Synthesis, antimicrobial activity, and molecular docking study of some novel cyclophanes with imino intra-annular functionality

Article abstract of DOI:10.1071/CH12326

The synthesis and structural characterisation of novel imino cyclophanes incorporating various spacer units is described. All the imino cyclophanes exhibit comparable antibacterial activity against Gram positive (Bacillus subtillus, Staphylococcus aureus)

Full text of DOI:10.1071/CH12326

CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH12326
Synthesis, Antimicrobial Activity, and Molecular
Docking Study of Some Novel Cyclophanes
with Imino Intra-Annular Functionality
A E
,
B
Perumal Rajakumar, Ramar Padmanabhan, Chandrasekaran
C
C
Ramprasath, Narayanasamy Mathivanan, Vaidhyanathan
Silambarasan, and Devadasan Velmurugan
D
D
A
Department of Organic Chemistry, University of Madras, Guindy Campus,
Chennai, 600 025, India.
B
Research and Development Centre, Orchid Chemicals and Pharmaceuticals Ltd,
Sozhanganallur, Chennai, 600 119, India.
C
Center for Advanced Studies in Botany, University of Madras, Guindy Campus,
Chennai, 600 025, India.
D
Center for Advanced Studies in Crystallography and Biophysics, University of Madras,
Guindy Campus, Chennai, 600 025, India.
E
Corresponding author. Email: perumalrajakumar@gmail.com
The synthesis and structural characterisation of novel imino cyclophanes incorporating various spacer units is
described. All the imino cyclophanes exhibit comparable antibacterial activity against Gram positive (Bacillus subtillus,
Staphylococcus aureus) and Gram negative (Escherchia coli, Klebsiella pneumonia) bacterial strains. The imino
cyclophanes also exhibit good antifungal activity against human pathogenic fungus, Candida albicans.
Manuscript received: 11 July 2012.
Manuscript accepted: 14 August 2012.
Published online: 12 September 2012.
Introduction
with additional donor atoms appended to the ring have attracted
considerable interest because of their capacity to bind and
transport metal ions.[9] Imino macrocycles have been greatly
attractive for their host–guest complexes and molecular recog-
nition.[10–12] Macrocycles containing hetero atoms are valuable
compounds with a broad spectrum of pharmacological activi-
ties.[13] Macrocyclic Schiff bases exhibit biological activity as
antibiotics, antiviral, antitumour, antifungal, antibacterial, and
anticancer agents because of their specific structure.[14] Schiff
bases have been reported as potential antimicrobial agents.[15]
Recently, the dicationic acridinophanes,[16] quinolino-
phanes,[17] carbazolophanes,[18] and imidazolophanes[19] using
various spacers like pyridine, m-terphenyl, and chiral binaphthol
have been reported from our laboratory. In a continuation of our
on-going investigation into the synthesis of various bioactive
cyclophanes, attempts were made to synthesise novel imino
cyclophanes by coupling between suitable bis-aldehydes and bis
(aminomethyl)m-terphenyl and then to study the bioactivity of
the synthesised cyclophanes. However, to the best of our
knowledge, such imino cyclophanes with bis(aminomethyl)m-
terphenyl as a spacer have not been reported in the literature.
Hence, we report herein the synthesis, antibacterial, and anti-
fungal activity of imino cyclophanes 1–7 (Fig. 1) from the
corresponding dialdehyde precursors 8, 9a, 9b, 9c, 10a, 10b, 11,
12, and 13.
Most antibacterials are semisynthetic modifications of various
natural compounds.[1] Some common b-lactam antibacterials
are penicillins, cephalosporins, penems, and carbapenems.[2]
Aminoglycosides are the antibacterials isolated from living
organisms whereas other antibacterials, such as sulfonamides,
quinolones, and oxazolidinones are obtained by chemical syn-
thesis. The development of bacterial resistance to existing
potent antimicrobial drugs is a major concern for managing
infections.[3] One of the concerns is severe infections caused by
multi drug-resistant (superbug) Gram-positive pathogens,
which result in high mortality rates especially in hospital set-
tings. The individual organisms responsible include methicillin-
resistant Staphylococcus aureus (MRSA), vancomycin resistant
Enterococcus faecalis (VRE), penicillin-resistant Streptococcus
pneumonia, and fluconazole resistant Candida albicans.[4] This
necessitates the continuing research for new classes of anti-
bacterial drugs.[5] There are several antibiotics effective against
these organisms but these organisms easily acquire resistance
against them. The development of resistance is inherent in the
mode of action of all antibiotics and hence poses challenges in
the development of new antimicrobial agents to overcome it.[6]
Some bacterial strains have been found to be resistant to all
currently available antibacterial agents.[7] Several anti-
microbials are well documented in the literature.[8] Macrocycles
Journal compilation Ó CSIRO 2012
www.publish.csiro.au/journals/ajc

B
P. Rajakumar et al.
N
N
N
N
N
N
N
N
N
N
3a ꢀ ortho
3b ꢀ meta
3c ꢀ para
1
2
3a, 3b, 3c
NO2
N
O
O
O
O
O
O
N
N
N
N
N
N
4a ꢀ ortho
4b ꢀ meta
5
6
4, 4a
O
O
N
N
7
Fig. 1. Structure of imino cyclophanes 1–7.
