Design and synthesis of C3-tethered 1,2,3-triazolo-β-carboline derivatives: Anticancer activity, DNA-binding ability, viscosity and molecular modeling studies

Article abstract of DOI:10.1016/j.bioorg.2015.11.005

A series of new DNA-interactive C3-tethered 1,2,3-triazolo-β-carboline derivatives have been synthesized via 'click' reaction and evaluated for their in vitro cytotoxicity as well as DNA binding affinity. Interestingly, these hybrids have displayed potent in vitro cytotoxicity in comparison to Harmine against the HT-29 (colon cancer) and HGC-27 (gastric cancer) cell lines. The compounds 7f, 7k, 7n and 7s appear to be more effective against the HGC-27 cell line, among which compound 7f showed the highest cytotoxicity (5.44 ± 0.58, IC₅₀ μM). The compounds 7e and 7f appear to be more active against the HT-29 cell line, among which compound 7f exhibited the highest cytotoxicity (3.67 ± 0.62, IC₅₀ μM). To gain more insight into the DNA-binding ability, spectroscopic techniques such as UV-Visible, fluorescence and circular dichroism studies were performed. Viscosity measurements and molecular docking studies substantiate that these compounds indeed bind to DNA via the minor groove.

Full text of DOI:10.1016/j.bioorg.2015.11.005

Accepted Manuscript
Design and synthesis of C3-tethered 1,2,3-triazolo-β-carboline derivatives: An-
ticancer activity, DNA-binding ability, viscosity and molecular modeling stud-
ies
Nagula Shankaraiah, Chetna Jadala, Shalini Nekkanti, Kishna Ram Senwar, Narayana
Nagesh, Shweta Shrivastava, V.G.M. Naidu, Manda Sathish, Ahmed Kamal
PII:
S0045-2068(15)30036-5
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.11.005
YBIOO 1859
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
21 July 2015
10 November 2015
21 November 2015
Please cite this article as: N. Shankaraiah, C. Jadala, S. Nekkanti, K.R. Senwar, N. Nagesh, S. Shrivastava, V.G.M.
Naidu, M. Sathish, A. Kamal, Design and synthesis of C3-tethered 1,2,3-triazolo-β-carboline derivatives: Anticancer
activity, DNA-binding ability, viscosity and molecular modeling studies, Bioorganic Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bioorg.2015.11.005
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Design and synthesis of C3-tethered 1,2,3-
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Leave this area blank for abstract info.
a,
a
a
a
c,*
Nagula Shankaraiah , Chetna Jadala , Shalini Nekkanti , Kishna Ram Senwar , Narayana Nagesh , Shweta
d
d
b
a,b*
Shrivastava , V. G. M. Naidu , Manda Sathish , Ahmed Kamal
A series of new DNA-interactive C3-tethered 1,2,3-triazolo-β-carboline derivatives have been synthesized via ‘click’
reaction and evaluated for their in vitro cytotoxicity as well as DNA binding affinity.
2

Bioorganic Chemistry
Design and synthesis of C3-tethered 1,2,3-triazolo-β-carboline derivatives:
Anticancer activity, DNA-binding ability, viscosity and molecular modeling
studies
a,
a
a
a
c,*
Nagula Shankaraiah , Chetna Jadala , Shalini Nekkanti , Kishna Ram Senwar , Narayana Nagesh ,
d
d
b
a,b*
Shweta Shrivastava , V. G. M. Naidu , Manda Sathish , Ahmed Kamal
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad-500 037, India
Medicinal Chemistry and Pharmacology , CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
b
c
CSIR-Centre for Cellular & Molecular Biology, Hyderabad-500 607, India
d
Pharmacology and Toxicology Department, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of new DNA-interactive C3-tethered 1,2,3-triazolo-β-carboline derivatives have been
synthesized via ‘click’ reaction and evaluated for their in vitro cytotoxicity as well as DNA
binding affinity. Interestingly, these hybrids have displayed potent in vitro cytotoxicity in
comparison to Harmine against the HT-29 (colon cancer) and HGC-27 (gastric cancer) cell lines.
The compounds 7f, 7k, 7n and 7s appear to be more effective against the HGC-27 cell line,
among which compound 7f showed the highest cytotoxicity (5.44±0.58, IC50 µM). The
compounds 7e and 7f appear to be more active against the HT-29 cell line, among which
compound 7f exhibited the highest cytotoxicity (3.67±0.62, IC50 µM). To gain more insight into
the DNA-binding ability, spectroscopic techniques such as UV-visible, flourescence and circular
dichroism studies were performed. Viscosity measurements and molecular docking studies
substantiate that these compounds indeed bind to DNA via the minor groove.
Keywords:
β-Carbolines
1
,2,3-Triazoles
Click reaction
Cytotoxicity
DNA-binding ability
Minor groove binder
Molecular modeling
2
009 Elsevier Ltd. All rights reserved.
1. Introduction
demonstrated that the presence of appropriate groups at the C1
and C3-positions of the β-carboline core enhanced antitumor as
well as DNA-binding ability. It was also observed that β-
Anticancer drugs that target DNA are one of the most
effective agents in clinical use and have enhanced the survival
3
carboline hybrids with substituted phenyl group and
heterocyclics at the C1 and C3-positions respectively, have
rates of patients when used alone or in combination with other
1
drugs. Lately, DNA interacting agents have been extensively
4
displayed potential antitumor properties.
studied with regard to cancer chemotherapy, which has led to the
development of many novel heterocyclic compounds. These
agents exert their biological profile by non-covalent and covalent
interactions in either the minor or major groove or between the
base pairs (intercalation) of the double helix. Anticancer drug
discovery has focused mainly on the development of novel DNA
Over the past few years, several molecules possessing 1,2,3-
triazole scaffold have been synthesized as useful
5
chemotherapeutic agents possessing antibacterial, antifungal,
6
7
antihelmintic and anticancer activities. Among the diverse
synthetic tools helpful for scaffold ligation, 1,2,3-triazole
formation by Cu(I)-catalyzed azide-alkyne cycloaddition
(CuAAC) reactions are significant owing to their reliability,
interacting
scaffolds
like
anthracyclines,
acridines,
2
anthraquinones, distamycins, β-carbolines and their derivatives.
Structural alteration of these bioactive scaffolds may result in the
development of new drugs with improved therapeutic properties.
