Synthesis, antibacterial and antioxidant properties of novel ethylenoindolophanes-a new class of cyclophanes

Article abstract of DOI:10.1039/c4ra00303a

Synthesis of indole based ethylenophanes has been achieved using direct N-arylation followed by McMurry coupling. All the ethylenophanes show antibacterial and antioxidant activity similar to that of commercially available drugs. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra00303a

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Synthesis, antibacterial and antioxidant properties
of novel ethylenoindolophanes – a new class of
Cite this: RSC Adv., 2014, 4, 21190
cyclophanes†
a
Perumal Rajakumar, Nagarathinam Venkatesana and Gunasekaran Mohanrajb
Received 12th January 2014
Accepted 11th March 2014
*
DOI: 10.1039/c4ra00303a
www.rsc.org/advances
Synthesis of indole based ethylenophanes has been achieved using cyclophanes, and various reagents based on transition metals
direct N-arylation followed by McMurry coupling. All the ethyl- such as samarium,18 cobalt19 and low valent titanium have been
enophanes show antibacterial and antioxidant activity similar to that of extensively used for the synthesis of cyclophanes.20,21 Inter- and
commercially available drugs.
intramolecular McMurry coupling for the synthesis of stilbe-
nophanes22 and indolophanes23 has been reported from our
laboratory. Although the antioxidant properties of carbazole
based dendrimers was reported recently from our laboratory,24
the synthesis and biological applications of bis-indolophanes is
still a rare observation. It is important to mention here that the
synthesis of indole based ethylenophanes by a direct arylation
route is not known so far, and hence we report herein the
synthesis and the antibacterial and antioxidant activity of the
indole based ethylenophanes 1, 1a, 2, 3 and 4 (Fig. 1).
Introduction
The indole moiety is a bioactive nucleus1 present in various
natural products.2 Cyclophanes with indole moieties are called
indolophanes,3 and have received much attention during recent
times due to their biological applications. Indole based cyclo-
phanes are infrequently encountered systems,4 though N-ary-
lindole has a key role with interesting biological activities, such
as anti-estrogenic,5 analgesic,6 antiallergy,7 cyclooxygenase
(COX)-1 inhibitory,8 neuroleptic,9 5-HT6 receptor antagonistic,10
FTase inhibitor (FTIs),11 and anti HIV-1 activity.12 Indole deriv-
atives extracted from marine sponges have been recently
reported to show antioxidant behavior.13,14 Although Ullmann-
type coupling of indole with aryl halides to synthesize N-ary-
lindole represents a straightforward and inexpensive route,15
the yields are poor, not reliable, and furthermore many
discouraging operational problems such as a high reaction
temperature, use of highly polar aprotic solvents, strong base,
requirement of a large amount of aryl halides and stoichio-
metric amounts of copper reagents restrict the application of
Ullmann coupling for the synthesis of indolophanes. Therefore,
the use of a mild and efficient method for generating the N-
arylindole moiety from indole is highly desirable.16 Further-
more, the ring closing step is oen crucial17 in the synthesis of
aDepartment of Organic Chemistry, University of Madras, Maraimalai Campus,
Chennai-600025, Tamil Nadu, India. E-mail: perumalrajakumar@gmail.com; Fax:
+91 044 22300488; Tel: +91 044 22351269 ext. 213
bCentre for Advanced Studies in Botany, University of Madras, Maraimalai Campus,
Chennai-600025, Tamil Nadu, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra00303a
Fig. 1 Structures of the ethylenoindolophanes 1–4.
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The purpose of the synthesis of ethylenophanes 1–4 is two- spectrum, dialdehyde 13 showed the methyl carbon attached to
fold. It would be of interest to examine the antioxidant prop- the benzene ring at d 21.63 and the aldehydic carbonyl at d
erties of indole based ethylenophanes containing electron 184.90 in addition to twelve aromatic signals. The structure of
donating functionalities and also to examine the antibacterial the dialdehyde 13 was conrmed from mass spectral and
activity of such ethylenophanes. Hence the antibacterial activity elemental analysis. Similarly, the structure of the dialdehydes
of the ethylenophanes 1–4 was assayed against Staphylococcus 13a–16 was also conrmed from spectroscopic and elemental
aureus, Bacillus cereus, Escherichia coli and Proteus vulgaris at analysis.
