potent anti-HIV-1 agents
•
Matloob Ahmad Sana Aslam Mujahid Hussain Bukhari
•
•
•
Catherine Montero Mervi Detorio
•
•
Masood Parvez Raymond F. Schinazi
Received: 23 March 2013 / Accepted: 13 August 2013 / Published online: 1 September 2013
Ó Springer Science+Business Media New York 2013
Abstract A new series of pyrazolobenzothiazine-based
carbohydrazides was prepared in a facile way, starting with
commercially available sodium saccharine. The final
products were sufficiently characterized by spectroscopic
techniques. In addition, compound 5k was confirmed with
X-ray crystallography as well. All the compounds were
screened for anti-HIV-1 and cytotoxicity activities. Over-
all, out of 15 compounds, seven exhibited good activity
with EC50 values \20 lM. Compounds 5c, 5d, 5g, 5j and
5k appeared as the potent anti-HIV-1 agents with EC50
values\5.0 lM. The structure–activity relationship would
facilitate the discovery of new molecules with better profile
of HIV inhibition activity.
Keywords Heterocyclic compounds ꢀ
Biological activity ꢀ 1,2-Benzothiazine 1,1-dioxide ꢀ
Pyrazolobenzothiazines ꢀ Anti-HIV-1 activity ꢀ
Cytotoxic studies
Introduction
This research article is dedicated to the late professor Dr. Hamid Latif
Siddiqui, our honorable teacher (MA, SA and MHB) and friend (MP
and RFS).
Human immunodeficiency virus (HIV) is a continuous
threat to the human being. This pathogen is reported to
have affected about 40 million people worldwide and is
responsible for acquired immune deficiency syndrome
(AIDS). However, this lethal infection has now been con-
verted into a chronic condition by using a combination
therapy of various antiviral drugs (Coffin et al., 1986; De
Clercq, 2006, 2007a, b). The first drug that showed
inhibitory effects to HIV was Zidovudine (AZT). Now
more than 20 drugs are approved for the treatment of HIV.
Combinations of various antiviral drugs that inhibit dif-
ferent stages in the viral replication are widely used for the
treatment of this infectious disease and are called highly
active antiretroviral therapy (HAART). A handsome
number of anti-HIV drugs contain heterocyclic ring sys-
tems as a basic skeleton such as Vicriviroc (I), Aplaviroc
(II) (Fig. 1). These heterocyclic scaffolds include pyrimi-
dines, pyrazoles, benzimidazoles heterocycles, etc. How-
ever, there is a continuous need for the development of new
and effective anti-HIV drugs with less toxicity (Gallant
et al., 2006; Mitsuya et al., 1985; Pozniak et al., 2006; De
Clercq, 2007a).
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0718-x) contains supplementary
material, which is available to authorized users.
M. Ahmad (&)
Department of Chemistry, Government College University,
Faisalabad 38000, Pakistan
e-mail: matloob_123@yahoo.com
S. Aslam ꢀ M. H. Bukhari
Institute of Chemistry, University of the Punjab,
Lahore 54590, Pakistan
S. Aslam
Department of Chemistry, Government College Women
University, Faisalabad, Pakistan
C. Montero ꢀ M. Detorio ꢀ R. F. Schinazi
Laboratory of Biochemical Pharmacology, Emory University
School of Medicine/Veterans Affairs Medical Center,
1760 Haygood Drive, Atlanta, GA 30322, USA
M. Parvez
Department of Chemistry, University of Calgary,
2500 University Drive NW, Calgary, AB T2N 1N4, Canada
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Med Chem Res (2014) 23:1309–1319
Benzothiazines are versatile biologically active hetero-
pyrazole ring as well as hydrazide formation. Thus, the
strategy provides an efficient way to form pyrazole and
benzothiazine hybrid compounds, i.e., pyrazolobenzothia-
zines (Scheme 1). This ring system is rarely reported in
literature and could be further explored for a broad range of
new derivatives possessing various functionalities and
pharmacophores resulting in worthwhile molecules. We
herein converted the hydrazide to respective imines by
treating it with a range of substituted benzaldehydes.
Spectroscopic tools were effectively utilized for the
characterization of products. Compound 1 was confirmed
by the appearance of N–CH2 moiety at d value of 5.13 ppm
in the 1H NMR and in FTIR, the peak at 1,715 cm-1
confirmed the presence of carbonyl group. Compound 2
was confirmed by the appearance of singlet at d value of
5.81 ppm (SO2NH). Moreover, in FTIR spectrum, ketone
peak is considerably shifted from 1,715 cm-1 (compound
1) to 1,615 due to the formation of b-diketone moiety (enol
form). Compound 3 bearing ester functionality was char-
acterized by IR peaks of carbonyl at 1,754 cm-1 and the
appearance of protons of –COOCH3 group at d 3.27 ppm
and N–CH2 protons at d 3.98 ppm in 1H NMR. Compound
4 (the precursor compound) was characterized sufficiently
by the appearance of CONH protons at 14.23 ppm and NH2
of carbohydrazide moiety at 4.42 ppm. The corresponding
carbohydrazones obtained by treatment with substituted
benzaldehydes were confirmed by the presence of CH=N
cyclic precursors exhibiting a broad spectrum of biological
activities. 1,2-Benzothiazine-based carboxamides are
excellent in their anti-inflammatory and analgesic activities
and are known as oxicams that include meloxicam, pir-
oxicam, and sudoxicam (Lomabardino et al., 1971; Marfat,
1985; Turck et al., 1995). Various other derivatives of
benzothiazines exhibit anticancer activity (Gupta et al.,
1993), endothelin receptor antagonistic activity (Berryman
et al., 1998), and calpain I inhibiting activity (Bihovsky
et al., 2004). While continuing our research in 1,2-benzo-
thiazine derivatives, we have explored N0-arylmethyli
dene-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzo-
thiazin-2(4H)-yl)acetohydrazides (Ahmad et al., 2010a, b),
N0-(substituted-2-chloroquinolin-3-yl)methylidene-4-hydroxy-
2H-1,2-benzothiazine-3-carbohydrazides 1,1-dioxides (Ahmad
et al., 2012), pyrazolobenzothiazine-based carboxamides as
antioxidants (Ahmad et al., 2013), and pyrazolobenzo-
thiazine-based pyrimidines as antibacterial (Bukhari et al.,
2012). Recently, we have reported various pyrazolobenzo-
thiazine derivatives as anti-HIV-1 agents (Aslam et al.,
2013).
The observation that heterocyclic ring systems act as
basic skeleton in various anti-HIV drugs as depicted in
Fig. 1 and biological significance of benzothiazine and
pyrazolobenzothiazine heterocycles led us to assume the
potent biological nature of our developed compounds. The
hypothesis proved with the discovery of some pyra-
zolobenzothiazine derivatives as HIV inhibitors with EC50
values less than 5 lM (Scheme 1).
