Copper(II) chloride mediated synthesis and DNA photocleavage activity of 1-aryl/heteroaryl-4-substituted-1,2,4-triazolo[4,3-a]quinoxalines

Article abstract of DOI:10.1016/j.ejmech.2011.10.032

A new class of photonucleases, 1-aryl/heteroaryl-4-substituted-1,2,4- triazolo[4,3-a]quinoxalines (4) was synthesized in a facile and efficient manner via copper(II) chloride mediated oxidative intramolecular cyclization of 2-(arylidenehydrazino)-3-substi

Full text of DOI:10.1016/j.ejmech.2011.10.032

European Journal of Medicinal Chemistry 46 (2011) 6083e6088
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Copper(II) chloride mediated synthesis and DNA photocleavage activity
of 1-aryl/heteroaryl-4-substituted-1,2,4-triazolo[4,3-a]quinoxalines
,
a,
Ranjana Aggarwal a , Garima Sumran 1, Virender Kumar a, Ashwani Mittal b
*
a Department of Chemistry, Kurukshetra University, Kurukshetra 136 119, India
b Biochemistry Department, University College, Kurukshetra University, Kurukshetra 136 119, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of photonucleases, 1-aryl/heteroaryl-4-substituted-1,2,4-triazolo[4,3-a]quinoxalines (4) was
synthesized in a facile and efficient manner via copper(II) chloride mediated oxidative intramolecular
cyclization of 2-(arylidenehydrazino)-3-substituted-quinoxalines (3). DNA cleavage potency of
Received 31 August 2011
Received in revised form
4 October 2011
Accepted 14 October 2011
Available online 21 October 2011
compounds 4aed (40
mg each) was quantitatively evaluated on supercoiled plasmid FX174 under UV
irradiation (312 nm, 15 W) without any additive. Compound 4c was found to be the most efficient DNA
photocleaver which had converted supercoiled DNA (form I) into the relaxed DNA (form II) at 30 mg and
the DNA photocleavage activity increases with increase in concentration of 4c.
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
Triazolo[4,3-a]quinoxalines
Copper(II) chloride
Oxidative cyclization
Plasmid DNA
Photocleavage
AM1 calculations
1. Introduction
sequence specific sites of DNA binding drugs and proteins or as site-
directed photonucleases for accessing structural and genetic
information. Wender et al. [7] demonstrated that benzotriazoles
when combined with a DNA recognition subunit can upon photo-
activation cleave DNA via radical mechanism in a potent and
selective fashion. The conjugated C]N bond in aromatic hetero-
The design of novel photonucleases, which selectively cleave
DNA under irradiation with a specific light under mild conditions
without any additives such as metals and reducing agents, is very
interesting from chemical and biological standpoints and offers
a significant potential in medicine especially in post-genome era
[1]. Research in this area has revealed that photocleavage is
a promising approach for new antitumor and antiviral therapeutic
strategies. Several heterocyclic DNA photocleaving molecules such
as thiazo- or thiadiazo-naphthalene carboxamides [2] and isoquino
[4,5-bc]acridines [3] exhibit antitumor activity whereas iso-
quinolino[5,4-ab]phenazine derivatives [4] could be used as topo-
isomerase I targeted antitumor agents. Other photosensitive DNA
cleavers which have been reported include antitumor agents, 3-
amino-1,2,4-benzotriazine 1,4-dioxide, (tirapazamine, WIN 59075,
SR4233) [5] and chlorobithiazoles derivatives structurally related to
bleomycin [6]. In addition, DNA photocleaving molecules find
potential use for the design of photofootprinting agents to map the
cycles was reported to generate the photoexcited [8] (nep*) state,
which would have radical character and could be able to cleave
DNA photochemically [8]. Triostin A and echinomycin, members of
the quinoxaline family of antitumor antibiotics, bind to DNA and
exhibit high level of cytotoxicity [9]. Prompted by these reports, it
seemed worthwhile to synthesize and explore the photochemical
DNA cleaving properties of 1,2,4-triazolo[4,3-a]quinoxalines as this
nucleus exhibits diverse pharmacological activities, viz., antitumor
[10], antimicrobial [11,12], and antidepressants [13].
Traditional routes to 1,2,4-triazolo[4,3-a]quinoxalines include
the reaction of 2-hydrazinoquinoxaline with acid chlorides/acids/
acid anhydrides at elevated temperature [14]; ring closure of 2-
hydrazino-3-chloroquinoxalines with triethyl orthoalkanoates
[14]; pyrolysis of hydrazones [14,15]; cyclodehydration of aroylhy-
drazine with phosphorus oxychloride [15]; reaction of 2-
chloroquinoxaline with mono or diacylhydrazine [14,16]; dehy-
drogenative cyclization of Schiff’s bases with DDQ [17]. Unfortu-
nately, such methods are often limited due to inevitably poor
conversions, multistep synthesis, harsh reaction conditions, long
* Corresponding author. Tel.: þ91 1744238734; fax: þ91 1744238277.
