Synthesis, spectral characterization and antitumor activity of phenothiazine derivatives

Article abstract of DOI:10.1002/jhet.3980

Different types of phenothiazine derivatives were synthesized by reactions of 10-alkyl-10H-phenothiazine-3-carbaldehydes. Structures of the prepared compounds were confirmed through spectroscopic techniques such as IR, ¹H NMR, ¹³C NMR and mass spectroscopy. All the compounds were studied for their antitumor activities.

Full text of DOI:10.1002/jhet.3980

Received: 10 January 2020
DOI: 10.1002/jhet.3980
Revised: 9 March 2020
Accepted: 12 March 2020
A R T I C L E
Synthesis, spectral characterization and antitumor activity
of phenothiazine derivatives
Kasi Venkatesan1
| Vardhineedi Sri Venkata Satyanarayana2
|
Amaravadi Sivakumar2 | Chitteti Ramamurthy3 | Chinnasamy Thirunavukkarusu3
1Department of Humanities and Sciences,
Abstract
CVR College of Engineering, Hyderabad,
India
Different types of phenothiazine derivatives were synthesized by reactions of
10-alkyl-10H-phenothiazine-3-carbaldehydes. Structures of the prepared com-
pounds were confirmed through spectroscopic techniques such as IR, 1H
NMR, 13C NMR and mass spectroscopy. All the compounds were studied for
their antitumor activities.
2Department of Chemistry, School of
Advanced Sciences, VIT University,
Vellore, India
3Department of Biochemistry and
Molecular Biology, School of Life
Sciences, Pondicherry University,
Pondicherry, India
Correspondence
Kasi Venkatesan, Department of
Humanities and Sciences, CVR College of
Engineering, Hyderabad, India.
Email: venkippk@gmail.com
Amaravadi Sivakumar, Department of
Chemistry, School of Advanced Sciences,
VIT University, Vellore, India.
Email: svkmr7627@gmail.com
1 | INTRODUCTION
2 and 10. The first reagents to be successfully used for the
treating psychosis were phenothiazine compounds. Pheno-
The chemistry of heteroatom such as sulfur, nitrogen con-
taining aromatic compounds is becoming a prominent
area of research in synthetic organic chemistry. The com-
bination in a molecule of two and more pharmacophores
is one of the predominant approaches to design of new
biologically important compounds, including natural alka-
loids. Heterocyclic compounds containing sulfur and nitro-
gen atoms have wide a range of biological activity and
occupies a special place among various heterocyclic com-
pounds. Bernthsen first identified the parent compound,
10H-dibenzo-1,4-thiazine(phenothiazine) in 1883.
The phenothiazine derivatives are profoundly studied in
various fields such as biological, chemical and medical
research owing to their pharmaceutical activity. Many of
the phenothiazine derivatives are used in analytical chemis-
try, especially the substitution at 3 and 7 positions (dyes), as
well as the substitution at position 10 alone and positions
thiazine derivatives are pharmaceutically important bioac-
tive heterocyclic compounds with various pharmaceutical
activities such as antibacterial,1,2 antioxidant,3–5 antifungal,6
tranquilizers,7 anti-inflammatory,8 antimalarial,9,10
antipsychotropic,11 antitubercular12,13 and antimicro-
bial.14 In addition, a number of these derivatives have
good anti-cancer activities that have given rise to an
excellent interest in the preparation and synthesis of
new phenothiazine compounds to investigate their anti-
cancer activities.15,16 Phenothiazine has been shown to
be an inhibitor of human cholinesterase, and these com-
pounds have acted as MDR (Multidrug Resistance Rever-
sal Agents) in several instances.17–19 Phenothiazine-based
dyes mainly increase the performance of dye-sensitized
solar cells.20 Recently phenothiazine has become promi-
nent in material science and biochemistry as marker for
proteins and DNA.21 Phenothiazine is found as the redox
J Heterocyclic Chem. 2020;1–7.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC
1

2
VENKATESAN ET AL.
active unit in donor–acceptor systems.22 The therapeutic
action of the phenothiazine drug has been reported which
blocks dopamine receptors in the brain23 with the interac-
tion of the side chain. The substituent present in the par-
ent compound enhances ability of phenothiazine
derivatives to mimic the preferred trans-α conformation of
dopamine (Data S1).
