Novel structural hybrids of pyrazolobenzothiazines with benzimidazoles as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.03.035

Two series of novel pyrazolobenzothiazine-based hybrid compounds were efficiently synthesized starting from saccharin sodium salt. Pyrazolo[4,3-c][1,2]benzothiazine scaffolds were N-arylated by using p-fluorobenzaldehyde, followed by the incorporation of

Full text of DOI:10.1016/j.ejmech.2014.03.035

European Journal of Medicinal Chemistry 78 (2014) 106e117
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel structural hybrids of pyrazolobenzothiazines with
benzimidazoles as cholinesterase inhibitors
,
Sana Aslam a b, Sumera Zaib c, Matloob Ahmad d, John M. Gardiner b, Aqeel Ahmad b,
Abdul Hameed e, Norbert Furtmann f, Michael Gütschow f, Jürgen Bajorath g,
,
Jamshed Iqbal c
*
a Department of Chemistry, Government College Women University, Faisalabad 38000, Pakistan
b School of Chemistry and Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, UK
c Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
d Department of Chemistry, Government College University, Faisalabad 38000, Pakistan
e HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan
f Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
g Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität,
Dahlmannstr. 2, D-53113 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel pyrazolobenzothiazine-based hybrid compounds were efficiently synthesized
starting from saccharin sodium salt. Pyrazolo[4,3-c][1,2]benzothiazine scaffolds were N-arylated by using
p-fluorobenzaldehyde, followed by the incorporation of a benzimidazole or similar ring systems by
treatment with arylenediamines. These phenylene-connected hybrid compounds were investigated as
potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Compounds 12d
and 12k were the most potent AChE inhibitors with IC50 values of 11 and 13 nM, respectively, while 6j
(IC50 ¼ 17 nM) proved to be the most active inhibitor against BuChE with remarkable selectivity for
BuChE over AChE. Molecular docking studies were also performed on human AChE and BuChE to suggest
possible binding modes in which the inhibitor’s extended structure is accommodated along the active
site gorge of both enzymes.
Received 1 January 2014
Received in revised form
9 March 2014
Accepted 12 March 2014
Available online 13 March 2014
Keywords:
Pyrazolobenzothiazine
Benzimidazole
Hybrid compounds
Cholinesterases
Docking studies
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
and BuChE have numerous physiological functions depending on
their localization and time of expression [4]. Therefore, both en-
Cholinergic neurons of the basal forebrain play an important
role in cognitive and behavioural functions, with extensive cortical
projections that modulate other neurotransmitters [1]. These
cholinergic systems are known to be widely compromised in Alz-
heimer’s disease (AD). In particular, AD is characterized by a fore-
brain cholinergic neuronal loss, a progressive cognitive decline and
neuropsychiatric disturbances in acetylcholine (ACh) [2]. The ac-
tivity of ACh in the brain is terminated by the hydrolytic action of
cholinesterases (ChEs). Inhibitors of these enzymes have hence
been developed to augment the activity of surviving cholinergic
neurons in patients with AD. All ChE inhibitors currently licensed
for AD inhibit acetylcholinesterase (AChE) and, to a varying extent,
butyrylcholinesterase (BuChE), a second ChE in the brain [3]. AChE
zymes are likely to be involved in regulating ACh levels and
represent legitimate therapeutic targets to ameliorate the cholin-
ergic deficit. BuChE may also have a role in the aetiology and pro-
gression of AD beyond regulation of synaptic ACh levels [5]. The
deficit of presynaptic markers is an indication of a neuronal
degeneration which is the real cause of the profound depletion of
ACh within the hippocampus, basal forebrain and cortical areas [6].
Since changes in cholinergic regulation are a contributing factor to
the cognitive dysfunction observed in AD patients, the selective
AChE inhibitor, donepezil, is widely utilized for symptomatic
treatment of patients [7].
AChE is principally associated with neurons and axons, while
BuChE is primarily expressed and secreted by glial cells within the
brain [8]. Studies indicate that specific neurons use BuChE rather
than AChE to cleave presynaptic ACh. Indeed, 10e15% of cholinergic
neurons in the human hippocampus and amygdala express BuChE
in their cell bodies and proximal dendrites, in lieu of AChE [9,10]. In
* Corresponding author.
E-mail addresses: drjamshed@ciit.net.pk, jamshediqb@gmail.com, jamshed.
iqbal@uni-bonn.de (J. Iqbal).
http://dx.doi.org/10.1016/j.ejmech.2014.03.035
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
107
the healthy human brain, AChE and BuChE are found in the ratio of
4:1. However, in the brains of AD patients, AChE activity can decline
by up to 45% during disease progression, reflecting the disappear-
ance of neurons and axons to which it is associated, while BuChE
activity can be elevated by up to 2-fold [10], thereby altering this
ratio considerably.
HIV [26,27], herpes (HSV-1 and HCMV) [28] and hepatitis C [29].
Various benzimidazoles also have a potential as antitumour [30,31]
or antiparasitic agents [32] and as angiotensin II receptor antago-
nists [33]. Derivatives containing this pharmacophore are also used
as ligands to transition metals, designed for biological system
modelling [34].
The benzothiazine ring systems have emerged as suitable het-
erocyclic templates for biologically active compounds. For example,
4H-3,1-benzothiazin-4-ones have been characterised as serine pro-
tease inhibitors [11] and as potent, dual-acting compounds targeting
adenosine A2A receptors and monoamine oxidase B [12]. Among the
isomeric 4H-1,3-benzothiazin-4-ones, extremely effective anti-
mycobacterial nitro derivatives have been explored [13]. 1,4-
Benzothiazine derivatives are known to possess anticancer [14] and
antibacterial [15] activities and act as activators of ATP-sensitive po-
tassium channels [16]. Antimicrobial and anti-inflammatory proper-
ties have been reported for 1,2-benzothiazine 1,1-dioxides [17e19].
Among the tricyclic benzothiazine derivatives, pyrazolo[4,3-c][1,2]
benzothiazine 5,5-dioxides possess anti-inflammatory [20] and anti-
viral [21] properties. Biological activity was demonstrated for similar
fused benzothiazine templates, e.g. for pyrazolo[4,3-c][2,1]benzo-
thiazine 4,4-dioxides acting as selective focal adhesion kinase in-
hibitors [22] and pyrazolo[4,3-b][1,4]benzothiazine 4,4-dioxides with
antimalarial properties [23]. Thus, these molecules exhibit a wide
range of biological activities and are considered to be privileged
structures for medicinal chemistry. With respect to the versatile
bioactivity profiles of pyrazolobenzothiazine pharmacophores, some
of us explored 2,4-dihydro-3,4-dimethylpyrazolo[4,3-c][1,2]benzo-
thiazine 5,5-dioxide derivatives as antioxidants and antimicrobial
agents [24,25].
On the basis of the biological activity profiles of the two phar-
macophores i.e. pyrazolobenzothiazine and benzimidazole, we
have hybridized these two ring systems into one unit. Our current
study describes the discovery and structureeactivity relationships
of novel pyrazolobenzothiazine derivatives as inhibitors of cholin-
esterases. The active site of cholinesterases is located at the bottom
of a deep and narrow gorge. Thus, the extended structure of the
inhibitors designed in this study might be accommodated along the
active site gorge, as it has been explored for several heterodimeric,
linker connected inhibitors of cholinesterases [35e37].
2. Chemistry
We designed the synthetic route to two novel series of hybrids
of benzimidazole-substituted pyrazolobenzothiazines as depicted
in Scheme 1. The initial N-alkylation of the commercially available
saccharin sodium salt with appropriate alkyl halides in DMF [25,38]
was carried out under optimized conditions, i.e. via a thermal
method, microwave irradiation and in an ultrasonic bath. Excellent
yields of the product were obtained under microwave irradiation
after a short reaction time. Then, the 1,2-benzothiazine nuclei (2
and 8) were prepared in an inert nitrogen atmosphere [39] via
GabrieleColman-type ring expansion of the five-membered ben-
zoisothiazole rings of compound 1 and 7 to the six-membered
thiazine ring, including the ring cleavage and ring closure steps.
The subsequent N-methylation of compounds 2 and 8 with
dimethyl sulphate in acetone was performed under ultrasonic
The benzimidazole ring system is another molecular template to
generate biologically active compounds. Benzimidazole derivatives
showed significant activities against a number of viruses including
OH
O
O
O
O
O
Cl
R
R
R
NaOCH3
N
NNa
NH
S
S
O
DMF, 110 °C
CH3OH, reflux
S
O
O
O
O
O
1 R = CH3
7 R = C6H5
2 & 8
H
O
N
NH
OH
S
O
F
N2H4 . H2O
C2H5OH, reflux
R
(CH3)2SO4
acetone
R
K2CO3, DMF
N
N
h exad ecyltriphen yl-
S
CH3
CH3
phosphonium bromide
O
O
O
O
4 & 10
3 & 9
R'
O
N
NH
H
H2N
R'
N
N
N
N
H2N
R
R
Na2S2O5, DMF
N
N
S
CH3
S
CH3
6a-h
R = CH3
O
O
O
O
5 & 11
12a-h R = C6H5
Scheme 1. Synthesis of pyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxides (6aeh and 12aeh).

108
S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
conditions for 10e15 min. 1H NMR data revealed the formation of
the alkylated products by the appearance of the NeCH3 peak at 3.11
and 2.89 ppm for compounds 3 and 9, respectively. Reactions of
compounds 3 and 9 with hydrazine monohydrate in refluxing
ethanol resulted in cyclization and gave the respective pyrazolo
[4,3-c][1,2]benzothiazine 5,5-dioxide scaffold (4 and 10). The IR
The final step was the condensation reaction of compounds 5
and 11 with a variety of substituted o-phenylenediamines in DMF in
the presence of sodium metabisulfite, which resulted in the tar-
geted compounds (Scheme 1), i.e. 2-[4-(substituted-1H-benzimi-
dazol-2-yl)phenyl]-3,4-dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]
benzothiazine 5,5-dioxides (6aeh) and 2-[4-(substituted-1H-ben-
zimidazol-2-yl)phenyl]-4-methyl-3-phenyl-2,4-dihydropyrazolo
[4,3-c][1,2]benzothiazine 5,5-dioxides (12aeh). The formation of
the benzimidazole structure was accomplished by the condensa-
tion reaction between the aldehyde and the diamine in the pres-
ence of sodium metabisulfite as an oxidizing agent. The
spectroscopic data of the final compounds were in agreement with
the proposed structures.
The last reaction step was similarly used for the preparation of
structurally modified compounds (Scheme 2). In 6i and 12i or 6j
and 12j, the benzimidazole core was exchanged for purine or
benzoxazole, when 5 or 11 were reacted with pyrimidine-4,5-
diamine or 2-aminophenol, respectively, in place of o-phenyl-
enediamine. Moreover, starting from benzimidazole-5-carboxylic
acid derivatives 6a and 12a, the corresponding ethyl esters (6k
and 12k) were prepared (Scheme 2).
stretching frequency for NH was observed at 3355 and 3368 cmꢀ1
,
and the C]N functionality was detected at 1594 and 1655 cmꢀ1. In
the 1H NMR spectra, the NH signal appeared as a broad singlet at
10.11 and 13.78 ppm for compounds 4 and 10, respectively.
The following key intermediates were prepared by N-arylation
of compounds 4 and 10 with 4-fluorobenzaldehyde in the presence
of anhydrous K2CO3 and hexadecyltriphenylphosphonium bromide
as phase transfer catalyst yielding 4-(3,4-dimethyl-5,5-
dioxidobenzo[e]pyrazolo[4,3-c][1,2]thiazin-2(4H)-yl)benzaldehyde
(5) and 4-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c]
[1,2]thiazin-2(4H)-yl)benzaldehyde (11), respectively. Absorption
peaks at 1727 and 1737 cmꢀ1 in the IR spectra and singlets at 10.03
and 10.23 ppm in the 1H NMR spectra appeared for the aldehyde
moiety of 5 and 11, respectively. The 13C NMR chemical shifts for the
aldehyde carbons were detected at 190.9 and 191.0 ppm.
Scheme 2. Synthesis of purine- and benzoxazole-based derivatives (6i and 12i; 6j and 12j) and of functionalized benzimidazole derivatives (6k and 12k).

S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
109
3. In vitro AChE and BuChE inhibitory activity
excellent AChE inhibition (IC50 54 nM). Thus, we obtained four new
inhibitors of AChE (6f, 12d, 12j, 12k) with an activity similar to that
of the reference compound donepezil.
Even more striking was the unexpected finding of the strong
activity (and selectivity) of some of our compounds against BuChE
(and over AChE). This was observed for the 1,3-benzoxazol-2-yl
compound 6j bearing a methyl group at position R. Inhibitor 6j
showed IC50 values of less than 20 nM and was thus much more
potent at BuChE than donepezil. This inhibitor exhibited an at least
400-fold stronger inhibition of BuChE than of AChE.
