L-Proline anchored multicomponent synthesis of novel pyrido[2,3-a] carbazoles; Investigation of in vitro antimicrobial, antioxidant, cytotoxicity and structure activity relationship studies

Article abstract of DOI:10.1016/j.ejmech.2011.09.024

The newly synthesized pyrido[2,3-a]carbazoles were prepared in good yields by multicomponent reactions under l-proline as promoter and structurally characterized. Few compounds showed significant activity toward both gram-positive, gram-negative bacterial

Full text of DOI:10.1016/j.ejmech.2011.09.024

European Journal of Medicinal Chemistry 46 (2011) 5580e5590
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
L-Proline anchored multicomponent synthesis of novel pyrido[2,3-a]carbazoles;
investigation of in vitro antimicrobial, antioxidant, cytotoxicity and structure
activity relationship studies
,
T. Indumathia, V.S. Jamal Ahamedb, Surk-Sik Moonb, Frank R. Fronczekc, K.J. Rajendra Prasada
*
a Department of Chemistry, Bharathiar University, Coimbatore, Tamil Nadu, India
b Department of Chemistry, Kongju National University, Kongju 314-701, Republic of Korea
c Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The newly synthesized pyrido[2,3-a]carbazoles were prepared in good yields by multicomponent
Received 16 August 2011
Received in revised form
5 September 2011
Accepted 15 September 2011
Available online 22 September 2011
reactions under L-proline as promoter and structurally characterized. Few compounds showed significant
activity toward both gram-positive, gram-negative bacterial strains. All compounds exerted negative
efficacy for antifungal activity except compounds 5f and 7f which showed moderate activity. All
compounds showed weak to moderate capacity for scavenging DPPH. The cytotoxicity was evaluated by
Sulforhodamine B assay against A-549, B16F10, HCT-15, SKMel2 and SKOV3 cell lines and compared with
standard drug cisplatin. Compound 5f outperformed cisplatin against A-549, HCT-15, SKMel2 and B16F10
cell lines. Compound 5e outranged cisplatin against A-549, HCT-15, and SKMel2 cell lines. 5b
outperformed cisplatin specifically against B16F10. The preliminary structure activity relationships were
carried out.
Keywords:
Pyrido[2,3-a]carbazoles
Antibacterial
Antifungal
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antioxidant
Cytotoxicity
Structure-activity relationship
1. Introduction
their biological activities and potential applications as pharmaco-
logical agents [3]. A large number of natural and synthetic carba-
The ongoing battle against cancer is far from over. Altering cell
fate with small synthetic compounds is one of the key strategies in
cancer therapy. The discovery of novel chemotherapeutics has
become one of the most important goals in medicinal chemistry. In
this context, the search for novel chemotherapeutic agents and
approaches to cancer treatment is an active research field stimulated
by the discovery of new biological targets and by the possibility of
obtaining new drugs with less undesirable side effects. Among
potential chemotherapeutic agents, heterocyclic compounds
represent an outstanding type of anticancer drug candidates. Novel
heterocyclic organic compounds have been widely studied as
promising agents able to induce cancer cells to undergo one of
several types of cell death, in particular, apoptosis [1,2].
zoles derivatives have been reported to exhibit diverse biological
activities such as anti-HIV [4], antimicrobial [5e9], anti inflam-
matory [10], antiencephalomycarditis [11], antiviral [12], antihis-
tamine and antiseratonin [13], anticonvulsant and diuretic [14],
fungicide and antinflammation inhibitor [15], trypanocidal [16],
vasodilator and
b-blocker [17], antidepressant [18], anticancer
[19,20], mosquitocidal [21].
It has been established that, ellipticine (5,11-dimethyl-6H-pyr-
ido[4,3-b]carbazole) naturally occurring alkaloid isolated from the
leaves of the tree Ochrosia elliptica Labill (Apocynaceae), has
powerful antiproliferative properties and its natural analogs.
9-methoxyellipticine, 9-hydroxyellipticine and the isomeric oliva-
cines show promise in the treatment of osteolytic breast cancer
metastases, kidney sarcoma, brain tumors and myeloblastic
leukemia [22e29]. A number of successful ellipticine analogs have
been designed and synthesized with improved cytotoxicity and
anticancer activities against a panel of cancer cell lines [30e32].
The derivative 9-hydroxy-2-methyl ellipticinium acetate (cellip-
tium), is currently undergoing extensive clinical trials with good
solubility, has found application in the treatment of breast, kidney
The heterocyclic carbazole motif is a privileged pharmacophore
scaffold found in many biologically active compounds of diverse
origins, from natural products to synthetic sources. Widespread
interests of chemists have been attracted to these structures due to
* Corresponding author.
E-mail address: prasad_125@yahoo.com (K.J. Rajendra Prasad).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.024

T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
5581
Scheme 2. Synthesis of novel pyrido[2,3-a]carbazoles.
protons resonate at
d 5.66. The total number of protons matched
perfectly with its structure. The 13C NMR spectrum revealed the
presence of 23 carbons. The structures of all compounds were
confirmed by elemental and spectral analyses. The same reaction
was carried out with substituted aldehydes which are represented
in Scheme 2 and Table 2. The crystal structure of 5a is represented
in Fig. 1 and the crystallographic data in Table 3.
Scheme 1. Annelated N-heterocycles as intercalating antitumor drugs.
In continuation of this work, in the second part, we reacted 6-
methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one 1, benzaldehyde 2a,
ethyl cyanoacetate 6, ammonium acetate 4 and L-Proline in dry
ethanol to yield 2-amino-8-methyl-4-phenyl-5,11-dihydro-6H-
pyrido[2,3-a]carbazole-3-carboxylic acid ethyl ester 7a. The IR
spectrum of 7a displayed absorption bands at 3321 (asym, NH2),
3293 (sym, NH2), 1721 (C]O). In its 1H NMR spectrum the amino
and thyroid cancer [33e35]. The interest in ellipticine and its
derivatives for clinical purposes is mainly due to their high effi-
ciency against several types of cancer, limited toxic side effects, and
complete lack of hematological toxicity [36]. Examples of ellipticine
and its analogs which act as good antitumor drugs are represented
in Scheme 1.
proton resonates at
d 5.15. The total number of protons matched
Prompted by the findings from the above mentioned studies, we
set out to explore and synthesize novel pyrido[2,3-a]carbazoles by
multicomponent reactions in excellent yields by simple and
straight forward manner. Furthermore, the newly synthesized
compounds were evaluated for their in vitro antimicrobial activity,
antioxidant assay and cytotoxicity against 5 cancer cell lines and
their primary structure-activity relationships have been
established.
perfectly with its structure. The 13C NMR spectrum revealed the
presence of 25 carbons. The structures of all compounds were
confirmed by elemental and spectral analyses. The same reaction
was carried out with substituted aldehydes which are represented
in Scheme 3 and Table 2. The crystal structure of 7a is represented
in Fig. 2 and the crystallographic data in Table 3.
2.1.1. Mechanism for the formation of pyrido[2,3-a]carbazoles
A plausible mechanism for the formation of pyrido[2,3-a]
carbazoles is represented in Scheme 4. Firstly, it involves the
Knoevenagel condensation followed by Michael addition and
prototrophic shift, finally aerial oxidation leads to pyrido[2,3-a]
carbazoles. The similar type of chemistry in some other simple
systems is reported in literature [39].
2. Results and discussions
2.1. Chemistry
This work was focused on the synthesis of novel pyrido[2,3-a]
carbazoles by multicomponent reactions. Multicomponent reac-
tions have proven to be a valuable asset in medicinal chemistry
drug design and drug discovery because of their simplicity, effi-
ciency and high selectivity. Herein, we report a simple and facile
multicomponent synthesis of pyrido[2,3-a]carbazoles in high
2.2. Pharmacology
2.2.1. Antimicrobial activity
yields, using
L
-proline as an organocatalyst. We optimized the
-proline as an
The newly synthesized pyrido[2,3-a]carbazoles were screened
for their in vitro antimicrobial activities against two Gram-positive
bacteria e Staphylococcus aureus and Enterococcus faecalis; Gram-
negative Escherichia coli and Klebsiella pneumoniae and fungal
strains e Aspergillus niger and Candida albicans by the agar well
diffusion method using Ciprofloxacin and Fluconazole as control
drugs for antibacterial and antifungal activities, respectively. The
pathogens were cultured on the nutrient agar medium (antibac-
terial activity) and sabouraud dextrose agar (antifungal activity).