The phenothiazine derivative's donor potential is very
high even in the ground state. There is practically the total
transfer of an electron to an acceptor with formation of CT
complexes. Consistent with the literature information,
substituents attached to the tricyclic phenothiazine ring
position C-2 and the alkyl bridge length linking the nitro-
gen atom at position 10 (N-10) of the tricyclic ring with
the terminal amine in the side chain determine phenothia-
zine activity against cancer cells.24,25 The activity is more
closely linked to the type of substituent in the phenothia-
zine ring than to the nature of the attached side chain.26
imidazoles 5a-b were synthesized by refluxing with N-
alkyl-phenothiazine-3-carbaldehyde. The synthetic path-
way is represented in Scheme 2.
The IR spectra of compounds 5a-b revealed the pres-
ence of absorption bands from 3423 to 3453 cm−1 for the
1
NH group and the H NMR spectra of compounds 5a-b
showed singlet at δ 12.5 to 12.6 ppm which also supports
the formation of imidazole NH proton. Benzimidazoles
substituted phenothiazine derivatives were prepared
using 10-alkyl-10H-phenothiazine-3-carbaldehyde 3a-b,
o-phenylenediamine and DMF (10 mL) using sodium
metabisulfite (10 mol%) catalyst and the reaction mass
was stirred at 80ꢀC for 7 hours. It created the desired
product 6a-b (Scheme 2).
1
In the H and 13C NMR spectra of compounds 6a-b
the disappearance of formyl hydrogen at δ 9.77 ppm and
carbonyl (C O) peak at δ 190.67 ppm indicates the for-
mation of benzimidazoles derivatives 6a-b. Compounds
3a-b were treated with 2-(9H-carbazol-9-yl) acet-
ohydrazide in the presence of catalytic acetic acid in
methanol at reflux temperature to provide Schiff base
derivatives27 7a-b (Scheme 2). The IR spectra of the com-
pound 7a the absorption peaks at 1645 and 1604 cm−1
corresponds respectively to the group of C O & C N. In
1H NMR compound spectrum 7a displays a singlet at
11.77 ppm indicating the NH group presence.
2 | RESULTS AND DISCUSSION
N-alkyl phenothiazine compounds were prepared via
reaction of alkyl iodide (ethyl, methyl), phenothiazine in
the presence of KOt-Bu in dry DMF at 70ꢀC-80ꢀC for a
period of 24 hours (Scheme 1). The compounds, 3a-b was
obtained via Vilsmeier Hack reaction from (2a-b)
10-alkyl phenothiazines with a yield of 78%.27
The compounds, 3-(10-methyl-10H-phenothiazin-
3-yl)-1-substituted phenylprop-2-en-1-one 4a-c were pre-
pared via Claisen-Schmidt reaction by condensation of
compound 3a with different substituted acetophenones
in the presence of alcoholic KOH at room temperature
(Scheme 1). In their IR spectrum, the 4a-c compounds
showed bands of absorption at 1653-1657 cm−1 due to
styril ketone, C O stretching frequency. In 1H NMR
spectrum of compounds 4a-c showed the protons
attached to the carbon atoms of α, β unsaturated ketone
moiety at δ 7.4 to 7.8 ppm, which were seen merged with
aromatic protons.
The singlet at 8.19 ppm due to N CH proton, the
disappearance of NH2 signal of compound 7a indicates
the formation of hydrazide to hydrazones 7a-b. Yields of
all the synthesized products with respect reaction time,
temperature and physical properties are given in Table 1.