In vitro inhibitory studies of the newly synthesized pyrazoloe
benzothiazines derivatives were carried out on AChE from Elec-
trophorus electricus (EeAChE) and BuChE from equine serum. Inhi-
bition potencies of compounds expressed as IC50 values are shown
in Table 1. These values suggest that most of these synthesized
compounds are potent inhibitors of cholinesterases.
Compounds 12aek with phenyl substitution in R position
mainly showed a lower potency against AChE and BuChE than the
methyl substituted analogues 6aek (see, for example IC50 values of
6f versus those of 12f). This was, however, not the case for the
representatives with an unsubstituted benzimidazole unit. The
phenyl derivative 12d was much more active on AChE than the
methyl derivative 6d, and both compounds exhibited the same IC50
for BuChE inhibition. Moreover, among the two compounds with
the rather large and polar substituent at the benzimidazole core, i.e.
ethoxycarbonyl, 12k was more potent towards AChE than the
analogous methyl derivative 6k. Inhibitor 12k exhibited moderate
selectivity for AChE over BuChE (13 nM versus 180 nM). Also
compound 12j having a phenyl residue at position R and a 1,3-
benzoxazol-2-yl group attached to the phenylene linker showed
4. Docking studies
The X-ray structures of human AChE (PDB ID 4BDT) and BuChE
(PDB ID 4BDS) were selected for the docking study because
available electric eel structures for AChE show low crystallographic
ꢀ
resolutions (>4 A) and structures of equine BuChE are not avail-
able at the moment. The active sites of AChE and BuChE show a
high degree of similarity despite the replacement of residues
Pro446, Tyr124 and Phe297 in the active site of AChE by residues
Met437, Gln119 and Val288 in BuChE. In addition, residue Tyr337
in AChE is replaced by the smaller Ala328 in BuChE. Depending on
Table 1
Enzyme inhibition results for the 2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxides 6aek and 12aek.
Compound
R
R0
IC50 ꢁ SEM (
mM) or
(% inhibition at 500
mM)
AChE
BuChE
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
12a
12b
12c
12d
12e
12f
12g
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
5-carboxy
5-chloro
5-bromo
H
5-methyl
5,6-dimethyl
5-nitro
1.13 ꢁ 0.1
1.02 ꢁ 0.3
0.026 ꢁ 0.001
(38.7)
0.541 ꢁ 0.01
2.91 ꢁ 0.5
0.049 ꢁ 0.003
0.043 ꢁ 0.006
0.041 ꢁ 0.008
0.273 ꢁ 0.031
(38.5)
7.53 ꢁ 0.9
0.041 ꢁ 0.008
0.492 ꢁ 0.04
1.14 ꢁ 0.1
5-methoxy
e
6.92 ꢁ 0.42
1.95 ꢁ 0.1
(35.5)
e
6.92 ꢁ 0.7
0.017 ꢁ 0.002
(29.5)
5-ethoxycarbonyl
5-carboxy
5-chloro
5-bromo
H
5-methyl
5,6-dimethyl
5-nitro
0.993 ꢁ 0.09
6.11 ꢁ 0.1
4.62 ꢁ 0.61
9.94 ꢁ 0.62
3.23 ꢁ 0.26
0.043 ꢁ 0.033
(31.8)
(34.1)
(30.7)
(37.3)
3.02 ꢁ 0.1
6.9 ꢁ 0.1
0.011 ꢁ 0.004
0.122 ꢁ 0.05
3.82 ꢁ 0.11
7.94 ꢁ 0.11
7.13 ꢁ 0.12
5.22 ꢁ 0.11
0.054 ꢁ 0.009
0.013 ꢁ 0.004
22.2 ꢁ 3.2
12h
12i
12j
12k
Neostigmine
Donepezil
5-methoxy
e
1.33 ꢁ 0.12
e
(36.5)
5-ethoxycarbonyl
0.18 ꢁ 0.01
49.6 ꢁ 6.11
6.41 ꢁ 0.34
0.032 ꢁ 0.003

110
S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
the co-crystallized compounds, different X-ray structures revealed
multiple side chain orientations of Tyr337. Therefore, the side
chain of Tyr337 in AChE was treated flexibly in our docking study,
while the corresponding residue Ala328 in BuChE has no side
chain flexibility.
Based on the results of the biological evaluation, compound 12d
was selected in order to investigate the possible interactions be-
tween the inhibitor and the active sites of both enzymes. The upper
part of Fig. 1 shows an overlay of the putative binding mode of the
best dual AChE/BuChE inhibitor 12d and the crystallographic ligand
Fig. 1. Putative binding modes. Overlays of the docking poses of compound 12d in the active sites of AChE (top, cyan) and BuChE (bottom, orange) with the crystallographic ligands
huprine W (top, brown colour) and tacrine (bottom, purple colour) are shown. The active site is displayed in line representation and important residues which differ in AChE and
BuChE are shown in stick representation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
111
huprine W in the active site of AChE. The pyrazolobenzothiazine
ring system of 12d adopts a comparable position to huprine W and
might interact with side chain atoms of several aromatic residues
including Tyr337 and Trp86. The phenyl substituent at the pyrazol
ring is directed into a hydrophobic subpocket formed by the resi-
dues Phe297, Ala204 and Phe123. The phenyl ring between the
pyrazolobenzothiazine and the benzimidazole substructures is in
Ltd). Acetylcholinesterase (AChE, EC 3.1.1.7, type VI-S from electric
eel), butylcholinesterase (BuChE, 3.1.1.8, from equine serum), ace-
tylthiocholine iodide (ATCI), S-butyrylthiocholine chloride (BTCCl),
5,50-dithio-bis(2-nitrobenzoic acid) (DTNB), neostigmine methyl-
sulphate and dimethylsulfoxide (DMSO) and donepezil hydro-
chloride were purchased from SigmaeAldrich (St. Louis, MO, USA).
p
e
p
stacking interaction distance to residue Tyr341. The benz-
imidazole moiety is placed in the mid-gorge area of the binding site
and might also form interactions with the residue Trp286.
6.1.1. General procedure for the synthesis of 2-(2-oxopropyl)-1,2-
benzisothiazol-3(2H)-one 1,1-dioxide (N-acetonylsaccharin) and 2-
(2-oxo-2-phenylethyl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide
(N-phenacylsaccharin) (1 & 7)
Saccharin sodium salt (0.222 mol) was mixed with chlor-
oacetone (0.25 mol) or phenacyl bromide (0.222 mol) for com-
pounds 1 and 7 respectively in dried DMF with continuous stirring.
The reaction mixture was heated for 3 h at 110 ꢂC under inert at-
mosphere, cooled to ambient temperature and poured on crushed
ice. The precipitate was filtered, washed with cold water and
recrystallized from ethanol. The reactions were performed under
30W microwave irradiation for 25 min and in ultrasonic bath for
40 min.
pep
The lower part of Fig. 1 shows an overlay of the possible binding
mode of compound 12d in the active site of BuChE and the co-
crystallized ligand tacrine. The predicted binding mode of 12d in
the active site of BuChE is very similar to the putative binding
conformation of this compound in complex with AChE. In case of
ꢀ
BuChE, the position of 12d is slightly moved upwards (w1.5 A) to
the entrance of the pocket. The exchange of residue Tyr337 (AChE)
to the smaller Ala328 (BuChE) allows a repositioning of 12d, which
enables a possible hydrogen bonding interaction of the benzimid-
azole substructure to the backbone carbonyl moiety of residue
Tyr332.
Plausible binding conformations could be generated for the best
dual inhibitor 12d of AChE and BuChE, which are consistent with
the presence of comparable potency of this compound against both
enzymes. The orientation of the pyrazolobenzothiazine ring system
correlates with the position of huprine W (AChE) and tacrine
(BuChE) in both predicted binding conformations.
6.1.1.1. 2-(2-Oxopropyl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide
(N-acetonylsaccharin, 1). Off white solid; yield: 86%; m.p.: 112e
114 ꢂC; FT-IR (KBr): 1717, 1584, 1355, 1185 cmꢀ1 1H NMR (CDCl3,
;
400 MHz) d: 2.24 (3H, s, CeCH3), 4.77 (2H, s, NeCH2) 7.57 (1H, d,
J ¼ 7.8 Hz, AreH), 7.66 (1H, t, J ¼ 7.4 Hz, AreH), 7.96 (2H, d,
J ¼ 7.6 Hz, AreH); MS m/z: 239.0 [Mþ].
5. Conclusion
6.1.1.2. 2-(2-Oxo-2-phenylethyl)-1,2-benzisothiazol-3(2H)-one 1,1-
dioxide (N-phenacylsaccharin, 7). Off white solid; yield: 83%; FT-IR
In this research article, we report on synthetic routes for the
hybridization of pyrazolobenzothiazine scaffolds with benzimid-
azole ring systems. Structural diversity of the final compounds was
ensured by using a range of substituted phenylenediamines and
other bifunctional aromatic amines to also introduce purine and
benzoxazole moieties. In light of the suggested role of BuChE in
central cholinergic transmission, its altered expression in the AD
brain, and its probable association with the development of
neuropathologic changes, it was hypothesized that high BuChE
activity would be detrimental in AD and that inhibiting this enzyme
would be of clinical value along with AChE. Some of our inhibitors
exhibit strong inhibition of BuChE and selectivity over AChE.
We identified compound 12d (2-[4-(1H-benzimidazol-2-yl)
phenyl]-4-methyl-3-phenyl-2,4-dihydropyrazolo[4,3-c][1,2]benzo-
thiazine 5,5-dioxide) as a highly potent, dual-acting inhibitor of
both cholinesterases. Computational analysis revealed plausible
putative binding modes of the extended structure of 12d along the
active site gorge of AChE and BuChE. Inhibitor 12d can be consid-
ered a promising lead for further investigations on this chemotype
to develop disease-modifying drugs for the treatment of AD.
(KBr): 1715, 1345, 1165 cmꢀ1 1H NMR (DMSO-d6) (400 MHz)
; d:
5.15 (2H, s, NeCH2), 7.47 (2H, t, J ¼ 7.4 Hz, AreH), 7.59 (1H, t,
J ¼ 7.4 Hz, AreH), 7.81e7.90 (2H, m, AreH), 7.92 (1H, d, J ¼ 6.6 Hz,
AreH), 7.94 (2H, d, J ¼ 6.8 Hz, AreH), 8.07 (1H, d, J ¼ 7.8 Hz, AreH);
13C NMR (DMSO-d6)
d: 51.3 (CH2), 123.2 (AreC), 124.0 (AreC), 126.3
(AreC), 129.2 (2C, Ar), 129.4 (2C, Ar), 133.5 (AreC), 134.8 (AreC),
135.3 (AreC), 137.0 (AreC), 141.1 (AreC), 169.5 (C]O), 191.3 (Are
CO); MS m/z: ESꢀ; 301.1 (Mþ), þES; 324.04 (Mþ þ Naþ).
6.1.2. General procedure for the synthesis of 3-acetyl-4-hydroxy-
2H-1,2-benzothiazine 1,1-dioxide 3-benzoyl-4-hydroxy-2H-1,2-
benzothiazine 1,1-dioxide (2 & 8)
Fresh sodium methoxide was first prepared by refluxing the
sodium metal (5.34 g, 0.232 mol) in 60 mL of freshly dried meth-
anol under inert atmosphere maintained by N2 gas. Subsequently,
N-acetonylsaccharine (5.0 g, 20.9 mmol) (1) or N-phena-
cylsaccharin (7) (10.0 g, 0.232 mmol) was added to the refluxing
methanolic solution. The reaction mixture immediately turned to
orange-red colour. The mixture was further refluxed for 20 min and
then cooled to room temperature and poured to ice cold 10% HCl
solution. The resulting white precipitate was filtered, washed with
excess water and dried. (Caution: atmosphere should be inert
otherwise traces of moisture could cause a drastic decrease in
yield).
6. Experimental section
6.1. Chemicals
All the chemicals were purchased from Merck, Sigma/Aldrich or
Fluka and used without purification. General melting points were
obtained on a Gallenkamp melting point apparatus and were un-
corrected. IR spectra were recorded in KBr pellets on Perkin Elmer
infrared spectrophotometer. 1H NMR spectra were recorded in
DMSO-d6, acetone-d6 and CDCl3 on a Bruker/XWIN NMR (300 and
400 MHz) apparatus, 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 a Clifton Ultrasonic Bath (29 T2A, 300W, DU-4; Nickel Electro
6.1.2.1. 3-Acetyl-4-hydroxy-2H-1,2-benzothiazine 1,1-dioxide (2).
