Synthesis of Aryl-1,2,4,5-tetrazinane-3-thiones, in vitro DNA binding studies, nuclease activity and its antimicrobial activity

Article abstract of DOI:10.1016/j.molstruc.2012.03.049

A series of small molecules with a tetrazine scaffold (5-8) was designed and synthesized as metal free DNA cleaving agent. These compounds (5-8) were characterized by using various spectroscopic (via; IR, ¹H, ¹³C NMR and ESI-MS) and analytical methods. The interaction studies of (5-8) with CT DNA were carried out by using various biophysical techniques, which showed high binding affinity of 7 and 8 towards CT DNA. Gel electrophoresis pattern demonstrated that the compounds are efficient artificial nuclease and prefers minor groove binding site. The mechanistic pattern showed that compounds follow hydrolytic pathway for DNA cleavage. In vitro antibacterial screening of these tetrazine-thiones (5-8) gives remarkable results both against gram-positive and gram-negative strains. These compounds also possess moderate antifungal activity when compared with the standard drugs.

Full text of DOI:10.1016/j.molstruc.2012.03.049

Journal of Molecular Structure 1020 (2012) 33–40
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Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis of Aryl-1,2,4,5-tetrazinane-3-thiones, in vitro DNA binding studies,
nuclease activity and its antimicrobial activity
a,
a
a
b
b
Sartaj Tabassum a, , Mehtab Parveen , Akhtar Ali , Mahboob Alam , Anis Ahmad , Asad U Khan ,
⇑
⇑
Rais Ahmad Khan a
a Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, India
b Interdisciplinary Biotechnology Unit, Aligarh Muslim University Aligarh 202 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of small molecules with a tetrazine scaffold (5–8) was designed and synthesized as metal free
DNA cleaving agent. These compounds (5–8) were characterized by using various spectroscopic (via;
IR, 1H, 13C NMR and ESI-MS) and analytical methods. The interaction studies of (5–8) with CT DNA were
carried out by using various biophysical techniques, which showed high binding affinity of 7 and 8
towards CT DNA. Gel electrophoresis pattern demonstrated that the compounds are efficient artificial
nuclease and prefers minor groove binding site. The mechanistic pattern showed that compounds follow
hydrolytic pathway for DNA cleavage. In vitro antibacterial screening of these tetrazine-thiones (5–8)
gives remarkable results both against gram-positive and gram-negative strains. These compounds also
possess moderate antifungal activity when compared with the standard drugs.
Received 16 January 2012
Received in revised form 22 March 2012
Accepted 22 March 2012
Available online 29 March 2012
Keywords:
Heterocyclic
Tetrazines
Nuclease activity
Antimicrobial
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
because of serious issues over the Lability and toxicity produced
due to the free radical generation of some transition metal during
Heterocyclic chemistry, as an applied science is an inexhaust-
ible resource of novel compounds [1]. The majority of pharmaceu-
tical products that mimic natural products with biological activity
are heterocycles. Therefore, researchers are on a continuous pur-
suit to design and produce better pharmaceuticals, by following
natural models. In the past decade, numerous small molecules pos-
sessing azine scaffold have been shown to exhibit a great variety of
pharmacological effects. Various reports have been published on
the application of such molecules, such as 5-lipoxygenase (5-LO)
inhibitors [2], herbicides, bactericides, fungicides, antimicrobials
[3], and gonadotropin-releasing hormone receptor (GnRH-R)
antagonists [4]. Even for fused azine compounds not only antitu-
mor and antimetastatic activities against a wide range of cancer
cells but also kinase inhibiting activities could be observed [5]. In
2007, cancer accounted for about 7.9 million death cases (around
13% of all deaths). Deaths from cancer worldwide are projected
to continue rising, with an estimated 12 million deaths in 2030 [6].
In the field of molecular biology and drug development, the
cleaving agents of nucleic acid have attracted extensive attention
due to their potential applications [7]. Metal complexes have been
widely investigated as cleaving agents of nucleic acids and found to
be reasonably efficient [8], but their use in pharmacy is restricted
the redox processes [9]. Göbel and co-workers have put forward
the idea of metal-free cleavage agents. Although, the metal-free
cleaving agents studies were mainly applied to active phosphodi-
esters, for example, nucleic acid mimic and RNA [10]. Under uncat-
alyzed physiological conditions, the phosphodiester bonds of DNA
are extremely stable and the half life of DNA for hydrolysis is esti-
mated to be about 200 million years [11]. Thus, developing new
metal free molecules for DNA cleavage is an area of active research.