DMSO was used as solvent and as negative control. The suscepti-
bility was assessed on the basis of diameter of zone of inhibition
against gram-positive, gram-negative bacteria and fungal strains.
Inhibition zones were measured and compared with the current
antimicrobial drugs ciprofloxacin for antibacterial and fluconazole
for antifungal activity. The diameter of zone of inhibition measured
in mm for antibacterial and antifungal tests are depicted in Table 4.
The minimum inhibitory concentration is represented in Table 5.
The antibacterial activity results revealed that the majority of
the synthesized compounds showed varying degrees of inhibition
against the tested microorganisms. It was found that compounds 5f
and 7f exhibited almost equipotent efficacy with standard drug
Ciprofloxacin against E. faecalis and E. coli, respectively and
reaction in different solvent conditions only using
L
organocatalyst which is portrayed in Table 1. Thereby we reacted 6-
methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one [37,38] 1, benzalde-
hyde 2a, malononitrile 3, ammonium acetate 4 and L-proline in dry
ethanol to yield 2-amino-8-methyl-4-phenyl-5,11-dihydro-6H-
pyrido[2,3-a]carbazole-3-carbonitrile 5a. The IR spectrum of 5a
displayed absorption bands at 3318 (asym, NH2), 3227 (sym, NH2),
2207 (C^N), 1627 (C]N). In its 1H NMR spectrum the amino
Table 1
Solvent screen for the synthesis of pyrido[2,3-a]carbazoles.
Entry
Catalysta
Solvent
Temperature (ꢀC)
Time (h)
Yield (%)
5a
7a
1
2
3
4
5
6
7
e
EtOH
EtOH
CH3CN
CH3OH
THF
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
3
1
3.5
3
4
72
85
65
71
43
49
40
70
81
61
69
35
37
32
L-Proline
L-Proline
L-Proline
L-Proline
L-Proline
L-Proline
DMF
Toluene
4
5
a
-10 mol % of catalyst was used.

5582
T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
Table 2
Synthesis of pyrido[2,3-a]carbazoles using
L
-Proline as catalyst.
Compounds
Aldehydes
Producta
Time(h)
1
Yield(%)b
5a
85
5b
5c
1
82
81
1.2
5d
5e
1
82
84
1.5
5f
1.5
83
81
81
7a
7b
1
1
7c
1.2
81
7d
1.2
82

T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
5583
Table 2 (continued )
Compounds
Aldehydes
Producta
Time(h)
Yield(%)b
7e
1.5
82
81
7f
1.5
a
The products were characterized by IR, NMR, mass and elemental analysis.
Isolated yields.
b
present at C4-position played a key role in the efficacy of the
compound in terms of antimicrobial activity.
exhibited good activity against all the other microorganisms with
the inhibition zone 26e29 mm at 25 g/ml. Compounds 5b, 5e, 7b
and 7e exhibited significant activity against all four microorgan-
isms with inhibition zone ranging between 21 and 26 mm at 25 g/
ml. Compounds 5c, 5d, 7c and 7d showed moderate activity with
inhibition zone ranging between 17 and 22 mm at 50 g/ml.
Compounds 5a and 7a showed least activity with inhibition zone
12e16 mm at 100 g/ml. The in vitro antifungal evaluation
m
ꢁ Thiophene core [40,41] at C4-position in compounds 5f and 7f
played a positive role in the antibacterial effectiveness, which
made them show good antibacterial activity against all the four
bacterial pathogens compared to all the other compounds.
ꢁ It is interesting to point out that 5e and 7e carrying the quin-
oline moiety [42] showed significant activity against both the
gram-positive and gram-negative bacteria. Comparatively, the
m
m
m
exhibited that, the activities for all compounds were relatively
weak compared to their antibacterial activities. Unexpectedly, all
target compounds exerted negative efficacy except compounds 5f
0
introduction of electron withdrawing group at C4 position
exhibited a significant activity than the electron donating
N(CH3)2 group at C40 position. The role of electron withdrawing
group in improving antimicrobial activities is reported in
literature [43].
and 7f which showed
a moderate activity. In general, the
compounds showed improved antibacterial activity when
compared to their antifungal activity. (Scheme 5)
ꢁ The presence of a pyridine moiety at the C4 position does not
induce a major improvement in antibacterial activity but
showed a moderate change in antibacterial activity against all
the four pathogens.
The structural antimicrobial activity relationship of the
synthesized compounds demonstrated the following assumptions
about the structural activity relationship:
ꢁ Compounds 5a and 7a with an unsubstituted aryl ring showed
ꢁ There is no notable difference in antimicrobial activity by
changing X ¼ CN to COOCH2CH3 at C3 but the substituent
least activity only at 100 mg/ml.
Fig. 1. X-ray crystal structure and atom numbering scheme for 5a as thermal ellipsoids at 50% probability level.

5584
T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
Table 3
those of the positive control, Vitamin C. Nearly all the tested
compounds showed weak to moderate capacity for scavenging
DPPH. All the compounds showed scavenging capacity beyond
Crystal data and structure refinement data.
5a
7a
140
scavenging capacity up to 600
show scavenging capacities as effective as the standards. The IC50
m
g/ml. Among them compound 5c did not show any radical
CCDC deposit number
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell Dimensions
a(Å)
831698
C23H18N4
350.41
824602
C25H23N3O2
397.46
mg/ml. However the samples did not
90.0(5)
90.0(5)
values are expressed in
represented in Fig. 3.
mg/ml of all compounds except 5c and 7c as
1.54178 Å
Monoclinic
P21/c
1.54178 Å
Monoclinic
P21/n
2.2.3. Cytotoxicity
6.9626(4)
7.9051(4)
32.9708(18)
90
90.215(2)
90
1814.70(17)
4
1.283
8.5394 (3)
13.9227 (4)
17.0618 (5)
90
95.6620(10)
90
2018.61 (11)
4
1.308
For evaluating the cytotoxicity of newly synthesized
compounds, 5 different cancer cell lines were utilized: HCT-15
(Human Colon tumor), B16F10 (Mouse Melanoma), A-549
(Human Lung adenocarcinoma), SKOV3 (Human Ovarian adeno-
carcinoma), SKMel2 (Human Skin Melanoma). The viability of the
cells was assessed by SRB (Sulforhodamine B) assay in vitro.
Cisplatin, a clinically used antitumor agent was also evaluated for
positive control. Three independent experiments in triplicate were
performed for determination of sensitivity to each compound; the
anticancer potency of these compounds was indicated by IC50 (the
concentration that causes a 50% reduction of the cell growth) values
that were calculated by linear regression analysis, expressed in
mean ꢂ SD. The results are summarized in Table 6.
b(Å)
c(Å)
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
Volume(Å3)
Z
Density (calculated) Mg/m3
Absorption coefficient(mmꢃ1)
F(000)
0.611
736
0.672
840
Theta range for data collection 2.68e69.40ꢀ
4.11e68.57ꢀ
ꢃ10 <¼ h <¼ 10,
ꢃ16 <¼ k <¼ 16,
ꢃ20 <¼ l <¼ 19
21053/3670
0.983
Index ranges
ꢃ8 <¼ h <¼ 7,
ꢃ9 <¼ k <¼ 5,
ꢃ36 <¼ l <¼ 40
11186/3317
0.973
Full-matrix least-
squares on F2
3317/0/255
Reflections collected/unique
Completeness to theta (max)
Refinement method
As shown in Table 6, some of the tested compounds showed
moderate to potent anticancer activities against all the tested cells.