Yellowish brownish amorphous material; yield: 80%; m.p.: 155 ꢂC;
IR (KBr): 3472, 3251, 1597, 1584, 1355, 1177 cmꢀ1 1H NMR (CDCl3,
;
400 MHz)
J ¼ 7.2 Hz, AreH), 7.69 (1H, t, J ¼ 7.4 Hz, AreH), 7.97 (2H, t, J ¼ 8.0 Hz,
AreH), 14.53 (1H, s, OH); 13C NMR (DMSO-d6)
: 21.5 (CeCH3), 69.2
d: 2.26 (3H, s, CeCH3), 5.87 (1H, s, SO2NH), 7.57 (1H, t,
d
(C-3, thiazine), 122.5 (AreC), 123.4 (AreC), 124.4 (AreC), 127.2 (Are
C), 129.2 (AreC), 133.4 (AreC), 162.7 (C-4, thiazine), 191.2 (C]O);
MS m/z: 239.03; [Mþ]. HR-MS (ESþ) calcd for C10H9NO4S, 239.0252;
found, 240.0326 (M þ Hþ).

112
S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
6.1.2.2. 3-Benzoyl-4-hydroxy-2H-1,2-benzothiazine 1,1-dioxide (8).
2.29 (3H, s, CeCH3), 3.12 (3H, s, NeCH3), 7.55 (1H, t, J ¼ 7.6 Hz, Are
H), 7.63 (1H, d, J ¼ 7.4 Hz, AreH), 7.97 (2H, t, J ¼ 8.0 Hz, AreH), 10.11
White crystalline solid; yield: 82%; m.p.: 156 ꢂC; FT-IR (KBr): 3353,
3133, 1625, 1358, 1157 cmꢀ1; 1H NMR (DMSO-d6) (400 MHz)
d
: 5.79
(1H, s, NH);13C NMR (CDCl3, 100 MHz)
d: 8.6 (CeCH3), 38.8 (Ne
(1H, s, SO2NH), 7.61 (1H, t, J ¼ 7.8 Hz, AreH), 7.71 (1H,d, J ¼ 7.8 Hz,
CH3), 121.8 (AreC), 123.3 (AreC), 124.2 (AreC), 128.8 (AreC), 129.3
(AreC), 131.1 (AreC), 132.9 (AreC), 133.2 (C]N, AreC), 134.5 (C]
CeSO2); MS m/z: 249.1 (Mþ). HR-MS (ESþ) calcd for C11H11N3O2S,
249.0572; found, 250.0650 (M þ Hþ).
AreH), 7.95e7.99 (5H, m, AreH), 8.15 (2H, t, J ¼ 4.6 Hz, AreH), 14.67
(1H, s, OeH); 13C NMR (DMSO-d6)
d: 110.1 (C-3, thiazine), 122.9
(AreC), 127.1 (AreC), 128.1 (AreC), 128.3 (AreC), 129.2 (AreC),
129.4 (2C, Ar), 132.2 (AreC), 132.3 (AreC), 134.0 (AreC), 137.1 (2C,
Ar), 167.6 (C-4, thiazine), 191.1 (C]O); MS m/z: (ESꢀ) 301.07 (Mþ),
(þES) 302.06 (Mþ þ Hþ).
6.1.4.2. 4-Methyl-3-phenyl-2,4-dihydrobenzo[e]pyrazolo[4,3-c][1,2]
thiazine 5,5-dioxide (10). Off white crystalline; yield: 87%; FT-IR
(KBr): 3368, 2965, 1655, 1337, 1165 cmꢀ1 1H NMR (DMSO-d6)
;
6.1.3. General procedure for the synthesis of 3-acetyl-4-hydroxy-2-
methyl-2H-1,2-benzothiazine 1,1-dioxide and 3-benzoyl-4-hydroxy-
2-methyl-2H-1,2-benzothiazine 1,1-dioxide (3 & 9)
(400 MHz)
d
: 2.91 (3H, s, NeCH3), 7.49 (1H, t, J ¼ 7.8 Hz, AreH), 7.61
(2H, q, J ¼ 8.4 Hz, AreH), 7.64e7.71 (1H, dq, J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz,
J3 ¼ 11.2 Hz, J4 ¼ 16.8 Hz, AreH), 7.73e7.79 (1H, dd, J1 ¼ 1.5 Hz,
J2 ¼ 8.4 Hz, AreH), 7.94e8.01 (3H, m, AreH), 8.17 (1H, d, J ¼ 7.6 Hz,
3-Acetyl-4-hydroxy-2H-1,2-benzothiazine
1,1-dioxide
(2)
(10.0 g, 42 mmol) or 3-benzoyl-4-hydroxy-2H-1,2-benzothiazine
1,1-dioxide (8) (10.0 g, 33.2 mmol) and aqueous sodium hydrox-
ide (2.66 g in 5 mL water) were mixed in 50 mL of acetone. The
mixture was allowed to stir at ambient temperature for 10 min.
Dimethyl sulphate (8.06 mL, 64 mmol) was added dropwise. The
mixture was placed in ultrasonic media for 15 min at ambient
temperatures. The resulting precipitate was obtained after addition
of cold dilute HCl (20 mL; 5%) and was filtered, washed with excess
water and then dried. The reaction was performed at room tem-
perature for 30e45 min.
AreH), 13.78 (1H, s, NeH); 13C NMR (DMSO-d6, 100 MHz)
d: 39.8
(NeCH3), 122.2 (AreC), 124.2 (AreC), 124.7 (AreC), 124.9 (AreC),
125.8 (AreC), 125.9 (AreC), 127.2 (AreC), 128.6 (AreC), 130.1 (Are
C), 130.3 (AreC), 130.4 (AreC), 134.2 (AreC), 134.4 (C]CeSO2),
138.2 (AreC), 147.4 (N]C, AreC); MS m/z: 312.4 (ESþ), HR-MS
(ESþ) calcd for C16H13N3O2S, 311.0728; found, 312.0801 (M þ Hþ).
6.1.5. General procedure for the synthesis of 4-(3,4-dimethyl-5,5-
dioxidobenzo[e]pyrazolo[4,3-c][1,2]thiazin-2(4H)-yl)benzaldehyde
(5) and 4-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c]
[1,2]thiazin-2(4H)-yl)benzaldehyde (11)
6.1.3.1. 3-Acetyl-4-hydroxy-2-methyl-2H-1,2-benzothiazine 1,1-
3,4-Dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine
5,5-dioxide (4) (12.50 g; 50.0 mmol) or 4-methyl-3-phenyl-2,4-
dioxide (3). White crystalline solid; yield: 89%; m.p.: 152 ꢂC; FT-
IR (KBr): 3475, 1601, 1588, 1349, 1191 cmꢀ1
;
1H NMR (CDCl3,
dihydrobenzo[e]pyrazolo[4,3-c][1,2]thiazine
5,5-dioxide
(10)
400 MHz)
J ¼ 7.4 Hz, AreH), 7.69 (1H, t, J ¼ 7.0 Hz, AreH), 7.94 (2H, t, J ¼ 7.5 Hz,
AreH), 12.34 (1H, s, OH); 13C NMR (CDCl3, 100 MHz)
: 21.5 (Ce
d
: 2.24 (3H, s, CeCH3), 3.11 (3H, s, NeCH3), 7.56 (1H, t,
(15.55 g; 50.0 mmol), 4-fluorobenzaldehyde (7.44 g; 60.0 mmol)
and anhydrous K2CO3 (8.30 g; 60.0 mmol) were dissolved in
dried DMF (25 mL) followed by the addition of hexadecyl-
triphenylphosphonium bromide (2.54 g; 5.0 mmol). The reaction
mixture was refluxed under the nitrogen atmosphere for 40 min.
Ice cold water was added and the resulting pale-yellow precipitate
was filtered, washed with excess water and recrystallized from
ethanol.
d
CH3), 38.7 (NeCH3), 117.9 (AreC), 123.4 (AreC), 124.4 (AreC), 127.2
(AreC), 129.2 (AreC), 133.4 (AreC), 136.7 (AreC), 161.2 (C-4, thia-
zine), 192.02 (C]O); MS m/z: 253.0 [Mþ]. HR-MS (ESþ) calcd for
C
11H11NO4S, 253.0409; found, 254.0471 (M þ Hþ).
6.1.3.2. 3-Benzoyl-4-hydroxy-2-methyl-2H-1,2-benzothiazine 1,1-
dioxide (9). Light yellow crystalline solid; yield: 85%; m.p.:ꢀ1147e
6.1.5.1. 4-(3,4-Dimethyl-5,5-dioxidobenzo[e]pyrazolo[4,3-c][1,2]thia-
zin-2(4H)-yl)benzaldehyde (5). Yellowish solid; yield: 73%; m.p.:
231 ꢂC; FT-IR (KBr): 3097, 2994, 2927, 2836, 2771, 1727, 1598, 1445,
148 ꢂC; FT-IR (KBr): 3470, 2970, 1610, 1586, 1357, 1184 cm
;
1H
NMR (CDCl3, 400 MHz)
d
: 2.89 (3H, s, NeCH3), 7.59 (2H, t, J ¼ 7.6 Hz,
AreH), 7.69 (1H,d, J ¼ 7.8 Hz, AreH), 7.73e7.77 (2H, dd, J1 ¼ 2.8 Hz,
J2 ¼ 8.4 Hz, AreH), 7.94e7.97 (3H, m, AreH), 8.17 (1H, t, J ¼ 4.6 Hz,
1359, 1244, 1161, 821, 725 cmꢀ1 1H NMR (DMSO-d6, 300 MHz)
; d:
2.49 (3H, s, CeCH3), 3.09 (3H, s, NeCH3), 7.52 (1H, t, J ¼ 7.5 Hz, Are
H), 7.62e7.70 (4H, dd, J1 ¼ 6.3 Hz, J2 ¼ 12.9 Hz, AreH), 7.91 (1H, d,
J ¼ 6.6 Hz, AreH), 7.95 (1H, d, J ¼ 8.4 Hz, AreH), 8.04 (1H, d,
AreH), 14.69 (1H, s, OeH); 13C NMR (DMSO-d6)
d: 39.8 (NeCH3),
121.9 (C-3, thiazine), 123.2 (AreC), 124.9 (AreC), 124.7 (AreC), 127.2
(AreC), 128.6 (AreC), 129.2 (AreC), 130.1 (AreC), 131.0 (AreC),
133.0 (AreC), 134.2 (AreC), 136.5 (AreC), 138.2 (AreC), 167.2 (C-4,
thiazine), 190.0 (C]O); MS m/z: 315.3 [Mþ].
J ¼ 7.2 Hz, AreH), 10.03 (1H, s, CH); 13C NMR (DMSO-d6, 75 MHz)
d:
11.0 (CeCH3), 40.0 (NeCH3), 124.3 (AreC), 124.8 (AreC), 124.9 (2C)
(AreC), 125.5 (AreC), 127.8 (AreC),129.3 (2C) (AreC), 130.9 (AreC),
132.6 (AreC), 133.0 (AreC), 133.6 (AreC), 135.5 (AreC), 139.7 (Are
C), 144.0 (N]C, AreC), 190.9 (eHC]O); MS m/z: (ESꢀ) 353 (Mþ),
6.1.4. General procedure for the synthesis of 3,4-dimethyl-2,4-
dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide and 4-methyl-
3-phenyl-2,4-dihydrobenzo[e]pyrazolo[4,3-c][1,2]thiazine 5,5-
dioxide (4 & 10)
(ESþ) 354.1 (M
þ
Hþ); HR-MS(þES) calcd for C18H15N3O3S,
353.0834; found, 354.0907 (M þ Hþ).
A
mixture of 1-(4-hydroxy-2-methyl-1,1-dioxido-2H-1,2-
6.1.5.2. 4-(4-Methyl-5,5-dioxido-3-aphenylbenzo[e]pyrazolo[4,3-c]
[1,2]thiazin-2(4H)-yl)benzaldehyde (11). Yellowish solid; yield:
69%; FT-IR (KBr): 3079, 2987, 2847, 2779, 1737, 1701, 1598, 1456,
benzothiazin-3-yl)ethanone (3) (5.0 g, 20.9 mmol) or 3-benzoyl-
4-hydroxy-2-methyl-2H-1,2-benzothiazine 1,1-dioxide (9) (5.0 g,
13.08 mmol) and hydrazine monohydrate (4.9 mL, 99 mmol) in
50 mL ethanol was refluxed for 3e4 h. Then the unreacted hydra-
zine monohydrate and ethanol was removed under vacuum. The
crude product was treated with ice cold water. The resulting pre-
cipitate was filtered and recrystallized from ethanol.