1
group which appeared in aromatic range as singlet in H
NMR and in 13C NMR, it was observed downfield in the
range of 140–144 ppm. CHN analysis data confirmed the
proposed structures (Table 1). The structure elucidation
was finalized with the X-ray crystal structure of compound
5k as represented compound of the series (Fig. 2).
Results and discussion
Chemistry
Crystal structure of compound 5k
N-Phenacylsaccharine (1), prepared by condensation of
saccharine with phenacyl bromide, was subjected to ring
expansion with sodium methoxide in MeOH to get
3-benzoyl-4-hydroxy-2H-1,2-benzothiazin-1,1-dioxide (2).
Compound 3 was obtained from compound 2 by reaction
with methyl chloroacetate, which was treated with hydra-
zine monohydrate that caused simultaneous ring closure to
The molecular structure of compound 5k is shown in
Fig. 2. The crystal data and structure refinement parame-
ters are listed in Table 2. The heterocyclic thiazine ring
adopts a twist chair conformation with atoms S1 and C1
˚
displaced by 0.819(2) and 0.261(3) A, respectively, from
the mean plane formed by the remaining ring atoms (N1//
C6–C8). The mean planes of the benzene rings C1–C6 and
Fig. 1 Structures of HIV co-
receptor antagonists Vicriviroc
(I) and Aplaviroc (II)
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Med Chem Res (2014) 23:1309–1319
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Scheme 1 Layout for the synthesis of 2-(5,5-dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)-N0-[arylmethylidene] acetohydraz-
ides (5a–o)
Table 1 CHN data for 2-(5,5-dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)-N0-[arylmethylidene]acetohydrazides (5a–o)
Sr. no.
Code no.
R
CHN analysis calculated (found)
C
H
N
1.
5a
5b
5c
5d
5e
5f
2-F
60.62 (60.75)
53.74 (53.89)
53.74 (53.87)
58.59 (58.71)
58.59 (58.72)
54.76 (54.91)
54.76 (54.90)
62.38 (62.29)
63.62 (63.51)
57.36 (57.47)
57.36 (57.49)
57.36 (57.48)
61.59 (61.73)
60.34 (60.75)
60.34 (60.74)
3.82 (3.97)
3.38 (3.51)
3.38 (3.50)
3.69 (3.86)
3.69 (3.85)
3.26 (3.39)
3.26 (3.38)
4.83 (4.77)
5.34 (5.25)
3.61 (3.75)
3.61 (3.76)
3.61 (3.78)
4.34 (4.47)
4.48 (4.63)
4.48 (4.65)
14.73 (14.87)
13.06 (13.21)
13.06 (13.20)
14.24 (14.41)
14.24 (14.42)
13.30 (13.50)
13.30 (13.49)
16.79 (16.63)
15.90 (15.65)
16.72 (16.87)
16.72 (16.85)
16.72 (16.86)
14.36 (14.43)
13.53 (13.70)
13.53 (13.72)
2.
3-Br
3.
4-Br
4.
3-Cl
5.
4-Cl
6.
2,4-(Cl)2
2,6 (Cl)2
4-NMe2
4-NEt2
2-NO2
3-NO2
4-NO2
4-OMe
3,4-(OMe)2
2,4-(OMe)2
7.
5g
5h
5i
8.
9.
10.
11.
12.
13.
14.
15.
5j
5k
5l
5m
5n
5o
˚
C10–C15 make dihedral angles 18.37(9) and 44.57(9)°,
respectively, with the mean plane of the pyrazolyl ring
(N2/N3/C7/C8/C9). The acetamide chain (O3/N4/N5/C17/
C18) linking is essentially planar (rms deviation 0.0056 A)
and forms dihedral angles with the mean planes of the ring
C19–C24 13.31(10)°. The nitro group (N6/O4/O5) is more
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Med Chem Res (2014) 23:1309–1319
Fig. 2 The molecular structure
of 5k with the atom numbering
scheme
Table 2 Crystal data and structure refinement parameters of compound 5k
Parameters
Values
Parameters
Values
CCDC No.
881055
Crystal size (mm)
T (K)
0.26 9 0.08 9 0.05
Moiety formula
Formula mass (g mol-1
Crystal system
C24 H18 N6 O5 S
502.50
173(2)
)
Index ranges
total data
h: -35 to 35; k: -14 to 9; l: -19 to 19
7,424
Monoclinic
C 2/c
Space group
Independent reflections
5,286 [R(int) = 0.013]
Unit cell dimensions
˚
a (A)
27.722(7)
11.221(3)
15.299(3)
101.549(16)
4,662.7(19)
2.30–27.47
8
Completeness to h = 27.5°
98.8 %
˚
b (A)
˚
c (A)
b (°)
Absorption correction
Multi-scan method
0.991 and 0.953
5,286/0/335
3
˚
V (A )
Max. and min. transmission
Data/restraints/parameters
Final R indices[I [/2r(I)]
Goodness-of-fit on F2
h Ranges for data collection (°)
Z
R1 = 0.0436; wR2 = 0.0990
1.08
-3
q
calc. (g cm-3
F(000)
)
1.432
˚
2,080
Largest diff. peak and hole
0.25 and -0.46 e.A
or less co-planar with the benzene ring (C19–C24) with a
enhanced activity. However, for bromo substitution,
4-bromo derivative was slightly more active than 3-bromo
derivative. Among the nitro-substituted compounds, fol-
lowing order was observed: 3-NO2 [ 2-NO2 [ 4-NO2
(Fig. 3). This order may be explained by the electron
withdrawing nature of nitro group as it withdraws elec-
trons from phenyl ring by inductive effect as well as by
resonance effect, but overall, mesomeric effect dominates
the inductive effect. Thus, NO2 group at 3-position
induces strong electron deficiency in the side chain con-
taining hydrazone moiety. This proposed explanation is
further supported by the X-ray data of 5k (3-Nitro) where
the nitro group (N6/O4/O5) is more or less co-planar with
the benzene ring which is the mandatory condition for
delocalization. Similarly, the order of biological activity
of 2-nitro- and 4-nitro-substituted compounds is as
expected. At ortho and para position, nitro group induces
withdrawal of electrons by inductive effect which is
2-NO2 [ 4-NO2.
dihedral angle between the mean planes being 9.6(11)°.
Pharmacology
Anti-HIV-1 activity
Among the series 5a–o, seven compounds were found to be
active against HIV-1 with EC50 values less than 20 lM
(Table 3). Structure–activity relationship confirmed that
the nature of substituent played an important role in the
biological activity of the compounds.
Substituents having dominant electron donating effect
are less active. Nitro- and halo-substituted compounds are
more active than methoxy- and dialkyamino-substituted
ones. All the methoxy- and N,N-dialkylamino-substituted
compounds were either weakly active or inactive.