Among them, compounds 5e and 5f were the most promising
compounds, which outranged the positive control cisplatin. The
most outstanding compound out of these, 5f which displayed
stronger cytotoxicity against A-549, HCT-15, SKMel2 and B16F10
cell lines with IC50 values 4.04 ꢂ 0.13, 3.45 ꢂ 0.07, 4.61 ꢂ 0.10 and
Full-matrix least-
squares on F2
3670/0/283
1.065
R1 ¼ 0.031,
wR2 ¼ 0.079
R1 ¼ 0.033,
wR2 ¼ 0.080
Data/restraints/parameters
Goodness-of-fit on F2
1.037
Final R indices [I > 2
s
(I)]
R1 ¼ 0.0363,
wR2 ¼ 0.0955
R1 ¼ 0.0383,
wR2 ¼ 0.0971
R indices (all data)
Largest diff. peak and hole
0.24 and ꢃ0.16e. Åꢃ3 0.23 and ꢃ0.16e. Åꢃ3
2.85 ꢂ 0.07
mg/ml which were 1.39, 2.31, 2.25 and 1.94 fold more
active than the positive control cisplatin which showed the IC50
values (5.62 ꢂ 0.28, 7.97 ꢂ 0.40, 10.41 ꢂ 1.16, 5.54 ꢂ 0.01 respec-
tively). It showed almost equipotent activity (IC50, 10.03 ꢂ 0.13)
with cisplatin (IC50, 9.61 ꢂ 0.28) against SKOV3 cell line. The next
most promising compound, 5e depicted stronger cytotoxicity
against A-549, HCT-15, SKMel2 cell lines with IC50 values
ꢁ It is worth noting that a thiophene moiety [44,45] substituted
at C4 position was endowed with certain antifungal activity.
ꢁ In general, it was observed that the hetero aryl rings and
electron rich aryl groups at C4 position showed better anti-
bacterial activity than the ones with a non-substituted aryl
ring.
3.72 ꢂ 0.16, 4.43 ꢂ 0.06 and 4.48 ꢂ 0.08
mg/ml which were 1.51, 1.79
and 2.32 fold more than the positive control cisplatin and almost
equipotent activity (IC50, 5.89 ꢂ 0.13) against B16F10. It showed
good activity with IC50 value 21.92 ꢂ 0.43 against SKOV3 cell line.
Furthermore, 5d exhibited significant cytotoxicity with the IC50
ꢁ The deduced patterns of antimicrobial activity of the newly
synthesized.
values ranging between 10.56 ꢂ 0.33 and 47.62 ꢂ 0.32
mg/ml
pyrido[2,3-a]carbazoles are in the following order:
antibacterial > antifungal.
against all cell lines; among them specifically it showed almost
equipotent activity with the positive control against SKOV3 cell
line. Next, we investigated the effect of electron withdrawing and
electron donating groups by introducing bromo and dimethyla-
mino substituents at C40 position. Compound 5b selectively showed
(IC50, 2.84 ꢂ 0.07) a twofold increased cytotoxicity compared with
the positive control against B16F10 cell line, significant activity
against, HCT-15 (IC50, 11.05 ꢂ 0.16) and a moderate activity against
A-549 and no activity against SKOV3 and SKMel2 cell lines.
Compound 5c showed moderate activity against B16F10, HCT-15
2.2.2. Antioxidant activity
The DPPH radical has been widely used to test the ability of
compounds as free radical scavengers or hydrogen donors to
evaluate the antioxidant activity. Hence, we carried out experi-
ments to explore the free radical scavenging ability of the novel
pyrido[2,3-a]carbazoles by their capacities for scavenging 1,1-
diphenyl-2-picrylhydrazyl free radical (DPPH) and compared with
Scheme 3. Synthesis of novel pyrido[2,3-a]carbazoles.

T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
5585
Fig. 2. X-ray crystal structure and atom numbering scheme for 7a as thermal ellipsoids at 50% probability level.
Table 4
and it was inactive against remaining three cell lines. Compound 5a
showed least cytotoxicity with IC50 value > 44 g/ml against A-549,
Antibacterial and antifungal activity of pyrido[2,3-a]carbazoles - in vitro activity-
zone of inhibition in mm.
m
B16F10 and HCT-15 cell lines and no activity against SKMel2 and
SKOV3 cell lines. (Scheme 6)
Compounds Gram-positive
Gram-negative
Fungi
S.aureusa E.faecalisb E.colic K.pneumoniaed A.nigere C.albicansf
In the second series compounds 7e and 7f rendered significant
cytotoxicity. Compound 7f, displayed IC50 values 23.40 ꢂ 2.20,
19.36 ꢂ 0.17, 22.51 ꢂ 0.67, 48.21 ꢂ 1.60 exhibited selective cyto-
toxicity against A-549, B16F10, HCT-15, SKMel2 cell lines and no
activity against SKOV3 cell line. Compound 7e, showed IC50 values
17.63 ꢂ 1.00, 20.46 ꢂ 1.47, 27.66 ꢂ 0.55, 18.60 ꢂ 0.06 against A-549
B16F10, SKMel2 and SKOV3 cell lines and no activity against HCT-
15. Compound 7d showed moderate activity with IC50 values
43.73 ꢂ 1.70, 29.40 ꢂ 1.94, 33.78 ꢂ 0.19, 24.92 ꢂ 0.87 against A-549,
B16F10, HCT-15 and SKMel2 cell lines and no activity against SKOV3
5a
5b
5c
5d
5e
5f
7a
7b
7c
7d
7e
7f
12
24
17
20
25
26
14
23
21
19
25
27
14
23
19
20
24
29
15
22
18
17
24
28
29
e
16
22
17
19
23
27
13
21
19
20
23
26
27
e
15
25
18
18
25
28
16
25
22
21
26
29
30
e
4
6
5
3
6
15
4
5
7
4
3
7
9
5
10
17
2
7
5
9
7
11
18
e
22
e
17
e
19
e
Ciprofloxacin 28
Fluconazole
Control
e
e
e
e
e
"-" no activity.
a
Staphylococcus aureus.
Enterococcus faecalis.
Escherichia coli.
Klebsiella pneumoniae.
Aspergillus niger.
b
c
d
e
f
Candida albicans.
Table 5
Minimum Inhibitory Concentration (MIC),
m
g/ml.
Compounds
Gram-positive
Gram-negative
Fungi
S.aureus E.faecalis E.coli K.pneumoniae A.niger C.albicans
5a
5b
5c
5d
5e
5f
7a
7b
7c
7d
7e
7f
100
25
50
50
25
25
100
25
50
50
25
25
100
25
50
50
25
25
100
25
50
50
25
25
100
25
50
50
25
25
100
25
50
50
25
25
100
25
50
50
25
25
100
25
50
50
25
25
>100
>100
>100
>100
>100
75
>100
> 100
>100
>100
>100
75
>100
>100
>100
>100
>100
75
>100
>100
>100
>100
>100
75
Ciprofloxacin
Fluconazole
12.5
e
12.5
e
12.5
e
12.5
e
e
e
12.5
12.5
"-" no activity.
Scheme 4. Mechanism for the formation of pyrido[2,3-a]carbazoles.

5586
T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
Scheme 5. Role of substituents in increasing the efficacy of antibacterial activity.
Scheme 6. 2-amino-3-cyanopyridine skeleton showing better cytotoxicity than 2-
amino-3-carboethoxypyridine.
Scheme 7. Role of substituents in increasing the efficacy of cytotoxicity.
also confirms that the cyano group is essential for the induction
of apoptosis and extreme cytotoxicity [52,53].
b) Next we investigated the effect of substituent at C4-position on
the 2-amino-3-cyanopyridine skeleton, among them
compound 5f outperformed cisplatin against A-549, HCT-15,
SKMel2 and B16F10 cell lines and showed equipotent activity
in case of SKOV3 cell line. It was due to the presence of thio-
phene moiety [54,55] which boosts the cytotoxic activity. The
next most promising compound, 5e which also outranged the
standard drug against A-549, HCT-15, SKMel2 and B16F10 cell
lines was due to the presence of quinoline moiety [56] which
enhances the cytotoxic activity. Furthermore, compound 5d
with the pyridine core [57] also showed significant cytotoxicity
Fig. 3. Free radical scavenging capacity of pyrido[2,3-a]carbazoles.
cell line. Compounds 7a, 7b and 7c were inactive against all cell
lines up to 50 g/ml. (Scheme 7)
m
The present study investigated the effect of several substituents
and from the results of cytotoxicity of the synthesized pyrido[2,3-a]
carbazoles, the following structure-activity relationships can be
derived:
with IC50 values ranging between 10.56
ꢂ
0.33 and
a) In general, it was observed that 2-amino-3-cyanopyridine
[46e50] skeleton showed excellent cytotoxicity when
compared to 2-amino-3-carboethoxypyridine skeleton. This
may be due to the stronger electron withdrawing ability of the
cyano group, which played an important role in anticancer
effect [51]. Literature survey showed that the antitumor
mechanism of action of cyano-substituted heterocycles occurs
via the induction of apoptosis through the inhibition of tubulin
polymerization. The structure-activity relationships analysis
47.62 ꢂ 0.32. The deducted pattern showed the cytotoxicity of
the order 5f > 5e > 5d.
c) Moreover, the investigations on the effect of electron with-
drawing and electron donating groups were carried out by
0
introducing bromo and 4-dimethylamino substituents at C4
position. The compound 5b exhibited better cytotoxicity
compared to 5c; in addition 5b portrayed selective cytotoxicity
against B16F10 cell line. It was observed that the electron
Table 6
In vitro cytotoxicity and IC50
(m
g/ml).
