Synthesis and biological evaluation of new tetra-aza macrocyclic scaffold constrained oxadiazole, thiadiazole and triazole rings

Article abstract of DOI:10.1002/ardp.201100181

A new series of N,N′-(benzene-1,3-diyldi-1,3,4-oxadiazole-5,2-diyl) bis{2-[(5-benzene-1,3-diyl-1,3,4-oxadiazol-2-yl)amino]acetamide}(macrocycle 1), N,N′-(benzene-1,3-diyldi-1,3,4-thiadiazole-5,2-diyl)bis{2-[(5-benzene-1, 3-diyl-1,3,4-thiadiazol-2-yl)amino

Full text of DOI:10.1002/ardp.201100181

240
Arch. Pharm. Chem. Life Sci. 2012, 345, 240–249
Full Paper
Synthesis and Biological Evaluation of New Tetra-aza
Macrocyclic Scaffold Constrained Oxadiazole, Thiadiazole
and Triazole Rings
B. Vinay Kumar1, H. S. Bhojya Naik1, D. Girija1, N. Sharath1, H. V. Sudeep2, and
H. Joy Hoskeri2
1 Department of Studies and Research in Industrial Chemistry, School of Chemical Sciences, Kuvempu
University, Shankaraghatta, India
2 Department of Studies and Research in Biotechnology and Bioinformatics, School of Biological Sciences,
Kuvempu University, Shankaraghatta, India
A new series of N,N0-(benzene-1,3-diyldi-1,3,4-oxadiazole-5,2-diyl)bis{2-[(5-benzene-1,3-diyl-1,3,4-oxadiazol-
2-yl)amino]acetamide}(macrocycle 1), N,N0-(benzene-1,3-diyldi-1,3,4-thiadiazole-5,2-diyl)bis{2-[(5-benzene-
1,3-diyl-1,3,4-thiadiazol-2-yl)amino]acetamide} (macrocycle 2) and S,S0-[benzene-1,3-diylbis(4H-1,2,4-
triazole-5,3-diyl)]bis{[(5-benzene-1,3-diyl-4H-1,2,4-triazol-3-yl)sulfanyl]ethanethioate}(macrocycle 3) was
synthesized from isophthalic dihydrazide (4) through a multistep reaction sequence. All the
synthesized compounds were screened for their inhibitory effect against four different bacterial
strains: P. aeruginosa ATCC-20852, K. pneumoniae MTCC-618, S. aureus ATCC- 29737, S. typhi MTCC- 3214.
The synthesized compounds showed a significant zone of inhibition and the results were comparable
with that of the standard drug ciprofloxacin. The synthesized compounds were further studied for their
possible in vitro antioxidant effects by DPPH scavenging, total antioxidant capacity, total reductive
capacity and H2O2 scavenging activity. The results indicated that the in vitro antioxidant activity for
all the three molecules was efficient when compared to the standards. The DNA interaction behavior of
macrocycles 1–3 with CT-DNA was investigated by the absorption spectra obtained (Kb constant for 1 is
4.53 ꢀ 104 Mꢁ1, for 2 is 5.75 ꢀ 104 Mꢁ1 and for 3 is 5.86 ꢀ 104 Mꢁ1). Based on the results it can be
interpreted that the reducing power effect of the newly synthesized compounds demonstrates a direct
effect on DNA binding and hence inhibiting the bacterial growth through their action on DNA by
inhibiting DNA replication or DNA transcription.
Keywords: Antibacterial activity / Antioxidant activity / DNA binding / Macrocyclic
Received: May 17, 2011; Revised: September 10, 2011; Accepted: September 20, 2011
DOI 10.1002/ardp.201100181
Introduction
articles and reviews that ascertain that compounds bearing
a 1,3,4-oxadiazole/thiadiazole and 1,2,4-triazole nucleus
possess significant anti-inflammatory activity [10–13]. The
development of synthetic macrocyclic methodology, contain-
ing five and six-membered heterocyclic rings as subunits has
led to the preparation of a range of such compounds which
have shown to possess very interesting biological properties
in a variety of fields. These macrocycles were found to exhibit
interesting host-guest complexation characteristics [14] and
have shown antibacterial and antitumor activities [15, 16] in
which the biological activity is highly dependent upon the
side chain substitution pattern.
Numerous macrocyclic systems exist, which contain nitro-
gen, sulphur and oxygen donor atoms, either as a thiazole,
oxazole, pyrrole or quinoline group or as an amine group
[1–8]. However, it is somewhat surprising that there is a
relative paucity of macrocycles containing five-membered
heterocyclic groups [9], considering that there are many
Correspondence: H. S. Bhojya Naik, Department of Studies and
Research in Industrial Chemistry, School of Chemical Sciences, Kuvempu
University, Shankaraghatta-577 451, India.
E-mail: hsb_naik@rediffmail.com
Fax: þ91-8282-257302
In order to demonstrate the potential antioxidant activity
of heterocyclic compounds, the interactions of nitrogen,
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Arch. Pharm. Chem. Life Sci. 2012, 345, 240–249 Tetra-aza Macrocyclic Scaffold Constrained Oxadiazole, Thiadiazole and Triazole Rings
241
sulfur and oxygen containing molecules with DNA are of
major biochemical and biological importance [17]. It has
been suggested that macrocyclic systems can chelate Fe(II)
or Fe(III) and prevent free radical production in the Fenton
reaction [18] and themselves can also intercalate in the DNA
duplex and react with free radicals in order to protect DNA
from oxidative damage [19, 20].
