Synthesis and biological evaluation of novel carbazolyl glyoxamides as anticancer and antibacterial agents

Article abstract of DOI:10.1039/c5ra27175d

A new library of 24 carbazolyl glyoxamides 14a-x were designed and synthesized from glyoxalic acids and arylamines in the presence of HATU as a coupling reagent under MW irradiation. The synthesized carbazolyl glyoxamides were evaluated for their in vitro anticancer and antibacterial activities. Of the synthesized carbazolyl glyoxamides, compounds 14l and 14q exhibited the most potent cytotoxicity towards a breast cancer cell line with IC₅₀ values of 9.3 and 9.8 μM, respectively. Further, caspase-3 assay for carbazolyl glyoxamides indicated that these compounds induced apoptotic cell death in Jurkat cells. Furthermore, some of the synthesized carbazolyl glyoxamides 14g, 14k, 14l and 14n exhibited comparable or even better antibacterial activity (MIC = 8-16 μg mL^-1) than chloramphenicol against the selected bacterial strains.

Full text of DOI:10.1039/c5ra27175d

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Synthesis and biological evaluation of novel carbazolyl
glyoxamides as anticancer and antibacterial agents
Venkataramana Reddy P O,a Mukund P Tantak,a Reyna Valdez,b Rajnish Prakash Singh,c Okram
Mukherjee Singh,d Rachna Sadanab*and Dalip Kumara*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A new library of 24 carbazolyl glyoxamides 14a-x were designed and synthesized from glyoxalic acids and arylamines in the
presence of HATU as a coupling reagent under MW irradiation. The synthesized carbazolyl glyoxamides were evaluated for
their in vitro anticancer and antibacterial activities. Of the synthesized carbazolyl glyoxamides, compounds 14l and 14q
exhibited the most potent cytotoxicity towards breast cancer cell line with IC50 values of 9.3 and 9.8 µM, respectively.
Further, caspase-3 assay for carbazolyl glyoxamides indicated that these compounds induced apoptotic cell death in Jurkat
cells. Furthermore, some of the synthesized carbazolyl glyoxamides 14g, 14k-l and 14n exhibited comparable or even
better antibacterial activity (MIC = 8-16 µg/mL) than chloramphenicol against the selected bacterial strains.
www.rsc.org/
inhibitor (IC50 = 2 µM).14 Further, carbazole sulfonamide (5)
exhibited significant cytotoxicity against leukemia cells (IC50
19 nM).15 Nakamura and co-workers reported the isolation of
the carbazomycins A-F ( ) from Streptoverticillium ehimense H
,
Introduction
=
Indole alkaloids are an important class of natural products and
medicinally important molecules, which elicit a wide range of
biological activities through diverse mechanisms.1, 2 Among the
indole containing heterocycles, the benzo[b]indole system is a
6
1051-MY 10 and these carbazole-based congeners were found
to possess promising antibacterial and antifungal activities.16-18
The current interest in carbazoles for clinical applications
arises mainly due to their high efficiencies against several
types of diseases, limited toxic side effects, and complete lack
of hematological toxicity.19 In addition to interesting and
useful biological applications, carbazole derivatives are also
used as organic materials due to their photorefractive,
photoconductive, whole transporting and light-emitting
properties.20
carbazole motif that is widely present in
a variety of
compounds obtained from natural and commercial sources
with anticancer and/or antibacterial properties (Fig. 1).3-6 For
example, Fujita et al investigated carbazole-based hydrazone,
HND-007 (1) and related compounds for their in vivo antitumor
activity suppressing the growth of various cancer cell lines
(IC50 ~ 1.3–4.6 μM).7 Pyridine fused carbazole derivative,
S16020 (2) exhibited potent cytotoxic effects (IC50 = 27.5 nM)
by virtue of its DNA intercalative and topoisomerase inhibition
properties.8-10 Multidrug resistant tumor cell lines are sensitive
H
N
N
H
O
H
O
N
N
O
HO
CH3
N
N
to S16020 and it is currently being evaluated in the clinical
12
O
stages.11,
Naturally occurring carbazole derivative,
3) was isolated from the ascidian Didemnum
NH
N
N
N
N
H
granulatimide (
Et
Granulatimide 3
Anticancer and
antibacterial agent
Carbazolylmethylene hydrazide-
hydrazone (HND-007) 1
Anticancer agent
S16020 2
Anticancer agent
granulatum and found to display anticancer and antibacterial
activities.13 Caulfield and co-workers had patented carbazolyl
MeO
chalcone
4 for its potential as a tubulin polymerization
OMe
OMe
OMe
OMe
OMe
O
O2
S
HO
N
OCH3
CH3
N
a.Department of Chemistry, Birla Institute of Technology and Science, Pilani 333
031, Rajasthan, India, Tel.: +91-1596-515238; fax: +91-1596-244183; E-mail:
dalipk@pilani.bits-pilani.ac.in.
