Novel benzothiazine-piperazine derivatives by peptide-coupling as potential anti-proliferative agents

Article abstract of DOI:10.1016/j.bmcl.2016.10.071

In an attempt to develop potential and selective anti-proliferative agents, a series of novel benzothiazine-piperazine derivatives 8a–i and 10a–g were synthesized by coupling of 2H-1,4-benzothiazin-3(4H)-one with various amines 7a–i and 9a–g in excellent yields and evaluated for their in vitro anti-proliferative activity against four cancer cell lines, HeLa (cervical), MIAPACA (pancreatic), MDA-MB-231 (breast) and IMR32 (neuroblastoma). In vitro inhibitory activity indicated that compounds 8a, 8d, 8g, 10a, 10b, 10e, 10f were found to be good anti-proliferative agents. Among them the derivatives 8g, 10e and 10f were found to be the most active members exhibiting remarkable growth inhibitory activity. Molecular docking was undertaken to investigate the probable binding mode and key active site interactions in HDAC8 and EHMT2 proteins. The docking results are complementary to the experimental results.

Full text of DOI:10.1016/j.bmcl.2016.10.071

Accepted Manuscript
Novel benzothiazin-piperazin derivatives by peptide-coupling as potential anti-
proliferative agents
Ramineni Venkatesh, Suresh Kasaboina, Deepthi Bidayat, Nishant Jain, Saritha
Jostna Tangeda, Rajashaker Bantu, Sridhara Janardhan, Lingaiah Nagarapu
PII:
S0960-894X(16)31109-X
http://dx.doi.org/10.1016/j.bmcl.2016.10.071
BMCL 24374
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
28 July 2016
6 October 2016
24 October 2016
Please cite this article as: Venkatesh, R., Kasaboina, S., Bidayat, D., Jain, N., Jostna Tangeda, S., Bantu, R.,
Janardhan, S., Nagarapu, L., Novel benzothiazin-piperazin derivatives by peptide-coupling as potential anti-
proliferative agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.10.071
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Bioorganic & Medicinal Chemistry Letters
Novel benzothiazin-piperazin derivatives by peptide-coupling as potential
anti-proliferative agents
Ramineni Venkatesha, Suresh Kasaboinaa, Deepthi Bidayata, Nishant Jainb, Saritha Jostna Tangedad,
Rajashaker Bantua, Sridhara Janardhanc*, Lingaiah Nagarapua*
b
aOrganic Chemistry Division-II (CPC), CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad- 500007, India. Centre for Chemical Biology,
CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500007, India. cCentre for Molecular Modeling, CSIR-Indian.Institute of Chemical
Technology, Tarnaka, Hyderabad 500007, India. dMedicinal Chemistry Division, S. N. V. Pharmacy Maha Vidyalay, Tarnaka, Hyderabad-500017, India.
Fax: + 91 40 27193382; Tel: +91 40 27191509; E-mail: lnagarapuiict@yahoo.com/nagarapu@iict.res.in
ARTICLE INFO
ABSTRACT
In an attempt to develop potential and selective anti-proliferative agents, a series of novel
benzothiazin-piperazin derivatives 8a-i and 10a-g were synthesized by coupling of 2H-1,4-
benzothiazin-3(4H)-one with various amines 7a-i and 9a-g in excellent yields and evaluated for
their in vitro anti-proliferative activity against four cancer cell lines, HeLa (cervical),
MIAPACA (pancreatic), MDA-MB-231 (breast) and IMR32 (neuroblastoma). In vitro inhibitory
activity indicated that compounds 8a, 8d, 8g, 10a, 10b, 10e, 10f were found to be good anti-
proliferative agents. Among them the derivatives 8g, 10e and 10f were found to be the most
active members exhibiting remarkable growth inhibitory activity. Molecular docking was
undertaken to investigate the probable binding mode and key active site interactions in HDAC8
and EHMT2 proteins. The docking results are complementary to the experimental results.
2009 Elsevier Ltd. All rights reserved.
Article history:
Received
Revised
Accepted
Available online
Keywords:
Peptide-coupling,
Anti-proliferative activity,
Molecular docking,
Benzothiazin-piperazin,
HDAC8,
EHMT2.