1
Spectral characterization such as IR, H NMR, 13C NMR
and HR-MS spectral data verified for the structures of the
newly prepared phenothiazine derivatives.
3 | ANTITUMOR ACTIVITY
3.1 | MTT assay
In the presence of ammonium acetate, glacial acetic
acid and benzil, phenothiazine substituted-4,5-diphenyl
MTT assay was used to determine cell viability of MCF-7
cancer cells in the presence of compounds (4a-c, 5a-b,
O
R
O
S
S
S
CHO
CH3-I, DMF
DMF
S
POCl3
N
H
N
t-BuOK, 800C
N
R
alc. KOH
N
1
2a
3a
4a-c
SCHEME 1 Synthetic pathway for the preparation of phenothiazine Chalcone

VENKATESAN ET AL.
3
SCHEME 2 Synthetic pathway for the preparation of phenothiazine compounds
TABLE 1
Data of synthesized phenothiazine compounds
Time (min.) Yield (%) m.p. (ꢀC)
compounds. The inhibition rates of the compound 4a at
1, 10 and 1000 μg against MCF-7 in vitro was 75.5 1.2,
42.98 0.6 and 20.74 3.0, respectively. A typical com-
parison is shown in Figure 1 for MCF-7 at different con-
centrations of compounds (4a-c, 5a-b, 6a-b and 7a-b).
Compound
R
4a
4b
4c
5a
5b
6a
6b
7a
7b
Methyl 160
Methyl 165
Methyl 170
Methyl 320
88
86
84
76
72
80
82
86
84
161-163
142-145
135-138
252-255
235-238
173-175
162-165
172-175
185-188
Ethyl
Methyl 400
Ethyl 410
Methyl 210
Ethyl 212
330
3.2 | LDH assay
Lactate dehydrogenase (LDH) release assay was per-
formed in MCF-7 cells to assess the cytotoxicity of com-
pounds (4a-c, 5a-b, 6a-b and 7a-b). This assay quantifies
cell death and lysis based on measuring the release of
LDH in the supernatant after the loss of membrane integ-
rity. A dose dependent increase of LDH release was
observed with compounds (4a-c, 5a-b, 6a-b and 7a-b) at
1, 10 and 1000 μg concentrations (Figure 2). Using the
LDH assay, we measured the optical density (at 340 nm)
of LDH release from MCF-7 cells upon the addition of
different concentrations of compounds (4a-c, 5a-b, 6a-b
and 7a-b). The LDH release increased with the concen-
tration of compounds (4a-c, 5a-b, 6a-b and 7a-b). The
highest LDH release (63.69 3.9 & 65.75 2.0) was
observed in the samples treated with compounds (4a, 4b)
at 1000 μg concentration.
6a-b and 7a-b) at different concentrations. After
24 hours of cell incubation following the addition of
compounds (4a-c, 5a-b, 6a-b and 7a-b) at 1, 10 and
1000 μg concentrations, MCF-7 cell viability decreases
as the compound concentration increases as indicated
in Table 2.
All the compounds showed good inhibition, but the
compounds 4a and 7a required only 10 μg and 1000 μg to
reach <50% inhibition compared to the remaining

4
VENKATESAN ET AL.
