Synthesis and biological evaluation of 1-(3-chloro-2-oxo-4-phenylazetidin- 1-yl)-3-(2-oxo-2-(10H-phenothiazin-10-yl)ethyl)urea derivatives

Article abstract of DOI:10.1007/s00044-012-0255-z

A new series of eleven novel 1-(3-chloro-2-oxo-4-phenylazetidin-1yl)-3-(2- oxo-2-(10H-phenothiazin-10-yl)ethyl)urea derivatives were synthesized by cyclocondensation of various Schiff bases of phenothiazine with chloroacetyl chloride in the presence of triethylamine. Various Schiff bases of phenothiazine were synthesized by condensation of 4-(2-oxo-2-(10H-phenothiazin-10-yl)ethyl semicarbazide with various aryl aldehydes. The synthesized compounds were characterized by IR, MASS and ¹H NMR spectral data and evaluated for in vitro antimicrobial, antitubercular, antioxidant and anticancer activities by disc diffusion method, MIC method, REMA, DPPH, FRAP and MTT assay method, respectively. All synthesized compounds showed moderate-to-significant anti-bacterial and anti-fungal activity and compound 4d, 4g, 4h and 4k showed good antioxidant activity with EC₅₀ value of 55, 57, 56 and 47 μg/ml tested by DPPH method. The compounds 4j at a concentration of 10 μg/ml showed inhibition against the growth of Mycobacterium tuberculosis and 4f showed significant activity against human cervical cancer cell line with IC₅₀ values of 18.26 μM.

Full text of DOI:10.1007/s00044-012-0255-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2578–2588
DOI 10.1007/s00044-012-0255-z
ORIGINAL RESEARCH
Synthesis and biological evaluation of 1-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-3-(2-oxo-2-(10H-phenothiazin-10-
yl)ethyl)urea derivatives
•
A. Rajasekaran K. Sheeja Devi
Received: 27 February 2012 / Accepted: 21 September 2012 / Published online: 4 October 2012
Ó Springer Science+Business Media New York 2012
Abstract A new series of eleven novel 1-(3-chloro-2-oxo-4-
phenylazetidin-1yl)-3-(2-oxo-2-(10H-phenothiazin-10-yl)ethyl)
urea derivatives were synthesized by cyclocondensation of var-
ious Schiff bases of phenothiazine with chloroacetyl chloride in
the presence of triethylamine. Various Schiff bases of pheno
thiazine were synthesized by condensation of 4-(2-oxo-2-(10H
-phenothiazin-10-yl)ethyl semicarbazide with various aryl
aldehydes. The synthesized compounds were characterized by
IR, MASS and 1H NMR spectral data and evaluated for in vitro
antimicrobial, antitubercular, antioxidant and anticancer activi-
ties by disc diffusion method, MIC method, REMA, DPPH,
FRAP and MTT assay method, respectively. All synthesized
compounds showed moderate-to-significant anti-bacterial and
anti-fungal activity and compound 4d, 4g, 4h and 4k showed
good antioxidant activity with EC50 value of 55, 57, 56 and
47 lg/ml tested by DPPH method. The compounds 4j at a
concentration of 10 lg/ml showed inhibition against the growth
of Mycobacterium tuberculosis and 4f showed significant
activity against human cervical cancer cell line with IC50 values
of 18.26 lM.
more popular as an area of research and provides a most
valuable molecular template for the development of agents
that are able to interact with a wide variety of biological
processes. They have been shown to possess a broad spec-
trum of biological activities as antinociceptive (Gildasio
et al., 2004), antimicrobial (Gautam et al., 2011; Wainwright
et al., 2011; Radadiya et al., 2005), anti-tumour (Krsti et al.,
2011; Plutal et al., 2010), antimalarial (Martha et al., 2002)
and anti-tubercular (Aaron et al., 2007) agents. The cyclic
2-azetidinone skeleton has been extensively used as a tem-
plate to build the heterocyclic structure fused to the four-
membered ring (Srivastava et al., 2010). The activity of the
famous antibiotics such as penicillins, cephalosporins and
carbapenems are attributed to the presence of 2-azetidinone
ring in them.
In continuation of our research in the synthesis of new
congeners by incorporating phenothiazine and azetidinone
moieties in a single molecular frame work (Rajasekaran et al.,
2010), herein, we report the synthesis and biological evalua-
tion of some new 1-(3-chloro-2-oxo-4-phenylazetidin-1-yl)-
3-(2-oxo-2-(10H-phenothiazin-10-yl)ethyl)urea derivatives.