Results and Discussion
protons appeared in the region d 7.28–7.81. The mass spectrum
of 1 showed the molecular ion peak [M þ H]þ at m/z 773.4. The
FT-IR spectrum showed the C¼N stretching frequency at
1639 cmꢀ1 for the imino cyclophane 1. The structure of the
cyclophane imine 1 was further confirmed from spectroscopic
and analytical data. In order to test the synthetic utility of
diamine 14 for the synthesis of imino cyclophanes, 1.0 equiv. of
m-terphenyldiamine 14[20] was condensed with 1.0 equiv. of
m-terphenyldialdehyde 8 in ethanol under high dilution
Reaction of 1.0 equiv. of m-xylylene diamine and 1.0 equiv. of
m-terphenyldialdehyde 8 in ethanol under high dilution condi-
tions at room temperature resulted in the formation of cyclo-
phane tetraimine 1 in 85 % yield (Scheme 1). The product slowly
precipitated out from the reaction mixture during the course of
the reaction. The 1H NMR spectrum of tetraimino cyclophane 1
displayed the N-methylene protons as a singlet at d 4.85 and the
N¼CH protons as a singlet at d 8.40. The rest of the aromatic

Cyclophanes with Imino Intra-Annular Functionality
C
conditions at room temperature. The reaction afforded the for-
mation of imino cyclophane 2 in 70 % yield (Scheme 1). The
structure of the cyclophane imine 2 has been confirmed from
spectroscopic and analytical data.
dilution conditions at room temperature (Scheme 3). During the
course of the reaction, the product slowly crystallized out from
the reaction mixture. The 1H NMR spectrum of the tetraimine
cyclophane 3b displayed the N-methylene protons as a singlet at
d 4.87 and the N¼CH protons appeared as a singlet at d 8.45. The
rest of the aromatic protons appeared in the region d 7.38–7.86.
The mass spectrum of 3b showed the molecular ion peak
[M þ H]þ at m/z 773.4. The FT-IR spectrum showed the C¼N
stretching frequency at 1642 cmꢀ1 for the imino cyclophane 3b.
The structure of imino cyclophane 3b was further confirmed
from spectroscopic and analytical data. Similarly the structures
of the imino cyclophanes 3a and 3c were also confirmed from
spectroscopic and analytical data.
In order to test the synthetic utility of the bis-oxyaldehydes
10a, 10b, 11, 12, and 13 for the synthesis of bis-oxyimino
cyclophanes 4a, 4b, 5, 6, and 7, 1.0 equiv. of m-terphenyldiamine
14 was condensed with 1.0 equiv. of the bis-oxyaldehydes 10a,
10b, 11, 12, and 13 in ethanol at room temperature under high
dilution conditions. The reaction afforded the bis-oxy cyclo-
phane diimines 4a, 4b, 5, 6, and 7 in 83, 87, 82, 86, and 71 %
yields (Scheme 3). The 1H NMR spectrum of tetraimine
Bis-oxyaldehydes 10a, 10b, 11, 12, and 13 (Fig. 2) required
for the synthesis of imino cyclophanes 4a, 4b, 5, 6, and 7 were
obtained in 86, 81, 93, 90, and 85 % yields after purification
by column chromatography by the reaction of 1.0 equiv. of
o-xylylene dibromide, m-xylylene dibromide, 1-nitro-3,5-bis
(bromomethyl)benzene, 2,6-(bisbromomethyl)pyridine, and
m-terphenyl dibromide with 2.1 equiv. of m-hydroxybenzalde-
hyde in the presence of 10.1 equiv. of potassium carbonate in
dimethylformamide (DMF) at 608C for 48 h (Scheme 2).[21] In
the 1H NMR spectrum of precyclophane 10b the O-methylene
protons appeared as a singlet at d 5.15 and the aldehyde protons
as a singlet at d 9.97. The rest of the aromatic protons appeared in
the region d 7.24–7.54. The mass spectrum of 10b showed the
molecular ion peak [M þ H]þ at m/z 347.1. The FT-IR spectrum
showed the carbonyl stretching frequency at 1695 cmꢀ1 for the
precyclophane 10b. The structure of dialdehyde 10b was further
confirmed from spectroscopic and analytical data. Similarly the
structures of the precyclophanes 10a, 11, 12, and 13 were also
confirmed from spectroscopic and analytical data.
O2N
11
10a, 10b
The synthetic pathway leading to imino cyclophane 3a, 3b,
4a, 4b, 5, 6, and 7 (Fig. 1) is outlined in Scheme 3. Cyclophane
tetraimines 3a, 3b, and 3c were prepared in ,81, 80, and 82 %
yields by the reaction of 1.0 equiv. of m-terphenyldiamine 14
and 1.0 equiv. of dialdehyde 9a, 9b, and 9c in ethanol under high
Br
Br
(i)
(i)
Br
Br
OH
CHO
Br
Br
OHC
CHO
N
8
(i)
(ii)
(i)
(i)
Br
Br
1
2
12
13
Scheme 1. Reagents and conditions: (i) m-xylylene diamine, ethanol, rt,
48 h, 1 (85 %) and (ii) m-terphenyl diamine (14), ethanol, rt, 48 h, 2 (70 %).
Scheme 2. Reagents and conditions: (i) K2CO3, DMF, 608C, 48 h, 10a
(86 %), 10b (81 %), 11 (93 %), 12 (90 %), and 13 (85 %).
O
O
CHO
OHC
9a, 9b, 9c
10a, 10b
OHC
CHO
CHO
CHO
8
9a ꢀ ortho
9b ꢀ meta
9c ꢀ para
10a ꢀ ortho
10b ꢀ meta
NO2
N
O
O
O
O
O
O
CHO
CHO
CHO
CHO
CHO
CHO
11
12
13
Fig. 2. Structure of dialdehydes 8–13.