TABLE 2
Antitumor activity of phenothiazine derivatives by
LDH and MTT methods in MCF-7 cells
Concentration Percentage of Percentage of
Compound (μg/well) viability released LDH
4a
1
75.55501 1.2 15.92357 3.0
42.98482 0.6 28.65605 2.6
20.74763 3.0 63.69 3.9
77.55468 1.7 26.75159 1.9
70.83333 1.8 48.21019 3.0
58.33333 2.2 65.7514 2.0
75.22037 2.4 29.7835 1.3
68.09909 0.9 35.24 2.7
56.97845 1.0 48.1257 1.0
76.76298 0.7 24.532 1.6
70.77212 0.1 31.3271 2.9
62.25106 0.9 46.1431 0.9
10
1000
1
4b
10
1000
1
4c
10
1000
1
5a
FIGURE 1 Antitumor activity of compounds (4a-c, 5a-b, 6a-b
and 7a-b) using MTT assay
10
1000
1
5b
73.6696 0.6
21.64605 1.2
10
60.74927 2.2 46.1531 0.8
60.71254 0.9 49.0341 0.5
80.78681 0.1 19.65605 1.93
73.84509 1.7 34.94268 2.7
52.78322 0.5 46.237 1.0
69.40908 1.0 28.02548 1.2
56.15736 0.4 36.94268 1.9
54.34215 0.02 39.49045 2.0
1000
1
6a
10
1000
1
6b
10
1000
1
7a
87.0715 0.7
14.023 1.9
10
64.66699 2.2 31.21019 1.8
47.91871 1.4 47.7707 1.6
1000
1
7b
76.5875 2.9
22.0951 3.0
10
73.16765 2.4 33.75796 2.8
68.0542 0.02 42.67516 3.0
FIGURE 2 Antitumor activity of compounds (4a-c, 5a-b, 6a-b
and 7a-b) using LDH assay
1000
Control
100
100
5 | EXPERIMENTAL SECTION
5.1 | General
4 | CONCLUSIONS
In this work, the synthesis of various heterocyclic
substituted phenothiazine derivatives prepared from N-
alkyl-phenothiazine-3-carbaldehyde using different
one step reaction conditions were reported. The struc-
tures of the derivatives have been confirmed through
spectral characterization such as IR, 1H NMR, 13C
NMR and mass spectra. All the prepared compounds
were checked for their antitumor activity against breast
cancer cell lines (MCF-7) using MTT and LDH assay
method. Among these compounds 4a and 7a showed
maximum activity of cell death at 10 and 1000 μg
concentrations.
All the chemicals were purchased from SD Fine (India),
Sigma-Aldrich (USA) and Sisco Research Laboratory
(SRL). On a Buchi-530 melting point unit, melting points
were determined using one end open capillary tubes and
the results were uncorrected. In Perkin Elmer model
1600 FT-IR RX1 spectrophotometer, IR spectra were
1
recorded using KBr discs. H NMR and 13C NMR spectra
were recorded on BRUKER AV-500 MHz and Bruker
Spectrospin DPX 400 MHz spectrometers using CDCl3
and DMSO-d6 as solvent and Tetramethylsilane (TMS) as
internal reference. Mass spectra were obtained using

VENKATESAN ET AL.
5
HRMS and the samples were dried under vacuum prior
to analysis.
Equimolar quantities of 10-methyl-10H-phenothiazine-
3-carbaldehyde (1 mmol) and substituted acetophenone
(1 mmol) were dissolved in little quantity of alcohol.
Sodium hydroxide solution (40℅) 5 mL was added slowly
and the reaction mixture stirred continuously for 3 hours
until the total mixture became cloudy. The total mixture
was discharged slowly into large amount of water with
continuous stirring and kept in refrigerator for 1 day. The
precipitate obtained was filtered, washed with water and
crystallized using ethanol to get pure products.
3-(10-methyl-10H-phenothiazin-3-yl)-1-phenylprop-
2-en-1-one (4a) mp 161-163ꢀC; IR (KBr) νmax: 3059,
1654, 1591, 1571, 1500, 1465, 1442, 1402, 1336, 1292,
1215, 1159 cm−1; 1H NMR (400 MHz, CDCl3, ppm),
δH = 8.06 (d, 2H, J = 7.2 Hz, CH and Ar H of phenyl
ring); 7.72 (s, 1H, Ar H of phenyl ring); 7.68 (s, 1H,
Ar H of phenyl ring); 6.78-7.58 (m, 10H, CH, Ar H of
phenyl and phenothiazine ring); 3.41 (s, 3H, CH3); 13C
NMR (100.645 MHz, CDCl3, ppm), δC = 190.44, 144.86,
143.91, 138.54, 132.75, 129.43, 129.22, 129.13, 128.71,
128.55, 128.26, 127.79, 127.36, 126.38, 123.21, 122.69,
199.99, 114.55, 114.20, 35.67; HRMS (EI): m/z [M+]
calcd. For C22H17NOS: 343.1031; found: 343.1031.
