Synthesis of novel carbazole chalcones as radical scavenger, antimicrobial and cancer chemopreventive agents

Article abstract of DOI:10.3109/14756366.2012.663365

A series of novel carbazole chalcones has been synthesised and evaluated for radical scavenging activity, cytotoxicity and antimicrobial activities. Compounds 12m, 12o and 12c exhibited good 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, compounds 12e, 12m and 12d were excellent hydroxyl radical scavengers and compounds 12a, 12e, 12g, 12n and 12m have shown inhibition of oxidative DNA damage induced by 2,2′-azobis (2-amidinopropane hydrochloride). Compounds 12j, 12i, 12n, 12c, 12m and 12e were most active against the selected cancer cell lines. Compounds 12a, 12e and 12m showed good antibacterial activity and compounds 12h and 12m have shown good antifungal activity. All the compounds were subjected for absorption, distribution, metabolism and excretion (ADME) predictions by computational method and found that these molecules could be considered as potential candidates for oral drug development.

Full text of DOI:10.3109/14756366.2012.663365

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 593–600
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.663365
research artIcle
Synthesis of novel carbazole chalcones as radical scavenger,
antimicrobial and cancer chemopreventive agents
Babasaheb P. Bandgar1, Laxman K. Adsul1, Shrikant V. Lonikar1, Hemant V. Chavan1,
Sadanand N. Shringare1, Sachin A. Patil1, Shivkumar S. Jalde1, Basawaraj A. Koti1,
Nagesh A. Dhole2, Rajesh N. Gacche2, and Amol Shirfule3
1Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur, Maharashtra,
2Biochemistry Research Laboratory, School of Life Sciences, S.R.T.M. University, Nanded, Maharashtra, and
3Food and Drug Toxicology Research Centre, National Institute of Nutrition, Hyderabad, Andhra Pradesh, India
abstract
A series of novel carbazole chalcones has been synthesised and evaluated for radical scavenging activity, cytotoxicity
and antimicrobial activities. Compounds 12m, 12o and 12c exhibited good 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavengingactivity,compounds12e,12mand12dwereexcellenthydroxylradicalscavengersandcompounds12a,12e,12g,
12n and 12m have shown inhibition of oxidative DNA damage induced by 2,2′-azobis (2-amidinopropane hydrochloride).
Compounds 12j, 12i, 12n, 12c, 12m and 12e were most active against the selected cancer cell lines. Compounds 12a, 12e
and 12m showed good antibacterial activity and compounds 12h and 12m have shown good antifungal activity. All the
compounds were subjected for absorption, distribution, metabolism and excretion (ADME) predictions by computational
method and found that these molecules could be considered as potential candidates for oral drug development.
Keywords: Chalcones, carbazole, anticancer, antimicrobial, antioxidant, Lipinski rule
Introduction
to malignancy. Damage to DNA by oxidative stress has
been widely accepted as a major cause of cancer4. DNA
mutation is a critical step in carcinogenesis and elevated
levels of oxidative DNA lesions have been observed in
various tumours. e discovery of the role of free radicals
in cancer, diabetes, cardiovascular diseases, autoimmuno
diseases, neurodegenerative disorders, aging and other
diseases has lead to new medical insight5,6. Minimum
oxidative damage may be an important approach to
the primary prevention or treatment of these diseases.
Antioxidants may stop the free radical formation or inter-
rupt an oxidising chain reaction. is has attracted a
great deal of research interest in therapeutic antioxidant-
based drug formulations. e development of synthetic
compounds capable of scavenging free radicals has been
a great success.