Keywords Phenothiazines ꢀ 2-Azetidinones ꢀ
MTT assay ꢀ REMA
Materials and methods
Introduction
General method for the synthesis of 2-chloro-1-(10H-
phenothiazin-10-yl)ethanone from phenothiazine (1)
(Ihara et al., 2011)
The chemistry of nitrogen–sulphur heteroatom containing
aromatic compounds particularly phenothiazine is becoming
Chloroacetyl chloride (0.02 mol) was added drop by drop at
0.5 °C to phenothiazine (0.01 mol) in dry benzene (50 ml)
and stirred for 2 h. Solution was kept at room temperature
where the solid separated was filtered and washed with
petroleum ether and recrystallized from chloroform. The IR
spectrum of compound 1 revealed a sharp strong absorption
A. Rajasekaran (&) ꢀ K. S. Devi
Department of Pharmaceutical Chemistry, KMCH College
of Pharmacy, Kalapatti Road, Coimbatore 641048,
Tamil Nadu, India
e-mail: rsekaran2001in@yahoo.co.in
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Med Chem Res (2013) 22:2578–2588
2579
band around 1,680 cm-1 due to the presence of carbonyl
function in the structure.
instrument employing electron impact ionisation technique.
Thin layer chromatography analyses were performed on
pre-coated silica gel plates. Elemental analysis was per-
formed on Heraeus Carlo Erba 1108, and the analyses
indicated by the symbols of the elements were within
0.4 % of theoretical values.
Preparation of 4-(2-oxo-2-(10H-phenothiazin-
10yl)ethyl)semicarbazide (2) (Sahoo et al., 2010)
A mixture of 2-chloro-1-(10H-phenothiazin-10-yl) ethanone
(0.01 mol) and semicarbazide (0.04 mol) in absolute ethanol
wasrefluxedfor5 h.Contentswere pouredintoicecoldwater.
The resulting solid was filtered, dried and recrystallised from
chloroform. The IR spectrum of 2 showed a band at
1,657 cm-1 for carbonyl group and showing two broad bands
in the region of 3,300–3,400 cm-1 and 3,100–3,400 cm-1 for
NH2 and NH frequencies, respectively.
1-(3-Chloro-2-oxo-4-phenylazetidin-1-yl)-3-(2-oxo-2-
(10H-phenothiazin-10-yl)ethyl)urea (4a)
IR (KBr disc) cm-1: 3340 (NH str), 3167, 1596 and 736
(aromatic str), 1718 (C=O str), 1596 and 1570 (phenothi-
azine ring) 1472 (C–N str), 811 (C–Cl) and 700 (C–S-C
str). 1H-NMR (DMSO-d6) 6.59–7.0 (m, 13H, Ar–H), 3.4 (s,
2H, CH2), 2.5(d, 1H, J = 5.9 Hz, C–CH–Cl)azetidinone
ring, 8.7 (s, 1H, NH). MS m/z 479 [M]?; Anal. Calcd for
C24H19ClN4O3S (478.95). C, 60.19; H, 4.00; N, 11.70.
Found: C, 60.03; H, 4.2; N, 11.40.
General method for the Schiff’s base (3a–k)
(Chopde et al., 2011)
Mixture of 4-(2-oxo-2-(10H-phenothiazin-10-yl)ethyl)semi
carbazide (0.01 mol) and substituted benzaldehyde
(0.01 mol) in ethanol containing acetic acid (0.5 ml) was
refluxed for 6 h. Excess of solvent was distilled off, con-
centrated and cooled. The solid thus separated was filtered,
washed and recrystallised from ethanol. The IR spectra of
compounds 3a–k showed strong absorption bands for car-
bonyl group at 1,650 cm-1, aromatic CH stretching at
3,100 cm-1 and aromatic C=C stretching at 1,600 and
1,500 cm-1. Compound 3f showed absorption bands for
nitro group at 1,315 and 1,515 cm-1. Compounds 3i
showed absorption bands for hydroxyl group at
1-(3-Chloro-2-oxo-4-styrylazetidin-1-yl)-3-(2-oxo-2-
(10H-phenothiazin-10-yl)ethyl)urea (4b)
IR (KBr disc) cm-1: 3340 (NH str), 3133,1596 and 736
(aromatic str), 1597 (C=O str), 1596 and 1570 (phenothi-
azine ring), 1472 (C–N str), 919 (CH=CH), 832 (C–Cl) and
1
686 (C–S–C str). H-NMR (DMSO-d6) 6.8–7.8 (m, 13H,
Ar–H), 3.3 (s, 2H, CH2) 2.5(d,1H, J = 5.9 Hz, C–CH–
Cl)azetidinone ring, 8.5 (s, 1H, NH). MS m/z 505 [M]?;
Anal. Calcd for C26H21ClN4O3S (504.99). C, 61.84; H,
4.19; N, 11.09. Found: C, 61.56; H, 4.06; N, 11.03.
3,280–3,450 cm-1
.
1-(3-Chloro-2-(4-methoxyphenyl)-4-oxoazetidin-1-yl)-3-
(2-oxo-2-(10H-phenothiazin-10-yl)ethyl)urea (4c)
General method for the azetidinones (4a–k)
(Jubie et al., 2009)
IR (KBr disc) cm-1: 3340 (NH str), 3133,1596 and 736
(aromatic str), 1597 (C=O str), 1596 and 1570 (phenothi-
azine ring), 1401 (C–N str), 1253 (C–O–C), 919 (CH=CH),
Chloroacetyl chloride (0.01 mol) was added drop wise to the
mixture of triethylamine (0.01 mol, 12.5 ml of dry 1,4 diox-
ane) and solution of substituted Schiff bases (0.005 mol). The
reaction mixture was stirred for 30 min and then refluxed for
5 h. A solid was obtained was recrystallized from a mixture of
ethanol and water. The physical parameters of the synthesized
compounds were reported in Table 1.