1362, 1204, 1162, 824, 723 cmꢀ1 1H NMR (DMSO-d6, 300 MHz)
; d:
3.23 (3H, s, NeCH3), 7.43e7.57 (2H, m, AreH), 7.67 (1H, t, J ¼ 7.5 Hz,
AreH), 7.75e7.83 (4H, dd, J1 ¼ 9.0 Hz, J2 ¼ 15.4 Hz, AreH), 7.91 (1H,
d, J ¼ 7.8 Hz, AreH), 7.97e8.05 (4H, dd, J1 ¼7.2 Hz, J2 ¼ 14.7 Hz, Are
H), 8.13 (1H, d, J ¼ 7.2 Hz, AreH), 10.23 (1H, s, CH); 13C NMR (DMSO-
d6, 75 MHz) d: 38.7 (NeCH3), 124.3 (2C) (AreC), 124.7 (AreC), 125.1
6.1.4.1. 3,4-Dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine
(AreC), 125.2 (AreC), 126.9 (AreC), 127.1 (AreC), 127.8 (AreC),
128.3 (AreC), 129.2 (AreC), 129.4 (AreC), 129.9 (2C) (AreC), 130.5
(AreC), 131.0 (AreC), 133.3 (AreC), 135.6 (AreC), 137.9 (AreC), 139.1
5,5-dioxide (4). Brownish yellow solid; yield: 81%; m.p.: 231 ꢂC; FT-
IR (KBr): 3355, 1594, 1315, 1156 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d:

S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
113
(AreC), 140.2 (AreC), 143.0 (N]C, AreC), 191.0 (eHC]O); MS m/z:
416.1 (ESþ, M þ Hþ); HR-MS (ESþ) calcd for C23H17N3O3S,
415.0991; found, 416.1064 (M þ Hþ).
128.3 (AreC), 128.5 (AreC), 128.9 (AreC), 129.0 (AreC), 129.6 (Are
C), 130.1 (AreC), 130.2 (AreC), 131.5 (AreC), 132.4 (AreC), 133.8
(AreC), 134.0 (AreC), 135.0 (AreC), 139.8 (AreC), 141.8 (N]C, Are
C), 162.9 (N]CeN, AreC); MS m/z: 518 (ESꢀ), 520 (M þ 2), 522
(ESþ); HR-MS (ESꢀ) calcd for C24H18BrN5O2S, 519.0365; found,
518.0286.
6.1.6. General procedure for the synthesis of pyrazolo[4,3-c][1,2]
benzothiazine 5,5-dioxides (6aek and 12aek) and purine- and
benzoxazole-containing derivatives (6i and 12i; 6j and 12j)
4-(3,4-Dimethyl-5,5-dioxidobenzo[e]pyrazolo[4,3-c][1,2]thiazin-
2(4H)-yl)benzaldehyde (5) (0.01 mol) or 4-(4-methyl-5,5-dioxido-3-
phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-2 (4H)yl)benzaldehyde
(11) (0.01 mol)andsodium metabisulfite (0.01 mol) were dissolved in
dried DMF (20 mL) and stirred at room temperature for 15e20 min.
Then, substituted o-phenylenediamine or pyrimidine-4,5-diamine
(for 6i and 12i) or 2-aminophenol (for 6j and 12j) (0.011 mol) was
added and stirred at 135e140 ꢂC for 6e8 h depending on the reac-
tivityof the aromaticamine. The reactionwasmonitored by TLC. After
completion, the reaction mixture was allowed to cool at room tem-
perature and poured into ice cold water. The resulting precipitate was
filtered, washed with water and recrystallized from ethanol.
6.1.6.4. 2-[4-(1H-Benzimidazol-2-yl)phenyl]-3,4-dimethyl-2,4-
dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide (6d). Light
brown powder; Yield: 83%; FT-IR (KBr): 3272, 2978, 2928, 1753,
1660, 1601, 1500, 1439, 1376, 1169, 1064, 847, 768, 744 cmꢀ1 1H
;
NMR (DMSO-d6, 300 MHz) d: 2.51 (3H, s, CeCH3), 3.06 (3H, s, Ne
CH3), 7.24 (2H, t, J ¼ 6.9 Hz, AreH), 7.57 (1H, d, J ¼ 7.8 Hz, AreH),
7.71 (2H, t, J ¼ 7.4 Hz, AreH), 7.82e7.88 (3H, m, AreH), 7.94 (1H, d,
J ¼ 7.8 Hz, AreH), 8.08 (1H, d, J ¼ 7.5 Hz, AreH), 8.37 (2H, d,
J ¼ 8.7 Hz, AreH), 13.07 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz)
d:
10.8 (CeCH3), 40.3 (NeCH3), 122.4 (AreC), 123.4 (AreC), 124.8 (Are
C), 125.1 (AreC), 125.5 (AreC), 125.8 (AreC), 127.0 (AreC), 128.3
(AreC), 128.6 (AreC), 128.9 (AreC), 129.1 (AreC), 129.8 (AreC),
130.2 (AreC), 130.5 (AreC), 131.2 (AreC), 132.5 (AreC), 133.7 (Are
C), 134.0 (AreC), 134.9 (AreC), 139.7 (AreC), 141.4 (N]C, AreC),
151.3 (N]CeN, AreC); MS m/z: 440 (ESꢀ), 442 (ESþ); HR-MS (ESꢀ)
calcd for C24H19N5O2S, 441.1259; found, 440.1179.
6.1.6.1. 2-[4-(5-Carboxy-1H-benzimidazol-2-yl)phenyl]-3,4-
dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide
(6a). Light brown powder; Yield: 87%; FT-IR (KBr): 3645, 3463,
3205, 3026, 2969,1738,1652,1495,1434,1372,1229,1167,1063, 847,
770, 745 cmꢀ1; 1H NMR (DMSO-d6, 300 MHz)
d
: 2.51 (3H, s, CeCH3),
6.1.6.5. 2-[4-(5-Methyl-1H-benzimidazol-2-yl)phenyl]-3,4-dimethyl-
3.06 (3H, s, NeCH3), 7.70 (2H, t, J ¼ 8.1 Hz, AreH), 7.82e7.96 (5H, m,
AreH), 8.08 (1H, d, J ¼ 7.5 Hz, AreH), 8.22 (1H, s, AreH), 8.41 (2H, d,
J ¼ 8.4 Hz, AreH), 13.29 (1H, s, NH); 13C NMR (acetone-d6, 100 MHz)
2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine
Light brown powder; Yield: 85%; FT-IR (KBr): 3272, 2969, 2921,
1739, 1609, 1507, 1439, 1375, 1170, 1064, 849, 773, 746 cmꢀ1 1H
NMR (acetone-d6, 400 MHz) : 2.35 (3H, s, AreCH3), 2.47 (3H, s, Ce
5,5-dioxide
(6e).
;
d
: 10.9 (CeCH3), 40.3 (NeCH3), 122.4 (AreC), 123.7 (AreC), 124.8
d
(AreC),125.1 (AreC),125.5 (AreC),125.8 (AreC),126.0 (AreC),128.4
(AreC),128.7 (AreC),128.9 (AreC),129.1 (AreC),129.8 (AreC),130.2
(AreC),130.5 (AreC),131.5 (AreC),132.5 (AreC),133.8 (AreC),134.0
(AreC),135.0 (AreC),139.8 (AreC),141.9 (N]C, AreC),152.9 (N]Ce
N, AreC),168.1 (C]O); MS m/z: 484 (ESꢀ), 486 (ESþ); HR-MS (ESꢀ)
calcd for C25H19N5O4S, 484.1158; found, 484.1073.
CH3), 3.00 (3H, s, NeCH3), 6.94e6.97 (1H, dd, J1 ¼1.2 Hz, J2 ¼ 8.0 Hz,
AreH), 7.30 (1H, s, AreH), 7.40 (1H, d, J ¼ 8.0 Hz, AreH), 7.56e7.60
(1H, dt, J1 ¼1.2 Hz, J2 ¼ 7.6 Hz, J3 ¼ 15.6 Hz, AreH), 7.68e7.72 (1H, dt,
J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz, J3 ¼ 16.4 Hz, AreH), 7.71e7.74 (2H, dd,
J1 ¼ 2.4 Hz, J2 ¼ 6.8 Hz, AreH), 7.78 (1H, q, J ¼ 4.0 Hz, AreH), 7.83
(1H, d, J ¼ 8.0 Hz, AreH), 7.99 (1H, d, J ¼ 8.4 Hz, AreH), 8.34 (1H, d,
J ¼ 8.4 Hz, AreH); 13C NMR (acetone-d6, 100 MHz)
d: 10.8 (CeCH3),
6.1.6.2. 2-[4-(5-Chloro-1H-benzimidazol-2-yl)phenyl]-3,4-dimethyl-
2,4 dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide (6b).
Off white powder; Yield: 82%; FT-IR (KBr): 3341, 3290, 3068, 2926,
1739, 1660, 1609, 1581, 1470, 1343, 1232, 1169, 1061, 848, 772,
20.7 (AreCH3), 40.3 (NeCH3), 122.4 (AreC), 123.4 (AreC), 124.8
(AreC), 125.1 (AreC), 125.5 (AreC), 125.8 (AreC), 126.0 (AreC),
128.2 (AreC), 128.6 (AreC), 128.1 (AreC), 129.2 (AreC), 129.8 (Are
C), 130.2 (AreC), 130.4 (AreC), 131.5 (AreC), 132.5 (AreC), 133.7
(AreC), 133.9 (AreC), 135.0 (AreC), 139.8 (AreC), 141.9 (N]C, Are
C), 152.0 (N]CeN, AreC); MS m/z: 454 (ESꢀ), 456 (ESþ); HR-MS
(ESꢀ) calcd for C25H21N5O2S, 455.1416; found, 454.1358.
746 cmꢀ1; 1H NMR (DMSO-d6, 300 MHz)
d: 2.51 (3H, s, CeCH3), 3.06
(3H, s, NeCH3), 7.24e7.27 (1H, dd, J1 ¼ 1.8 Hz, J2 ¼ 8.7 Hz, AreH),
7.63e7.73 (3H, m, AreH), 7.83 (1H, d, J ¼ 7.8 Hz, AreH), 7.88 (2H, d,
J ¼ 8.4 Hz, AreH), 7.94 (1H, d, J ¼ 7.8 Hz, AreH), 8.08 (1H, d, J ¼ 7.5 Hz,
AreH), 8.36 (2H, d, J ¼ 10.2 Hz, AreH), 13.28 (1H, s, NH); 13C NMR
6.1.6.6. 2-[4-(5,6-Dimethyl-1H-benzimidazol-2-yl)phenyl]-3,4-
dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide
(6f). Light yellow powder; Yield: 90%; FT-IR (KBr): 3021, 3004,
2968, 1742, 1608, 1505, 1441, 1375, 1216, 1169, 1061, 849, 771,
(acetone-d6, 100 MHz) d: 10.8 (CeCH3), 40.3 (NeCH3), 122.4 (AreC),
123.8 (AreC), 124.8 (AreC), 125.2 (AreC), 125.5 (AreC), 125.8 (Are
C),126.1 (AreC),128.5 (AreC),128.7 (AreC),128.8 (AreC),129.1 (Are
C),129.8 (AreC),130.2 (AreC),130.6 (AreC),131.5 (AreC),132.4 (Are
C),133.8 (AreC),134.0 (AreC),134.9 (AreC),139.8 (AreC),141.8 (N]
C, AreC), 152.9 (N]CeN, AreC); MS m/z: 474 (ESꢀ), 476 (ESþ); HR-
MS (ESꢀ) calcd for C24H18ClN5O2S, 475.0870; found, 474.0805.
746 cmꢀ1; 1H NMR (CDCl3, 300 MHz)
d: 2.31 (6H, s, 2XAreCH3), 2.36
(3H, s, CeCH3), 3.06 (3H, s, NeCH3), 7.45 (2H, s, AreH), 7.54 (3H, d,
J ¼ 7.4 Hz, AreH), 7.64 (1H, t, J ¼ 7.8 Hz, AreH), 7.94 (1H, d, J ¼ 7.5 Hz,
AreH), 8.03 (1H, d, J ¼ 7.8 Hz, AreH), 8.18 (2H, d, J ¼ 7.5 Hz, AreH);
13C NMR (acetone-d6,100 MHz)
d: 10.8 (CeCH3), 20.4 (AreCH3), 29.9
6.1.6.3. 2-[4-(5-Bromo-1H-benzimidazol-2-yl)phenyl]-3,4-dimethyl-
(AreCH3), 40.3 (NeCH3), 122.4 (AreC), 123.5 (AreC), 124.8 (AreC),
125.1 (AreC),125.5 (AreC),125.8 (AreC),125.9 (AreC),128.1 (AreC),
128.6 (AreC),128.9 (AreC),129.2 (AreC),129.8 (AreC),130.1 (AreC),
130.5 (AreC),131.4 (AreC),132.3 (AreC),133.7 (AreC),133.9 (AreC),
134.9 (AreC), 139.7 (AreC), 141.2 (N]C, AreC), 150.4 (N]CeN, Are
C); MS m/z: 468 (ESꢀ), 470 (ESþ); HR-MS (ESꢀ) calcd for
2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine
Brown powder; Yield: 84%; FT-IR (KBr): 3277, 3067, 2969, 2925,
1655, 1601, 1493, 1435, 1375, 1169, 1062, 845, 773, 745 cmꢀ1 1H
NMR (DMSO-d6, 300 MHz) : 2.51 (3H, s, CeCH3), 3.06 (3H, s, Ne
5,5-dioxide
(6c).