Antioxidant substances prevent or inhibit the natural
phenomena of oxidation. In the recent years, “antioxi-
dant research” has attracted several researchers because
of its broad applications in the management and amelio-
ration of a variety of human ailments1,2. e formation of
reactive oxygen species (ROS) is characteristic of aerobic
organisms that can normally defend themselves against
these highly reactive species using enzymes like superox-
ide dismutase and glutathione peroxidase, and naturally
occurring antioxidants such as α-tocopherol (vitamin
E), ascorbic acid (vitamin C) and β-carotene3. However,
in pathophysiological conditions, the excessive produc-
tion of ROS overwhelms the natural antioxidant defence
mechanisms. is imbalance is termed as oxidative
stress. High levels of free radicals can cause damage to
biomolecules such as lipids, proteins, enzymes and DNA
in cells and tissues, resulting in mutations that can lead
Carbazole and its derivatives are the important class
of heterocyclic compounds endowed with various
Address for Correspondence: Babasaheb P. Bandgar, Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur
University, Solapur 413 255, Maharashtra, India. Tel/Fax: 0091-217-2351300. E-mail: bandgar_bp@yahoo.com
(Received 08 December 2011; revised 28 January 2012; accepted 31 January 2012)
593

594 B. Bandgar et al.
pharmacological activities such as anticancer, antimi-
crobial, antiviral, anti-inflammatory and antioxidant
activity7. Most of carbazole heterocycles are of natural
and synthetic origins and have wide spread distribution
in terrestrial plants and micro-organisms. From the bio-
assay-guided approach, various carbazole alkaloids have
been isolated and are subjected for biological evaluation.
Naturally occurring carbazole derivatives, murrayafoline
A1 (Figure1), carbazomycinAandB2, haveshownpotent
antimicrobial activity8. Ellipticine 3, a pyridocarbazole,
shows potent antiproliferative activity; rebaccamycin 4,
an indolocarbazole derivative, acts as an antibiotic and
shows inhibition of topoisomerase I and carazostatin 5
is a potent natural antioxidant9–11. e isolation of these
molecules spurred the research to synthesise these mol-
ecules and its analogues to optimise these natural lead
molecules into a clinical molecule. Among the various
biological activities exhibited by carbazole derivatives of
the particular interest is anticancer activity12. e majority
of carbazole derivatives exert cytotoxicity via interaction
with DNA. Because these derivatives possess structural
features such as flat polycyclic and aromatic chro-
mophore, the optimal size for intercalating chromophore
is built by a tricyclic or tetracyclic ring system. erefore,
tricyclic planar carbazole chromophore is predestined for
intercalation into DNA. In addition to the DNA binding,
carbazole derivatives present antiproliferative effect by
other mechanisms, such as inhibition of angiogenesis13,
inhibition of cyclin-dependent kinase 414, inhibition of
microtubulin15, inhibition of check point kinase 116 and
inhibition of topoisomerase I and II17,18. Carvedilol 6, an
antihypertensive drug, acts as a nonspecific β-adrenergic
antagonist19. Carprofen 7, a nonsteroidal anti-inflamma-
tory drug, is a selective COX-2 inhibitor20.
In continuation of our studies in synthesising vari-
ous biologically active compounds21, in this study, we
have synthesised and characterised the novel carbazole
chalcones and evaluated for antioxidant, cytotoxicity and
antimicrobial activities. We also subjected all synthesised
compounds for ADME predictions by computational
method.
results and discussion
Chemistry
Chalcone is a unique template that is associated with
several biological activities. e biological activities
of chalcones are equally wide ranging. In fact, not
many structural templates can claim association with
a diverse range of pharmacological activities such as
antitumor, anti-inflammatory, antiplasmodial, immu-
nosuppression and antioxidant activities22,23. With the
great biological importance of carbazole derivatives,
we synthesised a series of carbazole chalcones from
Figure 1. Biologically active natural and synthetic carbazole derivatives.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of novel carbazole chalcones 595
3-formyl-9-methylcarbazole and various acetophe-
nones (Scheme 1). Carbazole (9) on methylation with
methyl iodide gave 9-methyl carbazole (10), which on
Vilsmeier–Haack formylation gave 3-formyl-9-methyl-
carbazole (11). is 3-formyl-9-methylcarbazole (11) on
Claisen–Schimdt condensation with various aromatic
and heteroaromatic acetophenones in aqueous sodium
hydroxide afforded titled compound (12a-o) in good to
excellent yields (Table 3, Supplementary information).
All the synthesised compounds were characterised by IR,
1H NMR, 13C NMR, CHN analysis and MS .
shown good radical scavenging activity (89.53%–82.30%)
as compared with the standard ascorbic acid (95.76%).