Nitrogen-containing heterocycles have always played a
major role in the pharmaceutical and agrochemical industries
because of their potent physiological properties which have
resulted in numerous applications.1 Benzothiazine derivatives are
an important class of natural products possessing a wide range of
biological and pharmaceutical activity due to presence of
nitrogen and sulphur axis, which is considered to be responsible
as one of the structural features to impart their activities.2, 3 They
are widely found in bioorganic and medicinal chemistry with
application in drug discovery. Benzothiazole moieties are part of
compounds showing numerous biological activities such as
antimicrobial,4-6 anticancer,7-9 anthelmintic,10 anti-diabetic11,
antihypertensive12 and an allosteric muscarine M1 receptor13.
They have also found application in industry such as anti-
oxidants, vulcanizations accelerators. Various benzothiazoles
such as 2-aryl benzothiazole received much attention due to
unique structure, and its use as radioactive amyloid imaging
agents14 and anticancer agents.15 It must be emphasized that
combination of benzothiazoles with other heterocyclic is a well
known approach to design new drug like molecules, which
allows achieving new pharmacological profile, activity and
toxicity lowering.
benzothiazines and piperazines, it was perceived that if both the
heterocyclic moieties are synergized in a single nucleus, the new
compounds obtained were likely to possess significant biological
activities. Recently, in an on-going program to discover and
develop potent new anti-proliferative agents, we have identified
several classes of molecules as novel tumor growth inhibitors.18-22
In an attempt to design new anticancer agents, we discovered
novel benzothiazin-piperazin derivatives 8a-i and 10a-g to elicit
combined antitumor efficacy/cytotoxicity against different cancer
cell lines in vitro.
Further, we have performed literature search for
investigation of enzymatic inhibition of benzothiazinone
derivatives for the cancer treatment. Histone-lysine N-
methyltransferase 2 (EHMT2, G9a) is a histone modifying
enzyme that specifically mono- and dimethylates 'Lys-9' of
histone H3 (H3K9me1 and H3K9me2, respectively) in
euchromatin.23 EHMT2 is over expressed in breast, prostate,
colon, bladder, ovarian, melanoma, lung, and liver cancers.24
Methylation of histones play a vital role in epigenetic
modification of chromatin that determines gene expression,
genomic stability, stem cell maturation, cell lineage development,
genetic imprinting, DNA methylation, and cell mitosis.25 Hence,
the dysregulation of EHMT2 plays an important role in the
growth regulation of cancer cells and it is considered a promising
therapeutic target for various types of cancer.26 The PUBCHEM
assays against EHTM2 inhibition have been reported for several
ChEBML compounds (eg. 1574417, 1548773, 589694, 1585706,
1459005, 1460888, 1541342, 1330971, 1481724 and 1539403)
having benzothiazinone moiety.27
The Piperazines are a broad class of chemical compounds
with many important pharmacological properties. This dinitrogen
moiety has been an inseparable component of plethora of drugs.
Piperazines have the chemical similarity with piperidine, a
constituent of piperazine in the black piper plant (Piper nigrum).
Piperazine was introduced into the medicine as a solvent for uric
16,17
acid.
Keeping in view the potential biological activities of

Histone deacetylase 8 (HDAC8) is a class I histone
deacetylase implicated as a therapeutic target for cancer. HDAC8
plays a vital role in transcriptional regulation, cell cycle
progression, developmental events anlong with the anti-parasitic
agents. In cancer, HDAC8 is a major 'epigenetic player' which is
linked to deregulated expression or interaction with transcription
factors critical to tumorigenesis. HDAC8 alters chromosome
structure and affects transcription factor access to DNA. It
catalyzes the deacetylation of lysine residues on the N-terminal
of the core histones (H2A, H2B, H3 and H4) and represses
transcription in large multiprotein complexes with transcriptional
co-repressors.28 Marek et.al., have been co-crystallized [PDB ID:
4BZ8]
(2R)-2-methyl-3-oxo-4H-1,4-benzothiazine-
6-
carbohydroxamic acid moiety with Schistosoma mansoni
HDAC8 protein. They have investigated that benzothiazinone
interfere with schistosome epigenetic mechanisms have a
capacity to induce apoptosis, mortality along with anti-parasitic
property. Hence, benzothiazine moiety can be investigated as
novel anti-cancer drugs and as new anti-parasitic agents.29
Molecular docking is a process can be used to find
energetically most stable position and orientation of ligand in a
protein cavity. The poses are prioritized based on their binding
strength for a given target and can be measured based on their
non-covalent interactions, rotational flexibility, size and shape.