Among the nitro- and halo-substituted compounds,
interestingly the substitution at 3-position results in
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Med Chem Res (2014) 23:1309–1319
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Table 3 Anti-HIV-1 and cytotoxic activities of 2-(5,5-dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)-N0-[arylmethylidene] acet-
ohydrazides (5a–5o)
Sr. no
R
Compound
Anti-HIV-1 activity in PBM cells (lM)a
Cytotoxicity (IC50, lM)a
EC50
EC90
PBM
CEM
Vero
1
4
[100
[100
6.0
[100
[100
35.9
[100
20.7
11.7
13.0
11.2
31.6
11.1
12.9
[100
52.2
47.8
22.0
C100
76.3
75.8
74.4
[100
15.1
15.4
6.8
[100
15.5
12.2
48.5
13.2
14.2
19.1
8.0
2
2-F
5a
5b
5c
5d
5e
5f
3
3-Br
4
4-Br
4.1
13.4
12.6
3.7
5
3-Cl
2.2
43.4
6
4-Cl
6.5
29.7
11.9
10.9
\1.0
28.9
12.5
2.0
7
2,4-(Cl)2
2,6 (Cl)2
4-NMe2
4-NEt2
2-NO2
3-NO2
4-NO2
4-OMe
3,4-(OMe)2
2,4-(OMe)2
AZT
46.0
2.7
95.4
8
5g
5h
5i
52.5
9
[100
27.2
4.8
[100
61.4
14.6
62.6
59.3
53.1
50.0
66.0
46.1
82.8
56.0
10
11
12
13
14
15
16
17
a
5j
47.3
5k
5l
3.3
30.2
15.2
20.8
31.6
48.6
17.5
14.3
25.1
42.0
46.6
41.0
0.0033
[100
81.0
5m
5n
5o
[100
80.6
0.031
All experiments were conducted in replicate
Fig. 3 Structure–activity
relationship (anti-HIV-1) among
the nitro-substituted compounds
5j, 5k, and 5l
Compounds 5c, 5d, 5g, 5j, and 5k bearing 4-Br, 3-Cl, 2,6
(Cl)2, 2-NO2, and 3-NO2 substituents, respectively, were
potentially active against HIV-1 with EC50 values 4.1, 2.2,
2.7, 4.8, and 3.3 lM, respectively (Fig. 4). However, sub-
stitutions by methoxy and N,N-dialkylamino groups resulted
in reduced activity especially at position-4, e.g., 4-NEt2 (5i)
and 4-OMe (5m) displayed an EC50 values of 27.2 and
42.0 lM, respectively. Chlorosubstituted compounds (5d
and 5g) appeared as the most potent anti-HIV agents among
the titled compound. These results support our recently
published related work (Aslam et al., 2013), where 3-chlo-
rothiophenyl- and 2,5-dichlorothiophenyl-substituted prod-
ucts (a, Fig. 5) were the most potent and in the series of
(2E)-3-(2-chloro-6-methyl/methoxyquinolin-3-yl)-1-(aryl)-
prop-2-en-1-ones where 2,5-dichlorothiophenyl-substituted
product (b, Fig. 5) was the most potent (Rizvi et al., 2013).
Cytotoxicity studies
Compounds were screened for their cytotoxicity in human
PBM, CEM, and Vero cells to determine their spectrum of
toxicity. CEM cells are a line of lymphoblastic cells orig-
inally derived from a child with acute lymphoblastic leu-
kemia whereas Vero cells are derived from African Green
monkey kidney cells.
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Med Chem Res (2014) 23:1309–1319
Chemical shifts are reported in ppm referenced to the
residual solvent signal. FT-IR spectra were recorded on a
Thermo Nicolet IR 200 spectrometer. Melting points were
recorded on a Gallenkamp melting point apparatus and are
uncorrected.
Synthesis of 2-(2-oxo-2-phenylethyl)-1,2-benzisothiazol-
3(2H)-one 1,1-dioxide (1)
Compound (1) was prepared following our reported proce-
dure (Ahmad et al., 2010c). Phenacyl bromide (0.122 mol)
was added to the suspension of sodium saccharine (25 g,
0.122 mol) in DMF (30 mL). The reaction mixture was
stirred at 110 °C for 3 h and was poured on crushed ice.
Precipitates formed were filtered, washed with excess water,
and were dried after re-crystallization from ethanol.
Fig. 4 Structures of the most potent anti-HIV-1 agents among
compounds 5a–o
In the series 5a–o, all the halogen-substituted compounds
were cytotoxic to human PBM, CEM, and Vero cells with
IC50 values\50 lM. Among nitro-substituted compounds,
5l (4-NO2) showed no toxicity to human PBM and Vero
cells. Methoxy-substituted compounds showed no toxicity
in primary human PBM and Vero cells except compound 5n
which was toxic to Vero cells (Table 3) (Fig. 6).
Yield: 84 %. FT-IR (KBr) vmax:1715; 1335; 1175 cm-1
;
1H NMR: (DMSO-d6, 400 MHz) d: 5.13 (s, 2H, N–CH2),
7.49 (t, 2H, J = 7.6 Hz, Ar–H), 7.61 (t, 1H, J = 7.6 Hz,
Ar–H), 7.83–7.91 (m, 2H, Ar–H), 7.94 (d, 1H, J = 6.8 Hz,
Ar–H), 7.96 (d, 2H, J = 6.8 Hz, Ar–H), 8.09 (d, 1H,
J = 8.0 Hz, Ar–H); 13C NMR (DMSO-d6): 51.3 (CH2),
123.1 (Ar–C), 124 (Ar–C), 126.3 (Ar–C), 129.1 (2C, Ar),
129.4 (2C, Ar), 133.5 (Ar–C), 134.8 (Ar–C), 135.3 (Ar–C),
137 (Ar–C), 141.1 (Ar–C), 169.5 (C=O), 191.2 (Ar–CO);
MS: ES-; 301.1 (M?), ?ES; 324.04 (M??Na?).
Conclusion
In summary, we herein report the anti-HIV-1 activity of new
acetohydrazides based on pyrazolobenzothiazine ring sys-
tem. Seven compounds were active against HIV-1 with EC50
values less than 20.0 lM, which indicates the potential of
these compounds as anti-HIV-1 agents. Among these seven
active compounds, 5c, 5d, 5g, 5j, and 5k were the potent anti-
HIV-1 agents with EC50 values less than 5.0 lM. Compound
5d appeared as the most potent of all having EC50 value of
2.2 lM. On these grounds, the structural modification of
these compounds could lead us for new candidates of anti-
HIV drugs with better inhibition profiles.
Synthesis of (4-hydroxy-1,1-dioxido-2H-1,2-benzothiazin-
3-yl) (phenyl)methanone (2)
Compound (2) was prepared according to our reported
methods (Ahmad et al., 2010d). Sodium methoxide was
freshly prepared by dissolving sodium metal (2.67 g,
0.116 mol) in 40 mL freshly dried methanol on reflux. The
inert atmosphere was maintained by N2 gas. Then, N-
phenacylsaccharin (1) (5.0 g, 0.0166 mmol) was added to
refluxing mixture of sodium methoxide and methanol. The
solution turned to orange red color suspension immediately.