E-mail address: ranjana67in@yahoo.com (R. Aggarwal).
Present address: Technology Education
Kurukshetra 136 119, India.
1
& Research Integrated Institutions,
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.032

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R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 6083e6088
reaction times at higher reaction temperature, side product
formation, sometimes tedious work up procedures, toxic and
expensive reagents. Alternatively, this ring system can be obtained
by oxidative cyclization of hydrazones with oxidant iodobenzene
diacetate [18,19] in order to overcome these limitations. Though
IBD itself is an efficient and environmentally benign oxidant, but it
is usually synthesized in the laboratory by four step procedure
involving harsh reaction conditions, passing of toxic chlorine gas
for hours, expensive reagents and cumbersome reaction work ups
[20]. Moreover, IBD is used in stoichiometric ratio for oxidation
reactions; its molecular weight being very high (MW 322) makes it
a cost-ineffective reagent. Therefore, development of an alterna-
tive, convenient, economically viable oxidant that requires mild
conditions and proceeds to high yields is still in demand.
a sharp singlet around
d 8.5e8.9 ppm for azomethine (N]CH) and
a broad singlet at 9.0e9.4 corresponding to resonance of NH as it
d
was exchangeable with D2O. IR spectra of 4 revealed the disap-
pearance of NH stretch, thus confirming the oxidation of 3 into 4.
An important characteristic feature in the 1H NMR spectra of 4 was
the disappearance of the signals at around
d 8.5e8.9 and 9.0e9.4
corresponding to azomethine (N]CH) and NH, respectively, of its
precursor hydrazones 3, thus indicating the successful triazole ring
formation. Also, 1H NMR spectra of 4aeh exhibited a signal at
d
3.1
for CH3 protons instead of
d 2.5 as shown in the NMR of 3aeh. This
downfield shift may be attributed to the lone pair effect of the
nitrogen of the triazole ring on the methyl protons at position-3 of
quinoxaline ring.
In recent years, CuCl2 (copper(II) chloride) has emerged as
a reagent of choice for various synthetically useful transformations
replacing successfully the mercury (II), thallium (III) and lead (IV)
compounds, due to its similarity in oxidation reactions, low toxicity,
ready availability, ease of handling and inexpensiveness. In this
regard, some recent applications include: CuCl2 catalyzed intra-
molecular CeH oxidation/acylation of formyl-N-arylformamides to
indoline-2,3-diones via O2 as terminal oxidant [21], CuCl2-medi-
ated oxidative cyclization of heterocyclically substituted aldimines
[22] and regioselective C-allylation of enaminones [23]. Herein, we
describe the copper(II) chloride mediated synthesis of fused 1,2,4-
triazolo[4,3-a]quinoxalines and their photocleavage activities.
3. Biological results and discussion
Out of the nine synthesized compounds, four triazolo[4,3-a]
quinoxalines 4aed, bearing 20-chlorophenyl, 20-nitrophenyl, 20-
thienyl and 20-furyl at position-1 were chosen for preliminary DNA
photocleavage studies. The choice of these compounds was made
due to their high solubility in DMSO and on the observation that
these rings play crucial role in cleaving DNA photochemically. It is
known that excitation of a haloarene can lead to homolytic cleavage
of a carbonehalogen bond, thereby generating a phenyl radical
[24], potentially capable of causing single-stranded lesions to DNA.
Photoreactivity of nifedipine (NIF), a nitroaromatic drug used in the
treatment of myocardial ischemia and hypertension, in vitro and in
vivo has been reported in Ref. [25]. Nitrobenzamido ligands linked
2. Chemistry
to 9-aminoacridine were found to photocleave DNA [26].
a-Ter-
2.1. Synthesis
thienyl (2,20:5,200-terthiophene), a naturally occurring secondary
plant metabolite, is an excellent singlet oxygen sensitizer, causes
phototoxicity to a number of insect species and photocleave DNA
[27]. In the present study compounds 4c and d were chosen to
compare the DNA photocleavage activity of thiophene and furan
rings as it has been reported earlier that compounds possessing
a thiono or thio group were found to possess significantly enhanced
DNA photocleavage compared with oxygen-containing counter-
parts [28].
The synthetic pathway of the target compounds (4aei) is out-
lined in Scheme 1. The key intermediate, 2-(arylidenehydrazino)-3-
substituted-quinoxalines (3aei), was prepared by the condensation
of 2-hydrazino-3-methyl/phenylquinoxaline (1a or 1b) with
various aromatic/heteroaromatic aldehydes (2) in refluxing
ethanol. Subsequent oxidative intramolecular cyclization of
a variety of 3 with 2 equiv of copper(II) chloride in absolute DMF
under nitrogen atmosphere provided the desired 1-aryl/heteroaryl-
4-substituted-1,2,4-triazolo[4,3-a]quinoxalines (4) in excellent
yield. The structure of compounds 4 was established by their
elemental analysis, mass and a careful comparison of their IR and
1H NMR spectra with those of their corresponding hydrazones 3.