A literature survey shows that there is evidence that anti-
Figure 1. Structures of macrocycles 1–3.
tumor activity is due to the intercalation between the base
pairs of DNA and interference with the normal functioning
of the enzyme topoisomerase II, which is involved in the
breaking and releasing of DNA strands [21]. The antitumor
drugs that intercalate in DNA are of growing interest in the
field of anticancer derivatives. Generally, they are character-
ized by planar chromospheres, which are often constituted
by three or four condensed rings, which can intercalate
support to their biological activity, antibacterial activity was
screened. Further to unmask and understand the mode of
action against the bacterial strains, we further evaluated the
antioxidant property, and ds-DNA interacting ability of the
synthesized compounds.
between base pairs. Results of these various binding studies Results and discussion
have been useful in designing new and promising anticancer
agents for clinical use [22, 23]. DNA binding and cleavage
studies of macrocyclic molecules have been recently reported
in the literature [24, 25].
Chemistry
The first stride from the synthesis of macrocycle 1 and 2 can be
achieved from the reaction of isophthalic dihydrazide 4 in
dioxane with 2.2 equiv. of cyanogen bromide in water in the
presence of 2.2 equiv. of sodium bicarbonate. The mixture was
stirred at room temperature for 8 h, this workup afforded the
5,5’-benzene-1,3-diylbis(1,3,4-oxadiazol-2-amine) 5 (Scheme 1)
with 77% yield [29] which was characterized from spectral
In view of the above biological importance and in continu-
ation of our previous work [26–28], we demonstrate a new
strategy to introduce oxadiazole or thiadiazole or triazole
rings into macrocycles employing the novel precursors 1, 2
and 3 (Fig. 1) without using high dilution conditions. In
Scheme 1. Reagents and conditions: (a) 2.2
equiv. cyanogen bromide, NaHCO3, dioxane,
RT, 8 h, 5, 77%, (b) 4.2 equiv. thiourea, THF,
120–1508C, 22 h, 6, 73%, (c) 2.2 equiv.
ClCH2COCl, DMF, 0–108C, 1 h, 7, 89%; 8,
84%, (d) triethylamine, 2.2 equiv. K2CO3,
THF, reflux, 10–12 h, 1, 68%; 2, 62%.
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and analytical data. Replacement of –O– by –S– in hetero-
cycles was performed as previously reported, viz., Bordners
preparation of pyrroles from furan and the transformation of
epoxides to episulfides by the action of thiocyanates or thio-
urea [30, 31]. The compound 5 was treated with twofold
excess thiourea in tetrahydrofuran. The reaction mixture
after workup gave a solid which was identified as 5,50-
benzene-1,3-diylbis(1,3,4-thiadiazol-2-amine) 6 (Scheme 1)
with 73% yield. The probable mechanism involved in the
formation of thiouronium salt which undergoes rearrange-
ment to form the mesomeric oxouronium salt via the oxa-
thiadiazepine derivative. Further, ring closure of the
oxouronium salt led to formation of thiadiazole by the elim-
ination of urea. The comparison of the IR spectra of com-
pounds 5 and 6 showed that the peak at 1087 cmꢁ1 can be
assigned to n(C–O–C) of the oxadiazole ring and the peak at
685 cmꢁ1 can be assigned to n(C–S–C) of the thiadiazole ring,
thus the compounds were characterized from spectral and
analytical data. Coupling of compound 5 or 6 with chloro-
acetyl chloride in DMF at 0–108C stirred for 1 h afforded the
bis chloroacetamide receptors 7 and 8 with 89% and 84%
yield [32], respectively (Scheme 1). The 1H NMR spectrum of 7
displayed the amide NH protons as singlet at 11.2 ppm and
CH2 protons as singlet at 4.9 ppm. The rest of the aromatic
protons appeared in the usual region. Molecular ion [MþH]þ
peaks of compound 7 and 8 were observed at different
intensities in positive ionization mode and confirmed the
molecular weights. Compound 7 and 8 which have halogen
atoms in their molecule show in their mass spectrum the
characteristic peaks corresponding to isotopic distribution
of the target macrocycles 1 and 2 was 16–18%, we also
observed (by TLC monitoring) the formation of unknown
secondary products. This may be attributed to reaction (as
by-products) as reported in the earlier literature. We next
applied a second strategy as depicted above in Scheme 1.
The macrocyclization step in this method involves the reac-
tion of compound 7 capping with compound 5 or 6 in THF
which was completely solubilized with triethylamine under
inert (N2) atmosphere in the presence of K2CO3. Later the
solution of 7 in THF was added dropwise for 30 min. The
reaction mixture was refluxed for 12 h and after workup gave
a solid which was identified as macrocycles 1 and 2 respect-
ively in reasonable yields [34]. The IR spectrum of the com-
pound 5 showed absorption bands at 3303 cmꢁ1 due to the
NH2 group, which was absent in the IR spectrum of the macro-
cycle 1. Similarly, the 1H NMR spectrum of compound 5
showed a broad signal at 5.2 ppm attributed to the NH2 group,
which was not present in the spectrum of compound 1. In the
1H NMR spectrum of compound 1, the signal corresponding to
protons of the methylene group appeared as doublet at
4.36 ppm and signals due to the amide NH group appeared
as singlet at 10.7 ppm. The attributions of the signals 13C NMR
of compound 1 resulted from the spectra. In compound 1, the
–
C O signal is observed at ꢂ168 ppm and the CH group
–
2
resonated at ꢂ35 ppm. The C(3) and C(5) heterocyclic carbon
resonated at ꢂ153 and ꢂ151 ppm, respectively, further con-
firming the formation of macrocycle 1. The structure of macro-
cycle 2 was also confirmed through spectral studies.
As an extension of this study, 2,20-(benzene-1,3-diyldi-
ethene-1,1-diyl)dihydrazine carbothioamide 9 (Scheme 2)
was obtained by the reaction of isophthalic dihydrazide 4 with
potassium isothiocyanate. The cyclization of compound 9 in
the presence of aqueous NaOH resulted in the formation of
5,50-benzene-1,3-diylbis(4H-1,2,4-triazole-3-thiol) 10 [35]. The
reaction of compound 10 with chloroacetyl chloride in the
presence of dimethyl sulfoxide produced S,S0-[benzene-1,3-
(
35Cl and 37Cl isotopes). The structure of compound 8 was
fully characterized by spectroscopic methods.
