Cu(ii), Ir(i) and CuO nanocatalyzed mild synthesis of luminescent symmetrical and unsymmetrical bis(triazolylmethyl)quinoxalines: Biocompatibility, cytotoxicity, live cell imaging and biomolecular interaction

Article abstract of DOI:10.1039/c9nj03131f

Quinoxaline derivatives play versatile roles in various fields of science. Thus different types of scaffolds have been designed with these quinoxaline moieties to make them more efficient for preparation of various biologically active materials in the pharmaceutical industry. The ability of quinoxaline moieties has been reinforced by introducing triazole rings into them with the aid of a "click reaction" following environmentally friendly green techniques in the presence of a Cu(ii) catalyst as well as in the presence of a CuO nanomaterial under microwave irradiation. The novelty of our work is being highly focussed on the introduction of unsymmetrical triazole rings with the isolation of mono triazolyl quinoxaline derivatives using an Ir(i) catalyst in aqueous medium and on the capability of the CuO nanomaterial in catalyzing a "click reaction" in various biological media. The role of our synthsized bis(trizolylmethyl)quinoxaline compounds as cytotoxic agents has been confirmed with clear evidence from a DNA and BSA binding study, stability study, viscosity study and MTT assay. Due to the detecting ability of live cancer cells, high cellular uptake, biocompatibility and high cytoselectivity of these scaffolds, ultimately compound 6g has been established as a cancer theranostic drug.

Full text of DOI:10.1039/c9nj03131f

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Cu(II), Ir(I) and CuO nanocatalyzed mild synthesis
of luminescent symmetrical and unsymmetrical
bis(triazolylmethyl)quinoxalines: biocompatibility,
cytotoxicity, live cell imaging and biomolecular
interaction†
Cite this:DOI: 10.1039/c9nj03131f
Santanu Maiti, Nilmadhab Roy,
C. C. Athira, E. Darsana Sreedhar, Sourav De,
Priyankar Paira
Lavanya Thilak Babu, Prithvi Moharana,
S. K. Ashok Kumar and
*
Quinoxaline derivatives play versatile roles in various fields of science. Thus different types of scaffolds
have been designed with these quinoxaline moieties to make them more efficient for preparation of
various biologically active materials in the pharmaceutical industry. The ability of quinoxaline moieties
has been reinforced by introducing triazole rings into them with the aid of a ‘‘click reaction’’ following
environmentally friendly green techniques in the presence of a Cu(II) catalyst as well as in the presence
of a CuO nanomaterial under microwave irradiation. The novelty of our work is being highly focussed
on the introduction of unsymmetrical triazole rings with the isolation of mono triazolyl quinoxaline
derivatives using an Ir(I) catalyst in aqueous medium and on the capability of the CuO nanomaterial in
catalyzing a ‘‘click reaction’’ in various biological media. The role of our synthsized bis(trizolyl-
methyl)quinoxaline compounds as cytotoxic agents has been confirmed with clear evidence from a DNA
and BSA binding study, stability study, viscosity study and MTT assay. Due to the detecting ability of live
cancer cells, high cellular uptake, biocompatibility and high cytoselectivity of these scaffolds, ultimately
compound 6g has been established as a cancer theranostic drug.
Received 16th June 2019,
Accepted 27th November 2019
DOI: 10.1039/c9nj03131f
rsc.li/njc
lower yield of products, long reaction time, less selectivity, and
use of expensive and toxic catalysts, which are not desirable
Nitrogen-containing heterocyclic compounds having quinoxaline today.
Introduction
1
8
derivatives are important in the pharmaceutical industry with
various biological activities such as antimicrobial, antitumor,
On the other hand, a simple but powerful tool that has
stimulated many scientific reactions is the discovery of the
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction
1
2
3
4
5
6,7
antiviral, anti-glucoma, anti-tuberculosis, anti-inflammatory,
8
19
20
and anticancer activities. Furthermore, they are potential by the Fokin/Sharpless and Meldal groups, where two indivi-
building blocks for the synthesis of organic semiconductors,
electroluminescent materials,
and also as ‘‘DNA intercalating agents‘‘ that have been modification, and bioconjugation.
9
dual molecular species get together, and form new molecules with
1
0
11
12
dehydroannulenes,
dyes
the properties of high regiospecificity, atom economy, surface
1
3
21–24
According to the original
reported already. There are several methods for synthesizing definition of ‘‘click chemistry’’, there is a possibility to obtain
21,25–27
quinoxaline derivatives. In the presence of an acid catalyst, aryl different regioisomers of triazoles.
Basically, in the presence
1
,2-diamines and 1,2-dicarbonyl compounds form quinoxaline of a Cu(I) or Cu(II) catalyst, 1,4-disubstituted 1,2,3-triazoles are
1
4–17
28,29
scaffolds, commonly by a condensation reaction.
However, produced exclusively,
but the formation of 1,5-disubstituted
the methods have many limitations like a requirement for triazoles and selected metal acetylides (Sn, Ge, Si, and Na) are
3
0–33
strong acidic conditions, difficulties in work-up procedures, needed.
Zhang et al. in 2005 studied the role of the catalyst,
34
which governs the regioselectivity of click reactions. Likewise,
the importance of copper nanoparticles in click chemistry was
highlighted by various researchers a few years ago.
This versatile (CuAAC) reaction has since been followed to
Department of Chemistry, School of Advanced Sciences, VIT, Vellore-632014,
Tamil Nadu, India. E-mail: priyankar.paira@vit.ac.in; Fax: +91-416-2243092;
Tel: +91-416-2243091
35
3
6–40
41–43
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nj03131f a great extent,
in different areas of biology
and in
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44
producing new materials. Moreover, ‘‘click chemistry’’ is consi- After completion of the reaction, the mixture was transferred
4
5–47
dered to have great impact in materials science,
in building novel structures of polymers
especially into a 250 ml beaker followed by addition of ethanol to remove
4
8,49
50
and dendrimers.
the desired product from the silica gel. Finally, the ethanol was
The unique template ‘triazole’ has been associated with anti- evaporated to isolate grey crystals of bis(bromomethyl)quin-
1
13
viral, anti-fungal, anti-tumour, anti-bacterial, anti-microbial, oxaline (3a). Compound 3a was characterized by H and
C
1
anti-inflammatory, anti-tubercular, anti-HIV, anti-malarial, NMR, IR and mass spectroscopy. In H NMR, the most downfield
5
1–54
cardiovascular and also CNS activities.
protons adjacent to nitrogens (H-1, H-4) are shown as double
Our objective of this project is to synthesize a group of doublet (dd) peaks at d 8.07 ppm. Another two aromatic protons
molecules having bistriazole and quinoxaline moieties in a (H-2, H-3) are displayed at d 7.79 ppm as dd and the last four
2
single domain. However, a perfectly green protocol is needed to aliphatic CH protons (H-9, H-10) are shown as a singlet peak at
1
3
develop the symmetrical and unsymmetrical bis(triazolylmethyl)- d 4.93 ppm. In the C NMR spectra, the most characteristic
quinoxalines. Microwave heating at controlled temperature and peaks of CH (C-9, C-10) were obained at d 30.5 ppm (see the
2
pressure may considerably reduce the reaction time without ESI†). Likewise, compound 3b was also characterized and used
5
5
promoting any side reactions. The other way of synthesis for further reactions. The coversion of bromide to azide (4a and
following green protocol is solid support method using silica 4b) was also performed by the reaction of 3a/3b with sodium
gel or alumina which possesses excellent ability to adsorb the azide in a microwave under a silica gel solid support method.
compounds on its surface without absorbing them and helps Blackish crystals of azidomethylquinoxalines were isolated after
5
6
1
them to take part in reaction under microwave irradiation.
an easy work up. Compounds 4a–b were characterized by H and
1
3
C NMR, IR and mass spectroscopy. There were no significant
differences observed in the H NMR spectra of the azides (4a–b)
and bromides (3a–b). In the C NMR spectra, the most charac-
teristic peaks of CH (C-9, C-10) in compounds 4a–b were
observed at d 52–53 ppm because of the presence of azide
1
1
3
Results and discussion
Synthesis and characterization
2
Microwave assisted Cu(II) catalyzed click reaction with a solid groups. The characteristic peaks of NQNQN stretching
ꢀ
1
support. At the outset, an ethanolic solution of o-phenylene- of compounds 4a and 4b were displayed at 2083 cm and
ꢀ1
diamine (1) was mixed with 1.1 equivalent of 1,4 dibromo-2,3- 2094 cm in the IR spectra.
dione (2) followed by the addition of an adequate amount of
To recognize the most efficient and appropriate conditions
silica gel (100–200 mesh) to make the slurry. After complete air for accomplishing the click reaction, a standardization technique
drying, the solid supported form of this reaction mixture was was applied. We have selected unsubstituted bis(azidomethyl)-
kept in a microwave for 10 min at 70 1C (Scheme 1). The progress quinoxaline (4a) and phenyl acetylene (5a) as the model substrates
of the reaction was monitored by TLC (3: 1 hexane/ethylacetate). for standardization of the click reaction. At the beginning, we
Scheme 1 Schematic representation of symmetrical and unsymmetrical bis(triazolylmethyl)quinoxalines.