The UVeVis absorption data of compounds 4aed were shown in
Table 1. It was found that compounds 4a, b, c and d showed
maximum absorption wavelength 358, 360, 344, 351 nm,
respectively.
3.1. DNA photocleavage
2.2. Results and discussion
In our initial explorations, DNA cleaving activities of 4aed
IR spectra of 3 showed characteristic absorption bands due to
the stretching vibrations of NeH and C]N at w3155e3364 and
1597e1642 cmꢀ1, respectively. The 1H NMR spectra of 3 displayed
toward plasmid
FX174 DNA under photoirradiative conditions was
monitored quantitatively by measuring the conversion of native
supercoiled DNA (Form I) via densitometry into the photocleave
product i.e. relaxed circular DNA (Form II) resulting from nicking of
the DNA backbone using Image J Software. These DNAs (Form I & II)
though are similar in their molecular weight, charge and size but
are very different in their conformations. Because of the tight
conformation of supercoiled DNA, it migrates faster through a gel
R'
R'CH
N
N
N
o
N
NHNH2
R
N
R'CHO
2
NH
N
CuCl2, 100 C
DMF, N2
EtOH
N
N
R
N
4
R
1a-b
R = CH3, Ph
3
g
a
e
f
3,4
Cl
c
b
d
h
i
Table 1
Absorption peaks of 4aed.
O2N
Me
Ph
S
O
a
R'
Compd
labs/nm (log 3 )
N
H
N
N
N
N
p-NO2Ph
Ph
4a
4b
4c
4d
255 (3.21), 358 (3.27), 416 (3.25)
255 (3.21), 360 (3.27), 386 (3.12)
261 (3.19), 344 (3.21), 449 (2.64)
257 (3.21), 351 (3.26), 436 (2.86)
F
CH3
Ph
R
CH3
CH3 CH3
CH3
CH3
CH3
CH3
a
Scheme 1. Synthesis of 1,2,4-triazolo[4,3-a]quinoxalines (4aei).
In CHCl3.

R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 6083e6088
6085
1
2
3
4
5
Form II
Form I
II%
I%
4
96
4
96
26
74
53
47
68
32
Fig. 1. Ethidium bromide stained agarose gel (1%) showing the DNA photocleavage of
l (60 g) X174 plasmid (in 0.04 M Triseborate, 0.114% acetic acid and 50 mM EDTA,
Fig. 3. Effect of concentration of compound 4c on the photocleavage of DNA. Lane 1:
control plasmid only þ UV þ DMSO, Lane 2: control DNA þ 40 g 4c in DMSO þ no
irradiation, Lanes 3, 4, 5: DNA þ UV þ 4c at the concentrations of 1.5 l (30 g), 4
(80 g) and 6 l (120 g) in DMSO, respectively.
3
m
m
F
m
pH 8.0, lirr 312 nm, 15 W, 45 min) by 20 mg/ml (DMSO) stock solution of 4a, 4b, 4c and
4d. Lane 1: control plasmid DNA only þ UV þ DMSO, Lane 2: control plasmid DNA þ no
m
m
ml
m
m
m
UV þ 40
m
g 4c in DMSO, Lanes 3e6: DNA þ UV þ 2
ml (40 mg) solution of 4c, 4a, 4b and
4d in DMSO, respectively.
the DNA was irradiated under light in the absence of 4c (lane 1) or
the mixture of DNA and the highest conc. of 4c (40 g) was left in
the dark (lane 2), the amount of the relaxed circular DNA remains
about 4%. With increase in 4c concentration (lanes 3e5) under
same assay conditions, nicked DNA (Form II) increases progres-
m
than relaxed circular DNA and due to the differences in their
migration rate they can be separated easily by gel electrophoresis.
3.2. Results
sively (26% Form II at a concentration as low as 30
mg and 68% form
II at concentration of 120 g) while Form I diminishes compared to
control DNA (lane 1) and dark control (lane 2).
m
Fig. 1 shows the gel electrophoresis pattern of 60
mg FX174
plasmid after incubation with 4 upon irradiation (lirr 312 nm, 15 W,
45 min), from which relative amounts of the two DNA forms were
evaluated (Fig. 2). Two control experiments conducted under same
conditions, (i) whether the DNA (Form I) was incubated with DMSO
3.3. Structureephotocleavage efficiency relationship based on
molecular orbital calculations
(2
ml) and UV radiations in absence of compound 4 (lane 1), (ii) or
incubation of the mixture of DNA and compound 4c (40
m
g,
The minimum energy structures for 4aed have been obtained
by performing geometry optimization using molecular mechanics
method and mmþ force field, which were used further for single
point level calculations. The minimum energy in the most stable
conformation, LUMO and HOMO orbital energies for compounds
4aed were obtained from AM1 force field semi-empirical quantum
calculations, using molecular modeling programs HyperChem 7.0
and are listed in Table 2. The relative UV-induced DNA photonicking
efficiency of compounds 4aed (the order is 4c > 4a > 4d > 4b,
Figs. 1 and 2) was compared with their highest occupied molecular
orbital and the lowest unoccupied orbital (HOMOeLUMO) gaps.