Docking follows first pose prediction followed by score the poses
using either empirical or force field based methods by performing
flexible or rigid docking. Pharmacophore modeling is a 3D
arrangement of features in set of aligned conformation of known
active compounds provides a hypothetical active site, which is
essential for a drug-like molecule to exert a particular biological
effect. Hence, in our present work we have performed molecular
docking study on a series of benzothiazine-piperazin derivatives
in both EHMT2 and HDAC8 proteins for understanding key
active site residues and explored all probable binding modes.
Also we have performed pharmacophore modeling to understand
important structural features that are responsible for better ligand
binding and can be used for virtual screening30-33. The identified
structural features from docking and pharmacophore modeling
could help in rational design of benzothiazin-piperazin
derivatives with enhanced potency for both EHMT2 and HDAC8
proteins in cancer treatment.
Scheme 1.Synthesis of novel benzothiazin-piperazin derivatives 8a-i, 10a-g.
Pharmacology
The in vitro anti-proliferative activity of the designed
compounds 8a-i and 10a-g was evaluated against four different
human cancer cell lines HeLa (cervical), MIAPACA (pancreatic),
MDA-MB-231 (breast) and IMR32 (neuroblastoma) summarized
in Table 1. The compounds were picked for an advanced assay
against four human cancer cell lines at five concentrations (0.01,
0.1, 1, 10, 100 µM). GI50 (growth inhibitory activity) was
calculated and these values corresponds to concentration of the
compound causing 50% decrease in net cell growth as compared
with standard Doxorubicin, Paclitaxel drugs. Results were
calculated for each of these parameters if the level of activity was
reached; however, if the effect was not achieved, the value was
expressed as greater or less than the maximum or minimum
concentration tested.
Based on the Table 1, all the synthesized compounds
showed significant cancer cell growth inhibition with GI50 values
ranging from <0.01 to >100 µM. In particular, the compounds
8a, 8d, 8g, 10a, 10b, 10e, 10f showed promising anti-
proliferative activity against the four human cancer cell lines.
Among them, compound 8e showed potent anti-proliferative
activity against the three human cancer cell lines He La
(cervical), MDA-MB-231 (breast), and IMR32 (neuroblastoma)
with GI50 values of 0.12, 0.63, and 0.83 µM, respectively.
Compounds 8g and 10f exhibited good growth inhibition, in
particular the neuroblastoma cell line and breast cancer cell lines
respectively with GI50 values in the range of <0.01 µM as
compared with the reference drugs Doxorubicin and Paclitaxel.
We observed that the substitution of simple phenyl ring played an
important role in exhibiting the anti-proliferative activity.
The synthesis of 3-(3-oxo-2H-benzo[b][1,4]thiazin-
4(3H)-yl)propanoic acid (6)34 was achieved by reaction of 2-
amino thiophenol (1) with chloroacetic acid (2) and sodium
acetate in ethanol at reflux temperature for 6 h. The obtained
solid, 2H-benzo[b][1,4]thiazin-3(4H)-one (3) was filtered and
dried. It was dissolved in DMF, K2CO3, ethyl 3-bromopropionate
(4) and DMAP (4-Dimethylaminopyridine) were added and
stirred overnight. The obtained product, ethyl 3-(3-oxo-2H-
benzo[b][1,4]thiazin-4(3H)-yl)propionate (5) was extracted with
chloroform. The above compound 5 was treated with 1N KOH-
EtOH (1:1) for 30 min to obtain 3-(3-oxo-2H-
benzo[b][1,4]thiazin-4(3H)-yl)propanoic acid (6) in 75.6% yield.
Subsequently, benzothiazin-piperzine derivatives 8a-i and 8a-g
have been achieved in excellent yields by the coupling of acid
compound
6
with N-phenylpiperzines 7a-i and 3-(4-
phenylpiperazin-1-yl)propan-1-amines 9a-g35 using EDC.HCl
and HOBt in DCM at room temperature for 12 h, as shown in
1
Scheme 1 and their structures were confirmed by H NMR, 13C
NMR, ESI-MS and IR spectral analysis.37

Molecular Modeling
Docking studies
AAHHR. Alignment of the set of active compounds from the
dataset using pharmacophore hypothesis (AAHHR) is shown in
Figure 1.
