FIELD OF INVENTION:
[0001] The present invention is a derivative of the compound of 5Hbenzo[
2,3][1,4]thiazepino[5,6-b] indoles having the formula:
Additionally, the present invention relates to a convenient and selective one-pot multicomponent
reaction process for the preparation of 5H-benzo[2,3][1,4]thiazepino[5,6-b]indoles
and the most active compound (11b) is characterized by the structure:
N
H
N
S
OH
C2H5O
11b
The compound is useful as an anti-hepatocellular carcinogenic agent in the prevention and/or
treatment of liver cancer.
BACKGROUND OF THE INVENTION:
[0002] Liver cancer is one of the leading causes of cancer-related deaths in the world today.
Hepatocellular carcinoma (HCC) is the most common form of liver cancer in adults which
generally starts as a single tumor that grows larger or as many small cancer nodules throughout
the liver. It is still a big challenge for the researchers and there is an immense need of exploration
and development of novel lead compound for cancer therapy.
[0003] For the majority of cancers, chemotherapy has become one of the methods that are being
adopted to treat cancer. Many compounds have been synthesized with this aim, but their clinical
use has been limited by their relatively high risk of toxicity, because they lack specificity and
produce adverse effects related to the impact on rapidly dividing noncancerous cells. Therefore,
N
H
N
S
R2 R3
R4
R1
R1, R2, R3, R4 = -H, -F, -Cl, -Br, -OH, -OCH3, -OC2H5
4
the current scenario highlights the great desideratum for the discovery and development of new
lead compounds of simple structure, exhibiting excellent anti-tumor property with minimal side
effects.
[0004] Indole ring is one of the widely reputed structural units to identify new drug candidate as
anti-proliferative agents. A representative member of this class is sunitinib (1A, Figure 1) which
is currently used in the clinics as a multi-targeting tyrosine kinase inhibitor for the treatment of
renal cell carcinoma (RCC) and gastrointestinal stromal tumor (GIST). Besides, a diverse variety
of Indolylazoles (1B, Figure 1) such as Labradorins 1 & 2 and indolylthiazoles are known for
their cytotoxic activities against human lung cancer. Marine indole alkaloids, Meridianins (1C,
Figure 1) and their synthetic analogues have shown prominent anticancer activities against breast
cancer.
[0005] Sunitinib (previously known as SU11248) is a multi-targeted receptor tyrosine kinase
inhibitor that was the first cancer drug for treatment of renal cell carcinoma (RCC) and
gastrointestinal stromal tumor (GIST).
[0006] Seven-membered heterocycles such as azepines have been the subject of much
investigation among synthetic chemists and pharmacologists not only because of variety of
bioactivities, but also due to some ubiquitous properties of these compounds. Naturally occurring
antimitotic agent colchicine (1D, Figure 1) and its synthetic analogues have been studied
extensively for cancer chemotherapy, however, they lacks in-vivo anticancer efficacy at its
maximum tolerated dose (MTD). The interest towards azepine analogs such as oxazepine,
thiazipine and diazipine, has been increased persistently, since they are a class of totally
synthetic pharmacological agents with psychotropic, antidepressant, anticonvulsant, anticancer,
antibacterial, anti-HIV and other bioactivities. Few scientific reports and patents have been
claimed that 1,4-benzothiazepine (BT-1)(1E, Figure 1) and 1,5-benzothiazepine (BT-2)(1F,
Figure 1) analogs are used for overcoming resistance to anticancer drugs as well as potentiators
for anti-cancer drugs (Figure 1). In addition, acis-stilbene natural product combretastatin A-4
(1G, Figure 1) is a lead compound of vascular disrupting agents targeting tumor blood vessels
which binds to the colchicine site and exerts potent cytotoxicity, particularly due to having a cisconfiguration
linking bridge and two relatively flexible six-membered hydrophobic rings with
the appropriate dihedral angle.
[0007] Moreover, WO 2006117221 A1 relates to the use of azapaullones, particularly in
combination with immunomodulating agents, in the prevention, and/or treatment of pancreatic
autoimmune disorders, e.g. type I diabetes or LADA and neurodegenerative disorders.
[0008] US 2010/0286120 provides a compound having a superior serotonin 5-HT2C receptor
activating action, which is represented by the formula:
5
wherein A is —OR1 or —S(O)pR2, R1 and R2 are the same or different and each is a hydrogen
atom, a hydrocarbon group optionally having substituent(s), or a heterocyclic group to optionally
having substituent(s), and p is 0, 1 or 2, or a salt thereof. The compounds are useful as an agent
for the treatment or prophylaxis of a lower urinary tract symptom, obesity and/or organ prolapse
etc., and the like.
[0009] US20090318412 provides a tricyclic heterocyclic compound having a serotonin 5-HT2C
receptor activation action and the like. A 5-HT2C receptor activator containing a compound
represented by the formula (I):
wherein each symbol is as defined in the specification, or a salt thereof or a prodrug thereof,
useful as a drug for the treatment or prophylaxis of stress urinary incontinence, obesity, pelvic
organ prolapse, and the like.
[0010] WO2008037746 provides new progesterone receptor modulators (I) and (II) which are
(cis)-8-fluorodibenzo[b,f]pyrido[1,2-d]oxazepine-1-amine compounds and uses thereof.