8
greenness and atom economy. ‘Click’ reaction has emerged as
powerful modular reaction towards the synthesis of diverse
compounds and have found widespread applications in
The β-carboline alkaloids display a broad spectrum of
biological and pharmacological properties including DNA
intercalation and minor grove binding, resulting in antitumor
activity. Previous Structure-Activity Relationship (SAR) analysis
9
10
combinatorial drug discovery, material science, and bio-
11
conjugate chemistry. In view of the biological importance of
1,2,3-triazoles and β-carbolines, it was of considerable interest to
—
——
*
Author to whom correspondence should be addressed. Dr. N. Shankaraiah, E-mail: shankar.niperhyd@gov.in;
shankarnbs@gmail.com; Tel.: +91-40-23073741; fax: +91-40-23073751; Dr. N. Nagesh, E-mail: nagesh@ccmb.res.in; Dr
Ahmedkamal, E-mail: ahmedkamal@iict.res.in
3

develop novel conjugates incorporating both the ring systems,
such a combination could enhance the DNA interacting ability.
linked and N-linked alkynes were used, which were prepared
from substituted phenols and amines respectively. Finally, the
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction
was performed to afford C3-linked 1,2,3-triazolo-β-carboline hy
brids 7a–t in 70–95% yields as shown in Table 1. The chemical
Table 1. Synthesis of C3-linked 1,2,3-triazolo-β-
carboline hybrids 7a–t
1
2
3
4
a
compound
R
R
R
R
yield (%)
7
a
OCH
OCH
3
3
OCH
OCH
3
3
OCH
OCH
3
3
95
7
b
90
7
c
OCH
OCH
3
3
OCH
OCH
3
3
OCH
OCH
3
3
88
93
7
d
7
e
OCH
OCH
3
3
OCH
OCH
3
3
OCH
OCH
3
3
80
75
7
f
Scheme 1. Synthesis of various C3-tethered triazolo-β-carboline
derivatives 7a–t.
7g
OCH
OCH
3
3
OCH
OCH
3
3
OCH
OCH
3
3
70
87
7
h
Moreover, the 1,4-substituted 1,2,3-triazole moiety built
between the scaffolds can mimic the trans geometry of an amide
bond, resulting in the conversion of a nonpeptidic molecule to a
peptidomimetic with rigidity and protease stability, valuable in
drug discovery. In this context, we have synthesized a library of
hybrids that are diversified at positions C1 and C3 of the β-
carboline scaffold, which possess potential antitumor activity and
investigated their mode of DNA interaction. Since, 1,2,3-
triazoles are one of the most flexible, easy to introduce and
attractive connecting units, herein, we have designed and
synthesized a series of novel C3-linked 1,2,3-triazolo-β-carboline
hybrids by employing ‘click’ approach. The focus of this
investigation was to explore the optimal structural requirements
of the compounds for antitumor activities, and further
development of more potent β-carboline hybrids.
7
i
OCH
H
3
OCH
OCH
3
3
OCH
OCH
3
3
90
93
7j
7
k
H
OCH
3
OCH
3
84
7
l
H
H
OCH
OCH
3
3
OCH
OCH
3
3
85
85
7
m
2. Results and discussion
2
.1. Chemistry
7
n
H
H
H
OCH
OCH
Cl
3
3
OCH
OCH
H
3
3
82
90
95
The synthetic route for the novel C3-linked 1,2,3-triazolo-β-
carboline hybrids was outlined in Scheme 1. The β-carboline ring
system was obtained from the commercially available L-
tryptophan (1), which was esterified with SOCl in methanol to
2
provide L-tryptophan methyl ester. Methyl 1-(3, 4, 5 substituted
phenyl)-9H-pyrido[3,4-b] indole-3-carboxylates (3a–c) were
prepared by the condensation of L-tryptophan methyl ester with
7o
7
7
p
q
3,4,5-substituted benzaldehydes 2a–c via Pictet-Spengler
H
H
Cl
Cl
H
H
88
82
condensation followed by dehydrogenation with sulphur in
refluxing xylene to afford the fully unsaturated compounds 4a–c.
Later, the ester group at C3-position was reduced into the
corresponding alcohols by LiAlH
mesylated with mesyl chloride by using Et
7
r
4
in dry THF. The alcohol was
N. Then, the
3
compounds 5a–c were made to react with sodium azide in DMF
to yield substituted 9H-pyrido indolyl azides 6a–c. For the
generation of 1,2,3-triazoles, different substituted alkynes i.e., O-
7s
7t
H
H
Cl
Cl
H
H
85
90
4

a
Isolated yields.
1
structures of all the new compounds were characterized by H,
C NMR, HRMS, and IR spectroscopic techniques.
lines while the compound 7n with dimethoxy phenyl substituent
at C1-position and 1,2,3-triazolo naphthalimide at C3-position
exhibited cytotoxicities similar to that of 7f. Similarly,
compounds 7e and 7k possessing phthalimide groups at C3-
position have shown significant activity on colon cancer
(5.79±1.85, IC50 µM and 20.18±2.42, IC50 µM respectively) and
gastric cancer (6.27±0.93, IC50 µM and 20.18±2.42, IC50 µM
respectively) cell lines. From these results, we could infer that the
presence of a phthalimide or naphthalimide pharmacophore
linked through a 1,2,3-triazole at C3-position played a key role in
the modulation of cytotoxic potencies.
13
2.2. MTT assay
The novel β-carbolines bearing substituted 1,2,3-triazole
moieties at C3-position have been evaluated for their in vitro
cytotoxicity against PC-3 (prostate cancer), HeLa (cervical
carcinoma), MCF-7 (breast cancer), HT-29 (Colon cancer), and
HGC-27 (Human gastric carcinoma) cell lines by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay and compared with that of Harmine and Hoechst 33258.
The results of this cytotoxicity data expressed as IC50 values were
summarized in Table 2. These compounds have displayed
significant in vitro cytotoxicity in comparison to Harmine against
the HT-29 and HGC-27 cell lines. The compounds 7f, 7k, 7n and
2.3. Circular dichroism (CD) studies
Circular dichroism (CD) studies provide information on
changes in the DNA conformation upon interaction with
derivatives. DNA is a very important biological macromolecule
which plays a major role in the cell proliferation and other bio
logical activities. Hence, changes in the DNA topology will
provide information on the nature of DNA-ligand interactions
and conformational changes on its interaction with DNA. The
CD spectrum of CT-DNA exhibits a positive band at 275 nm and
a negative band at 245 nm due to π–π base stacking and right-
hand helicity, the characteristic profile of B form DNA. In the
present study, the positive CD exhibited hypochromocity initially
and on increasing the ratio between CT-DNA and 7f from 1:1 to
1:2, the positive band showed slight hyperchromicity. This
indicates that 7f unfolds DNA at lower DNA: derivative ratio and
on increasing the derivative concentration, it tends to stabilize the
7
(
s appear to be more effective against the gastric carcinoma
HGC-27) cell line with IC50 values lower than 10 µM, among
which compound 7f displayed the highest cytotoxicity
5.44±0.58, IC50 µM). Compound 7f was also potently cytotoxic
(
against the colon cancer (HT-29) cell line (3.67±0.62, IC50 µM)
compared to the standard drug Harmine (4.28±1.15, IC50 µM).