Two strategies were attempted for the synthesis of macro-
cycles 1 and 2. In the first strategy, compound 5 or compound
6 was chosen as the starting material as described by Da-Ming
Du and coworkers [33]. After the cyclization process, the yield
Scheme 2. Reagents and conditions: (a) 2.2
equiv. KSCN, HCl, water, reflux, 6 h, 9, 86%,
(b) 4 N NaOH, water, reflux, 4 h, 10, 68%,
(c) 2.2 equiv. ClCH2COCl, DMSO, RT, 14 h,
11, 71%, (d) DMSO, RT, 26 h, 3, 58%.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 240–249 Tetra-aza Macrocyclic Scaffold Constrained Oxadiazole, Thiadiazole and Triazole Rings
243
Table 2. Minimum inhibitory concentrations (in mg/mL) of
compounds using macrodilution method.
diylbis(4H-1,2,4-triazole-5,3-diyl)]bis(chloroethanethioate) 11.
The macrocyclization step in this method involves the reac-
tion of compound 10 in dimethyl sulfoxide treated with
Bacterial strains
Compounds
2
Ciprofloxacin
compound 11 and then the reaction mixture was stirred at
room temperature for 12 h, this workup gave a solid precipi-
tate, which was identified as macrocycle 3 with 58% yield. The
IR spectrum of the compound 3 showed strong absorption
band at 1681 cmꢁ1 characteristic of carbonyl group. The
1
3
Pa ATCC-20852
Kp MTCC-618
Sa ATCC-29737
St MTCC-3214
82
148
66
122
132
102
122
64
82
82
62
2
2
4
2
absorption due to the –NH group appeared at 3304 cmꢁ1
,
54
while the –SH peak at 2576 cmꢁ1 was absent in the IR
spectrum of the macrocycle 3.
Similarly, the 1H NMR spectrum of compound 10 showed a
broad signal at 13.9 ppm attributed to the –SH group which
was not present in the spectrum of compound 3. The attri-
butions of the 13C NMR signals of compound 3 resulted from
the test bacteria, compounds 1 and 3 were more effective
against S. typhi with inhibition zone diameter (IZD) of
15.2 ꢃ 0.60 and 15.2 ꢃ 0.44 respectively. K. pneumoniae was
least susceptible to compound 1 (10.8 ꢃ 0.17). Compound 2
showed moderate activity against all the test organisms with
IZD ranging from 13.17 ꢃ 0.44 to 13.8 ꢃ 1.17. In addition,
the above three compounds were found to be effective at
different dilutions based on the activity. The MIC data was
varying against different test organisms (Table 2). MIC of
synthesized compound 1 was found to be 148 mg/mL for
K. pneumoniae. It was comparatively lower against the other
strains, viz. P. aeruginosa, S. aureus and S. typhi. The MIC of
compound 2 was higher for all the strains. Compound 3
had MIC of 62 mg/mL and 64 mg/mL for S. typhi and
P. aeruginosa respectively. It was found to be 82 mg/mL for
both K. pneumoniae and S. aureus.
–
the spectra. In compound 3, the C O signal is observed at
–
ꢂ193 ppm and the CH2 group resonated at ꢂ34 ppm. The
C(3) and C(5) heterocyclic carbon resonated at ꢂ153 and
ꢂ152 ppm, respectively, this further confirmed the for-
mation of macrocycle 3. The structure of macrocycle 3 was
fully characterized spectroscopically.
The synthesis of macrocycles 1, 2 and 3 is of interest due to
the presence of carbonyl functionalities in the annular cavity.
Hence, its electron-deficient nature would alter the position of
the charge transfer complexation band. Also functional group
transformations can be carried out on the carbonyl group.
Biological activity
In vitro antioxidant activity
Antibacterial activity
In the DPPH assay for free radical scavenging the most potent
molecule was found to be compound 1 which showed the
maximum percentage inhibition of 88.9% at 100 mg/mL con-
centration. It was followed by compound 2 which also exhib-
ited good scavenging activity ranging from 74.8% to 77.8% at
different concentrations. Compound 3 demonstrated activity
with values of 52.3% to 74.3% (Fig. 2).
The synthesized macrocyclic compounds were tested for
their in vitro antibacterial activity against three gram nega-
tive and one gram positive standard strains of bacteria. The
compounds were found to be effective against gram positive
and gram negative strains based on the values obtained
through relative zone of inhibition (Table 1). However,
slightly varying levels of effect were observed among the
synthesized compounds. The results were significant and were
comparable with the standard antibiotic drug ciprofloxacin.
On the basis of minimum inhibitory activity shown against
The FRAP assay is to measure the reductive ability of anti-
oxidant and it was evaluated by the transformation of Fe(III)
to Fe(II) in the presence of the synthesized compounds. The
results of reducing power assays of the synthesized macro-
Table 1. Antibacterial activity of synthesized compounds against standard bacterial strains (inhibition zone in mm).
Bacterial strains
Compounds
2
Ciprofloxacin
1
3
Pa ATCC-20852
Kp MTCC-618
Sa ATCC-29737
St MTCC-3214
13.50 ꢃ 0.76ꢄ
10.83 ꢃ 0.17ꢄꢄ
14.83 ꢃ 0.44ꢄ
15.17 ꢃ 0.60ꢄ
13.50 ꢃ 0.79ꢄ
13.17 ꢃ 0.44ꢄꢄ
13.83 ꢃ 1.17ꢄ
13.33 ꢃ 0.88ꢄ
14.83 ꢃ 0.44ꢄ
14.00 ꢃ 1.04ꢄ
13.67 ꢃ 0.88ꢄ
15.17 ꢃ 0.44ꢄꢄ
20.00 ꢃ 0.58
21.00 ꢃ 0.87
20.17 ꢃ 1.01
20.77 ꢃ 0.54
ꢄ
The values are the means of three experiments ꢃ S.E. P <0.01 vs. standard.