H
N
N
H
CH3
Et
CH3
b.Department of Natural Sciences, University of Houston – Downtown, Houston, TX
77002, United States, Tel.: 001-7132265562; fax: 001-7132237476; E-mail:
sadanar@uhd.edu.
Carbazolyl chalcone 4
Anticancer agent
Carbazolyl sulfonamide 5
Carbazomycin B 6
Antibacterial agent
Anticancer agent
c. Department of Biological Sciences, Birla Institute of Technology and Science, Pilani
333031, Rajasthan, India.
Fig. 1 Carbazole analogues as anticancer and antibacterial
agents
d.Department of Chemistry, Manipur University, Canchipur, Imphal-795003,
Manipur, India.
Glyoxamide is an important structural unit found in many
e. † Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
biologically active compounds and synthetic drug
candidates,21,
22
especially those with anticancer and
) destabilizes
antibacterial properties. For example, Indibulin (
7
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tubulin polymerization by arresting tumor cell growth at the reagents under conventional as well as microwave (MW)
G2/M phase. Indibulin is also active in multidrug resistant irradiation conditions generated 14a in low yield (40%, Table 1.
tumor cell lines and its oral formulation is currently being entries 3-6). Finally, the reaction of 13a and
examined in clinical trials.23 Structurally similar, conscinamides aryl/heteroarylamines in the presence of O-(7-azabenzotriazol-
A-C (
8) containing an indolic enamide fragment, were isolated 1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate
from marine sponge Coscinoderma sp and found to display (HATU) at 70 °C under MW irradiation led to 14a in 70% yield.
antitumor activity against a human prostate cancer cell line Scope and generality of this developed protocol was further
(IC50 = 7.6 µg/mL)24 and partial cytoprotection against HIV.25 demonstrated by coupling glyoxalic acid 13 with various
More recently, Singh et al. have described the synthesis of arylamines and a series of carbazolyl glyoxamides 14a-x was
different bis(indole)glyoxamides 9 as potent antibacterial prepared in 70-91% yields. Structures of the newly synthesized
candidates.26 In our continued efforts to find out biologically glyoxamides were well characterized by using spectroscopic
active indole-based molecules, recently, we have prepared α- techniques including IR, NMR (1H and 13C) and HRMS. HPLC
cyano bis(indolyl)chalcones,27 2-arylamino-5-(3
oxadi-azoles,28 5-(3
5-(3
-indolyl)-1,3,4-thiadiazoles30 and indolyl-1,2,4-triazoles31 spectra of 14a-x, characteristic peaks at ~1690 and 1655 cm−1
′
-indolyl)-1,3,4- analysis of synthesized carbazolyl glyoxamides 14a-x indicated
′
-indolyl)-1,3,4-thiadiazoles,29 2-arylamino- the purity of all the compounds was greater than 97%. In IR
′
as potent anticancer agents. Very recently, we have identified were assigned to the carbonyl and amide functionalities,
2-(3′-indolyl)-N-arylthiazole-4-carboxamides as antibacterial respectively. Further, in 13C NMR spectra of 14a-x, the carbons
and anticancer agents.32 Encouraging anticancer and of carbonyl and amide functionalities were resonated at ~180
antibacterial activities of glyoxamides and pivotal roles of (CO) and ~160 ppm (NHCO).
O
OEt
carbazole scaffold in bioactive compounds prompted us to
a
b
investigate their new analogues. In this paper, we report a
series of 24 carbazolyl glyoxamides 14a–x by incorporating
important scaffolds, glyoxamide and carbazole in a single
molecule (Fig. 2).