Interestingly, the compounds 7f and 7n were found to be
significantly cytotoxic against all the five human cancer cell lines
investigated with IC50 values of lower than 27 µM. All these
hybrids have shown lower activity against the breast cancer cell
line with IC50 values greater than that of harmine (3.52±0.78, IC50
µM). In the prostate cancer (PC-3) cell line, only the hybrids 7f
and 7n have displayed significant cytoxicities compared to that
of harmine (24.45±3.93, IC50 µM) while the other hybrids have
displayed no activity. Interestingly, compounds 7f, 7h, 7n and 7s
were almost twice as cytotoxic as Hoechst 33258 in the MCF-7
cell line.
12
DNA-derivative complex. The negative band intensities have
gradually increased indicating decrease in the DNA helicity on β-
carboline’s interaction with DNA. On the other hand CD spectra
obtained on 7n and 7s interaction with DNA was different. The
positive peak at 275 nm exhibited hyperchromicity.
Hyperchromicity of the CD positive band indicates stabilization
The most potent compound 7f with a trimethoxy phenyl at C1-
position and 1,2,3-triazolo methyl phthalimide at C3-position,
has displayed good to moderate IC50 values on all the cancer cell
13
of CT-DNA on 7n and 7s interaction. The positive CD band int-
Table 2. Cytotoxic (IC50 μM) evaluation of 1, 2, 3-triazolo-β-carboline derivatives 7a–t
a
Compound
PC-3
MCF-7
HeLa
HT-29
HGC-27
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
>25
>25
>25
>25
>25
22.4±2.42
>25
>25
>25
>25
>25
>25
>25
18.5±2.46
>25
>25
>25
>25
>25
>25
>25
>25
>25
26.5±0.79
>25
29.6±1.00
>25
>25
>25
>25
>25
19.7±3.28
>25
>25
>25
>25
22.0±2.45
>25
>25
>25
>25
>25
>25
15.6±0.19
>25
>25
>25
>25
>25
>25
>25
23.7±2.59
>25
>25
>25
>25
>25
>25
>25
>25
5.79±1.85
3.67±0.62
>25
>25
>25
>25
20.18±2.42
>25
>25
7.46±1.69
>25
>25
>25
>25
15.55±2.32
>25
>25
>25
>25
>25
>25
5.44±0.58
>25
>25
>25
>25
6.27±0.93
>25
>25
8.67±1.28
>25
>25
>25
>25
9.95±1.52
>25
>25
>25
>25
>25
t
Harmine
Hoechst 33258
24.45±3.93
N.D
3.52±0.78
52.2±2.1
14.59±2.52
0.02±0.005
4.28±1.15
N.D
10.56±1.75
N.D
a
PC-3 (Prostate cancer), MCF-7 (Breast cancer), HeLa (Cervical cancer), HT-29 (Colon cancer), HGC-27 (Human gastric carcinoma). Half
maximal inhibitory concentration values (IC50) are the mean ±SD of three individual experiments performed in duplicate. Harmine (natural
product) was included as reference standards. ND: not determined.
5

Figure 1. CD spectra of hybrids 7f and 7n (a, b) with CT-DNA in the absence or presence of increasing amounts of 7f and 7n.
ensities did not change even on increasing 7n and 7s
concentration. The CD spectra of 7f after interaction with CT-
DNA exhibited couple of isodichroic points at 257 nm and 288
nm, manifesting that the interaction between the 7f molecules
and DNA occurs in multiple steps and more than one species co-
exists in equilibrium with each other. The CD spectra obtained
when 7f and 7n interact with CT-DNA were shown in Figure 1a
and 1b respectively.
1
4
(a)
2.4. UV-Visible spectral studies
The results from the UV-Visible spectral studies indicate the
mode of DNA-ligand interaction. In the present study, on
addition of equal increments of CT-DNA to 7f solution, the
absorption band has showed continuous increase in its intensity
and the hyperchromicity is more evident at 220 nm. Such
continuous hyperchromicity of the absorption band is an
indication of electrostatic binding to CT-DNA. Compounds 7n
and 7s have also exhibited absorption bands at 875 nm and 570
nm respectively and the absorption band of these derivatives also
exhibited hyperchromicity on interaction with CT-DNA,
manifesting electrostatic binding or partial unfolding of CT-DNA
(
b)
1
5,16
exposing more bases to the incident light.
On the other hand,
Figure 2. UV–visible spectra of harmine and 7f (a,b).
when CT-DNA was added to Harmine solution, the absorption
band at 303 nm has shown hypochromisity and red shift of about
2
5 nm (shifed to 328 nm). This indicates that Harmine
(a)
intercalates between the bases of CT-DNA. Since the UV-Visible
spectral pattern of 7f, 7s and 7n derivative was same, a
representative UV-Visible spectral profile obtained when 7f -
interacts with CT-DNA was shown in Figure 2.
2.5. Fluorescence emission spectra
Fluorescence titration is yet another valuable technique for
understanding the binding mode of small molecules with DNA
and to study the electronic environment around the DNA-
complex at comparatively lower concentrations. Since the
complexes are fluorescent, their interaction with DNA can be
monitored at low concentration with this study. Emission spectra
of 7f, 7n and 7s show prominent peaks at 512.5 nm, 875 nm and
(b)
573 nm respectively. On addition of equal increments of CT-
DNA to all the β-carboline derivatives, the emission peak
intensities decreased gradually, indicating that the derivative
17
molecules bind externally to the surface of CT-DNA. The
fluorescence hypochromicity occurs due to the dissipation of
energy by excited derivative molecules to the surrounding water
molecules. Whereas when CT-DNA was added to Harmine
solution, the fluorescence emission band at 418 nm exhibited
hyperchromicity. This shows that Harmine will intercalate
between the bases of CT-DNA. The representative fluorescence
emission spectrum obtained after the addition of increasing
concentrations of CT-DNA to 7f is shown in Figure 3.
Figure 3. Fluorescence spectra of harmine and 7f (a,b) in the presence
of increasing amount of CT-DNA.
6

2.6. DNA interaction study by viscosity measurements
Spectroscopic studies provide information about the type of
ligand binding to DNA. However, these studies alone cannot
illustrate the nature of ligand binding to DNA. Hence, relative
viscosity experiments were carried out to have a clear
understanding on 7f, 7s and 7n derivative-DNA interaction.
Intercalation of β-carboline derivative molecules between the two
stakes of DNA results in an increase of the viscosity of DNA
18
solutions. Very small variation in viscosity is observed in case
19
of molecules that bind to the surface of DNA or groove binders,
whereas a reduction in the relative viscosity is typically observed
20
with covalent DNA-binding.
Figure 5. DNA minor groove binding of 7f with DNA. DNA is shown in
ribbon form and 7f in yellow tube form.