D
P. Rajakumar et al.
cyclophane 4b displayed the N-methylene protons as a singlet at
d 4.85, O-methylene protons as a singlet at d 5.10, and the
N¼CH protons appeared as a singlet at d 8.37. The rest of the
aromatic protons appeared in the region d 7.05–7.77. The mass
spectrum of 4b showed the molecular ion peak [M þ H]þ at m/z
599.2. The FT-IR spectrum showed the C¼N stretching fre-
quency at 1642 cmꢀ1 for the imino cyclophane 4b. The structure
of imino cyclophane 4b was further confirmed from spectro-
scopic and analytical data. Similarly the structures of the imino
cyclophanes 4a, 5, 6, and 7 were also confirmed from spectro-
scopic and analytical data.
The in vitro antimicrobial activity of the imino cyclophanes
was determined by an agar well diffusion assay against the Gram
positive human pathogenic bacteria Bacillus subtilis (ATCC
6633) and Staphylococcus aureus (ATCC 12600), the Gram
negative bacteria Escherichia coli (ATCC 11775) and Klebsiel-
la pnuemonia (ATCC 13883), and the human fungal pathogen
Candida albicans (obtained from the culture collections of
Biocontrol and Microbial Metabolites Laboratory, Centre for
Advanced Studies in Botany, University of Madras, Chennai,
India). The primary screening of the antimicrobial activity of the
imino cyclophanes was carried out by an agar well diffusion
method[22] using Mueller Hinton agar medium. The minimum
inhibitory concentration (MIC) for the all the imino cyclophanes
1–7 against the same microorganisms was determined using a
microdilution susceptibility method.[22] Vancomycin and flu-
conazole were used as standards for antibacterial and antifungal
activity, respectively. The imino cyclophanes 1–7 were tested
for their in vitro antimicrobial activity at three different con-
centrations of 50, 75, and 100 mg mLꢀ1. The results revealed that
the majority of the imino cyclophanes showed varying degrees
of inhibition against the tested human pathogenic microorgan-
isms. The results also revealed that, in general, the inhibitory
activity against the Gram-negative bacteria was higher than the
Gram-positive bacteria. All the imino cyclophanes 2, 3a, 3b, 3c,
4a, 4b, 6, and 7 showed significant antimicrobial activity against
the test pathogens. Furthermore, all the imino cyclophanes
showed a good inhibitory effect against the fluconazole resistant
C. albicans (Fig. 3).
The MIC values were determined as the lowest concentration
that completely inhibited visible growth of the microorganisms
(Table 1). The relationship between the structure of the imino
cyclophanes and antimicrobial activity revealed that the imino
cyclophane 6 exhibited better antibacterial and antifungal activity
against the test pathogens Bacillus subtillus, Staphylococcus
aureus, Escherchia coli, and Klebsiella pneumonia and the MIC
values are comparable to that of the standard drug vancomycin. In
fact, fluconazole did not show any inhibitory activity against the
tested pathogen Candida albicans. However all the imino cyclo-
phanes showed remarkable activity against Candida albicans.
All the imino cyclophanes were evaluated for their antifungal
activity against C. albicans and their MIC was determined. All
the tested cyclophanes showed good inhibitory effect against the
fluconazole resistant C. albicans (Table 1 and Fig. 3). In order to
ensure that the solvent had no effect on bacterial and fungal
growth a control test was performed with DMSO (dimethyl
sulfoxide) which revealed no inhibiting activity.
3a ꢀ ortho
4a ꢀ ortho
3b ꢀ meta
4b ꢀ meta
3c ꢀ para
4a, 4b
3a, 3b, 3c
(i)
9a, 9b, 9c
10a, 10b
(i)
(i)
(i)
To study the mechanism of antimicrobial activity of the
imino cyclophanes 1–7, docking studies were carried out using a
GLIDE application. Imino cyclophanes were tested against the
enzyme DNA gyrase B (PDB ID: 2Y3P). These studies revealed
that the imino cyclophanes 1–7 showed good inhibition of DNA
gyrase B enzyme and also showed strong hydrogen bonding
with the hydrophophic residues of side chain A of the enzyme
dimer DNA gyrase B. In general, the binding of ligand (com-
pounds or drugs) towards the enzyme DNA gyrase B affects its
catalytic activity. Prior to unwinding, the DNA binds to the side
chain A of DNA gyrase B. As the DNA gyrase B is necessary for
the DNA unwinding, the binding of the imino cyclophanes
5
7
13
11
14
NH2
NH2
(i)
12
6
Scheme 3. Reagents and conditions: (i) ethanol, rt, 48 h, 3 (81 %), 3a
(80 %) and 3b (82 %), 4 (83 %), 4a (87 %), 5 (82 %), 6 (86 %), and 7 (71 %).
MIC of Iminocyclophanes [µg mLꢁ1
]
3.0
2.5
2.0
1.5
1.0
0.5
0
Human pathogens
B. subtilus K. pneumonia
E. coli
S. aures
C. albicans
Fig. 3. Minimum inhibitory concentration (MIC) of imino cyclophanes 1–7.