O
N
H
N
R
N
R
12
10
11
O
H
O
OH
MeO
OMe
R1
N
H
c
d
O
N
O
O
O2
S
OMe
O
N
H
N
N
R
NH
R
13
N
H
N
N
14a-x (
70-91%)
Me
3
Granulatimide
anticancer and
R= H, CH3, C2H5, 4-ClC6H4CH2
Carbazolyl sulfonamides 5
anticancer agent
antbacterial agent
Scheme 1 Reagents and conditions: (a) CH3I/C2H5I/4-
N
N
HN
O
ClC6H4CH2Cl, KOH, DMF, rt, 13 h; (b) Cl(CO)2OEt, AlCl3, DCM, 0
°C to rt, 3 h; (c) LiOH, THF:H2O (1:1), rt, 2 h; (d) R1NH2, HATU,
DIPEA, DMF, 70 °C, 45 min, MW.
O
H
O
O
N
O
R1
Br
O
N
N
N
R
Cl
Carbazolyl glyoxamides 14a-x
Indibulin 7
Anticancer activity
N
anticancer agent
Br
Twenty four synthesized glyoxamides were evaluated for their
anticancer activities towards human T lymphocyte (Jurkat),
histiocytic lymphoma (U937), and breast (MCF-7 and MDA-
MB-231) cancer cell lines (Table 2). Structure–activity
relationship (SAR) studies of carbazolyl glyoxamides 14a-x
were demonstrated by varying aryl/heteroarylamines and
substitution at 9-NH position of carbazoles. Initial screening of
compounds 14a-x at 10 µM concentration indicated that
compounds 14i-m and 14q displayed about 50% cell survival
(Table 2). N-Methyl and N-ethyl congeners of the carbazole
were reported to possess significant cytotoxicity, therefore,
derivatives 14r-x were synthesized.7, 15 Unfortunately, analogs
14r-x exhibited low activity against the tested tumor cell lines.
The IC50 values of selected carbazolyl glyoxamides 14i-m and
14q are summarized in Table 3. Compound 14i, bearing 4-
chlorobenzyl and N,N′-dimethylaminophenyl moieties was
found to show moderate activity. Cytotoxicity was retained by
the replacement of a phenyl ring in 14a with heteroaryl
moieties such as 6-quinolyl (14j) and 2-(5-methyl)thiazolyl
O
H
O
R
N
O
N
N
O
H
R
N
Coscinamides A-C 8
anticancer agent
9
Bis(indole) glyoxamide
antibacterial agent
H
Fig. 2 Design of carbazolyl glyoxamides 14a–x
Results and discussions
Synthesis and characterization
Carbazolyl glyoxamides 14a–x were synthesized from the
reaction of carbazole 10 with ethyl chlorooxoacetate in the
presence of anhydrous AlCl3 followed by ester hydrolysis using
LiOH to afford glyoxalic acid 13 in good yield (Scheme 1). For
the coupling of 13 with aryl/heteroaryl amines, we optimized
the reaction conditions by varying temperatures, solvents, and
reagents as illustrated in Table 1. Initial efforts by using thionyl
chloride or oxalyl chloride failed to produce 14a (Table 1,
entries 1-2). Subsequent efforts by employing well known
carbodiimides including DCC, CDI and EDCI.HCl as coupling
(
14k).
2 | J. Name., 2012, 00, 1-3
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Table 1 Optimization of reaction conditions for the synthesis of carbazolyl glyoxamide 14a
O
O
H
N
OH
NH2
O
O
N
R
N
R
conditions
13b
14a
R = 4-ClC6H4CH2
Entry Reagent
Solvent
Base
Temperature
(°C)
Conventional heating
Microwave irradiation
Time (h)
Yield (%)
Time
(min.)