Abbreviations: Pa, Pseudomonas aeruginosa; Kp, Klebsiella pneumoniae; Sa, Staphylococcus aureus; St, Salmonella typhi.
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Figure 4. Phosphomolybdenum antioxidant assay.
Figure 2. Free radical scavenging using DPPH.
Figure 5. Hydroxyl radical scavenging.
Figure 3. Ferric ion reducing/antioxidant power.
cyclic compounds are summarized in Fig. 2. It is evident that
the ability of compound 1 to reduce Fe(III) was better than that
of the standard BHT at 100 mg/mL concentration. On the other
hand, the compounds 2 and 3 were less effective in exhibiting
reducing activity at the same concentration (Fig. 3).
The total antioxidant capacity of the compounds was eval-
uated at three different concentrations, viz. 100, 150 and
200 mg/mL. The results indicated that the compound 1
showed very good antioxidant ability, which was similar
to the standard ascorbic acid. Compounds 2 and 3 demon-
strated moderate activity (Fig. 4).
often characterized through absorption spectra followed by
the changes in the absorbance and shift in the wavelength.
The absorption spectra of macrocycles 1–3 in the absence and
presence of CT-DNA are illustrated in Fig. 6a–c, respectively.
As shown in Fig. 6, the interaction of macrocycles 1–3 with
CT-DNA was monitored by the blue shift (the hypsochromic
effect) in UV–visible spectra. The absorption maximum wave-
length was shifted from 370 nm in the spectra of macrocycle
3 to 338 nm in the spectra of CT-DNA/macrocycle 3 (Fig. 6c).
Progressive addition of DNA led to strong hypochromism in
the absorption intensities in all the macrocycles studied. The
hypochromicity, characteristic of intercalation [36], has been
usually attributed to the interaction between the electronic
states of the compound chromophores and those of the DNA
bases [37].
The hydroxyl radical scavenging activity was not appreci-
able at 100 mg/mL concentration of the compounds.
Compound 1 exhibited efficient activity with 31.75%. The
percentage inhibition was much lower for the other two
synthesized compounds (Fig. 5).
The percentage hypochromicity for the macrocycles 1–3
was determined from (ef ꢁ eb)/ef ꢀ100, where ef is the extinc-
tion coefficient of the free compound and eb is the extinction
coefficient of the bound compound. The percentages of hypo-
chromism of macrocycles 1–3 were found to be 22.4, 21.3 and
DNA binding studies (absorption spectral studies)
The DNA binding studies of the macrocyclic compounds 1–3
(the structures of the compounds are shown in Fig. 1) were
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Arch. Pharm. Chem. Life Sci. 2012, 345, 240–249 Tetra-aza Macrocyclic Scaffold Constrained Oxadiazole, Thiadiazole and Triazole Rings
245
18.1, respectively. The half-reciprocal plots for binding of
macrocycles 1–3 with CT DNA were presented (Fig. 6a–c).
The compounds exhibited similar absorption spectra pertain-
ing to chromophore.
To further illustrate the DNA binding strength, the intrin-
sic binding constant Kb was determined for macrocycles 1–3
which were found to be 4.53 ꢀ 104 Mꢁ1, 5.75 ꢀ 104 Mꢁ1 and
5.86 ꢀ 104 Mꢁ1, respectively. The binding constants of these
compounds were lower in comparison to those observed for
typical classical intercalators (ethidium-DNA, 1.4 ꢀ 106 Mꢁ1
)
[38].
This investigation on biological activity of the newly syn-
thesized macrocyclic compounds was by evaluating their
effect against bacteria, antioxidant effect by free radical
scavenging and reducing power effect. This reducing power
effect is in direct corroboration with their effect on DNA
binding. Thus it can be interpreted that the antibacterial
effect elicited by the synthesized macrocyclic compounds
is by binding to the DNA. Most of the antibiotics such as
the fluoroqinolones (e.g. ciprofloxacin) inhibit procaryotic
(not eucaryotic) DNA replication, and rifamycins inhibit bac-
terial (not eucaryotic) DNA transcription. The potential anti-
bacterial and antioxidant activities of heterocyclic compounds
by their interactions with DNA are of major biochemical and
biological importance [17]. These chemical compounds that
bind to DNA molecule can induce various effects on the
microbial growth by inhibiting the binding of DNA dependant
RNA polymerase to the DNA or cleaving the genome or form-
ing DNA bulky adducts and thus hindering the growth of the
bacteria. Further, it can also be interpreted that the reducing
power effect of the newly synthesized compounds can
demonstrate a direct effect on DNA binding and hence inhib-
iting the bacterial growth through their action on DNA by
inhibiting DNA replication or DNA transcription.
Experimental
Chemicals and instrumentation
All chemicals used for the synthesis were of analytical grade and
procured from HiMedia Laboratories Pvt. Ltd., and isophthalic
dihydrazide was purchased from Sigma Chemical Co., U.S.A., E.