Cyclophanes with Imino Intra-Annular Functionality
E
Table 1. Antimicrobial activity (minimal inhibitory concentration (MIC) in lg mL21) of imino cyclophanes 1 7 against human
pathogens
]
NA: not applicable; –: no inhibition
Imino cyclophanes
B. subtilus
K. pneumonia
E. coli
S. aureus
C. albicans
1
1.25
1.25
1.25
1.25
0.75
1.00
1.25
0.75
0.31
0.75
0.39
NA
1.25
1.25
1.25
1.25
1.50
1.00
2.50
0.75
0.63
0.75
0.19
NA
1.25
2.50
1.25
2.50
1.50
1.00
2.50
1.50
0.16
1.50
0.02
NA
2.50
2.50
2.50
2.50
0.75
1.00
10.00
0.75
1.25
1.50
0.002
NA
2.50
1.25
2.50
1.25
1.50
0.50
2.50
1.50
0.63
1.50
NA
–
2
3a
3b
3c
4a
4b
5
6
7
Vancomycin
Fluconazole
Control
–
–
–
–
–
Thr 88(A)
Val 44(A)
Arg 91(A)
ND1
Gln 267(A)
CE1
CB
CG
O
Ser 172(A)
3.16
NE2
C1
CA
C
C3
N1
C6
C2
CD2
C4
C7
C39
N
C37
C5
His 45(A)
O2
C9
CE
C38
N
NZ
C41
C40
O1
C8 C11
CG
2.90
CA
LyS 42(A)
O
NH1
C13
C36
CD
N3
CB
NE
C10
CZ
C
CG
C35
C14
NH2
CB
CD
C30
C12
3.17
Val 176(A)
C31
C15
Arg 32(A)
C32
CA
C
C33
N2
C34
N
O
C20
C18
C16
C24
C29
C25
C26
C19
C23
C22
C21
C17
C27
C28
Ala 175(A)
Fig. 4. Ligplot results: Binding of ligand–imino cyclophane 6 on A-chain amino acids of DNA gyrase B (PDB ID: 2Y3P) with three hydrogen bonds and
seven hydrophobic interactions.
inhibits the interaction of the DNA enzyme complex.[23] Imino
cyclophanes were docked at the active site of the receptor. The
imino cyclophane 6 showed strong hydrogen bond interactions
with Lys42 (A), Arg32 (A), and His45 (A) and hydrophophic
interactions with Val44 (A), Thr88 (A), Ser172 (A), Arg91 (A),
and Gln116 (A) (Fig. 4). The Glide energy (binding energy) is
ꢀ46.64 kcal molꢀ1 and the glide score (docking score) is ꢀ3.26
(Table 2 and Fig. 4). The imino cyclophane 4b also showed

F
P. Rajakumar et al.
Table 2. Interaction of imino cyclophanes with the target enzyme DNA
washed with water (2 ꢁ 250 mL) and then dried over anhydrous
sodium sulfate. Removal of the ether gave the corresponding
dialdehyde as a crude material, which was purified by column
chromatography (SiO2).
gyrase B
D: donor; A: acceptor
Imino
Glide score Glide energy
[kcal molꢀ1
Hydrogen bond
3,30-(1,2-Phenylenebis(methylene))bis-oxy)
cyclophane
]
˚
Distance [A]
DꢂꢂꢂA
dibenzaldehyde (10a)
3a
4b
ꢀ5.02
ꢀ4.04
ꢀ57.42
ꢀ49.74
–
–
Yield 86 %; mp 798C; nmax (KBr)/cmꢀ1 1696, 1680; dH
(400 MHz, CDCl3) 9.98 (s, 2H), 7.51–7.55 (m, 2H), 7.46–7.48
(m, 5H), 7.39–7.44(m, 3 H), 7.21–7.24 (m, 2H), 5.24 (s, 4H); dC
(100 MHz, CDCl3) 192.0, 159.1, 137.9, 134.6, 130.2, 129.3,
128.8, 123.9, 122.1, 113.2, 68.3; MS (ESI) m/z: 364.1 ([M þ
NH4]þ); Anal. Calc. for C22H18O4: C 76.29, H 5.24. Found:
C 76.36, H 5.28 %.
His45[N–HꢂꢂꢂN]
Arg91[O–HꢂꢂꢂN]
Lys42[N–HꢂꢂꢂO]
Arg32[N–HꢂꢂꢂN]
His45[N–HꢂꢂꢂO]
His45[N–HꢂꢂꢂN]
3.14
2.94
2.90
3.17
3.16
3.23
6
7
ꢀ3.26
ꢀ46.64
ꢀ6.27
ꢀ70.22
3,30-(1,3-Phenylenebis(methylene))bis-oxy)
dibenzaldehyde (10b)
strong hydrogen bonding with the residues His45 (A) and
Arg91 (A) and hydrophophic interactions with Arg91 (A),
Gln267 (A), Thr88 (A), and Asp87 (A). The Glide energy
(binding energy) is ꢀ49.74 kcal molꢀ1 and the glide score
(docking score) is ꢀ4.04.
Yield 91 %; mp 758C; nmax (KBr)/cmꢀ1 1695, 1681; dH
(400 MHz, CDCl3) 9.97 (s, 2H), 7.54 (br s, 1H), 7.48 (d, 6H,
J 1.8), 7.42–7.46 (m, 3H), 7.24–7.27 (m, 2H), 5.15 (s, 4H); dC
(100 MHz, CDCl3) 192.0, 159.2, 137.9, 136.9, 130.2, 129.1,
127.3, 126.5, 123.8, 122.2, 113.2, 70.0; MS (ESI) m/z: 347.1
([M þ H]þ); Anal. Calc. for C22H18O4: C 76.29, H 5.24. Found:
C 76.35, H 5.28 %.