3. Conclusion
In conclusion, an efficient access to the construction of a
novel C3-tethered 1,2,3-triazolo-β-carboline hybrids with
promising cytotoxicity and DNA interacting property has been
accomplished. Derivatives 7f and 7n were found to be the most
potent cytotoxic agents with IC50 values lower than 27 µM
against the panel of human cancer cell lines investigated (PC-3,
MCF, Hela, HT-29, HGC-27). Molecular docking studies have
shown that these derivatives bind to DNA through minor groove
binding. To further strengthen the DNA interacting ability of
these congeners, DNA-binding assays were performed including
circular dichroism, fluorescence titration, UV–visible and
viscometric titration studies which indicated strong electrostatic
binding of the derivatives into DNA. Based on the cytotoxicity
and DNA-binding affinity, the Structure Activity Relationships
(SARs) for this new class of derivatives were proposed and it
was observed that the presence of a phthalimide or naphthalimide
pharmacophore linked through a 1,2,3-triazole at the C3-position
enhanced the cytotoxicity and DNA-binding potential.
Figure 4. Relative viscosity experiment of hybrids 7f, 7n and 7s with CT-
DNA. EtBr, Harmine and Hoechst 33258 were used as controls.
Ethidium bromide (EtBr), a well-known DNA intercalater
increases the relative viscosity strongly by lengthening the DNA
double helix. Whereas relative viscosity remains almost unaltered
in case of well-known groove binders like Hoechst 33258. In the
present experiment, EtBr was taken as a control and Hoechst
33258 was considered as positive control. On slowly increasing
the concentration of the β-carboline derivatives, the relative
viscosity of 7f, 7n and 7s complex solutions did not change
much. However, when EtBr is added to CT-DNA, enhancement
in viscosity of the solution was noticed. Similarly, when Harmine
was added to CT-DNA solution, gradual enhancement of
viscosity was noticed, indicating intercalation of Harmine with
DNA (like EtBr). Further, on addition of Hoechst 33258 also not
much change was observed. The lesser variation in viscosity
suggests that these derivative molecules, bind to the surface of
DNA through minor like Hoechst 33258. Among the β-carboline
derivatives studied, 7f showed marginal increase in viscosity
after its interaction with DNA, indicating better interaction of 7f
hybrid with CT-DNA (Figure 4). The data obtained from
viscosity studies supports the results obtained in spectroscopic
studies.
4. Experimental
4.1. General
1
13
H NMR and C NMR spectra are recorded on Varian Gemini
00 or Varian Unity 400 or Varian Inova 500 or Bruker Avance
00 MHz Making a solution of samples in CDCl solvent using
3
2
3
Tetramethylsilane (TMS) as the internal standard unless
otherwise mentioned, and are given in the  scale. The standard
abbreviations s, d, t, m, dd refer to singlet, doublet, triplet,
multiplet and doublet of a doublet respectively. Infrared (IR)
spectra are recorded on Perkin-Elmer Infrared–683 or 1310 with
NaCl optics. Spectra were calibrated against the polystyrene
2.7. Molecular Modelling
Molecular docking simulations were carried out using XP
21
Glide 6.0 (Schrödinger 2013) with default settings into DNA
duplex. The docked poses for the one of the best scored hybrids
7f, shown in Figure 5, depict its binding along the minor groove.
-1
absorption at 1610cm . Samples were scanned neat, KBr wafers
Interestingly, the 1,2,3-triazole ring forms a kink in the molecule
akin to the peptide bond and shapes it in such a way that it orients
itself in a curved manner along the minor groove. Further, the
docked poses were stabilized electronically by hydrogen-bonds
between the carbonyl oxygens of the phthalimide and the side
chains of the DNA base pairs. The aromatic ring of the
phthalimide was also observed to be involved in side wise π-π
stacking with the base pairs. The results obtained from molecular
docking studies were consistent with the DNA binding studies as
well as in vitro cytotoxicity data.
or in chloroform as a thin film. Mass spectra recorded on CEC-
21-110B, Finnegan Mat 1210 or MICROMASS-7070
spectrometers operating at 70eV using a direct inlet system. If
necessary, FABMS is recorded. Melting points are determined on
an Electro thermal melting point apparatus and are uncorrected.
All reactions are monitored by thin layer chromatography (TLC)
carried out on 0.25 mm E. Merck silica gel plates (60F-254) with
UV light, iodine as probing agents. Acme (India) silica gel (finer
than 200 mesh) is used for flash chromatography. Column
chromatography was performed by using silica gel 60-120, 100-
7

2
00 mesh. The reactions wherever anhydrous conditions needed
3-((4-((4-Methoxyphenoxy) methyl)-1H-1, 2, 3-triazol-1-yl)
methyl)-1-(3, 4, 5-trimethoxyphenyl)-9H-pyrido [3, 4-b]
indole (7d). 93% Yield; Mp: 176–179 C; FT-IR (cm ) 3368,
are carried out under the positive pressure of nitrogen atmosphere
using dry and freshly distilled solvents. All solvents and reagents
were purified by standard techniques. All evaporation of solvents
was carried out under reduced pressure on Heidolph rotary
evaporator below 45 °C. Yield reported are isolated yields of
material judged homogeneous by TLC and NMR spectroscopy.
The names of all compounds given in the experimental section
were taken from Chem. ultra, version 11.0. All computational
modeling are performed on a desktop PC with a Red Hat
Enterprise linux version 5.0 using Maestro software version 9.5.
o
-1
3152, 2990, 2960, 2931, 2827, 2361, 1717, 1625, 1581, 1508,
1
1453, 771, 721, 612, 521; H NMR (300 MHz, CDCl
3
): δ 8.6
(brs, 1H), 8.12 (d, 1H, J = 7.55 Hz), 7.82 (d, 2H, J = 3.77 Hz),
7.46 (t, 2H) 7.34(t, 1H), 7.12 (s, 2H), 6.23 (s, 2H), 5.90 (s, 2H),
13
5.17 (s, 2H),3.90(s, 18H); C NMR (75 MHz, CDCl ):  157.8,
3
153.1, 152.9, 151.3, 148.2, 142.3, 141.3, 137.8, 132.4, 130.5,
123.3, 122.9, 121.4, 115.3, 111.1, 107.9, 103.0, 72.3, 60.8, 56.1,
55.8, 57.1; HRMS (ESI): m/z calcd. for C31
found 552.2297 [M+H]
30
H O
5
N
5
552.2247,
+
4
4
.2. Chemistry
2-((1-((1-(3, 4, 5-Trimethoxyphenyl)-9H-pyrido [3, 4-b]
.2.1 General procedure for the synthesis of 7a–t.
indol-3-yl) methyl)-1H-1, 2, 3-triazol-4-yl) methyl)
o
To a stirred solution of alkyne (1.0 mmol) and β-carboline-azide
a–c (1.2 mmol) in tert-butanol-water (1:1) (20 mL),
isoindoline-1, 3-dione (7e). 80% Yield; Mp:168–171 C; FT-IR
-1
6
(cm ) 3354, 3157, 3061, 2941, 2834, 2361, 1947, 1763, 1678,
.
