Molecular docking and antiviral activity of N-substituted benzyl/phenyl-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetamides

Article abstract of DOI:10.1016/j.bmcl.2015.01.007

Two series of fifteen N-substituted benzyl/phenyl-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetamides were screened for anti-HIV-1 activity and cytotoxicity. The compounds 6a, 6d, 6e, 6g and 6i from the series 6a-i of benzylami

Full text of DOI:10.1016/j.bmcl.2015.01.007

Accepted Manuscript
Molecular docking and anti-viral screening of N-substituted benzyl/phenyl-2-
(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acet-
amides
Matloob Ahmad, Sana Aslam, Syed Umar Farooq Rizvi, Muhammad
Muddassar, Usman Ali Ashfaq, Catherine Montero, Olivia Ollinger, Mervi
Detorio, John. M. Gardiner, Raymond F. Schinazi
PII:
S0960-894X(15)00009-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.01.007
BMCL 22347
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 August 2014
3 January 2015
5 January 2015
Please cite this article as: Ahmad, M., Aslam, S., Farooq Rizvi, S.U., Muddassar, M., Ashfaq, U.A., Montero, C.,
Ollinger, O., Detorio, M., Gardiner, n.M., Schinazi, R.F., Molecular docking and anti-viral screening of N-
substituted
benzyl/phenyl-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetamides,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.01.007
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Molecular docking and anti-viral screening of N-
substituted benzyl/phenyl-2-(3,4-dimethyl-5,5-
dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-
yl)acetamides
Leave this area blank for abstract info.
Matloob Ahmad, Sana Aslam, Syed Umar Farooq Rizvi, Muhammad Muddassar, Usman Ali Ashfaq, Catherine
Montero, Olivia Ollinger, Mervi Detorio, John M. Gardiner* and Raymond F. Schinazi
R
R
HN
HN
N
O
N
N
O
N
6g
6i
N
N
S
S
O
O
O
O
6
7
9 examples
EC50 = 16.7-57.5 µM
6 examples
EC50 = 6.3-7.6 µM
Benzylamides 6g and 6i docked to HIV-1 NNRTI
binding site, superimposition with nevirapine (yellow)

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Molecular docking and anti-viral screening of N-substituted benzyl/phenyl-2-
(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetamides
Matloob Ahmad,a Sana Aslam,b Syed Umar Farooq Rizvi,c Muhammad Muddassar,d Usman Ali Ashfaq,e
∗
Catherine Montero,f Olivia Ollinger,f Mervi Detorio,f John. M. Gardinerg, and Raymond F. Schinazif
aDepartment of Chemistry, Government College University, Faisalabad-38000, Pakistan.
bDepartment of Chemistry, Government College Women University, Faisalabad, Pakistan.
cInstitute of Chemistry, University of the Punjab, Lahore-54590, Pakistan.
dDepartment of Biosciences, COMSATS Institute of Information Technology, Park Road, Islamabad, Pakistan.
eDepartment of Bioinformatics and Biotechnology, Government College University, Faisalabad-38000, Pakistan.
fLaboratory of Biochemical Pharmacology, Emory University School of Medicine/Veterans Affairs Medical Center, 1760 Haygood Drive, Atlanta, GA 30322,
USA.
gSchool of Chemistry and Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, UK.
The research work is dedicated to Prof. Dr. Hamid Latif Siddiqui (Late) who was our beloved teacher (MA, SA & SUFR) and our friend and colleague (MD,
JMG & RFS).
ARTICLE INFO
ABSTRACT
Article history:
Received
Two series of fifteen N-substituted benzyl/phenyl-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-
c][1,2]benzothiazin-2(4H)-yl)acetamides were screened for anti-HIV-1 activity and cytotoxicity.
The compounds 6a, 6d, 6e, 6g and 6i from the series 6a-i of benzylamides and 7a, 7b, 7c, 7d
and 7e from the series 7a-f of anilides were identified as effective anti-HIV-1 agents with EC50
values < 20 µM. Among these compounds that displayed anti-HIV-1 activity, 6a, 6e, 6g and 6i
showed no toxicity in human PBM, CEM and Vero cells, with the exception of 6a which
displayed toxicity in Vero cells. Molecular docking of these compounds provided insight into
the molecular mechanism and it was found that 6e, 6g and 6i bound deeply in the NNRTI
binding pocket of the HIV-1 reverse transcriptase, using RT-bound nevirapine X-ray data and
molecular docking for validation, showing the potential of these new structures as inhibitors of
this viral enzyme.
Revised
Accepted
Available online
Keywords:
Pyrazolobenzothiazine
anti-HIV activity
1,2-benzothiazine 1,1-dioxide
Reverse Transcriptase
Cytotoxicity
2009 Elsevier Ltd. All rights reserved.
Pyrazolobenzothiazines are based on the benzothiazine
skeleton, many structurally-varied derivatives of which exhibit
yl)acetamides,15
yl)methylidene-4-hydroxy-2H-1,2-benzothiazine-3-
N'-(substituted-2-chloroquinolin-3-
a
diverse range of biological activities. Various
pyrazolobenzothiazine derivatives have been reported to be
carbohydrazides 1,1-dioxides to be effective antioxidants6 and
pyrazolobenzothiazine-based chalcones as antibacterials.16
1,2
antipyretic,
antihypertensive,3
analgesic
and
anti-inflammatory,
anti-oxidant
anti-depressant,4
and
N
HN
F
antibacterial agents.5,6 Recently, compounds based on this ring
system have been reported as inhibitors of HCV replication (a,
Figure 1)7 and as anti-HIV agents (b, Figure 1).8
H
N
N
N
Cl
S
N
NHSO2Me
S
O
O
N
O
N
S
O
N
Other derivatives of benzothiazines have been reported to
act as anti-inflammatory,9 antimalarial,10 antiallergic,11 anti-
thrombotic,12 antidepressants13 and antibacterial agents.14 In
our prior work on other 1,2-benzothiazines, we have reported
N'-arylmethylidene-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-
O
O
H
Cl
(a, EC50 = 3.6 µM)
(b, EC50 = 3.2 µM)
Figure 1. Structures of antiviral agents belonging to
pyrazolobenzothiazine ring system.7,8
c][1,2]benzothiazin-2(4H)-yl
substitutedbenzyl/phenyl-2-(3,4-dimethyl-5,5-
acetohydrazides,5
N-
Human immunodeficiency virus (HIV) is responsible for
Acquired Immunodeficiency Syndrome (AIDS) and is treated
d—io—xid—opyrazolo[4,3-c][1,2]benzothiazin-2(4H)-
∗ Corresponding author. Tel.: +44-161-306-4530; e-mail: gardiner@manchester.ac.uk