Whereas, compounds 12f, 12h, 12k, 12l, 12a and 12e
have shown moderate DPPH radical scavenging activ-
ity (79.03%–70.76%). e structure activity relationship
(SAR) study reveals electron-withdrawing substituents
on B ring increases DPPH radical scavenging activity,
while electron-donating substituents decreases the activ-
ity with the exception of compound 12c. Replacement
of aromatic B ring by heteroaromatic ring enhances the
DPPH radical scavenging activity.
Hydroxyl radical is known to react with all compo-
nents of DNA molecule, damaging both the purine and
pyrimidine bases and also the deoxyribose backbone26.
Hydroxyl radical scavenging ability of the synthesised
chalcones is presented in Table 1. e results reveal that
compounds 12e, 12m and 12d have shown excellent
hydroxyl radical scavenging activity (91.72%–87.59%)
as compared with the standard ascorbic acid (88.34%).
While compounds 12a, 12j, 12l, 12g, 12i and 12n
have shown good hydroxyl radical scavenging activity
(86.84%–71.42%) and compounds 12k, 12h, 12f and 12o
are moderate OH radical scavengers (69.54%–54.51%).
In general, an electron-withdrawing substituent on
B ring increases hydroxyl radical scavenging activity,
whereas electron-donating substituent on B ring reduces
the activity except in case of 12d. Replacing aromatic B
ring of 12a by heterocyclic ring, in particular thiophene,
decreases the OH radical scavenging activity. e highest
occupied molecular orbital (HOMO), a parameter repre-
senting the molecular electron-donating ability, is also
a good index predicting antioxidant activity27. e com-
pounds possessing higher energy of HOMO (EHOMO) and
Biological evaluation
All the synthesised compounds were evaluated for DPPH
radical scavenging activity (Table 1). e DPPH radical
scavenging method is a well-known method for deter-
mining antioxidant activity24,25. Among the tested chal-
cones, compounds 12m, 12o, 12c, 12g, 12n and 12i have
Scheme 1. Reagents and conditions: (i) DMF, NaH, CH3I, rt, 3h;
(ii) DMF, POCl3, 80°C, 4h; (iii) Substituted acetophenones, NaOH,
ethanol, rt, 24–36h.
Table 1. Radical scavenging activity of carbazole chalcones.
DPPH radical scavenging activity
Hydroxyl radical scavenging activity
Inhibition (%)
72.69
18.65
83.84
61.92
70.76
79.03
83.07
76.34
82.30
59.61
75.57
73.65
89.53
82.88
87.50
95.76
IC50 (μg/mL)
70.15
ND
Inhibition (%)
86.84
32.33
69.54
87.59
91.72
61.65
78.19
63.90
74.06
83.83
63.90
79.32
88.34
71.42
54.51
88.34
IC50 (μg/mL)
51.10
ND
Compounds
R
12a
H
12b
4-OCH3
3,4-OCH3
2,4,6-OCH3
4-F
12c
52.49
ND
ND
12d
38.60
35.15
ND
12e
ND
12f
2,4-F
67.50
60.45
68.15
61.45
ND
12g
3,4-F
56.10
ND
12h
4-Cl
12i
2,4-Cl
3,4-Cl
2,5-Cl
4-Br
ND
12j
54.34
ND
12k
ND
12l
ND
56.15
36.63
ND
12m
12n
4-NO2
2-iophene
2-pyridine
—
47.10
60.45
50.65
45.94
12o
Ascorbic acida
ND
48.45
e results summarised for inhibition (%) are mean values of two parallel experiments.
Inhibition (%) activity tested at 100 μg.
IC50 values represent mean standard deviation from three different experiments.
ND, Not determined.
aStandard.
© 2013 Informa UK, Ltd.