different concentrations. Similarly, the molecular docking of
The dialdehydes 13–16, on treatment with TiCl4 (20 equiv.)
the indole based ethylenophanes has been carried out using and Zn (40 equiv.) in dry THF under reuxing conditions for
Autodock version 4 with Lamarckian genetic algorithm as a trial 12 h, gave the directly linked ethylenoindolophanes 1, 1a, 2, 3
study to nd the mode of action of such compounds. The eth- and 4 in 17%, 18%, 15%, 20% and 15% yields respectively
ylenophanes were subjected to docking studies with the bacte- (Scheme 1). The 1H NMR spectrum of the indolophane 1
rial enzyme Topoisomerase IV (PDB ID: 3FV5). The results showed a six proton singlet at d 2.41 for the methyl group
obtained from the docking studies support the existence of attached to the benzene ring, a four proton singlet at d 6.84 for
interactions between ethylenophanes and protein. The results the olenic proton and a two proton singlet at d 6.53 for inner
are shown in 3D form using Pymol soware. Furthermore, the protons of the benzene ring in addition to other aromatic
antioxidant behavior of the ethylenophanes 1–4 was deter- protons. In the 13C NMR spectrum, the indolophane 1 showed
mined on the basis of their free radical scavenging activity by a the methyl carbon at d 21.58 in addition to thirteen other
spectroscopic assay method25 and was measured in vitro by signals in the aromatic region. Furthermore, the structure of the
using the stable 1,1-diphenyl-2-picryl hydrazyl (DPPH).26 The ethylenoindolophane 1 was conrmed from the appearance of
antioxidant activity of the ethylenophanes is comparable to that the molecular ion peak at m/z 692 in the FAB-mass spectrum.
of the standard antioxidant viz. ascorbic acid.
Similarly the structures of the ethylenoindolophanes 1a, 2–4
were also characterized thoroughly from the spectral and
analytical data. The yields of all the synthesized compounds are
given in Table 1. In general, the yields of McMurry coupling in
Results and discussion
Synthesis of indole based ethylenophanes 1–4 was achieved by aliphatic systems are always good, but in the case of aromatic
McMurry coupling of the bisindole dialdehyde moiety which, in molecules, especially in a conjugated system, the yields are low
turn, was obtained from bisindole. The bisindole was obtained due to the p-bond conjugation, which leads to polymerization
by the N-arylation of the indole with various dibromides using along with the cyclised dimer molecules. In our case the
Buchwald's procedure.27 The McMurry coupling in the current formation and removal of unwanted inorganic materials (Ti
study leads to the reductive coupling of two carbonyl groups to complex) is also responsible for low yields of the McMurry
form a macrocycle by an intermolecular dimerization rather coupling (target) product.
than an intramolecular cyclization process.
The N-arylation of 1.0 equiv. dibromides 5–8 with 2.0 equiv.
of indole in the presence of CuI, trans-1,2-diaminocyclohexane
and K3PO4 in toluene for 24 h under reuxing conditions
afforded the bisindoles 9–12 in 64–85% yields. The 1H NMR
spectrum of the bisindole 9 showed a three proton singlet at d
2.52 for the methyl group attached to the benzene ring, and a set
of two proton doublets at d 7.63 (J ¼ 8.1 Hz) and 7.69 (J ¼ 7.5 Hz)
for the protons at the C4 and C2 positions of the indole moiety
respectively, and the proton at C3 of the indole moiety appeared
as a doublet at d 7.36 in addition to the other aromatic proton.
In the 13C NMR spectrum, bisindole 9 showed the methyl
carbon attached to the benzene ring at d 21.6 and the C3 carbon
of the indole moiety at d 104.1 in addition to eleven aromatic
signals. Furthermore, the structure of the bisindole 9 was
conrmed from elemental analysis and by the appearance of
the molecular ion peak at m/z 322 in the mass spectrum (EI).
Similarly the structures of the bisindoles 9a–12 were also
conrmed from spectroscopic and elemental analysis.