The dataset compounds were docked into the prepared
3SFH and 3K5K crystal structures using Glide module of
Schrödinger suite.36 The prepared protein structures were used
for grid generation using the default value of protein atom scaling
(1.0) within a cubic box centered on the co-crystal ligand. No
constraints were imposed and extra precision (XP) flexible
docking of ligands was carried out with default value of ligand
atom scaling (0.8). The post docking minimization has been
carried out and maximum of 10 poses per ligand was saved. The
obtained docked complex structures were analyzed and the
compounds were prioritized by using docking score, interactions
with active site residues.
Molecular Docking:
In order to explore the binding mode and understanding
of key active site residues, molecular docking study has been
performed. The synthesized benzothiazin-piperazin hybrids were
docked into the active site of HDAC8 and EHMT2 proteins. In
the present study, the docking result of compound 10e in HDAC8
and EHMT2 proteins are discussed.
HDAC8: From the docking study, we observed that the non-
covalent hydrophobic interactions as well as electrostatic
interactions are more predominating for strong binding towards
HDAC8 protein. The oxygen of 3-oxo-2,3-dihydro-
benzo[1,4]thiazin-4-yl moiety was coordinated to Zn+2 along with
aspartic acid (Asp178) and histidine residue (His180). The
carbonyl oxygen of acetamide moiety showed electrostatic
interaction with methionine (Met 274, 4.00 Å) and histidine
(His180, 3.06 Å) residues. The aromatic phenyl ring of 4H-
benzo[1,4]thiazin-3-one showed strong hydrophobic interactions
with tyrosine (parallel-displaced <π>-<π> stacking; Tyr306),
tryptophan (parallel-displaced <π>-<π> stacking; Trp141) and
isoleucine (Ile34) residues. Thiomorpholinyl propyl moiety of the
molecule was involved in hydrophobic interactions with
phenylalanine (Phe208, Phe152) residues.
Pharmacophore modeling
In order to identify the essential structural features of
the ligand responsible for its potency, we have generated a ligand
based pharmacophore model from a dataset comprising of 8a-i
and 10a-g compounds using HeLa cell-line activity. A total of
428 conformations thus obtained were used to generate
pharmacophore hypotheses. The series of compounds have
shown maximum variation in their growth inhibition (pGI50: 4 to
6.92). The common pharmacophore hypothesis (CPH) was
obtained based on 11 as highly active compounds (pGI50 ≥ 4.5)
from the dataset and then inactive molecules (pGI50 < 4.5) were
aligned with it. The identified best CPH consists of two acceptors
(A), two hydrophobic (H) and one aromatic (R) features i.e
Table-1: GI50 (µM)a values of the tested compounds against four human cancer cell lines.)
S.No
Compound
HeLa
MIAPACA
MDA-MB-
IMR32
IMR32
pGI50
(HeLa)
5.08
5.38
4
231
1
2
8.4±0.2
4.2±0.1
>100
68.1±0.2
>100
5.6±0.9
1.0±0.0
>100
0.34±0.0
46.4±0.2
>100
0.34±0.0
46.4±0.2
>100
8a
8b
3
51.5±0.6
56.8±0.4
>100
8c
4
17.7±0.8
5.4±0.4
61.0±0.6
41.9±0.8
11.9±0.4
18.5±0.4
>100
3.8±0.3
2.2±0.6
1.6±0.0
5.4±0.1
6.6±0.2
6.0±0.3
>100
0.1±0.0
10.0±0.1
>100
0.1±0.0
10.0±0.1
>100
4.75
5.27
4.21
4.38
4.92
4.73
4
8d
5
8e
6
>100
8f
7
>100
8g
<0.01
<0.01
8
34.9±0.0
>100
>100
>100
8h
9
21.7±0.4
0.46±0.0
>100
21.7±0.4
0.46±0.0
>100
8i
10
11
12
13
14
15
16
17
>100
10a
10b
10c
10d
10e
10f
10g
0.97±0.0
5.7±0.5
1.4±0.0
0.12±0.0
2.17±0.8
56.2±0.3
59.2±0.5
>100
2.0±0.0
20.4±0.1
4.6±0.4
0.63±0.1
<0.01
6.01
5.24
5.85
6.92
5.66
4.25
7.08
1.4±0.0
1.4±0.1
0.82±0.0
>100
1.4±0.0
1.4±0.1
0.82±0.0
>100
>100
83.3±0.8
>100
>100
27.6±0.2
1.5±0.0
1.5±0.0
0.025±0.003
Doxorubicin 0.083±0.001 0.095±0.004 0.073±0.0025 0.025±0.003
18
7.41
Paclitaxel 0.039±0.004 0.06±0.002 0.092±0.0031 0.086±0.004
0.086±0.004
aGI50: 50% Growth inhibition, concentration of drug resulting in a 50% reduction in net rotein increase compared with control cells.