Merck, Germany, Sarabhai Merck Company, India. Thin layer
chromatography (TLC) was performed throughout the reaction
to optimize the reaction for purity and completion of reaction on
Merck silica gel GF254 aluminium sheets using a mixture of
different polar and nonpolar solvents in varying proportions and
spots were observed using iodine as visualizing agent. Melting
points were measured on a Mel-Temp apparatus and are uncor-
rected. Mass spectra were recorded on an Agilent 6320 ion trap
mass spectrometer. IR spectra were recorded on a Shimadzu IR-
470 spectrometer. 1H NMR spectra were recorded on a Bruker
DRX-300 Avance spectrometer 300.13 MHz; chemical shifts
(d scale) are reported in parts per million (ppm). Signals were
characterized as s (singlet), d (doublet), t (triplet), m (multiplet),
Figure 6. Absorption spectral traces of (a) macrocycle 1, (b) macro-
cycle 2 and (c) macrocycle 3 in Tris-HCl buffer upon addition of CT-
DNA. Inset: Plots of [DNA]/(eaꢁef) versus [DNA] for the titration of
CT-DNA with macrocycles 1–3.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 240–249
br s (broad signal) and Ar (aromatic). The 13C NMR spectra were
recorded at 75.47 MHz; chemical shifts (d scale) are reported in
parts per million (ppm). The elemental analyses were performed
at Cochin University, Sophisticated Test and Instrumentation
Center, Kochi, Kerala, India. All the final products are
new compounds, which were characterized by IR, 1H NMR,
13C NMR spectra and mass spectral data.
N,N0-(Benzene-1,3-diyldi-1,3,4-thiadiazole-5,2-diyl)-
bis(2-chloroacetamide) 8
Yield 84%; mp 2538C; IR (KBr): n (cmꢁ1): 3243 (amide NH stretch.),
–
–
1657 (amide CO stretch.), 1613 (C N), 1464 (C C ring stretch.),
–
–
791 (C–Cl stretch); 1H NMR (300 MHz, DMSO-d6) d (ppm): 10.7 (bs,
2H, 2 NH), 8.11 (t, 1H, ArH), 7.98 (dd, 2H, ArH), 7.78 (t, 1H, ArH.),
4.7 (s, 4H, 2 CH2); (LC-MS) m/z: 430 [MþH]þ, 432 [MþH]þ, 434
[MþH]þ. Anal. Calcd. for C14H10Cl2N6O2S2: C, 39.17; H, 2.35; N,
19.58. Found: C, 39.19; H, 2.36; N, 19.55.
Synthesis of 5,50-benzene-1,3-diylbis(1,3,4-oxadiazol-2-
amine) 5
To a stirring solution of compound 4 (1.44 g, 7.42 mmol) in
dioxane (24 mL) sodium bicarbonate (1.25 g, 14.8 mmol) in
water (16 mL) was added at room temperature. The mixture
was stirred at room temperature for 5 min and cyanogens bro-
mide (1.63 g, 15.4 mmol) was added. After 8 h water (60 mL) was
added to the mixture and the precipitate was removed by filtra-
tion and recrystallized from ethanol to afford compound 5.
Yield 77%; mp 1378C; IR (KBr): n (cmꢁ1): 3303 (NH2 stretch.),
Synthesis of N,N0-(benzene-1,3-diyldi-1,3,4-oxadiazole-
5,2-diyl)bis{2-[(5-benzene-1,3-diyl-1,3,4-oxadiazol-2-yl)-
amino]acetamide} 1
Compound 5 (1.22 g, 5 mmol) in tetrahydrofuron (THF, 25 mL)
was completely solubilized with triethylamine under inert (N2)
atmosphere. To this, K2CO3 (0.69 g, 5 mmol) was added. Later the
solution of 7 (1.99 g, 5 mmol) in THF (25 mL) was added drop by
drop for 30 min. The reaction mixture was refluxed for 10–12 h.
The progress of the reaction mixture was monitored by TLC. The
reaction mixture was then desolventized in a rotavapour and the
compound was extracted in ethyl acetate. The ethyl acetate layer
was washed with water and dried over anhydrous Na2SO4. The
solid was obtained by further desolventation in a rotary evapor-
ator at 508C. The product was separated and purified by column
chromatography, using mixture of ethyl acetate/hexane (80:20)
and gave compound 1.
–
–
3057 (aromatic CH stretch.), 1608 (C N), 1489, 1462 (C C ring
–
–
stretch.), 1087 (C–O–C stretch); 1H NMR (300 MHz, DMSO-d6)
d (ppm): 8.06 (t, 1H, ArH), 7.89 (dd, 2H, ArH), 7.64 (t, 1H, ArH),
5.28 (bs, 4H, 2 NH2); (LC-MS) m/z: 245 [MþH]þ. Anal. Calcd. for
C10H8N6O2: C, 49.18; H, 3.30; N, 34.41. Found: C, 49.12; H, 3.33; N,
34.39.
Synthesis of 5,50-benzene-1,3-diylbis(1,3,4-thiadiazol-2-
amine) 6
Yield 68%; mp 1058C; IR (KBr): n (cmꢁ1): 3286 (amide NH
A mixture of 5 (1.22 g, 5 mmol), thiourea (1.52 g, 20 mmol)
dissolved in tetrahydrofuran (10 mL) was heated at 120–1508C
in an oil bath for 22–26 h. After the reaction was completed,
the reaction mixture was extracted with dichloromethane. The
organic layer was washed with water, brine solution and dried
over anhydrous Na2SO4. The resultant solid was recrystallized
using methanol yielding compound 6.
–
–
stretch.), 1647 (amide CO stretch.), 1589 (C N), 1478 (C C ring
–
–
stretch.); 1H NMR (300 MHz, DMSO-d6) d (ppm): 10.7 (bs, 2H, 2
CONH), 8.15 (t, 2H, ArH), 7.94 (dd, 4H, ArH), 7.68 (t, 2H, ArH), 5.24
(s, 2H, 2 NH), 4.36 (d, 4H, 2 CH2); 13C NMR (75 MHz, DMSO-d6) d
(ppm): 168.37, 153.04, 151.26, 136.99, 128.74, 127.01, 124.86,
35.62; (LC-MS) m/z: 569 [MþH]þ. Anal. Calcd. for C24H16N12O6:
C, 50.71; H, 2.84; N, 29.57. Found: C, 50.68; H, 2.87; N, 29.55.