Conclusion
A series of novel imino cyclophanes were synthesised and
assayed for their antibacterial activity against the Gram positive
human pathogenic bacteria B. subtilis and S. aureus, the Gram
negative bacteria E. coli and K. pneumonia, and the humanfungal
pathogen C. albicans. Vancomycin and fluconazole were used as
standards for antibacterial and antifungal activity, respectively.
The imino cyclophane 6 showed good antibacterial activity with
a MIC of 0.16–1.25 mg mLꢀ1 against the tested bacterial strains
and exhibited the most potent inhibitory activity against E. coli
with 0.16 mg mLꢀ1 and B. subtilus with 0.31 mg mLꢀ1. The
antibacterial assay results also showed that the imino cyclophane
6 could be developed as a potential antmicobial agent against
B. subtilus, E. coli, K. pneumonia, and C. albicans.
3,30-(5-Nitro-1,3-phenylenebis(methylene))bis-oxy)
dibenzaldehyde (11)
Yield 93 %; mp 1408C; nmax (KBr)/cmꢀ1 1698, 1684; dH
(400 MHz, CDCl3) 9.99 (s, 2H), 8.32 (d, 2H, J 0.6), 7.87 (s, 1H),
7.48–7.55 (m, 6H), 7.27–7.30 (m, 2H), 5.25 (s, 4H); dC
(100 MHz, CDCl3) 191.8, 158.7, 148.8, 139.1, 137.9, 131.5,
130.4, 124.5, 122.1, 121.7, 112.8, 68.7; MS (ESI) m/z: 409.0
([M þ NH4]þ); Anal. Calc. for C22H17NO6: C 67.51, H 4.38,
N 3.58. Found: C 67.58, H 4.42, N 3.61 %.
3,30-(Pyridine-2,6-diylbis(methylene))bis-oxy)
dibenzaldehyde (12)
Experimental
General
Yield 90 %; mp 1468C; nmax (KBr)/cmꢀ1 1690, 1682; dH
(400 MHz, CDCl3) 9.98 (s, 2H), 7.76–7.80 (m, 1H), 7.46–7.51
(m, 8H), 7.25–7.30 (m, 2H), 5.28 (s, 4H); dC (100 MHz, CDCl3)
191.9, 159.0, 156.3, 137.9, 137.8, 130.3, 123.7, 121.8, 120.5,
113.9, 70.8; MS (ESI) m/z: 348.1 ([M þ H]þ); Anal. Calc. for
C21H17NO4: C 72.61, H 4.93, N 4.03. Found: C 72.68; H 4.97,
N 4.07 %.
All the reagents and solvents employed were of the best grade
available and were used without further purification. The
melting points were determined by using a Metler Toledo
melting point apparatus by open capillary tube method and were
uncorrected. Spectroscopic data were recorded by the following
instruments: UV/Vis: Shimadzu 2550 spectrophotometer. IR:
Perkin-Elmer series 2000 FTIR spectrophotometer. NMR:
Bruker Avance 400 MHz. Mass spectrometry: electrosray
ionisation (ESI) PerkinElmer Sciex, API 3000 mass spectrom-
eter and fast atom bombardment (FAB) mass spectra Jeol SX
102/DA-6000 mass spectrometer. The elemental analysis for the
compounds was carried out using an Elementar Vario EL III
elemental analyzer. Pre-coated silica gel plates from Merck
were used for TLC. Column chromatography was carried out
using silica gel (100–200 mesh) purchased from ACME.
3,30-(1,10:30,10-Terphenyl-4,40-dylbis(methylene))bis
(oxy)dibenzaldehyde (13)
Yield 85 %; mp 1068C; nmax (KBr)/cmꢀ1 1697, 1682; dH
(400 MHz, CDCl3) 9.98 (s, 2H), 7.81 (br s, 1H), 7.68 (d, 4H, J
8.2), 7.56–7.60 (m, 2H), 7.53–7.55 (m, 4H), 7.45–7.51 (m, 7H),
7.25–7.30 (m, 2H), 5.18 (s, 4H); dC (100 MHz, CDCl3) 192.1,
159.3, 141.3, 141.1, 137.9, 135.6, 130.2, 129.4, 128.1, 127.6,
126.3, 126.1, 123.8, 122.22, 113.3, 70.0; MS (ESI) m/z: 516.3
([M þ NH4]þ); Anal. Calc. for C34H26O4: C 81.91, H 5.26.
Found: C 81.98, H 5.31 %.
General Procedure for the Synthesis of Bis-Oxy
Aldehydes (10–13)
A mixture of dibromide (10 mmol), m-hydroxybenzaldehyde
(21 mmol), and potassium carbonate (101 mmol) in anhydrous
DMF (100 mL) was stirred under nitrogen for 48 h at 608C. The
reaction mixture was poured into water (600 mL) and stirred.
The resulting precipitate was filtered off, washed with water,
and dissolved in diethyl ether (300 mL). The organic layer was
General Procedure for the Synthesis of Imino
Cyclophanes (1–7)
A solution of diamine (15 mmol) in ethanol (400 mL) and a
solution of dialdehyde (15 mmol) in ethanol (400 mL) were
simultaneously added dropwise to a well stirred volume of

Cyclophanes with Imino Intra-Annular Functionality
G
ethanol (800 mL) for 6–8 h. After the addition was complete the
reaction mixture was stirred for another 48 h. The precipitated
solid was filtered off, washed with ethanol, and dried.