bPositive control. pGI50 (HeLa): -log [GI50 (HeLa)] used for pharmacophore modeling

EHMT2: From the docking results, we observed that the
hydrophobic interactions were more predominating than
electrostatic interactions for binding of the molecules. The active
site was mostly composed of the hydrophobic residues which
includes tyrosine (Tyr1085, Tyr1067), phenyl alanine (Phe1110,
Phe1138, Phe1158, Phe1087), proline (Pro1121, Pro1011), and
isoleucine (Ile1136, Ile1009, Ile1120). The oxygen of 3-oxo-2,3-
dihydro-benzo[1,4]thiazin-4-yl moiety showed electrostatic
interactions with surrounding arginine residue (Arg1157) and
nitrogen of propionamide linker between 3-oxo-2,3-dihydro-
benzo[1,4]thiazin-4-yl moiety and thiomorpholine moiety
showed hydrogen bond interaction with aspartic acid (Asp1083,
3.22 Å). The sulphur atom of thiomorpholine moiety showed
hydrogen bond interaction with aspartic acid (Asp1074, 3.26) and
nitrogen atom thiomorpholine moiety oriented towards another
aspartic acid (Asp1078, 4.54 Å). The aromatic phenyl ring of 4H-
benzo[1,4]-thiazin-3-one showed strong hydrophobic interactions
with tyrosine (parallel-displaced π-π stacking, Tyr1154), phenyl
alanine (T-shaped π-π stacking, Phe1158) and also interacting
with other two phenyl alanine residues (Phe1087, Phe1152). The
probable binding mode of most potent compound 8e of the
benzothiazin-piperazin hybrids in EHMT2 and HDAC8 is shown
in Figure 2. The active site residues around 4Å of the most potent
compound 8e of the series are shown in Table 2.
HDAC8
EHMT2
Figure 2: The probable binding mode compound 10e (orange) in the active
site of HDAC8 and EHMT2 and the hydrogen bond interactions are shown in
pink colour dotted lines, metal Zn+2 in HDAC8 is shown in yellow colour, the
important residues are shown in green colour and protein cartoon is
represented in wheat colour. The 2D protein-ligand interaction diagram is
shown in the picture.
In conclusion, a new series of benzothiazin-piperazin
derivatives 8a-i and 10a-g have been synthesized and evaluated
for their anti-proliferative activity. The most active compounds of
8a, 8d, 8g, 10b, 10e and 10f displayed broad spectrum antitumor
activity against HeLa, MDA-MB-231 and IMR32 cancer cell
lines at the GI50 levels, together with a mild to moderate
cytotoxic activity. The computational techniques such as
pharmacophore modeling and molecular docking studies have
been applied to support our experimental results. We have been
identified five featured pharmacophore (AAHHR) and explored
the probable binding mode and identified key active site residues
around potent compound 10e in HDAC8 and EHMT2 proteins.
The preliminary results could offer an excellent framework in
this field that may lead to the discovery of new potent antitumor
agents.
A
Aromatic
Hydrogen bond Acceptor
B
Acknowledgements
Hydrophobic
The authors gratefully acknowledged to DST-
SERB/EMEQ-078/2013 for financial assistance. RV thanks
CSIR, New Delhi for award of research fellowship. We thank to
Dr. G. Narahari Sastry, Centre for Molecular modeling, CSIR-
IICT, Hyderabad for his valuable suggestions and support.
Figure 1. A: Common pharmacophore hypothesis (AAHHR) and its
pharmacophoric distances (Å) are shown in pink colour. B: Alignment of
active data set compounds on AAHHR.
References and notes
Table 2: The active site residues around 4Å of the most potent
compound 10e.