NA
NA
NA
NA
60
Yield (%)
1
2
3
4
5
6
7
8
9
SOCl2
DCM
DCM
THF
TEA
RT
RT
RT
RT
RT
70
60
70
70
12
15
30
20
14
12
15
12
12
Trace
Trace
Trace
Trace
30
NA
(COCl)2
DCC
TEA
NA
TEA
NA
CDI
THF
TEA
NA
EDCI.HCl/HOBt
EDCI.HCl/HOBt
HATU
THF
DIPEA
TEA
40
DMF
THF
Trace
Trace
40
60
Trace
Trace
70
DIPEA
DIPEA
TEA
60
HATU
DMF
DMF
45
HATU
20
45
40
RT: room temperature; NA: not attempted
O
Table 2 In vitro cytotoxicity of carbazolyl glyoxamides 14a-x
H
N
R1
O
N
R
Percentage cell survival (@ 10 µM)
Compound
14a
14b
14c
R
R1
Jurkat
U937
MCF-7
MDA-MB-231
82.3±7.5
83.5±6.8
80.5±5.5
78.6±6.8
72.5±2.2
70.9±5.2
88.9±6.4
90.2±2.2
62.8±3.5
63.5±2.9
64.8±1.9
75.2±2.4
61.3±2.0
72.6±8.3
84.9±10.5
98.2±5.4
65.2±4.5
94.7±6.2
92.3±3.9
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
H
C6H5
71.5±10.7
70.2±0.8
71.3±26.7
71.3±14.4
69.8±14.7
64.5±1.4
118.4±26.4
83.1±19.6
56.5±6.9
56.5±1.6
51.3±0.5
83.4±41.0
52.4±5.9
60.7±0.7
77.9±9.4
94.9±6.3
60.5±6.2
95.31±7.2
99.77±6.6
93.7±5.3
92.3±2.2
100.4±10.8
102.3±9.7
100.4±10.8
100±5.9
88.5±5.2
79.5±3.4
82.1±2.2
67.2±8.5
85.5±5.3
98.8±2.9
83.2±6.7
59.1±14.8
78.8±1.2
60.3±17.4
62.2±5.8
64.4±8.7
67.4±5.8
72.1±6.9
85.6±6.5
92.8±5.2
65.8±8.5
109.7±10.4
107.5±9.3
91.3±10.9
104.7±9.5
106.9±7.4
93.6±4.2
95.7±7.2
101.5±9.5
31.8±1.1
69.5±7.8
67.1±7.4
74.6±0.9
70.9±10.4
67.3±4.6
73.7±19.9
93.3±5.6
66.3±5.4
70.5±5.8
68.0±7.0
58.4±6.6
48.5±4.0
59.1±13.3
59.8±19.9
89.4±17.3
60.5±10.6
50.0±7.3
104.2±6.7
72.1±5.4
4-CH3C6H4
4-CH3OC6H4
3-CH3OC6H4
3,4-(CH3O)2C6H3
3,4,5-(CH3O)3C6H2
4-FC6H4
14d
14e
14f
14g
14h
14i
4-pyridyl
4- (CH3)2N,N-C6H4
6-quinolyl
14j
14k
14l
2-(5-methyl)thiazolyl
C6H5
14m
14n
14o
14p
14q
14r
H
3,4,5-(CH3O)3C6H2
3-CH3OC6H4
4-CH3OC6H4
4-pyridyl
H
H
H
H
3,4-(CH3O)2C6H3
C6H5
CH3
14s
CH3
3,4,5-(CH3O)3C6H2
C6H5
14t
C2H5
114.2±11.3 94.5±5.2
106.9±12.1 96.5±6.6
14u
14v
14w
14x
C2H5
3,4-(CH3O)2C6H3
3,4,5-(CH3O)3C6H2
4- (CH3)2N,N-C6H4
6-quinolyl
C2H5
78.1±8.8
79.7±7.6
73.8±8.5
100±3.2
38.2±10.6
88.8±7.1
89.9±9.2
83.9±8.9
100±2.2
40.2±3.5
C2H5
C2H5
Control (negative)
Doxorubicin (positive)
21.2±5.6
The activity data represent mean values
± SD of experiments conducted in triplicates at three independent times
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Table 3 IC50 values (µM) of selected carbazolyl glyoxamides
Antibacterial activity
S. No Jurkat
U937
MCF-7
MDA-MB-231
In light of interesting antibacterial activities of many
carbazole containing natural and synthetic compounds, the
newly synthesized carbazolyl glyoxamides 14a-x were
screened for their antibacterial activity.27 All the compounds
were tested for their in vitro antibacterial activities against
Gram-positive bacteria including Staphylococcus aureus
(MTCC 96) and Bacillus subtilis (MTCC 121), and Gram-
negative bacteria including Escherichia coli (MTCC 1652) and
Pseudomonas putida (MTCC102) with respect to
chloramphenicol, a standard drug. The Minimum Inhibitory
Concentrations (MICs) and Zone of Inhibition (ZOI) for
compounds 14a-x were determined in vitro by the modified
broth micro-dilution values method as given in Table 4.