For the nucleic acid hydrolysis, the key functionality at the ac-
tive site in natural nuclease via, staphylococcal nuclease (SNase)
[12] and bovine pancreatic ribonuclease (RNase A) [13], is the po-
sitive charge group (guanidinium or ammonium), because of the
binding ability to negative groups through hydrogen bonding and
electrostatic interaction [14] in biological molecules with the phos-
phate of DNA or RNA [15,16]. It is reported in literature, that some
of the compounds with positive charge groups (ammonium or
guanidinium) as nuclease mimics for cleavage of phosphodiester,
among those few of them were identified as efficient cleavers of
RNA [9,17]. Furthermore, as ammonium or guanidinium group is
positively charged, the corresponding compounds might also be
with a reasonable aqueous solubility which could be helpful for
cell infiltration at physiological conditions.
Herein, we report the synthesis of novel Aryl-1,2,4,5-tetraz-
inane-3-thiones (5–8) as metal free cleaving agents. The presence
of ANH and AOH groups in the molecules can cooperatively
participate in the interaction with DNA via hydrogen bonding.
⇑
Corresponding authors. Mobile: +91 9358255791 (S. Tabassum).
E-mail addresses: tsartaj62@yahoo.com (S. Tabassum), mehtab.organic2009@
gmail.com (M. Parveen).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.03.049

34
S. Tabassum et al. / Journal of Molecular Structure 1020 (2012) 33–40
Furthermore, these compounds have also been screened for anti-
bacterial and antifungal activity. It is indeed gratifying to note that
these compounds (5–8) show significantly good antibacterial
activity against both gram (+ve) as well as gram (Àve) and fungal
strains, selected for the study.
22.3 (CH3), 76.8 (HNACANH), 117.6–159.2 (6C, Caromatic), 178.3
(C@S); MS ES+: [M + H]+Á m/z: 225.
2.2.4. 6-(2,4-Dihydroxyphenyl)-6-methyl-1,2,4,5-tetrazinane-3-
thione (8)
Yellow solid; yield: 75%, m.p. 260–270 °C; Anal. Calc. for
C9H12N4O2S; C, 44.99; H, 5.03; N, 23.32; O, 13.32; S, 13.4; found:
C, 45.03; H, 5.09; N, 23.37; O, 7.15; S, 14.39; IR (KBr)
2. Experimental
t :
cmÀ1
3384 (N–H), 3549, 3555 (AOH aromatic),1635 (conjugated C@C),
1457 (NAN), 1520 (CAN), 1185 cmÀ1 (C@S); for 1H NMR (CDCl3):
1.58 (s, 3H), 4.46 (br, 2H, NHACANH), 6.51–7.45 (m, 3Haromatic),
8.96 (s, 2H, ANHACSANH), 9.10, 9.16 (s, 2H, PhAOH) and 13C
NMR (CDCl3);); 22.5 (CH3), 76.9 (HNACANH), 125.3–158.1 (6C,
Caromatic), 178.7 (C@S).MS ES+: [M + 2H]+Á m/z:242.
2.1. Materials and methods
Melting points were measured on a digital Kofler block appara-
tus and are uncorrected. IR spectra were recorded on Interspec
2020 FT-IR Spectrometer SpectroLab in KBr and only noteworthy
absorptions are noted, its values are given in cmÀ1 1H and 13C
.
NMR spectra were run in CDCl3 on a Bruker Avance 400 MHz
instrument with TMS as internal standard and its values are given
in ppm (d). Mass spectra were recorded on a JEOL D-300 mass
spectrometer. Thin layer chromatography (TLC) plates with
0.5 mm layer of silica gel G and exposed to iodine vapors to check
the purity as well as the progress of reaction. All the chemicals
used were of analytical grade and were purchased from Sigma Al-
drich, Merck and Otto-kemi Pvt. Ltd. The solvents were purified
prior to use.
2.3. Binding studies
DNA binding experiments that include absorption spectral
traces, luminescence experiments conformed to the standard
methods [18,19] and practices previously adopted by our labora-
tory [20,21].