There seems to exist a correlation between relative DNA photo-
cleavage efficiency and the energy gap between LUMO and HOMO
prepared in DMSO) was left in the dark (lane 2), do not result in
DNA damage. However, compounds 4aed caused single strand
nicking of DNA with the conversion of Form I to Form II at
concentrations as low as 40 mg when irradiated by light without any
further additives (lanes 3e6). It was established through control
experiment that in the absence of light (lane 2), no cleavage occurs,
confirming that UV light functioned as a trigger to initiate these
triazoloquinoxaline derivatives for the DNA strand scission.
On irradiation with UV light, 1-(20-thienyl)-4-methyl-1,2,4-
triazolo[4,3-a]quinoxaline (4c, lane 3) resulted in most efficient
DNA cleavage (41% Form II) than that of 4a, 4b and 4d (Fig. 1, lanes
4e6) of the same series and also compared to control DNA where
no DNA damage was observed (lane 1). Therefore, it was decided to
(DELeH). For example, the most prominent DNA photocleaver 4c
study the effect of concentration, varying from 30
m
g to 120
m
g, on
was found to have the largest HOMOeLUMO gap while the least
effective compound 4b has the minimum HOMOeLUMO gap. This
observation is in close agreement with the literature reports
describing the relationship between photocleavage ability and
the photocleavage ability of compound 4c on supercoiled DNA. It is
clearly evident from gel electrophoresis (Fig. 3) and bar graph
(Fig. 4) representing the % of DNA cleavage that DNA-nicking is
directly proportional to concentration of compound 4c. The original
HOMOeLUMO gap [29]. Though
DELeH for compounds 4aed is
plasmid
F
X174 DNA has about 4% relaxed circular DNA and 96%
showing variations, but this difference is not significant enough to
draw a relationship between structure and DNA photocleavage
efficiency of 4aed to their excited-state properties. The observed
fact in the present study that photonicking efficiency of compound
supercoiled DNA (as in lane 1). In the control experiments, whether
120
100
120
100
80
80
Form I
60
Form II
60
40
20
0
Form I
Form II
40
20
0
Cnt 1
Cnt 2
4c
4a
4b
4d
Cnt 1
Cnt 2
4c(30 ug)
4c(80 ug)
4c(120ug)
Fig. 2. Quantitative analysis of relative intensities of cleavage bands obtained by
densitometry assay of lanes 1e6 shown in Fig. 1 using Image J Software. Reported
values reflect the average of three experiments and results are expressed as
mean ꢁ S.D.
Fig. 4. Scanning densitometry results of effect of concentration of 4c on the photo-
cleavage of DNA. Reported values reflect the average of three experiments and results
are expressed as mean ꢁ S.D.

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R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 6083e6088
5.1.1. 2-(20-Chlorobenzylidenehydrazino)-3-methylquinoxaline (3a)
M.p. 170 ꢂC (Lit. [19] m.p. 169e170 ꢂC); yield 92%.
Table 2
The minimum energy and frontier orbitals energies for 4aed.
Compd
Ea
LUMOa
HOMOa
DELeH
5.1.2. 2-(20-Nitrobenzylidenehydrazino)-3-methylquinoxaline (3b)
M.p. 184e186 ꢂC (Lit. [19] m.p. 184e186 ꢂC); yield 92%.
4a
4b
4c
4d
ꢀ3503.517296
ꢀ3665.214847
ꢀ3117.678469
ꢀ3113.266902
ꢀ1.344616
ꢀ1.759671
ꢀ1.180223
ꢀ1.161189
ꢀ9.097906
ꢀ9.275171
ꢀ8.947033
ꢀ8.870054
7.75339
7.51550
7.76681
7.708865
5.1.3. 2-(20-Thiophenemethylidenehydrazino)-3-methylquinoxaline
(3c)
a
E (minimum energy of molecule in least strained conformation, kcal/mol) and
the frontier orbital energies (ev) were obtained from AM1 force field calculations.
M.p. 163e164 ꢂC (Lit. [19] m.p. 162e164 ꢂC); yield 80%.
4c with 20-thienyl moiety was much stronger than its oxygen-
containing counterpart 4d, further supports the earlier observa-
tion that the presence of sulfur promotes DNA photocleaving ability
more efficiently than its oxo-counterpart [28].