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Table 1 Optimization of the click reaction
a
c
Entry
Catalyst
Additive
Solvent
Condition
Time
Yield
1
2
3
4
5
6
7
8
9
CuSO
CuSO
CuSO
CuSO
[Ir(cod)Cl]
[Ir(cod)Cl]
[Ir(cod)Cl]
[Rh(COD)Cl]
[Rh(COD)Cl]
[Rh(COD)Cl]
[Ru(p-cymene)Cl
[Ru(p-cymene)Cl
[Ru(p-cymene)Cl
4
4
4
4
NaAsc
NaAsc
NaAsc
NaAsc
—
—
TBAB
—
Ethanol
Ethanol
rt
80 1C
rt
24 h
18 h
24 h
24 h
24 h
1 h
48 h
24 h
1 h
48 h
24 h
1 h
48 h
15 min
15 min
15 min
92
90
85
88
Trace
Trace
Trace
H
2
O
DMF
DMF
DMF
rt
2
2
2
100 1C
MW
rt
100 1C
MW
rt
100 1C
MW
rt
MW
Solid support in MW
rt
b
[R]
H
2
O
e
2
2
2
DMF
DMF
NI
—
TBAB
—
NI
NI
NI
NI
NI
90
95
96
10
11
12
13
14
15
16
H
2
O
2
]
2
]
2
]
2
2
2
DMF
DMF
—
TBAB
d
H
2
O
CuSO
CuSO
CuO
4
NaAsc
NaAsc
NaAsc
DMF
—
H O
2
f
4
a
b
c
d
e
f
4
a : 5a = 1 : 2.5. 420 watt, 100 1C. Isolated yield. Tetra butyl ammonium bromide. Not isolated. Silica gel.
followed the standard click reaction protocol to isolate bis(4-
phenyltriazolylmethyl)quinoxaline (6a) (Table 1, entry 1).
Regardless of the high yield, the major drawbacks of this
method are the long reaction time and tedious isolation steps.
We could not improve the reaction time by changing the
solvent and reaction temperature (entries 2–4). Concurrently,
when we use other catalysts like Ir(I), Rh(I), and Ru(II) we could
not find a satisfactory yield of the product under normal
heating and microwave irradiation (entries 5–13). Pleasingly,
4
CuSO (5 mol%) afforded the desired bis-triazole product with a
high yield under microwave irradiation using DMF as a reaction
solvent (entry 14). After turning our attention to microwaves, we
performed the same reaction using CuSO4 as a catalyst and
Fig. 1 TEM images of the synthesized CuO nanoparticles.
silica gel as a solid support (entry 15). In brief, bis(azidomethyl)- stirring at 80 1C for 10 min. The brown black precipitate of
quinoxaline 4a (50 mg, 0.2 mmol) and 2.5 equivalent of phenyl the CuO nanomaterials was dried at room temperature.
acetylene 5a (102 mg, 0.5 mmol) were dissolved in ethanol and The formation of CuO nanomaterials was confirmed by IR
4
then an aqueous solution of CuSO (5 mol%, 0.01 mmol, 2.5 mg) spectroscopy, XRD, SEM, EDX and TEM analysis (Fig. S1–S4,
and sodium ascorbate (NaAsc) (5 mol%, 0.01 mmol, 2 mg) were ESI,† and Fig. 1 and 2).
added to it. Afterward, a suitable amount of silica gel was added to
Catalytic amounts of the CuO nanomaterials were used for
make the slurry. Lastly, the solid supported reaction mixture was the synthesis of compound 6a in the presence of sodium
dried and kept stirring in a microwave at 420 watt (100 1C) for ascorbate in aqueous medium at ambient temperature. Surpris-
15 min. After completion of the reaction, the silica gel was washed ingly, compound 6a was formed with high yield within 15 min
with ethanol and the crude bis-triazole (6a) was obtained of the reaction under green conditions.
1
after evaporation of ethanol. Moreover, the crude triazole was
In H NMR, the two most downfield protons adjacent to the
recrystallized from a hexane–ethylacetate (1 : 1) mixture and bis(4- two nitrogen atoms of the quinoxaline ring displayed a multi-
phenyltriazolylmethyl)quinoxaline (6a) was isolated as brown fine plet at d 8.06–8.09 ppm. The characteristic singlet peak of
crystals with a 95% yield. Compound 6a was characterized by triazole aromatic protons was observed at d 8.02 ppm. The
1
13
H and C NMR, IR and mass spectroscopy.
To know the application of nanomaterials in this reaction, rings are displayed in the range of d 7.32–7.80 ppm. Lastly, the
we intrigued our attention on the synthesis of CuO nanomaterial. characteristic four aliphatic CH protons are observed as a
Briefly, an ethanolic solution of 0.2 M Cu(CH COO) was mixed singlet at d 6.13 ppm. In LCMS, the characteristic molecular
remaining aromatic protons of the phenyl ring and quinoxaline
2
3
2
+
with 0.01 mol NaOH followed by addition of 0.5 g of polyethylene ion peak of compound 6a was displayed as [M + H] , 445. After
glycol (PEG). The reaction mixture was kept in a microwave for complete characterization of compound 6a, the scope of the
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Table 2 Synthesis of 2,3-bis((4-aryl-1H-1,2,3-triazol-1-yl)methyl)quinoxaline
(6a–6i, 8)
a,b
c
d
Azide Alkyne Product
Reaction time
Yield
4a
4a
4a
4a
5a
5b
5c
5d
15
95/96
96/97
95/95
92/94
10
10
20
Fig. 2 Elemental analysis of the CuO nanoparticles.
chemistry has been extended by using 4a and 4b as the azide
source and various aromatic acetylenes (5a–e) as the alkyne
source for the click reaction (Scheme 1 and Table 2). It is
notworthy that disubstituted aliphatic alkyne 7 also afforded a
similar triazole product (8) following the same reaction proto-
1
col (Table 2). The structure of compound 8 was confirmed by H 4a
5e
20
94/95
1
3
1
and C NMR, IR and mass spectroscopy. In the H NMR
spectra, the characteristic peaks of the four methyl groups are
exhibited in the downfield region (d 3.87 and 4.02 ppm) as two
singlets. Similarly, quinoxaline aromatic protons and aliphatic
4
4
4
4
b
b
b
b
a
5a
5b
5c
5d
7
20
15
15
20
15
95/96
95/97
96/98
92/95
90/94
CH
2
protons were displayed at d 7.71–7.88 ppm and 6.35 ppm
respectively. In the IR spectra, the characteristic CQO stretch-
ing of the ester groups is observed at 1722 cm . The char-
acteristic molecular ion peak of compound 8 was found at
ꢀ
1
+
[M + H] , 525. The expected mechanism of the click reaction is
depicted in Fig. S5 (ESI†).