4
1
CuSO
were added and the mixture was stirred at room temperature for
2 h. The progress of the reaction was monitored by TLC. The
2
5H O catalyst (1 mol%) and sodium ascorbate (5 mol%)
3
1585, 1505, 1423, 935, 785, 691; H NMR (300 MHz, CDCl ): δ
8.6 (brs, 1H), 7.92 (s, 2H), 7.84 (s, 2H), 7.52 (t, 1H, J = 7.6 Hz),
7.56 (s, 1H), 7.25 (s, 1H), 7.32 (t, 1H, J = 6.7 Hz ) 5.84 (s, 2H),
1
13
resulting reaction mixture was washed with water (20 mL) and
extracted with ethyl acetate (60 mL). The organic layer was dried
with sodium sulfate and filtered. The filtrate was evaporated at
reduced pressure, which was purified by silica gel column
chromatography with a gradient elution of ethyl acetate/n-hexane
to afford a desired products 7a–t.
3
4.99 (s, 2H), 3.96(s, 9H); C NMR (75 MHz, CDCl ):  168.2,
157.8, 153.1, 148.2, 141.9, 141.3, 137.8, 132.4, 132.5, 131.9,
130.7, 130.5, 129, 127.8, 127.5, 123.3, 122.9, 121.4, 121.7,
119.8, 111.1, 107.9, 100.8, 60.8, 57.1, 56.1; HRMS (ESI): m/z
+
27 5 6
calcd. for C32H O N 575.2043, found 575.2022 [M+H]
5
-Methyl-2-((1-((1-(3, 4, 5-trimethoxyphenyl)-9H-pyrido
3
-((4-((3, 4, 5-Trimethoxyphenoxy) methyl)-1H-1, 2, 3-
[3, 4-b] indol-3-yl) methyl)-1H-1, 2, 3-triazol-4-yl) methyl)
o
triazol-1-yl) methyl)-1-(3, 4, 5-trimethoxyphenyl)-9H-pyrido
3, 4-b] indole (7a). 95%Yield; Mp: 143–145 C; FT-IR (cm )
316, 3155, 2938, 2360, 1585, 1504, 1465, 1451, 1395, 1231,
192, 1126, 812, 747, 611, 438; H NMR (300 MHz, CDCl
.6 (brs, 1H), 8.12 (d, 1H, J = 7.5 Hz), 7.82 (d, 2H, J = 3.8 Hz),
.46 (t, 2H), 7.34 (t, 1H), 7.12 (s, 2H), 6.23 (s, 2H), 5.90 (s, 2H),
.17 (s, 2H), 3.90 (s, 18H); C NMR (75 MHz, CDCl
53.1, 152.9, 151.2, 148.2, 142.3, 141.3, 137.8, 132.4, 131.5,
isoindoline-1, 3-dione (7f). 75% Yield; Mp: 143–145 C; FT-IR
o
-1
-1
[
(cm ) 3354, 3157, 3061, 2941, 2834, 2361, 1947, 1763, 1678,
1
3
1
8
7
5
1
1
6
6
1585, 1505, 1423, 935, 785, 691; H NMR (300 MHz, CDCl
3
): δ
1
3
): δ
8.6 (brs, 1H), 7.92 (s, 2H), 7.84 (s, 2H), 7.52 (t, 1H, J = 6.3 Hz ),
7.56 (s, 1H), 7.25 (s, 1H), 7.32 (t, 1H, J = 7.5 Hz ) 5.84 (s, 2H),
13
4.99 (s, 2H), 3.96 (s, 9H), 2.43 (s, 3H); C NMR (75 MHz,
CDCl ):  168.2, 157.8, 153.1, 148.2, 141.9, 141.3, 137.8, 132.4,
13
3
):  157.6,
3
132.5, 131.9, 130.7, 130.5, 129, 127.8, 127.5, 123.3, 122.9,
121.4, 121.7, 119.8, 111.1, 107.9, 100.8, 60.8, 57.1, 56.1, 20.9;
30.5, 123.3, 122.9, 121.7, 121.4, 119.8, 111.1, 100.8, 93.7, 72.3,
0.8, 57.1, 56.1; HRMS (ESI): m/z calcd. for C33
12.2458, found 612.2441 [M+H]
34
H N
5
O
7
HRMS (ESI): m/z calcd. for C33
589.2180 [M+H]
29
H O
5
N
6
589.2199, found
+
+
3
-((4-((4-Allyl-2-methoxyphenoxy) methyl)-1H-1, 2, 3-
3-((4-((3, 4-Dihydroquinolin-1(2H)-yl) methyl)-1H-1, 2, 3-
triazol-1-yl) methyl)-1-(3, 4, 5-trimethoxyphenyl)-9H-pyrido
triazol-1-yl) methyl)-1-(3, 4, 5 trimethoxyphenyl)-9H-pyrido
3, 4-b] indole (7b). 90% Yield; Mp: 150–153 C; FT-IR (cm )
316, 3141, 2937, 2832, 2367, 1623, 1583, 1508, 1465, 1427,
396, 1364, 1327, 914, 848; H NMR (300 MHz, CDCl
o
-1
o
-1
[
3
1
[3, 4-b] indole (7g). 70% Yield; Mp: 183–185 C; FT-IR (cm )
1
3384, 2925, 2366, 1219, 1043, 772; H NMR (300 MHz,
1
3
): δ 8.56
3
CDCl ): δ 8.63 (brs, 1H), 8.07 (d, 1H, J = 7.9 Hz), 7.96 (d, 1H, J
(
7
(
2
brs, 1H), 8.02 (d, 1H, J = 8.1 Hz), 7.82 (s, 2H), 7.46 (s, 3H)
.32 -7.16 (m, 1H), 7.04 (s, 1H), 6.90 (d, 1H, J = 8.4 Hz), 6.61
d, 2H, J = 6.9 Hz), 5.81 (s, 2H), 5.91-5.72 (m, 1H), 5.22 (s,
= 7.5 Hz), 7.59 (t, 1H, J = 8.2 Hz), 7.53 (s, 1H), 7.46 (t, 1H, J =
8.0 Hz), 7.30 (t, 1H, J = 7.52 Hz), 7.09 (s, 1H), 6.95 (t, 1H, J =
7.2 Hz), 6.90 (d, 1H, J = 7.5 Hz), 6.64 (d, 1H, J = 8.2 Hz), 6.54
(t, 1H, J = 7.52 Hz), 5.84 (s,2H), 4.58 (s, 2H), 3.93 (s, 9H), 3.35
(t, 2H, J = 5.7 Hz), 2.72 (t, 2H, J = 6.4 Hz), 1.90-1.94 (m, 2H);
C NMR (75 MHz, CDCl ):  154.3, 146.3, 144.3, 141.8, 133.8,
3
133.3, 133.2, 131.0, 129.3, 129.0, 128.7, 128.5, 127.2, 123.0,
122.3, 122.1, 121.8, 120.7, 116.5, 112.0, 111.3, 105.7, 76.8, 61.1,
13
H), 4.95 (t, 2H), 3.87 (s, 12H), 3.27 (d, 2H, J = 6.6); C NMR
3
(75 MHz, CDCl ):  157.8, 153.1, 149.7, 148.2, 142.3, 141.3,
13
1
1
37.8, 136.5, 133.2, 132.4, 130.5, 123.3, 122.9, 122.3, 121.4,
15.9, 111.1, 100.8, 72.6, 60.8, 57.1, 56.1, 39.8; HRMS (ESI):
+
34 5 5
m/z calcd. for C34H O N 592.2560, found 592.2537 [M+H]
5
6.5, 49.8, 47.5, 29.9, 28.1, 27.9, 22.4; HRMS (ESI): m/z calcd.