2.4. Nuclease activity
Cleavage experiments were performed with the help of Axygen
electrophoresis supported by Genei power supply with a potential
range of 50–500 V, visualized and photographed by Vilber-INFIN-
ITY gel documentation system. Cleavage experiments of super-
2.2. General method for the preparation of Aryl-1,2,4,5-tetrazinane
-3-thiones
To a solution of acetophenone (1–4) (10.0 mmol) in ethanol
(15 ml) was added thiocarbohydrazide (TCH) (10 mmol) in acetic
acid (20 ml) and the reaction mixture was stirred at room temper-
ature for 2–3 h. The yellowish/brownish colored precipitate so ob-
tained was filtered out and washed thoroughly with water, cold
ether and air dried. It was crystallized with chloroform ethanol
to obtain the corresponding pure solid compound (5–8).
coiled pBR322 DNA (300 ng) by 7 and 8 (10–50 lM) in (5 mM
Tris–HCl/50 mM NaCl), buffer at pH 7.2 were carried out and the
reaction followed by agarose gel electrophoresis. The samples were
incubated for 1 h at 37 °C. A loading buffer containing 25% bromo-
phenol blue, 0.25% xylene cyanol, 30% glycerol was added and elec-
trophoresis was carried out at 60 V for 1 h in Tris–HCl buffer using
1% agarose gel containing 1.0 lg/mL ethidium bromide. The reac-
tion was also monitored upon addition of various radical inhibitors
and/or activators such as DMSO, tert-butyl alcohol (TBA), sodium
azide (NaN3), superoxide dismutase (SOD), mercaptopropionic acid
(MPA), glutathione (GSH), H2O2; groove binders-methyl green and
DAPI.
2.2.1. 6-Methyl-6-phenyl-1,2,4,5-tetrazinane-3-thione (5)
White colored solid; yield: 65%, m.p. 210–212 °C; Anal. Calc. for
C9H12N4S; C, 51.90; H, 5.81; N, 26.90; S, 15.39; found: C, 51.84; H,
5.86; N, 26.95; S, 15.45; IR (KBr)
t
cmÀ1: 3382 (NAH), 1635 (con-
jugated C@C), 1515 (CAN), 1455 (NAN), 1179 (C@S); for 1H NMR
(ppm, CDCl3): 1.75 (s, 3H), 4.32 (br, 2H, NHACANH), 7.52–7.91
(m, 5Haromatic), 8.77 (s, 2H, ANHACSANH) and 13C NMR (CDCl3);
21.2 (CH3), 74.7 (HNACANH), 126.2–149.0 (6C, Caromatic), 178.1
(C@S). MS ES+: [M]+Á m/z: 208.
2.5. Antimicrobial activity
2.5.1. Antibacterial studies
The newly prepared compounds were screened for their anti-
bacterial activity against Escherichia coli (ATCC-25922), Methicillin
resistant Staphylococcus aureus (MRSA + Ve), Pseudomonas aerugin-
osa (ATCC-27853), Streptococcus pyogenes and Klebsiella pneumo-
niae (Clinical isolate) bacterial strains by disk diffusion method
[22,23]. A standard inoculums (1–2 Â 107 c.f.u./ml 0.5 McFarland
standards) was introduced onto the surface of sterile agar plates,
and a sterile glass spreader was used for even distribution of the
inoculums. The disks measuring 6 mm in diameter were prepared
from Whatman No. 1 filter paper and sterilized by dry heat at
140 °C for 1 h. The sterile disks previously soaked in a known con-
centration of the test compounds were placed in nutrient agar
medium. Solvent and growth controls were kept. Ciprofloxacin
was used as positive control whereas DMSO was used as negative
control. The plates were inverted and incubated for 24 h at 37 °C.
The susceptibility was assessed on the basis of diameter of zone
of inhibition against gram-positive and gram-negative strains of
bacteria. Inhibition zones were measured and compared with the
controls.
2.2.2. 6-(3-Hydroxyphenyl)-6-methyl-1,2,4,5-tetrazinane-3-thione (6)
Cream colored solid; yield: 65%, m.p. 255–257 °C; Anal. Calc. for
C9H12N4OS; C, 48.18; H, 5.40; N, 24.88; O, 7.15; S, 14.25; found: C,
48.20; H, 5.44; N, 24.94; O, 7.12; S, 14.35; IR (KBr)
t
cmÀ1: 3385
(N–H), 3549 (AOH aromatic), 1637 (conjugated C@C), 1460
(NAN), 1517 (CAN), 1185 (C@S); for 1H NMR (CDCl3-
400 MHz ppm): 1.51 (s, 3H), 4.52 (br, 2H, NHACANH), 6.76–7.21
(m, 4Haromatic), 9.10 (s, 2H, ANHACSANH), 9.98 (s, 1H, PhAOH)
and 13C NMR (CDCl3); 22.1 (CH3), 76.2 (HNACANH), 116.7–148.4
(6C, Caromatic), 178.3 (C@S). MS ES+[M + Na]+Á m/z: 247.