Ir(I) and Cu(II) catalysed mono and unsymmetrical triazole synthesis
In this section, our aim was to develop efficient and environ-
mentally benign reaction conditions for the preparation of
unsymmetrical bis-triazoles. To accomplish this, we have arbi-
trarily chosen Ir(I) as a metal catalyst and water as the reaction
medium. Briefly, compound 4a was treated with aryl acetylene
2
(5b–d) in the presence of a catalytic amount of [Ir(cod)Cl] in
water for 48 h at room temperature. After completion of the
reaction, the pure mono triazolylquinoxaline (9b–d) was iso-
lated via filtration followed by crystallization of the crude
products (Scheme 2). The azide and alkyne are spring loaded,
high-energy compounds and their cycloaddition is thermody-
4
a
4
Azide (50 mg), alkyne (2.5 equiv.), CuSO (5 mol%), NaAsc (2.5 mol%),
b
namically favourable with a relatively strong driving force. silica gel, MW, 420 watt, 100 1C. Azide (50 mg), alkyne (2.5 equiv.),
c
2
CuO nano (catalytic amount), NaAsc (2.5 mol%), H O, rt. Reaction
However, because of the high activation energy, they could
not react spontaneously by simple mixing. Fundamentally, the
catalysts assist the formation of a transition state either by
changing the activation energy through electronic restructuring
time is determined by TLC, for CuO in all cases reaction time = 15 min.
d
Isolated yield for both the methods.
and/or by carrying the reactants into close proximity. The most in the alkyne and thereby stabilise the transition state (Fig. 3).
stable transition state will be that transition state where the As a consequence we get a 1,4-disubstituted triazole ring after
5
7
bulky group with poor p-donating capability will tend to remain reductive elimination. There are two adjacent azide groups
away from iridium to avoid strong steric crowding arising from which expect two simultaneous cycloaddition reactions catalysed
the bulky cyclooctadiene group and another substituted group by [Ir(m-cl)cod] . But, the actual scenario is different where we get
2
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Scheme 2 Ir(I) and Cu(II) catalysed synthesis of unsymmetrical bis-
(triazolylmethyl)quinoxaline analogues.
Fig. 4 The plausible pathway for Ir-COD catalysed mono triazolyl ring
formation.
increased (hyperchromic shift) and the wavelength maximum was
also shifted to around 255 nm (red shift). Compound 6g and 10a
experience a non-covalent electrostatic binding interaction with
Fig. 3 Catalytic cycle for the Ir(I) catalysed click reaction.
the mono triazole product (9b–d) instead of the bis triazole CT-DNA, which can demonstrate the mechanism of action and
product; due to the preference of the 1,4-addition mode, strong effectiveness of the drugs. Furthermore, the intrinsic binding
steric crowding will arise from the bulky cyclooctadiene group constant (K ) of the compounds (6g and 10a) was obtained using
b
during further cycloaddition with another azide group in close eqn (i) (ESI†) and from the slope of [DNA]/(e ꢀ e ) vs. [DNA]
a
f
proximity to another azide group (Fig. 4 and Fig. S6, ESI†). linear plots (Fig. S7, ESI†). K for 6g and 10a was observed as
b
1
5
ꢀ1
5
ꢀ1
The structures of compounds 9b–d were confirmed by H and 2.35 ꢁ 10
M
and 2.78 ꢁ 10
M . The significant hyper-
13
1
C NMR, IR and mass spectroscopy. For example, in the H NMR chromism and high binding constant for compounds 6g and
spectra of compound 9b, the most characteristic two distinct 10a suggest a strong electrostatic mode of binding between the
singlet peaks of non-identical CH were obtained at d 4.86 and aromatic chromophores and the base pairs of DNA. However,
2
5.98 ppm. The characteristic CH proton of triazole was obtained at both the compounds also showed good Kapp values (eqn (ii), ESI†)
d 7.92 as a sharp singlet. In LCMS, the characteristic molecular which are 100 fold lower than the literature value of classical
+
7
ꢀ1
ion signal was displayed as [M + H] , 343.
intercalators, such as EtBr–DNA (K
= 1.0 ꢁ 10
M )
EtBr
58
Then we turned our attention to the synthesis of mixed (Table 3). The strong hyperchromism and intrinsic binding
triazolylquinoxaline analogues. The mono triazolyl products constant (K ) in the UV titration experiment and good K value
b
app
(9b–d) were further treated with various acetylenes (5a–c) under in the EtBr displacement assay suggest that these compounds
microwave irradiation. We have isolated a pure crystalline form bind with DNA in electrostatic as well as intercalative mode.
of mixed triazolyl quinoxaline scaffolds (10a–e) following a
similar experimental protocol to that discussed in the previous
1
13
section. All the compounds are characterized by H and
C
Table 3 DNA-binding data for complexes 6g and 10a with CT-DNA
NMR, IR and mass spectroscopy.
a
b
ꢀ1
b
c
ꢀ1
Compounds
K
(M
)
% hyperchromism
K
app (M
)
5
5
5
Biological study
6g
2.35 ꢁ 10
54
59
2.6 ꢁ 10
5
10a
2.78 ꢁ 10
3.2 ꢁ 10
DNA binding study. The absorption bands of compound 6g
and 10a were observed in the range of 244–246 nm while calf
thymus DNA (CT-DNA) alone appears at 258 nm. With increasing
a
b
K , intrinsic DNA binding constant from UV-visible absorption titra-
b
tion. Hyperchromism of the DNA-bound complexes measured from
c
UV-vis titration.
Kapp, apparent DNA binding constant from competi-
the CT-DNA concentration, the absorption peaks are gradually tive displacement from fluorescence spectroscopy.
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Table 4 Binding parameters for interaction of compound 6g and 10a
Table 5 Preliminary MTT cytotoxicity screening of symmetrical and
with BSA
unsymmetrical bis(triazolylmethyl)quinoxalines at 24 h of drug exposure
a
ꢀ1
b
q
ꢀ1 ꢀ1
c
ꢀ1
d
a
b
Compound
K
BSA (M
)
K
(M
s
)
K (M
)
n
IC50 (mM)
SF
3
4
11
5
5
6
1
g
0a
4.7 ꢁ 10
4.7 ꢁ 10
1.2 ꢁ 10
0.87
0.98
Compound
Caco-2
HeLa
HEK 293
Caco-2
HeLa
12
3.5 ꢁ 10
3.5 ꢁ 10
2.3 ꢁ 10
6a
35.8 ꢂ 1.4
25.8 ꢂ 1.4
26.4 ꢂ 0.8
30.0 ꢂ 1.2
28.6 ꢂ 1.5
23.5 ꢂ 0.9
20.0 ꢂ 1.8
20.1 ꢂ 0.6
25.5 ꢂ 0.8
30.6 ꢂ 0.9
21.6 ꢂ 1.5
22.2 ꢂ 1.4
26.2 ꢂ 1.7
26.4 ꢂ 1.1
26.8 ꢂ 1.6
—
25.6 ꢂ 1.2
17.7 ꢂ 0.7
16.2 ꢂ 1.2
18.1 ꢂ 1.1
17.2 ꢂ 1.3
15.8 ꢂ 1.1
12.0 ꢂ 1.2
12.9 ꢂ 1.6
17.4 ꢂ 1.6
24.1 ꢂ 1.1
14.6 ꢂ 1.2
16.6 ꢂ 1.4
19.2 ꢂ 1.1
18.8 ꢂ 0.9
19.7 ꢂ 1.3
—
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
—
42.8
43.9
43.7
43.3
43.5
44.2
45
44.9
43.9
43.2
44.6
44.5
43.8
43.8
43.7
—
43.9
45.6
46.2
45.5
45.8
46.3
48.2
47.8
45.7
44.1
46.8
46.0
45.2
45.3
45.0
—
a
c
b
K
BSA, Stern–Volmer quenching constant.
K, binding constant with BSA. n, number of binding sites.
K
q
, quenching rate constant. 6b
d
6c
6
6
6
6
6
d
e
f
g
h
BSA binding study. The emission intensity of BSA at lem
50 nm decreases gradually with increasing the concentration
of compounds 6g and 10a, which confirms that interactions 6i
of compounds 6g and 10a with BSA have occurred. The Stern–
Volmer quenching constant for BSA (KBSA) and bimolecular
quenching rate constant (K
obtained using the Stern–Volmer eqn (iii) (ESI†) and corres-
ponding Stern–Volmer plots (Table 4). The K
two compounds were on the order of 10 –10
=
3
8
1
1
0a
0b
) for compounds 6g and 10a are 10c
q
1
1
0d
0e
q
values of these
DMSO
1
1
12
ꢀ1 ꢀ1
M
s
, which Cisplatin
19.8 ꢂ 1.2
14.5 ꢂ 0.9
450
450
42.5
43.2
43.4
42.6
RAPTA-C
15.4 ꢂ 1.1
18.8 ꢂ 0.8
were higher enough than the maximum scatter collision
1
0
ꢀ1 ꢀ1
59
a
quenching constant (2.0 ꢁ 10
M
s
) of BSA quenchers.