596 B. Bandgar et al.
energy of lowest unoccupied molecular orbital (ELUMO
)
were found to be effective agents for stabilising the DPPH
and hydroxyl radicals; the antioxidant effect of carve-
dilol mainly resides in carbazole moiety28 and N-ethyl
carbazole derivatives acts as electron donors29; all these
properties will explain the high antioxidant activity of the
compounds under investigation.
e IC50 values for radical scavenging activities of the
tested compounds are listed in Table 1. e compounds
12m, 12o and 12c exhibited low IC50 values (47.10–52.49
μg/mL) for DPPH radical scavenging activity. However,
the tested compounds did not show DPPH radical scav-
enging activity as effective as the standard ascorbic acid
(IC50 = 45.94 μg/mL). In addition, IC50 values for hydroxyl
radical scavenging activity, compounds 12e, 12m and
12d, have shown low IC50 values (35.15–38.60 μg/mL) as
compared with the standard ascorbic acid (IC50 = 48.45
μg/mL). IC50 value is defined as the concentration of the
sample causing 50% decrease in absorbance; a lower IC50
value would reflect greater radical scavenging activity of
the sample.
Figure 2. Agarose gel electrophoresis pattern of pUC 18 DNA
strand breakage by 2,2′-azobis (2-amidinopropane hydrochloride)
(AAPH) and inhibited by carbazole chalcones. Supercoiled
plasmid DNA was incubated with AAPH and/or carbazole
chalcones in phosphate-buffered saline (pH 7.4) at 37°C for 1h.
Lane 1: DNA strand breakage induced by AAPH at 10mM; Lane
2: control super coiled DNA; Lane 3: compound 12g; Lane 4: 12a;
Lane 5: 12e; Lane 6: 12n and lane 7: 12m.
Permanent modification of genetic material result-
ing from oxidative damage incidents represents the first
step involved in mutagenesis, carcinogenesis and aging.
In fact, it is well established that free radical-mediated
DNA damage occurs in various cancer tissues. Till date,
more than 100 products have been identified from the
oxidation of DNA. ROS-induced DNA damage involves
single- or double-stranded DNA breaks, purine, pyrimi-
dine or deoxyribose modifications and DNA cross links.
DNA damage can result either in arrest or induction of
transcription, induction of signal transduction path-
ways, replication errors and genomic instability, all of
which are associated with carcinogenesis30,31. In case of
plasmid, damage of DNA results in a cleavage of one of
the phosphodiester chains of the supercoiled DNA and
produces a relaxed open circular form. Further cleavage
near the first breakage results in linear double-stranded
DNA molecules. e formation of circular form of DNA
is indicative of single-strand breaks and the formation of
linear form of DNA is indicative of double-strand breaks32.
We have evaluated antioxidant activity of novel carba-
zole chalcones against 2,2′-azobis (2-amidinopropane
hydrochloride) (AAPH)-induced plasmid pUC18 DNA
strand breakage using agarose gel electrophoresis analy-
sis (Figure 2)33. Compounds 12a, 12e, 12g, 12n and 12m
have shown inhibition of AAPH-induced DNA breakage.
e DNA protection is in the order 12g > 12e > 12n > 12a
> 12m. ese results confirm the antioxidant activity
exhibited by these compounds by DPPH, OH methods as
well as AAPH-induced DNA damage.
effective against selected cell lines. ese compounds
inhibit the proliferation of selected three cancer cell lines
with highest selectivity (3.85–2.06) than the normal cell
lines. Whereas compounds 12f, 12k and 12o showed
preferential cytotoxicity against the Hep 3BPN7 and HL60
P58. e SAR studies with respect to cytotoxicity revealed
that compounds with electron-withdrawing groups on
aromaticringBshowhighercytotoxicitythancompounds
containing electron-donating groups with the exception
of compound 12c. Position of substituents on aromatic
ring B influences the cytotoxicity; compound with mono-
fluoro substituent (12e) shows cytotoxicity against all
the three cancer cell lines. However, compounds with
difluoro substituent (12f and 12g) were cytotoxic to only
liver and leukaemia cell line. e bioisosteric replace-
ment of aromatic ring (12a) by heteroaromatic ring (12n)
increases cytotoxicity.