A549a
Compounds
B16F10b
HCT15c
SKMel2d
SKOV3e
5a
5b
5c
5d
5e
5f
7a
49.24 ꢂ 0.65
34.97 ꢂ 3.50
NA
16.13 ꢂ 0.64
3.72 ꢂ 0.16
4.04 ꢂ 0.13
NA
47.58 ꢂ 1.22
2.84 ꢂ 0.07
34.57 ꢂ 0.11
13.71 ꢂ 0.59
5.89 ꢂ 0.13
2.85 ꢂ 0.07
NA
44.85 ꢂ 0.39
11.05 ꢂ 0.16
43.53 ꢂ 0.58
47.62 ꢂ 0.32
4.43 ꢂ 0.06
3.45 ꢂ 0.07
NA
NA
NA
NA
NA
NA
NA
16.61 ꢂ 0.66
4.48 ꢂ 0.08
4.61 ꢂ 0.10
NA
10.56 ꢂ 0.33
21.92 ꢂ 0.43
10.03 ꢂ 0.13
NA
7b
NA
NA
NA
NA
NA
7c
NA
NA
NA
NA
NA
7d
7e
7f
43.73 ꢂ 1.70
17.63 ꢂ 1.00
23.40 ꢂ 2.20
5.62 ꢂ 0.28
29.40 ꢂ 1.94
20.46 ꢂ 1.47
19.36 ꢂ 0.17
5.54 ꢂ 0.01
33.78 ꢂ 0.19
24.92 ꢂ 0.87
27.66 ꢂ 0.55
48.21 ꢂ 1.60
10.41 ꢂ 1.16
NA
NA
18.60 ꢂ 0.06
22.51 ꢂ 0.67
7.97 ꢂ 0.40
NA
Cisplatin
9.61 ꢂ 0.28
NA-Compounds that are not active are up to 50 mg/ml.
Each data point represents mean ꢂ SD from three different experiments performed in triplicate.
a
Human Lung adenocarcinoma.
Mouse Melanoma.
Human Colon tumor.
Human Skin Melanoma.
b
c
d
e
Human Ovarian adenocarcinoma.

T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
5587
withdrawing group [58] enhances the anticancer activity
compared to electron donating group.
d) In the second series (7ae7f) the compounds holding the hetero
aryl rings (thiophene, quinoline, pyridine moiety) at C4 position
showed significant cytotoxicity but comparatively lower than
the compounds having the 2-amino-3-cyanopyridine skeleton.
graphite monochromatized Cu Kalpha radiation. The purity of the
products was tested by TLC with plates coated with silica gel-G,
petroleum ether and ethyl acetate were used as developing
solvents.
4.2. Synthesis
In general, it was observed that 2-amino-3-cyanopyridine
skeleton along with the hetero aryl rings and electron rich aryl
groups increase the potency of cytotoxicity when compared to
electron poor aryl system and unsubstituted aryl groups.
4.2.1. General procedure for synthesis of compounds 5aef
A mixture of 6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one (1,
0.01 mol), aromatic aldehydes (2aef, 0.01 mol), malononitrile (3,
0.01 mol), ammonium acetate (4, 0.01 mol) and
L-proline
(0.001 mol) in dry ethanol (15 mL) was heated under reflux for 1 h.
After completion of the reaction, the excess of solvent was evapo-
rated. The residue was dissolved in ice\water and extracted with
ethyl acetate. Combined organic layers were dried over anhydrous
sodium sulfate. It was then purified on a silica gel column (eluent -
petroleum ether: ethyl acetate (96: 4)). The pure product was
recrystallised from ethanol.
3. Conclusions
We have designed and synthesized novel pyrido[2,3-a]carba-
zoles by L-proline catalyzed multicomponent reaction and struc-
turally characterized by spectroscopic and X-ray crystallographic
method. Furthermore, they were subjected to biological evaluation
and preliminary structure-activity relationship. All compounds
were examined for their in vitro antimicrobial activities against four
bacteria and two fungi. Compounds 5b, 5e, 5f, 7b, 7e and 7f holding
the thiophene moiety and the quinoline moiety at C4 position,
electron withdrawing group in the phenyl ring showed significant
antibacterial activity compared to all the other compounds. All
compounds showed negative antifungal activity except,
compounds 5f and 7f. All the compounds showed weak to
moderate capacity for free radical scavenging. Cytotoxicity studies
were carried out by SRB assay method against A-549, B16F10, HCT-
15, SKMel2 and SKOV3 cell line. In particular compound 5f out-
performed the standard drug cisplatin against A-549, HCT-15,
SKMel2 and B16F10 cell lines and compound 5e outperformed
the standard drug cisplatin against A-549, HCT-15 and SKMel2 cell
lines. Compound 5b outranged cisplatin specifically against B16F10,
all the other compounds showed weak to moderate activity. The
structure-activity relationship study revealed that the compounds
holding the 2-amino-3-cyanopyridine skeleton along with thio-
phene moiety, quinoline moiety and electron withdrawing aryl
group at C4-position showed good cytotoxicity compared to 2-
amino-3-carboethoxypyridine skeleton. In summary, on the basis
of preliminary antibacterial and cytotoxicity results, we suppose
that these compounds exert antibacterial action due to their cyto-
toxicity. Taken together, compounds reported in this paper could
serve as potential leads for cytotoxic activity and further investi-
gations are warranted to know the mechanism of action of these
molecules. Studies on the mechanism of action of these compounds
are in progress and will be reported upon in the future.
4.2.1.1. 2-Amino-8-methyl-4-phenyl-5,11-dihydro-6H-pyrido[2,3-a]
carbazole-3-carbonitrile (5a). Orange solid; yield: 85%; m.p.
235e237 ꢀC; IR (KBr, cmꢃ1
,
n
): 3318, 3227, 2207 (C^N), 1627 (C]
N); 1H NMR (
d, ppm, CDCl3): 9.24 (b s, 1H, N11-H), 7.54e7.50 (m, 3H,
H-7, H-9, H-10), 7.35e7.32 (m, 4H, H-20, 30, 50 & 60), 7.10 (d t,1H, H-40,
Jo ¼ 7.1 Hz, Jm ¼ 2 Hz), 5.66 (s, 2H, NH2), 2.86 (m, 2H, H-5), 2.79 (m,
2H, H-6), 2.51 (s, 3H, CH3); 13C NMR (d,0 ppm, CDCl3): 158.3, 152.1,
0
150.0, 1306.3, 136.1, 132.3, 129.4 (C3 & C5 ), 129.3, 128.9, 128.4, 126.9
0
(C2 & C6 ), 126.4, 119.3, 119.2, 118.8 (C^N), 117.2, 111.4, 88.58, 29.6,
25.1, 21.4 (CH3); MS: m/z (%) 350 (Mþ, 100); Anal.Calcd.For
C23H18N4: C, 78.83; H, 5.18; N, 15.99; Found: C, 78.80; H, 5.21; N,
16.01%.
4.2.1.2. 2-Amino-4-(40-bromo-phenyl)-8-methyl-4-phenyl-5,11-dihydro-
6H-pyrido[2,3-a]carbazole-3-carbonꢃit1rile (5b). Orange solid; yield:
82%; m.p. 243e245 ꢀC; IR (KBr, cm
, n): 3433, 3352, 2196 (C^N),
1610 (C]N); 1H NMR (
d, ppm, CDCl3): 9.35 (b s, 1H, N11-H),
7.57e7.45 (m, 3H, H-7, H-9, H-10), 7.32e7.29 (m, 4H, H- 20, 30, 50 &
60), 5.46 (s, 2H, NH2), 2.82 (m, 2H, H-5), 2.75 (m, 2H, H-6), 2.53 (s,
3H, CH3); 13C NMR (
d, ppm, CDCl3): 161.4, 160.3, 153.3, 135.3, 133.1,
0
0
0
0
132.1 (C3 & C5 ), 131.2, 131.0, 129.4 (C2 & C6 ), 125.8, 123.0, 122.4,
121.0, 119.8, 119.4, 117.7 (C^N), 111.7, 90.2, 28.5, 27.9, 22.2 (CH3);
MS: m/z (%) 429 (Mþ, 100), 431 (Mþ2); Anal.Calcd.For C23H17BrN4:
C, 64.35; H, 3.99; N, 13.05 Found: C, 64.31; H, 4.02; N, 13.02%.