The mixture was refluxed for half an hour and then poured on
ice cold 10 % HCl. The white precipitates formed were col-
lected and washed with excess water and dried.
Experimental
Chemistry
Yield: 80 %. Appearance: white crystalline solid,
m.p.156 °C; FT-IR (KBr) vmax: 3157; 1615; 1358;
All the chemicals were purchased from Alfa Aesar and
were used without purification. 1H NMR spectra were
recorded on a Bruker DPX-400 instrument at 400 MHz.
1
1151 cm-1; H NMR: (DMSO-d6, 400 MHz) d: 5.81 (s,
1H, SO2NH), 7.64 (t, 1H, J = 8.0 Hz, Ar–H), 7.73 (d, 1H,
Fig. 5 Structures of the most
potent compounds among our
previously reported work
(Aslam et al., 2013; Rizvi et al.,
2013)
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Med Chem Res (2014) 23:1309–1319
1315
Fig. 6 Structure–activity
relationship among methoxy-
substituted compounds for
cytotoxicity screening against
human PBM cells
J = 8.0 Hz, Ar–H), 7.94–7.98 (m, 5H, Ar–H), 8.17 (t, 2H,
J = 3.6 Hz, Ar–H), 14.63 (s, 1H, 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), 129.5
(2C, Ar), 132.1 (Ar–C), 132.3 (Ar–C), 134 (Ar–C), 137.1
(2C, Ar), 151.6 (C-4, thiazine), 191.1 (C=O); LRMS (EI):
ES-; 301.07 (M?), ?ES; 302.06 (M??H?).
and hydrazine monohydrate (4.9 mL, 99 mmol) in 50 mL eth-
anol was refluxed for 4 h. Then the unreacted hydrazine
monohydrate and ethanol was removed under vacuum. The
crude product then dissolved in ice cold water. The resulted
precipitates were filtered, recrystallized with ethanol, and finally
dried.
Appearance: light dirty yellow; m.p. 190–192 °C; FT-IR
(KBr) vmax: 3378; 1685; 1333; 1162 cm-1 1H NMR:
;
Synthesis of methyl [4-hydroxy-1,1-dioxido-3-
(DMSO-d6, 400 MHz) d: 4.06 (s, 2H, N–CH2), 4.42 (d, 2H,
J = 12.4 Hz, NH2), 7.50–7.66 (m, 3H, Ar–H), 7.73–7.85
(m, 4H, Ar–H), 7.97–8.03 (dd, 2H, J1 = 7.2 Hz,
J2 = 20.4 Hz, Ar–H), 8.98 (s, 1H, NH), 14.23 (s, 1H, N–
H); 13C NMR (DMSO-d6): 51.3 (CH2), 126.9 (2C, Ar),
127.5 (Ar–C), 128.1 (Ar–C), 128.7 (Ar–C), 128.9 (2C, Ar),
129.2 (Ar–C), 131.1 (Ar–C), 131.3 (Ar–C), 131.6 (Ar–C),
133.1 (Ar–C), 134.2 (Ar–C), 134.4 (Ar–C), 136.2 (Ar–C),
166.8 (C=O); MS m/z: 369.1 (ES-), 392.10 (ES?,
M??Na?).
(phenylcarbonyl)-2H-1,2-benzothiazin-2-yl]acetate (3)
(4-Hydroxy-1,1-dioxido-2H-1,2-benzothiazin-3-yl)
(phenyl)methanone (2) (6.0 g, 0.020 mol) and anhydrous
potassium carbonate (3.31 g, 0.024 mol) were dissolved in
acetonitrile (30 mL). Then methyl chloroacetate (2.60 g,
0.024 mol) was added and allowed to reflux for 8 h. The
completion of reaction was monitored by TLC. The solvent
was removed under vacuum. Resulted residues were dis-
solved in cold water and maintain the neutral pH by dil. HCl
to get the dirty yellow crystalline product.
Synthesis of 2-(5,5-dioxido-3-phenylpyrazolo[4,3-
c][1,2]benzothiazin-4(2H)-yl)-N0-[arylmethylidene]
acetohydrazides (5a–o)
Appearance: dirty yellow; m.p. 165 °C; FT-IR (KBr)
v
max: 1754; 1328; 1179 cm-1 1H NMR (DMSO-d6,
;
400 MHz) d: 3.27 (s, 3H, –COOCH3), 3.98 (s, 2H, N–
CH2), 7.63 (t, 1H, J = 8.0 Hz, Ar–H), 7.74 (d, 1H,
J = 8.0 Hz, Ar–H), 7.90–7.95 (m, 5H, Ar–H), 8.16 (t, 2H,
J = 4.2 Hz, Ar–H), 14.65 (s, 1H, O–H); 13C NMR
(DMSO-d6): 43.2 (CH2), 51.9 (O–CH3), 114.1 (C-3, thia-
zine), 122.1 (Ar–C), 127.2 (Ar–C), 128.6 (2C, Ar), 128.9
(Ar–C), 129.3 (Ar–C), 129.5 (Ar–C), 132.1 (Ar–C), 133.2
(Ar–C), 134.6 (Ar–C), 134.8 (Ar–C), 138.1 (Ar–C), 156.7
(C-4, thiazine), 167.4 (C=O, ester), 190.3 (Ar–CO); MS m/z:
ES-; 373.09 (M?), ?ES; 374.09 (M??H?).
Equimolar quantities of acetohydrazides 4 and corre-
sponding benzaldehydes were refluxed in ethyl alcohol for
4–5 h. After completion of reaction, as indicated by TLC,
the contents of the flask were concentrated on rotary
evaporator. The precipitates formed were filtered, washed
with excess ethanol, and dried.
2-(5,5-Dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothiazin-
4(2H)-yl)-N0-[(2-fluorophenyl)methylidene]acetohydrazide
(5a) White powder; m.p. 239–240 °C; FT-IR (KBr) vmax
:
3727; 3229; 1684; 1587; 1324; 1157 cm-1 1H NMR
;
Synthesis of 2-(5,5-dioxido-3-phenylpyrazolo[4,3-
c][1,2]benzothiazin-4(2H)-yl)acetohydrazide (4)
(DMSO-d6, 400 MHz) d: 4.70 (s, 2H, N–CH2), 7.17–7.25
(m, 3H, Ar–H), 7.39 (t, 2H, J = 6.8 Hz, Ar–H), 7.55 (d,
2H, J = 8.4 Hz, Ar–H), 7.60–7.67 (m, 3H, Ar–H), 7.91 (d,
2H, J = 8.4 Hz, Ar–H),8.01 (s, 1H, C–H), 8.06 (d, 1H,
A mixture of methyl [4-hydroxy-1,1-dioxido-3-(phenylcarbon-
yl)-2H-1,2-benzothiazin-2-yl]acetate (3) (5.0 g, 13.4 mmol)
123

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Med Chem Res (2014) 23:1309–1319
J = 6.8 Hz, Ar–H), 11.41 (s, 1H, N–H), 14.27 (s, 1H, N–
H); 13C NMR (DMSO-d6): 51.2 (CH2), 115.5 (Ar), 119.1
(Ar–C), 123.7 (Ar–C), 124.4 (Ar–C), 126.9 (Ar–C), 127.5
(Ar–C), 127.7 (Ar–C), 127.9 (Ar–C), 128.7 (Ar–C), 128.9
(Ar–C), 129.3 (Ar–C), 129.5 (Ar–C), 130.8 (Ar–C), 132.5
(Ar–C), 132.7 (Ar–C), 133.1 (Ar–C), 134.9 (Ar–C), 135.4
(Ar–C), 137.5 (Ar–C), 139.1 (Ar–C), 142.8 (N=CH), 148.9
(Ar–C), 167.7 (C=O); MS m/z: 475.15 (ES-, M?), 476.14
(?ES, M?H?).