2.2.3. 6-(4-Hydroxyphenyl)-6-methyl-1,2,4,5-tetrazinane-3-thione
(7)
Yellow solid; yield: 70%, m.p. 253–255 °C; Anal. Calc. for
C9H12N4OS; C, 48.18; H, 5.40; N, 24.88; O, 7.15; S, 14.35; found:
C, 48.22; H, 5.35; N, 24.92; O, 7.12; S, 14.40; IR (KBr)
t :
cmÀ1
3385 (N–H), 3549 (AOH aromatic),1639 (conjugated C@C), 1457
(NAN), 1520 (CAN), 1185 cmÀ1 (C@S); for 1H NMR (CDCl3): 1.65
(s, 3H), 4.45 (br, 2H, NHACANH), 6.79–7.45 (m, 4Haromatic), 9.14
(s, 2H, ANHACSANH), 9.85 (s, 1H, PhAOH) and 13C NMR (CDCl3):
Minimum inhibitory concentrations (MICs) were determined by
broth dilution technique. The nutrient broth, which contained log-
arithmic serially two fold diluted amount of test compound and

S. Tabassum et al. / Journal of Molecular Structure 1020 (2012) 33–40
35
controls were inoculated with approximately 5 Â 105 c.f.u./ml of
actively dividing bacteria cells. The cultures were incubated for
24 h at 37 °C and the growth was monitored visually and spectro-
photometrically. To obtain the minimum bacterial concentration
(MBC), 0.1 ml volume was taken from each tube and spread on agar
plates. The number of c.f.u. was counted after 18–24 h of incuba-
tion at 35 °C. MBC was defined as the lowest drug concentration
at which 99.9% of the inoculums were killed.
NMR, 13C NMR, ESI MS) and analytical methods. IR spectrum of
the compounds (5–8) exhibited important diagnostic bands at
3382–3385 cmÀ1 and 1179–1185 cmÀ1 attributed to NAH and
C@S stretching frequencies respectively. Moreover the absorption
band at 3500–3549 cmÀ1 along with aromatic overtone was as-
cribed to the aromatic phenols. The lack of band for carbonyl group
in the IR spectra of all the synthesized compounds and appearance
of weak bands at 1455–1460 cmÀ1 (NAN) gave a clear signal for
the formation of 1,2,4,5-tetrazine-3-thiones [25].
The 1H NMR spectra of the title compounds displayed signal asso-
ciated with the methyl protons attached to benzyl carbon as a sharp
singlet’s integrating for three protons at around 1.75–1.51 ppm. A
broad singlet’s integrating for two protons each observed at
ꢀ4.32–4.52 ppm was assigned to NHACANH. The downfield singlet
of two protons at 9.10–8.77 ppm was attributed to NHACSANH and
is due to the influence of C@S groups. The phenolic AOH in case of
(6–8) was resonating as in independent singlets at 9.98 ppm,
9.85 ppm and 9.10, 9.16 ppm respectively. Aromatic protons were
2.5.2. Antifungal studies
Antifungal activity was also done by disk diffusion method. For
assaying antifungal activity Candida albicans, Aspergillus fumigatus,
Penicillium marneffei and Trichophyton mentagrophytes (recultured)
in DMSO by agar diffusion method [22–24]. Sabourands agar media
was prepared by dissolving peptone (1 g), D-glucose (4 g) and agar
(2 g) in distilled water (100 ml) and adjusting pH to 5.7. Normal sal-
ine was used to make a suspension of spore of fungal strain for law-
ning. A loopful of particular fungal strain was transferred to 3 ml
saline to get a suspension of corresponding species. Twenty millili-
ters of agar media was poured into each Petri dish. Excess of suspen-
sion was decanted and the plates were dried by placing in an
incubator at 37 °C for 1 h. Using an agar punch, wells were made
and each well was labeled. A control was also prepared in triplicate
and maintained at 37 °C for 3–4 days. The fungal activity of each
compound was compared with Greseofulvin as standard drug. The
cultures were incubated for 48 h at 35 °C and the growth was mon-
itored. To obtain the minimum fungicidal concentration (MFC),
0.1 ml volume was taken from each tube and spread on agar plates.