(m, 8H), 7.32–7.44 (m, 7H), 7.28–7.30 (m, 3H), 7.02–7.06 (m,
2H), 5.20 (s, 4H), 4.85 (s, 4H); dC (100 MHz, CDCl3) 162.1,
159.1, 141.7, 138.6, 137.8, 135.2, 130.4, 129.9, 129.3, 128.7,
127.6, 126.2, 126.1, 126.0, 122.2, 118.3, 113.3, 70.0, 64.8; MS
(ESI) m/z: 599.2 ([M þ H]þ), Anal. Calc. for C42H34N2O2:
C 84.25, H 5.72, N 4.68. Found: C 84.57, H 5.78, N 4.76 %.
3,9,13,19-Tetraimine-1,6,11,16(1,3),5,7,15,17(1,4)-
octabenzenacycloeicosanaphane (1)
Yield 85 %; mp 1758C; nmax (KBr)/cmꢀ1 1641, 1605; dH
(400 MHz, CDCl3) 8.40 (s, 4H), 7.80 (8H, d, J 8.2), 7.74 (br s,
2H), 7.61 (d, 8H, J 8.2), 7.56 (br s, 2H), 7.54 (d, 2H, J 1.6), 7.46–
7.48(m, 2H), 7.34–7.36 (m, 8H), 4.85 (s, 8H); dC (100 MHz,
CDCl3) 161.6, 143.0, 138.6, 137.2, 135.5, 129.6, 128.7, 128.2,
126.6, 126.0, 125.6, 124.5, 65.3; MS (ESI) m/z: 773.4
([M þ H]þ), Anal. Calc. for C56H44N4: C 87.01, H 5.74, N
7.25. Found: C 87.25, H 5.77, N 7.31 %.
3,9-Diimine-12,16-dioxa-1,6,11,14(1,3),5,7(1,4)-
hexabenzenacyclohexadecanaphane (4b)
Yield 87 %; mp 1598C; nmax (KBr)/cmꢀ1 1642, 1579; dH
(400 MHz, CDCl3) 8.37 (s, 2H), 7.77 (s, 1H), 7.60 (d, 4H, J 6.4),
7.46–7.52 (m, 5H), 7.39–7.41 (m, 8H), 7.32 (br s, 4H), 7.05 (br s,
2H), 5.10 (s, 4H), 4.85 (s, 4H); dC (100 MHz, CDCl3) 162.1,
159.2, 141.7, 140.1, 138.6, 137.8, 137.4, 129.8, 128.6, 127.5,
127.3, 126.7, 126.1, 126.0, 122.2, 118.3, 115.0, 113.0, 70.0, 64.8;
MS (ESI) m/z: 599.2 ([Mþ H]þ), Anal. Calc. for C42H34N2O2:
C 84.25, H 5.72, N 4.68. Found: C 84.59, H 5.79, N 4.75 %.
4,9-Dimine-1,7(1,3),2,6,8,12(1,4)-
hexabenzenacyclododecanaphane (2)
Yield 75%; mp 1828C; nmax (KBr)/cmꢀ1 1638, 1601; dH
(400 MHz, CDCl3) 8.43 (s, 2H), 7.79 (t, 2H, J 1.7), 7.62 (d, 8H,
d, J 8.2), 7.58 (t, 2H, J 1.7), 7.56 (d, 2H, J 1.5), 7.50–7.52 (m, 2H),
7.41 (d, 8H, J 8.2), 4.86 (s, 4H); dC (100 MHz, CDCl3) 165.6,
143.3, 137.9, 137.3, 135.4, 129.9, 129.6, 128.5, 128.2, 127.2, 126.4,
65.0; MS (ESI) m/z: 539.3 ([M þ H]þ), Anal. Calc. for C40H30N2:
C 89.19, H 5.61, N 5.20. Found: C 89.45, H 5.67, N 5.27 %.
3,9-Diimine-12,16-dioxa-1,6,11(1,3),5,7(1,4)-
pentaabenzena-14(5-nitro-1,3)
benzenacyclohexadecanaphane (5)
Yield 82 %; mp 1578C; nmax (KBr)/cmꢀ1 1643, 1581; dH
(400 MHz, CDCl3) 8.39 (s, 2H), 8.28 (s, 2H), 7.75 (s, 1H), 7.58–
7.66 (m, 4H), 7.49–7.52 (m, 4H), 7.45 (m, 2H), 7.37–7.41 (m,
4H), 7.30–7.35 (m, 4H), 7.03–7.15 (m, 2H), 5.20 (s, 4H), 4.85 (s,
4H); dC (100 MHz, CDCl3) 162.3, 161.1, 158.8, 142.1, 140.7,
138.4, 138.0, 129.4, 129.1, 128.7, 128.0, 127.6, 126.2, 125.5,
122.9, 121.7, 118.5, 111.8, 69.1, 64.8; MS (ESI) m/z: 644.2
([M þ H]þ), Anal. Calc. for C42H33N3O4: C 78.36, H 5.17,
N 6.53. Found: C 78.62, H 5.26, N 6.03 %.