(I) (II)
It relates to (cis)-8-fluorodibenzo[b,f]pyrido[1,2-d] oxazepine-1-amine derivatives that are
modulators of progesterone receptors, to their application in the field of contraception, hormone
replacement therapy (HRT) or therapy of gynaecological disorders, as well as adjuvant therapy
in cancer and other diseases, and to methods for the making and use of such compounds.
[0011] US2009163473 relates to (pyrido/thieno)-[f]-oxazepine-5-one derivatives having the
general
Formula I
6
wherein R1, R2 and R3 are independently H or (C1-4)alkyl; Ar represents a fused thiophene or
pyridine ring optionally substituted with one or more substituents selected from (C1-4)alkyl,
(C1-4)alkyloxy, (C1-4)alkyloxy, (C1-4)alkyl, CF3, halogen, nitro, cyano, NR4R5, NR4COR6,
and CONR4R5; R4 and R5 are independently H or (C1-4)-alkyl; or R4 and R5 form together
with the nitrogen atom to which they are bound a 5- or 6-membered saturated heterocyclic ring,
optionally containing a further heteroatom selected from O, S or NR6; R6 is (C1-4)alkyl; A
represents the residue of a 4-7 membered saturated heterocyclic ring, optionally containing an
oxygen atom, the ring being optionally substituted with 1-3 substituents selected from (C1-
4)alkyl, (C1-4)alkyloxy, hydroxy, halogen and oxo; or a pharmaceutically acceptable salt
thereof. The invention also relates to pharmaceutical compositions comprising said derivatives,
and to the use of these (pyrido/thieno)-[f]-oxazepine-5-one derivatives in the treatment of
neurological diseases and psychiatric disorders which are responsive to enhancement of synaptic
responses mediated by AMPA receptors in the central nervous system.
[0012] US6977252 B1 provides use of compounds represented by the following general formula
[1] or salts or pro drugs thereof:
where R1 represents hydrogen atom or lower alkoxy group; R2 represents hydrogen atom, lower
alkoxy group and optionally substituted phenyl group. The present invention relates to use of
1,4-benzothiazepine derivatives as a drug for overcoming a resistance to an anticancer drug or as
a drug for enhancing an effect of an anticancer drug. The present invention also relates to
treatment for a cancer, and relates to a pharmaceutical composition and a pharmaceutical kit,
each of which is useful particularly for treating a cancer having an acquired resistance to an
anticancer drug. Further, the present invention relates to a method for treating cancer, which
includes administrating an anticancer drug in combination with an agent for overcoming the
resistance to the anticancer drug or an agent for enhancing the effect of the anticancer drug.
[0013] Likewise, EP 0433683 A2 provides the use of benzothiazepine compounds represented
by the following general formula [1]:
7
N
S
Y
O
Q
R
R''
(I)
Wherein, Q is H or halo;R is H, lower alkoxy, lower haloalkyl, CN, lower alkyl or halo; YisOR′,
wherein R′ is H or alkylacyl, and R˝ is 2-[di(lower alkyl)amino]ethyl (R˝1), 3-[di(lower
alkyl)amino]propyl (R˝2), 2-(pyrrolidino)ethyl (R˝3), 3-(pyrrolidino)propyl (R˝4), 2-
(piperidino)ethyl (R˝5), 3-(piperidino)propyl (R˝6), 2-(morpholino)ethyl (R˝7), 3-
(morhpolino)propyl (R˝8) or (N-pyridinium)alkyl with a suitable counter ion being present
(+R˝9-X).The present invention relates to the use of benzothiazepine compounds for preparing a
pharmaceutical composition for treating cancer by potentiating an anti-cancer drug effect in a
subject.
[0014] It is noted that one of our previous patents was filed with the title “Indole fused
pyridooxazepine and its synthesis for the treatment of hepatocellular carcinoma” (Application
No. 201611016493) having a general formula:
N
H
N
O
N
R1
R2
R4
R5
R3
wherein R1, R2, R3, R4, R5 are independently selected from -H, -OH, -Cl, -Br, -F, -OCnH2n+1,
and -CnH2n+1. The present invention relates to our previously filed invention in a manner
thatoxygen containing oxazepinering is isosterically replaced bysulphur containingthiazepine
ringand pyridine ring is replaced by benzene. This modification leads to the formation of 5Hbenzo[
2,3][1,4]thiazepino[5,6-b]indole as a newer chemical entity for the treatment of liver
cancer.
[0015] In view of the above background, there arises an urgent need to develop compounds for
the treatment of hepatocellular carcinoma (Liver cancer) with improved efficacy as well as
decreased side effects.
8
OBJECT OF INVENTION
[0016] The main object of the invention is to develop a compound for the treatment of
hepatocellular carcinoma (Liver cancer) with improved efficacy as well as decreased side effects.
[0017] Another object of the invention is to provide Indole fused benzothiazepines with a
flexible substituted phenyl ring as novel lead compounds for the treatment of hepatocellular
carcinoma (HCC).
SUMMARY OF INVENTION:
[0018] Indole and seven-membered azepine rings have been individually reported for their
potential benefits in the prevention of different cancers.
[0019] Indole fused benzothiazepines incorporated with a flexible substituted phenyl ring were
synthesized by an efficient one-pot multi-component reaction as novel lead compounds for the
treatment of hepatocellular carcinoma (HCC).