5.2 | LDH assay
The LDH assay was performed 1 day before the assay
with 1 in 104 cells seeded in a 96-well plate and the com-
pounds were analyzed in triplicates in the untreated con-
fluent culture medium, by using a commercial kit
(Agappe Diagnostics, India) based on LDH pyruvate
transformation at pH 7.5, in the presence of coenzyme
NADH. The decrease in absorbance (A) at 340 nm, shows
the NADH to NAD+ transition that correlates with the
LDH activity. The change in absorbance was measured
over a period of 0.5 to 4.5 minutes in the presence or
absence of different doses of compounds (1/10/1000 μg/
well), and the relative A/min was determined. The
change in absorbance was translated to LDH interna-
tional units per liter (U/L) by means of the following
equations: ΔA/minꢁ(tVꢁ1000/EMCꢁlꢁsV), where l is path
length of the light, tV is the total volume, sV is the sam-
ple volume and EMC is the NADH extinction micromo-
lar coefficient (6.22 cm2 μmol at 340 nm).
1-(4-methoxyphenyl)-3-(10-methyl-10H-phenothiazin-
3-yl)prop-2-en-1-one (4b) mp 142-145ꢀC; IR (KBr) νmax:
3145, 1653, 1589, 1463, 1400, 1330, 1257, 1184,
1
5.3 | Cell viability assay
1124 cm−1; H NMR (400 MHz, CDCl3, ppm), δH = 8.02
(dd, 2H, J = 2.0 & 6.8 Hz, o, o'- Ar H of phenyl ring);
7.71 (s, 1H, Ar H of phenyl ring); 6.77-7.67 (m, 10H, CH,
Ar H of phenyl and phenothiazine ring); 3.88 (s, 3H,
OCH3); 3.38 (s, 3H, CH3); 13C NMR (100.645 MHz,
CDCl3, ppm), δC = 188.63, 163.45, 147.75, 144.92, 143.02,
131.46, 130.84, 129.66, 129.07, 127.77, 127.36, 126.28,
124.05, 123.15, 122.17, 119.89, 114.52, 114.18, 113.94,
55.61, 35.64; HRMS (EI): m/z [M+] calcd. For
C23H19NO2S: 373.1136; found: 373.1135.
1-(2-hydroxyphenyl)-3-(10-methyl-10H-phenothiazin-
3-yl)prop-2-en-1-one (4c) mp 135-138ꢀC; IR (KBr) νmax:
3450, 1657, 1627, 1593, 1571, 1498, 1465, 1401, 1336,
1290 cm−1; 1H NMR (400 MHz, CDCl3, ppm), δH = 12.94
(s, 1H, OH); 7.926 (d, 1H, J = 7.6 Hz, CH); 6.81-7.84 (m,
12H, CH, Ar H of phenyl and phenothiazine ring); 3,42
(s, 3H, CH3); 13C NMR (100.645 MHz, CDCl3, ppm),
δC = 193.61, 163.71, 148.37, 144.75, 144.60, 136.35,
129.71, 129.69, 129.15, 127.87, 127.42, 126.52, 124.22,
123.35, 122.65, 120.26, 118.94, 118.74, 117.89, 114.65,
114.25, 114.25, 35.76; HRMS (EI): m/z [M+] calcd. For
C22H17NO2S: 359.0980; found: 359.0979.