3,9,13,19-Tetraimine-1,11(1,2),6,16(1,3),5,7,15,
17(1,4)-octabenzenacycloeicosanaphane (3a)
Yield 81 %; mp 1798C; nmax (KBr)/cmꢀ1 1641, 1601; dH
(400 MHz, CDCl3) 8.45 (s, 4H), 8.17–8.29 (m, 2H), 7.75–7.89
(m, 6H), 7.49–7.61 (m, 14H), 7.38–7.48 (m, 12H), 4.86 (s, 8H);
dC (100 MHz, CDCl3) 161.3, 140.4, 137.7, 137.2, 130.2, 129.7,
129.4, 129.2, 127.6, 126.6, 126.0, 64.7; MS (ESI) m/z: 773.4
([M þ H]þ), Anal. Calc. for C56H44N4: C 87.01, H 5.74, N 7.25.
Found: C 87.27, H 5.80, N 7.30 %.
3,9-Diimine-12,16-dioxa-1,6,11(1,3),5,7(1,4)-
pentaabenzena-14(1,3)pyridinacyclo-
hexadecanaphane (6)
Yield 86 %; mp 1558C; nmax (KBr)/cmꢀ1 1643, 1581; dH
(400 MHz, CDCl3) 8.36 (s, 2H), 7.64–7.73 (s, 3H), 7.59–7.64
(m, 4H), 7.51–7.55 (m, 4H), 7.42–7.47 (m, 3H), 7.47–7.40 (m,
3H), 7.24–7.43 (m, 4H), 7.05 (d, 2H, J 7.8), 5.23 (s, 4H), 4.84 (s,
4H); dC (100 MHz, CDCl3) 162.9, 161.7, 156.4, 141.8, 138.8,
138.2, 137.5, 130.9, 130.8, 130.3, 128.7, 128.1, 127.9, 127.3,
122.6, 122.2, 111.7, 68.9, 64.1; MS (ESI) m/z: 600.4 ([M þ
H]þ), Anal. Calc. for C41H33N3O2: C 82.11, H 5.55, N 7.01.
Found: C 82.42, H 5.63, N 7.12 %.
3,9,13,19-Tetraimine-1,6,11,16(1,3),5,7,15,17(1,4)-
octabenzenacycloeicosanaphane (3b)
Yield 80 %; mp 1768C; nmax (KBr)/cmꢀ1 1596, 1516; dH
(400 MHz, CDCl3) 8.45 (s, 4H), 7.85 (d, 2H, J 1.8), 7.84 (d, 2H,
J 1.6), 7.75–7.79 (m, 2H), 7.60 (d, 10H, J 8.2), 7.57–7.56 (m,
4H), 7.43–7.51 (m, 4H), 7.39 (d, 8H, J 8.2), 4.86 (s, 8H); dC
(100 MHz, CDCl3) 161.6, 141.7, 140.1, 138.5, 136.7, 130.5,
130.0, 129.4, 129.1, 128.7, 127.6, 126.2, 64.9; MS (ESI) m/z:
773.4 ([M þ H]þ), Anal. Calc. for C56H44N4: C 70.36, H 5.33,
N 6.00. Found: C 70.50, H 5.41, N 6.08 %.
3,9-Diimine-12,18-dioxa-1,6,11,15(1,3),5,7,14,16(1,4)-
octabenzenacyclooctadecanaphane (7)
3,9,13,19-Tetraimine-6,16(1,3),1,5,7,11,15,17(1,4)-
Yield 71 %; mp 1548C; nmax (KBr)/cmꢀ1 1643, 1581; dH
(400 MHz, CDCl3) 8.39 (s, 2H), 7.74–7.77 (m, 2H), 7.59–7.64
(m, 8H), 7.46–7.55 (m, 12H), 7.41–7.42 (m, 4H), 7.28–7.35 (m,
4H), 7.02–7.07 (m, 2H), 5.12 (s, 4H), 4.86 (s, 4H); dC (100 MHz,
CDCl3) 163.7, 160.7, 140.8, 139.7, 137.8, 137.0, 135.6, 129.8,
129.7, 129.5, 128.1, 127.8, 127.6, 126.8, 126.2, 121.5, 116.6,
112.2, 68.6, 64.3; MS (ESI) m/z: 751.3 ([M þ H]þ), Anal. Calc.
for C54H42N2O2: C 86.37, H 5.64, N 3.73. Found: C 86.72,
H 5.73, N 3.82 %.
octabenzenacycloeicosanaphane (3c)
Yield 92 %; mp 2078C; nmax (KBr)/cmꢀ1 1642, 1600; dH
(400 MHz, CDCl3) 8.45 (s, 4H), 7.85 (2H, J 1.8), 7.84 (2H, J
1.6), 7.75 (t, 2H, J 1.4), 7.60 (d, 10H, J 8.2), 7.57 (t, 2H, J 1.4),
7.56 (d, 2H, J 1.6), 7.43–7.51 (m, 4H), 7.39 (d, 8H, J 8.2), 4.86
(s, 8H); dC (100 MHz, CDCl3) 162.6, 138.4, 137.6, 136.7, 129.6,
129.4, 129.1, 127.5, 126.6, 126.0, 65.1; MS (ESI) m/z: 773.4
([M þ H]þ), Anal. Calc. for C56H44N4: C 87.01, H 5.74, N 7.25.
Found: C 87.29, H 5.81, N 7.31 %.