[0020] A feasible one pot efficient synthetic approach for the proposed derivatives will make it
cost effective.While considering all the newly synthesized compounds together, it may be
concluded that the fusion of 5H-benzo[2,3][1,4]thiazepino[5,6-b]indoles with substituted
hydrophobic phenyl ring as flexible side chain, establish an important pharmacophoric structure.
[0021] The positions 2, 3 and 4 of the phenyl side chain are the key reactive sites which could be
altered with different groups to elicit valuable anticancer profiles. 3-hydroxy-4-ethoxy phenyl
substituted 5H-benzo[2,3][1,4]thiazepino[5,6-b]indoleled to compound 11b, exhibited
excellentenhancement in cytotoxic potential with GI50= 17.8.
[0022] Substitution on the vacant site of indole nucleus using different derivatives of 2-oxindole
in the reaction procedure, may enhance the scope of the invention and open a new avenue for
further drug discovery.The synthesized compounds containing a novel pharmacophore
incorporating 5H-benzo[2,3][1,4]thiazepino[5,6-b]indoles, have never been synthesized prior to
this invention in our knowledge. Thus the present scaffold may be emerged as an anticancer lead
for the future.
[0023] In further another embodiment, the present invention provides a pharmaceutical
composition comprising an effective amount of compound of 5H-benzo[2,3][1,4]thiazepino[5,6-
9
b]indole and its derivatives or pharmaceutically acceptable salt thereof and a pharmaceutical
acceptable additive.
[0024] In still another embodiment, the present invention provides a use of the compounds of
5H-benzo[2,3][1,4]thiazepino[5,6-b]indole and its derivatives for the preparation of medicament
for preventing and treating Hepatocellular carcinoma.
[0025] In another embodiment, the present invention provides a method for the treatment of
Hepatocellular carcinoma which comprises administering the compound of 5Hbenzo[
2,3][1,4]thiazepino[5,6-b]indole and its derivatives to a mammal.
BRIEF DESCRIPTION OF DRAWINGS:
Figure 1 represents rational approach to design phenyl substituted
5Hbenzo[2,3][1,4]thiazepino[5,6-b]indoles.
Figure 2 represents survival curves of Hep-G2 for 5H-benzo[2,3][1,4]thiazepino[5,6-
b]indoles(2b, 4b, 5b 7b, 8b, 9b, 11b, 13b, 16b) and adriamycin (ADR).
Figure 3 represents microscopic pictures of in-vitro testing of active compound(11b)and
standard drug, adriamycin(ADR)on Human hepatoma cell line Hep-G2.
DETAILED DESCRIPTION OF INVENTION
[0026] In spite of the availability of aplethora of research manifesting the importance of indole
andazepine derivatives as anticancer agents,the indole-fused benzothiazepine rings remains
widely unexplored for cancer therapy to date.Inspired by the promising findings and the rational
approach of drug designing, a novel series of compounds containing an indole fused
benzothiazepine ring system with a flexible six-membered hydrophobic ring have been designed
and synthesized to discover an important pharmacophoric structure with powerful anticancer
potentials. Few representative chemical structures of important compounds possessing indole,
seven-membered ring, benzothiazepine, six-membered flexible ring and our synthesized
prototype containing these fragments have been presented in Figure 1, which endow promising
anticancer potentials.
[0027] Now-a-days, highly efficient and multi-component reactionsstrategies have extensively
been focused for the preparation of compounds of medicinal value. Thus, a feasible one pot
efficient synthetic approach has been developed for the synthesis of5Hbenzo[
2,3][1,4]thiazepino[5,6-b]indolesby exploiting the reaction of 2-oxindole with substituted
aromatic aldehyde and 2-amino thiophenol in presence of conc. HCl.The presented selective
method is well suited for synthesis of pure compounds and delivers novel candidates in a simple
10
way and just in a single-step.Moreover, the possibility of introducing a variety of substituents at
different positions of the 5H-benzo[2,3][1,4]thiazepino[5,6-b]indole ring system can be achieved
by this method.This method is efficient for the substances used in this experiment and has
several advantages including good yields of products, reduced reaction times and environment
friendly.
[0028] A total of nine synthesized compounds have been screened for their anticancer potential
via in vitro anticancer testing on the Hep-G2 cell line. The result demonstrated that
compound11bhas been found to have excellent potential against hepatocellular carcinoma.This
newly synthesized lead structure have broad scope to undergo further structural modifications on
the basis of the structural activity relationship, molecular docking or QSAR modelling to
improve its potency, lipophilicity and to minimize the side-effects. These pharmacophoric
structures can also be tested to other biological activities in future.
N
H
N
S
R2 R3
R4
R1
Designed scafold
1
2
3
4
5
6
[0029] Chemistry:
In an effort to synthesize a total of nine compounds (Table 1), 2-oxindole, an aromatic
aldehyde and 2-amino thiophenol underwent an acid-catalyzed three-component reaction to
constitute a rapid and facile synthesis of 5H-benzo[2,3][1,4]thiazepino[5,6-b]indoles. The onepot
efficient synthetic route andplausiblereaction mechanism are delineated in Scheme
1andScheme 2. The first step in the mechanism is believed to be the condensation between the
aldehyde and 2-amino phenol. The intermediate so formed acts as an electrophile for the
nucleophilic addition on the methylene group of 2-oxindole, particularly through the formation
of enol tautomer. The resulting adduct undergoes condensation between >C=O and NH2 to give
the cyclized product. The mechanism involved is similar to that of modifiedBiginelli reaction.