Vilsmeier formylation of the bisindoles 9–12 with DMF and
ꢀ
Scheme 1 Reagents and conditions: (i) Indole (2.0 equiv.), CuI, trans-
1,2-diaminocyclohexane, K3PO4, toluene, 120 ꢀC, 24 h; 9 (82%); 9a
(85%); 10 (73%); 11 (80%); 12 (64%); (ii) POCl3 (2.2 equiv.), DMF (8.8
equiv.), 0 ꢀC to RT, 3 h; 13 (78%); 13a (72%); 14 (56%); 15 (80%); 16 (56%);
(iii) TiCl4 (20 equiv.), Zn (40 equiv.), Py (2 drops), THF, reflux, 12 h, 1
POCl3 at 0 C to RT afforded the dialdehydes 13–16 in 72% to
84% yields. The 1H NMR spectrum of the dialdehyde 13 showed
a three proton singlet at d 2.61 for the methyl group attached to
the benzene ring, and a singlet at d 10.10 for the aldehydic
protons in addition to other aromatic protons. In the 13C NMR (17%); 1a (18%); 2 (15%); 3 (20%); 4 (15%).
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Table 1 The yields of all of the synthesized compoundsa
natural compounds.30 Topoisomerase IV is an important
enzyme in the process of decatenation of multiply linked
daughter chromosomes during the end stage of DNA replica-
tion, thus helping in the division of cells. Topoisomerase IV is
one of the type II topoisomerases in bacteria, which is respon-
sible for unlinking or deactivating DNA following DNA replica-
tion. Topoisomerase IV helps in removal of interlinking in the
two newly replicated DNA strands and thus helps in the segre-
gation of chromosomes into daughter cells to complete cell
division. When the catalytic property of this enzyme is affected,
the cell division is affected, thus leading to the death of the cell.
Thus we have chosen Topoisomerase IV as a target for ethyl-
enophanes for computational study. The ethylenophanes 1–4
bind to the active pocket of the enzyme Topoisomerase IV and
thus inhibit its function, which substantiates the wet lab
experimental results. All ve ethylenophanes show good
binding energy and have hydrophobic interactions with the
amino acids in the active pockets. The ethylenophane 1a has
strong hydrophobic interactions (Fig. 2) with Topoisomerase IV
with high binding energy (ꢁ9.04 kcal molꢁ1) when compared to
other compounds. The three ethylenophanes viz. 1, 2, 3 show
good binding energy with the enzyme and compound 4 shows
the lowest binding energy and the least hydrophobic interac-
tions. Ethylenophane 1a has hydrophobic interactions with
Pro75 (A), Ile90 (A), Thr163 (A), Met74 (A), Gly73 (A), Arg132 (A),
Arg72 (A), Gly71 (A), Gly162 (A), Asp70 (A), Glu46 (A), Asp69 (A),
Ser43 (A), Asn42 (A), Val67 (A) and Val165 (A). The interactions
of the other compounds are diagrammatically represented
using Pymol soware. Even though the interactions of ethyl-
enophanes 1, 2, 3 and 4 with the enzyme show the same
hydrophobic interactions, ethylenophane 1a is considered to be
Yields (%)
N-arylindoles
Dialdehydes
Cyclophane
9 (82%)
9a (85%)
10 (73%)
11 (80%)
12 (64%)
13 (78%)
13a (72%)
14 (56%)
15 (80%)
16 (56%)
1 (17%)
1a (18%)
2 (15%)
3 (20%)
4 (15%)
a
Notes: yields are given for all new compounds aer column
purication (column chromatography on silica gel). N-aryl indoles:
CHCl3–Hexane (1 : 4, v/v) as eluent. Dialdehydes: Chloroform–
methanol (99 : 1) as eluent. Cyclophanes: CHCl3–Hexane (1 : 4, v/v) as
eluent.
Antibacterial activity
Minimal inhibitory concentration (MIC) was taken as the lowest
concentration to arrest the bacterial growth.28 The inhibition of
bacterial growth was determined visually by lack of turbidity. All
the indole based ethylenophanes were effective against the
tested bacterial pathogens, similar to the commercial antibiotic
streptomycin. Ethylenophane 1 was very active against Staphy-
lococcus aureus whereas ethylenophane 1a was very active
against Escherichia coli and Proteus vulgaris and against other
tested bacterial pathogens with good inhibition zones. Ethyl-
enophane 2 was fairly active against Gram negative bacterial
pathogens and less potent for Gram positive bacteria. Ethyl-
enophanes 3 and 4 are less active against Gram positive bacte-
rial pathogens. The antibacterial activities of ethylenophanes 1
and 1a are promising and hence they could be potential anti-
microbial drugs. Minimum inhibitory concentrations (MIC) of
the indole based ethylenophanes 1–4 are shown in Table 2.