Cancer chemoprevention is regarded as a promising
avenue for cancer control and has become increasingly
important in recent years. Tumourigenesis is a multistep
process that begins with cellular transformation, pro-
gresses to hyperproliferation and culminates in the acqui-
sition of invasive potential, angiogenic properties and
establishment of metastatic lesions. Chemoprevention
was described as the use of natural or synthetic chemi-
cals allowing suppression, retardation or inversion of
carcinogenesis. Substantial evidence exists to support
the role of both oxygen and organic-free radical interme-
diates in the bimolecular interactions, which contribute
to the initiation, promotion and/or progression stages
of chemical carcinogenesis34. ere are many reports on
the synthetic chalcones as chemopreventive agents. First
report on the chalcones as chemopreventive agents by
Anto et al.,35 described that compounds with good anti-
oxidant activity showed good antitumor activity. In the
present case, compounds 12m, 12e and 12a exhibited
Cytotoxicity of the synthesised compounds was tested
against selected three cancer cell lines such as Hep 3BPN7
(liver cancer cell line), HL60 P58 (leukaemia cancer cell
line), HeLa-B75 (cervix cancer cell line) and one normal
cell line PN-15C-12 (a normal chang liver cell line). e
results obtained are summarised in Table 2. Compounds
12j, 12i, 12n, 12c, 12m and 12e were found to be most
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of novel carbazole chalcones 597
Table 2. Cytotoxicity of carbazole chalcones.
Compounds
Hep 3BPN7
70.33
63.40
72.80
65.60
69.40
62.40
61.53
67.86
66.93
65.10
61.40
70.73
69.46
70.60
67.66
64.60
SRHep 3BPN7
1.63
3.56
2.37
2.27
2.12
2.92
3.16
1.81
2.94
3.86
4.36
1.61
2.18
2.54
2.28
1.20
HL60 P58
57.73
62.52
59.32
49.07
56.80
57.89
55.79
60.50
55.12
51.17
66.63
64.28
58.31
59.07
62.85
54.08
SRHL60 P58
1.34
3.51
1.93
1.70
1.73
2.58
2.86
1.61
2.42
3.03
4.73
1.46
1.83
2.12
2.12
1.00
HeLa B75
75.44
71.26
73.31
78.39
77.09
62.27
56.31
80.58
76.74
78.73
56.31
75.85
74.89
73.89
65.36
78.66
SRHeLa B75
1.75
4.01
2.39
2.72
2.35
2.78
2.89
2.15
3.37
4.67
3.99
1.73
2.35
2.66
2.20
1.46
PN-15C-12
42.95
17.78
30.69
28.85
32.80
22.39
19.49
37.54
22.79
16.86
14.09
43.87
31.88
27.79
29.64
53.85
SRaverage
1.57
3.69
2.23
2.23
2.06
2.76
2.97
1.85
2.90
3.85
4.36
1.60
2.12
2.44
2.20
1.22
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
Methotrexate
e results are the mean values of n=2.
SR=Inhibition of cancer cell line (%)/Inhibition of normal cell line (%).
Hep 3BPN7, liver cancer cell line; HL60 P58, leukaemia cancer cell line; HeLa-B75, cervix cancer cell line; PN-15C-12, normal liver cell
line, compounds tested at 100 μg; SR, selectivity ratio.
good antioxidant activity, and these compounds also pre-
sented significant cytotoxicity, which reinforces the idea
of designing antioxidant-based cancer chemoprevention
agents and these compounds could be considered as
chemopreventive agents.
good antifungal activity, while the rest of the compounds
were inactive against C. albicans.
About 30% of oral drugs fail in development due to
poor pharmacokinetics36. Among the pharmacokinetic
properties, a low and highly variable bioavailability is
indeed the main reason for stopping further develop-
ment of the drug. An in-silico model for predicting oral
bioavailability is very important, both in the early stage
of drug discovery to select the most promising com-
pounds for further optimisation and in the later stage
to identify candidates for further clinical development.