Compounds containing N-chlorobenzyl carbazole and C6H5
14i
14j
14k
14l
10.5 ± 2.1
29.2 ± 3.9
15.1 ± 4.8
17.5 ± 8.2
18.3 ± 7.8
23.3 ± 10
29.2 ± 9.2
0.15 ± .05
23.5 ± 8.2
18.9 ± 3.8
12.2 ± 5.4
9.3 ± 4.3
11.5 ± 5.5
9.8 ± 2.8
0.35 ± .15
17.9 ± 5.7
18.7 ± 7.7
20.1 ± 8.4
31.2 ± 11.4
14.7 ± 5.4
18.5 ± 5.4
0.5 ± .20
11.3 ± 3.6
10.2 ± 2.9
11.8 ± 3.3
14m 12.1 ± 1.8
14q 17.8 ± 3.2
DX-1 0.25 ± .11
*Bold value indicates IC50 > 10 µM; DX-1 = Doxorubicin
Compound 14l with N-H free carbazole and N-phenyl
glyoxamide was identified as the most active member of the
series with IC50 values between 9.3 to 31.2 µM. Also,
compound 14l was found to be 2-3 fold more cytotoxic
towards MCF-7 (IC50 = 9.3 µM) and Jurkat (IC50 = 11.8 µM)
cells. No significant change in activity was observed by the
replacement of a phenyl group in compound 14l with a
trimethoxyphenyl (14m) and dimethoxyphenyl (14q) groups
except for 14q (IC50 = 9.8 µM; MCF-7). However, compound
(
14a), CH3C6H4 (14b) CH3OC6H4 (14c), (CH3O)3C6H2 (14f) and
N,N′-(CH3)2C6H4 (14i) substituents in the glyoxamide fragment
were found to display moderate activity against the tested
bacterial strains. Interestingly, introduction of an electron-
withdrawing fluoro group in the phenyl ring resulted in 14g
endowed with potent antibacterial activity against all tested
bacterial strains with MIC values ranging between 8 to16
µg/mL. Replacement of a N-phenyl ring in 14a with heteroaryl
groups such as 4-pyridyl (14h) and 6-quinolyl (14j) led to
inactive derivatives; except 14k bearing 2-(5-methyl)thiazolyl)
moiety exhibited comparable antibacterial activity against B.
cereus and E. coli bacterial strains (ZOI = 16-19 mm; MIC = 16
µg/mL). Compound 14l with carbazole N-H and a phenyl
moiety on the amide part displayed improved activity when
14q was found to be equipotent (compounds 14l vs 14q
)
against MCF-7 cell line (IC50 = 9.8 µM). From the structural
variation it was realized that carbazole-N-H with its appended
glyoxamide
bearing
phenyl,
dimethoxyphenyl
and
trimethoxyphenyl units increases cytotoxicity.
To further characterize the mode of cellular death by
carbazolyl glyoxamides, apoptosis induction studies for the
selected compounds 14i-m and 14q were performed on Jurkat
cells by the caspases 3/7 activation method. Caspases
belonging to a family of cysteine proteases are known to play
an essential role in apoptosis.33 Out of these caspases,
caspase-3 is an effector caspase that cleaves multiple proteins
in cells leading to apoptotic cell death. Therefore, activation of
caspase 3 pathway is a hallmark of apoptosis and can be used
in cellular assay to quantify activator. Of the carbazolyl
compared to the corresponding N-substituted carbazole 14a
.
Replacement of a phenyl ring in 14l by methoxyphenyl (14m-o
and 14q) or 4-pyridyl (14p) moiety led to a decrease in the
activity except for 14n (MIC = 8-16 µg/mL). Alkylations of
carbazole N-H resulted in compounds 14r-x with moderate
activity against the tested bacterial strains.
glyoxamides tested, compounds 14i, 14k, 14l and 14q showed
Cell viability assay
4-5-fold enhancement in caspase level compared to the
control (Fig 3). These results imply that carbazolyl glyoxamides
induced apoptosis in Jurkat cells via caspase-3-dependent
pathway.