by combination antiretroviral therapy.17,18,19,20
Such
decreasing electronegativity and increasing atomic size of the
substituent. Among methoxy-substituted compounds, the
following order was observed; 2-MeO > 4-MeO > 3-MeO with
EC50 values less than 20 µM. Moreover other electron donating
group e.g. methyl substitution at 4-position in compound 6i
showed significant anti-HIV-1 activity with EC50 of 9.9 µM
whereas 6h (2-Me) was totally inactive with EC50 greater than
100 µM.
antiretroviral therapy uses a combination of various antiviral
drugs and such therapies have now converted AIDS from a
routinely fatal, to
a chronic disease which can be
controlled.21,22 However, there remains important impetus for
the discovery of new anti-HIV leads to address the future risks
of resistance to current generations of drugs.23 Recently, we
have evaluated pyrazolobenzothiazine derivatives family
having hydrazone moiety in the side chain as anti-HIV agents.8
Herein we report the further extension of this previous work by
exploring anti-HIV-1 potential of the titled carboxamides
(Scheme 1) providing new SAR information and low-toxicity
agents with good anti-HIV activity.
In the series of anilides (7a-f), compounds 7a, 7b, 7c, 7d
and 7e were effective inhibitors of HIV-1. It was found that
compound 7a bearing an unsubstituted phenyl moiety
displayed comparable anti-HIV-1 activity (EC50 of 6.8 µM) to
the halogen-substituted compounds (7b-7d) and the mono-
methoxy bearing 7e. Compound 7d having 3-bromo moiety
showed the most potent inhibitory activity against HIV-1
amongst these compounds with an EC50 of 6.3 µM, whilst the
2-bromo derivative 7c and methoxy-substituted compound 7e
both exhibited similar activities with EC50 values of 6.5 µM
and 7.2 µM, respectively. Whilst Table 1 illustrates that the
amide derivatives with either no or one aryl substituent
(electron-withdrawing substituents such as chloro and bromo
or an electron-donating methoxy group) had significantly
higher (at least 10-fold) anti-HIV activities than 7f containing
two electron-donating groups. It is worthwhile to mention here
that all anti-HIV compounds from anilide (7a-e) series were
significantly toxic to normal human PBM, CEM and Vero cell
lines.
Pyrazolobenzothiazine 5,5-dioxide precursor
1
was
prepared from sodium saccharine as a starting material,5,24 and
thence to the target compounds 6a-i and 7a-f, as previously
reported (Scheme 1).15
Scheme 1. Synthesis of N-substituted benzyl/phenyl-2-(3,4-dimethyl-5,5-
OH
O
OH
O
NH
N
(ii)
(i)
NH
N
N
S
S
S
O
O
O
O
O
O
(1)
(2)
(3)
MeO2C
HO2C
N
N
N
N
Compounds were also evaluated for cytotoxicity in primary
human PBM, CEM and Vero cells to determine their spectrum
of toxicity. CEM cells are a line of lymphoblastic cells
originally derived from a child with acute lymphoblastic
leukemia whereas Vero cells are derived from African Green
monkey kidney cells. In the series 6a-i, none of compounds
showed cytotoxicity in primary human PBM cells except 6f,
which was toxic to all the three cell systems used. In addition,
6c was toxic to CEM cells and 6a to Vero cells and 6d was
toxic to both CEM and Vero cells. However, among the series
of compounds 7a-f, five compounds i.e. 7a-e showed acute
toxicity in primary human PBM, CEM and Vero cells, with the
exception of 7e which showed no toxicity in Vero cells.
Compound 7f showed no toxicity in the cell systems used with
IC50 values greater than 100 µM.
(iii)
(iv)
N
S
N
S
O
O
O
O
(4)
(v)
(5)
(vi)
R
R
HN
HN
O
N
O
N
N
N
N
N
S
S
O
O
O
O
To propose a mode of inhibitory action, these synthetic
compounds were investigated by docking into the HIV-1
reverse transcriptase NNRTI drug-binding site. The best fit
compounds 6e, 6g and 6i were selected on the bases of their
anti-HIV activity and lower toxicity. The best conformations
from docking of the compounds were selected on the basis of
their Fred chemguass score25 shown in Table 2 and visual
inspection. Fred docking tool uses the shapes and implicit
solvent interaction terms to pose and score small molecules in
protein binding sites. Interestingly, we have found a good
relationship between docking scores and anti-HIV activities of
these compounds which would be consistent with their
predicted plausible binding modes shown in figure 2. The
compound 6i, 6e and 6g possessed the best scores in
descending order. Compound 6i having methyl group
substitution at the tail phenyl ring was ranked best with Fred
docking score of -13.775. Similarly nevirapine, a potent
clinical anti-HIV NNRTI compound, was also docked with the
NNRTI binding pocket of HIV-1 reverse transcriptase to
validate the docking procedure (Figure 3). In docking
(6a-i)
(7a-f)
a R = 2-Cl
b R = 2-F
c R = 4-F
d R = 2-Br
e R = 2-OMe
f R = 3-OMe
g R = 4-OMe
h R = 2-Me
i R = 4-Me
a R = H
b R = 2-F
c R = 2-Br
d R = 3-Br
e R = 3-OMe
f R = 2,4-(OMe)2
dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetamides. Reagents:
(i) (CH3)2SO4, NaOH(aq) Acetone (ii) N2H4.H2O, EtOH (iii)
,
BrCH2COOMe/K2CO3; DMF (iv) NaOH (aq), MeOH (v) Borane-THF
complex/Ar-CH2-NH2; toluene-THF (1:2) (vi) SOCl2; Ar-NH2.
All the fifteen compounds belonging to both series i.e. (6a-i
& 7a-f) were tested for their anti-HIV-1 activity and ten
compounds exhibited moderate activity (Table 1). Among the
benzylamides (6a-i), five compounds i.e. 6a, 6d, 6e, 6g and 6i
were found to be active against HIV-1 with EC50 values less
than 20 µM. It was observed that among halo-substituted
derivatives electronegativity and atomic size of the substituent
play an important role i.e. the activity enhanced with