In-silico prediction of oral bioavailability is pioneered
by Lipinski37 “rule of five”; if two out of the following
parameters are out of the range, then poor absorption
or permeability is possible: the molecular weight is over
500, hydrogen bond donors are more than 5, the calcu-
lated octanol–water partition coefficient is over 5 (c Log
P), hydrogen bond acceptors are more than 10. Drug
likeness can be deduced as a delicate balance among
the molecular properties of compound that directly
influence its pharmacodynamics and pharmacokinet-
ics and ultimately affect their absorption, distribution,
metabolism and excretion in human body like a drug38.
us, to obtain a good oral drug candidate, we have
subjected a series of novel carbazole chalcones to the
prediction of Lipinski rule of five and other properties
and results are shown in Table 5 (Supplementary infor-
mation). e results revealed that all the compounds
satisfy the Lipinski rule of five. Compounds 12h, 12i, 12j,
12k and 12l have c Log P value more than 5; however,
these compounds have molecular weight, hydrogen
bond donor, hydrogen bond acceptor within the range
of Lipinski rule of five, and these compounds have low
polar surface area (PSA). Hence, these compounds do
not pose absorption and permeation difficulty.
e newly synthesised compounds were screened for
their antibacterial activity against two Gram-negative
strains Escherichia coli (NCIM 2065), Proteus vulgaris
(NCIM2813)andoneGram-positivestrainStaphylococcus
aureus (NCIM 2120) (Table 4, Supplementary informa-
tion). Most of the compounds are active against P. vul-
garis and E. coli, some of the compounds are inactive
against S. aureus. Results show that compounds 12a,
12e and 12m inhibited all the three bacterial growth.
Whereas compounds 12b, 12g and 12h inhibited P. vul-
garis and E. coli selectively, but compounds 12c and 12o
are active against S. aureus. e SAR study indicates that
the presence of electron-withdrawing groups on B ring is
necessary for antibacterial activity; furthermore, replace-
ment of aromatic B ring by heteroaromatic ring slightly
decreases the antibacterial activity.
Pathogenic yeasts of the genus Candida can cause
life-threatening infections in immuno-deficient patients.
is is because of the higher number of patients with
malignancies treated with intensive immunosuppres-
sive therapy regimens as well as their improved survival
from formerly fatal bacterial infections and the rising
number of patients undergoing allogenic hematopoietic
stem cell or organ transplantation. ere is an urgent
need for novel fungicidal compounds with low toxicity.
Synthesised compounds were screened for antifungal
activity against Candida albicans (NCIM 3100) using flu-
conazole as standard, and the results are given in Table
4, Supplementary information. e antifungal screening
data revealed that compounds 12h and 12m showed
© 2013 Informa UK, Ltd.

598 B. Bandgar et al.
Hydrogen bonding capacity has also been identified
as an important parameter for drug permeability. e
results (Table 5, Supplementary information) reveal
that compounds under study possessing less number
of hydrogen bond donor and hydrogen bond accep-
tor; PSA provides good correlation with experimental
transport data. It has been successfully applied for the
prediction of intestinal absorption. PSA equal to or
less than 140 will have high probability of good oral
bioavailability39. Compound having low PSA could be
a good oral drug candidate. Navia et al.,40 has postu-
lated the desirability of molecular flexibility for mem-
brane permeation. Molecular flexibility is measured
by number of rotatable bonds. Compounds with 10 or
fewer rotatable bonds will have probability of good oral
bioavailability. e compounds under study have the
number of rotatable bonds less than 10. Overall, all the
synthesised carbazole chalcones meet the requirement
of the Lipinski rule and other physicochemical proper-
ties, and may considered as good candidates for oral
drug development.
and were used as supplied unless otherwise stated.
in layer chromatography (TLC) was performed on
silica gel-coated plates for monitoring the reactions.
Synthesis of 9-methyl carbazole (10)
Carbazole 9 (4.1 g, 25 mmol) was dissolved in 50mL
DMF and NaH (1.8 g, 75 mmol) was added to it. e
solution was stirred for 10 min and methyl iodide (7.05 g,
50 mmol) was added with constant stirring and stirring
continued at room temperature for 3 h. After completion
of reaction (TLC), the reaction mass was poured over
crushed ice. e obtained pale yellow solid of 9-methyl
carbazole 10 was filtered, washed with water and dried.
us, the 9-methyl carbazole 10 (mp: 97°C, yield 94%)
obtained was sufficiently pure and used further without
purification.