2-((1-((1-(3, 4, 5 Trimethoxyphenyl)-9H-pyrido [3, 4-b]
+
33 3 6
for C33H O N 561.2614, found 561.25893 [M+H]
indol-3-yl) methyl)-1H-1, 2, 3-triazol-4-yl) methyl)-1H benzo
[
de]isoquinoline-1, 3(2H)-dione (7c). 88% Yield; Mp: 199–203
3-((4-((Biphenyl-4-yloxy) methyl) -1H-1, 2, 3 -triazol-1-yl)
methyl) -1- (3, 4, 5-trimethoxyphenyl) -9H-pyrido [3, 4-b]
indole (7h). 87% Yield; Mp: 198–201 C; FT-IR (cm ) 3312,
o
-1
C; FT-IR (cm ) 3320, 2927, 2362, 1695, 1656, 1625, 1586,
1
o
-1
1
8
8
502, 1384, 1326, 1236, 848; H NMR (300 MHz, CDCl
3
): δ
.58 (brs, 1H), 8.54 (d, 2H, J = 6.4 Hz), 8.18 (d, 2H, J = 7.6 Hz),
.03 (d, 1H, J = 8.3 Hz) 7.73 (d, 2H, J = 15.6 Hz), 7.70 (d, 2H, J
3057, 3061, 2934, 2874, 2361, 1897, 1628, 1486, 1467, 1450,
1
1384, 1287, 1270, 1243, 1112, 861, 792; H NMR (300 MHz,
=
8.30 Hz), 7.67 (s, 2H), 7.46 (d, 2H, J = 7.6 Hz), 7.25(s, 1H),
3
CDCl ): δ 8.66 (brs, 1H), 8.10 (d, 2H, J = 8.30 Hz), 7.72 (s, 2H),
13
7
(
.04 (d, 1H, J = 8.30 Hz), 5.82 (s, 2H), 5.52 (s, 9H); C NMR
75 MHz, CDCl ): 159.6, 157.8, 153.1,141.3, 140.2, 137.5,
7.51(s, 2H), 7.54 (t, 1H, J = 8.30 Hz), 7.40 (t, 1H,J = 6.79 Hz),
7.29 (s, 2H), 7.15 (s, 2H), 7.11(s, 1H), 7.04 (d, 1H, J = 8.30 Hz),
3
13
1
1
37.9, 137.8, 132.4, 130.7, 130.5, 129.5, 128.1, 125.6, 123.6,
23.3, 122.9, 121.4, 111.1, 107.9, 103, 60.8, 57.1, 56.1, 44.5;
5.90 (s, 2H), 5.25 (s, 2H), 3.94(s, 9H); C NMR (75 MHz,
CDCl ):  157.9, 153.1, 142.3, 141.3, 140.8, 137.8, 133.1, 132.4,
130.5, 130.1, 129.2, 127.9, 127.6, 123.3, 122.9, 121.7, 121.4,
3
HRMS (ESI): m/z calcd. for C36
6
29
H O
5
N
6
625.2199, found
+
25.2184 [M+H]
8

1
14.8, 119.8, 111.1, 107.9, 72.3, 60.8, 57.1, 56.1; HRMS (ESI):
181.1, 148.4, 148.1, 146.5, 137.2, 135.8, 135.0, 130.0, 129, 128,
120.2, 115.6, 115.4, 109.2, 109.1, 108.0, 107.0, 106.3, 102.1,
+
m/z calcd. for C36
32 4 5
H O N 598.2454, found 598.24693 [M+H]
6
8.5, 61.1, 58.1, 56.2, 53.0, 46.3, 44.2, 42.1, 36.7, 30.0; HRMS
5
,
6-Dichloro-2-((1-((1-(3, 4, 5-trimethoxyphenyl)-9H-
(
[
ESI): m/z calcd. for C33 562.2454, found 562.24331
M+H]
32 4 5
H O N
pyrido [3, 4-b] indol-3-yl) methyl)-1H-1, 2, 3-triazol-4-yl)
methyl) isoindoline-1, 3-dione (7i). 90% Yield; Mp: 193–195
C; FT-IR (cm ) 3128, 2962, 2360, 1780, 1719, 1622, 1585,
506, 1447, 1425, 1396, 1327, 1223, 858, 800, 749, 727, 601; H
+
o
-1
2-((1-((1-(3, 4-Dimethoxyphenyl)-9H-pyrido [3, 4-b] indol-
1
1
3-yl) methyl)-1H-1, 2, 3-triazol-4-yl) methyl)-1H-benzo
NMR (300 MHz, CDCl
3
): δ 8.6 (brs, 1H), 8.07 (s,2H), 7.92 (s,
H), 7.84 (s, 2H), 7.52 (t, 1H, J = 8.30 Hz), 7.56(s, 1H), 7.25 (s,
H), 7.32 (t, 1H, J = 7.0 Hz ) 5.84(s, 2H), 4.99 (s, 2H), 3.96 (s,
[de]isoquinoline-1, 3(2H) - dione (7n). 82% Yield; Mp:182–
185 C; FT-IR (cm ): 3376, 3157, 2922, 2851, 2361, 1701,
o
-1
2
1
9
1
1
1
1662, 1627, 1590, 1515, 1438, 1402, 1385, 1358, 1235, 1140,
1
3
1
H); C NMR (75 MHz, CDCl
3
):  168.2, 157.8, 153.1, 148.2,
1029, 961, 846, 777, 739; H NMR (300 MHz ,CDCl3,) δ 8.58
41.9, 141.3, 137.8, 132.4, 132.5, 131.9, 130.7, 130.5, 129,
27.8, 127.5, 123.3, 122.9, 121.4, 121.7, 119.8, 111.1, 107.9,
00.8, 60.8, 57.1, 56.1; HRMS (ESI): m/z calcd. for
(brs, 1H), 8.54 (d, 2H, J = 6.0 Hz ), 8.18 (d, 2H, J = 7.5 Hz ),
8.03 (d, 1H, J = 8.3 Hz ) 7.73 (d, 2H, J = 15.6 Hz ), 7.70 (d, 2H,
J = 8.3 Hz ), 7.67 (s, 2H), 7.46 (d, 2H, J = 7.5 Hz ), 7.25(s, 1H),
7.04 (d, 1H, J = 8.30 Hz ), 5.82 (s, 2H), 5.52 (s, 6H); HRMS
+
C
32 2
H25Cl O
5
N
6
643.1263, found 643.1258 [M+H]
(
5
ESI): m/z calcd. for
95.20767[M+H]
C
35
H
27
O
4
N
6
595.2094, found
1
-(3,
4-Dimethoxyphenyl)-3-((4-((3,