Yield 74%; mp 2048C; IR (KBr): n (cmꢁ1): 3301 (NH2 stretch.),
–
–
3077 (aromatic CH stretch.), 1597 (C N), 1484 (C C ring stretch.),
–
–
Synthesis of N,N0-(benzene-1,3-diyldi-1,3,4-thiadiazole-
5,2-diyl)bis{2-[(5-benzene-1,3-diyl-1,3,4-thiadiazol-2-yl)-
amino]acetamide} 2
685 (C–S–C stretch); 1H NMR (300 MHz, DMSO-d6) d (ppm): 8.14
(t, 1H, ArH), 7.94 (dd, 2H, ArH), 7.72 (t, 1H, ArH), 5.8 (bs, 4H, 2
NH2); (LC-MS) m/z: 277 [MþH]þ. Anal. Calcd. for C10H8N6S2: C,
43.46; H, 2.92; N, 30.41. Found: C, 43.49; H, 2.88; N, 30.38.
Similar to macrocycle 1 procedure, except compound 6 and 8
were used instead of compound 5 and 7, which gave compound 2.
Yield 62%; mp 1588C; IR (KBr): n (cmꢁ1): 3152 (amide NH
General procedure for compounds 7 and 8
–
–
stretch.), 1659 (amide CO stretch.), 1602 (C N), 1442 (C C ring
–
–
Compound 5 (1.22 g, 5 mmol) or compound 6 (1.38 g, 5 mmol)
was dissolved in DMF (20 mL), and the solution was cooled to 08C.
To this cold solution, chloro acetylchloride (1.56 mL, 20 mmol)
in dioxane (5 mL) was added dropwise during a period of
60 minutes, with vigorous stirring at 0–108C. Compound 7 or
compound 8 separated out as a solid from the reaction mixture.
It was filtered, washed with water and dried, affording com-
pound 7 or compound 8, respectively.
stretch.); 1H NMR (300 MHz, DMSO-d6) d (ppm): 9.34 (bs, 2H, 2
CONH), 8.08 (t, 2H, ArH), 7.87 (dd, 4H, ArH), 7.62 (t, 2H, ArH), 5.29
(bs, 2H, 2 NH), 4.00 (s, 4H, 2 CH2); 13C NMR (75 MHz, DMSO-d6) d
(ppm): 167.79, 152.68, 150.49, 135.92, 128.43, 127.57, 125.04,
35.05; (LC-MS) m/z: 633 [MþH]þ. Anal. Calcd. for C24H16N12O2S4:
C, 45.56; H, 2.55; N, 26.56. Found: C, 45.59; H, 2.52; N, 26.53.
Synthesis of 2,20-(benzene-1,3-diyldiethene-1,1-diyl)-
dihydrazinecarbothioamide 9
N,N0-(Benzene-1,3-diyldi-1,3,4-oxadiazole-5,2-diyl)-
bis(2-chloroacetamide) 7
A suspension of compound 4 (1.94 g, 10 mmol), potassium
thiocyanate (30 mmol), hydrochloric acid (16 mL) and water
(50 mL) was refluxed for 8 h. The solution was cooled and a
white solid appeared. This was filtered and recrystallized from
dimethyl sulfoxide/water (1:2) affording compound 9.
Yield 86%; mp 1878C; IR (KBr): n (cmꢁ1): 3246 (NH stretch.),
Yield 89%; mp 1768C; IR (KBr): n (cmꢁ1): 3397 (amide NH stretch.),
–
–
1652 (amide CO stretch.), 1601 (C N), 1450 (C C ring stretch.),
–
–
781 (C–Cl stretch); 1H NMR (300 MHz, DMSO-d6) d (ppm): 11.2
(bs, 2H, 2 NH), 8.25 (t, 1H, ArH), 8.08 (dd, 2H, ArH), 7.96 (t, 1H,
ArH), 4.9 (s, 4H, 2 CH2); (LC-MS) m/z: 398 [MþH]þ, 400 [MþH]þ, 402
[MþH]þ. Anal. Calcd. for C14H10Cl2N6O4: C, 42.43; H, 2.54; N,
21.16. Found: C, 42.45; H, 2.51; N, 21.19.
1682 (C O stretch.), 1310 (C S); 1H NMR (300 MHz, DMSO-d6)
–
–
–
d (ppm): 10.2 (bs, 4H, 2 NH2), 9.82 (s, 2H, 2 NH), 9.73 (s, 2H, 2 NH),
–
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Arch. Pharm. Chem. Life Sci. 2012, 345, 240–249 Tetra-aza Macrocyclic Scaffold Constrained Oxadiazole, Thiadiazole and Triazole Rings
247
8.19 (t, 1H, ArH), 7.91 (dd, 2H, ArH), 7.84 (t, 1H, ArH); (LC-MS)
m/z: 313 [MþH]þ. Anal. Calcd. for C10H12N6O2S2: C, 38.45; H, 3.87;
Test microorganisms
The standard microbial strains of P. aeruginosa ATCC-20852,
K. pneumoniae MTCC-618, S. aureus ATCC-29737, S. typhi MTCC-
3214 were used for antibacterial evaluation. The bacteria were
maintained on agar slants at 48C and sub-cultured into nutrient
broth by a picking-off technique for 24 h before use.
N, 26.90. Found: C, 38.49; H, 3.84; N, 26.86.
Synthesis of 5,50-benzene-1,3-diylbis(4H-1,2,4-triazole-3-
thiol) 10
A solution of compound 9 (3.12 g, 10 mmol) in 2 N NaOH was
refluxed for 5 h. The resulting solution was cooled to room
temperature and acidified to pH 3–4 with 37% HCl. The precipi-
tate thus formed was filtered, washed with water and recrystal-
lized from ethanol/water (1:1) giving compound 10.