Thus, these compounds of 4 represent promising new class of
photonucleases which may be useful as tools for probing DNA
structure and for photodynamic chemotherapy. Further experi-
ments aimed to study the mode of action and sequence specific
photolytic DNA cleavage are under progress.
5.1.4. 2-(20-Furanylmethylidenehydrazino)-3-methylquinoxaline
(3d)
M.p. 162e163 ꢂC (Lit. [19] m.p. 160e162 ꢂC); yield 78%.
5.1.5. 2-(40-Fluorobenzylidenehydrazino)-3-methylquinoxaline (3e)
M.p. 192e193 ꢂC; yield 82%; IR: 3155 (NeH str), 1597 (C]N); 1H
NMR (CDCl3) d: 2.59 (s, 3H, CH3), 7.11e7.13 (m, 1H, quinox-6-H),
7.14e7.19 (t, 2H, Jo ¼ 8.7 Hz, Ph-30, 50-H), 7.36e7.40 (t, 1H,
Jo ¼ 6.9 Hz, quinox-7-H), 7.66e7.69 (d, 1H, Jo ¼ 7.8 Hz, quinox-5-H),
7.82e7.86 (dd, 2H, Jo ¼ 8.7 Hz, J(m)HF ¼ 5.4 Hz, Ph-20, 60-H), 8.11e8.14
(d, 1H, Jo ¼ 8.4 Hz, quinox-8eH), 8.57 (s, 1H, N]CeH), 9.36 (s, 1H,
4. Conclusion
NH); 13C NMR (CDCl3)
d: 21.21, 114.83, 115.76e116.05 (d,
2JCeF ¼ 21.75 Hz, Ph-30, 50-C), 123.05, 128.46, 129.20, 129.74e129.86
(d, 3JCeF ¼ 9.0 Hz, Ph-20, 60-C), 131.06, 131.10, 131.50, 133.47, 146.71,
154.86, 162.46e165.79 (d, 1JCeF ¼ 249.75 Hz, Ph-40-C). HRMS (m/z):
280.1132 (Mþ) (C16H13N4F (Mþ) requires 280.1124). Anal. Calcd. for
C16H13N4F: C, 68.57; H, 4.64; N, 20.0. Found: C, 68.69; H, 4.53; N,
19.87%.
In summary, the present work provides a general and efficient
route to
a
new class of DNA photocleaving fused tri-
azoloquionoxalines 4 by CuCl2 mediated oxidative cyclization of 2-
(arylidenehydrazino)-3-substituted-quinoxalines. The readily
available, non-toxic and inexpensive CuCl2 offers a valuable alter-
native as an oxidizing agent. The data from preliminary studies
revealed that 1,2,4-triazolo[4,3-a]quinoxalines exhibit promising
DNA photocleaving activities and the cleavage efficiency was
substrate and concentration dependent.
5.1.6. 2-(1H-Indol-30-ylmethylidenehydrazino)-3-
methylquinoxaline (3f)
M.p. 202 ꢂC; yield 87.5%; IR: 3297 (NeH str), 1617 (C]N); 1H
5. Experimental
NMR (CDCl3) d: 2.60 (s, 3H, CH3), 7.10e7.24 (m, 2H, quinox-6-H, 7-
H), 7.35e7.46 (m, 4H, Indole-40, 50, 60, 70-H), 7.56 (s,1H, Indole-20-H),
7.60e7.62 (d, 1H, Jo ¼ 7.8 Hz, quinox-5-H), 7.63e7.66 (d, 1H,
Jo ¼ 7.8 Hz, quinox-8-H), 8.55 (s, 1H, N]CeH), 8.84 (s, 1H, Indole-
NH), 9.04 (s, 1H, NH). Anal. Calcd. for C18H15N5: C, 71.76; H, 4.98;
N, 23.25. Found: C, 71.99; H, 5.21; N, 23.65%.
Melting points were determined in open capillaries using
a melting point apparatus and are uncorrected. The IR spectra were
recorded on a Buck Scientific IR M-500 spectrophotometer in KBr
discs (nmax in cmꢀ1). 1H NMR spectra in CDCl3 were recorded on
a Bruker instrument at 300 MHz using TMS as an internal standard.
5.1.7. 2-(10,30-Diphenyl-1H-pyrazol-40-ylmethylidenehydrazino)-3-
methylquinoxaline (3g)
Chemical shifts are recorded in d values and coupling constants J in
Hz. High resolution mass spectra (HRMS) were recorded on a Kratos
MS-50 spectrometer in EI mode. The electronic absorption spectra
M.p. 204e205 ꢂC; yield 76.9%; IR: 3342 (NeH str), 1620 (C]N);
of compounds 4aed (dissolved in chloroform to give 10ꢀ3
M
1H NMR (CDCl3)
d: 2.55 (s, 3H, CH3), 7.03e7.06 (m, 1H, quinox-6-H),
solutions) were recorded on a Smart UV-2202 UVeVis systronics
double beam spectrophotometer (700-200 nm) at room tempera-
ture. Data were reported in lmax/nm. Hydrazines 1aeb were
prepared according to the literature procedure [14]. 1,3-
Diphenylpyrazole-4-carboxaldehyde and 1-(p-nitrophenyl)-3-
methylpyrazole-4-carboxaldehyde were prepared from the appro-
priated phenylhydrazones via the VilsmeiereHaack reaction
according to the literature procedure [30]. Rest aldehydes (2) were
commercially available.