simulations, we observed that the compounds efficiently
docked in the binding pocket of HIV-1 reverse transcriptase,
thus consistent with their experimental anti-HIV activity.
Table 1. Anti-HIV-1 and cytotoxic activity of N-substituted benzyl/phenyl-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-
c][1,2]benzothiazin-2(4H)-yl)acetamides.
Anti-HIV-1 Activity in Human PBM cells* (µM)
Cytotoxicity* (IC50, µM)
Code
R
EC50
EC90
PBM
CEM
Vero
6a
6b
6c
6d
6e
6f
2-Cl
2-F
15.9
57.5
22.4
16.7
17.2
21.1
18.6
≥100
9.9
28.5
≥100
≥100
87.5
60.3
92.0
≥100
74.2
87.5
>100
63.1
≥100
18.7
>100
>100
52.2
7.7
68.4
82.0
41.9
38.1
>100
28.8
>100
>100
>100
<1.0
9.5
38.7
96.1
>100
36.5
>100
31.1
73.0
>100
63.1
6.8
4-F
2-Br
2-OMe
3-OMe
4-OMe
2-Me
4-Me
H
6g
6h
6i
77.7
16.3
35.4
15.7
15.3
17.3
7a
7b
7c
7d
7e
7f
6.8
2-F
7.6
18.0
2.4
28.2
8.2
2-Br
6.5
6.0
3-Br
6.3
8.1
5.2
7.2
3-OMe
2,4-(OMe)2
7.2
10.5
>100
9.3
≥100
>100
>100
>100
AZT
0.0033
0.031
>100
14.3
56.0
*All experiments were conducted in replicate.
Table 2.
Docking scores of anti-HIV-1 compounds with HIV-1 reverse
transcriptase.
Post-docking analysis also indicated that compound 6g, 6e
and 6i had good hydrophobic interactions with Leu100,
Val106, Tyr-181, and Tyr-188. All these compounds have
been gorged well in the hydrophobic binding pocket of HIV-1
reverse transcriptase, shown in figure 2. We have also
observed that these compounds have t-type π-π stacking
between phenyl ring of head group and Trp-229 residue,
which might be responsible for their good binding affinity
with HIV-1 reverse transcriptase.
Sr. No.
Compound
Fred Chemguass Score
-13.644
-12.323
1
2
3
4
6e
6g
6i
-13.775
Nevirapine
-14.104
Figure-2: Binding modes of the compound 6e (magenta), 6g (cyan) and 6i (purple) in HIV-1
reverse transcriptase NNRTI binding pocket. Nevirapine (Yellow) is the co-crystal structure with in HIV-1 reverse transcriptase.