Finally, structures of the synthesized compounds were established by IR, 1HNMR, 13C
NMR spectroscopy and mass spectrometry.The formation of Indole fused benzothiazepine
derivatives was supported by the presence of -N=C and >C-S-C˂ stretching band (1600-1700
cm-1 and 600-800 cm-1), and absence of –OH stretching band (3500-3600 cm-1) in the IR spectra.
In addition, appearance of two azepinic>CH- peaks in aliphatic region (δ = 1.5-4.0) of 1H NMR
spectra also confirms the formation of thiazepine ring in the reaction. Further, mass spectra were
used to confirm the assigned molecular weight of compounds in form of their stable fragments.
11
NH
HS
O H2N
NH
N
S
+
Conc. HCl, Ethanol
Reflux
5H-benzo[2,3][1,4]thiazepino[5,6-b]indoles
O
H
R3
R1
R1
R3
R4 R4
R R2 2
Scheme 1: One pot efficient synthetic route to the synthesized compounds.
Table 1: Shows the group R of the synthesized compounds:
Compound
code
R1 R2 R3 R4
2b –H –H –OH –H
4b –H –H –Cl –H
5b –H –H –Br –H
7b –H –OCH3 –OCH3 –OCH3
8b –H –H –OCH3 –H
9b –H –OCH3 –H –H
11b –H –OH –OC2H5 –H
13b –H –Cl –H –H
16b –F –H –H –H
12
HS
H2N
O
+
S
OH
H H
H2N
Condensation
H+
_ H2O
S
H2N
H
_ H+
N
O
H
S
N
O
H
H2N
_ H2O
NH
N
S
H
R1
R2
R3
R4
R1
R2
R3
R4
R1
R2
R3
R4
R1
R2 R3
R4
R1
R2
R3
R4
Scheme 2: The plausible reaction mechanism for the synthesized compounds
[0030] General experimental procedures for characterization of synthesized compound:
The chemicals and reagents were procured from Sigma Aldrich Chemicals and used without
further purification. The progress of the reaction was monitored by thin layer chromatography on
silica gel G plates using iodine vapors and UV light as visualizing agents. Melting points was
determined on melting point apparatus and is uncorrected.Infrared (IR) spectra were recorded on
Thermo Scientific Nicolet 6700 FT-IR spectrometerand the values are expressed in cm-1and only
noteworthy absorption levels are listed. Nuclear magnetic resonance spectroscopy (NMR)
spectra were assessed on Bruker 800 MHz NMR spectrometer (1H 800 MHz, 13C 200 MHz)
NMR spectrometers (Rheinstetten, Germany) processed in Topspin-2.1. The chemical shifts are
expressed as δppm. Mass spectroscopy (MS) data was acquired by hybrid triple quadrupolelinear
ion trap mass spectrometer (QTRAP MS) equipped with Turbo Ion Spray electrospray ionization
(ESI) source (3200 QTRAP, Applied Biosystems, Foster City, CA, USA) (MS ranges from
100.0-750.0).The purity of the compound was evaluated by thin layer chromatography by using
various non-aqueous solvents.
[0031] The following examples define the invention by way of illustration which does not limit
the scope of the invention.
Example 1:
A solution of 2-oxindole (0.40 g, 3.0 mmol), appropriate aromatic aldehyde (3.0 mmol) and 2-
amino thiophenol(0.375 g, 3.0 mmol) in methanol (15 mL) with catalytic amount of conc. HCl
(1.5 mL) was placed in 100 mL round bottom flask and heated under reflux for 10-12 h
approximately. After completion of the reaction, the mixture was allowed to stand at room
13
temperature overnight. The solid products so-formed was collected by filtration, dried and
recrystallized with methanol to obtain 5H-benzo[2,3][1,4]thiazepino[5,6-b]indoleand its
derivatives.
The progress of reaction was monitored by thin layer chromatography (TLC), using the solvent
system 40% ethyl acetoacetate: n-hexane. TLC revealed just a single spot which proved the
presence of a single product.
The structure of synthesized compounds was confirmed by nuclear magnetic resonance
spectroscopy (NMR, 1H and 13C), mass spectroscopy (MS) and Fourier transform infrared
spectroscopy (FTIR) in the following examples.
Example 2:
N
H
N
S
HO
4-(12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indol-12-yl)phenol (2b)
Yield: 70 %; Melting range (°C): 181-184;Rf = 0.57;
IR (KBr) (νmax/cm-1): 3349.4 (O-H str), 3194.5 (N-H str), 2709.7 (C-H str, aromatic), 1687.3
(C=N str), 1600.0 (N-H bend), 1440.6 (C=C str, aromatic), 1302.0 (C-N str), 751.0 (C-S-C str);
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)10.51 (s, 1H, -NH-),8.14 (d, 1H, ArH), 8.01 (d, 2H,
ArH), 7.57 (t, 1H, ArH), 7.47 (q, 1H, ArH), 7.22 (m, 2H, ArH), 7.05 (d, 2H, ArH), 6.99 (t, 2H,
ArH), 6.90 (d, 1H, ArH)4.24 (s, 1H, -OH), 3.27 (s, 1H, >CH-), 2.75 (s, 1H, >CH-);
13C-NMR (DMSO-d6, 200 MHz): δ (ppm)176.84, 168.10, 161.20, 153.88, 144.14, 134.44,
130.62, 129.53, 127.89, 126.95, 126.20, 125.40, 124.81, 124.24, 122.62, 121.59, 116.61, 115.30,
109.61, 40.34, 36.19;
MS (EI): m/z 345.21 [M+1]+.