1
832 (C–Cl) and 686 (C–S–C str). H-NMR (DMSO-d6)
6.5–7.6 (m, 12H, Ar–H), 3.82 (s, 3H, OCH3), 3.3 (s, 2H,
CH2) 2.5(d, 1H, J = 5.9 Hz, C–CH–Cl)azetidinone ring,
8.8 (s, 1H, NH). MS m/z 509 [M]?; Anal. Calcd for
C25H21ClN4O4S (508.98). C, 58.99; H, 4.16; N, 11.01.
Found: C, 58.79; H, 4.05; N, 11.30.
All the chemicals were of synthetic grade and com-
mercially procured from sigma Aldrich Chemicals. The
melting points of all the synthesized compounds were
determined using capillary tubes with Thermionic model
C-LMP-1-Campbell melting point apparatus and are
uncorrected. IR spectra were determined on JASCO FTIR
4100 using KBr pellets and wave number was reported in
1-(3-Chloro-2-(4-(dimethylamino)phenyl)-4-oxoazetidin-1-
yl)-3-(2-oxo-2-(10H-phenothiazin-10-yl)ethyl)urea (4d)
IR (KBr disc) cm-1: 3410 (NH str), 3150,1542 and 737
(aromatic str), 1620 (C=O str), 1596 and 1570 (phenothiazine
ring), 1470 (C–N str), 1312 (CN of N(CH3)2, 827 (C–Cl) and
1
cm-1. H NMR spectra (in CDCl3) on AV-III-400 Fourier
1
Transform NMR instrument using TMS as internal stan-
dard; the chemical shifts (d) were reported in ppm. Mass
spectra were recorded on JOEL SX 102-GC MATE
686 (C–S–C str). H-NMR (DMSO-d6) 6.59–7.0 (m, 12H,
Ar–H), 2.9 (s, 6H, N(CH3)2), 3.4 (s, 2H, CH2) 2.5(d, 1H,
J = 5.9 Hz, C–CH–Cl)azetidinone ring, 8.5 (s, 1H, NH). MS
123

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Med Chem Res (2013) 22:2578–2588
Table 1 Physical and analytical data of synthesized compounds
Code no.
R
Melting range (°C)
*Rf
Molecular formula
C24H19ClN4O3S
Mol. wt.
Colour
% yield
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
106–110
159–162
141–144
170–173
150–155
186–190
165–169
165–167
149–152
155–160
168–172
0.66
0.69
0.49
0.58
0.72
0.65
0.63
0.54
0.76
0.55
0.58
478.95
504.99
508.98
522.02
524.98
523.95
513.4
Pale pink
Green
65.50
85.38
82.72
71.11
67.71
55.33
76.44
79
H
C26H21ClN4O3S
4-OCH3
4-N(CH3)2
4-OH, 3-OCH3
4-NO2
2-Cl
C25H21ClN4O4S
C26H24ClN5O3S
C25H21ClN4O5S
C24H18ClN5O5S
Bright green
Blackish brown
Bluish green
Black
C
C
C
C
C
24H18Cl2N4O3S
24H18Cl2N4O3S
24H19ClN4O4S
24H18Cl2N4O3S
24H18ClFN4O3S
Pink
4-Cl
513.4
Pale brown
Pale green
Green
4-OH
3-Cl
494.95
513.4
66.07
69.33
72.86
4j
4k
a
3-F
496.94
Pale pink
Mobile phase: 4a–k—benzene:chloroform (1:9); detecting agent: 4a–k—iodine vapours
m/z 522 [M]?; Anal. Calcd for C26H24ClN5O3S (522.02). C,
59.82; H, 4.63; N, 13.42. Found: C, 59.62; H, 4.32; N, 13.17.
(d, 1H, J = 5.9 Hz, C–CH–Cl)azetidinone ring 8.6 (s, 1H,
NH). MS m/z 513 [M]?; Anal. Calcd for
C24H18Cl2N4O3S(513.4). C, 56.15; H, 3.53; N, 10.91. Found:
C, 56.02; H, 3.27; N, 10.77.
1-(3-Chloro-2-(4-hydroxy-3-methoxyphenyl)-4-
oxoazetidin-1-yl)-3-(2-oxo-2-(10H-phenothiazin-10-
yl)ethyl)urea (4e)
1-(3-Chloro-2-(4-chlorophenyl)-4-oxoazetidin-1-yl)-3-(2-
oxo-2-(10H-phenothiazin-10-yl)ethyl)urea (4h)
IR (KBr disc) cm-1: 3340 (OH str), 3154 (NH), 2932,1595
and 736 (aromatic str), 1750 (C=O str), 1596 and 1570
(phenothiazine ring), 1400 (C–N str), 1120 (C–O–C), 821
IR (KBr disc) cm-1: 3340 (NH str), 3133,1517 and 737
(aromatic str), 1590 (C=O str), 1596 and 1570 (phenothi-
azine ring), 1470 (C–N str), 816 (C–Cl) and 655 (C–S-C
(C–Cl) and 686 (C–S–C str). 1H-NMR (DMSO-d6
d
1
6.68–7.0 (m, 11H, Ar–H), 3.3 (s, 3H, OCH3), 3.4 (s, 1H,
OH), 2.5 (d, 1H, J = 5.9 Hz, C–CH–Cl)azetidinone ring,
8.7(s, 1H, NH) MS m/z 525 [M]?; Anal. Calcd for
C25H21ClN4O5S (524.98). C, 57.20; H, 4.03; N, 10.67.