7.63–7.70 (dd, 2H, J1 = 7.2 Hz, J2 = 18.4 Hz, Ar–H),
7.75 (s, 1H, CH), 7.83–7.88 (m, 4H, Ar–H), 7.99–8.07 (dd,
2H, J1 = 7.6 Hz, J2 = 24.4 Hz, Ar–H), 11.47 (s, 1H, NH),
14.25 (s, 1H, NH); 13C NMR (DMSO-d6): 51.3 (CH2),
120.9 (Ar–C), 122.7 (Ar–C), 123.8 (Ar–C), 124.4 (Ar–C),
126.4 (Ar–C), 127.2 (Ar–C), 127.7 (Ar–C), 128.1 (Ar–C),
128.4 (Ar–C), 128.7 (Ar–C), 129.0 (Ar–C), 129.7 (Ar–C),
130.9 (Ar–C), 132.5 (Ar–C), 132.9 (Ar–C), 133.4 (Ar–C),
133.8 (Ar–C), 134.7 (Ar–C), 135.9 (Ar–C), 137.7 (Ar–C),
138.7 (Ar–C), 142.9 (N=CH), 168.1 (C=O); MS m/z:
492.11 (ES-), 514.09 (ES?, M?Na?).
N0-[(3-Bromophenyl)methylidene]-2-(5,5-dioxido-3-phenyl
pyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)acetohydrazide
(5b) Off white powder; m.p. 217–219 °C; FT-IR (KBr)
N0-[(4-Chlorophenyl)methylidene]-2-(5,5-dioxido-3-phe-
nylpyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)acetohyd-
razide (5e) Light yellow powder; m.p. 252–253 °C; FT-
v
max: 3741; 3303; 1694; 1609; 1326; 1158 cm-1; 1H NMR:
(DMSO-d6, 400 MHz) d: 4.69 (s, 2H, N–CH2), 7.28 (t, 2H,
J = 7.6 Hz, Ar–H), 7.34 (d, 1H, J = 8.4 Hz, Ar–H), 7.55
(d, 2H, J = 7.6 Hz, Ar–H), 7.62 (s, 1H, CH), 7.65 (d, 1H,
J = 8.4 Hz, Ar–H), 7.74 (s, 1H, CH), 7.85 (d, 4H,
J = 10.4 Hz, Ar–H), 8.00–8.07 (dd, 2H, J1 = 7.2 Hz,
J2 = 24.0 Hz, Ar–H), 11.48 (s, 1H, NH), 14.26 (s, 1H,
NH); 13C NMR (DMSO-d6): 51.7 (CH2), 118.3 (Ar–C),
121.5 (Ar–C), 123.8 (Ar–C), 124.5 (Ar–C), 126.9 (Ar–C),
127.6 (Ar–C), 127.7 (Ar–C), 128.0 (Ar–C), 128.6 (Ar–C),
128.9 (Ar–C), 129.1 (Ar–C), 129.8 (Ar–C), 131.8 (Ar–C),
132.2 (Ar–C), 132.7 (Ar–C), 133.2 (Ar–C), 134.7 (Ar–C),
135.5 (Ar–C), 135.7 (Ar–C), 137.5 (Ar–C), 138.1 (Ar–C),
142.8 (N=CH), 168.9 (C=O); MS m/z:534.1, 536.06 (ES-),
558.04, 560.04 (?ES, M?Na?) .
1
IR (KBr) vmax: 3315; 1685; 1609; 1328; 1155 cm-1; H
NMR (DMSO-d6, 400 MHz) d: 4.68 (s, 2H, N–CH2), 7.16
(t, 2H, J = 8.4 Hz, Ar–H), 7.42 (s, 1H, CH), 7.52 (t, 2H,
J = 6.8 Hz, Ar–H), 7.58 (t, 2H, J = 6.8 Hz, Ar–H),
7.76–7.90 (m, 5H, Ar–H), 7.99–8.07 (dd, 2H, J1 = 7.2 Hz,
J2 = 24.0 Hz, Ar–H), 11.26 (s, 1H, NH), 14.25 (s, 1H,
NH); 13C NMR (DMSO-d6): 51.4 (CH2), 121.1 (Ar–C),
122.7 (Ar–C), 123.8 (Ar–C), 124.5 (Ar–C), 126.7 (Ar–C),
127.1 (Ar–C), 127.7 (Ar–C), 128.3 (Ar–C), 128.7 (Ar–C),
129.1 (Ar–C), 129.3 (Ar–C), 129.8 (Ar–C), 130.8 (Ar–C),
132.5 (Ar–C), 132.8 (Ar–C), 133.7 (Ar–C), 134.2 (Ar–C),
134.8 (Ar–C), 135.7 (Ar–C), 137.9 (Ar–C), 139.1 (Ar–C),
142.8 (N=CH), 168.3 (C=O); MS m/z: 492.1 (ES-), 514.09
(ES?, M?Na?).
N0-[(4-Bromophenyl)methylidene]-2-(5,5-dioxido-3-phe-
nylpyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)acetohyd-
razide (5c) Off white powder; m.p. 254–255 °C; FT-IR
(KBr) vmax: 3325; 1685; 1609; 1328; 1154 cm-1; 1H NMR:
(DMSO-d6, 400 MHz) d: 4.68 (s, 2H, N–CH2), 7.29 (t, 2H,
J = 7.2 Hz, Ar–H), 7.50–7.55 (m, 5H, Ar–H), 7.73 (s, 1H,
CH), 7.83–7.88 (m, 4H, Ar–H), 8.03–8.07 (dd, 2H,
J1 = 8.0 Hz, J2 = 24.8 Hz, Ar–H), 11.31 (s, 1H, NH),
13.90 (s, 1H, NH); 13C NMR (DMSO-d6): 51.4 (CH2),
119.8 (Ar–C), 122.7 (Ar–C), 123.9 (Ar–C), 124.5 (Ar–C),
126.7 (Ar–C), 127.4 (Ar–C), 127.7 (Ar–C), 128.1 (Ar–C),
128.5 (Ar–C), 128.7 (Ar–C), 129.1 (Ar–C), 129.9 (Ar–C),
130.8 (Ar–C), 132.7 (Ar–C), 133.1 (Ar–C), 133.7 (Ar–C),
134.5 (Ar–C), 135.4 (Ar–C), 135.9 (Ar–C), 137.7 (Ar–C),
138.2 (Ar–C), 142.9 (N=CH), 168.9 (C=O); MS m/z:
534.05, 536.05 (ES-), 558.03, 560.06 (ES?, M?Na?).