Although antibacterial (ZOI & MIC) assays might show a
potential to kill pathogenic micro-organisms, concentration vs
time curves provide more insight about the rate of
antibacterial activity. To determine the rate of bactericidal
activity of the two most active compounds 14g and 14l, time-
kill studies were performed. The interactive time and the
change in the number of microorganisms for compounds 14g
and 14l is presented in Fig. 4A-D.
The percent population reduction at different time interval
was calculated to demonstrate the change of the population of
microorganism respect to concentration dependent dose. The
compound 14g showed high bacteriostatic effect against E. coli
in the first hours of incubation.
Fig. 3 Carbazolyl glyoxamides 14i-m and 14q induced caspase
activation in Jurkat cells
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O
Table 4 In vitro antibacterial activities of carbazolyl glyoxamides 14a-x
H
N
R1
O
N
R
Compound
R
R1
Gram
B. cereus
MIC
-
positive bacteria
Gram
-
negative bacteria
S. aureus
E. coli
ZOI
14
P.putida
ZOI
16
15
16
17
15
15
18
15
16
15
16
17
15
18
14
15
16
13
14
15
14
15
15
12
21
ZOI
16
-
MIC
MIC
64
ZOI
15
-
MIC
14a
14b
14c
14d
14e
14f
4-ClC6H4CH2 C6H5
32
>32
-
32
-
4-ClC6H4CH2 4-CH3C6H4
4-ClC6H4CH2 4-CH3OC6H4
4-ClC6H4CH2 3-CH3OC6H4
4-ClC6H4CH2 3,4-(CH3O)2C6H3
4-ClC6H4CH2 3,4,5-(CH3O)3C6H2
4-ClC6H4CH2 4-FC6H4
>32
32
16
64
15
-
32
-
17
>16
>16
32
15
-
>16
-
16
14
>64
>32
16
17
17
18
17
18
16
19
18
17
19
14
16
18
13
13
14
-
64
>64
>8
>32
>32
15
16
16
19
16
16
18
18
17
16
18
15
16
14
14
15
17
16
-
>16
32
16
32
64
64
>16
8
18
32
14g
14h
14i
20
8
4-ClC6H4CH2 4-pyridyl
128
64
16
32
4-ClC6H4CH2 4-(CH3)2N,N-C6H4
4-ClC6H4CH2 6-quinolyl
4-ClC6H4CH2 2-(5-methyl)thiazolyl
14
32
14j
>32
16
>128 15
32
14k
14l
>8
19
19
16
16
17
18
17
15
17
16
16
14
16
13
22
16
H
C6H5
16
>8
>8
14m
14n
14o
14p
14q
14r
14s
14t
14u
14v
14w
14x
H
3,4,5-(CH3O)3C6H2
3-CH3OC6H4
4-CH3OC6H4
4-pyridyl
>16
>8
>16
16
32
64
8
H
16
H
64
>64
>64
>32
64
128
32
>64
>32
>64
32
>32
>16
>16
-
H
32
H
3,4-(CH3O)2C6H3
C6H5
128
>32
64
>16
32
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
3,4,5-(CH3O)3C6H2
C6H5
32
32
>32
64
64
>16
>32
>64
32
3,4-(CH3O)2C6H3
3,4,5-(CH3O)3C6H2
4- (CH3)2N,N-C6H4
6-quinolyl
-
>16
>32
32
15
14
14
21
>32
>64
32
16
14
21
32
64
16
>32
16
Chloramphenicol
32
16
*ZOI (in mm) and MIC (in µg/mL) values, bold values indicate comparable or even better antibacterial activity than
chloramphenicol.
Moreover, the highest reduction (85%) in E. coli population
From the structure-activity relationship (SAR) studies of
was observed at 8 h of incubation with compound 14g (Fig. carbazolyl glyoxamides it implies that a combination of
4A). Compound 14g was also showed about >70% reduction in carbazole N-H and glyoxamide unit possessing p-fluorophenyl
viable cells of S. aureus (Fig. 4B). Compound 14l with free and methoxyphenyl substituents are beneficial for the activity
carbazole N-H and a phenyl moiety in amidic part led to (Fig. 6). All the synthesized compounds demonstrated well to
significant inhibition of bacterial growth after
4 h of moderate cytotoxicity against a panel of cancer cell lines and
incubation. It is evident that within 8 h, compound 14l excellent to moderate antibacterial activity towards tested
exhibited almost 80% reduction in the viability of P. Putida bacterial strains. Particularly, compound 14l with carbazole N-
(Fig. 4C). Similarly, bacterostatic effect of compound 14l H and phenyl moietiy in glyoxamide part exhibited potent
against S. aureus reveals about 70% reduction of viable cell at cytotoxicity and antibacterial activities. Exclusively, with
8 h of incubation (Fig. 4D). Thus the results of present study carbazole N-H and dimethoxyphenyl moieties, compound 14q
revealed that 14g and 14l were capable of inhibiting the was found to be the most active against the tested cancer cell
bacterial growth within few hours of initial interactions.