4.2.1.3. 2-Amino-4-(40-dimethylamino-phenyl)-8-methyl-5,11-dihydro-
6H-pyrido[2,3-a]carbazole-3-carbonitrile (5c). Orange solid; yield: 81%;
m.p. 248e250 ꢀC; IR (KBr, cmꢃ1
, n): 3428, 3317, 2204 (C^N), 1614 (C]
4. Experimental protocols
N); 1H NMR (
d, ppm, CDCl3): 9.22 (b s, 1H, N11-H), 7.71e7.41 (m, 3H, H-
7, H-9, H-10), 7.36e7.31 (m, 4H, H-20, 30, 50 & 60), 5.53 (s, 2H, NH2), 3.13
4.1. General
(s, 6H, N(CH3)2), 2.87 (m, 2H, H-5), 2.72 (m, 2H, H-6), 2.51 (s, 3H, CH3);
13C NMR (
d
, ppm, CDCl3): 162.4, 159.4, 153.2, 144.1, 137.0, 132.4 (C3
&
0
0
0
Melting points (m.p.) were determined on Mettler FP 51appa-
ratus (Mettler Instruments, Switzerland) and are uncorrected. They
are expressed in degree centigrade (ꢀC). A Nicolet Avatar Model FT-
IR spectrophotometer was used to record the IR spectra
(4000e400 cmꢃ1). 1H NMR and 13C NMR spectra were recorded on
Bruker AV 500 (500 MHz (1H) and 125 MHz (13C)) spectrometer
using tetramethylsilane (TMS) as an internal reference. The
chemical shifts are expressed in parts per million (ppm). Mass
spectra (MS), were recorded on Auto Spec EI þ shimadzu QP 2010
PLUS GCeMS mass spectrometer. Microanalyses were performed
on a Vario EL III model CHNS analyzer (Vario, Germany) at the
Department of Chemistry, Bharathiar University. X-ray diffraction
C5 ),0127.6, 125.8, 124.4, 124.1, 123.2, 123.0, 121.5, 118.4 (C^N), 113.6 (C2
& C6 ), 112.5, 109.7, 88.5, 43.5 (N(CH3)2), 29.2, 27.4, 21.7 (CH3); MS: m/z
(%) 393 (Mþ, 100); Anal.Calcd.For C25H23N5: C, 76.31; H, 5.89; N, 17.80.
Found: C, 76.28; H, 5.91; N, 17.83%.
4.2.1.4. 2-Amino-8-methyl-4-pyridin-3-yl-5,11-dihydro-6H-pyrido
[2,3-a]carbazole-3-carbonꢃit1rile (5d). Orange solid; yield: 82%; m.p.
255e257 ꢀC; IR (KBr, cm
, n): 3468, 3372, 2193 (C^N),1614 (C]
N); 1H NMR ( , ppm, CDCl3): 9.22 (b s, 1H, N11-H), 8.71 (s, 1H, H-20),
d
8.52e8.48 (m, 3H, H- 40, 50 and 60), 7.60e7.47 (m, 3H, H-7, H-9, H-
10), 5.65 (s, 2H, NH2), 2.88 (m, 2H, H-5), 2.67 (m, 2H, H-6), 2.57 (s,
3H, CH3); 13C NMR (
d, ppm, CDCl3): 161.9, 160.8, 151.4, 149.3, 148.7,
measurements were performed on
a
Bruker Kappa Apex-II
134.6, 134.4, 133.5, 132.2, 131.4, 127.2, 123.7, 123.2, 121.4, 120.1, 118.0
diffractometer equipped with an Oxford Cryostream chiller and
(C^N), 112.2, 109.5, 90.6, 29.2, 27.7, 21.3 (CH3); MS: m/z (%) 351

5588
T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
(Mþ, 100); Anal.Calcd.For C22H17N5: C, 75.19; H, 4.88; N, 19.93.
Found C, 75.22; H, 4.84; N, 19.97%.
C25H22N3O2Br: C, 63.03; H, 4.65; N, 8.82. Found: C, 63.00; H, 4.63; N,
8.80%.
4.2.1.5. 2-Amino-4-(2-chloro-quinolin-3-yl)-8-methyl-5,11-dihydro-
4.2.2.3. 2-Amino-4-(40-dimethylamino-phenyl)-8-methyl-5,11-
dihydro-6H-pyrido[2,3-a]carbazole-3-carboxylic acid ethyl ester
6H-pyrido[2,3-a]carbazole-3-carbonꢃit1rile (5e). Yellow solid; yield:
84%, m.p. 260e264 ꢀC; IR (KBr, cm
,
n
d
): 3422, 3361, 2175 (C^N),
: 8.77 (b s, 1H, N11-H), 8.20
(7c). Yellow solid; yield: 81%; m.p. 265e267 ꢀC; IR (KBr, cmꢃ1
,
n
):
1622 (C]N); 1H NMR (
d
, ppm, CDCl3)
3327, 3274, 1734 (C]O); 1H NMR (
d
, ppm, CDCl3): 9.11 (b s, 1H, N11-
(s, 1H, H-40), 8.01e7.87 (m, 4H, H- 50, 60, 70 & 80), 7.72e7.54 (m, 3H,
H), 7.77e7.44 (m, 3H, H-7, H-9, H-10), 7.36e7.33 (m, 4H, H-20, 30, 50
& 60), 5.23 (s, 2H, NH2), 4.38 (q, 2H, CH2CH3, J ¼ 7.5 Hz), 3.13 (s, 6H,
N(CH3)2), 2.92 (m, 2H, H-5), 2.87 (m, 2H, H-6), 2.47 (s, 3H, CH3), 1.32
H-7, H-9, H-10), 5.29 (s, 2H, NH2), 2.94 (m, 2H, H-5), 2.72 (m, 2H,
H-6), 2.60 (s, 3H, CH3).13C NMR (
d, ppm, CDCl3): 161.7, 161.1, 155.0,
150.5, 147.1, 135.3, 134.0, 132.1, 131.1, 131.0, 129.3, 128.4, 127.4,
126.5, 126.4, 125.4, 123.3, 121.8, 119.3, 118.5 (C^N), 111.8, 109.6,
91.2, 28.7, 27.5, 21.3 (CH3); MS: m/z (%) 435 (Mþ, 100); Anal.Calcd.
For C26H18ClN5: C, 71.64; H, 4.16; N, 16.07. Found: C, 71.61; H,
4.20; N, 16.04%.
(t, 3H, CH2CH3, J ¼ 7.5 Hz); 13C NMR (
d
, ppm, CDCl3):0 166.3 (C]O),
0
158.0, 157.2, 150.2, 143.3, 134.5, 132.1, 131.9, 127.9 (C3 & C5 ), 126.0,
0
0
123.5, 122.6, 121.4, 120.9, 114.2 (C2 & C6 ), 113.1, 112.2, 103.2, 59.2,
42.4 (N(CH3)2), 28.3, 27.1, 21.3 (CH3), 13.2; MS: m/z (%) 440 (Mþ,
100); Anal.Calcd.For C27H28N4O2: C, 73.61; H, 6.41; N, 12.72; Found:
C, 73.65; H, 6.39; N, 12.68%.
4.2.1.6. 2-Amino-8-methyl-4-thiophen-2-yl-5,11-dihydro-6H-pyrido
4.2.2.4. 2-Amino-8-methyl-4-pyridin-3-yl-5,11-dihydro-6H-pyrido
[2,3-a]carbazole-3-carboxylic acid ethyl ester (7d). Yellow solid;
[2,3-a]carbazole-3-carbonitrile (5f). Orange solid; yield: 83%; m.p.