Yield 67%; mp 2898C; IR (KBr): n (cmꢁ1): 3194 (NH stretch.),
Preparation of culture medium and inoculation
Nutrient agar (Hi Media, India) was used as the bacteriological
medium. The media were sterilized by autoclaving at 1208C for
20 min. Under aseptic conditions, in the laminar air flow 15 mL
of culture medium was dispensed into pre-sterilized petridishes
to yield a uniform depth of 4 mm. After solidification of
the medium, the microbial cultures were inoculated by spread
plating technique.
–
2576 (SH stretch), 1599 (C N stretch.), 1034 (N–N stretch);
–
1H NMR (300 MHz, DMSO-d6) d (ppm): 13.8 (bs, 2H, 2 SH), 11.4
(s, 2H, 2 NH), 8.33 (t, 1H, ArH), 8.09 (dd, 2H, ArH), 7.87 (t, 1H, ArH);
(LC-MS) m/z: 277 [MþH]þ. Anal. Calcd. for C10H8N6S2: C, 43.46; H,
2.92; N, 30.41. Found: C, 43.48; H, 2.89; N, 30.43.
Primary screening by agar well diffusion
Preliminary screening for all the macrocyclic compounds was
performed at fixed concentrations of 10 mg/mL by dissolving in
10% DMSO. Pure DMSO was taken as the negative control and
10 mg/mL of ciprofloxacin (fluoroquinolone antibiotic) was used
as the positive control. Wells were prepared in the agar plates
using a sterile cork borer of 6.0 mm diameter. 1000 mg/100 mL of
each compound were loaded in the corresponding wells. The
plates were allowed to stand at room temperature for 1 h for
extract to diffuse into the agar and then they were incubated at
378C for 18 h. Subsequently, the plates were examined for
microbial growth inhibition and the inhibition zone diameter
(IZD) was measured to the nearest millimeter. The experiment
was performed in triplicates.
Synthesis of S,S0-[benzene-1,3-diylbis(4H-1,2,4-triazole-
5,3-diyl)]bis(chloro-ethanethioate) 11
Compound 10 (1.38 g, 5 mmol) dissolved in dimethyl sulfoxide
(30 mL) was treated with chloro acetylchloride (1.56 mL,
20 mmol) and the mixture was stirred at room temperature
for 14 h. The reaction mixture was then poured into ice water.
The precipitate obtained was filtered, washed with water and
then with ethyl acetate. The compound was purified by recrys-
tallization using ethanol giving compound 11.
Yield 71%; mp 2268C; IR (KBr): n (cmꢁ1): 3243 (NH stretch.),
–
–
1637 (C O stretch), 1613 (C N stretch.), 1018 (N–N stretch), 754
–
–
(C–Cl stretch); 1H NMR (300 MHz, DMSO-d6) d (ppm): 11.3 (s, 2H, 2
NH), 8.02 (t, 1H, ArH), 7.79 (dd, 2H, ArH), 7.54 (t, 1H, ArH), 4.4
(s, 4H, 2 CH2); (LC-MS) m/z: 430 [MþH]þ, 432 [MþH]þ, 434 [MþH]þ.
Anal. Calcd. for C14H10Cl2N6O2S2: C, 39.17; H, 2.35; N, 19.58.
Found: C, 39.15; H, 2.38; N, 19.56.
Determination of minimum inhibitory concentration
(MIC) of synthesized compounds
Minimum inhibitory concentration (MIC) is the lowest concen-
tration of any compound at which it will inhibit the visible
growth of microorganisms after overnight incubation.
Minimum inhibitory concentrations are important in diagnostic
laboratories to confirm the resistance of microorganisms to
antimicrobial agents and also to monitor the activity of new
antimicrobial agents. The MIC of the newly synthesized macro-
cyclic compounds was tested against bacterial strains through a
microdilution tube method [40]. In this method, the test con-
centrations of chemically synthesized compounds were made
from 148 to 0.25 mg/mL in the sterile tubes Nos. 1–11. Mueller
Hinton Broth (MHB) medium was prepared and 100 mL sterile
MHB was poured into each sterile tube followed by addition of
200 mL compound in tube 1. Twofold serial dilutions were
carried out from tube 1 to tube 11 and excess broth (100 mL)
was discarded from the last tube No. 11. 0.5 McFarland standard
was made, which is visually comparable to a microbial suspen-
sion of approximately 1.5 ꢀ 108 cells/mL. To each tube, 100 mL
of standard inoculum was added. Ciprofloxacin (antibacterial
drug) was used as control. All the tubes were incubated for
24 h at 378C.
Synthesis of S,S0-[benzene-1,3-diylbis(4H-1,2,4-triazole-
5,3-diyl)]bis{[(5-benzene-1,3-diyl-4H-1,2,4-triazol-3-yl)-
sulfanyl]ethanethioate} 3
Compound 10 (1.38 g, 5 mmol) dissolved in dimethyl sulfoxide
(30 mL) was treated with compound 11 (2.15 g, 5 mmol) and the
mixture was stirred at room temperature for 26 h. The reaction
mixture was then poured into ice water. The precipitate obtained
was filtered, washed with water and then with ethyl acetate. The
compound was purified by recrystallization using ethanol giving
compound 3.
Yield 58%; mp 3038C; IR (KBr): n (cmꢁ1): 3304 (NH stretch.),
–
–
–
1681 (C O stretch.), 1587, (C N), 1486 (C C ring stretch.);
–
–
–
1H NMR (300 MHz, DMSO-d6) d (ppm): 10.7 (bs, 4H, 4 NH), 8.42
(t, 2H, ArH), 8.23 (dd, 4H, ArH), 8.07 (t, 2H, ArH), 4.38 (s, 4H,
2 –S–CH2–CO); 13C NMR (75 MHz, DMSO-d6) d (ppm): 193.28,
153.65, 152.78, 137.68, 129.57, 127.86, 125.43, 34.57; (LC-MS)
m/z: 633 [MþH]þ. Anal. Calcd. for C24H16N12O2S4: C, 45.56; H,
2.55; N, 26.56. Found: C, 45.51; H, 2.58; N, 26.58.