Example 3:
14
N
H
N
S
Cl
12-(4-chlorophenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indole (4b)
Yield: 68 %; Melting range (°C): 193-195;Rf = 0.54;
IR (KBr) (νmax/cm-1): 3409.9 (N-H str), 3055.4 (C-H str, aromatic), 1691.0 (C=N str), 1591.0 (NH
bend), 1471.1 (C=C str, aromatic), 1311.5 (C-N str), 827.0 (C-Clstr), 755.4 (C-S-C str);
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)8.25 (d, 1H, -NH-), 8.19 (dd, 4H, ArH), 7.72 (d, 3H,
ArH), 7.64 (t, 1H, ArH), 7.56 (t, 1H, ArH), 7.27 (m, 1H, ArH), 7.01 (t, 1H, ArH), 6.89 (d, 1H,
ArH), 3.54 (s, 1H, >CH-), 3.46 (s, 1H, >CH-);
13C-NMR (DMSO-d6, 200 MHz): δ (ppm) 176.82, 166.45, 153.93, 144.15, 136.50, 135.04,
132.15, 129.94, 129.35, 127.90, 127.28, 126.23, 124.83, 123.45, 122.92, 121.58, 109.57, 40.33,
36.19;
MS (EI): m/z 364.97 [M+1]+.
Example 4:
NH
N
S
Br
12-(4-bromophenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indole (5b)
Yield: 77 %; Melting range (°C):167-170;Rf = 0.63;
IR (KBr) (νmax/cm-1): 3334.2 (N-H str), 3253.4 (C-H str, aromatic), 1690.8 (C=N str), 1616.9 (NH
bend), 1471.9 (C=C str, aromatic), 1395.4 (C-N str), 827.8 (C-Br str), 754.6 (C-S-C str);
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)8.25 (d, 1H, -NH-), 8.12 (dd, 3H, ArH), 7.87 (d, 3H,
ArH), 7.65 (t, 1H, ArH), 7.57 (t, 2H, ArH), 7.31 (t, 1H, ArH), 7.25 (m, 1H, ArH), 6.85 (t, 1H,
ArH),3.45 (s, 2H, >CH-);
15
13C-NMR (DMSO-d6, 200 MHz): δ (ppm)166.58, 153.94, 135.02, 132.88, 132.48, 129.53,
127.31, 126.26, 125.37, 123.46, 122.95, 40.33;
MS (EI): m/z 408.95 [M+1]+.
Example 5:
NH
N
S
H3CO
H3CO
OCH3
12-(3,4,5-trimethoxyphenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indole (7b)
Yield: 80 %; Melting range (°C): 166-169; Rf = 0.55;
IR (KBr) (νmax/cm-1): 3440.5 (N-H str), 3001.6 (C-H str, aromatic), 2941.6 (C-H str, aliphatic),
1583.6 (C=N str), 1517.5 (N-H bend), 1485.7 (C=C str, aromatic), 1331.7 (C-N str), 1127.7 (CO-
C str), 760.8 (C-S-C str);
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)8.22 (d, 1H, -NH-), 8.16 (d, 1H, ArH), 7.63 (t, 2H,
ArH), 7.55 (t, 2H, ArH), 7.42 (s, 3H, ArH), 7.15 (t, 2H, ArH), 4.00 (s, 6H, 2-OCH3), 3.84 (s, 3H,
1-OCH3), 3.68 (d, 1H, >CH-), 3.25 (d, 1H, >CH-);
13C-NMR (DMSO-d6, 200 MHz): δ (ppm)167.58, 153.95, 153.86, 140.66, 135.03, 128.79,
127.13, 125.90, 123.22, 122.74, 104.88, 60.70, 56.58, 40.33;
MS (EI): m/z 419.10 [M+1]+.
Example 6:
NH
N
S
H3CO
12-(4-methoxyphenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indole (8b)
Yield: 65 %; Melting range (°C): 188-190;Rf = 0.60;
16
IR (KBr) (νmax/cm-1): 3442.2 (N-H str), 3062.6 (C-H str, aromatic), 2820.4 (C-H str, aliphatic),
1694.9 (C=N str), 1599.1 (N-H bend), 1480.7 (C=C str, aromatic), 1310.6 (C-N str), 1227.0 (CO-
C str), 756.4 (C-S-C str);
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)8.23 (d, 5H, ArH), 8.15 (d, 1H, -NH-), 7.64 (t, 2H,
ArH), 7.50 (dt, 4H, ArH), 7.24 (m, 1H, ArH), 3.54 (s, 1H, >CH-), 3.45 (s, 3H, -OCH3), 3.07 (s,
1H, >CH-);
13C-NMR (DMSO-d6, 200 MHz): δ (ppm)166.57, 164.98, 163.74, 153.98, 135.02, 130.11,
130.06, 129.98, 127.90, 127.20, 126.05, 124.83, 123.33, 122.86, 121.59, 117.03, 116.91, 36.19;
MS (EI): m/z 359.83 [M+1]+.