Found: C, 57.52; H, 4.24; N, 10.82.
str). H-NMR (DMSO-d6) 6.52–7.57 (m, 12H, Ar–H), 3.4
(s, 2H, CH2), 2.5 (d, 1H, J = 5.9 Hz, C–CH–Cl)azetidi-
none ring 8.5 (s, 1H, NH). MS m/z 513 [M]?; Anal. Calcd
for C24H18Cl2N4O3S (513.4). C, 56.15; H, 3.53; N, 10.91.
Found: C. 56.34; H, 3.71; N, 10.65.
1-(3-Chloro-2-(4-nitrophenyl)-4-oxoazetidin-1-yl)-3-(2-
oxo-2-(10H-phenothiazin-10-yl)ethyl)urea (4f)
1-(3-Chloro-2-(4-hydroxyphenyl)-4-oxoazetidin-1-yl)-3-
(2-oxo-2-(10H-phenothiazin-10-yl)ethyl)urea (4i)
IR (KBr disc) cm-1: 3490 (NH str), 3143, 1578 and 736
(aromatic str), 1699 (C=O str), 1596 and 1570 (phenothi-
azine ring), 1517 and 1341 (N=O), 1467 (C–N str), 816 (C–
Cl) and 688 (C–S–C str). 1H-NMR (DMSO-d6) 6.8–7.5 (m,
12H, Ar–H), 3.5 (s, 2H, CH2) 2.5(d, 1H, J = 5.9 Hz, C–
CH–Cl)azetidinone ring, 8.8 (s, 1H, NH). MS m/z 524
[M]?; Anal. Calcd for C24H18ClN5O5S (524.06). C, 55.02;
H, 3.46; N, 13.37. Found: C, 55.37, H, 3.65, N, 13.58.
IR (KBr disc) cm-1: 3340 (OH str), 3132 (NH), 2932,1595
and 736 (aromatic str), 1596 and 1570 (phenothiazine
ring), 1590 (C=O str), 1470 (C–N str), 827 (C–Cl) and 686
1
(C–S–C str). H-NMR (DMSO-d6) 6.7–7.8 (m, 12H, Ar–
H), 3.4 (s, 1H, OH), 3.6 (s, 2H, CH2), 2.5 (d, 1H,
J = 5.9 Hz, C–CH–Cl)azetidinone ring, 8.4 (s, 1H, NH).
MS m/z 495 [M]?; Anal. Calcd for C24H19ClN4O4S
(494.95). C, 58.24; H, 3.87; N, 11.32. Found: C, 58.12; H,
3.62; N, 11.20.
1-(3-Chloro-2-(2-chlorophenyl)-4-oxoazetidin-1-yl)-3-(2-
oxo-2-(10H-phenothiazin-10-yl)ethyl)urea (4g)
1-(3-Chloro-2-(3-chlorophenyl)-4-oxoazetidin-1-yl)-3-(2-
oxo-2-(10H-phenothiazin-10-yl)ethyl)urea (4j)
IR (KBr disc) cm-1: 3346 (NH str), 3127, 1578 and 737 (aro-
matic str), 1721 (C=O str), 1596 and 1570 (phenothiazine ring),
1
1463 (C–N str), 816 (C–Cl) and 688 (C–S–C str). H-NMR
IR (KBr disc) cm-1: 3346 (NH str), 3127,1595 and 736
(aromatic str), 1671 (C=O str), 1596 and 1570 (phenothia-
zine ring), 1460 (C–N str), 837 (C–Cl) and 656 (C–S–C str).
(DMSO-d6) 6.52–7.26 (m, 12H, Ar–H), 3.32 (s, 2H, CH2), 2.5
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Med Chem Res (2013) 22:2578–2588
2581
1H-NMR (DMSO-d6) 6.6–7.8 (m, 12H, Ar–H), 3.7 (s, 2H,
CH2), 2.5 (d, 1H, J = 5.9 Hz, C–CH–Cl)azetidinone ring
8.8 (s, 1H,NH). MS m/z 513 [M]?; Anal. Calcd for
C24H18Cl2N4O3S (513.4). C, 56.15; H, 3.53; N, 10.91.
Found: C, 56.05; H, 3.22; N, 10.72.
Monococus purpureus, Aspergillus niger and Trichophyton
rubrum by disc diffusion method and MIC method.