N0-[(2,4-Dichlorophenyl)methylidene]-2-(5,5-dioxido-3-
phenylpyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)acet-
ohydrazide (5f) Off white powder; m.p. 237–238 °C; FT-
IR (KBr) vmax: 3648; 3313; 1686; 1601; 1324; 1157 cm-1
;
1H NMR (DMSO-d6, 400 MHz) d: 4.69 (s, 2H, N–CH2),
7.36–7.61 (m, 5H, Ar–H), 7.65 (s, 1H, CH), 7.76–7.88 (m,
4H, Ar–H), 7.99 (d, 2H, J = 7.2 Hz, Ar–H), 8.05 (d, 1H,
J = 8.0 Hz, Ar–H), 11.48 (s, 1H, NH), 14.25 (s, 1H, NH);
13C NMR (DMSO-d6): 51.3 (CH2), 122.8 (Ar–C), 123.9
(Ar–C), 124.7 (Ar–C), 126.9 (Ar–C), 127.1 (Ar–C), 127.8
(Ar–C), 128.1 (Ar–C), 128.7 (Ar–C), 129.0 (Ar–C), 129.5
(Ar–C), 129.8 (Ar–C), 131.8 (Ar–C), 132.5 (Ar–C), 132.7
(Ar–C), 134.1 (Ar–C), 134.9 (Ar–C), 135.3 (Ar–C), 135.5
(2C, Ar), 137.7 (Ar–C), 139.8 (Ar–C), 140.8 (N=CH),
168.7 (C=O); MS m/z: 526.06 (ES-), 550.04 (ES?,
M?Na?).
N0-[(3-Chlorophenyl)methylidene]-2-(5,5-dioxido-3-phenyl
pyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)acetohydrazide
N0-[(2,6-Dichlorophenyl)methylidene]-2-(5,5-dioxido-3-
phenylpyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)acet-
ohydrazide (5g) Off white powder; m.p. 254–255 °C;
FT-IR (KBr) vmax: 3222; 1692; 1606; 1324; 1158 cm-1; 1H
NMR: (DMSO-d6, 400 MHz) d: 4.63 (s, 2H, N–CH2), 7.35
(t, 2H, J = 8.0 Hz, Ar–H), 7.47 (d, 4H, J = 8.0 Hz, Ar–
(5d) White powder; m.p. 218–219 °C; FT-IR (KBr) vmax
:
3305; 1693; 1609; 1326; 1158 cm-1; 1H NMR (DMSO-d6,
400 MHz) d: 4.74 (s, 2H, N–CH2), 7.33 (t, 1H, J = 5.2 Hz,
Ar–H), 7.37–7.44 (dd, 2H, J1 = 7.6 Hz, J2 = 18.4 Hz,
Ar–H), 7.47 (s, 1H, CH), 7.49 (d, 1H, J = 6.8 Hz, Ar–H),
123

Med Chem Res (2014) 23:1309–1319
1317
2-(5,5-Dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothia-
zin-4(2H)-yl)-N0-[(2-nitrophenyl)methylidene]acetohydraz-
ide (5j) Light yellow powder; m.p. 274 °C; FT-IR (KBr)
H), 7.61–7.70 (m, 3H, Ar–H), 7.79–7.86 (m, 3H, Ar–H),
8.07 (s, 1H, CH), 11.46 (s, 1H, N–H), 14.25 (s, 1H, N–H);
13C NMR (DMSO-d6): 51.3, 122.9 (Ar–C), 124.0 (Ar–C),
124.8 (Ar–C), 126.8 (Ar–C), 127.1 (Ar–C), 127.7 (Ar–C),
128.0 (2C, Ar), 129.1 (Ar–C), 129.5 (Ar–C), 129.8 (Ar–C),
131.7 (Ar–C), 132.4 (2C, Ar), 132.7 (Ar–C), 134.2 (Ar–C),
135.1 (Ar–C), 136.5 (2C, Ar), 137.5 (Ar–C), 139.8 (Ar–C),
142.7 (N=CH), 168.9 (C=O); MS m/z: 524.07 (ES-),
548.05 (ES?, M?Na?).
v
max: 3226; 1688; 1588; 1322; 1156 cm-1 1H NMR
;
(DMSO-d6, 400 MHz) d: 4.69 (s, 2H, N–CH2), 7.52–7.69
(m, 5H, Ar–H), 7.78–7.89 (m, 4H, Ar–H), 7.98 (d, 2H,
J = 8.0 Hz, Ar–H), 8.06 (d, 2H, J = 7.6 Hz, Ar–H), 8.16
(s, 1H, CH=N), 11.55 (s, 1H, NH), 14.27 (s, 1H, NH); 13C
NMR (DMSO-d6): 51.3 (CH2), 122.7 (Ar–C), 124.0
(Ar–C), 124.7 (Ar–C), 126.7 (Ar–C), 127.1 (Ar–C), 127.7
(Ar–C), 128.0 (Ar–C), 128.3 (Ar–C), 128.7 (Ar–C), 129.5
(Ar–C), 129.8 (Ar–C), 131.7 (Ar–C), 132.5 (Ar–C), 132.7
(Ar–C), 134.6 (Ar–C), 135.1 (Ar–C), 135.5 (Ar–C), 135.9
(Ar–C), 137.5 (Ar–C), 139.7 (Ar–C), 142.5 (N=CH), 147.7
(Ar–C), 170.1 (C=O); MS m/z: 502.14 (ES-), 525.11
(ES?, M?Na?).
N0-[4-(Dimethylamino)phenyl]methylidene-2-(5,5-dioxido-
3-phenylpyrazolo[4,3-c][1,2]benzothiazin-4(2H)-yl)acet-
ohydrazide (5h) Dirty yellow; m.p. 255–257 °C; FT-IR
(KBr) vmax: 3747; 3322; 1685; 1598; 1322; 1154 cm-1; 1H
NMR (DMSO-d6, 400 MHz) d: 2.91 (s, 3H, N–CH3), 2.94
(s, 3H, N–CH3), 4.64 (s, 2H, N–CH2), 6.62 (t, 2H,
J = 8.4 Hz, Ar–H), 7.16 (t, 1H, J = 5.6 Hz, Ar–H),
7.32–7.35 (dd, 1H, J1 = 4.4 Hz, J2 = 7.6 Hz, Ar–H),
7.51–7.56 (dd, 2H, J1 = 4.8 Hz, J2 = 15.2 Hz, Ar–H),
7.61 (s, 1H, CH), 7.64 (v 2H, J = 8.4 Hz, Ar–H),
7.79–7.90 (m, 4H, Ar–H), 8.02–8.05 (dd, 1H, J1 = 8.0 Hz,
J2 = 25.2 Hz, Ar–H), 11.01 (s, 1H, NH), 14.23 (s, 1H,
NH); 13C NMR (DMSO-d6): 40.7 (2C, N(CH3)2), 51.4
(CH2), 115.1 (2C, Ar), 122.7 (Ar–C), 124.1 (Ar–C), 124.7
(Ar–C), 126.8 (Ar–C), 127.3 (Ar–C), 127.5 (Ar–C), 128.1
(Ar–C), 128.5 (Ar–C), 129.1 (Ar–C), 129.5 (Ar–C), 129.8
(Ar–C), 131.7 (Ar–C), 132.4 (Ar–C), 132.7 (Ar–C), 134.7
(Ar–C), 135.1 (Ar–C), 137.5 (Ar–C), 139.8 (Ar–C), 142.7
(N=CH), 151.9 (Ar–C), 168.7 (C=O); MS m/z: 500.2 (ES-
), 523.17 (ES?, M?Na?).