Example 7:
N
H
N
S
OCH3
12-(3-methoxyphenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indole (9b)
Yield: 66 %; Melting range (°C): 187-190;Rf = 0.66;
IR (KBr) (νmax/cm-1): 3415.1 (N-H str), 3061.9 (C-H str, aromatic), 2995.5 (C-H str, aliphatic),
1605.8 (C=N str), 1519.3 (N-H bend), 1481.8 (C-N str), 1434.9 (C=C str, aromatic), 1257.1 (CO-
C str), 758.4 (C-S-C str);
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)8.18 (d, 1H, -NH-), 8.12 (q, 3H, ArH), 7.60 (t, 2H,
ArH), 7.51 (t, 2H, ArH), 7.27 (d, 1H, ArH), 7.20 (d, 2H, ArH), 7.00 (d, 1H, ArH), 6.90 (d, 1H,
ArH),3.93 (s, 3H, -OCH3), 3.54 (s, 1H, >CH-), 3.46 (s, 1H, >CH-);
13C-NMR (DMSO-d6, 200 MHz): δ (ppm)167.53, 162.25, 154.12, 134.69, 129.35, 127.00,
125.98, 125.58, 122.94, 122.67, 115.22, 55.96;
MS (EI): m/z 359.15 [M+1]+.
Example 8:
17
NH
N
S
OH
C2H5O
5-(12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indol-12-yl)-2-ethoxyphenol (11b)
Yield: 78 %; Melting range (°C): 194-197;Rf = 0.60;
IR (KBr) (νmax/cm-1): 3439.7 (O-H str), 3068.7 (N-H str), 3068.7 (C-H str, aromatic), 2981.5 (CH
str, aliphatic), 1688.5 (C=N str), 1593.8 (N-H bend), 1506.1 (C=C str, aromatic), 1434.8 (C-N
str), 1303.5 (C-O-C str), 761.4 (C-S-C str);
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)10.82 (s, 1H, -NH-),8.23 (d, 2H, ArH), 8.16 (d, 3H,
ArH), 8.08 (d, 2H, ArH), 7.66 (m, 2H, ArH), 7.56 (s, 2H, ArH), 5.46 (s, 1H, -OH), 5.07 (d, 2H,
>CH-), 3.42 (s, 2H, -OC2H5), 2.76 (s, 3H, -OC2H5);
13C-NMR (DMSO-d6, 200 MHz): δ (ppm) 169.42, 166.05, 153.78, 135.17, 135.08, 134.57,
131.84, 131.55, 131.12, 129.27, 128.09, 127.33, 126.81, 126.48, 126.38, 123.57, 121.69, 110.06;
MS (EI): m/z 389.11 [M+1]+.
Example 9:
N
H
N
S
Cl
12-(3-chlorophenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indole (13b)
Yield: 82 %; Melting range (°C): 191-193;Rf = 0.60;
IR (KBr) (νmax/cm-1): 3399.5 (N-H str), 3191.9 (C-H str, aromatic), 1702.8 (C=N str), 1616.1 (NH
bend), 1466.6 (C-N str), 1324.1 (C=C str, aromatic), 783.9 (C-Clstr), 753.8 (C-S-C str);
18
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)8.15 (d, 1H, -NH-), 8.08 (d, 2H, ArH), 7.70 (s, 2H,
ArH), 7.58 (t, 4H, ArH), 7.48 (t, 2H, ArH), 7.07 (d, 2H, ArH), 4.22 (s, 1H, >CH-), 3.46 (s, 1H,
>CH-);
13C-NMR (DMSO-d6, 200 MHz): δ (ppm)168.15, 153.83, 150.89, 147.73, 144.14, 134.51,
127.90, 126.96, 125.44, 124.54, 122.65, 122.60, 121.77, 116.48, 111.76, 109.59, 64.49, 36.19,
15.18;
MS (EI): m/z 363.24 [M+1]+.
Example 10:
N
H
N
S
F
12-(2-fluorophenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indole (16b)
Yield: 70 %; Melting range (°C): 188-190;Rf = 0.65;
IR (KBr) (νmax/cm-1): 3451.4 (N-H str), 3187.6 (C-H str, aromatic), 1710.0 (C=N str), 1615.1 (NH
bend), 1457.5 (C-N str), 1323.0 (C=C str, aromatic), 1094.6 (C-F str), 751.7 (C-S-C str);
1H-NMR (DMSO-d6, 800 MHz): δ (ppm)10.77 (s, 1H, -NH-), 7.84 (t, 1H, ArH), 7.63 (d, 2H,
ArH), 7.44 (dt, 3H, ArH), 7.34 (t, 3H, ArH), 6.97 (m, 3H, ArH), 4.23 (d, 1H, >CH-), 3.25 (d, 1H,
>CH-);
13C-NMR (CDCl3, 100 MHz): δ (ppm)168.56, 160.76, 159.52, 143.61, 132.48, 131.07, 130.99,
130.29, 127.90, 125.21, 123.16, 122.80, 121.79, 121.11, 116.59, 116.48, 110.68, 40.33;
MS (EI): m/z 346.91 [M]+.
Example 11:
Preliminary in-vitro anticancer screening:
In vitro anticancer activity was carried out against human hepatoma cell line (Hep-G2).
Adriamycin (ADR) was used as a reference drug. Cell viability in the presence of test samples
was measured by the SRB assay.The relationship between surviving fraction and drug
concentration was plotted to obtain the survival curve of Hep-G2 cells.