The number of c.f.u. was counted after 48 h of incubation at 35 °C.
MFC was defined as the lowest drug concentration at which 99.9%
of the inoculums were killed. The minimum inhibitory concentra-
tion and minimum fungicidal concentration are given in Table 4.
appeared as a multiplet in each case at around 7.52–6.51 ppm. 13
C
NMR signals are in good agreement with proposed structure of the
prepared compounds and the characteristic signal exhibited at
178.1–178.7 ppm due to C@S. The mass spectral data of these com-
pounds showed molecular ion peaks M+Á at m/z 208 for compound
(5), m/z 225 (M + H)+ for compound (6), at m/z 247 (M + Na)+ for
compound (7), and at m/z 242 (M + 2H)+ for compound (8).
3.2. Binding studies
3.2.1. Absorption titration
The interaction of Aryl-1,2,4,5-tetrazinane-3-thiones (5–8) with
CT DNA was monitored by spectrophotometric titrations in 5 mM
Tris–HCl/50 mM NaCl, buffer of pH = 7.4 at 25 °C. The UV spectra
of the compounds in methanol showed a significant absorption
bands at 205, 260 nm (5), 205, 297 nm (6), 205, 307 nm (7), 206,
3. Results and discussion
254 and 327 nm (8), these bands are attributed to p–
p⁄ transitions.
3.1. Characterization
The absorption spectra of compounds (5–8), in presence and in the
absence of CT-DNA are shown in Fig. 1a–d. On increasing the con-
centration of CT DNA, there was concomitant increase in the absorp-
tion intensity – ‘hyperchromism’ up to 25–40% with a blue shift of
2–4 nm. This strong ‘‘hyperchromic’’ effect with a moderate red
shift is suggestive of the compounds possessing a higher propensity
All the four compounds (Scheme 1) were prepared by stirring
acetophenone/substituted acetophenones and thiocarbohydrazide
(TCH) in the mixture of ethanol and acetic acid. The structures of
these compounds were characterized by spectroscopic (IR, 1H
Scheme 1. Synthesis of novel Aryl-1,2,4,5-tetrazinane-3-thiones.

36
S. Tabassum et al. / Journal of Molecular Structure 1020 (2012) 33–40
(a) Compound 5
(b) Compound 6
(c) Compound 7
(d) Compound 8
Fig. 1. Variation of UV–vis absorption for tetrazines (a) compound 5, (b) compound 6, (c) compound 7 and (d) compound 8, with increase in the concentration of CT DNA in
buffer 5 mM Tris –HCl/50 mM NaCl, pH = 7.2 at room temperature.
for DNA binding via non-intercalative binding via, electrostatic and/
or hydrogen bonding. Nevertheless, DNA double helix possesses
many hydrogen bonding sites positioned on the edges of the DNA
bases, it is quite amenable that the ANH groups and AOH groups
of the compounds participate in hydrogen bonding with the DNA
base pairs [26]. Thus, these compounds bind and electrophilically
activate the anionic phosphodiester through hydrogen bonding
and electrostatic interaction, whereas hydroxyl groups working as
nucleophilic group in the intermolecular transphosphorylation
[27].
3.2.2. Fluorescence studies
3.2.2.1. Steady-state emission titration. The emission spectra of the
compound (5–8) in the absence and in presence of increasing
amounts of CT-DNA are depicted in Fig. 2a–d. In the absence of
CT-DNA, all four compounds emits moderate luminescence in
Tris–HCl buffer at 25 °C with a maxima appearing at 290–
340 nm when excited at 260–327 nm. However, the subsequent
addition of CT-DNA from 0 to 0.33 Â 10À4 M causes gradual
enhancement in the fluorescence intensity of the compounds with
no apparent change in the shape and position of the emission
bands. This implies that the compounds strongly interact with
CT-DNA probably due to the inaccessibility of the solvent water
molecules to reach the hydrophobic environment inside the DNA
helix, and the mobility of the compounds is restricted at the
binding site ultimately leading to decrease in vibrational mode
of relaxation. However, this enhancement is much less as
compared to classical intercalators [28,29]. The binding constant
(K) estimated for compounds 5–8 by Scatchard equation were
7.92 Â 103 MÀ1, 1.31 Â 104 MÀ1, 1.99 Â 104 MÀ1, and 3.5 Â 104
MÀ1 respectively.