Docking results
In order to study the biological mode of action of indole based
ethylenophanes, a docking study was performed.29 All ve
ethylenophanes were docked with the bacterial enzyme top-
oisomerase IV (PDB ID: 3FV5). We chose this target because the
topoisomerases are the target of many antibiotics and other
Table 2 The range of minimum inhibitory concentration (MIC) values
of the ethylenoindolophanes 1–4
MIC (mg mLꢁ1
)
Staphylococcus
aureus
Bacillus
cereus
Escherichia
coli
Proteus
vulgaris
Cyclophanes
1
1a
2
3
7.81
15.62
31.25
62.50
62.50
62.50
3.90
15.62
7.81
15.62
7.81
15.62
31.25
62.50
62.50
3.90
15.62
15.62
31.25
3.90
15.62
31.25
31.25
7.81
4
Streptomycin
Control
NIa
NIa
NIa
NIa
Fig. 2 Binding of ligand (Ethylenophane 1a) with the A chain amino
acids of the bacterial enzyme Topoisomerase IV (PDB ID: 3FV5).
a
NI – no inhibition.
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a good lead, based on its high binding energy and high anti- streptomycin for antibacterial and ascorbic acid for antioxidant
bacterial activity, which is further supported by experimental activity, and hence ethylenophane 1a may be a potential lead
results.
molecule for the synthesis of drugs with both antibacterial and
antioxidant activities. Synthesis of similar types of ethyl-
enophanes and antibacterial and antioxidant studies are
underway.
Antioxidant activity
The DPPH assay is an easy, rapid and sensitive method to screen
the antioxidant activity of the given test compounds.31 In the
present study the antioxidant capacity of the ethylenophanes 1–
4 were analyzed by a DPPH assay. All ve ethylenophanes
showed signicant scavenging of the free radical generated by
DPPH. The free radical scavenging property of all the ethyl-
enophanes was tested from the lowest concentration of 10 mg
mLꢁ1 up to the highest concentration of 50 mg mLꢁ1. The
scavenging activity increases with increasing concentration of
the ethylenophanes. The free radical scavenging properties of
all ethylenoindolophanes are given in Fig. 3. The ethylenophane
1a showed excellent free radical scavenging activity at all
concentrations with an IC50 value of 28 mg mLꢁ1 and it is more
effective than the other four ethylenophanes. Ascorbic acid
(vitamin C) was used as a standard to measure the free radical
scavenging by DPPH assay. The IC50 values of all compounds
and the standard are given in Fig. 4.
Acknowledgements
The authors thank CSIR, New Delhi, India, for nancial assis-
tance, DST-FIST for providing NMR specialities to the depart-
ment and SAIF, IIT Madras for mass spectral data. NV thanks
DST, CSIR, New Delhi for a fellowship.
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scavenging activity and was measured in vitro using 1,1-
diphenyl-2-picrylhydrazyl (DPPH). Stable free radical
existing in DPPH is usually utilized for the detection of the
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28 Antibacterial activity: Gram positive and Gram negative
bacterial cultures were used for the antibacterial study.
The cultures of human pathogenic bacteria Staphylococcus
aureus, Bacillus cereus, Escherichia coli and Proteus vulgaris
used in this study were obtained from the Culture
Collections of Bio control and Microbial Metabolites Lab,
Centre for Advanced Studies in Botany, University of
Madras and maintained on Nutrient Agar (NA) consisting
of the following (g Lꢁ1): beef extract 1.0; yeast extract 2.0;
peptone 5.0; NaCl 5.0; agar 15.0; distilled H2O 1 L; pH 7.2
in slants or Petriplates at room temperature (28 ꢂ 2 ꢀC).
The minimum inhibitory concentration was determined by
517 nm using a Beckman spectrophotometer. The
percentage of radical scavenging ability was determined at
different concentrations of compounds with D-ascorbic
acid as standard. DPPH absorbs at 517 nm, and its
concentration is reduced by the existence of an
antioxidant.
A dose response curve was plotted to
determine the IC50 values. The following formula is used
to calculate the percentage of radical scavenging.
Acontrol ꢁ Asample
Radical scavengingð%Þ ¼
ꢃ 100
Acontrol
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