262e264 ꢀC; IR (KBr, cmꢃ1
,
n
): 3456, 3350, 2210 (C^N), 1634 (C]
, ppm, CDCl3): 9.12 (b s, 1H, N11-H),7.66e7.51 (m, 3H,
H-7, H-9, H-10), 7.33e7.26 (m, 3H, H- 30, 40 & 50), 5.42 (s, 2H, NH2),
2.90 (m, 2H, H-5), 2.85 (m, 2H, H-6), 2.49 (s, 3H, CH3); 13C NMR (
yield: 82%; m.p. 270e272 ꢀC; IR (KBr, cmꢃ1
,
n
): 3453, 3323, 1710
N); 1H NMR (
d
(C]O); 1H NMR (
d, ppm, CDCl3): 9.11 (b s, 1H, N11-H), 8.52 (s, 1H, H-
20), 8.43e8.35 (m, 3H, H- 40, 50 and 60), 7.62e7.42 (m, 3H, H-7, H-9,
H-10), 5.23 (s, 2H, NH2), 4.29 (q, 2H, CH2CH3, J ¼ 7.2 Hz), 2.82 (m,
2H, H-5), 2.74 (m, 2H, H-6), 2.44 (s, 3H, CH3), 1.30 (t, 3H, CH2CH3,
d
,
ppm, CDCl3): 161.2, 160.5, 150.4, 142.5, 134.5, 132.2, 131.7, 127.5,
127.2, 125.3, 124.4, 122.8, 121.7, 120.2, 118.2 (C^N), 113.3, 111.4, 91.2,
29.5, 28.3, 22.0 (CH3); MS: m/z (%) 356 (Mþ,100). Anal.Calcd.For
C21H16N4S: C, 70.76; H, 4.52; N, 15.72, S, 9.00. Found: C, 70.72; H,
4.49; N, 15.70; S, 9.03%.
J ¼ 7.2 Hz); 13C NMR (
d, ppm, CDCl3): 168.1 (C]O), 157.3, 149.2,
148.3, 145.4, 134.1, 134.0, 133.5, 131.9, 130.2, 125.2, 124.5, 123.3,
120.9, 119.1, 112.2, 109.9, 106.2, 105.5, 61.1, 28.8, 27.6, 22.2 (CH3),
13.2; MS: m/z (%) 398 (Mþ, 100); Anal.Calcd.For C24H22N4O2: C,
72.34; H, 5.57; N, 14.06; Found: C, 72.38; H, 5.54; N, 14.10%.
4.2.2. General procedure for synthesis of compounds 7aef
A mixture of 6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one (1,
0.01 mol), aromatic aldehydes (2aef, 0.01 mol), ethyl cyanoacetate
4.2.2.5. 2-Amino-4-(2-chloro-quinolin-3-yl)-8-methyl-5,11-dihydro-
6H-pyrido[2,3-a]carbazole-3-carboxylic acid ethylꢃe1ster (7e). Yellow
(3, 0.01 mol), ammonium acetate (4, 0.01 mol) and L-proline
solid; yield: 81%, m.p. 265e267 ꢀC. IR (KBr, cm
, n): 3432, 3362,
(0.001 mol) in dry ethanol (15 mL) was heated under reflux for 1 h.
After completion of the reaction, the excess of solvent was evapo-
rated. The residue was dissolved in ice\water and extracted with
ethyl acetate. Combined organic layers were dried over anhydrous
sodium sulfate. It was then purified on a silica gel column (eluent-
petroleum ether: ethyl acetate (99: 1)). The pure product was
recrystallised from ethanol.
1722 (C]O); 1H NMR (CDCl3)
d: 8.86 (b s, 1H, N11-H), 8.32 (s, 1H, H-
40), 7.85e7.79 (m, 4H, H- 50, 60, 70 & 80), 7.68e7.43 (m, 3H, H-7, H-9, H-
10), 5.33 (s, 2H, NH2), 4.31 (q, 2H, CH2CH3, J ¼ 7.2 Hz), 2.84 (m, 2H, H-
5), 2.69 (m, 2H, H-6), 2.51 (s, 3H, CH3),1.41 (t, 3H, CH2CH3, J ¼ 7.2 Hz);
13C NMR (
d, ppm, CDCl3): 166.6 (C]O), 158.7, 157.8, 155.5, 147.2,
146.1, 137.2, 135.2, 134.4, 132.2, 131.1, 130.2, 127.7, 127.2, 126.9, 126.5,
125.2, 123.3, 121.3, 120.2, 112.2, 111.5, 106.6, 62.2, 28.5, 27.2, 21.9
(CH3),13.0; MS: m/z (%) 482 (Mþ,100); Anal.Calcd.For C28H23ClN4O2:
C, 69.63; H, 4.80; N, 11.60 Found: C, 69.60; H, 4.78; N, 11.55%.
4.2.2.1. 2-Amino-8-methyl-4-phenyl-5,11-dihydro-6H-pyrido[2,3-a]
carbazole-3-carboxylic acid etꢃh1yl ester (7a). Yellowsolid; yield: 81%;
m.p. 224e226 ꢀC; IR (KBr, cm
, n
): 3321, 3293,1721(C]O); 1H NMR
4.2.2.6. 2-Amino-8-methyl-4-thiophen-2-yl-5,11-dihydro-6H-pyrido
(d, ppm, CDCl3): 9.24 (b s, 1H, N11-H), 7.61e7.42 (m, 3H, H-7, H-9, H-
[2,3-a]carbazole-3-carboxylic acid ethyl ester (7f). Yellow solid;
10), 7.40e7.36 (m, 4H, H-20, 30, 50 & 60), 7.15 (d t, 1H, H-40, Jo ¼ 7.0 Hz,
Jm ¼ 1.5 Hz), 5.15 (s, 2H, NH2), 4.32 (q, 2H, CH2CH3, J ¼ 7.2Hz), 2.86 (m,
2H, H-5), 2.74 (m, 2H, H-6), 2.52 (s, 3H, CH3), 1.39 (t, 3H, CH2CH3,
yield: 81%; m.p. 276e278 ꢀC; IR (KBr, cmꢃ1
, n): 3421, 3335,1715 (C]
O); 1H NMR (
d, ppm, CDCl3): 9.07 (b s, 1H, N11-H), 7.87e7.51 (m, 3H,
H-7, H-9, H-10), 7.46e7.39 (m, 3H, H-30, 40 & 50), 5.65 (s, 2H, NH2),
4.32 (q, 2H, CH2CH3, J ¼ 7.1 Hz), 2.91 (m, 2H, H-5), 2.84 (m, 2H, H-6),
J ¼ 7.2 Hz); 13C NMR (
d, ppm, CDCl3): 168.5 (C]O),163.9,157.5,151.0,
2.47 (s, 3H, CH3), 1.31 (t, 3H, CH2CH3, J ¼ 7.1 Hz); 13C NMR (
d, ppm,
140.4,136.0,135.3,132.8,129.9,129.4,129.3,129.1,126.9,126.6,125.7,
119.5,119.1,113.0,111.3,105.4, 60.2, 25.3, 23.5, 21.3,13.7; MS: m/z (%)
397 (Mþ,100); Anal.Calcd.For C25H23N3O2: C, 75.54; H, 5.83; N,10.57
Found: C, 75.50; H, 5.87; N, 10.60%.
CDCl3): 166.6 (C]O), 147.6, 142.2, 141.3, 139.2, 135.3, 132.2, 131.1,
128.8, 127.4, 126.6, 123.1, 123.0, 122.2, 121.1, 112.7, 115.0, 111.4, 60.7,
28.7, 27.2, 22.4 (CH3), 13.0; MS: m/z (%) 403 (Mþ, 100); Anal.-
Calcd.For C23H21N3O2S: C, 68.46; H, 5.25; N, 10.41, S, 7.95; Found: C,
68.40; H, 5.20; N, 10.37, S, 7.98%.