lines and less potent towards tested bacterial strains.
The toxicity of potent compounds 14i-m and 14q was However, compound 14g with N-chlorobenzylcarbazole and
evaluated using LDH (Lactate dehydrogenase) assay. The LDH fluorophenyl units, and analogue 14n having carbazole N-H
activity shows that all the tested compounds 14i-m and 14q and 3-methoxyphenyl moieties, were the most active
exhibited lower toxicity than the standard drug, doxorubicin carbazolyl glyoxamides against the tested bacteria but
which justifies the potential use of these compounds as anti- exhibited low cytotoxicity towards tested cancer cell lines.
bacterial agents (Fig. 5).
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Fig. 4 Cell viability assay of 14g and 14l against selected bacteria
Conclusions
In summary, we have synthesized various carbazolyl
glyoxamides from readily available glyoxalic acids 13 and
arylamines by employing HATU as
a coupling reagent.
Synthesized glyoxamides were assessed for their cytotoxicity
which enabled us to identify 14l and 14q as the most potent
compounds against MCF-7 cells with IC50 values of 9.3 µM and
9.8 µM, respectively. Preliminary mechanism of action studies
indicated that carbazolyl glyoxamides induced apoptosis in
Jurkat cells via caspase-3 and-7 activation. In addition,
antibacterial activity evaluation led us to compounds 14g, 14k-
Fig. 5 Cytoxicity induced by carbazolyl glyoxamides in terms of
l
and 14n with significant potency against Gram-positive and
Gram-negative bacteria (MIC = 8-16 µg/mL and ZOI = 16-20
mm). Antibacterial activities of potent compounds 14g 14k-l
LDH release
P.putida
O
,
H
phenyl
N
and 14n were found to be comparable to the reference drug,
chloramphenicol. Cell viability assay revealed that analogues
14g and 14l were capable of inhibiting the bacterial growth
within few hours of initial interactions. Interesting activity
results indicate that the identified potent carbazolyl
glyoxamides 14g and 14l can be exploited further to develop
either highly specific or potent antibacterial/ anticancer
Selective
R1
inhibition
p-flurophenyl
O
N
E.coli
R
N-H good
Good for cytotoxicity
(phenyl & 3,4-dimethoxyphenyl)
for activity
Fig. 6 Structure-activity relationship of carbazolyl glyoxamides
14a-x
6 | J. Name., 2012, 00, 1-3
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agents, or if required both of these properties could be NMR (101 MHz, CDCl3) δ 186.1, 159.8, 157.0, 144.2, 141.1,
incorporated in the same molecule.
134.6, 133.7, 130.1, 129.7, 129.2, 127.7, 126.9, 126.1, 125.1,
123.6, 123.1, 121.6, 121.1, 121.0, 114.4, 109.5, 108.7, 55.5,
46.2; IR (KBr, v, cm-1): 3348, 3055, 2924, 1682, 1643, 1620,
1582, 1528, 1443, 1327, 1250, 1149; Anal. RP-HPLC tR = 4.368
min, purity 98.67%; HRMS (ESI+) calculated for C28H22ClN2O3 [M
+ H]+, 469.1319; Found 469.1310 and 491.1129 [M+Na]+.
Experimental
General procedure for the synthesis of carbazolyl
glyoxamides (14a-x).
To a 10 mL microwave tube was added carbazole glyoxalic acid
13 (0.275 mmol), HATU (0.12 g, 0.317 mmol), N,N-
diisopropylethyl-amine (0.09 g, 0.687 mmol) and an
appropriate aryl/heteroarylamine (0.303 mmol) in DMF (2 mL).