Disc diffusion method
The growth media (Muller Hinton Agar media for bacterial
growth and potato dextrose agar media for fungal growth)
were prepared and sterilized in autoclave at 15 psi for
15 min. These media were poured into petri plates under
standard conditions and allowed to solidify. Standardized
bacterial inoculum was spread uniformly over the surface
of medium using a sterile non-absorbent cotton swab, and
finally, the swab was passed around the edge of the med-
ium. The inoculated Petri plates were closed with the lid
and allowed to dry at room temperature. The sample
impregnated discs and standard discs were placed on the
inoculated agar medium. All Petri plates were incubated at
37 °C for 24 h. After the incubation, diameter of zone of
inhibition produced by the sample and standard was mea-
sured (Ginnis and Rinaldi, 2001; Rokade and Dongare,
2010). The details are tabulated in Table 2.
1-(3-Chloro-2-(3-fluorophenyl)-4-oxoazetidin-1-yl)-3-(2-
oxo-2-(10H-phenothiazin-10-yl)ethyl)urea (4k)
IR (KBr disc) cm-1: 3346 (NH str), 2920,1596 and 736
(aromatic str), 1718 (C=O str), 1596 and 1570 (phenothi-
azine ring), 1472 (C–F), 1401 (C–N str), 876(C–Cl) and
1
656 (C–S–C str). H-NMR (DMSO-d6) 6.8–7.5 (m, 12H,
Ar–H), 3.3 (s, 2H, CH2), 2.5 (d, 1H, J = 5.9 Hz, C–CH–
Cl)azetidinone ring, 8.6 (s, 1H, NH). MS m/z 497 [M]?;
Anal. Calcd for C24H18ClFN4O3S (496.94). C, 58.01; H,
3.65; N, 11.27. Found: C, 58.24; H, 3.82; N, 11.48.
Biological activity
Antibacterial and antifungal activity
Minimum inhibitory concentration method
All the synthesized compounds have been screened for anti-
bacterial activity against Gram-positive bacteria Micrococcus
luteus. Staphylococcus aureus, Bacillus subtilis and Staphy-
lococcus albus; Gram-negative bacteria Pseudomonas aeru-
ginosa, Salmonella paratyphi, Escherichia coli and Vibreo
cholera; and antifungal activity against Candida albicans,
The serial dilution of compound solutions were made from
the stock (1,000 lg/ml) using Muller Hinton broth for
bacteria and potato dextrose broth for fungi using the
method.
Table 2 Antimicrobial activity of titled compounds by disc diffusion method
Zone of inhibition in mm
Bacteria
Fungi
C.a
Compound
P.a
E.c
S.p
V.c
M.l
S.a
B.s
S.a
M.p
A.n
T.r
4a
7
5
5
7
5
7
7
6
4
5
–
3
5
8
7
4
4
4
9
7
7
6
5
7
8
6
8
3
6
4
6
4
8
4b
4c
7
8
8
9
7
7
10
8
7
6
10
8
6
4d
12
10
–
7
6
8
5
8
7
7
7
4e
10
15
8
10
7
10
5
8
6
5
10
14
12
7
7
5
4
4f
3
5
4
13
10
12
10
7
9
5
4g
7
12
5
6
7
7
5
8
10
6
4h
5
19
5
18
9
5
7
5
12
8
4i
18
10
11
12
10
10
12
5
10
11
11
13
11
11
13
12
11
8
4j
5
8
7
6
4k
6
9
7
10
7
7
Control
Ciprofloxacin
Clotrimazole
No zone of inhibition
22
–
20
–
18
–
15
–
16
–
20
–
25
–
23
–
–
–
–
–
4
–
20
22
P.a Pseudomonas aeruginosa; E.c Escherichia coli; S.p Salmonella paratyphii; V.c Vibreo cholera; M.l Micrococcus luteus; S.a Staphylococcus
aureus; B.s Bacillus subtilis; S.a Staphylococcus albus. C.a Candida albicans; M.p Monococus purpureus; A.n Aspergillus niger; T.r Tricho-
phyton rubrum
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Med Chem Res (2013) 22:2578–2588
The tubes were labelled 1–8 and 1 ml of broth was
200 ll. The final concentrations of the test molecules were 1, 10
and 100 lg/ml. Growth control wells contained medium and
M. tuberculosis H37Rv alone. Rifampicin (1.0 lg/ml) served
as positive control for inhibition of growth. Negative control
wells contained the highest volume of DMF used in test wells
without any compound. After incubation at 37 °C for 7 days,
15 ll of 0.01 % resazurin (Sigma, St. Louis. MO, USA) solu-
tion in sterile water was added to the first growth control wells
and incubated for 24 h. Once the first set of growth controls
turned pink, the dye solution was added to the second set of
growth controls and the test wells, and incubated for 24 h at
37 °C. Blue colour in the wells containing the test compounds
indicated inhibition of growth and pink indicated lack of inhi-
bition of growth of M. tuberculosis. The details are tabulated in
Table 4.
added to the first 5 tubes and 8th tube, and then added
0.5 ml broth to 6th and 7th tubes. One ml of different
synthesized compounds was added to the 1st tube, mixed
and transferred 1 ml serially up to tube 6. Mixed and
transferred 0.5 ml to the 7th tube so that each tube, 1–7
contained 1 ml diluted extracts. The 8th tube served as the
control. With a standardized micro pipette, added a drop of
the diluted broth culture approximately 0.01 ml of the test
organism to all tubes, including the control, gently mixed
and incubated at 37 °C for 18 h. After incubation the tur-
bidity was observed. The highest dilution of particular
compound showing no turbidity was recorded. This was
taken as the end point, and this dilution was considered to
contain the concentration of drug equivalent to MIC
(Walsh et al., 1990) (Table 3).