To study quantitatively, the binding ability of 5–8 compounds
with CT DNA, the intrinsic binding constant Kb values were deter-
mined. The binding constant Kb value follows the order
8
(6.72 Â 104 MÀ1) > 7 (5.40 Â 104 MÀ1) > 6 (1.99 Â 104 MÀ1) > 5
(0.76 Â 103 MÀ1). From the results of the binding constants, it
was concluded that the compound 8 revealed a stronger binding
affinity for DNA double helix. The relative difference in the Kb val-
ues can be associated with the number and the position of AOH
groups in the compounds, which facilitates the hydrogen bonding
and provides better binding affinity.

S. Tabassum et al. / Journal of Molecular Structure 1020 (2012) 33–40
37
Wavelength (nm)
Wavelength (nm)
(b) Compound 6
(a) Compound 5
Wavelength (nm)
(c) Compound 7
Wavelength (nm)
(d) Compound 8
Fig. 2. Emission spectra of tetrazines (a) compound 5, (b) compound 6, (c) compound 7 and (d) compound 8, in the absence and in presence of CT DNA in buffer 5 mM Tris–
HCl/ 50 mM NaCl, pH = 7.2 at 25 oC.
3.2.2.2. Ethidium bromide displacement assay. To further investigate
the mode and extent of binding of compounds (5–8), ethidium bro-
mide displacement assay was carried out [30,31]. The molecular
fluorophore EthBr emits intense fluorescence in presence of CT
DNA due to its strong intercalation between the adjacent DNA base
pairs. Addition of second molecule, which binds to DNA more
strongly than EthBr, would quench the DNA-induced EthBr by
either replacing the EthBr and/or by accepting the excited-state
electron of the EthBr through a photoelectron transfer mechanism
[32]. The extent of emission quenching of the EthBr bound to DNA
would reflect the extent of DNA binding affinity of compounds. On
addition of 5–8, to CT DNA pretreated with EthBr ([DNA]/[Eth-
Br] = 1) a decrease in emission intensity was observed. The emis-
sion intensity of EthBr-DNA in the absence and in presence of 5–
8, is depicted in Fig. 3a–d. As there is no complete quenching of
emission intensity of the EthBr-DNA adduct induced by all the
compounds, thus the intercalative mode of binding is ruled out.
The non-replacement based quenching has been suggested with
DNA-mediated electron transfer from the excited ethidium bro-
mide to acceptor compounds. Furthermore, quantitatively quench-
ing extent i.e.; Ksv was evaluated for compounds 5–8 by following
values obtained follows the order 8 > 7 > 6 > 5. These results are
consistent with the findings obtained from absorption spectral
studies.
3.3. Nuclease activity
The DNA cleaving ability of compound 7 and 8 was studied by
agarose gel electrophoresis using supercoiled pBR322 plasmid
DNA as a substrate [33,34]. The activity of 7 and 8 was assessed
by the conversion of DNA from Form I to Form II or Form III. A con-
centration-dependent DNA cleavage by 7 and 8 was performed.
With the increase of concentration of 7 and 8, DNA was converted
from Form I (supercoiled form) to Form II (nicked circular form),
however no conversion to form III (Linear form) was observed in
both the compounds. At a 30
cient nuclease activity whereas 8 showed significantly good activ-
ity at 20 M. At still higher concentrations there was complete
lM concentration, 7 exhibited effi-
l
conversion of SC form into NC form as depicted in Figs. 4 and 5.
To predict the mechanism of pBR322 plasmid DNA cleavage by
7 and 8, comparative reactions were carried out in the presence of
various radical inhibitors or trappers [35,36] such as singlet oxygen
scavenger sodium azide (NaN3), hydroxyl radical scavengers via;
dimethylsulfoxide (DMSO) t-butyl alcohol (TBA) and superoxide
the Stern–Volmer equation and were found to be 1.9 Â 102 MÀ1
,
2.56 Â 103 MÀ1, 3.68 Â 103 MÀ1, 5.56 Â 104 MÀ1, respectively, the

38
S. Tabassum et al. / Journal of Molecular Structure 1020 (2012) 33–40
Wavelength (nm)
Wavelength (nm)
(a)
(b)
Wavelength (nm)
Wavelength (nm)
(c)
(d)
Fig. 3. Quenching spectra of ethidium bromide bound DNA, with increasing concentration of tetrazines (a) compound 5, (b) compound 6, (c) compound 7 and (d) compound
8, in buffer 5 mM Tris–HCl/ 50 mM NaCl, pH = 7.2 at room temperature.[Complex] (6.60–33.30) lM.