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) was used for determination of cell viabil-
ity. In 96-well plates, the MCF-7 (1 × 104 cells/well) cells
were grown in 5% CO2 at 37ꢀC in the RPMI medium
(containing 100 μg/mL penicillin, 10% FBS and 100 μg/
mL streptomycin). After overnight incubation, different
concentrated compounds (1/10/1000 μg/well) replaced the
RPMI medium in each well and incubated for 24 hours.
Afterwards, 20 μL of MTT (5 mg/mL in PBS) was subse-
quently applied to each well and the cells were incubated
at 37ꢀC for an additional 4 hours. Then the supernatants
were carefully removed and 100 μL of dimethyl sulfoxide
(DMSO) was added to each well. The plates were shaken
for another 10 minutes, and the Microplate Reader
(Molecular Devices CA) recorded the absorbance values at
570 nm. The percentage of each concentration's inhibition
was calculated using the following formula:
Percentage of inhibition = Control O:D
−Test O:D × 100=Control O:D:
Procedure for the preparation of 3-(4,5-diphenyl-1H-
imidazol-2-yl)-10-alkyl-10H-phenothiazine 5(a-b).
Procedure for the preparation of 3-(10-methyl-10H-
phenothiazin-3-yl)-1-substituted phenylprop-2-en-1-one 4
(a-c).
A mixture of 10-alkyl-10H-phenothiazine-3-carbaldehyde
(1 mmol), ammonium acetate (10 mmol), benzil (1 mmol),
and glacial acetic acid (15 mL) was refluxed for 6 hours.

6
VENKATESAN ET AL.
The progress of reaction was checked by TLC. After reac-
tion was completed, the reaction mass was cooled to room
temperature and then discharged into cold water (200 mL)
and neutralized with ammonium hydroxide. The obtained
solid was filtered, washed with water and crystallized from
alcohol.
3-(4,5-diphenyl-1H-imidazol-2-yl)-10-methyl-10H-
phenothiazine (5a) mp. 252-255ꢀC; IR (KBr) νmax: 3421,
3057, 2962, 1602, 1462, 1450, 1334, 1257, 1139,
1072 cm−1; 1H NMR (400 MHz, DMSO-d6, ppm),
δH = 12.56 (s, 1H, NH); 7.92 (d, 1H, J = 8.0 Hz, Ar H of
phenyl ring); 7.87 (s, 1H, Ar H of phenyl ring); 6.98-7.55
(m, 15H, Ar H of phenothiazine and phenyl ring); 3.35
(s, 3H, CH3); 13C NMR (100.612 MHz, DMSO-d6, ppm),
δC = 145.14, 144.88, 144.78, 136.97, 135.18, 131.05,
128.62, 128.27, 123.28, 122.69, 122.30, 121.55, 114.77,
114.72, 35.26; HRMS (EI): m/z [M+] calcd. For
C28H21N3S: 431.1456; found: 431.1456.
3-(4,5-diphenyl-1H-imidazol-2-yl)-10-ethyl-10H-pheno-
thiazine (5b) mp. 235-238ꢀC; IR (KBr) νmax: 3421, 3132,
2987, 2935, 1604, 1462, 1382, 1328, 1251, 1136, 1072 cm−1;
1H NMR (400 MHz, DMSO-d6, ppm), δH = 12.61 (s, 1H,
NH); 7.89 (d, 1H, J = 8.4 Hz, Ar H of phenyl ring); 7.84
(s, 1H, o- Ar H of phenyl ring); 6.93-7.52 (m, 15H, Ar H
of phenothiazine and phenyl ring); 3.95 (d, 2H,
J = 6.0 Hz, CH2); 1.32 (t, 3H, J = 5.4 Hz, CH3); 13C NMR
(100.612 MHz, DMSO-d6, ppm), δC = 144.76, 144.25,
143.83, 128.38, 127.73, 127.68, 127.08, 124.72, 124.60,
123.49, 123.00, 122.58, 122.29, 115.54, 115.41, 41.20, 12.61;
HRMS (EI): m/z [M+] calcd. For C29H23N3S: 445.1613;
found: 445.1613.