7.14e7.16 (m, 1H, quinox-7-H), 7.31e7.39 (m, 3H, Ph-H), 7.49e7.54
(m, 5H, Ph-H), 7.58e7.61 (m, 2H, Ph-H), 7.64e7.66 (d,1H, Jo ¼ 7.8 Hz,
quinox-5-H), 8.01e8.04 (m, 1H, quinox-8-H), 8.49 (s, 1H, pyrazole-
50-H), 8.66 (s, 1H, N]CeH), 9.30 (s, 1H, NH). Anal. Calcd. for
C25H20N6: C, 74.25; H, 4.95; N, 20.79. Found: C, 74.63; H, 5.21; N,
20.99%.
5.1.8. 2-[30-Methyl-10-(p-nitrophenyl)-1H-pyrazol-40-
ylmethylidenehydrazino]-3-methylquinoxaline (3h)
M.p. 252e253 ꢂC; yield 79.8%; IR: 3342 (NeH str), 1620 (C]N),
5.1. General procedure for the synthesis of 2-(arylidenehydrazino)-
1527 (NO2), 1350 (NO2); 1H NMR (CDCl3) : 2.3 (s, 3H, pyrazole-30-
d
3-substituted-quinoxalines (3aei)
CH3), 2.43 (s, 3H, CH3), 7.09e7.12 (m, 1H, quinox-6-H), 7.15e7.18 (m,
1H, quinox-7-H), 7.58e7.61 (m, 1H, quinox-5-H), 7.69e7.72 (d, 2H,
Jo ¼ 9.0 Hz, Ph-20, 60-H), 8.07e8.10 (m, 1H, quinox-8-H), 8.29e8.31
(d, 2H, Jo ¼ 9.0 Hz, Ph-30, 50-H), 8.54 (s, 1H, pyrazole-50-H), 8.96 (s,
1H, N]CeH), 9.28 (s, 1H, NH). Anal. Calcd. for C20H17N7O2: C, 62.01;
H, 4.39; N, 25.32. Found: C, 61.99; H, 4.38; N, 25.55%.
2-Hydrazino-3-methylquinoxaline (1a) (0.6 g, 3.4 mmol) was
dissolved in 20 ml ethanol containing three drops of glacial acetic
acid and appropriate aldehyde (2) (3.4 mmol). The reaction mixture
was heated under reflux for 1 h and then mixture was allowed to
cool at room temperature. The crude product thus obtained was
collected by filtration, dried and recrystallized from ethanol to
afford 2-(arylidenehydrazino)-3-substituted-quinoxalines 3.
2-Benzylidenehydrazino-3-phenylquinoxaline (3i) was synthe-
sized by the same method.
5.1.9. 2-Benzylidenehydrazino-3-phenylquinoxaline (3i)
M.p. 118e120 ꢂC (Lit. [14] m.p. 118 ꢂC); yield 73%; IR: 3342 (NeH
str), 1636 (C]N); 1H NMR (CDCl3)
d: 7.14e7.21 (m, 2H, quinox-6-H,
7-H), 7.37e7.55 (m, 10H, Ph-H), 7.61e7.64 (d, 1H, Jo ¼ 7.8 Hz,

R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 6083e6088
6087
quinox-5-H), 8.05e8.07 (d, 1H, Jo ¼ 8.4 Hz, quinox-8-H), 8.87 (s, 1H,
5.2.7. 1-(10,30-Diphenyl-1H-pyrazol-40-yl)-4-methyl-1,2,4-triazolo
[4,3-a]quinoxaline (4g)
M.p. 168e170 ꢂC; yield 88%; IR: 1620 (C]N); 1H NMR (CDCl3)
d:
]CeH), 9.40 (s, 1H, NH). Anal. Calcd. for C21H16N4: C, 77.77; H, 4.93;
N, 17.28. Found: C, 77.84; H, 4.96; N, 17.32%.
3.1 (s, 3H, CH3), 7.18e7.26 (m, 2H, quinox-6-H, 7-H), 7.46e7.54 (m,
5H, Ph-H), 7.56e7.59 (m, 3H, Ph-H), 7.68e7.71 (d, 1H, Jo ¼ 8.1 Hz,
quinox-5-H), 7.89e8.01 (m, 2H, Ph-H), 8.09e8.12 (d, 1H, Jo ¼ 8.1 Hz,
quinox-8-H), 8.41 (s, 1H, pyrazole-50-H). Anal. Calcd. for C25H18N6:
C, 74.62; H, 4.47; N, 20.89. Found: C, 74.28; H, 4.66; N, 20.44%.