Antitubercular activity
Antioxidant activity
The anti-TB activity of the compounds was tested by resazurin
microplate assay (Martin et al., 2003). Mycobacterium tuber-
culosis H37Rv was grown in Middlebrook 7H9 broth (Difco
BBL, Sparks, MD, USA) supplemented with 10 % OADC
(Becton-Dickinson, Sparks, MD, USA) and 0.5 % glycerol.
The optical density of the bacterial culture was adjusted to
McFarland 1.0 unit and 50 ll from this suspension was used as
the inoculums. Stock solutions of the test compounds were
prepared in dimethyl formamide (DMF) and were added to
fresh medium in the wells of a 96-well microplate to which
50 ll inoculum was added making the total assay volume
DPPH radical scavenging method
Sample stock solutions (1.0 mg/ml) were diluted to appro-
priate final concentrations in ethanol. An ethanolic solution
of 1 ml of DPPH (0.3 mM) was added to 0.5 ml of the
compound and allowed to react at room temperature in a dark
place for 30 min. After 30 min, the absorbance values were
measured at 518 nm. All the measurements were taken as a
triplicate values. From the average of the absorbance values,
lower absorbance of the reaction mixture indicates higher
free radical scavenging activity (Mensor et al., 2000). The
Table 3 Minimum inhibitory concentration of synthesized compounds against bacteria by broth dilution method
MIC (lg/ml)
Bacteria
P.a
Fungi
C.a
Compound
E.c
S.p
V.c
M.l
S.a
B.s
S.a
M.p
A.n
T.r
4a
250
125
125
62.5
125
500
250
250
62.5
125
125
62.5
–
500
250
125
250
125
62.5
250
62.5
250
500
250
62.5
–
500
125
125
250
125
125
125
250
62.5
125
125
125
–
250
125
125
125
125
250
250
62.5
125
250
125
125
–
250
250
250
250
250
250
250
250
62.5
250
250
125
–
250
500
250
125
250
250
500
62.5
125
250
250
62.5
–
250
250
250
125
250
250
250
125
125
125
62.5
62.5
–
250
125
62.5
125
250
250
250
250
125
125
125
62.5
–
500
250
250
250
125
62.5
62.5
125
62.5
62.5
125
–
250
250
250
250
250
62.5
125
62.5
500
250
125
–
250
250
125
125
250
500
125
125
62.5
250
250
–
250
125
250
250
250
250
125
250
250
250
125
–
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
Ciprofloxacin
Clotrimazole
62.5
62.5
125
62.5
P.a Pseudomonas aeruginosa; E.c Escherichia coli; S.p Salmonella paratyphii; V.c Vibrio cholera; M.l Micrococcus luteus; S.a Staphylococcus
aureus; B.s Bacillus subtilis; S.a Staphylococcus albus. C.a Candida albicans; M.p Monococus purpureus; A.n Aspergillus niger; T.r Tricho-
phyton rubrum
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Med Chem Res (2013) 22:2578–2588
2583
120
100
80
60
40
20
0
Table 4 In vitro antitubercular screening by REMA
No.
Compound
Concentration lg/ml
1
10
100
1
2
3
4
5
4d
N
N
N
P
N
N
P
P
P
P
4f
4d
4j
std
Rifampicin
Control
P
N
N
P inhibition; N no inhibition
DPPH radical scavenging capability was calculated using the
following equation:
25
50
conc µg
75
100
% inhibition ¼ ðABScontrol ꢁ ABStestÞ=ABScontrol ꢂ 100
where,
Fig. 1 DPPH radical scavenging activity of compound 4d, measured
at 517 nm
ABSControl ¼ Absorbance of ethanol þ DPPH
ABSTest ¼ Absorbance of DPPH þ compound =standard
120
100
80
The percentage antioxidant activity (% inhibition) was
extrapolated against concentration of the compound and
EC50 was determined graphically. The results are tabulated
in Table 5 and Figs. 1, 2, 3, 4.
4g
60
40
20
0
std
FRAP radical scavenging method
FRAP assay was carried out 0.2 ml of the compound is added to
3.8 ml of FRAP reagent, the reaction mixture was incubated at
37 °C for 30 min and the increase in absorbance at 593 nm is
measured. FeSO4 is used for calibration. Quantitative calcula-
tion for each sample was done by plotting linear calibration
curve (Benzie and Strain 1996). The antioxidant activities were
expressed as the concentration of antioxidants having a ferric
reducing ability equivalent to that of 1 mM/l FeSO4. The
equation used for absorbance was as (y) = 0.274x ? 0.114
[r2 = 0.974]. The results are tabulated in Table 5and Figs. 5, 6.