IC50 is the concentration at which 50% of cells underwent cytotoxic
b
cell death due to organoruthenium & cisplatin treatment. SF (selectivity
factor) = ratio of IC50 for HEK-293/IC50 for all the cancer cell lines. HEK-293
is generally selected as the model for healthy cells for evaluation of the
The binding affinity (K) of 6g and 10a was calculated from
5
ꢀ1
Scatchard plot (Fig. S8, eqn (iv), ESI†) analysis (1.2 ꢁ 10 M
,
5
ꢀ1
2
.3 ꢁ 10 M ), which showed strong binding propensity of the selectivity of chemotherapeutic drugs.
tested complexes with BSA, which is required for transport of
protein-bound complexes in biological systems.
Live cell imaging and bioorthogonality study
Stability study. For novel therapeutic requirements, a
chemical entity has to be stable within the cells in the internal A bio-imaging study was performed with the most potent and
atmosphere. In the presence of 5% DMSO in phosphate buffer selective compound 6g among all the synthesized compounds
media, the stability was measured over a period of 24 h at using the HeLa cell line. After incubating the cancer HeLa cells
regular time intervals for compounds 6g and 10a. While with compound 6g (20 mM) and control precursors (4b and 5b,
executing this study, we observed that in MTT medium, the 20 mM) for 2 h strong red fluorescence was visualized in live
absorbance vs. wavelength curves (Fig. S9, ESI†) for compounds cells from the compound 6g set under a fluorescence micro-
6
g and 10a at the beginning and after 24 h are almost the same. scope using a green filter (excitation filter 480–550 nm) (Fig. 5).
Hence, both the compounds are stable in MTT medium However, we could not find any fluorescence from the precursor
5% DMSO in phosphate buffer).
sets. This is evidence of fluorogenic behavior of compound 6g and
(
Cytotoxicity study
The cytotoxicity of all the synthesized symmetrical and unsym-
metrical triazoles (6a–i, 8, 10a–e) (Table 5) was evaluated by
standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) bioassay with a panel of cancer cell lines such
as human epitheloid cervix carcinoma (HeLa), a human colorectal
adenocarcinoma cell line (Caco-2) and normal human embryonic
kidney cells (HEK-293) in triplicate. These cells were incubated
with triazoles along with cisplatin, and RAPTA-C as a standard
positive control drug at concentration 1 to 200 mM for 24 h. Most
of the scaffolds exhibited comparable potency with the positive
controls in all the cell lines with respect to the normal kidney
cells. However, in terms of selectivity, the synthesized scaffolds
showed much better efficiency than the control drugs. In HeLa
cells the IC50 values for these compounds were in the range
of 12–25 mM and in the Caco-2 cell line IC50 is slightly higher
(
20–35 mM). It was also observed that compound 6g and 6h
Fig. 5 Fluorescence images of live HeLa cells with (a) compound 4b
20 mM in PBS buffer); incubation time 2 h, (b) compound 5b (20 mM in PBS
buffer); incubation time 2 h and (c) compound 6g (20 mM in PBS buffer);
incubation time 2 h. Scale bar 400 mm.
showed the best cytoselectivity among all the synthesized scaf-
folds. Lipophilic –Br and hydrophobic substituted phenyl groups
facilitate the cellular delivery of these drugs.
(
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a
Table 6 Analysis of the biocompatibility of the method
purchased from Sigma-Aldrich Limited. All the organic solvents
used for chemical synthesis were procured from E. Merck
b
c
Entry
Condition
Conversion
Yield
1
13
(
India) of analytical grade. H NMR and C NMR spectra were
measured using a Bruker DPX spectrometer at 400 MHz and
00 MHz respectively using tetramethylsilane as an internal
1
2
3
4
5
6
7
8
9
H
H
H
2
2
2
O/THF
O
O/Ct-DNA
96
99
95
98
99
99
98
98
99
96
96
95
95
96
91
90
89
88
1
Cell cultured media (DMEM)
PBS
H O/glutathione (GSH)
2
Fetal bovine serum (FBS)
Bovine serum albumin (BSA)
Cell lysates (HeLa)
standard and the chemical shifts have been reported in ppm
units. Abbreviations are as follows: s, singlet; d, doublet;
dd, double doublet; t, triplet; m, multiplet. The melting points
of triazole derivatives were measured using an Elchem Micro-
processor based DT apparatus using open capillary tubes and
a
Reaction conditions: 4a :5a = 1 : 2.5, CuO (catalytic), NaAsc (2.5 mol%), are uncorrected. The synthesized compounds were also charac-
additive, in the selected milieu, and the resulting mixture was stirred for
5 min. Determined by H NMR analysis of the crude reaction mixture
terized using a Shimadzu LCMS-4000 LC-MS instrument, having a
b
1
1
c
4000 triple quadrupole MS, using methanol as the solvent. Thin
layer chromatography was performed on pre-coated silica gel 60
F254 aluminium sheets (E. Merck, Germany) using a solvent
system of a hexane/ethyl acetate (3 : 1) mixture and the spots
were identified when exposed to UV light. Microwave model
CATA-R was used for synthesizing all the compounds. All the
experiments were performed in compliance with the relevant
laws and institutional biosafety guidelines. This study was
performed in strict accordance with the NIH guidelines (NIH
Publication No. 85-23 Rev. 1985).
using an internal standard. Isolated yield of 6a.
the significant cellular uptake of the compound in HeLa cells.
As the same compound showed the highest cytoselectivity in
cancer cells this one could be a good choice for cancer therano-
stics among all the reported compounds. We next explored the
bioorthogonality of the chemistry by performing the reaction in
the presence of biologically relevant conditions with various
biomolecular additives (Table 6 and Fig. S10, ESI†). Pleasantly,
the CuO nanomaterial catalysed click reaction could be effi-
ciently carried out in the presence of DNA (entry 3), PBS buffer
General procedure for the synthesis of 2,3-bis((4-aryl-1H-
1,2,3-triazol-1-yl)methyl)quinoxaline (6a–i). 2,3-Bis(azidomethyl)-
(
entry 5), and excess glutathione (entry 6). The desired triazole
quinoxaline (4a–b) (50 mg) was dissolved in ethanol, and
was obtained in excellent yields. Moreover, frequently used
culture cell media, such as DMEM, fetal bovine serum (FBS),
bovine serum albumin (BSA) and HeLa cell lysates are also
excellent reaction media for the synthesis of triazole 6a, with
high yields (entries 4 and 7–9).
5
mol% of CuSO
4
, 2.5 mol% of sodium ascorbate and
2.5 equivalents of acetylene (5 or 10) were added to it. To the
reaction mixture an adequate amount of silica gel was added to
make the slurry. The silica gel supported reaction mixture was
dried under a vacuum. Finally, the mixture was kept in a
microwave vessel and kept stirring at 420 watt (100 1C) for
Viscosity measurement
10–15 min. The progress of the reaction was monitored by TLC.
In general, for identification of the binding mode of drugs
with DNA a hydrodynamic method like a viscosity study has
been conducted. Binding via intercalation needs adjacent
base pair separation to have binding of drug molecules into
the DNA double helix, which in turn leads to an increase of the
length of DNA and its viscosity. However, interaction of the
drug via groove or electrostatic binding does not change
the relative viscosity of DNA, as the molecule does not change
After complete conversion to the product the solid supported
reaction mixture was treated with a 1 : 1 hexane–ethyl acetate
solvent mixture repeatedly and then filtered. After filtration the
filtrate was evaporated to dryness. Further, the crude product
was recrystallized from 3 : 1 hexane–ethyl acetate. Fine brown
needle like crystals of compound 6a–i appeared in the pure
form with 90–96% yield.
the length of DNA on binding. The analysis of the graphical Characterization of 1,2,3-traizolyl methyl quinoxaline (6a–i)
representation of viscosity studies for 6g and 10a reveals that
2,3-Bis((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)quinoxaline (6a).
for a 20–60 mM concentration of 6g it indicates the intercala-
tive mode of binding with DNA and from 60 mM onwards
it exhibits electrostatic or groove binding (Fig. S11, ESI†).