Form II
Form I
CT
NaN3 DMSO TBA SOD
CT 10
20
30
40
50 µM
Fig. 4. Agarose gel electrophoresis patterns of pBR322 plasmid DNA (300 ng)
cleaved by tetrazine (7) (10–50 M), after 1 h incubation time (concentration
dependent) Lane 1: DNA control; Lane 2: 10 M 7 + DNA; Lane 3: 20 M 7 + DNA;
Lane 4: 30 M 7 + DNA. Lane 5: 40 M 7 + DNA; Lane 6: 50
(5 mM Tris –HCl/50 mM NaCl, pH = 7.2 at 25 °C).
Fig. 6. Agarose gel electrophoresis pattern for the cleavage of pBR322 supercoiled
DNA (300 ng) by 7 (30 M), in presence of different radical scavengers. Lane 1: DNA
control; Lane 2: 7 + NaN3 + DNA. Lane 3: 7 + DMSO + DNA; Lane 4: 7 + t-butyl
alcohol + DNA; Lane 5: 7 + SOD + DNA, in buffer (5 mM Tris –HCl/ 50 mM NaCl,
pH = 7.2 at 25 °C).
l
l
l
l
l
l
l
M 7 + DNA, in buffer
Form II
Form I
CT
NaN3 DMSO TBA SOD
CT
10
20
30
40
50 µM
Fig. 5. Agarose gel electrophoresis patterns of pBR322 plasmid DNA (300 ng)
cleaved by (8) (10–50 M), after 1 h incubation time (concentration dependent)
Lane 1: DNA control; Lane 2: 10 M 8 + DNA; Lane 3: 20 M 8 + DNA; Lane 4:
30 M 8 + DNA. Lane 5: 40 M 8 + DNA; Lane 6: 50 M 8 + DNA, in buffer (5 mM
Tris –HCl/50 mM NaCl, pH = 7.2 at 25 °C).
Fig. 7. Agarose gel electrophoresis pattern for the cleavage of pBR322 supercoiled
DNA (300 ng) by compound 8 (30 M) in presence of different radical scavengers.)
Lane 1: DNA control; Lane 2: 8 + NaN3 + DNA. Lane 3: 8 + DMSO + DNA; Lane 4:
8 + t-butyl alcohol + DNA; Lane 5: 8 + SOD + DNA, in buffer (5 mM Tris –HCl/ 50 mM
NaCl, pH = 7.2 at 25 °C).
l
l
l
l
l
l
l

S. Tabassum et al. / Journal of Molecular Structure 1020 (2012) 33–40
39
Table 3
Antifungal activity of Tetrazines (5–8) Positive control (greseofulvin) and negative
control (DMSO) measured by the Halo Zone Test (Unit, mm). Diameter of zone of
inhibition (mm).
Compounds
CA
AF
TM
PM
5
6
7
8
19.1 0.2
18.5 0.4
17.2 0.2
16.2 0.5
30.0 0.2
–
20.6 0.2
18.9 0.2
18.2 0.4
17.2 0.2
27.0 0.2
–
18.5 0.3
18.4 0.4
17.1 0.2
16.0 0.5
24.0 0.3
–
13.9 0.4
13.2 0.2
13.1 0.3
13.2 0.1
20.0 0.5
–
CT
DAPI
MG
Fig. 8. Agarose gel electrophoresis pattern for the cleavage of pBR322 supercoiled
DNA (300 ng) by 7, in presence of minor groove (DAPI) and major groove binding
agent (methyl green).
Standard
DMSO
⁄CA; Candida albicans, AF; Aspergillus fumigatus, TM; Trichophyton mentagrophytes,
PM; Penicillium marneffei.
Table 4
MIC and MFC results of Tetrazines (5–8) positive control, Greseofulvin.
Compounds
CA
AF
TM
PM
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
CT
DAPI
MG
5
6
7
8
12.5
12.5
12.5
25
25
25
25
50
50
12.5
12.5
25
25
12.5
25
25
100
100
25
12.5
12.5
25
50
6.25
25
25
50
100
25
12.5
12.5
50
50
12.5
25
100
100
100
25
Fig. 9. Agarose gel electrophoresis pattern for the cleavage of pBR322 supercoiled
DNA (300 ng) by 8, in presence of minor groove (DAPI) and major groove binding
agent (methyl green).