25
50
conc µg
75
100
Fig. 2 DPPH radical scavenging activity of compound 4g, measured
at 517 nm
120
100
80
Anticancer activity
4g
60
40
20
0
Anticancer activity of samples determined by MTT assay
method. The human cervical cancer cell line (HELA) was
std
Table 5 DPPH and FRAP radical scavenging activity of titled
compounds
Compound
DPPH (EC50 lg/ml)
FRAP (R2 value)
25
50
75
100
conc µg
4d
55
57
56
47
40
–
0.956
0.586
0.567
0.967
–
4g
Fig. 3 DPPH radical scavenging activity of compound 4g, measured
at 517 nm
4h
4k
obtained from National Centre for Cell Science (NCCS), Pune.
The cells were grown in Eagles minimum essential medium
containing 10 % foetal bovine serum (FBS). For screening
Ascorbic acid
Ferrous sulphate
0.981
123

2584
Med Chem Res (2013) 22:2578–2588
120
100
80
60
40
20
0
y = 0.004x + 0.863
R² = 0.981
1.4
1.2
1
std
4k
0.8
0.6
0.4
0.2
0
Linear
(std)
4k
std
Linear
(4k)
y = 0.001x + 0.245
R² = 0.967
0
50
100
150
conc
25
75
50
conc µg
100
Fig. 6 FRAP radical scavenging activity of compound 4g, measured
at 593 nm
Fig. 4 DPPH radical scavenging activity of compound 4k, measured
at 517 nm
Results and discussion
experiment, the cells were seeded into 96-well plates in 100 ll
of medium containing 5 % FBS, at plating density of 10,000
cells/well and incubated at 37 °C, 5 % CO2, 95 % air and
100 % relative humidity for 24 h prior to addition of samples.
The samples were solubilized in dimethylsulphoxide and
diluted in serum free medium. After 24 h, 100 ll of the med-
ium containing the samples at various concentrations (6.25,
12.5, 25, 50 and 100 lM) were added and incubated at 37 °C,
5 % CO2, 95 % air and 100 % relative humidity for 48 h.
Triplicate was maintained and the medium containing without
extracts were served as control. After 48 h, 15 ll of MTT
(5 mg/ml) in phosphate-buffered saline was added to each well
and incubated at 37 °C for 4 h. The medium with MTT was
then flicked off and the formed formazan crystals were solu-
bilized in 100 ll of DMSO and then measured the absorbance
at 570 nm using microplate reader (Mosmann 1983; Monks
et al., 1991). The % cell inhibition was determined using the
following formula. The results are tabulated in Table 6 and
represented in Figs. 7, 8, 9, 10, 11 and 12
Chemistry
A series of eleven 1-(3-chloro-2-oxo-4-phenylazetidin-1-
yl)-3-(2-oxo-2-(10H-phenothiazin-10-yl)ethyl) urea deriv-
atives have been synthesized by a four-step reaction as
described in Scheme. In the first step, 2-chloro-1-(10H-
phenothiazin-10-yl)ethanone has been prepared from phe-
nothiazine and chloroacetyl chloride using dry benzene as
solvent followed by the treatment with semicarbazide to
form 4-(2-oxo-2-(10H-phenothiazin-10yl)ethyl) semicarb-
azide. Schiff bases of phenothiazines were synthesized by
the condensation of 4-(2-oxo-2(10H-phenothiazin-10yl)
semicarbazide with various substituted aromatic aldehydes.
Finally, a series of substituted azetidinones have been syn-
thesized by cyclocondensation of various Schiff bases of
phenothiazine with chloroacetyl chloride in the presence of
Table 6 Percentage cell inhibition produced by titled compounds at
varying concentrations
% cell inhibition ¼ 100 ꢁ abs ðsampleÞ=abs ðcontrolÞ
ꢂ 100
Code
Conc (lM)
% cell inhibition
IC50 (lM)
4f
6.25
12.5
25
23.61624
41.82042
56.45756
73.18573
89.5449
18.26
50
y = 0.004x + 0.863
1.4
1.2
1
R² = 0.981
100
6.25
12.5
25
std
4h
4i
0.123001
5.904059
15.91016
33.57934
44.28044
0.123001
5.04305
111.2
4d
0.8
0.6
0.4
0.2
0
50
Linear
(std)
y = 0.000x + 0.063
R² = 0.956
100
6.25
12.5
25
84.11
Linear
(4d)
0
50
100
150
19.06519
37.39237
53.01353
conc
50
Fig. 5 FRAP radical scavenging activity of compound 4d, measured
at 593 nm
100
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Med Chem Res (2013) 22:2578–2588
2585
100
80
60
40
20
0
0.5
1.0
1.5
2.0
2.5
Log 10 concentration (µM)
Fig. 7 Graph plotted between the percentage growth inhibition and
log concentration of compound 4f (R2 = 0.9922)
Fig. 10 Inhibition of HELA by compound 4f (25 lM)
Fig. 11 Inhibition of HELA by compound 4f (50 lM)
Fig. 12 Inhibition of HELA by compound 4f (100 lM)
Fig. 8 Inhibition of HELA by compound 4f (6.5 lM)
Fig. 9 Inhibition of HELA by compound 4f ((12.5 lM)
triethyl amine. All the titled compounds yielded the products
in the range of 57–85 %. The melting points of compounds
4a–k were observed in the range of 106–190 °C. All com-
pounds showed only one spot of migration from the origin on
TLC plates, thereby, confirming their purity (Scheme 1).