1. Hagan, D. O.; Nat. Prod. Rep. 2000, 17, 435.
2. Morgenstern, H.; Glazer, W. M.; Wagner, R.; Doucette.
J.; Keyzer, H.; Eckert, G. M.; Forrest, I. S.; Gupta, R.
R.; Gutmann, F.; Molnar, J.; Malabar, F. L.; Krieger
Publishing Co.USA. 1992, 379.
Compound 10e (active site residues around
Target
4Å)
ILE34, ARG37, GLY140, TRP141, HIS142,
HIS143, GLY151, PHE152, CYS153, ASP178,
HIS180, PHE208, GLN263, ASP267, MET274,
GLY303, GLY304, GLY305, TYR306, ILE34,
ARG37, GLY140, TRP141, HIS142, HIS143,
GLY151, PHE152, CYS153, ASP178, HIS180,
PHE208, GLN263, ASP267, MET274, GLY303,
GLY304, GLY305, TYR306
TYR1067, ASP1074, ALA1077, ASP1078,
ASP1083, SER1084, TYR1085, LEU1086,
PHE1087, ASP1088, CYS1098, PHE1152,
TYR1154, ARG1157, PHE1158, ILE1161
3. Gupta, R.; Gupta, A.; Hetero Letters. 2011, 1(4), 351.
4. Murthi, Y.; Pathak, D.; J. Pharm. Res. 2008, 7(3), 153.
5. Rajeeva, B.; Srinivasulu, N.; Shantakumar, S.; Eur. J.
Chem.2009, 6(3), 775.
6. Maharan, M.; William, S.; Ramzy, F.; Sembel, A.;
Molecules. 2007, 12. 622.
7. Stanton, H. L. K.; Gambari, R.; Chung, H. C.; Johny, C.
O. T.;Filly, C.; Albert. S. C. C.; Bioorg. Med. Chem.
2008, 16, 3626.
HDAC8
EHMT2

8. Wang, M.; Gao, M.; Mock, B.; Miller, K.; Sledge, G.;
Hutchins, G.; Zheng, Q.; Bioorg. Med. Chem. 2006, 14,
8599.
9. Hutchinson, I.; Chua, M. S.; Browne, H. L.; Trapani, V.;
Bradshaw, T. D.; Westwell, A. D.; J. Med. Chem. 2001,
44, 1446.
10. Sreenivasa, M.; Jaychand, E.; Shivakumar, B.;
Jayrajkumar, K.; Vijaykumar, J.; Arch. Pharm. Sci. and
Res. 2009, 1(2), 150.
11. Pattan, S.; Suresh, C.; Pujar, V.; Reddy, V.; Rasal, V.;
Koti, B.; Ind. J. Chem. 2005, 44B, 2404.
28. Buggy, J. J.; Sideris, M. L.; Mak, P.; Lorimer, D. D.;
McIntosh, B.; Clark, J. M.; Biochem. J. 2000. 350. 199-
205. Chakrabarti, A.; Oehme, I.; Witt, O.; Oliveira, G.;
Sippl, W.; Romier, C.; Pierce, R. J.; Jung, M.; Trends
Pharmacol Sci. 2015, Jul. 36(7), 481-92.
29. Marek, M.; Kannan, S.; Hauser, A.; Moraes Mourao, M.;
Caby, S.; Cura, V.; Stolfa, D.A.; Schmidtkunz, K.;
Lancelot, J.; Andrade, L.; Renaud, J.; Oliveira, G.; Sippl,
W.; Jung, M.; Cavarelli, J.; Pierce, R.J.; Romier, C, Plos
Pathog, 2013, 9, 03645.
30. Janardhan, S.; Srivani, P.; Sastry, G. N.; QSAR and
Combinatoria Science. 2006, 25, 860.
31. Janardhan, S.; Srivani, P.; Sastry, G. N.; Curr. Med.
Chem. 2006, 13, 1169.
12. Violetta, C.; Fausto, S.; Oriana, T.; Arnaldo, F.;
Bioorganic & Medicinal Chemistry Letters. 2000, 10,
465 - 468.
13. Anette, G. S.; Morten, H.; Gitte, K. M.; Krestian, L.;
32. Badrinarayan, P.; Sastry, G. N.; Comb. Chem.High
Throughput Screen. 2011, 14, 840.