121.56, 121.19, 115.68, 115.44, 114.81, 35.95; HRMS (EI):
m/z [M+] calcd. For C20H15N3S: 329.0987; found:
329.0987.
3-(1H-benzo[d]imidazol-2-yl)-10-ethyl-10H-pheno-
thiazine (6b) mp. 162-165ꢀC; IR (KBr) νmax: 3441, 2980,
2929, 2854, 1629, 1602, 1581, 1460, 1373, 1330, 1253,
1
1180, 1138 cm−1; H NMR (500 MHz, DMSO-d6, ppm),
δH = 8.10 (dd, 2H, J = 2.0 & 8.5 Hz, p- Ar H of phenyl
ring); 7.93 (d, 1H, J = 2.0 Hz, NH); 7.67-7.70 (m, 2H, C-2,
C-6 Ar H of ptz ring); 7.36 (q, 2H, J = 4.5 Hz, Ar H of
phenyl ring); 6.97-7.48 (m, 5H, Ar H of phenothiazine
ring); 4.01 (q, 4H, J = 6.5 Hz, CH2); 1.31 (t, 3H,
J = 6.75 Hz, CH3); 13C NMR (125.757 MHz, DMSO-d6,
ppm), δC = 149.86, 147.47, 143.47, 135.95, 128.48, 127.63,
127.49, 125.75, 124.32, 123.72, 123.69, 122.13, 120.68,
116.36, 116.06, 114.75, 41.96, 12.97; HRMS (EI): m/z
[M+] calcd. For C21H17N3S: 343.1143; found: 343.1142.
Procedure for the preparation of 2-(9H-carbazol-9-yl)-
N0-((10-methyl-10H-phenothiazin-3-yl)methylene)acet-
ohydrazide 7(a-b).
In a 50 mL round bottomed flask a mixture of 10-alkyl-
10H-phenothiazine-3-carbaldehyde 3(a-b) (1 mmol), 2-(9H-
carbazol-9-yl)acetohydrazide (1 mmol), 1 mL of glacial
acetic acid and methanol 25 mL were taken. The resulting
solution was refluxed for 3-4 hour. The progress of the reac-
tion was supervised by thin layer chromatography. After
reaction was completed, the reaction mass could cool at
room temperature and the solid separated was filtered off
and washed with large amount of methanol (40 mL) and
dried at room temperature.
2-(9H-carbazol-9- yl)-N0- ([10-methyl-10H-phenothiazin-
3-yl] methylene) acetohydrazide (7a) mp 172-175ꢀC; IR
(KBr) νmax: 3224, 3072, 2872, 1674, 1598, 1575, 1463, 1382,
1257, 1151 cm−1; 1H NMR (500 MHz, DMSO-d6, ppm),
δH = 11.63 (s, 1H, NH), 8.19 (s, 1H, N CH), 8.16-8.17 (split
peaks, 1H, C5-Ar H of carbazole ring), 7.99 (s, 1H,
C4-Ar H of carbazole ring), 7.65 (d, 1H, J = 2.0 Hz,
C1-Ar H of carbazole ring), 6.98-7.59 (m, 12H, Ar H of
carbazole and phenothiazine ring), 5.17 (s, 2H, NCH2), 3.36
(s, 3H, CH3); 13C NMR (125.757 MHz, DMSO-d6, ppm),
δC = 164.38, 147.27, 147.68, 146.91, 145.66, 144.99, 143.44,
141.34, 141.16, 129.01, 128.94, 128.40, 127.94, 127.60, 127.33,
126.18, 126.07, 125.39, 124.97, 123.37, 122.73, 121.98, 121.91,
120.55, 119.55, 119.34, 115.41115.17, 115.03, 109.88, 109.82,
44.15, 35.84; HRMS (EI): m/z [M+] calcd. For C28H22N4OS:
462.1514 found: 462.1513.