Likewise, compound 10a also shows intercalation into the
DNA molecule up to a 60 mM concentration and after that
concentration it presents either electrostatic or groove binding
with DNA (Fig. S12, ESI†).
Yield: 95%; mp: 180–185 1C; R 0.44 (1 : 1 ethyl-acetate : hexane);
f
1
H NMR (400 MHz, CDCl ): d 8.06–8.09 (m, 2H, CH, H-1, H-4),
3
8.02 (s, 2H, CH, H-12, H-14), 7.81 (d, J = 7.2 Hz, 6H, CH, H-2,
H-3, H-16, H-20, H-22, H-26), 7.40 (t, J = 7.2 Hz, 4H, CH, H-17,
13
2
H-19, H-23, H-25), 6.13 (s, 4H, CH , H-9, H-10); C NMR
(
(
(
400 MHz, DMSO-d
6
): d 149.4 (2 ꢁ CQN, 7, C-8), 147.0
2 ꢁ CQC–N, C-11, C-13), 140.9 (2 ꢁ C–N, C-5, C-6), 131.3
2 ꢁ CH, C-12, C-14), 131.2 (2 ꢁ C, C-15, C-21), 129.4 (4 ꢁ CH,
C17, C-19, C-23, C-25), 129.14 (2 ꢁ CH, C-18, C-24), 128.4
Experimental
Materials and methods
(
2 ꢁ CH, C-1, C-4), 125.7 (2 ꢁ CH, C-2, C-3), 123.3 (4 ꢁ CH,
ꢀ1
C-16, C-20, C-22, C-26), 52.41 (2 ꢁ CH ); IR (cm , KBr): 3082
2
3
The chemicals 4-bromo-o-phenylene diamine, 1,4-dibromo (sp C–H stretching), (CH
2
asymmetric stretching), 1637 (CQC),
2
2,3-butane dione, acetylene derivatives, copper acetate, poly- 1442 (CH bending), 1292 (C–N stretching), 758 (C–H stretching
ethylene glycol, and Ru(II), Ir(I), Rh(I) and Cu(II) catalysts were of the o-disubstituted group); LC-MS (CH
3
OH): m/z 445.4
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0
[
[
M + H] ; HRMS (ESI): m/z calcd for C26
H
20
N
8
: 445.1811
3,3 -((Quinoxaline-2,3-diylbis(methylene))bis(1H-1,2,3-triazole-
1,4-diyl))dianiline (6e). Yield: 95%; mp: 220–222 1C, R 0.32 (ethyl-
,3-Bis((4-(p-tolyl)-1H-1,2,3-triazol-1-yl)methyl)quinoxaline (6b). acetate); H NMR (400 MHz, CDCl ): d 8.06–8.08 (m, 2H, CH,
+
+
M + H] ; found: 445.1816 [M + H] .
f
1
2
3
Yield: 95%, mp: 190–195 1C; R 0.46 (1 : 1 ethyl-acetate : hexane); H-1, H-4), 7.94 (s, 2H, CH, H-12, H-14), 7.80–7.83 (m, 2H, CH, H-2,
f
1
H NMR (400 MHz, CDCl ): d 8.07 (dd, 2H, J = 6.4 Hz, J = 3.6 Hz, H-3), 7.22 (s, 2H, CH), 7.14–7.18 (m, 4H, CH, H-20, H-26, H-16,
3
1
2
2
ArH, H-1, H-4), 7.98 (s, 2H, CH, H-12, H-14), 7.80 (dd, 2H, H-22), 6.65 (d, 2H, J = 7.2 Hz, CH, H-18, H-24), 6.09 (s, 4H, CH ,
1
3
J
1
= 6.4 Hz, J
CH, H-16, H-20, H-22, H-26) 7.20 (d, J = 7.6 Hz, 4H, CH, H-17, 2 ꢁ C–NH
H-19, H-23, H-25), 6.10 (s, 4H, (H-9, H-10), CH
), 2.36 (s, 6HH-27, 140.9 (2 ꢁ C–N, C-5, C-6), 131.4 (2 ꢁ CH, C-12, C-14), 129.9 (2 ꢁ C,
): d 149.4 (2 ꢁ CQN, C-15, C-21), 129.0 (4 ꢁ CH, C-1, C-2, C-3, C-4), 122.9 (2 ꢁ CH, C-19,
2
= 3.6 Hz, ArH, H-2, H-3), 7.72 (d, J = 7.6 Hz, 4H, H-9, H-10); C NMR (400 MHz, DMSO-d
6
): d 149.2 (2 ꢁ CQN,
2
, C-7, C-8, C-17, C-23), 147.7 (2 ꢁ CQC–N, C-11, C-13),
2
13
3 6
H-28, CH ); C NMR (400 MHz, DMSO-d
C-7, C-8), 147.1 (2 ꢁ CQC–N, C-11, C-13), 140.9 (2 ꢁ C–N, C-5, C-25), 114.4 (2 ꢁ CH, C-20, C-26), 113.8 (2 ꢁ CH, C-16, C-22), 111.1
ꢀ1
C-6), 137.7 (2 ꢁ C–Me, C-18, C-24), 132.1 (CH, C-12), 131.3 (CH, (2 ꢁ CH, C-18, C-24), 52.1 (2 ꢁ CH , C-9, C-10); IR (cm , KBr):
2
3
C-14), 129.9 (4 ꢁ CH, C-17, C-19, C-23, C-25), 129.7 (C, C-15), 129.1 3468 (N–H symmetric), 3369 (symmetric stretching), 3134 (sp
(
C, C-21), 128.4 (2 ꢁ CH, C-2, C-3), 125.6 (4 ꢁ CH, C-16, C-20, C-22, C–H stretching), 2976 (C–H Ar stretching), 1616 (CQC stretching),
C-26), 122.8 (2 ꢁ CH, C-1, C-2), 52.4 (2 ꢁ CH
2
, C-9, C-10), 21.3 1454 (CH
2
bending), 1276 (C–N stretching), 786, 761 (C–H stretch-
OH): m/z 475.2
ꢀ
1
(2 ꢁ CH
3
, C-27, C-28); IR (cm , KBr): 3246 (N–H symmetric and ing of the o-di-substituted group); LC-MS (CH
3
3
+
+
asymmetric stretching), 2972 (sp C–H stretching), 2860 (C–H Ar [M + H] ; HRMS (ESI): m/z calcd for C26
22
H N10: 475.2029 [M + H] ;
+
stretching), 2750 (CH
1
7
(
C
2
asymmetric stretching), 1654 (CQC), 1498, found: 475.2023 [M + H] .
404.92 (CH bending), 1226 (C–N stretching), 808 (CH bending), 6-Bromo-2,3-bis((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)quin-
54.54 (C–H stretching of the o-di-substituted group); LC-MS oxaline (6f). Yield: 87%; mp: 265–270 1C; R 0.48 (1 : 1 ethylace-
2
f
+
1
CH OH): m/z 473.1 [M + H] . HRMS (ESI): m/z calcd for tate : hexane); H NMR (400 MHz, DMSO-d ): d 8.69 (brs, 2H,
3
6
+
+
28
H
24
N
8
: 473.2124 [M + H] ; found: 473.2 [M + H] .
CH, H-12, H-14), 8.18 (brs, 1H, CH, H-4), 7.88–7.93 (m, 6H, CH,
2
,3-Bis((4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)- H-1, H-2, H-16, H-20, H-22, H-26), 7.46 (brm, 4H, CH, H-17,
quinoxaline (6c). Yield: 96%, mp: 238–245 1C, R
ethyl-acetate : hexane); H NMR (400 MHz, DMSO-d
f
0.41 (1 : 1 H,H-19, H-23, H-25), 7.35–7.37 (brm, 2H, CH, H-18, H-25), 6.30
1
ꢀ1
6
): d 8.53 (brs, 4H, CH
2
, H-9, H-10); IR (KBr, cm ): 3406 (N–H symmetric
(
s, 2H, CH, H-12, H-14), 7.95–7.97 (m, 2H, CH, H-1, H-4), stretching), 3236 (N–H asymmetric stretching), 3091 (C–H
7
.78–7.83 (m, 6H, CH, H-2, H-3, H-16, H-20, H-22, H-26), 7.00 stretching), 1614 (CQC stretching), 1479, 1431, 601 (C–Br
+
(
d, J = 8.8 Hz, 4H, CH, H-17, H-19, H-23, H-25), 6.22 (s, 4H, stretching); LC-MS (CH3OH): m/z 523.5 [M + H] ; HRMS (ESI):
1
3
+
CH , H-9, H-10), 3.78 (s, 6H, –OCH , H-27, H-28); C NMR m/z calcd for C H BrN : 523.0916 [M + H] ; found: 523.0920
2
3
26 19
8
+
(
(
(
400 MHz, DMSO-d
2 ꢁ CQN, C-7, C-8), 146.9 (2 ꢁ CQC–N, C11, C-13), 140.9
6
): d 159.5 (2 ꢁ C–OMe, C-18, C-24), 149.5 [M + H] .