2-(5,5-Dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothiazin-
4(2H)-yl)-N0-[(3-nitrophenyl)methylidene]acetohydrazide
(5k) Dirty yellow powder; m.p. 248–249 °C; FT-IR
(KBr) vmax: 3319; 1689; 1599; 1328; 1158 cm-1; 1H NMR
(DMSO-d6, 400 MHz) d: 4.74 (s, 2H, N–CH2), 7.42 (d, 2H,
J = 7.6 Hz, A–r–H), 7.64 (t, 2H, J = 7.2 Hz, Ar–H),
7.81–7.89 (m, 5H, Ar–H), 8.04 (s, 1H), 8.11 (t, 1H,
J = 10.0 Hz, Ar–H), 8.20 (t, 2H, J = 8.4 Hz, Ar–H), 8.37
(d, 1H, J = 10.0 Hz, Ar–H), 11.75 (s, 1H, NH), 14.03
(s, 1H, NH); 13C NMR (DMSO-d6): 51.3 (CH2), 122.5
(Ar–C), 124.1 (Ar–C), 124.7 (Ar–C), 126.8 (Ar–C), 127.0
(Ar–C), 127.7 (Ar–C), 128.1 (Ar–C), 128.4 (Ar–C), 128.7
(Ar–C), 129.4 (Ar–C), 129.8 (Ar–C), 131.9 (Ar–C), 132.5
(Ar–C), 132.8 (Ar–C), 134.5 (Ar–C), 135.0 (Ar–C), 135.4
(Ar–C), 135.8 (Ar–C), 137.5 (Ar–C), 139.8 (Ar–C), 142.7
(N=CH), 148.9 (Ar–C), 169.9 (C=O); MS m/z: 502.14
(ES-), 525.11 (ES?, M?Na?).
N0-[4-(Diethylamino)phenyl]methylidene-2-(5,5-dioxido-3-
phenylpyrazolo[4,3 c][1,2]benzothiazin-4(2H)-yl)acet-
ohydrazide (5i) Dirty yellow; m.p. 255–257 °C; FT-IR
(KBr) vmax: 3747; 3322; 1685; 1598; 1322; 1154 cm-1; 1H
NMR (DMSO-d6, 400 MHz) d: 1.10 (s, 3H, CH3), 1.13 (s,
3H, CH3), 2.87 (s, 2H, N–CH2), 2.90 (s, 2H, N–CH2), 4.60
(s, 2H, N–CH2), 6.60 (t, 2H, J = 8.4 Hz, Ar–H), 7.15 (t,
1H, J = 5.6 Hz, Ar–H), 7.31–7.35 (dd, 1H, J1 = 4.4 Hz,
J2 = 7.6 Hz, Ar–H), 7.50–7.54 (dd, 1H, J1 = 4.8 Hz,
J2 = 15.2 Hz, Ar–H), 7.59 (s, 1H, CH), 7.62 (t, 2H,
J = 8.4 Hz, Ar–H), 7.81–7.90 (m, 4H, Ar–H), 8.01–8.03
(dd, 2H, J1 = 8.0 Hz, J2 = 25.2 Hz, Ar–H), 11.02 (s, 1H,
NH), 14.20 (s, 1H, NH); 13C NMR (DMSO-d6): 12.9 (2C,
CH3(ethyl)), 44.5 (2C, CH2(ethyl)), 51.3 (N–CH2), 115.1
(2C, Ar), 122.7 (Ar–C), 124.0 (Ar–C), 124.8 (Ar–C), 126.8
(Ar–C), 127.1 (Ar–C), 127.7 (Ar–C), 128.0 (Ar–C), 128.5
(Ar–C), 129.1 (Ar–C), 129.4 (Ar–C), 129.7 (Ar–C), 131.7
(Ar–C), 132.5 (Ar–C), 132.7 (Ar–C), 134.5 (Ar–C), 135.0
(Ar–C), 137.5 (Ar–C), 139.7 (Ar–C), 142.5 (N=CH), 151.4
(Ar–C), 168.7 (C=O); MS m/z:528.2 (ES-), 551.17 (ES?,
M?Na?).
2-(5,5-Dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothia-
zin-4(2H)-yl)-N0-[(4-nitrophenyl)methylidene]acetohyd-
razide (5l) Light yellow powder; m.p. 238–240 °C; FT-
IR (KBr) vmax: 3748; 3321; 1688; 1587; 1327; 1155 cm-1
;
1H NMR (DMSO-d6, 400 MHz) d: 4.74 (s, 2H, N–CH2),
7.42 (d, 2H, J = 7.2 Hz, Ar–H), 7.51–7.66 (m, 5H, Ar–H),
7.80–7.87 (dd, 4H, J1 = 8.4 Hz, J2 = 18.8 Hz, Ar–H),
8.01 (s, 1H, CH), 8.16 (t, 2H, J = 9.4 Hz, Ar–H), 11.58 (s,
1H, NH), 14.27 (s, 1H, N-H); 13C NMR (DMSO-d6): 51.3
(CH2), 122.7 (Ar–C), 124.0 (Ar–C), 124.7 (Ar–C), 126.7
(Ar–C), 127.1 (Ar–C), 127.7 (Ar–C), 128.1 (Ar–C), 128.5
(Ar–C), 128.7 (Ar–C), 129.5 (Ar–C), 129.7 (Ar–C), 132.0
(Ar–C), 132.4 (Ar–C), 132.7 (Ar–C), 134.7 (Ar–C), 135.0
(Ar–C), 135.7 (Ar–C), 135.9 (Ar–C), 137.5 (Ar–C), 139.8
(Ar–C), 142.5 (N=CH), 150.4 (Ar–C), 170.1 (C=O); MS
m/z: 502.16 (ES-), 541.16 (ES?, M ? K?).