4,
5
+
trimethoxyphenoxy) methyl)-1H-1, 2, 3-triazol-1-yl) methyl)-
H-pyrido [3, 4-b] indole (7j). 93% Yield; Mp: 143–145 C;
FT-IR (cm ) 3313, 3139, 2928, 1601, 1507, 1450, 1401, 1317,
222, 811, 745, 607, 438; H NMR (300 MHz, CDCl
o
9
3-((4-((Biphenyl-4-yloxy) methyl)-1H-1, 2, 3-triazol-1-yl)
-1
methyl)-1-(3, 4-dimethoxyphenyl)-9H-pyrido [3, 4-b] indole
o -1
(7o). 90% Yield; Mp: 175–178 C; FT-IR (cm ) 3319, 3140,
2929, 1606, 1514, 1449, 1401, 1113, 832, 744, 697, 606; H
1
1
(
3
): δ 8.6
1
brs, 1H), 8.12 (d, 1H, J = 7.55 Hz), 7.82 (d, 2H, J = 3.77 Hz),
7
5
.46 (t, 2H, J = 7.5 Hz) 7.34 (t, 1H), 7.12 (s, 2H), 6.23 (s, 2H),
.90 (s, 2H), 5.17 (s, 2H), 4.02 (s, 9H), 3.90 (s, 6H); C NMR
3
NMR (300 MHz, CDCl ): δ 8.66 (brs, 1H), 8.10 (d, 2H, J = 8.30
13
Hz), 7.72 (s, 2H), 7.51(s, 2H), 7.54 (t, 1H, J = 8.30 Hz), 7.40 (t,
1H,J = 6.79 Hz), 7.29 (s, 2H), 7.15 (s, 2H), 7.11(s, 1H), 7.04 (d,
(
1
3
75 MHz, CDCl ):  57.6, 153.1, 152.9, 151.2, 148.2, 142.3,
13
41.3, 137.8, 132.4, 131.5, 130.5, 123.3, 122.9, 121.7, 121.4,
1H, J = 8.30 Hz), 5.90 (s, 2H), 5.25 (s, 2H), 3.94(s, 6H);
NMR (75 MHz, CDCl ):  157.9, 153.1, 142.3, 141.3, 140.8,
137.8, 133.1, 132.4, 130.5, 130.1, 129.2, 127.9, 127.6, 123.3,
C
1
19.8, 111.1, 100.8, 93.7, 72.3, 60.8, 57.1, 56.1; HRMS (ESI):
3
+
32 6 5
m/z calcd. for C32H O N 582.2353, found 582.2453 [M+H]
1
5
22.9, 121.7, 121.4, 114.8, 119.8, 111.1, 107.9, 72.3, 60.8, 57.1,
6.1; HRMS(ESI): m/z calcd. for C35 568.2349, found
2
-((1-((1-(3, 4-Dimethoxyphenyl)-9H-pyrido [3, 4-b] indol-
27 4 6
H O N
3
-yl) methyl)-1H-1, 2, 3-triazol-4-yl) methyl) isoindoline-1, 3-
dione (7k). 84% Yield; Mp: 165–168 C; FT-IR (cm ) 3354,
+
o
-1
568.2403[M+H]
3
1
1
1
2
1
1
1
157, 3061, 2941, 2834, 2361, 1947, 1763, 1678, 1585, 1505,
423, 935, 785, 691; H NMR (300 MHz, CDCl ): δ 8.6 (brs,
3
H), 7.92 (s, 2H), 7.84 (s, 2H), 7.52 (t, 1H, J = 8.3 Hz), 7.56 (s,
1-(4-Chlorophenyl)-3-((4-((3, 4, 5-trimethoxyphenoxy)
methyl)-1H-1, 2, 3-triazol-1-yl) methyl)-9H-pyrido [3, 4-b]
indole (7p). 95% Yield; Mp: 135–138 C; FT-IR (cm ) 3314,
1
o
-1
H), 7.25 (s, 1H), 7.32 (t, 1H, J = 7.5 Hz) 5.84(s, 2H), 4.99 (s,
3140, 2961, 2364, 1773, 1628, 1595, 1505, 1503, 1449, 1406,
13
1
H), 3.96 (s, 9H); C NMR (75 MHz, CDCl
3
):  168.2, 157.8,
1321, 1224, 1123, 1049, 802, 738, 699,610; H NMR (300 MHz,
53.1, 148.2, 141.9, 141.3, 137.8, 132.4, 132.5, 131.9, 130.7,
30.5, 129, 127.8, 127.5, 123.3, 122.9, 121.4, 121.7, 119.8,
3
CDCl ): δ 8.66 (brs, 1H), 8.02 (d, 2H, J = 7.5 Hz), 7.20 (s, 1H),
7.65 (d, 1H, J = 8.3 Hz ) 7.63 (s, 2H), 7.60 (t, 2H, J = 8.3 Hz),
11.1, 107.9, 100.8, 60.8, 57.1, 56.1; HRMS (ESI): m/z calcd. for
7.50 (s, 1H), 7.21 (s, 2H), 6.12 (s, 2H), 5.32 (s,2H), 5.20(s, 2H),
+
13
C
31 25
H O
4
N
6
545.1937, found 545.1987 [M+H]
3.83 (s, 9H); C NMR (75 MHz, CDCl
3
):  157.8, 152.9, 151.3,
1
1
48.3, 140.8, 139.5, 134.1, 134.4, 129.5, 129.0, 123.2, 122.9,
11.6, 111.1, 107.9, 72.3, 60.8, 57.1, 56.1; HRMS (ESI): m/z
3
-((4-((4-Bromophenoxy) methyl)-1H-1, 2, 3-triazol-1-yl)
methyl)-1-(3, 4-dimethoxyphenyl)-9H-pyrido [3, 4-b] indole
7l). 85% Yield; Mp: 148–150 C; FT-IR (cm ) 3338, 3140,
+
o
-1
27 4 6
calcd. for C35H O N 556.1752, found 556.1792[M+H]
(
2
8
1
928, 1624, 1578, 1560, 1513, 1487, 1450, 1425, 1400, 1274,
3-((4-((4-Allyl-2-methoxyphenoxy) methyl)-1H-1, 2, 3-
triazol-1-yl) methyl)-1-(4-chlorophenyl)-9H-pyrido [3, 4-b]
indole (7q). 88% Yield; Mp: 146–149 C; FT-IR (cm ) 3144,
1
14, 759, 605, 503; H NMR (300 MHz, CDCl
3
):  8.66 (brs,
o
-1