4.2.2.2. 2-Amino-4-(40-bromo-phenyl)-8-methyl-5,11-dihydro-6H-
pyrido[2,3-a]carbazole-3-carboxylic acid ethyl ester (7b). Yellow
solid; yield: 81%; m.p. 240e242 ꢀC; IR (KBr, cmꢃ1
,
n
): 3425, 3392,
4.3. In vitro antimicrobial assay
1721 (C]O); 1H NMR (
d, ppm, CDCl3): 9.01 (b s, 1H, N11-H),
7.54e7.45 (m, 3H, H-7, H-9, H-10), 7.35e7.30 (m, 4H, H-20, 30, 50 &
6’), 5.15 (s, 2H, NH2), 4.30 (q, 2H, CH2CH3 J ¼ 7 Hz), 2.83 (m, 2H, H-
5), 2.71 (m, 2H, H-6), 2.49 (s, 3H, CH3), 1.34 (t, 3H, CH2CH3, J ¼ 7 Hz);
The bacterial pathogens S. aureus, E. faecalis, E. coli and Klebsiella
pneumonia and the fungi A. niger and C. albicans were procured from
PSG Hospital, Coimbatore, India. The synthesized compounds were
evaluated for antimicrobial activity by agar well diffusion method
[59,60]. The bacteria and fungi were tested on nutrient agar and
Sabouraud Dextrose agar media respectively. The medium was
sterilized by autoclaving at 120 ꢀC. About 30 ml of the agar medium
13C NMR (
d, ppm, CDCl3): 167.7 (C]O), 158.5, 158.0, 151.1, 138.1,
0
0
0
0
134.4, 132.3 (C3 & C5 ), 132.0, 131.5, 129.0 (C2 & C6 ), 123.2, 122.9,
122.0, 121.8, 120.1, 112.2, 109.5, 104.4, 60.1, 28.4, 27.6, 21.1 (CH3),
13.2. MS: m/z (%) 476 (Mþ, 100), 478 (Mþ2); Anal.Calcd.For

T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
5589
with the respective strains of bacteria and fungi was transferred
Appendix. Supplementary information
aseptically in to each sterilized Petri plate. The plates were left at
room temperature for solidification. A well of 6 mm diameter was
made using a sterile cork borer. The test drugs were added at
The representative 1H NMR, 13C NMR and mass spectra of
compound 5a have been given in the supplementary material.
Crystallographic data for the compounds 5a and 7a have been
deposited at the Cambridge Crystallographic Data Centre as CCDC
number 831698 and 824602.Copy of this information can be ob-
tained as free of charge from The Director, CCDC, 12 Union Road,
Cambridge, CB2 IEZ, UK (fax: ¼ 44 1223 336033; email:deposit@
ccdc.cam.ac.ukor http://www.ccdc.cam.ac.uk.
concentrations of 0 (control), 25, 50, 75, 100
mg/mL Ciprofloxacin
(30 g/ml) and Fluconazole (30 g/ml) was used as positive control.
m
m
The antibacterial assay plates were incubated at 37 ꢂ 2 ꢀC for 24 h,
antifungal assay plates were incubated at 28 ꢂ 2 ꢀC for 48 h. After
the incubation period, the diameter of inhibition zone was
measured as an indicator for the activity of the compounds.
Dimethyl sulphoxide was used both as a solvent and as a negative
control. No inhibition zone was observed in control (i.e. for DMSO).
Appendix. Supplementary material
4.4. Free radical scavenging assay
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.09.024.
Radical scavenging activities were determined by 2,2-diphenyl-
1- picrylhydrazyl (DPPH) assay. Various concentrations of the
experimental complexes were taken and the volume was adjusted
References
to 100 mL with ethanol. About 5 mL of 0.1 mM ethanolic solution of
DPPH was added to the aliquots of samples and standard (Vitamin C)
in a 96-well plate. After incubation for 30 min with shaking at 24 ꢀC
changes in absorbance were measured in a 96-well micro plate
reader at 517 nm. The concentration required for 50% decrease in the
[1] I. Caleta, M. Kralj, M. Marjanovic, B. Bertosa, S. Tomic, G. Pavlovic, K. Pavelic,
G. Karminski-Zamola, J. Med. Chem. 52 (2009) 1744e1756.
[2] M. Hranjec, I. Piantanida, M. Kralj, L. Suman, K. Pavelic, G. Karminski-Zamola,
J. Med. Chem. 51 (2008) 4899e4910.
[3] H.J. Knölker, K.R. Reddy, Chem. Rev. 102 (2002) 4303e4427.
[4] K. Hirata, C. Ito, H. Furukawa, M. Itoigawa, L.M. Cosentino, K.H. Lee, Bioorg.
Med. Chem. Lett. 9 (1999) 119e122.
absorbance of a control solution of DPPH was expressed as IC50
.
[5] K. Thevissen, A. Marchand, P. Chaltin, E.M.K. Meert, B.P.A. Cammue, Curr. Med.
Chem. 16 (2009) 2205e2211.
4.5. In vitro cytotoxicity
[6] P. Rajakumar, K. Sekar, V. Shanmugaiah, N. Mathivanan, Eur. J. Med. Chem. 44
(2009) 3040e3045.
[7] P. Rajakumar, K. Sekar, V. Shanmugaiah, N. Mathivanan, Bioorg. Med. Chem.
Lett. 18 (2008) 4416e4419.
[8] T.A. Choi, R. Czerwonka, W. Froehner, M.P. Krahl, K.R. Reddy, S.G. Franzblau,
H.J. Knoelker, Chem. Med. Chem. 1 (2006) 812e815.
[9] M.M. Rahman, A.I. Gray, Phytochemistry 66 (2005) 1601e1606.
[10] A.A. Andre, G.H. Leslie, A.D. Thomas, K. Jacqueline, R.M. Rene, J. Med. Chem. 19
(1976) 787e792.
[11] W.L. Albrecht and R.W. Fleming, U. S. Patent, 3,932,456 (1976); Chem. Abstr.
84 (1976) 150499t.
[12] W.L. Albrecht and R.W. Fleming, U. S. Patent. 3,932,424 (1976); Chem. Abstr.
84, (1976) 150498s.
[13] V.I. Shvedov, L.B. Altukova, A.N. Grinev, Khim. Farm. Zh 3 (1970) 28;
Chem. Abstr. 72 (1970) 12476j.
[14] A. Shoeb, F. Anwer, R.S. Kapil, S.P. Popli, P. Dua, B.N. Dhawan, J. Med. Chem. 16
(1973) 425e427.
[15] H. Biere, H. Ahrens, C. Rufer, E. Schroeder and H. Koch, German Patent,
2,337,154 (1975); Chem. Abstr. 82 (1975) 156068j.
[16] N. Poliakoff, S.M. Albonico, M. Alvarez, J.C. Pacca, M.J. Vervego, J. Med. Chem.
16 (1973) 1411e1413.
[17] F. Wiedemann, W. Kampe, M. Theil, G. Sponer, E. Roesch, K. Dietmann, Chem.
Abstr 92 (1980) 128716e German Offen. 2,815,926 (1979).
[18] V.I. Shvedov, L.B. Altukova, M.D. Mashkovskii, A.N. Grinev, Khim. Farm. Zh 6
(1972) 14e17 Chem. Abstr. 78 (1973) 92397w.
[19] N. Haider, R. Jbara, F. Khadami, R. Wanko, Heterocycles 48 (1998) 1609e1622.
[20] E. Lescot, G. Muzard, J. Markovits, J. Belleney, B.P. Roques, J.B. Le Pecq, J. Med.
Chem. 29 (1986) 1731e1737.
[21] R.S. Ramsewak, M.G. Nair, G.M. Strasburg, D.L. DeWitt, J.L. Nitiss, J. Agric. Food
Chem. 47 (1999) 444e447.
[22] M. Stiborová, C.A. Bieler, M. Wiessler, E. Frei, Biochem. Pharmacol. 62 (2001)
1675e1684.
[23] K. Fang, S.P. Chen, C.W. Lin, W.C. Cheng, H.T. Huang, Lung Cancer 63 (2009)
227e234.
[24] P.L. Kuo, Y.L. Hsu, C.H. Chang, C.C. Lin, Cancer Lett. 16 (2005) 789e795.
[25] M. Hagg, M. Berndtsson, A. Mandic, R. Zhou, M.C. Shoshan, S. Linder, Mol.
Cancer Ther. 3 (2004) 489e497.
4.5.1. Cell lines and cell culture
The A-549 (Human Lung adenocarcinoma), SKMel2 (Human
melanoma), and B16F10 (Mouse melanoma), SKOV3 (Human
Ovarian adenocarcinoma) and HCT-15 (Human colon tumor) cell
lines were purchased from Korean Cell Line Bank (Chongno-gu,
Seoul, Republic of Korea). Cells were cultured in RPMI-1640 media,
supplemented with 10% heat-inactivated fetal bovine serum (FBS),
1 mM NaHCO3, 2 mM L-glutamine. All cell lines were maintained in
culture at 37 ꢀC in an atmosphere of 5% CO2.