Experimental Procedure for the Sulforhodamine B (SRB) Assay:
19
Hep-G2 cells were grown in Roswell Park Memorial Institute media (RPMI 1640) containing
10% fetal bovine serum and 2 mM L-glutamine in T-75 flask at 37°C, 5% CO2, 95% air and
100% relative humidity for 24 h. After growing, 100 μL cells containing media was inoculated
into 96 well plates at a concentration of 5×103 cells/well. Separately, all the compounds to be
tested were solubilized in dimethyl sulfoxide at 100 mg/ml and diluted to 1 mg/ml using water
and stored frozen prior to use. Next day, 100 μL of compounds containing media was added in
each well (10, 20, 40 and 80 μg/ml) and incubated at standard conditions for 48 h. To terminate
the reaction, 50 μL of the cold 30% trichloroacetic acid (TCA) was added and incubated at 4°C
for 1 h. The supernatant was discarded; the plates were washed five times with tap water and air
dried. Further, 50 μl of SRB solution at 0.4% (w/v) in 1% acetic acid was added to each of the
wells and incubated for 20 minutes at room temperature. After staining, the residual dye was
removed by washing five times with 1% acetic acid and the plates were air dried. The bound
stain was subsequently eluted with 10 mMtrizma base and the absorbance was read on a plate
reader at a wavelength of 540 nm with 690 nm reference wavelength. The results were obtained
in triplicate on separate plates and finally the average values were determined from these three
experiments.
The percent growth inhibition was calculated using the formula [(Ti-Tz)/(C-Tz)] × 100 %. The
abbreviations used in formula were considered as; Time zero (Tz), control growth (C), and test
growth in the presence of drug at the four concentration levels (Ti).
Example 12:
Pharmacology: SRB assay using Hep-G2 cells
A total of nine newly synthesized compounds were investigated for anticancer effect using Hep-
G2 cells where we found that 11bwas most active on this particular cell line with GI50 value17.8
μg/mL(Table 2 and 3).Effects of the synthesized compounds and the standard drug adriamycin
(ADR) on human hepatoma cell line (Hep-G2 cells) are demonstrated asgraphical representation
(Figure 2).The microscopic pictures (Figure 3) are showing the effects of treatment with the most
active compound 11b and adriamycin (ADR) on Hep-G2 human hepatoma cell line.
The in vitro anticancer study revealed that one compound out of total ninenewly synthesized
compounds has been found active against liver cancer.
While considering all the newly synthesized compounds together, it has been concluded that the
fusion of indolering with benzothiazepine ring, with a six-membered phenyl ring as flexible side
chain, establish an important pharmacophore and the positions 2, 3, 4 and 5 of the phenyl side
chain are the key reactive sites which could be altered with different groups to elicit valuable
anticancer profiles. Although most of the synthesized compounds with various substitutions have
considerable efficacy to inhibit the growth of cancerous cells, however, the concomitant
substitutions with -OH group at position 2 and –OC2H5 group at position 3 of phenyl ring
directly attached to5H-benzo[2,3][1,4]thiazepino[5,6-b]indoles led to compound 11b
withexcellent enhancement in anticancer activity on Hep-G2 cell line.
20
In addition, substitution on the vacant site of indole nucleus using different derivatives of 2-
oxindole in the reaction procedure, can open a new avenue for further drug discovery and
expected to more enhancement of anticancer activity in future drug design perspective.
Close examination of the data represented in Figure 2 and Table 2, showed that at higher
concentrations, % control growth of compound 11b is comparable to that ofthe standard drug,
adriamycin.
Interestingly, the growth curve of in vitro data suggested that, at ˂ 80 μg/ml concentration of
active compound (11b), the % control growths are 50% or below 50%, but they do not fall in the
negative value of % control growth. Thus for the future, it might be expected that all the active
compounds of the series will kill the cancerous cell while minimizing the normal cell death.
Human Hepatoma Cell Line Hep-G2
% Control Growth
Drug Concentrations (μg/ml)
Average Values
Compounds Code 10 20 40 80
2b 104.4 105.8 89.1 50.2
4b 113.2 100.8 58.0 29.8
5b 118.7 113.2 76.9 40.8
7b 109.8 104.2 94.9 84.9
8b 123.5 120.0 110.5 87.5
9b 108.5 105.0 83.7 71.6
11b 57.7 47.7 21.6 -49.0
13b 97.5 84.8 49.8 23.1
16b 91.2 80.6 88.0 61.8
ADR -25.37 -32.38 -29.09 -13.77
Table 2: Drug Concentrations (μg/ml) vs. % Control Growth
Compounds Code Drug concentrations (μg/ml)
LC50 TGI GI50
2b NE NE >80
4b NE NE 58.6
5b NE NE 69.9
7b NE NE >80
8b NE NE NE
9b NE NE >80
11b NE 50.1 17.8
21
13b >80 >80 50.4
16b NE NE >80
ADR NE <10 <10
Table 3: Drug concentrations (μg/ml) required to kill/control growth of Hep-G2 cells
Definitions:
“NE” stands for “Not Effective” even at the concentration >80 μg/mL
GI50 = Concentration of drug causing 50% inhibition of cell growth
LC50 = Concentration of drug causing 50% cell kill
TGI = Concentration of drug causing total inhibition of cell growth
ADR = Adriamycin, Positive control compound
*GI50 value of ≤ 20μg/ml (or 2 μmolar) is considered to demonstrate activity.