Antimicrobial Activity
3,9-Diimine-12,16-dioxa-1,6,11(1,3),5,7(1,4),14(1,2)-
hexabenzenacyclohexadecanaphane (4a)
Agar Well Diffusion Assay
Yield 83 %; mp 1548C; nmax (KBr)/cmꢀ1 1641, 1579; dH
(400 MHz, CDCl3) 8.33 (s, 2H), 7.55–7.57 (m, 4H), 7.45–7.51
The antimicrobial activity of imino cyclophanes 1–7 against
human pathogenic bacteria was done by an agar well diffusion

H
P. Rajakumar et al.
assay. The human pathogenic Gram-positive bacteria B. subtilis
and S. aureus and Gram-negative bacteria E. coli and K. pneu-
monia were obtained from American Type Culture Collection.
The human pathogens were inoculated into 5 mL of sterile
Muller–Hinton broth, and incubated at 378C for 8 h. The cultures
were swabbed on the surface of sterile nutrient agar plates using
a sterile cotton swab. Agar wells were prepared with the help of a
sterilized cork borer (9 mm diameter). All the imino cyclo-
phanes 1–7 were dissolved in 10 % DMSO which did not affect
the microorganism’s growth, according to our control experi-
ments. Using a micropipette, 50 mL of different concentrations
of imino cyclophanes (50, 75, and 100 mL) were added to each
well in the plate. Commercial antibiotic, vancomycin was used
as a positive reference standard to compare the efficiency of the
test cyclophanes. The plates were incubated at 378C for 24 h.
The antibacterial activities was determined by the zone of
inhibition and are shown in Table S1 in the Supplementary
Material.
Molecular Docking Studies
The synthesised imino cyclophanes were subjected to a
molecular docking study using the GLIDE application of the
Schro¨dinger package, LLC (New York) version 9.0 and Induced
Fit Docking (IFD): one of the modules in the package was
employed as a primary docking engine.[24,25] A hierarchical
search protocol was utilised by the docking algorithm of the IFD
program. The scoring function is called a GLIDE score, for
computing the binding affinity is an extension of an empirically
based Chem-Score function of Eldridge et al.[26] Optimized
potential for liquid simulation (OPLS) – a molecular mechanics
potential energy function, was used throughout the calcula-
tions.[27] The extra precision mode of GLIDE, which has higher
penalties for unfavourable and unphysical interactions, was used
for docking. Computations were carried out on a Linux system
with a Cent OS 5 computer platform. Interaction pictures were
generated using Ligplot.[28] Imino cyclophanes 3a, 4b, 6, and 7
were analysed for their mechanism of antimicrobial action using
the above method. The estimated binding energy for each
molecule was compared along with the crucial hydrogen and
hydrophobic bonds between the protein and ligand to substan-
tiate the experimental results. The interactions were graphically
represented in 2D using the Ligplot software (Figs S1–S3 in the
Supplementary Material).
Antifungal Activity
The test human fungal pathogen C. albicans was maintained in
Sabouraud’s dextrose agar (SDA). The C. albicans was inocu-
lated in Sabouraud’s dextrose broth and incubated at 378C for
8 h. The pathogen was swabbed on the sterile SDA plates using a
sterile swab and wells were made using a sterile cork borer
(9 mm diameter). The imino cyclophanes 1–7 were tested for
their antifungal activity and their MIC was determined. The
imino cyclophanes at the concentrations of 50, 75, and 100 mL of
10 % DMSO was used for this study with fluconazole as a
positive control. The plates were incubated at 378C for 24 h. The
antifungal activity was determined by the zone of inhibition and
are shown in Table S1 in the Supplementary Material.
Supplementary Material
Zone of inhibition data for antimicrobial activity (Table S1),
1
Ligplot results (Figs S1–S3), and copies of H and 13C NMR
spectra are available on the Journal’s website.
Acknowledgements
The authors thank DST, New Delhi, for financial assistance, RSIC, CDRI,
Lucknow for MS, DST-FIST for providing the NMR facility for the
Department of Organic Chemistry, University of Madras. R.P. thanks
Orchid Chemicals and Pharmaceuticals Ltd, for providing the laboratory and
analytical facilities.
Minimum Inhibition Assay
The MIC was determined by the methods used by Gabrielson
et al.[22b] A sterile 96-well plate was used for the experiment.
A volume of 100 mL of test material in 10 % (v/v) DMSO or
sterile water was pipetted into the first row of the plate. To all
other wells 50 mL of Muller–Hinton broth or normal saline was
added. Serial dilutions were performed using a multichannel
pipette and the concentration of compound for serial dilution was
10mg in the first row and tips were discarded after use such that
each well had 50 mL of the test material in serially descending
concentrations. Aliquots of 10 mL of bacterial suspension
(5 ꢁ 106 cfu mLꢀ1) were then added to each well to achieve a
concentration of 5 ꢁ 105 cfu mLꢀ1. Each plate was wrapped
loosely with cling film to ensure that bacteria did not become
dehydrated. Each plate had a positive control (vancomycin in
serial dilution) in the first column at the last two columns C1 and
C2. C1 contained nutrient broth, bacterial suspension and indi-
cator, and C2 contained nutrient broth and indicator). The plates
were prepared in triplicate, and placed in an incubator at 378C for
18–24 h. Finally, to each well 10 mL of 2,3-bis(2-methoxy-4-
nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt
(MTT) indicator solution was added. After incubation, the colour
change was assessed visually. The MTT tetrazolium salt assay
measures the cells ability to convert the tetrazolium salt into the
fromazan product. Any colour changes from yellow to purple
were recorded as positive. The lowest concentration was taken as
the MIC value. The bar diagram of MIC value obtained for
various imino cyclophanes is shown in Fig. 3.
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