;
d
CH3), 7.35e7.38 (1H, dd, J1 ¼ 1.8 Hz, J2 ¼ 8.7 Hz, AreH), 7.59 (1H, d,
J ¼ 8.7 Hz, AreH), 7.71 (1H, t, J ¼ 7.8 Hz, AreH), 7.83 (2H, d,
J ¼ 6.3 Hz, AreH), 7.88 (2H, d, J ¼ 8.7 Hz, AreH), 7.94 (1H, d,
J ¼ 7.8 Hz, AreH), 8.08 (1H, d, J ¼ 7.5 Hz, AreH), 8.36 (2H, d,
J ¼ 8.7 Hz, AreH), 13.29 (1H, s, NH); 13C NMR (acetone-d6, 100 MHz)
C26H23N5O2S, 469.1572; found, 468.1485.
6.1.6.7. 2-[4-(5-Nitro-1H-benzimidazol-2-yl)phenyl]-3,4-dimethyl-
2,4-dihydro pyrazolo [4,3-c][1,2] benzothiazine 5,5-dioxide (6g).
Yellow powder; Yield: 85%; FT-IR (KBr): 3113, 3095, 2929, 1655,
d: 10.8 (CeCH3), 40.3 (NeCH3), 122.3 (AreC), 123.6 (AreC), 124.8
(AreC), 125.1 (AreC), 125.5 (AreC), 125.8 (AreC), 126.0 (AreC),
1600, 1494, 1438, 1374, 1243, 1170, 1064, 1006, 849, 772, 745 cmꢀ1
;

114
S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
1H NMR (acetone-d6, 400 MHz)
d: 2.48 (3H, s, CeCH3), 2.99 (3H, s,
C), 126.7 (AreC), 128.7 (AreC), 128.8 (AreC), 129.2 (AreC), 129.4
NeCH3), 7.20 (1H, t, J ¼ 5.2 Hz, AreH), 7.47 (1H, d, J ¼ 2.4 Hz, AreH),
7.57e7.61 (1H, dt, J1 ¼1.2 Hz, J2 ¼ 7.6 Hz, J3 ¼ 15.2 Hz, AreH), 7.69e
7.73 (1H, dt, J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz, J3 ¼ 15.2 Hz, AreH), 7.80 (2H, t,
J ¼ 8.4 Hz, AreH), 7.99 (1H, d, J ¼ 7.6 Hz, AreH), 8.06e8.09 (2H, dd,
J1 ¼ 2.0 Hz, J2 ¼ 8.8 Hz, AreH), 8.35 (1H, d, J ¼ 8.4 Hz, AreH), 8.43
(AreC), 129.5 (AreC), 129.7 (AreC), 130.8 (AreC), 132.9 (AreC),
133.0 (AreC), 135.1 (AreC), 141.3 (N]C, AreC), 155.3 (N]CeN, Are
C); MS m/z: 443 (ESꢀ), 445 (ESþ); HR-MS (ESꢀ) calcd for
C24H18N4O3S, 442.1100; found, 443.1183.
(1H, s, AreH), 12.98 (1H, s, NH); 13C NMR (acetone-d6, 100 MHz)
d:
6.1.6.11. Ethyl 2-(4-(3,4-dimethyl-5,5-dioxidobenzo[e]pyrazolo[4,3-c]
[1,2] thiazin-2(4H)-yl)phenyl)-1H-benzo[d]imidazole-5-carboxylate
(6k).
10.9 (CeCH3), 40.4 (NeCH3), 122.4 (AreC), 123.7 (AreC), 124.8 (Are
C), 125.1 (AreC), 125.6 (AreC), 125.8 (AreC), 126.0 (AreC), 128.3
(AreC), 128.6 (AreC), 128.8 (AreC), 129.0 (AreC), 129.5 (AreC),
130.0 (AreC), 130.2 (AreC), 131.5 (AreC), 132.5 (AreC), 133.8 (Are
C), 134.0 (AreC), 134.9 (AreC), 139.8 (AreC), 141.8 (N]C, AreC),
154.0 (N]CeN, AreC); MS m/z: 485 (ESꢀ), 487 (ESþ); HR-MS
(ESꢀ) calcd for C24H18N6O4S, 486.1110; found, 485.1039.
2-[4-(5-Carboxy-1H-benzimidazol-2-yl)phenyl]-3,4-dimethyl-2,4-
dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide (6a) (1.0 g)
was dissolved in ethanol (20 mL) and a catalytic amount of conc.
H2SO4 was added. The reaction mixture refluxed for 30e40 min and
completion of the reaction was confirmed by TLC. The solvent was
removed under vacuum. The product was dissolved in ice cold water
and a pH 7e8 was adjusted by using 5% NaHCO3. The formed pre-
cipitate was filtered, washed with water and dried. Off white pow-
der; yield: 70%; FT-IR (KBr): 3380, 2990, 2937,1722,1630,1605,1506,
1439, 1369,1296,1170,1001, 843, 767, 743 cmꢀ1; 1H NMR (DMSO-d6,
6.1.6.8. 2-[4-(5-Methoxy-1H-benzimidazol-2-yl)phenyl]-3,4-
dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide
(6h). Light brown powder; Yield: 89%; FT-IR (KBr): 3019, 2973,
2929, 2912, 1731, 1600, 1506, 1475, 1375, 1232, 1160, 847, 773,
746 cmꢀ1; 1H NMR (DMSO-d6, 400 MHz)
d: 2.44 (3H, s, CeCH3), 3.01
400 MHz)
d
: 1.45 (3H, t, J ¼ 7.2 Hz, CeCH3), 2.60 (3H, s, CeCH3), 3.01
(3H, s, NeCH3), 3.87 (3H, s, OeCH3), 7.01e7.04 (1H, dd, J1 ¼ 2.0 Hz,
J2 ¼ 8.8 Hz, AreH), 7.20 (1H, d, J ¼ 2.0 Hz, AreH), 7.41 (1H, d, J ¼ 8.4 Hz,
AreH), 7.59 (1H, s, AreH), 7.65 (1H, d, J ¼ 8.8 Hz, AreH), 7.73 (1H, t,
J ¼ 8.0 Hz, AreH), 7.86 (1H, t, J ¼ 7.6 Hz, AreH), 7.97 (1H, d, J ¼ 8.4 Hz,
AreH), 8.09 (1H, d, J ¼ 7.6 Hz, AreH), 8.25 (1H, d, J ¼ 8.4 Hz, AreH),
8.36 (1H, d, J ¼ 8.4 Hz, AreH), 12.97 (1H, s, NH); 13C NMR (DMSO-d6,
(3H, s, NeCH3), 4.46 (2H, q, J ¼ 7.2 Hz, OeCH2), 7.62 (1H, t, J ¼ 9.2 Hz,
AreH), 7.81 (1H, t, J ¼ 7.6 Hz, AreH), 7.94 (1H, t, J ¼ 7.6 Hz, AreH),
8.07e8.09 (4H, dd, J1 ¼ 8.0 Hz, J2 ¼ 20.4 Hz, AreH), 8.17 (1H, t,
J ¼ 8.4 Hz, AreH), 8.23 (1H, s, AreH), 8.51 (2H, t, J ¼ 8.8 Hz, AreH),
12.98 (1H, s, NH); 13C NMR (DMSO-d6,100 MHz)
d: 10.5 (CeCH3),14.2
(H2CeCH3), 40.0 (NeCH3), 60.7 (OeCH2),114.6 (AreC),122.4 (AreC),
124.5 (AreC),124.7 (AreC),125.0 (AreC),125.3 (AreC),125.8 (AreC),
127.1 (AreC),128.4 (AreC), 128.9 (AreC),129.5 (AreC),130.0 (AreC),
130.2 (AreC), 130.5 (AreC), 131.5 (AreC),132.5 (AreC), 133.6 (AreC),
134.0 (AreC), 134.5 (AreC), 139.8 (AreC), 141.9 (N]C, AreC), 150.4
(N]CeN, AreC),162.2 (C]O); MS m/z: 512 (ESꢀ), 514 (ESþ); HR-MS
(ESþ) calcd for C27H23N5O4S, 513.1471; found, 514.1548 (M þ Hþ).
100 MHz) d: 10.4 (CeCH3), 40.1 (NeCH3), 55.7 (OeCH3),113.8 (AreC),
123.8 (AreC),124.4 (AreC),124.5 (AreC),125.0 (AreC),127.3 (AreC),
127.8 (AreC),128.5 (AreC),129.0 (AreC),129.8 (AreC),130.1 (AreC),
130.2 (AreC), 131.9 (AreC), 132.4 (AreC), 133.6 (AreC), 134.0 (AreC),
134.2 (AreC), 138.4 (AreC), 140.6 (AreC), 148.7 (N]C, AreC), 152.3
(N]CeN, AreC),157.0 (AreC); MS m/z: 470 (ESꢀ), 472 (ESþ); HR-MS
(ESꢀ) calcd for C25H21N5O3S, 471.1365; found, 470.1303.
6.1.6.12. 2-(4-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-
c][1,2] thiazin-2(4H)-yl)phenyl)-1H-benzo[d]imidazole-5-carboxylic
acid (12a). Brown powder; Yield: 86%; FT-IR (KBr): 3650, 2944,
6.1.6.9. 3,4-Dimethyl-2-[4-(9H-purin-8-yl)phenyl]-2,4-
dihydropyrazolo[4,3-c][1,2]
benzothiazine
5,5-dioxide
(6i).
Brown powder; Yield: 83%; FT-IR (KBr): 3062, 2927, 2873, 1699,
1663, 1600, 1490, 1436, 1375, 1342, 1245, 1169, 1063, 849, 772,
2920, 1648, 1490, 1437, 1382, 1289, 1094, 967, 845 cmꢀ1 1H NMR
;
(acetone-d6, 400 MHz)
d
: 2.98 (3H, s, NeCH3), 7.02 (1H, d, J ¼ 7.6 Hz,
746 cmꢀ1
;
1H NMR (acetone-d6, 400 MHz)
d: 2.47 (3H, s, CeCH3),
AreH), 7.37 (1H, t, J ¼ 7.6 Hz, AreH), 7.29e7.33 (2H, dd, J1 ¼ 6.0 Hz,
J2 ¼ 9.2 Hz, AreH), 7.53e7.57 (2H, dd, J1 ¼ 6.4 Hz, J2 ¼ 12.8 Hz, Are
H), 7.65 (1H, d, J ¼ 6.8 Hz, AreH), 7.69 (1H, t, J ¼ 8.8 Hz, AreH), 7.89e
7.93 (2H, m, AreH), 7.95 (2H, d, J ¼ 7.6 Hz, AreH), 8.01 (1H, d,
J ¼ 7.6 Hz, AreH), 8.08 (1H, d, J ¼ 8.8 Hz, AreH), 8.24 (1H, t,
J ¼ 7.6 Hz, AreH), 8.33 (1H, d, J ¼ 9.2 Hz, AreH), 12.83 (1H,s,OH),
2.98 (3H, s, NeCH3), 7.47 (1H, d, J ¼ 2.4 Hz, AreH), 7.56e7.60 (1H, dt,
J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz, J3 ¼ 15.6 Hz, AreH), 7.67e7.72 (1H, dt,
J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz, J3 ¼ 18.8 Hz, AreH), 7.82 (1H, t, J ¼ 8.4 Hz,
AreH), 7.99 (1H, d, J ¼ 7.6 Hz, AreH), 8.40 (2H, d, J ¼ 8.8 Hz, AreH),
8.46 (1H, d, J ¼ 8.8 Hz, AreH), 8.76 (1H, s, AreH), 8.95 (1H, s, AreH),
11.57 (1H, s, NH); 13C NMR (acetone-d6, 100 MHz)
d: 10.8 (CeCH3),
13.01 (1H, s, NH); 13C NMR (acetone-d6, 100 MHz)
d: 40.3 (NeCH3),
40.4 (NeCH3), 122.4 (AreC), 123.6 (AreC), 124.9 (AreC), 125.1 (Are
C), 125.5 (AreC), 127.0 (AreC), 128.5 (AreC), 128.8 (AreC), 129.7
(AreC), 130.1 (AreC), 130.3 (AreC), 133.6 (AreC), 134.0 (AreC),
135.0 (AreC), 139.6 (AreC), 141.6 (N]C, pyr AreC), 147.8 (N]C,
purine AreC), 151.8 (NeC]N, purine AreC), 155.8 (NeC]C, purine
AreC), 157.9 (N]CeN, AreC); MS m/z: 442 (ESꢀ), 444 (ESþ); HR-
MS (ESꢀ) calcd for C22H17N7O2S, 443.1164; found, 442.1084.