The structure of intermediate compounds 3a–k were con-
firmed by the presence of characteristic peaks in the regions of
1,690 cm-1 for C=O stretching, 3,350 cm-1 for NH stretch-
ing, 3,080, 1,600 and 790 cm-1 for aromatic stretching and
1,569 cm-1 for N=CH stretching. The presence of phenothi-
azine ring in compounds 4b–k is confirmed by the IR
absorption bands in the region 1,596 and 1,570 cm-1 (Kure
and Morris, 1976). The compounds of 4a–k showed
123

2586
Med Chem Res (2013) 22:2578–2588
Scheme 1
characteristic peaks in the regions of 1,657 cm-1 for C=O
stretching for azetidinone, 3,340 cm-1 for NH stretching,
3,129 cm-1, 1,592 cm-1 and 737 cm-1 for aromatic
stretching, 1,467 cm-1 for C–N stretching and 827 cm-1 for
C–Cl stretching.
for two protons of CH2 group and a singlet at d 8.5 for one NH
proton confirming the synthesized derivatives. Compounds
4a–k were verified from their elemental analysis and molec-
ular ion peaks (M?.).
Compounds containing NO2 group showed absorption
bands at 1,518, 1,343 cm-1 for the N=O stretching, and the
peaks at 650–740 cm-1 could be assigned to C–S–C stretch-
ing. The presence of hydroxyl group was confirmed by the
Antibacterial and antifungal activity
The synthesized compounds 4a–k were screened in vitro
antimicrobial activity. All the synthesized compounds have
shown moderate to significant activity against the pathogenic
bacteria and fungi. Compounds (4h) showed significant
antibacterial activity against Gram-negative organisms P.
aeruginosaand V. cholerae(zoneofinhibition19and 18 mm,
1
appearance of broad peak at 3,430–3,161 cm-1. H NMR
signals showed multiplet signals in the range of d 6.52–7.5 for
the protons of aromatic ring, doublets in the range of d 2.5 for
one proton of –CH–Cl group of azetidinone, singlet at d 3.35
123

Med Chem Res (2013) 22:2578–2588
2587
respectively, MIC 62.5 lg/ml). This may be due to the pres-
ence of electron-withdrawing chlorophenyl group on ring
nitrogen of azetidinone moiety, which is in correlation with
reported azetidinone moiety possess antimicrobial action
(Singh et al., 2011) or the presence of phenothiazine could be
another important reason for its antibacterial activity (Upad-
hyay et al., 2009) and (4i) showed comparable activity as that
of standard ciprofloxacin against E. coli (zone of inhibition
18 mm, MIC 62.5 lg/ml). urea (4f) was found to show
excellent activity against C. albicans and M. purpurea
(14 mm, 13 mm zone of inhibition, respectively, MIC
62.5 lg/ml). The compound is expected to be highly active
due to the presence highly electronegative nitro group in
addition to the presence of azetidinone linked with urea
moiety. The compounds (4g), (4 h) and (4j) which showed
equipotent activity against C. albicans, M. purpurea and A.
niger (10 mm zone of inhibition respectively, MIC 62.5 lg/
ml) which may be due to the ortho, para and meta substitution
of chloro substituted phenyl group (Kumar et al., 2010).
Conclusion
From all these studies, it is apparent that the phenothiazine
and structurally related compounds possessing three ben-
zene rings are often endowed with potent antimicrobial
action. All synthesized compounds showed moderate to
significant antibacterial and antifungal activity. Among the
compounds tested for antimicrobial action, compound 4h
showed potent activity against E. coli, V. chlorea and A .
niger which produced 19, 18 and 12 mm of zone inhibi-
tion. The same fact is reiterated in MIC studies of the
compound, where it exhibited inhibitory concentration of
62.5 g/ml. The compound 4j exhibited significant antimy-
cobacterial activity at a concentration of 10 lg/ml.
Compound 4d, 4g, 4h and 4k showed good antioxidant
activity with EC50 value of 55, 57, 56 and 47 lg/ml tested by
DPPH method. Compounds 4j at a concentration of 10 lg/
ml showed inhibition against the growth of M. tuberculosis
and 4f showed significant activity against HELA with IC50
values of 18.26 lM.
Antimycobacterial activity
The results obtained, encouraged us to pursue further
research in the synthesis of many derivatives of titled
compounds to perform in vivo trials in experimental ani-
mals to broaden their pharmacological assessment and
receptor interactions.
In vitro antitubercular screening of three compounds which
showed significant antibacterial and antifungal activity were
studied by REMA which revealed the compounds 4j showed
significant antimycobacterial activity at a concentration of
10 lg/ml. The bulkier group of compound (4d) and electron-
withdrawing group of compound (4f) possessed antimyco-
bacterial activity at a concentration of 100 lg/ml. This activity
may be due to electron-withdrawing substitution at position 4
of aryl ring attached to azetidinone (Mitchell et al., 2007).
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