4.5.2. In vitro assay
Cells were plated in 96-multiwell plate (104 cells/well) for 24 h.
The compounds to be tested were dissolved in dimethyl sulph-
oxide. Solutions of different concentration of the compounds under
test (1.56, 3.12, 6.25, 12.5, 25 and 50) were added to the cell
monolayer. Triplicate wells were prepared for each individual
concentration. The cytotoxicity assay was done against B16F10
(5 ꢄ 104 cells/mL), A-549 (1 ꢄ 105 cells/mL), SKMel2 (1 ꢄ 105 cells/
mL), SKOV3 (1 ꢄ 105 cells/mL), and HCT-15 (1 ꢄ 105 cells/mL) cell
lines using a colorimetric SRB assay method [61]. Exponentially
growing cells were harvested and suspended in the culture media
(100
37 ꢀC in humidified 5% CO2, the cells were treated with varying
concentrations of test compounds (100 L) and incubated further
mL, RPMI-1640) in a 96-well plate. After 24 h of incubation at
m
for 48 h under the same conditions. The cells were fixed with 50%
trichloroacetic acid and stained for 30 min with SRB. The unbound
dye was removed by 1% acetic acid, and the protein-bound dye was
extracted with 10 mM tris base (pH 10.5) for 5 min. The optical
density was measured at 520 nm in a micro plate reader to deter-
mine cell growth inhibition. The results were expressed as the
concentration at which there was 50% inhibition (IC50).
[26] M. Sainbury, D.E.V. Wilman, Ed.; Blackie: Glasgow and London, UK, (1990).
[27] R.J. Howorko, M. Pelczynska, A. Nasulewicz, J. Wietrzyk, A. Opolski, Arch.
Pharm. Chem. Life Sci. 338 (2005) 556e561.
[28] A. Segall, H. Pappa, R. Causabon, G. Martin, R. Bergoc, M.T. Pizzorono, Eur. J.
Med. Chem. 30 (1995) 165e169.
[29] G.W. Gribble, A. Brossi, Ed.; Academic Press: New York. 39 (1990) 239e343.
[30] M.G. Ferlin, C. Marzano, V. Gandin, S.D. Acqua, L.D. Via, Chem. Med. Chem. 4
(2009) 363e377.
Acknowledgment
[31] V. Gaddam, R. Nagarajan, Tetrahedron Lett. 50 (2009) 1243e1248.
[32] M. Dracinsky, J. Sejbal, B. Rygerova, M. Stiborova, Tetrahedron Lett. 48 (2007)
6893e6895.
[33] J.G. Rouesse, T.L. Chevalier, P. Caille, J.M. Mondesir, H. Sancho-Garnier, F. May-
Levin, M. Spielmann, R.D. Jager, J.L. Amiel, Cancer Treat. Rep. 69 (1985)
707e708.
We thank the IIT Madras, Chennai, India for their support in
spectral studies and the Biotechnology Department Bharathiar
University, Coimbatore, India for undertaking the antimicrobial
studies.

5590
T. Indumathi et al. / European Journal of Medicinal Chemistry 46 (2011) 5580e5590
[34] P. Juret, A. Tanguy, A. Girard, J.Y.L. Talaer, J.S. Abbatucci, N. Dat-Xuong, J.B. Le
Pecq, C. Paoletti, Eur. J. Cancer 14 (1978) 205e206.
[50] D.R. Anderson, S. Hegde, E. Reinhard, et al., Bioorg. Med. Chem. Lett. 15 (2005)
1587e1590.
[35] S. Kenney, D.T. Vistica, H. Linden, M.R. Boyd, Biochem. Pharmacol. 49 (1995)
23e32.
[51] L. Xiaolian, W. Qianqian, Q. Yang, L. Yanjie, Z. Yingli, Q. Xuhong, C. Jingnan,
Bioorg. Med. Chem. 18 (2010) 3279e3284.
[36] C. Auclair, Arch. Biochem. Biophys. 259 (1987) 1e14.
[37] B.P.J. Patel, Indian J. Chem. 21B (1982) 20e23.
[38] G.D. Shah, B.P.J. Patel, Indian J. Chem. 18B (1979) 451e453.
[39] M.N. Jachak, D.B. Kendre, A.B. Avhale, R.B. Toche, V.J. Medhane, Org. prepa-
ration Procedure Int 38 (3) (2006) 313e324.
[40] M.C. Silva Lourenco, F. Rodrigues Vicente, M.G. de Oliveira Henriques,
A.L. Peixoto Candea, R.S. Borges Goncalves, T.C. Nogueira, M.G. de Lima Fer-
reira, M.V. Nora de Souza, Bioorg. Med. Chem. Lett. 17 (2007) 6895e6898.
[41] A.M. Khalil, M.A. Berghot, M.A. Gouda, Eur. J. Med. Chem. 44 (2009)
4434e4440.
[52] W. Kemnitzer, J. Kuemmerle, S. Jiang, H.Z. Zhang, N. Sirisoma, S. Kasibhatla,
C. Crogan-Grundy, B. Tseng, J. Drewe, S.X. Cai, Bioorg. Med. Chem. Lett. 18
(2008) 6259e6264.
[53] W. Kemnitzer, S. Jiang, Y. Wang, S. Kasibhatla, C. Crogan-Grundy, M. Bubenik,
D. Labrecque, R. Denis, S. Lamothe, G. Attardo, H. Gourdeau, B. Tseng, J. Drewe,
S.X. Cai, Bioorg. Med. Chem. Lett. 18 (2) (2008) 603e607.
[54] K. Starcevic, M. Krali, I. Piantanida, L. Suman, K. Pavelic, G.K. Zamola, Eur. J.
Med. Chem. 41 (2006) 925e939.
[55] Y.D. Wang, S. Johnson, D. Powell, J.P. McGinnis, M. Miranda, S.K. Rabindran,
Bioorg. Med. Chem. 15 (2007) 3763e3766.
[42] M. Kidwai, K.R. Bhushan, P. Sapra, R.K. Saxena, R. Gupta, Bioorg. Med. Chem. 8
(2000) 69e72.
[56] I.A. Saleh, M.A. Areej, M.A. Ahmed, N. Eman, M.G. Mostafa, Eur. J. Med. Chem.
45 (2010) 738e744.
[43] P. Sharma, N. Rane, V.K. Gurram, Bioorg. Med. Chem. Lett. 14 (2004)
4185e4190.
[44] J. Saravanan, S. Mohan, J.J. Roy, Eur. J. Med. Chem. 45 (2010) 4365e4369.
[45] G.F.Q. Chan, G.H.N. Towers, J.C. Mitchell, Phytochemistry 14 (1975) 229e2296.
[46] H.M. Kanjariya, T.V. Radhakrishnan, K.R. Ramchandran, P. Hansa, Indian J.
Chem. Sect. B. 43 (2004) 1569e1573.
[47] D.R. Anderson, N.W. Stehle. WO 2004055015 A1, 2004.
[48] E.J. Reinhard, S.A. Kolodziej, D.R. Anderson, et al., WO 2004054505 A2, 2004.
[49] S.X. Cai, H. Zhang, W.E. Kemmitzer, S. Jiang, J.A. Drewe, R. Storer,
WO2002092076 A1, 2002.
[57] K. Radha, T. Pritam, M.J. Kang, T.C. Jeong, M.N. Jung, H.L. Kim, N. Younghwa,
W.J. Cho, K. Youngjoo, E.S. Lee, Bioorg. Med. Chem. 18 (2010)
3066e3077.
[58] B.C. Seong, K. Yon, M.I. Young Yun, Z.A. Byung, Arch. Pharm. Res. 5 (2004)
485e494.
[59] C. Perez, M. Paul, P. Bazerque, Acta Biol. Med. Exp. 15 (1990) 113e115.
[60] D. Srinivasan, N. Sangeetha, T. Suresh, P. Lakshmana, J. Ethnopharmacol 74
(2001) 217e220.
[61] M.A.A. Rahman, S.C. Cho, J. Song, H.T. Mun, S.S. Moon, Planta Med. 73 (2007)
1089e1094.