6-Bromo-2,3-bis((4-(p-tolyl)-1H-1,2,3-triazol-1-yl)methyl)-
2 ꢁ C–N, C-5, C-6), 131.3 (2 ꢁ CH, C-12, C-14), 129.1 (2 ꢁ C, quinoxaline (6g). Yield: 94%, mp: 285–287 1C, R 0.42 (1 : 1
): d 8.60
2 ꢁ CH, C-2, C-3), 122.2 (2 ꢁ CH, C-1, C-4), 114.8 (4 ꢁ CH, (brs, 2H, CH, H-12, H-14), 8.20 (brs, 1H, CH, H-4), 7.89–7.93
f
1
C-15, C-21), 127.1 (4 ꢁ CH, C-16, C-20, C-22, C-26), 123.7 ethylacetate : hexane); H NMR (400 MHz, DMSO-d
6
(
C-17, C-19, C-23, C-25), 55.6 (2 ꢁ OMe, C-27, C-28), 52.4 (m, 2H, CH, H-1, H-2), 7.7 (d, J = 6.8 Hz, 4H, CH, H-16, H-20,
ꢀ
1
(
2 ꢁ CH , C-9, C-10); IR (cm , KBr): 3371 (N–H symmetric H-22, H-26), 7.26 (d, J = 6.8 Hz, 4H, CH, H-17, H-19, H-23, H-25),
2
3
and asymmetric stretching), 3028 (sp C–H stretching), 2833 6.25 (brs, 4H, CH , H-9, H-10), 2.36 (s, 6H, CH , H-27, H-28); IR
2
3
ꢀ1
(
C–H Ar stretching), 1616 (CQC), 1498, 1460 (CH
2
bending), (KBr, cm ): 3419 (N–H symmetric and asymmetric stretching),
247 (C–N stretching), 821 (CH bending) p-disubstituted, 2974 (C–H Ar stretching), 1602 (CQC stretching), 1462 (CH
61 (C–H stretching of the o-di-substituted group); LC-MS bending), 1292 (C–N stretching), 808 (C–H bending), 721 (C–Br
1
7
2
+
+
(
CH
3
OH): m/z 505.2 [M + H] ; HRMS (ESI): m/z calcd for stretching); LC-MS (CH
3
OH): m/z 551.0 [M + H] , 553.7
+
+
+
C
28
H
2
24
N
8
O
2
: 505.2022 [M + H] ; found: 505.2028 [M + H] .
[M + 2 + H] ; HRMS (ESI): m/z calcd for C26 19BrN : 551.1229
H
8
+
+
,3-Bis((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)quinox- [M + H] ; found: 551.1234 [M + H] .
aline (6d). Yield: 95%; mp: 172–177 1C, R 0.26 (ethyl acetate); 6-Bromo-2,3-bis((4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-
H NMR (400 MHz, CDCl ): d 8.56 (s, 2H, CH, H-19, methyl)quinoxaline (6h). Yield: 95%; mp: 257–260 1C, R 0.44
f
1
3
f
1
H-24), 8.38 (s, 2H, CH, H-12, H-14), 8.16 (d, 2H, J = 6.4 Hz, (1 : 1 ethylacetate : hexane); H NMR (400 MHz, DMSO-d
6
): d
CH, H-16, H-21), 8.06–8.09 (m, 2H, CH, H-17, H-22), 7.80– 8.52 (brs, 2H, CH, H-12, H-14), 8.19 (s, 1H, CH, H-4), 7.91
7
.83 (m, 2H, CH, H-18, H-23), 7.78 (d, 2H, J = 8.0 Hz, CH, H-1, (d, 2H, J = 6.8 Hz, CH, H-1, H-2), 7.78 (d, J = 6.4 Hz, 4H, CH,
ꢀ
1
H-4), 7.32 (brs, 2H, CH, H-2, H-3) 6.16 (s, 4H, CH); IR (cm
KBr): 3082 (sp C–H stretching), 2970 (C–H Ar stretching), H-23, H-25), 6.23 (s, 4H, CH
,
H-16, H-20, H-22, H-26), 7.00 (d, 4H, J = 6.8 Hz, CH, H-17, H-19,
3
, H-9, H-10), 3.77 (s, 6H, CH ,
3
2
1
ꢀ
2
833 (CH2 asymmetric stretching), 1598 (CQC), 1570 (C–H H-27, H-28); IR (KBr, cm ): 3385 (N–H symmetric and
bending), 1450 (CH2 bending), 1290 (C–N stretching), 783 asymmetric stretching), 3086 (C–H stretching), 2951 (C–H Ar
CH bending), 756 (C–H stretching of the o-di-substituted stretching), 2833 (CH2 asymmetric stretching), 1616 (CQC
(
+
group); LC-MS (CH bending),
18
m/z calcd for C24H N10: 447.1716 [M + H] ; found: 447.1721 1249 (C–N stretching), 821 (C–H bending), 732.95 (C–Br
3
OH): m/z 447.1 [M + H] ; HRMS (ESI): stretching), 1562 (C–H bending), 1498, 1462 (CH
2
+
+
+
[
M + H] .
3
stretching); LC-MS (CH OH): m/z 583.0 [M + H] ; HRMS (ESI):
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+
1
m/z calcd for C28
H
23BrN
8
O
2
: 583.1127 [M + H] ; found: (1 : 3 ethylacetate : hexane); H NMR (400 MHz, CDCl
3
): d
+
583.1132 [M + H] .
8.12–8.14 (m, 1H, CH, H-1), 8.07–8.10 (m, 1H, CH, H-4), 7.92
6-Bromo-2,3-bis((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)- (s, 1H, CH, H-12), 7.81–7.83 (m, 2H, CH, H-2, H-3), 7.69 (d, J =
quinoxaline 5(6i). Yield: 90%; mp: 295–297 1C, R 0.31 (ethyl- 8.4 Hz, 2H, CH, H-14, H-18), 7.20 (d, J = 8.0 Hz, 2H, CH, H-15,
f
1
acetate); H NMR (400 MHz, DMSO-d ): d 8.69–8.73 (m, 2H, CH, H-17), 5.98 (s, 2H, CH , H-9), 4.85 (s, 2H, CH , H-10), 2.36
6
2
2
ꢀ
1
3
H-17, H-23), 8.62 (brs, 2H, CH, H-19, H-24), 8.16 (brs, 1H, CH, (s, 3H, CH
3
, H-19); IR (KBr, cm ): 3099 (sp C–H stretching),
asymmetric stretching),
CH, H-2, H-18, H-24), 7.37 (brs, 3H, CH, H-1, H-12, H-14), 6.33 2108 (NQNQN stretching), 1496, 1448 (CH bending), 1226
, H-9, H-10); IR (KBr, cm ): 3535 (N–H symmetric), (C–N stretching), 810 (CH bending), 754 (C–H stretching of the
361 (asymmetric stretching), 2956 (C–H Ar stretching), 1600 o-di-substituted group).
CQC stretching), 1421 (CH bending), 1228 (C–N stretching), 2-(Azidomethyl)-3-((4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-
08 (C–H bending), 617 (C–Br stretching); LC-MS (CH OH): yl)methyl)quinoxaline (9c). Yield: 85%, mp: 195–198 1C, R
H-4), 8.09 (d, J = 8 Hz, 2H, CH, H-16, H-21), 7.86–7.94 (m, 3H, 2970 (C–H Ar stretching), 2866 (CH
2
2
ꢀ
1
(
3
(
8
s, 4H, CH
2
2
3
f
+
+
1
m/z 524[M + H] , 526 [M + H] ; HRMS (ESI): m/z calcd for 0.71 (1 : 3 ethylacetate : hexane); H NMR (400 MHz, CDCl ): d
3
+
+
C
24
H
17BrN10: 524.0821 [M + H] ; found: 524.0826 [M + H] .