116.9 (AreC), 119.0 (AreC), 120.2 (AreC), 122.4 (AreC), 123.4 (Are
C), 123.7 (AreC), 124.0 (AreC), 124.8 (AreC), 125.1 (AreC), 125.5
(AreC), 125.8 (AreC), 126.0 (AreC), 128.4 (AreC), 128.7 (AreC),
128.9 (AreC), 129.1 (AreC), 129.8 (AreC), 130.2 (AreC), 130.5 (Are
C), 131.5 (AreC), 132.5 (AreC), 133.8 (AreC), 134.0 (AreC), 135.0
(AreC), 139.8 (AreC), 141.9 (AreC), 143.7 (N]C, AreC), 152.9 (N]
CeN, AreC), 168.2 (C]O); MS m/z: 546 (ESꢀ), 548 (ESþ); HR-MS
(ESꢀ) calcd for C30H21N5O4S, 547.1314; found 546.1249.
6.1.6.10. 2-[4-(1,3-Benzoxazol-2-yl)phenyl]-3,4-dimethyl-2,4-
dihydropyrazolo[4,3-c][1,2]benzothiazine
Yellowish powder; Yield: 87%; FT-IR (KBr): 3098, 3034, 2958, 1749,
1601, 1509, 1438, 1374, 1232, 1169, 1064, 843, 768, 747 cmꢀ1 1H
NMR (CDCl3, 400 MHz) : 2.45 (3H, s, CeCH3), 3.06 (3H, s, NeCH3),
5,5-dioxide
(6j).
6.1.6.13. 2-[4-(5-Chloro-1H-benzimidazol-2-yl)phenyl]-4-methyl-3-
phenyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide
(12b). Brown powder; Yield: 86%; FT-IR (KBr): 3113, 3075, 2953,
;
d
1656, 1601, 1490, 1436, 1369, 1234, 1170, 1059, 847, 771 cmꢀ1 1H
;
6.85e6.88 (1H, dt, J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz, J3 ¼ 15.2 Hz, AreH), 6.97
(1H, d, J ¼ 8.8 Hz, AreH), 7.27e7.30 (4H, dd, J1 ¼ 1.2 Hz, J2 ¼ 8.0 Hz,
AreH), 7.50e7.55 (2H, m, AreH), 7.61 (1H, t, J ¼ 8.0 Hz, AreH), 7.67
(1H, t, J ¼ 8.4 Hz, AreH), 7.90 (1H, d, J ¼ 8.8 Hz, AreH), 8.01 (1H, t,
NMR (acetone-d6, 400 MHz) d: 2.98 (3H, s, NeCH3), 7.08e7.10 (1H,
d, J ¼ 2.0 Hz, AreH), 7.09 (1H, m, AreH), 7.13 (1H, q, J ¼ 2.4 Hz, Are
H), 7.18 (1H, d, J ¼ 7.2 Hz, AreH), 7.33 (1H, t, J ¼ 7.2 Hz, AreH), 7.37
(1H, s, AreH), 7.43e7.46 (2H, dd, J1 ¼ 8.0 Hz, J2 ¼ 15.6 Hz, AreH),
7.49 (1H, q, J ¼ 6.8 Hz, AreH), 7.56e7.58 (1H, dt, J1 ¼ 1.2 Hz,
J2 ¼ 5.2 Hz, J3 ¼ 8.0 Hz, AreH), 7.59e7.60 (1H, dd, J1 ¼ 1.2 Hz,
J2 ¼ 4.4 Hz, AreH), 7.70 (1H, q, J ¼ 8.4 Hz, AreH), 7.83 (1H, t,
J ¼ 8.4 Hz, AreH); 13C NMR (CDCl3, 100 MHz)
d: 10.6 (CeCH3), 40.0
(NeCH3), 115.3 (AreC), 115.9 (AreC), 120.2 (AreC), 124.2 (AreC),
124.8 (AreC), 124.9 (AreC), 125.0 (AreC), 125.1 (AreC), 125.8 (Are

S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
115
J ¼ 8.0 Hz, AreH), 7.89e7.91 (1H, dd, J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz, AreH),
7.96 (1H, d, J ¼ 8.0 Hz, AreH), 8.00e8.26 (1H, dd, J1 ¼ 1.2 Hz,
J2 ¼ 8.4 Hz, AreH), 8.07 (1H, d, J ¼ 7.6 Hz, AreH), 8.11 (1H, d,
(AreC), 123.0 (AreC), 123.3 (AreC), 124.2 (AreC), 124.3 (AreC),
124.7 (AreC), 124.9 (AreC), 125.3 (AreC), 125.3 (AreC), 125.6 (Are
C), 126.0 (AreC), 126.8 (AreC), 127.2 (AreC), 127.8 (AreC), 128.0
(AreC), 129.0 (AreC), 129.1 (AreC), 129.2 (AreC), 129.6 (AreC),
129.6 (AreC), 130.4 (AreC), 132.3 (AreC), 132.9 (AreC), 135.7 (Are
C), 139.4 (AreC), 140.5 (AreC), 145.6 (N]C, AreC), 152.5 (N]CeN,
AreC); MS m/z: 516 (ESꢀ), 518 (ESþ); HR-MS (ESꢀ) calcd for
J ¼ 8.8 Hz, AreH), 8.30 (2H, q, J ¼ 8.4 Hz, AreH) 13.13 (1H,s,NH);13
C
NMR (acetone-d6, 100 MHz) d: 40.3 (NeCH3), 116.9 (AreC), 118.9
(AreC), 123.5 (AreC), 123.7 (AreC), 124.1 (AreC), 124.5 (AreC),
124.8 (AreC), 124.8 (AreC), 125.2 (AreC), 125.5 (AreC), 125.8 (Are
C), 126.0 (AreC), 127.4 (AreC), 128.2 (AreC), 128.7 (AreC), 128.9
(AreC), 129.1 (AreC), 129.9 (AreC), 130.0 (AreC), 130.5 (AreC),
131.0 (AreC),131.6 (AreC),133.6 (AreC),134.1 (AreC),134.9 (AreC),
139.8 (AreC), 141.9 (N]C, AreC), 152.6 (N]CeN, AreC); MS m/z:
536 (ESꢀ), 538 (ESþ), HR-MS (ESꢀ) cacld for C29H20ClN5O2S,
537.1026; found, 536.0950.
C30H23N5O2S, 517.1572; found, 516.1495.
6.1.6.17. 2-[4-(5,6-Dimethyl-1H-benzimidazol-2-yl)phenyl]-4-
methyl-3-phenyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-
dioxide (12f). Dirty yellow powder; Yield: 81%; FT-IR (KBr): 3067,
2980, 1737, 1631, 1508, 1438, 1350, 1171, 1062, 849, 773 cmꢀ1 1H
;
NMR (CDCl3, 300 MHz) d: 2.81 (6H, s, CeCH3), 2.89 (3H, s, NeCH3),
6.1.6.14. 2-[4-(5-Bromo-1H-benzimidazol-2-yl)phenyl]-4-methyl-3-
phenyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide
(12c). Brown powder; Yield: 84%; FT-IR (KBr): 3403, 2926, 1652,
7.30e7.33 (2H, m, AreH), 7.41 (2H, d, J ¼ 6.3 Hz, AreH), 7.50 (3H, t,
J ¼ 6.0 Hz, AreH), 7.82 (2H, t, J ¼ 6.0 Hz, AreH), 7.89 (1H, t,
J ¼ 9.0 Hz, AreH), 8.01e8.04 (2H, m, AreH), 8.15 (2H, d, J ¼ 8.4 Hz,
1491, 1462, 1386, 1344, 1253, 1131, 1086, 913, 848, 747 cmꢀ1
;
1H
AreH), 13.06 (1H, s, NH); 13C NMR (CDCl3, 75 MHz)
d: 29.7 (¼Ce
NMR (acetone-d6, 400 MHz)
d: 2.97 (3H, s, NeCH3), 7.18e7.26 (2H,
CH3), 31.5 (¼CeCH3), 40.4 (NeCH3), 116.8 (AreC), 123.1 (AreC),
123.1 (AreC), 124.1 (AreC), 124.4 (AreC), 124.7 (AreC), 125.0 (Are
C), 125.2 (AreC), 125.4 (AreC), 125.7 (AreC), 126.0 (AreC), 126.8
(AreC),127.4 (AreC),127.6 (AreC),127.8 (AreC),128.9 (AreC),129.1
(AreC), 129.2 (AreC), 129.5 (AreC), 129.7 (AreC), 130.4 (AreC),
132.2 (AreC), 132.8 (AreC), 135.8 (AreC), 139.7 (AreC), 141.6 (N]C,
AreC), 150.6 (AreC), 151.7 (N]CeN, AreC); MS m/z: 530 (ESꢀ), 532
(ESþ); HR-MS (ESꢀ) calcd for C31H25N5O2S, 531.1729; found,
530.1631.
m, AreH), 7.34 (1H, d, J ¼ 6.4 Hz, AreH), 7.44e7.47 (2H, dd,
J1 ¼ 3.2 Hz, J2 ¼ 8.4 Hz, AreH), 7.68e7.73 (4H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 12.8 Hz, AreH), 7.84 (1H, s, AreH), 7.97e8.03 (2H, dd,
J1 ¼ 7.6 Hz, J2 ¼ 18.0 Hz, AreH), 8.07e8.13 (2H, dd, J1 ¼ 7.2 Hz,
J2 ¼ 16.8 Hz, AreH), 8.31 (2H, q, J ¼ 8.4 Hz, AreH) 13.14 (1H, s, NH);
13C NMR (acetone-d6, 100 MHz)
d: 40.3 (NeCH3), 117.7 (AreC),
123.5, 123.7, 124.1 (AreC), 124.5 (AreC), 124.8 (AreC), 125.0 (AreC),
125.3 (AreC), 125.5 (AreC), 125.8 (AreC), 126.1 (AreC), 126.3 (Are
C), 126.5 (AreC), 127.4 (AreC), 128.3 (AreC), 128.5 (AreC), 128.9
(AreC),129.9 (AreC),130.0 (AreC),130.5 (AreC),131.0 (AreC),131.6
(AreC), 133.6 (AreC), 134.0 (AreC), 134.9 (AreC), 139.8 (AreC),
141.8 (N]C, AreC), 152.8 (N]CeN, AreC); MS m/z: 580 (ESꢀ), 582
(ESþ) (M þ 2); HR-MS (ESꢀ) calcd for C29H20BrN5O2S, 581.0521;
found, 580.0452.
6.1.6.18. 2-[4-(5-Nitro-1H-benzimidazol-2-yl)phenyl]-4-methyl-3-
phenyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide
(12g). Light brown powder; Yield: 85%; FT-IR (KBr): 3456, 3093,
1748, 1647, 1509, 1438, 1340, 1248, 1171, 1062, 850, 772 cmꢀ1 1H
;
NMR (CDCl3, 400 MHz)
d: 2.92 (3H, s, NeCH3), 7.09 (1H, d,
J ¼ 8.0 Hz, AreH), 7.30 (1H, t, J ¼ 3.2 Hz, AreH), 7.34e7.41 (2H, dt,
J1 ¼ 2.0 Hz, J2 ¼ 8.0 Hz, J3 ¼ 16.4 Hz, AreH), 7.48 (2H, q, J ¼ 8.0 Hz,
AreH), 7.55e7.60 (4H, dd, J1 ¼ 8.0 Hz, J2 ¼ 16.4 Hz, AreH), 7.90 (1H,
t, J ¼ 8.8 Hz, AreH), 7.94 (1H, t, J ¼ 8.4 Hz, AreH), 8.01 (2H, d,
J ¼ 8.0 Hz, AreH), 8.09 (1H, t, J ¼ 11.2 Hz, AreH), 8.45 (1H, s, AreH),
6.1.6.15. 2-[4-(1H-Benzimidazol-2-yl)phenyl]-4-methyl-3-phenyl-
2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine
Brown powder; Yield: 85%; FT-IR (KBr): 2998, 2935, 1661, 1598,
1496, 1437, 1347, 1276, 1171, 1062, 887, 746 cmꢀ1 1H NMR (CDCl3,
400 MHz)
5,5-dioxide
(12d).