33. Reddy, A. S.; Pati, S. P.; Kumar, P. P.; Pradeep, H. N.;
Sastry, G. N.; Curr. Protein Pept. Sci. 2007, 8, 329.
34. Chande, M. S; & Suryanarayan, V.; Chemistry of
Heterocyclic Compounds. 2003, 39, 1094.
35. Nagarapu, L.; Saidulu, K.; Srujana, R.; Bhaskar, K.;
Rajesh Kumar, M.; Vijayacharan, G.; Akkewar, D. M.;
Bioorg. Med. Chem.Lett. 2015, 7, 1643.
36. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T.
A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E.
H.;Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S.; J. Med.Chem. 2004, 47, 1739.
Christoffer, B.; Niels, P.; Claus, T.; Benny, B. A.; J.
Med. Chem. 2010. 53, 6386 - 6397.
14. Reddy, P.; Lin, Y.; Chang, H.; Arcivoc, xvi, 2007, 113.
15. Heo, Y.; Song, Y.; Kim, B.; Heo. J.; Tetrahedron
Letters. 2006, 47, 3091.
16. Atherden. L. H.; ‘Bentley and Driver’s Text Book of
Pharmaceutical
Chemistry’
Oxford
Medicinal
Publication 8th Edition. 2004, 372, 204.
17. Beckett, A. H.; and Stenlake, J. B.; 2003, ‘Practical
Pharmaceutical Chemistry’ CBS Publishers and
Distributors, IVth edition.
37. General procedure for the synthesis of 4-(3-oxo-3-(4-
phenylpiperazin-1-yl) propyl)- 2H-benzo[b] [1,4]
thiazin-3(4H)-one derivatives (8a-i and 10a-g): To the
stirred solution of 3-(3-oxo-2H-benzo[b][1,4]thiazin-
4(3H)- yl)propanoic acid (100 mg, 0.448 mol) in DMF,
N- substituted piperzines 7a-i (0.538 mol) and 3-(4-
substitutedpiperazin-1-yl)propan-1-amine 9a-g (0.538
mol) was added at room temperature, followed by the
18. Venkatesh, R.; Suresh, K.; Gaikwad, H. K.; Janardhan,
S.; Bantu, R.; Nagarapu, L.; Sastry, G. N.; Banerjee, S.
K.; Eur. J. Med. Chem. 2015, 96, 22.
19. Venkatesh, R.; Ramaiah, M. J.; Gaikwad, H. K.;
Janardhan, S.; Bantu, R.; Nagarapu, L.; Sastry, G. N.;
Ganesh, A. R.; Bhadra, M.; Eur. J. Med. Chem. 2015,
94, 87.
20. Nagarapu, L.; Gaikwad, H. K.; Bantu, R.; Manikonda,
S. R.; Eur. J. Med. Chem. 2011, 46, 2152.
addition
of
EDC.HCl
(1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide) (102 mg) and HOBt
(60.5 mg), the stirring was continued for 12 h. After
completion of the reaction (TLC analysis), small amount
of ice cold water (10 mL) was added to the reaction
mixture and then extracted with chloroform (2 x 10 mL).
The chloroform layer was separated, dried over Na2SO4
and evaporated under vacuum to give corresponding
derivatives 8a-i and 10a-g in good yields.4-(3-(4-(4-
Nitrophenyl)piperazin-1-yl)-3-oxopropyl)-2H-
benzo[b][1,4]thiazin-3(4H)-one (8a). Yield 76%; mp
126-129 oC; IR (KBr) υ in cm-1: 1325, 1446, 2868, 2912;
1H NMR (300 MHz, CDCl3): δ 2.79 (t, J = 7.7 Hz, 2H,
CH2), 3.40 (s, 2H, CH2), 3.42-3.51 (m, 4H, 2xCH2), 3.69
(m, 2H, CH2), 3.79 (m, 2H, CH2), 4.33 (t, J = 7.7 Hz, 2H,
CH2), 6.82 (d, J = 9.2 Hz, 2H, Ar-H), 6.99-7.10 (m, 1H,
Ar-H), 7.27 (d, J = 6.4 Hz, 2H, Ar-H), 7.37 (d, J = 7.5
Hz, 1H, Ar-H), 8.14 (d, J = 9.0 Hz, 2H, Ar-H); 13C NMR
(75 MHz, CDCl3): δ 31.2, 31.5, 40.7, 41.9, 44.7, 46.7,
46.9, 112.8, 117.6, 123.7, 125.8, 127.4, 128.5, 139.0,
139.0, 154.2, 165.2, 169.1; ESI MS:m/z = 449 [M+Na]+;
HRMS calculated for C21H22O4N4NaS : 449.1254, Found
:449.1268.