5.2. General procedure for cyclization of hydrazones (3aei) to 1-
aryl/heteroaryl-4-methyl-1,2,4-triazolo[4,3-a]quinoxalines (4aei)
5.2.1. 1-(20-Chlorophenyl)-4-methyl-1,2,4-triazolo[4,3-a]
quinoxaline (4a)
5.2.8. 1-(30-Methyl-10-(p-nitrophenyl)-1H-pyrazol-40-yl)-4-methyl-
1,2,4-triazolo[4,3-a]quinoxaline (4h)
To a solution of hydrazone (3a) (0.48 g, 1.6 mmol) in 20 ml
absolute DMF at 50 ꢂC was added a solution of CuCl2 (0.43 g,
3.2 mmol) in 10 ml warm absolute DMF. The reaction mixture
was stirred at 50 ꢂC for 20 min and then heated at 100 ꢂC for 1 h
under nitrogen. After cooling to approximately 30 ꢂC, the mixture
was concentrated to 5 ml in vacuo. Then a solution of 50 ml
water, 25 ml concentrated ammonia and 8.0 g NaCl was added to
the residue in order to remove the copper ions as a water-soluble
complex. The mixture was stirred at 40 ꢂC for 20 min in presence
of air, cooled to room temperature and the precipitated solid was
collected by filtration. The solid was treated again with dilute
ammonia solution (20 ml). The crude product obtained was
washed with water and recrystallized with chloroform to get pure
1-(20-chlorophenyl)-4-methyl-1,2,4-triazolo[4,3-a]quinoxaline
(4a).
M.p. 210e212 ꢂC; yield 87%; IR: 1620 (C]N), 1527 (NO2), 1350
(NO2); 1H NMR (CDCl3) : 2.4 (s, 3H, pyrazole-30-CH3), 3.11 (s, 3H,
d
CH3), 7.05e7.08 (m, 1H, quinox-6-H), 7.19e7.22 (m, 1H, quinox-7-
H), 7.55e7.58 (d, 2H, Jo ¼ 8.1 Hz, Ph-20, 60-H), 7.71e7.73 (d, 1H,
Jo ¼ 7.8 Hz, quinox-5-H), 8.01e8.04 (d, 1H, Jo ¼ 7.8 Hz, quinox-8-H),
8.25e8.27 (d, 2H, Jo ¼ 8.4 Hz, Ph-30, 50-H), 8.49 (s, 1H, pyrazole-50-
H). Anal. Calcd. for C20H15N7O2: C, 62.33; H, 3.89; N, 25.45. Found: C,
62.30; H, 3.82; N, 25.64%.
5.2.9. 1,4-Diphenyl-1,2,4-triazolo[4,3-a]quinoxaline (4i)
M.p. 235e237 ꢂC (Lit. [14] m.p. 235 ꢂC); yield 78%; IR: 1632 (C]
N); 1H NMR (CDCl3)
d
: 7.33e7.38 (t, 1H, Jo ¼ 8.1 Hz, quinox-6-H),
7.54e7.57 (t, 1H, Jo ¼ 8.1 Hz, quinox-7-H), 7.60e7.66 (m, 6H, Ph-H),
7.68e7.71 (d, 1H, Jo ¼ 8.1 Hz, quinox-5-H), 7.75e7.78 (m, 2H, Ph-H),
8.19e8.22 (d, 1H, Jo ¼ 8.4 Hz, quinox-8-H), 8.88e8.91 (m, 2H, Ph-H).
Anal. Calcd. for C21H14N4: C, 78.26; H, 4.34; N,17.39. Found: C, 78.27;
H, 4.49; N, 17.33%.
M.p. 197e198 ꢂC (Lit. [19] m.p. 198e200 ꢂC); yield 87%.
All other compounds (4bei) were synthesized according to the
procedure mentioned above.
5.2.2. 1-(20-Nitrophenyl)-4-methyl-1,2,4-triazolo[4,3-a]quinoxaline
(4b)
6. Biological activity
M.p. 245e247 ꢂC (Lit. [19] m.p. 246e248 ꢂC); yield 91%.
6.1. Materials and methods
5.2.3. 1-(Thiophen-20-yl)-4-methyl-1,2,4-triazolo[4,3-a]quinoxaline
The DMSO, ethylenediaminetetraacetic acid (EDTA) and
(4c)
ethidium bromide (EtBr) were purchased from SigmaeAldrich,
USA. Bacteriophage plasmid, FX174, was procured from Invitrogen,
M.p. 184 ꢂC (Lit. [19] m.p. 184e186 ꢂC); yield 89%.