;
d
: 2.89 (3H, s, NeCH3), 7.03 (1H, d, J ¼ 8.0 Hz, AreH), 7.13
13.13 (1H, s, NH); 13C NMR (CDCl3, 100 MHz)
d: 40.3 (NeCH3),120.0
(2H, q, J ¼ 3.2 Hz, AreH), 7.18 (1H, d, J ¼ 7.2 Hz, AreH), 7.26e7.33
(4H, dd, J1 ¼ 8.0 Hz, J2 ¼ 20.4 Hz, AreH), 7.38 (1H, t, J ¼ 7.2 Hz, Are
H), 7.46 (2H, d, J ¼ 8.4 Hz, AreH), 7.50 (1H, t, J ¼ 8.0 Hz, AreH), 7.90
(2H, t, J ¼ 7.6 Hz, AreH), 8.11 (2H, d, J ¼ 8.4 Hz, AreH), 8.16 (1H, d,
(AreC), 123.5 (AreC), 123.7 (AreC), 124.2 (AreC), 124.6 (AreC),
124.8 (AreC), 124.9 (AreC), 125.1 (AreC), 125.1 (AreC), 125.2 (Are
C), 125.5 (AreC), 126.7 (AreC), 127.6 (AreC), 127.9 (AreC), 128.3
(AreC), 128.5 (AreC), 129.0 (AreC), 129.2 (AreC), 129.5 (AreC),
129.8 (AreC),131.0 (AreC),131.6 (AreC),133.1 (AreC),134.2 (AreC),
135.0 (AreC), 139.8 (AreC), 141.9 (N]C, AreC), 152.9 (N]CeN, Are
C); MS m/z: 547 (ESꢀ), 549 (ESþ); HR-MS (ESꢀ) calcd for
J ¼ 8.4 Hz, AreH) 13.10 (1H, s, NH); 13C NMR (CDCl3, 100 MHz)
d:
40.0 (NeCH3), 115.4 (AreC), 115.9 (AreC), 123.1 (AreC), 124.2 (Are
C), 124.4 (AreC), 124.7 (AreC), 124.9 (AreC), 125.2 (AreC), 125.3
(AreC), 125.4 (AreC), 126.1 (AreC), 126.8 (AreC), 127.4 (AreC),
127.6 (AreC), 128.0 (AreC), 128.9 (AreC), 129.1 (AreC), 129.2 (Are
C), 129.5 (AreC), 129.8 (AreC), 130.4 (AreC), 132.4 (AreC), 132.9
(AreC), 135.9 (AreC), 139.5 (AreC), 140.6 (N]C, AreC), 150.6 (Are
C), 152.7 (N]CeN, AreC); MS m/z: 502 (ESꢀ), 504 (ESþ); HR-MS
(ESꢀ) calcd for C29H21N5O2S, 503.1416; found, 502.1357.
C29H20N6O4S, 548.1267; found, 547.1182.
6.1.6.19. 2-[4-(5-Methoxy-1H-benzimidazol-2-yl)phenyl]-4-methyl-3
phenyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide
(12h). Brown powder; Yield: 84%; FT-IR (KBr): 3100, 3089, 1662,
1478, 1369, 1349, 1232, 1160, 1063, 1006, 970, 848, 774, 748 cmꢀ1
;
1H NMR (CDCl3, 300 MHz)
d: 2.91 (3H, s, NeCH3), 3.70 (3H, s, Oe
6.1.6.16. 2-[4-(5-Methyl-1H-benzimidazol-2-yl)phenyl]-4-methyl-3-
phenyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide
(12e). Off white powder; Yield: 82%; FT-IR (KBr): 3084, 2982,
CH3), 6.80 (1H, t, J ¼ 9.0 Hz, AreH), 7.03 (1H, d, J ¼ 6.0 Hz, AreH),
7.27 (1H, s, AreH), 7.33 (2H, t, J ¼ 5.7 Hz, AreH), 7.39 (2H, t,
J ¼ 5.7 Hz, AreH), 7.43e7.50 (4H, m, AreH), 7.90 (1H, t, J ¼ 5.7 Hz,
AreH), 7.96 (2H, q, J ¼ 5.1 Hz, AreH), 8.04 (1H, t, J ¼ 5.7 Hz, AreH),
8.13 (1H, d, J ¼ 6.0 Hz, AreH), 13.11 (1H, s, NH); 13C NMR (CDCl3,
1736, 1663, 1597, 1437, 1347, 1229, 1170, 1062, 848, 772 cmꢀ1 1H
;
NMR (CDCl3, 400 MHz) d: 2.81 (3H, s, CeCH3), 2.89 (3H, s, NeCH3),
7.02 (1H, q, J ¼ 8.4 Hz, AreH), 7.26e7.34 (2H, m, AreH), 7.41 (1H, t,
J ¼ 8.0 Hz, AreH), 7.47 (1H, t, J ¼ 8.0 Hz, AreH), 7.52 (1H, t,
J ¼ 8.4 Hz, AreH), 7.56e7.59 (4H, dd, J1 ¼1.2 Hz, J2 ¼ 8.8 Hz, AreH),
7.60e7.64 (1H, dt, J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz, J3 ¼ 16.4 Hz, AreH), 7.90
(1H, t, J ¼ 8.0 Hz, AreH), 7.94 (2H, d, J ¼ 7.2 Hz, AreH), 7.99 (1H, t,
J ¼ 8.4 Hz, AreH), 8.08 (1H, d, J ¼ 8.4 Hz, AreH), 13.05 (1H, s, NH).
75 MHz) d: 40.4 (NeCH3), 55.8 (OeCH3),123.0 (AreC),123.5 (AreC),
123.7 (AreC), 124.2 (AreC), 124.6 (AreC), 124.8 (AreC), 125.0 (Are
C), 125.0 (AreC), 125.1 (AreC), 125.2 (AreC), 125.5 (AreC), 126.8
(AreC), 127.8 (AreC), 127.9 (AreC), 128.2 (AreC), 128.4 (AreC),
129.0 (AreC), 129.1 (AreC), 129.3 (AreC), 129.8 (AreC), 130.6 (Are
C), 131.4 (AreC), 133.0 (AreC), 134.4 (AreC), 134.8 (AreC), 137.8
(AreC), 141.9 (N]C, AreC), 151.7 (N]CeN, AreC); MS m/z: 532
13C NMR (CDCl3, 100 MHz)
d: 29.7 (CeCH3), 40.4 (NeCH3), 117.6

116
S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
(ESꢀ), 534 (ESþ); HR-MS (ESꢀ) calcd for C30H23N5O3S, 533.1522;
(AreC), 124.2 (AreC), 124.3 (AreC), 124.4 (AreC), 124.5 (AreC),
124.8 (AreC), 125.2 (AreC), 125.4 (AreC), 125.6 (AreC), 126.2 (Are
C), 126.8 (AreC), 127.6 (AreC), 127.9 (AreC), 128.3 (AreC), 128.8
(AreC), 129.0 (AreC), 129.2 (AreC), 129.3 (AreC), 129.9 (AreC),
131.4 (AreC), 133.1 (AreC), 134.9 (AreC), 135.2 (AreC), 139.6 (Are
C), 141.5 (N]C, AreC), 151.6 (N]CeN, AreC), 162.8 (C]O); MS m/z:
574 (ESꢀ), 576 (ESþ); HR-MS (-ES) calcd for C32H25N5O4S, 575.1627;
found, 574.1542.
found, 532.1440.
6.1.6.20. 4-Methyl-3-phenyl-2-[4-(9H-purin-8-yl)phenyl]-2,4-
dihydropyrazolo[4,3c][1,2]benzothiazine
5,5-dioxide
(12i).
Yellowish powder; Yield: 85%; FT-IR (KBr): 3348, 3069, 2854, 1660,
1614, 1487, 1436, 1369, 1345, 1233, 1171, 1062, 1006, 969, 851, 773,
747 cmꢀ1 1H NMR (CDCl3, 300 MHz)
; d: 2.91 (3H, s, NeCH3), 7.15
(1H, d, J ¼ 5.7 Hz, AreH), 7.36 (1H, d, J ¼ 5.4 Hz, AreH), 7.42 (1H, t,
J ¼ 5.4 Hz, AreH), 7.48e7.55 (2H, m, AreH), 7.65 (1H, t, J ¼ 6.6 Hz,
AreH), 7.76 (1H, d, J ¼ 6.0 Hz, AreH), 7.90 (1H, t, J ¼ 5.4 Hz, AreH),
7.97 (2H, d, J ¼ 5.4 Hz, AreH), 8.02 (1H, t, J ¼ 5.7 Hz, AreH), 8.16 (1H,
d, J ¼ 6.3 Hz, AreH), 8.35 (1H, d, J ¼ 6.3 Hz, AreH), 8.98 (1H, s, ¼Ne
CeH), 9.15 (1H, s, ¼NeCeH), 11.76 (1H, s, NH); 13C NMR (CDCl3,
6.2. Determination of AChE and BuChE inhibitory activity
Ellman’s method was used to evaluate the inhibitory activities
of newly synthesized compounds against EeAChE and equine
BuChE [40]. The inhibitors studied were first dissolved in DMSO
(final concentration of DMSO was 1% in the assay) and assayed at
a 0.5 mM final concentration for initial screening. The com-
pounds exhibiting more than 50% inhibition were further tested
by making eight to ten serial dilutions in assay buffer (50 mM
TriseHCl, 0.1 M NaCl and 0.02 M MgCl2, pH 8.0). Composition of
75 MHz) d: 40.0 (NeCH3), 123.7 (AreC), 124.3 (AreC), 124.4 (AreC),
124.6 (AreC), 124.8 (AreC), 125.0 (AreC), 125.4 (AreC), 126.8 (Are
C), 127.6 (AreC), 127.9 (AreC), 128.2 (AreC), 128.5 (AreC), 128.8
(AreC), 129.0 (AreC), 129.2 (AreC), 129.4 (AreC), 129.8 (AreC),
130.5 (AreC), 132.4 (AreC), 133.0 (AreC), 134.2 (AreC), 135.0 (Are
C), 139.8 (AreC), 142.1 (N]C, AreC), 145.5 (N]CeN, AreC), 155.5
(N]C, AreC); MS m/z: 504 (ESꢀ), 506 (ESþ); HR-MS (ESꢀ) calcd for
the reaction mixture was: 20
mL assay buffer, 10 mL of test com-
pound and 10 L of enzymes (0.5 and 3.4 U/mg of EeAChE or
m
C
27H19N7O2S, 505.1321; found, 504.1246.
equine BuChE, respectively). A 10 min-pre-incubation of the re-
action mixture at 25 ꢂC was carried out. At the end of the pre-
6.1.6.21. 2-[4-(1,3-Benzoxazol-2-yl)phenyl]-4-methyl-3-phenyl-2,4-
dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide (12j).
Brown powder; Yield: 83%; FT-IR (KBr): 3085, 2963, 1651, 1596,
1490, 1437, 1371, 1236, 1170, 845, 772 cmꢀ1 1H NMR (CDCl3,
400 MHz)
incubation period, 10 mL of 1 mM acetylthiocholine iodide or
butyrylthiocholine chloride were added to the respective EeAChE
or equine BuChE enzyme solution to start the enzymatic re-
actions. The mixtures were incubated for 15 min at 25 ꢂC. The
change in the absorbance was measured at 405 nm to determine
the amount of the product using a microplate reader (Bio-Tek
ELx800). Neostigmine and donepezil were used as standard in-
hibitors. Assays were performed with a blank containing all of
the components except enzyme or substrate in order to account
for a non-enzymatic reaction. Each concentration was analysed in
triplicate. Enzyme dilution buffer consisted of 50 mM TriseHCl
buffer containing 0.1% (w/v) bovine serum albumin (BSA) and pH
8.0. The linear regression parameters were determined for each
curve and the IC50 values were measured. The computer program
GraphPad Prism 5.0 (San Diego, CA, USA) was used to analyse the
data.
;
d
: 2.89 (3H, s, NeCH3), 7.05 (1H, d, J ¼ 8.0 Hz, AreH), 7.15
(2H, q, J ¼ 6.8 Hz, AreH), 7.21 (1H, d, J ¼ 7.2 Hz, AreH), 7.27e7.32
(4H, dd, J1 ¼ 4.8 Hz, J2 ¼ 8.4 Hz, AreH), 7.38 (1H, t, J ¼ 7.2 Hz, AreH),
7.44 (2H, d, J ¼ 8.4 Hz, AreH), 7.51 (1H, t, J ¼ 8.0 Hz, AreH), 7.90 (2H,
t, J ¼ 7.6 Hz, AreH), 8.11 (2H, d, J ¼ 8.0 Hz, AreH), 8.15 (1H, d,
J ¼ 8.0 Hz, AreH); 13C NMR (CDCl3, 100 MHz)
d: 40.3 (NeCH3), 123.4
(AreC), 123.7 (AreC), 124.2 (AreC), 124.5 (AreC), 124.7 (AreC),
124.9 (AreC), 125.0 (AreC), 125.1 (AreC), 125.2 (AreC), 125.5 (Are
C), 126.7 (AreC), 127.6 (AreC), 127.9 (AreC), 128.2 (AreC), 128.5
(AreC), 128.9 (AreC), 129.1 (AreC), 129.4 (AreC), 129.8 (AreC),
130.9 (AreC), 131.5 (AreC), 133.1 (AreC), 134.2 (AreC), 134.7 (Are
C), 139.7 (AreC), 141.8 (N]C, AreC), 150.9 (AreC), 151.8 (N]CeO,
AreC); MS m/z: 503 (ESꢀ); HR-MS (ESꢀ) calcd for C29H20N4O3S,
504.1256; found, 503.0575.