CLAIMS
We claim,
1. “5H-benzo[2,3][1,4]thiazepino[5,6-b]indoles for the treatment of liver cancer” relates
to aconvenient and selective one-pot multi-component reaction process for the
preparation of 5H-benzo[2,3][1,4]thiazepino[5,6-b]indoles and the most active compound
(11b) is useful as an anti-hepatocellular carcinogenic agent in the prevention and/or
treatment of liver cancer.
2. A 5H-benzo[2,3][1,4]thiazepino[5,6-b]indole compound and its derivatives having the
general formula:
N
H
N
S
R2 R3
R4
R1
Wherein, R1, R2, R3, R4 are independently selected from H, F, Cl, Br, OH, OCnH2n+1,
and CnH2n+1.
3. The compound as claimed in claim 2, wherein n ranges from 1 to 3.
4. The invention as claimed in claim 1, wherein a feasible one pot efficient synthetic
approach has been developed for the synthesis of5H-benzo[2,3][1,4]thiazepino[5,6-
b]indolesby exploiting the reaction of 2-oxindole with substituted aromatic aldehyde and
2-amino thiophenol in presence of conc. HCl.
5. The process as claimed in claim 4, wherein the aromatic aldehyde is selected from 4-
hydroxybenzaldehyde, 4- chlorobenzaldehye, 4-bromobenzaldehyde, 3,4,5-
trimethoxybenzaldehyde, 4-methoxybenzaldehyde, 3-methoxybenzaldehyde, 3-hydroxy-
4-ethoxybenzaldehyde, 3-chlorobenzaldehyde and 2- fluorobenzaldehyde.
6. The process as claimed in claim 4, wherein the ratio of 2-oxindole, aromatic aldehyde
and 2-amino thiophenolis 1: 1: 1.
7. The process as claimed in claim 4, wherein the heating is performed at a temperature in
the range of 95°C to 100°C and the reaction mixture allowed to stand for a period of 10
to 12 hrs.
8. The compound as claimed in any of the preceding claims, wherein the yield of the pure
compounds ranges from about 65-80%.
9. The invention as claimed in claim 1 and 4, wherein a total of nine synthesized
compounds have been screened for their anticancer potential via in vitro anticancer
testing on the Hep-G2 cell line.
23
10. The invention as claimed in claim 1, wherein a total of nine newly synthesized
compounds were investigated for anticancer effect using Hep-G2 cells where11b was
found to be most active on this particular cell line with GI50 value 17.8 μg/mL; the
effects of the synthesized compounds and the standard drug adriamycin (ADR) on human
hepatoma cell line (Hep-G2 cells) are demonstrated asgraphical representation which
show the effects of treatment with the most active compound 11b and adriamycin (ADR)
on Hep-G2 human hepatoma cell line.
11. The invention as claimed in claim 1 and 10, wherein the properties of 11b compound are
shown,
NH
N
S
OH
C2H5O
5-(12,12a-dihydro-5H-benzo[2,3][1,4]thiazepino[5,6-b]indol-12-yl)-2-ethoxyphenol
(11b)
Yield: 78 %; Melting range (°C): 194-197;Rf = 0.60;IR (KBr) (νmax/cm-1): 3439.7 (O-H
str), 3068.7 (N-H str), 3068.7 (C-H str, aromatic), 2981.5 (C-H str, aliphatic), 1688.5
(C=N str), 1593.8 (N-H bend), 1506.1 (C=C str, aromatic), 1434.8 (C-N str), 1303.5 (CO-
C str), 761.4 (C-S-C str); 1H-NMR (DMSO-d6, 800 MHz): δ (ppm)10.82 (s, 1H, -
NH-),8.23 (d, 2H, ArH), 8.16 (d, 3H, ArH), 8.08 (d, 2H, ArH), 7.66 (m, 2H, ArH), 7.56
(s, 2H, ArH), 5.46 (s, 1H, -OH), 5.07 (d, 2H, >CH-), 3.42 (s, 2H, -OC2H5), 2.76 (s, 3H, -
OC2H5); 13C-NMR (DMSO-d6, 200 MHz): δ (ppm) 169.42, 166.05, 153.78, 135.17,
135.08, 134.57, 131.84, 131.55, 131.12, 129.27, 128.09, 127.33, 126.81, 126.48, 126.38,
123.57, 121.69, 110.06; MS (EI): m/z 389.11 [M+1]+.
12. The invention as claimed in claim 1 and 9, wherein compound 11b has been found to
have excellent potential against hepatocellular carcinoma and this lead structure have
broad scope to undergo further structural modifications on the basis of the structural
activity relationship, molecular docking or QSAR modelling to improve its potency,
lipophilicity and to minimize the side-effects.
13. The invention as claimed in claim 1, wherein the fusion of 5Hbenzo[
2,3][1,4]thiazepino[5,6-b]indoles with substituted hydrophobic phenyl ring as
flexible side chain, establish an important pharmacophoric structure, in particular for the
treatment of liver cancer.
24
14. The invention as claimed in claim 1 and 11, wherein 3-hydroxy-4-ethoxy phenyl
substituted 5H-benzo[2,3][1,4]thiazepino[5,6-b]indoleled to compound 11b, exhibited
excellent enhancement in cytotoxic potential with GI50= 17.8.
15. The invention as claimed in claim 1, 11 and 12, wherein the synthesized compounds
containing a novel pharmacophore incorporating 5H-benzo[2,3][1,4]thiazepino[5,6-
b]indoles, may be emerged as an anticancer lead for the future.