8.09–8.10 (m, 1H, CH, H-1), 8.05–8.06 (m, 1H, CH, H-4), 7.86
0
Tetramethyl
1,1 -(quinoxaline-2,3-diylbis(methylene))bis- (s, 1H, CH, H-12), 7.79 (t, J = 4 Hz, 2H, CH, H-2, H-3), 7.70 (d, J =
(
1H-1,2,3-triazole-4,5-dicarboxylate) (8). Yield: 95%; mp: 178– 8.8 Hz, 2H, CH, H-14, H-18), 6.90 (d, J = 8.4 Hz, 2H, CH, H-15,
1
1
80 1C; R
CDCl ): d 7.88 (dd, 2H, J
.74 (dd, 2H, J = 6.4 Hz, J = 3.6 Hz, CH, H-2, H-3), 6.35 (s, 4H, 2927 (C–H Ar stretching), 2835 (CH2 asymmetric stretching),
f
0.52 (1 : 1 ethyl-acetate : hexane); H NMR (400 MHz, H-17), 5.94 (s, 2H, CH
2 2
, H-9), 4.83 (s, 2H, CH , H-10), 3.79
ꢀ1
3
3
1
= 6.4 Hz, J = 3.2 Hz, CH, H-1, H-4), (s, 3H, CH , H-19); IR (KBr, cm ): 3138 (sp C–H stretching),
2
3
7
1
2
CH , H-9, H-10), 4.02 (s, 6H, CH , H-14, H-20), 3.87 (s, 6H, CH , 2100 (NQNQN stretching), 1614 (CQC), 1562 (C–H bending),
2
3
3
1
3
H-16, H-22); C NMR (400 MHz, CDCl ): 160.4 (2 ꢁ CQO, C-13, 1498, 1460 (CH2 bending), 1246 (C–N stretching), 827 (CH
3
C-21), 159.0 (2 ꢁ CQO, C-15, C-19), 145.5 (2 ꢁ CQN, C7, C-8), bending), 761 (C–H stretching of the o-di-substituted group).
1
(
41.0 (2 ꢁ C–N, C-12, C-17), 140.0 (2 ꢁ C–N, C-5, C-6), 131.2
2-(Azidomethyl)-3-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)-
2 ꢁ C–N, C-11, C-18), 131.1 (2 ꢁ C–H, C-2, C-3), 128.9 (2 ꢁ C–H, quinoxaline (9d). Yield: 85%, mp: 195–198 1C, R
f
0.44 (1 : 1
): d 8.55 (brs,
2
, C-9, C-10); IR (KBr, cm ): 2956 (sp C–H 1H, CH, H-17), 8.33 (brs, 1H, CH, H-14), 8.07–8.12 (m, 3H, CH,
1
C-1, C-4), 53.5 (2 ꢁ CH
3
, C-14, C-20), 52.9 (2 ꢁ CH
3 3
, C-16, C-22), ethylacetate : hexane); H NMR (400 MHz, CDCl
ꢀ1
3
5
2.1 (2 ꢁ CH
stretching), 1722 (CQO stretching), 1616 (CQC), 1564 (C–H H-1, H-4, H-15), 7.80–7.82 (m, 3H, CH, H-2, H-3, H-16), 7.22
bending), 1469, 1433 (CH bending), 1226 (C–N stretching), 779 (s, 1H, CH, H-12), 6.01 (s, 2H, CH , H-9), 4.83 (s, 2H, CH , H-10);
2
2
2
ꢀ1
(
C–H stretching of the o-di-substituted group); LC-MS (CH OH): IR (KBr, cm ): 3319 (N–H symmetric and asymmetric stretching),
m/z 525 [M + H] ; HRMS (ESI): m/z calcd for C22
25.1404 [M + H] ; found: 525.1408 [M + H] .
3
+
3
H
20
N
8
O
8
:
3080 (sp C–H stretching), 2100 (NQNQN stretching), 1598
(CQC stretching), 1419 (CH bending), 1294 (C–N stretching),
83, 758 (C–H stretching of the o-di-substituted group); LC-MS
+
+
5
2
7
General procedure for the synthesis of unsymmetrical
bis(triazolylmethyl)quinoxaline analogues (10a–e)
+
(CH
3
OH): m/z 344.0 [M + H] .
-((4-Phenyl-1H-1,2,3-triazol-1-yl)methyl)-3-((4-(p-tolyl)-1H-
2
2
,3-Bis(azidomethyl)quinoxaline (4a) (50 mg) was dissolved in 1,2,3-triazol-1-yl)methyl)quinoxaline (10a). Yield: 93%, mp:
1
water, and 5 mol% of [Ir(cod)Cl] , and 1.05 equivalents of 150–155 1C, R 0.44 (1 : 1 ethylacetate : hexane); H NMR (400 MHz,
2
f
acetylene (5b–d) were added to it. The reaction mixture was CDCl ): d 8.05 (brs, 3H, CH, H-1, H-4, H-19), 7.98 (s, 2H, CH, H-12,
3
stirred under room temperature for 48 h. After that, the volume H-14), 7.81 (d, J = 6.8 Hz, 3H, CH, H-16, H-20, H-22), 7.69 (d, J =
of water was reduced and the mono triazole products (9b–d) 7.2 Hz, 1H, CH, H-18), 7.38–7.41 (m, 3H, CH, H-17, H-2, H-3), 7.33
were isolated by simple filtration. 20 mg of the mono triazole (d, J = 6.8 Hz, 2H, CH, H-23, H-25), 7.20 (d, J = 7.6 Hz, 1H, CH, H-26),
13
products (9b–d) were dissolved in ethanol, and 5 mol% of 6.12 (s, 4H, CH
2
, H-9, H-10), 2.36 (s, 3H, CH
3
, H-27); C NMR
CuSO , 1.1 mol% of sodium ascorbate and 1.1 equivalents of (100 MHz, DMSO-d
4
6
): d149.0 (2 ꢁ CQN, C-7, C-8), 147.1 (CQC–N,
acetylenes (5a–c) were added. To the reaction mixture an C-11), 147.0 (CQC–N, C-13), 140.9 (2 ꢁ C–N, C-5, C-6), 137.7 (C-Me,
adequate amount of silica gel was added to make the slurry. C-24), 131.30 (C–C, C-15), 131.2 (2 ꢁ CH, C-12, C-14), 129.9
The silica gel supported reaction mixture was dried under a (C–C, C-21), 129.4 (2 ꢁ CH, C-23, C-25), 129.1 (CH, C-18), 128.4
vacuum. Finally, the mixture was kept in a microwave vessel (4 ꢁ CH, C-1, C-2, C-3, C-4), 125.7 (2 ꢁ CH, C-17, C-19), 125.6
and kept stirring at 420 watt (100 1C) for 20 min. The progress (2 ꢁ CH, C–), 123.3 (2 ꢁ CH, C-16, C-20), 122.8 (2 ꢁ CH, -22,
of the reaction was monitored by TLC. After complete conver- C-26), 52.4 (2 ꢁ CH
2 3
, C-9, C-10), 21.3 (CH , C-27); IR (KBr,
ꢀ
1
3
sion to the product the solid supported reaction mixture was cm ): 3082 (sp C–H stretching), 2924 (C–H Ar stretching),
treated with a 1 : 1 hexane–ethylacetate solvent mixture repeat- 2852 (CH asymmetric stretching), 1614 (CQC), 1463, 1460
edly and then filtered. After filtration the filtrate was evaporated (CH2 bending), 1224 (C–N stretching), 808 (CH bending),
2
ꢀ
1
to dryness. Further, the crude product was recrystallized from 758.02 cm
(C–H stretching of the o-disubstituted group);
+
3
: 1 hexane–ethyl acetate. Fine needle shaped crystals of com- LC-MS (CH OH): m/z 459.2 [M + H] ; HRMS (ESI): m/z calcd
3
+
+
22 8
pound 10a–e were obtained in the pure form with 90–96% yield. for C27H N : 459.1967 [M + H] ; found: 459.1971 [M + H] .