6.3. Docking
6.1.6.22. Ethyl 2-(4-(4-methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo
[4,3-c][1,2]thiazin-2(4H)-yl)phenyl)-1H-benzo[d]imidazole-5-
carboxylate (12k).
The X-ray structures of human AChE (PDB ID 4BDT) [41] bound
to huprine W and of human BuChE (PDB ID 4BDS) [41] bound to
tacrine were obtained from the RCSB Protein Data Bank [42].
AutoDock 4.2 [43] was used for flexible ligand docking into the
template X-ray structures. The side chain of residue Tyr337 in the
active site of AChE was also treated flexibly during docking while all
other residues were fixed in their crystallographic conformation.
For BuChE, where residue Ala328 replaces Tyr337 in AChE, the
entire active site was kept rigid. Prior to the calculations, crystal-
lographic water and ligand molecules were removed from the
template structures and hydrogen atoms were added using the
Molecular Operating Environment (MOE 2012.10) [44]. AutoDock
Tools [43] was used to add atomic partial charges. Selected hybrid
molecules of pyrazolobenothiazines and benzimidazoles were
docked into the active sites of AChE and BuChE using the La-
marckian Genetic Algorithm in AutoDock 4.2. High-scoring docking
poses were selected as putative binding modes on the basis of vi-
sual inspection.
2-(4-(4-Methyl-5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]
thiazin-2(4H)-yl)phenyl)-1H-benzo[d]imidazole-5-carboxylic acid
(12a) (1.0 g) was dissolved in ethanol (20 mL) and a catalytic
amount of conc. H2SO4 was added. The reaction mixture was
allowed to reflux for 30e40 min. Completion of the reaction was
confirmed by TLC. The solvent was removed under vacuum. The
resulting residue was dissolved in ice cold water and a pH of 7e8
was adjusted by using 5% NaHCO3. The precipitate formed was
filtered, excessively washed with water and dried. Off white pow-
der; yield: 71%; FT-IR (KBr): 3060, 2928, 2858, 1713, 1608, 1505,
;
1453, 1350, 1282, 1184, 1061, 848, 771 cmꢀ1 1H NMR (CDCl3,
300 MHz)
d: 1.34 (3H, t, J ¼ 5.7 Hz, CeCH3), 2.97 (3H, s, NeCH3), 4.22
(2H, q, J ¼ 5.7 Hz, OeCH2), 7.03 (1H, d, J ¼ 5.7 Hz, AreH), 7.36 (1H, t,
J ¼ 5.7 Hz, AreH), 7.27e7.32 (2H, dd, J1 ¼4.5 Hz, J2 ¼ 10.2 Hz, AreH),
7.55e7.60 (2H, dd, J1 ¼ 4.2 Hz, J2 ¼ 10.8 Hz, AreH), 7.63 (1H, d,
J ¼ 5.1 Hz, AreH), 7.69 (1H, t, J ¼ 6.6 Hz, AreH), 7.87e7.91 (3H, m,
AreH), 7.94 (1H, d, J ¼ 5.4 Hz, AreH), 8.02 (1H, d, J ¼ 5.7 Hz, AreH),
8.08 (1H, d, J ¼ 6.6 Hz, AreH), 8.26 (1H, t, J ¼ 5.7 Hz, AreH), 8.32
(1H, d, J ¼ 6.9 Hz, AreH), 8.49 (1H, s, NH); 13C NMR (CDCl3, 75 MHz)
Acknowledgements
S.A. acknowledges the financial support by HEC, Pakistan for
research visit to School of Chemistry, University of Manchester.
d: 29.7 (CH3), 40.4 (NeCH3), 55.0 (OeCH2),114.6 (AreC),116.8,123.8

S. Aslam et al. / European Journal of Medicinal Chemistry 78 (2014) 106e117
117
U.H. Danielson, J. Nevts, V. Cecchetti, Journal of Medicinal Chemistry 56 (2013)
2270e2282.
[22] N. Tomita, Y. Hayashi, S. Suzuki, Y. Oomori, Y. Aramaki, Y. Matsushita,
M. Iwatani, H. Iwata, A. Okabe, Y. Awazu, O. Isono, R.J. Skene, D.J. Hosfield,
H. Miki, T. Kawamoto, A. Hori, A. Baba, Bioorganic & Medicinal Chemistry
Letters 23 (2013) 1779e1785.
We also acknowledge School of Chemistry, University of Man-
chester for spectral analyses of compounds. N.F. is supported by a
fellowship from the Jürgen Manchot Foundation, Düsseldorf,
Germany.
[23] A. Barazarte, G. Lobo, N. Gamboa, J.R. Rodrigues, M.V. Capparelli, A. Alvarez-
Larena, S.E. Lopez, J.E. Charris, European Journal of Medicinal Chemistry 44
(2009) 1303e1310.
Appendix A. Supplementary data
[24] M. Ahmad, H.L. Siddiqui, J.M. Gardiner, M. Parvez, S. Aslam, Medicinal
Chemistry Research 22 (2013) 794e805.
[25] M. Ahmad, H.L. Siddiqui, M. Zia-ur-Rehman, M. Parvez, European Journal of
Medicinal Chemistry 45 (2010) 698e704.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.035.
[26] A.R. Porcari, R.V. Devivar, L.S. Kucera, J.C. Drach, L.B. Townsend, Journal of
Medicinal Chemistry 41 (1998) 1252e1262.
References
[27] T. Roth, M.L. Morningstar, P.L. Boyer, S.H. Hughes, R.W. Buckheit, C.J. Michejda,
Journal of Medicinal Chemistry 40 (1997) 4199e4207.
[1] E. Giacobini, International Journal of Geriatric Psychiatry 18 (2003) S1eS5.
[2] C. Geula, M.M. Mesulam, Alzheimer Disease and Associated Disorders
(Suppl. 2) (1995) 23e28.
[3] D.K. Lahiri, M.R. Farlow, K. Sambamurti, N.H. Greig, E. Giacobini, L.S. Schneider,
Current Drug Targets 4 (2003) 97e112.
[4] H. Soreq, S. Seidman, Nature Reviews Neuroscience 2 (2001) 294e302.
[5] N.H. Greig, T. Utsuki, Q. Yu, X. Zhu, H.W. Holloway, T. Perry, B. Lee,
D.K. Ingram, D.K. Lahiri, Current Medical Research and Opinion 17 (2001)
159e165.
[6] D.H. Small, C.A. McLean, Journal of Neurochemsitry 73 (1999) 443e449.
[7] D.H. Small, S.S. Mok, J.C. Bornstein, Nature Reviews Neuroscience 2 (2001)
595e598.
[8] M. Mesulam, A. Guillozet, P. Shaw, B. Quinn, Neurobiology of Disease 9 (2002)
88e93.
[9] S. Darvesh, D.L. Grantham, D.A. Hopkins, Journal of Comparative Neurology
393 (1998) 374e390.
[10] S. Darvesh, D.A. Hopkins, C. Geula, Nature Reviews Neuroscience 4 (2003)
131e138.
[11] U. Neumann, M. Gütschow, Bioorganic Chemistry 23 (1995) 72e88.
[12] A. Stößel, M. Schlenk, S. Hinz, P. Küppers, J. Heer, M. Gütschow, C.E. Müller,
Journal of Medicinal Chemistry 56 (2013) 4580e4596.
[13] G. Manina, M.R. Pasca, S. Buroni, E. De Rossi, G. Riccardi, Current Medicinal
Chemistry 17 (2010) 3099e3108.
[14] R.R. Gupta, P.K. Dev, M.L. Sharma, C.M. Rajoria, A. Gupta, M. Nyati, Anticancer
Drugs 4 (1993) 589e592.
[15] S. Sabatini, G.W. Kaatz, G.M. Rossolini, D. Brandini, A. Fravolini, Journal of
Medicinal Chemistry 51 (2008) 4321e4330.
[16] A. Martelli, G. Manfroni, P. Sabbatini, M.L. Barreca, L. Testai, M. Novelli,
S. Sabatini, S. Massari, O. Tabarrini, P. Masiello, V. Calderone, V. Cecchetti,
Journal of Medicinal Chemistry 56 (2013) 4718e4728.
[17] M. Ahmad, S.U.F. Rizvi, H.L. Siddiqui, S. Ahmad, M. Parvez, R. Suliman, Me-
dicinal Chemistry Research 21 (2012) 2340e2348.
[18] J. Wang, D. Limburg, J. Carter, G. Mbalaviele, J. Gierse, M. Vazquez, Bioorganic
& Medicinal Chemistry Letters 20 (2010) 1604e1609.
9
[28] M.T. Migawa, J.-L. Girardet, J.A. Walker, G.W. Koszalka, S.D. Chamberlain,
J.C. Drach, L.B. Townsend, Journal of Medicinal Chemistry 41 (1998) 1242e1251.
[29] S.R. LaPlante, A. Jakalian, N. Aubry, Y. Bousquet, J.M. Ferland, J. Gillard,
S. Lefebvre, M. Poirier, Y.S. Tsantrizos, G. Kukolj, P.L. Beaulieu, Angewandte
Chemie International Edition 43 (2004) 4306e4311.
[30] W.A. Denny, G.W. Rewcastle, B.C. Baguley, Journal of Medicinal Chemistry 33
(1990) 814e819.
[31] J.S. Kim, B. Gatto, C. Yu, A. Liu, L.F. Liu, E.J. LaVoie, Journal of Medicinal
Chemistry 39 (1996) 992e998.
[32] J. Valdez, R. Cedillo, A. Hernandez-Campos, L. Yepez, F. Hernandez-Luis,
G. Navarrete-Vazquez, A. Tapia, R. Cortes, M. Hernandez, R. Castillo, Bioorganic
& Medicinal Chemistry Letters 12 (2002) 2221e2224.
[33] Y. Kohara, K. Kubo, E. Imamiya, T. Wada, Y. Inada, T. Naka, Journal of Medicinal
Chemistry 39 (1996) 5228e5235.
[34] T. Fekner, J. Gallucci, M.K. Chan, Journal of the American Chemical Society 126
(2004) 223e236.
[35] P.W. Elsinghorst, W. Härtig, D. Gündisch, K. Mohr, C. Tränkle, M. Gütschow,
Current Topics in Medicinal Chemistry 11 (2011) 2731e2748.
[36] C. Galdeano, E. Viayna, P. Arroyo, A. Bidon-Chanal, J.R. Blas, D. Muñoz-Torrero,
F.J. Luque, Current Pharmaceutical Design 16 (2010) 2818e2836.
[37] P.W. Elsinghorst, W. Härtig, S. Goldhammer, J. Grosche, M. Gütschow, Organic
& Biomolecular Chemistry 7 (2009) 3940e3946.
[38] M. Ahmad, H.L. Siddiqui, M. Azam, I.H. Bukhari, M. Parvez, Acta Crystallog-
raphica E66 (2010) o616.
[39] M. Ahmad, H.L. Siddiqui, U.F. Rizvi, S. Ahmad, M. Parvez, Acta Crystallog-
raphica E66 (2010) o862.
[40] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Feather-Stone, Biochemical
Pharmacology 7 (1961) 88e95.
[41] F. Nachon, E. Carletti, C. Ronco, M. Trovaslet, Y. Nicolet, L. Jean, P.Y. Renard,
Biochemical Journal 453 (2013) 393e399.
[42] P.W. Rose, C. Bi, W.F. Bluhm, C.H. Christie, D. Dimitropoulos, S. Dutta,
R.K. Green, D.S. Goodsell, A. Prlic, M. Quesada, G.B. Quinn, A.G. Ramos,
J.D. Westbrook, J. Young, C. Zardecki, H.M. Berman, P.E. Bourne, Nucleic Acids
Research 41 (2013) D475eD482.
[43] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, Journal of Computational Chemistry 30 (2009) 2785e2791.
[44] Molecular Operating Environment (MOE), (2012).10; Chemical Computing
Group Inc., Montreal, Canada.
[19] M. Zia-ur-Rehman, J.A. Choudary, S. Ahmad, H.L. Siddiqui, Chemical and
Pharmaceutical Bulletin 54 (2006) 1175e1178.
[20] P.K. Sharma, S.N. Sawhney, Bulletin of the Chemical Society of Japan 66 (1993)
3843e3846.
[21] M.L. Barreca, G. Manfroni, P. Leyssen, J. Winquist, N. Kaushik-Basu,
J. Paeshuyse, R. Krishnan, N. Iraci, S. Sabatini, O. Tabarrini, A. Basu,