Synthesis of 3-formyl-9-methyl carbazole (11)
9-methyl carbazole 10 (1.81 g, 10 mmol) was dissolved
in dry DMF (20 mL) under anhydrous condition. It was
cooled to 0°C, and POCl3 (1.87 mL) was added drop
wise for 30 min and stirring continued for 4 h at 80°C.
After completion of reaction (TLC), reaction mass was
poured over crushed ice (50 g), basified with NaOH,
extracted with chloroform and dried over anhydrous
Na2SO4. Organic layer was concentrated and purified
through silica gel column using chloroform as elut-
ing solvent to yield product 11 (1.15 g). Yield: 55%;
off white solid; mp: 76°C–78°C; IR (cm−1): 3035, 2704,
conclusion
In summary, we have synthesised a series of novel carba-
zole chalcones and evaluated for cytotoxicity, antioxidant
and antimicrobial activities. ADME prediction revealed
that the compounds could be considered as good candi-
dates for drug development. Compounds 12m, 12o and
12c exhibited good DPPH radical scavenging activity,
compounds 12e, 12m and 12d were excellent hydroxyl
radical scavengers, compounds 12g, 12e, 12n, 12a and
12m inhibited the oxidative DNA damage induced by
AAPH. Compounds 12j, 12i, 12n, 12c, 12m and 12e
have shown promising cytotoxicity against selected can-
cer cell lines, and they were highly selective to cancer
cell line than the normal cell line; SAR study revealed
that electron-withdrawing groups increase cytotoxicity.
Compounds 12a, 12e and 12m showed good antibacte-
rial activity and compounds 12h and 12m have shown
good antifungal activity. Overall, compound 12m, 12e
and 12a can be considered as novel carbazole chalcones
as lead compounds for the development of novel chemo-
preventive agents.
1
1685, 1626, 1591, 1570, 1050, 750; H NMR (300 MHz,
DMSO-d6, δ in ppm): 3.91 (s, 3H, NCH3), 7.35 (t, 1H,
J = 8.2 Hz, ArH), 7.40–7.60 (m, 3H, ArH), 7.98 (d, 1H,
J = 8.2 Hz, ArH), 8.17 (d, 1H, J = 8.2 Hz, ArH), 8.60 (s, 1H,
ArH), 10.1 (s, 1H, −CHO); m/z = 210.20 (m + 1); Anal.
Calcd for C14H11NO: C, 80.36; H, 5.30; N, 6.69%. Found:
C, 80.34; H, 5.31; N, 6.71%.
Synthesis of chalcones (13a–o)
A mixture of 3-formyl-9-methyl carbazole 7 (1 mmol)
and substituted acetophenones (1 mmol) was dissolved
in 20 mL ethanol. To this mixture, sodium hydroxide
(20%, 1 mL) was added at 0°C–10°C. e reaction mix-
ture was stirred at room temperature for 24–36 h. en,
this reaction mixture was poured over crushed ice and
stirred. e solid thus obtained was filtered, washed
with water and dried. e solid products were purified
by recrystallisation using methanol to afford pure com-
pounds 12a-o.
experimental
General
Melting points were recorded in open capillaries with
electrical melting point apparatus and were uncor-
rected. IR spectra (KBr disks) were recorded using
3-(9-Methyl-9H-carbazol-3-yl)-1-phenyl-propenone (12a)
MP: 148°C–149°C; IR (cm−1): 3062, 2933, 2780, 1699, 1644,
1279, 1050, 750; 1H NMR (300 MHz, DMSO-d6, δ in ppm):
3.93 (s, 3H, NCH3), 7.27 (t, 1H, J= 7.5 and 7.8 Hz, ArH),
7.47–7.72 (m, 6H, ArH and CH=CH), 7.96 (s, 2H, ArH),
8.02 (d, 1H, J= 8.7 Hz, ArH), 8.15–8.26 (m, 3H, ArH), 8.74
(s, 1H, ArH); 13C NMR (100 MHz, DMSO-d6, δ in ppm):
189.8, 144.4, 139.5, 138.2, 137.0, 135.2, 131.1, 130.2, 128.5,
127.0, 125.5, 122.0, 120.6, 111.3, 109.8, 29.3; m/z = 312 (m
a
Perkin–Elmer 237 spectrophotometer. 1H NMR
(300 MHz) and 13C NMR (100 MHz) spectra were
recorded on a Bruker Avance spectrometer using
DMSO-d6 as solvent. Mass spectra were recorded on a
Shimadzu LCMS-QP 1000 EX. Elemental microanalysis
was performed on Carlo Erba 1108 (CHN) Analyser. All
the reagents and solvents used were of analytical grade
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of novel carbazole chalcones 599
+ 1); Anal. Calcd for C22H17NO: C, 84.86; H, 5.50; N, 4.50%.