Docking of nevirapine with HIV-1 reverse transcriptase
showed that it had interactions with Leu100, Val106, Glu138,
Tyr181, Tyr188, Gly190, Phe227 and Trp229 (Figure 3). These
results are consistent with prior work and with the X-ray data for
this drug and RT. 26,27,28
incubator at 37 °C with 5% CO2 for human PBM cells. The
results are also summarized in Table I.
Molecular Docking To further evaluate the mechanism of
antiviral activity of the reported compounds, a cheminformatics
based approach was used employing molecular docking
simulations to understand the mechanism of antiviral activity.
The three-dimensional crystal structure of the HIV-1 reverse
transcriptase was obtained from the Protein Data Bank using
PDB ID: 3V8132. The 3D structure of the downloaded protein
was prepared by removing the water molecules and other
heteroatoms from the structure using PDB2RECEPTOR utility
implemented in Openeye software suits. Similarly Openeye Fred
docking tool was used for docking simulations and its FRED
Chemgauss4 score was used for ranking the poses of each docked
compound. Maestro software was used for the drawing and 3D
conversion of the structures of synthesized compounds. Prior to
docking simulations, 200 conformations of each compound were
generated using the OMEGA software. The binding modes of the
compounds were analyzed using the Pymol visualization
program.
Figure 3: Superposition of docked pose (Green) of Nevirapine over its co-
crystal structure (Yellow).
In conclusion, most of the compounds evaluated were active
as anti-HIV-1 agents i.e., 6a, 6d, 6e, 6g and 6i from the series
6a-i of benzylamides and 7a, 7b, 7c, 7d, 7e from the series 7a-h
of anilides. Among these compounds that displayed anti-HIV-1
activity, 6e, 6g and 6i showed no toxicity in primary human
PBM, CEM and Vero cells. Furthermore, the mechanism of
effective antiviral compounds was proposed from molecular
docking and interaction analysis studies with HIV-1 RT as the
target of drug action. These evaluations indicate the potential of
the pyrazolobenzothiazine ring system as a lead structure group
for the potential development of new anti-HIV agents, and in
particular would select compounds of type 6 over type 7 based on
the differential toxicity data.
Acknowledgments
The authors (MA, SA, SUFR) are grateful to Higher
Education Commission, Pakistan and Institute of Chemistry,
University of the Punjab, Lahore, for financial assistance. We are
also thankful to School of Chemistry, University of Manchester,
for spectral measurements and SBI laboratory, RIKEN, Japan for
docking simulation facilities. This work was also supported in
part by NIH grant 2P30-AI-050409 and the Department of
Veterans Affairs (to RFS).
Supplementary Material
Supplementary data (representative experimental procedures,
characterization data, and copies of spectra) associated with this
article can be found, in the online version, at http://….
Experimental Protocols
Anti-HIV-1 In order to evaluate the synthesized
compounds for their in vitro antiviral effects in primary human
peripheral blood mononuclear (PBM) cells, we used
a
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using at least two different donor cells in duplicate or triplicate.
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