Standard
6.25
⁄MIC (
the compound to inhibit the growth of fungus completely.
lg/ml) = minimum inhibitory concentration, i.e. the lowest concentration of
c
MFC(lg/ml) = minimum fungicidal concentration, i.e., the lowest concentration of
Table 1
the compound for killing the fungus completely.
Antibacterial activity of Tetrazines (5–8).
Compounds Diameter of zone of inhibition (mm)
Gram positive bacteria Gram negative bacteria
radical can also be ruled out in both the compounds. Therefore,
DNA cleavage promoted by 7 and 8 might mainly (be or come)
through the non-oxidative pathway thus it is very possible that
the phosphodiester bond of DNA would have been cleaved by 7
and 8 via phosphoryl transfer reactions [10,15].
To probe the interacting site of 7 and 8 with pBR322 plasmid
DNA, The DNA was treated with DAPI or methyl green [37,38] prior
to the addition of 7 and 8. DAPI (minor groove binder) was added
to the reaction mixture, significant inhibition was observed in the
cleavage pattern. Whereas in presence of methyl green (major
groove binder), the cleavage was not suppressed (Figs. 8 and 9).
Thus electrophoretic pattern demonstrates that both compounds
7 and 8 shows preferential affinity towards the minor groove of
the DNA.
S.
MRSA*
P.
K.
E. coli
Pyogenes
aeruginosa pneumoniae
5
6
7
8
22.1 0.2 19.4 0.8
21.3 0.3 19.3 0. 6 24.3 0.3
20.5 0.5 19.2 0.6
20.4 0.5 18.5 0.5
23.0 0.2 22.0 0.2
27.1 0.6
18.6 .3
18.4 0.4
18.2 0.2
17.8 0.5
19.0 0.3
–
24.3 0.5
23.9 0.3
22.7 0.2
21.9 0.5
27.0 0.4
–
24.1 0.6
22.8 0.4
32.0 0.2
–
Standard
DMSO
–
–
Positive control (standard); ciprofloxacin and negative control (DMSO) measured by
the Halo Zone Test (Unit, mm).
Methicillin resistant Staphylococcus aureus (MRSA + Ve).
*
dismutase (SOD) as superoxide anion inhibitor as shown in Figs. 6
and 7. When the hydroxyl radical inhibitor DMSO and TBA were
added to the reaction mixture no evident inhibition of the nuclease
activity was observed, suggesting non involvement of hydroxyl
radical in the cleavage process. Similarly, in the presence of radical
scavengers like NaN3 and SOD, the cleavage showed no inhibition
in the presence of sodium azide with 7 and 8, thus the presence
of singlet oxygen can be ruled out where as in case of SOD the
cleavage was not subdued therefore the presence of superoxide
3.4. Antimicrobial activity
The in vitro antimicrobial activity of tetrazine-thiones deriva-
tives (5–8) were tested using the bacterial culture of S. pyogenes,
MRSA, P. aeruginosa, K. peneumoniae, E. coli and fungal culture of
C. albicans, A. fumigatus, T. mentagrophytes, P. marneffei. The
in vitro study results demonstrated that all the compounds were
Table 2
MIC and MBC results of Tetrazines (5–8) positive control, ciprofloxacin.
Compounds
Gram positive bacteria
Gram negative bacteria
S. Pyogenes
MRSA*
P.aeruginosa
K. pneumoniae
E. coli
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
5
6
7
8
12.5
12.5
25
25
12.5
25
25
50
50
25
12.5
12.5
25
25
6.25
50
25
100
100
12.5
25
25
50
50
50
50
100
100
25
12.5
25
25
25
6.25
25
50
100
100
25
12.5
25
25
25
6.25
25
50
50
50
25
Standard
12.5
c
MBC (
MIC (
l
g/ml) = minimum bacterial concentration, i.e., the lowest concentration of the compound for killing the bacteria completely.
*
lg/ml) = minimum inhibitory concentration, i.e. the lowest concentration of the compound to inhibit the growth of bacteria completely.

40
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DNA binding studies revealed that these compounds interact with
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Acknowledgement
The CSIR, New Delhi, is also thanked for the award of a Research
Associateship to M.A.
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