USA. Stock solution of compound 4 (20 mg/ml) were prepared in
DMSO and stored in brown containers in the refrigerator. All gel
electrophoresis experiments [31] were performed in 1 ꢃ TAE buffer
(0.04 M Triseborate, 0.114% acetic acid and 50 mM EDTA, pH 8.0).
The cleavage efficiency was quantified via densitometric analysis
using the Image J Software (recommended by NIH, USA).
5.2.4. 1-(Furan-20-yl)-4-methyl-1,2,4-triazolo[4,3-a]quinoxaline
(4d)
M.p. 160e162 ꢂC (Lit. [19] m.p. 162e164 ꢂC); yield 87%.
5.2.5. 1-(40-Fluorophenyl)-4-methyl-1,2,4-triazolo[4,3-a]
quinoxaline (4e)
M.p. 232e234 ꢂC; yield 90%; IR: 1605 (C]N); 1H NMR (CDCl3)
d:
6.2. Photocleavage of plasmid DNA by 4
3.09 (s, 3H, CH3), 7.31e7.34 (t, 1H, Jo ¼ 6.9 Hz, quinox-6-H),
7.35e7.40 (t, 2H, Jo ¼ 8.7 Hz, Ph-30, 50-H), 7.50e7.53 (d, 1H,
Jo ¼ 8.1 Hz, quinox-7-H), 7.59e7.61 (d, 1H, Jo ¼ 8.4 Hz, quinox-5-H),
7.71e7.76 (dd, 2H, Jo ¼ 8.7 Hz, J(m)HF ¼ 5.4 Hz, Ph-20, 60-H),
The photoinduced DNA cleaving activities of 4a, b, c and d were
assayed with a supercoiled, covalently closed, circular
FX174
double-stranded DNA (form I), in presence of UV light, which is
a very sensitive molecular biology tool for detection of any changes
in DNA. Experiments were carried out under aerobic conditions in
8.06e8.09 (d,1H, Jo ¼ 8.1 Hz, quinox-8-H); 13C NMR (CDCl3)
d: 21.15,
2
115.57, 116.40e116.70 (d, JCeF ¼ 22.5 Hz, Ph-30, 50-C), 124.34,
125.68, 127.70, 128.19,130.26, 132.13e132.24 (d, 3JCeF ¼ 8.25 Hz, Ph-
20, 60-C), 136.60, 145.0, 149.09, 152.86, 162.65e165.99 (d,
1JCeF ¼ 250.5 Hz, Ph-40-C). HRMS (m/z): 280.1132 (Mþ) (C16H13N4F
(Mþ) requires 280.1124). Anal. Calcd. for C16H11N4F: C, 69.06; H,
3.95; N, 20.14. Found: C, 69.03; H, 3.88; N, 20.18%.
six eppendorf microtubes containing 60
mg (3 ml) FX174 plasmid
DNA and 40 g of 4 (20 mg/ml, dissolved in DMSO) in 0.04 M
m
Triseborate, 0.114% acetic acid and 50 mM EDTA at pH 8.0. The
solutions were then irradiated at room temperature for 45 min at
312 nm using an ultraviolet lamp (15 W). Following irradiation,
the reaction was stopped by removing the UV light and 5
ml of
5.2.6. 1-(1H-Indol-30-yl)-4-methyl-1,2,4-triazolo[4,3-a]quinoxaline
(4f)
6ꢃ glycerol loading dye (containing 0.25% bromophenol blue in 30%
glycerol) in TAE buffer was added and the resulting mixture was
loaded onto a 1% agarose gel. Electrophoresis was carried out for 1 h
at 110 V in TAE buffer and gel was incubated in 1% ethidium bromide
(3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) solu-
tion for 10 min. DNA cleavage was indicated by the formation of
relaxed circular DNA (form II). DNA in the gel was visualized by
a UV transilluminator and photographs were taken with a digital
photocamera. The relative densities of various DNA bands on
M.p. 188e190 ꢂC; yield 73.6%; IR: 3297 (NeH str), 1616 (C]N);
1H NMR (CDCl3)
d: 3.07 (s, 3H, CH3), 7.09e7.13 (m, 1H, quinox-6-H),
7.18e7.21 (m,1H, quinox-7-H), 7.31e7.52 (m, 4H, Indole-40, 50, 60, 70-
H), 7.61 (s, 1H, Indole-20-H), 7.75e7.77 (d, 1H, Jo ¼ 8.4 Hz, quinox-5-
H), 8.01e8.03 (d, 1H, Jo ¼ 8.1 Hz, quinox-8-H), 10.49 (s, 1H, Indole-
NH). Anal. Calcd. for C18H13N5: C, 72.24; H, 4.34; N, 23.41. Found: C,
72.56; H, 4.72; N, 23.78%.

6088
R. Aggarwal et al. / European Journal of Medicinal Chemistry 46 (2011) 6083e6088
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