2
-(Azidomethyl)-3-((4-(p-tolyl)-1H-1,2,3-triazol-1-yl)methyl)-
2-((4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-3-((4-
quinoxaline (9b). Yield: 90%; mp: 130–135 1C, R
f
0.74 phenyl-1H-1,2,3-triazol-1-yl)methyl)quinoxaline (10b). Yield: 90%,
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1
m.p: 250–255 1C, R
400 MHz, CDCl
f
0.48 (1 : 1 ethylacetate : hexane); H NMR Conclusions
(
3
): d 8.07–8.09 (m, 2H, CH, H-1, H-4), 8.03
Based on our thorough experimental study, we are close
to achieving all our desired products in high yield. The for-
mation of symmetrical and unsymmetrical bis(triazolylmethyl)-
quinoxaline analogues only by using green methodologies in
the presence of Cu(II) and Ir(I) catalysts, and a CuO nano
catalyst has been revealed with supporting experimental evi-
dence. The introduction of a versatile nature in the same
scaffold with the formation of two different triazole rings, we
hope, will lead the scientific mind towards the discovery of
new materials in the near future. Meanwhile, for biological
applications, most of the compounds exhibited higher cyto-
selectivity in cancer cell lines than the control drug. The mode
of action of these drugs and their transporting properties were
supported by DNA and BSA binding experiments. Considering
the fluorescence property in live cells and high cytoselectivity of
compound 6g, it can be predicted that it will be established as a
cancer theranostic drug.
(
s, 1H, H-12), 7.93 (s, 1H, CH-14), 7.81–7.83 (m, 4H, CH, H-16,
H-20, H-23, H-24), 7.74 (d, J = 8.8 Hz, 2H, CH, H-2, H-3), 7.41 (t, J =
7
.6 Hz, 2H, CH, H-17, H-19), 7.35 (d, J = 7.2 Hz, 1H, CH, H-18),
.93 (d, J = 8.4 Hz, 2H, CH, H-22, H-26), 6.12 (s, 4H, CH , H-9,
, H-27); IR (KBr, cm ): 3340 (N–H sym-
metric and asymmetric stretching), 2935 (C–H Ar stretching),
833 (CH asymmetric stretching),1647 (CQC stretching), 1562
C–H bending), 1429 (CH bending), 705 (C–H stretching of the
o-disubstituted group); LCMS (CH OH): m/z 475 [M + H] ; HRMS
6
2
ꢀ
1
H-10), 3.83 (s, 3H, CH
3
2
(
2
2
+
3
+
(ESI): m/z calcd for C H N O: 475.1917 [M + H] ; found: 475.1921
27 22 8
+
[M + H] .
2-((4-Phenyl-1H-1,2,3-triazol-1-yl)methyl)-3-((4-(pyridin-2-yl)-
1
2
H-1,2,3-triazol-1-yl)methyl)quinoxaline (10c). Yield: 92%; mp:
1
30–335 1C; R
f
0.35 (1 : 1 ethylacetate : hexane); H NMR
(400 MHz, CDCl
3
): d 8.07 (brs, 3H, CH, H-16, H-17, H-19),
8
.02 (s, 1H, CH, H-18), 7.81 (d, J = 6.8 Hz, 6H, CH, H-1, H-4, H-2,
H-3, H-21, H-25), 7.40 (t, J = 7.2 Hz, 3H, CH, H-22, H-23, H-24),
7
3
ꢀ1
.32 (d, J = 6.8 Hz, 2H, CH), 6.15 (d, 4H, CH); IR (KBr, cm ):
3
412 (N–H symmetric and asymmetric stretching), 3082 (sp
List of abbreviations
C–H stretching), 1600 (CQC), 1562 (C–H bending), 1450, 1421
(
CH
2
bending), 1292 (C–N stretching), 783, 756 (C–H stretching NMR
Nuclear magnetic resonance
Liquid chromatography mass spectrometry
High resolution mass spectrometry
Parts per million
+
of the o-disubstituted group); LCMS (CH
HRMS (ESI): m/z calcd for C25
4
3
OH): m/z 446 [M + H] ; LCMS
: 446.1763 [M + H] ; found: HRMS
+
H
19
N
9
+
46.1768 [M + H] .
ppm
2
-((4-(Pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)-3-((4-(p-tolyl)- MLCT
Metal to ligand charge transfer
3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
Glutathione
1
2
(
H-1,2,3-triazol-1-yl)methyl)quinoxaline (10d). Yield: 90%, mp: MTT
42–245 1C; Rf 0.36 (1 : 1 ethylacetate : hexane); H NMR
400 MHz, CDCl
1
3
): d 8.38 (s, 1H, CH, H-19), 8.07 (m, 3H, CH, GSH
H-16, H-17), 7.96 (s, 1H, CH, H-18), 7.79–7.82 (m, 4H, CH, TLC
Thin layer chromatography
Singlet
H-22, H-24, H-21, H-25), 7.69 (d, J = 7.6 Hz, 2H, CH, H-1, H-4),
s
7
CH
.21 (d, J = 8.0 Hz, 3H, CH), 6.15 (s, 2H, CH
2
, H-9), 6.10 (s, 2H,
d
Doublet
Broad singlet
1
3
2
, H-10), 2.35 (s, 3H, CH, H-26); C NMR (100 MHz, DMSO- brs
d ): d 149.5 (2 ꢁ CQN, C-7, C-8), 149.3 (CQN, C–), 147.03
t
m
Triplet
Multiplet
Optical density
Calf-thymus DNA
Bovine serum albumin
Human serum albumin
Ethidium bromide
Density functional theory
Intrinsic binding constant
Stern–Volmer quenching constant
Apparent binding constant
Overall binding constant
6
(
2 ꢁ CQC–N, C–), 140.9 (CH), 140.8 (2 ꢁ C–N), 137.1 (C–C),
1
31.29 (C–C), 131.2 (2 ꢁ CH), 129.5 (CH), 129.0 (2 ꢁ CH), 128.4 OD
(
(
2 ꢁ CH), 125.6 (2 ꢁ CH), 125.3 (2 ꢁ CH), 122.9 (2 ꢁ CH), 52.4 CT-DNA
ꢀ1
2 ꢁ CH
2
), 21.31 (CH
3
); IR (KBr, cm ): 3309 (N–H symmetric BSA
and asymmetric stretching), 1635 (CQC stretching), 1421 HSA
CH bending), 1327 (C–N stretching); LCMS (CH OH): m/z EtBr
60 [M + H] ; HRMS (ESI): m/z calcd for C26
(
4
2
3
+
H
21
N
9
: 460.1920 DFT
+
+
[
M + H] ; found: 460.1926 [M + H] .
Kb
2
-((4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-3-((4- Ksv
pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)quinoxaline (10e). Kapp
0.38 (1 : 1 ethylacetate :
): d 8.55 (brs, 1H, CH,
H-19), 8.36 (brs, 2H, CH, H-16, H-17), 8.15 (d, J = 5.6 Hz, 2H,
(
Yield: 88%, mp: 278–280 1C, R
hexane); H NMR (400 MHz, CDCl
f
K
1
3
CH, H-1, H-4), 8.05–8.08 (m, 3H, CH, H-2, H-3), 7.91 (s, 1H, Conflicts of interest
CH, H-18), 7.79–7.82 (m, 5H, CH, H-22, H-24, H-21,
There are no conflicts to declare.
H-25), 6.14 (s, 2H, CH, H-12, H-14), 6.09 (s, 2H, CH , H-9,
2
ꢀ
1
H-10), 3.82 (s, 3H, CH , H-26); IR (KBr, cm ): 3084,
3
1
602 (CQC stretching), 1452 (CH2 bending), 1134 (C–N
Acknowledgements
+
stretching); LC-MS (CH
ESI): m/z calcd for C26
3
OH): m/z 476.0 [M + H] ; HRMS
+
(
4
H
21
N
9
O: 476.1869 [M + H] ; found: The authors are grateful to VIT University for providing VIT
SEED funding. We acknowledge DST, New Delhi, India for a
+
76.1874 [M + H] .
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DST-FIST project. We also acknowledge DST-SERB, India for a 25 M. V. Gil, M. J. Arevalo and O. Lopez, Synthesis, 2007, 1589.
young scientist grant (YSS/2014/000842) and DST-EMR grant 26 C. R. Becer, R. Hoogenboom and U. S. Schubert, Angew.
(EMR/2017/000816).
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