Found: C, 84.89; H, 5.53; N, 4.47%.
fraction was calculated. Methotrexate was used as refer-
ence drug. e inhibition of cell viability was calculated
as follows:
DPPH radical scavenging activity
T
e DPPH radical scavenging activity of chalcones was
measured according to the procedure described by
Blois with minor modification41. e reaction mixture
contained test compound with equal volume of DPPH
radical (10−4 M in absolute ethanol) solution. After 20 min
reaction time, the absorbance was recorded at 517nm
using UV–visible spectrophotometer. Ascorbic acid was
used as a standard antioxidant agent. For determination
of IC50 value, various concentrations of test compounds
were taken, and the concentration required for 50%
decrease in the absorbance of a control solution of DPPH
was expressed as IC50.
%inhibition rate = 1 − ×100
C
Where T - absorbance of treated cells, C - absorbance of
untreated cells
Antimicrobial activity (agar diffusion method)
e antimicrobial activity was tested by agar diffusion
method44. e test organism was inoculated on nutri-
ent agar plates and spread uniformly using a sterile glass
spreader. Wells of 6mm in diameter were made on the
nutrient agar using a sterile cork borer. In each well, the test
compound (100 μg) was added. e plates were incubated
at 37°C for 24h for bacteria. e above procedure is fol-
lowed for fungal assays; the media used was yeast peptone
dextrose agar medium incubated at 30°C for 24h. Each test
was performed in duplicate and the results were shown as
means. e zone of inhibition was measured in millimeter,
while standard antibiotics were used as positive control.
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity was determined by
the earlier reported method42. e reaction cocktail con-
tained 60 μL of 1 mM FeCl3, 90 μL of 1 mM 1,10-phenan-
throline, 2.4 mL of 0.2 M phosphate buffer (pH 7.8), 150
μL of 0.17 M H2O2 and 1.5 mL of test compound in DMSO.
Reaction mixture was kept at room temperature for 5 min
incubation, and absorbance was measured at 560 nm
using spectrophotometer. Ascorbic acid was used as a
reference compound.
Calculation of drug-likeness properties
ephysicochemicalpropertiessuchasmolecularweight,
c Log P, hydrogen bond acceptors (HBA), hydrogen bond
donors (HBD) and drug likeness of the synthesised car-
bazole chalcones are studied from online Osiris property
explorer for drug bioavailability of chemical compounds45.
PSA, molecular volume and quantum chemical descrip-
tors such as EHOMO and ELUMO of synthesised compounds
were calculated by using a Bio Med CaChe 6.1 (BioMed
CAChe 6.1, Fujitsu Limited, Chiba, Japan).
Inhibition of AAPH-induced oxidative DNA damage
e inhibition of AAPH-induced oxidative DNA damage
by novel carbazole chalcones was assessed by measur-
ing the conversion of super coiled pUC 18 plasmid DNA
to open circular forms by gel electrophoresis33. pUC 18
DNA (2 μL) was incubated with test compounds (8 μL)
at 100 μg concentration, and AAPH in phosphate-buff-
ered saline (PBS) (8 μL) at pH 7.4 and 37°C for 1 h. After
incubation, the sample (10 μL) was mixed with 2 μL of
gel-loading buffer (0.13% bromophenol blue and 30%
[W/V] glycerol) and immediately loaded onto prestained
0.8% agarose gel with ethidium bromide (10 μg/mL)
and the gel electrophoresis was performed for 1h. Clear
fluorescent plasmid DNA bands were observed and
photographed under gel documentation system. (UVItec
Cambridge, UK).
Declaration of interest
e authors report no conflicts of interest.
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