In vitro antibacterial activity of synthesized
macrocycles 1–3
The macrocyclic compounds 1–3 were tested for antibacterial
activity by the agar well diffusion method [39].
Statistical analysis
The results of the antibacterial study are expressed as
mean ꢃ SEM of three replicates in each test. The data were
evaluated by one-way analysis of variance (ANOVA) followed by
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

248
B. Vinay Kumar et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 240–249
Tukey’s multiple pairwise comparison tests to assess the statisti-
cal significance. The data were considered at P <0.01.
Hydroxyl radical scavenging
This assay was determined according to the previously reported
method [45] with a slight modification. The assay is based on
quantification of the degradation product of 2-deoxyribose by
condensation with TBA. Hydroxyl radical was generated by the
Fe3þ-ascorbate-EDTA-H2O2 system (the Fenton reaction). The reac-
tion mixture contained, in a final volume of 1 mL, 2-deoxy-2-
ribose (2.8 mM); KH2PO4–KOH buffer (20 mM, pH 7.4); FeCl3
(100 mM); EDTA (100 mM); H2O2 (1.0 mM); ascorbic acid
(100 mM) and the test compounds 1, 2 and 3 at a concentration
of 100 mg/mL. After incubation for 1 h at 378C, 0.5 mL of the
reaction mixture was added to 1 mL 2.8% TCA, then 1 mL 1%
aqueous TBA was added and the mixture was incubated at 908C
for 15 min to develop the color. After cooling, the absorbance
was measured at 532 nm against an appropriate blank solution.
All tests were performed six times. Percentage inhibition was
evaluated by comparing the test and blank solutions.
In vitro antioxidant activity
The macrocycles 1–3 were evaluated for their antioxidant
activity using assays such as DPPH scavenging, total antioxidant,
ferric ion reducing and hydroxyl radical scavenging.
Determination of free radical scavenging using the
DPPH radical
The DPPH free radical scavenging activity was assessed
according to Okada
& Okada method [41]. An ethanolic
solution of DPPH (0.05 mM) (300 mL) was added to 40 mL of
compound solution with different concentrations (0.01–
0.1 mg/mL). The DPPH solution was freshly prepared and kept
in the dark at 48C. Ethanol 96% (2.7 mL) was added and the
mixture was shaken vigorously. The mixture was left to
stand for 5 min and the absorbance was measured using a
spectrophotometer at 517 nm. Ethanol was used to zero the
UV-visible absorption studies
The concentration of CT-DNA per nucleotide [C(p)] was
measured using its known extinction coefficient at 260 nm
(6600 Mꢁ1 cmꢁ1) [46]. The absorbance at 260 nm (A260) and
280 nm (A280) for CT-DNA were measured in order to check
the purity level. The ratio A260/A280 was found to be 1.8 to 1.9,
indicating that CT-DNA was satisfactorily free from protein.
Buffer (5 mM tris-(hydroxymethyl)aminomethane, pH 7.2,
50 mM NaCl) was used for the absorption experiments.
Absorption titration experiments were carried out by varying
DNA concentration (0 to 100 mM) and maintaining the com-
pound concentration constant (0.5 mM). Absorption spectra were
recorded after each successive addition of DNA and equilibration
(approximately 10 min). In order to obtain the intrinsic binding
constant, Kb:
spectrophotometer.
A blank sample containing the same
amount of ethanol and DPPH was also prepared. All determi-
nations were performed in triplicate. The radical scavenging
activities of the tested samples, expressed as percentage of
inhibition, were calculated according to the following
equation [42]
Percent of DPPH inhibition ¼ ½ðAB ꢁ AAÞ=ABꢅ ꢀ 100
(1)
where AA and AB are the absorbance values of the test and of the
blank sample, respectively. A percent inhibition versus concen-
tration graph was plotted.
½DNAꢅ=ð"a ꢁ "f Þ ¼ ½DNAꢅ=ð"b ꢁ "f Þ þ 1=Kbð"b ꢁ "f Þ
where ea, ef and eb are the apparent, free and bound compound
extinction coefficients, respectively. A plot of [DNA]/(ea–ef) versus
[DNA] gave a slope of 1/(eb–ef) and an intercept y equal to 1/Kb(eb–
ef), where Kb is the ratio of the slope to the intercept y [47].
(2)
Ferric ion reducing/antioxidant power assay (FRAP)
Antioxidant activity was determined by ferric ion reducing anti-
oxidant power assay (FRAP) as described by Oyaizu method [43].
2 mL of compound 1, 2 and 3 (100 mg/mL) were mixed with
phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and 0.1% potassium
ferricyanide (2.5 mL). The mixture was incubated at 508C for
20 min. Aliquots of 10% trichloroacetic acid (2.5 mL) were added
to the mixture, which was then centrifuged at 3000 rpm for
10 min. The upper layer of solution (2.5 mL) was mixed with
distilled water (2.5 mL) and a freshly prepared ferric chloride
solution (0.5 mL, 0.1%). The absorbance was measured at
700 nm. BHA was taken as standard.
This work is supported by Major Research Project (F.No.33-322/2007 (SR)
dated: 28-02-2008) from the University Grants Commission, New Delhi.
The authors have declared no conflict of interest.
Phosphomolybdenum antioxidant assay
References
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measured at 695 nm using a UV visible spectrophotometer
against blank after cooling to room temperature. The anti-
oxidant activity was expressed as the number of gram
equivalents of ascorbic acid.
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