123

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Med Chem Res (2014) 23:1309–1319
2-(5,5-Dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothiazin-
4(2H)-yl)-N0-[(4-methoxyphenyl)methylidene]acetohydraz-
ide (5m) Off white powder; m.p. 255–257 °C; IR (KBr)
(Ar–C), 160.7 (Ar–C), 161.1 (Ar–C), 168.5 (C=O); MS
m/z: 547.58 (M?).
v
max: 3717; 3229; 1674; 1587; 1324; 1157 cm-1; 1H NMR
X-ray crystallographic studies
(DMSO-d6) d: 3.75 (s, 3H, O–CH3), 4.68 (s, 2H, N–CH2),
6.93 (d, 2H, J = 5.6 Hz, Ar–H), 7.24 (t, 1H, J = 7.6 Hz,
Ar–H), 7.47–7.69 (m, 3H, Ar–H), 7.74 (s, 1H, CH),
7.78–7.89 (m, 5H, Ar–H), 8.00–8.08 (dd, 2H, J1 = 7.2 Hz,
J2 = 24.0 Hz, Ar–H), 11.34 (s, 1H, NH), 14.25 (s, 1H,
NH); 13C NMR (DMSO-d6): 48.5 (O–CH3), 51.6 (CH2),
111.1 (Ar–C), 116.5 (Ar–C), 122.5 (Ar–C), 124.3 (Ar–C),
126.5 (Ar–C), 127.1 (Ar–C), 127.7 (Ar–C), 128.1 (Ar–C),
128.7 (Ar–C), 129.1 (Ar–C), 129.4 (Ar–C), 129.7 (Ar–C),
130.7 (Ar–C), 132.5 (Ar–C), 132.7 (Ar–C), 134.7 (Ar–C),
135.0 (Ar–C), 135.3 (Ar–C), 137.7 (Ar–C), 139.5 (Ar–C),
142.7 (N=CH), 161.4 (Ar–C), 168.7 (C=O); MS m/z:
487.17 (ES-, M?), 488.16 (ES?, M ? H?).
A pale yellow needle-shaped crystals of 5k was used for
data collection. The crystals were coated with Paratone 8277
oil (Exxon) and mounted on a glass fiber. All measurements
were made on a Nonius Kappa CCD diffractometer with
graphite-monochromated Mo-Ka radiation. Details of crys-
tal data and structure refinement have been provided in
Table 2. 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 multi-scan
method (Hooft et al., 1998; Otwinowski and Minor, 1997).
The structure was solved by the direct methods (Altomare
et al., 1993) and expanded using Fourier techniques
(Beurskens et al., 1994). The non-hydrogen atoms were
refined anisotropically. Nitro group in 5k was disordered.
Hydrogen atoms were included at geometrically idealized
positions and were not refined. 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 and 0.0990 (all data) for 5k,
and goodness of fit, S = 1.079. The weighting schemes were
based on counting statistics and the final difference Fourier
maps were essentially featureless. The figures were plotted
with the aid of ORTEP-3 (Farrugia, 1997).
2-(5,5-Dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothiazin-
4(2H)-yl)-N0-[(3,4,5-trimethoxyphenyl)methylidene]acet-
ohydrazide (5n) Off white powder; m.p. 279–281 °C;
FT-IR (KBr) vmax: 3610; 3230; 1692; 1604; 1323;
1
1121 cm-1; H NMR: (DMSO-d6, 400 MHz) d: 3.69 (s,
3H, O–CH3), 3.79 (s, 3H, O–CH3), 3.83 (s, 3H, O–CH3),
4.66 (s, 2H, N–CH2), 6.76 (t, 2H, J = 8.8 Hz, Ar–H), 7.03
(t, 1H, J = 8.8 Hz, Ar–H), 7.34 (d, 1H, J = 8.0 Hz, Ar–
H), 7.54–7.67 (m, 4H, Ar–H), 7.77–7.88 (m, 3H, Ar–H),
7.94 (s, 1H, CH), 11.09 (s, 1H, NH), 14.24 (s, 1H, NH); 13
C
NMR (DMSO-d6): 49.8 (2C, OCH3), 51.5 (CH2), 54.1
(OCH3), 113.5 (Ar–C), 115.7 (Ar–C), 122.7 (Ar–C), 124.1
(Ar–C),126.4 (Ar–C), 127.1 (Ar–C), 127.7 (Ar–C), 128.0
(Ar–C), 128.5 (Ar–C), 129.1 (Ar–C), 129.5 (Ar–C), 129.8
(Ar–C), 131.3 (Ar–C), 132.4 (Ar–C), 132.7 (Ar–C), 135.0
(Ar–C), 135.5 (Ar–C), 135.9 (Ar–C), 137.5 (Ar–C), 142.5
(N=CH), 149.7 (2C, Ar), 168.3 (C=O); MS m/z: 547.2
(M?).
Anti-HIV-1 assay
The synthesized compounds were screened for their
in vitro antiviral effects in human peripheral blood mono-
nuclear (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 discon-
tinuous gradient centrifugation 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 3.
2-(5,5-Dioxido-3-phenylpyrazolo[4,3-c][1,2]benzothiazin-
4(2H)-yl)-N0-[(2,3,4-trimethoxyphenyl)methylidene]acet-
ohydrazide (5o) Off white powder; m.p. 275–277 °C;
FT-IR (KBr) vmax: 3619; 3250; 1682; 1588; 1324;
1
1156 cm-1; H NMR (DMSO-d6, 400 MHz) d: 3.67 (s,
3H, O–CH3), 3.74 (s, 3H, O–CH3), 3.77 (s, 3H, O–CH3),
4.67 (s, 2H, N–CH2), 6.80 (d, 2H, J = 18.4 Hz, Ar–H),
7.46–7.67 (m, 5H, Ar–H), 7.70 (s, 1H, CH), 7.82 (t, 2H,
J = 7.2 Hz, Ar–H), 8.01–8.07 (dd, 2H, J1 = 6.8 Hz,
J2 = 22.0 Hz, Ar–H), 11.25 (s, 1H, N–H), 14.25 (s, 1H, N–
H); 13C NMR (DMSO-d6): 51.6 (CH2), 51.8 (OCH3), 52.3
(OCH3), 55.1 (OCH3), 105.5 (2C, Ar), 111.9 (Ar–C), 122.7
(Ar–C), 124.0 (Ar–C), 125.9 (Ar–C), 127.1 (Ar–C), 127.8
(Ar–C), 128.1 (Ar–C), 128.7 (Ar–C), 129.0 (Ar–C), 129.4
(Ar–C), 129.8 (Ar–C), 132.5 (Ar–C), 132.7 (Ar–C), 135.1
(Ar–C), 135.5 (Ar–C), 137.7 (Ar–C), 142.5 (N=CH), 149.9
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
123

Med Chem Res (2014) 23:1309–1319
1319
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37 °C with 5 % CO2 for human PBM cells (Stuyver et al.,
2002). The results are summarized in Table 3.
Acknowledgments The authors (MA, SA, and MHB) 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 compounds has been deposited at
Cambridge Crystallographic Data Centre with CCDC numbers 881054.
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