The tube was sealed with a pressure cap and placed in the
microwave cavity. The sample was irradiated for 45 min at 70
°C and then allowed to cool at room temperature. The residue
was poured into ice-cold water (30 mL) and stirred for 20 min
at room temperature. The solid so obtained was filtered, dried
and purified by column chromatography on silica gel using
Acknowledgements
The authors acknowledge the financial support received from
DBT (No. BCIL/NER-BPMC/2012/1549) New Delhi, India.
Dr. Rachna Sadana thanks to University of Houston-Downtown
(UHD) for providing organized research and creative activity
(ORCA) funds.
Notes and references
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2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-2-oxo-N-phenyl-
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Yellow solid; Yield 70%; M.p: 191-193 °C; 1H NMR (400 MHz,
CDCl3) δ 9.47 (d, J = 1.3 Hz, 1H), 9.15 (s, 1H), 8.58 (dd, J = 8.8,
1.7 Hz, 1H), 8.25 (dd, J = 7.6, 1.1 Hz, 1H), 7.78 (dd, J = 8.6, 1.0
Hz, 2H), 7.54-7.50 (m, 1H), 7.45 (dd, J = 8.3, 7.5 Hz, 3H), 7.41–
7.36 (m, 3H), 7.26 (t, J = 2.6 Hz, 2H), 7.08 (d, J = 8.6 Hz, 2H),
5.55 (s, 2H); 13C NMR (101 MHz, CDCl3) δ 185.9, 160.0, 144.2,
141.2, 136.9, 134.6, 133.7, 129.8, 129.3, 129.2, 127.7, 127.0,
126.2, 125.2, 125.0, 123.6, 123.1, 121.1, 121.1, 120.0, 109.5,
108.8, 46.24; IR (KBr, v, cm-1): 3335, 3090, 3052, 2916, 1682,
1653, 1589, 1520, 1435, 1307, 1250, 1134, 1011, 825, 795;
Anal. RP-HPLC tR = 4.641 min, purity 98.55%; HRMS (ESI+)
calculated for C27H20ClN2O2 [M+H]+, 439.1213; Found 439.1208
and 461.1025 [M+Na]+.
2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-2-oxo-N-(p-tolyl)
acetamide (14b).
1
Yellow solid; Yield 72%; M.p: 172-173 °C; H NMR (400 MHz,
CDCl3) δ 9.48 (d, J = 1.6 Hz, 1H), 9.09 (s, 1H), 8.58 (dd, J = 8.8,
1.7 Hz, 1H), 8.27–8.23 (m, 1H), 7.67–7.64 (m, 2H), 7.53-7.49
(m, 1H), 7.44–7.35 (m, 3H), 7.26 (t, J = 2.2 Hz, 3H), 7.24 (s, 1H),
7.09 (d, J = 8.6 Hz, 2H), 5.55 (s, 2H), 2.39 (s, 3H); 13C NMR (101
MHz, CDCl3) δ 186.0, 159.9, 144.2, 141.1, 134.9, 134.6, 134.3,
133.7, 129.9, 129.7, 129.2, 127.7, 126.9, 126.1, 125.1, 123.6,
123.1, 121.1, 121.0, 119.9, 109.5, 108.7, 46.2, 21.0; IR (KBr, v,
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1443, 1327, 1265, 1149; Anal. RP-HPLC tR = 5.317 min, purity
98.10%; HRMS (ESI+) calculated for C28H21ClN2O2 [M+H]+,
453.1369; Found 453.1365 and 475.1183 [M+Na]+.
2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N-(4-methoxyphenyl)
-2-oxoacetamide (14c).
1
Yellow solid; Yield 72%; M.p: 168-170 °C; H NMR (400 MHz,
CDCl3) δ 9.45 (d, J = 1.5 Hz, 1H), 9.04 (s, 1H), 8.56 (dd, J = 8.8,
1.7 Hz, 1H), 8.22 (d, J = 7.7 Hz, 1H), 7.69–7.65 (m, 2H), 7.51-
7.47 (m, 1H), 7.42–7.32 (m, 4H), 7.25–7.23 (m, 1H), 7.06 (d, J =
8.5 Hz, 2H), 6.97–6.94 (m, 2H), 5.52 (s, 2H), 3.84 (s, 3H); 13C
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Graphical Abstract