Procedure for the preparation of 3-(1H-benzo[d]
imidazol-2-yl)-10-alkyl-10H-phenothiazine 6(a-b).
In a 50 mL round bottom flask, a mixture of o-
phenylenediamine (1 mmol) and 10-alkyl-10H-phenothia-
zine-3-carbaldehyde (1 mmol) in DMF (10 mL) were mixed
and stirred in the presence of sodium metabisulfite
(Na2S2O5) catalyst (10 mol%) at 80ꢀC for 7 hours. The pro-
gress of reaction mixture was monitored by TLC. After reac-
tion was completed, the reaction mass was cooled and then
discharged into ice cooled water and the solid obtained was
filtered, washed with excess of H2O and dried. Column
chromatography was used to purify the crude product.
3-(1H-benzo[d]imidazol-2-yl)-10-methyl-10H-pheno-
thiazine (6a) mp. 173-175ꢀC; IR (KBr) νmax: 3419, 1629,
1
1581, 1460, 1340, 1259, 1087 cm−1; H NMR (500 MHz,
DMSO-d6, ppm), δH = 8.04 (dd, 1H, J = 2.0 & 8.5 Hz, p-
Ar H of phenyl ring); 7.96 (d, 1H, J = 2.0 Hz, NH);
7.01-7.36 (m, 8H, Ar H of phenothiazine and phenyl
ring); 7.68 (q, 2H, J = 3.25 Hz, C-2, C-6 Ar H of pheno-
thiazine ring); 3.32 (s, 3H, CH3); 13C NMR (125.757 MHz,
DMSO-d6, ppm), δC = 150.00, 148.31, 144.64, 136.25,
128.59, 127.55, 127.42, 125.52, 124.18, 123.74, 123.23,
2-(9H-carbazol-9-yl)-N0-([10-ethyl-10H-phenothiazin-
3-yl]methylene)acetohydrazide(7b).
mp 185-188ꢀC; IR (KBr) νmax: 3219, 3062, 2964, 2914,
1681, 1597, 1546, 1485, 1460, 1325, 1244, 1211, 1155,
1080 cm−1
;
1H NMR (500 MHz, DMSO-d6, ppm),
δH = 11.62 (s, 1H, NH), 8.16, (d, 2H, J = 7.5 Hz, N=CH &
C5-Ar H of carbazole ring), 7.97 (s, 1H, C4-Ar H of

VENKATESAN ET AL.
7
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carbazole ring), 6.95-7.60 (m, 14H, Ar H of phenothia-
zine and carbazole ring), 5.17 (s, 1H, N-CH2), 3.95 (q, 2H,
J = 6.5 Hz, CH2), 1.29-1.34 (m, 3H, CH3); 13C NMR
(125.757 MHz, DMSO-d6, ppm), δC = 169.17, 146.87,
146.12, 144.02, 143.42, 141.33, 141.15, 128.86, 128.26,
127.58, 127.54, 126.18, 126.07, 125.25, 123.73, 123.28,
122.73, 122.67, 120.55, 119.55, 119.34, 116.44, 115.73,
109.87, 44.13, 41.83, 13.02; HRMS (EI): m/z [M+] calcd.
For C29H24N4OS: 476.1671; found: 476.1671.
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ACKNOWLEDGMENTS
The authors are grateful to the management of VIT Uni-
versity, Vellore, for encouraging and providing the neces-
sary facilities to conduct this research, and to the
recording of the spectral analysis by SAIF IIT Madras
and SIF VIT University.
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ORCID
Kasi Venkatesan https://orcid.org/0000-0001-8010-0009
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How to cite this article: Venkatesan K,
Satyanarayana VSV, Sivakumar A, Ramamurthy C,
Thirunavukkarusu C. Synthesis, spectral
characterization and antitumor activity of
phenothiazine derivatives. J Heterocyclic Chem.
2020;1–7. https://doi.org/10.1002/jhet.3980