21. Nagarapu, L.; Gaikwad, H. K.; Bantu, R.;. Manda, K. M
.; Kalivendi, S. V.; Med. Chem. Res. 2013, 22, 165.
22. Yadagiri, B.; Holagunda, U. D.; Bantu. R.; Nagarapu, L.;
Kumar, C. G.; Pombala, S. U.; Sridhar, B.; Eur. J. M.
Chem. 2014, 79, 260.
23. Milner, C. M.; Campbell, R. D.; Biochem J. 1993,
290(3), 811–8.
24. Huang, J.; Dorsey, J.; Chuikov, S.; Pérez-Burgos, L.;
Zhang, X.; Jenuwein, T.; Biol Chem. 2010, 285, 9636–
41.
25. Sawan, C.; Herceg, Z.; Ushijima, T.; Herceg, Z.; In
Ushijima T, Herceg Z. Epigenetics and Cancer, Part A,
Boston: Academic Press. 2010, 70, pp. 57–85.
26. Cho, H. S.; Kelly, J. D.; Hayami, S.; Toyokawa, G.;
Takawa, M.; Yoshimatsu, M.; Neoplasia. 2011, 13(8),
676–84.
27. Bento, A. P.; Gaulton, A.; Hersey, A.; Bellis, L. J.;
Chambers, J.; Davies, M.; Krüger, F. A.; Light, Y.; Mak,
L.; McGlinchey, S.; Nowotka, M.; Papadatos, G.; Santos,
R.; and Overington, J. P.; Nucleic Acids Res., 2014, 42,
1083-1090.

N-(3-(4-(4-Nitrophenyl)piperazin-1-yl)propyl)-3-(3-oxo-
2H-benzo[b][1,4] thiazin-4(3H)-yl)propanamide (10a).
Yield 69%; mp 145-148 oC; IR (KBr) υ in cm-1: 825, 1373,
1665, 3006, 3213; 1H NMR (300 MHz, CDCl3): δ 1.08-1.39
(m, 2H, CH2), 2.03 (s, 2H, CH2), 2.35 (bs, 2H, CH2), 2.56
(t, J = 6.5 Hz, 2H, CH2), 2.68 (t, J = 7.4 Hz, 2H, CH2),
3.27-3.33 (m, 2H, CH2), 3.38 (s, 2H, CH2), 3.45 (d, J = 1.2
Hz, 2H, CH2), 3.54 (m, 2H, CH2), 4.29 (t, J = 7.7 Hz, 2H,
CH2), 6.86 (d, J = 9.3 Hz, 1H, Ar-H), 6.99-7.06 (m, 1H, Ar-
H), 7.24 (bs, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 7.35 (d, J = 7.3
Hz, 1H, Ar-H), 8.14 (d, J = 9.3 Hz, 1H, Ar-H), 9.07 (bs,
1H, Ar-H), 9.44 (bs, 1H, Ar-H); 13C NMR (75 MHz,
CDCl3) : δ 33.1, 36.5, 40.5, 43.6, 49.9, 52.9, 56.1, 56.6,
115.9, 116.1, 122.5, 123.6, 124.4, 127.3, 128.3, 132.5,
133.6, 139.0, 139.6, 153.8, 157.1, 165.1, 170.0; ESI MS :
m/z = 484 [M+H]+; HRMS calculated for C24H30O4N5S :
484.2013, Found : 484.2018.
Supplementary data
Copies of 1H NMR and 13C NMR, HRMS spectra of
compounds.

Research Highlights
 A new templates of benzothiazin-piperazin
derivatives 8a-i and 10a-g were efficiently
synthesized.
 Compound 10e obviously suppressed cancer
cell proliferation and migration.
 Compounds 8g, 10f shown GI50 values <0.01
µM against IMR32, MDA-MB-231 cell lines.
 Compound 10e was well docked in HDAC8
and EHMT2 proteins.