H), 8.10 (d, 2H, J = 8.30 Hz), 7.72 (s, 2H), 7.62(t, 1H, J = 8.3
Hz), 7.51(s, 2H), 7.54 (t, 1H, J = 8.30 Hz), 7.40 (t, 1H, J = 6.79
Hz), 7.29 (s, 2H), 7.15 (s, 2H), 7.11(s, 1H), 7.04 (d, 1H, J = 8.30
Hz), 6.82 (t, 1H, J = 8.3 Hz), 5.90 (s, 2H), 5.25 (s, 2H), 3.94(s,
2924, 2853, 2352, 1625, 1451,1465, 1429, 1397, 1383, 1221,
1
1135, 913, 733, 602, 453; H NMR (300 MHz, CDCl
3
): δ 8.66
(brs, 1H), 8.02 (d, 2H, J = 7.5 Hz), 7.20 (s, 1H), 7.65 (d, 1H, J =
8.3 Hz ) 7.63 (s, 2H), 7.60 (t, 2H, J = 8.3 Hz), 7.50 (s, 1H), 7.21
13
6
1
1
5
5
3
H); C NMR (75 MHz, CDCl ):  157.9, 153.1, 142.3, 141.3,
40.8, 137.8, 133.1, 132.4, 130.5, 130.1, 129.2, 127.9, 127.6,
23.3, 122.9, 121.7, 121.4, 114.8, 119.8, 111.1, 107.9, 72.3, 60.8,
13
(s, 2H), 6.12 (s, 2H), 5.32 (s,2H), 5.20(s, 2H); C NMR (75
MHz, CDCl ):  157.8, 149.7, 148.2, 146.8, 142.3, 141.3, 139.5,
3
7.1, 56.1;
70.1141, found 570.1187 [M+H]
HRMS (ESI): m/z calcd. for C29
H
25BrO
3
N
5
136.5, 133.2, 132.9, 132.4, 129.3, 129.0, 123.3, 122.9, 122.3,
121.4, 119.8, 115.9, 114.1, 112.3, 111.1, 107.9, 56.1, 39.8;
+
27 4 6
HRMS (ESI): m/z calcd. for C35H O N 536.1853, found
3
-((4-((4-Allyl-2-methoxyphenoxy) methyl)-1H-1, 2, 3-
+
536.1820[M+H]
triazol-1-yl) methyl)-1-(3, 4-dimethoxyphenyl)-9H-pyrido [3,
-b] indole (7m). 85% Yield; Mp: 152–155 C; FT-IR (cm )
317, 3145, 3002, 2923, 2853, 2837, 2360, 1729, 1671, 1625,
589, 1468, 1514, 1029, 1024, 758, 619, 469, 438; H NMR (300
o
-1
4
3
1
2-((1-((1-(4-Chlorophenyl)-9H-pyrido [3, 4-b] indol-3-yl)
methyl)-1H-1, 2, 3-triazol-4-yl) methyl) isoindoline-1, 3-dione
o -1
(7r). 82% Yield; Mp: 150–153 C; FT-IR (cm ) 3317, 3143,
1
MHz, CDCl
3
): δ 8.58 (brs, 1H), 8.54 (d, 2H, J = 6.04 Hz), 8.18
2360, 1770, 1713, 1625, 1563, 1396, 1321,1111, 742, 713, 609,
530; H NMR (300 MHz, CDCl3,): δ 8.6 (brs, 1H), 7.92 (s, 2H),
1
(d, 2H, J = 7.55 Hz), 8.03 (d, 1H, J = 8.30 Hz ) 7.73 (d, 2H, J =
1
7
5
5.6 Hz), 7.70 (d, 2H,J = 8.30 Hz), 7.67 (s, 2H), 7.46 (d, 2H, J =
.55 Hz), 7.25(s, 1H), 7.04 (d, 1H, J = 8.30 Hz), 5.82 (s, 2H),
7.84 (s, 2H), 7.52 (t, 1H, J = 8.3 Hz), 7.56 (s, 1H), 7.25 (s, 1H),
13
7.32 (t, 1H, J = 7.5 Hz) 5.84(s, 2H), 4.99 (s, 2H); C NMR (75
MHz, CDCl ):  157.9, 153.1, 142.3, 141.3, 140.8, 137.8, 133.1,
13
.52 (s, 6H), 3.87(s,6H); C NMR (75 MHz, CDCl
3
)  182.5,
3
9

1
1
32.4, 130.5, 130.1, 129.2, 127.9, 127.6, 123.3, 122.9, 121.7,
21.4, 114.8, 119.8, 111.1, 107.9, 72.3, 60.8, 57.1, 56.1; HRMS
of 25 µM of derivatives was prepared in DMSO and 25 µM CT
DNA were prepared in 100 mM KBPES buffer (30 mM
Potassium Phosphate with 100 mM KCl, pH 7.0). UV-visible
absorption titrations were done by adding CT-DNA stock
solution (25 µM) in 100 mM KBPES buffer to the quartz cuvette
containing approximately 25 µM complex solution. Preparation
of CT-DNA and complexes were done on the same day of
performing the experiment. Titrations were carried out until the
derivatives absorption band remains at a fixed wavelength upon
successive additions of CT-DNA. In the present experiment, a
well-known β-carboline molecule namely Harmine was
considered as control. UV-visible titration was carried out
between Harmine-CT-DNA also under the same experimental
conditions. 25 µM of Harmine was dissolved in 1:1
(
5
ESI): m/z c+alcd. for
C
35
27
H O
4
N
6
519.1336, found
19.1296[M+H]
2-((1-((1-(4-Chlorophenyl)-9H-pyrido [3, 4-b] indol-3-yl)
methyl)-1H-1, 2, 3-triazol-4-yl) methyl) isoindoline-1, 3-dione
o
-1
(7s). 85% Yield; Mp: 145–148 C; FT-IR:(cm ) 3311, 3131,
1
2
925, 2854, 2359, 1401, 1122, 736, 603, 504; H NMR (300
MHz, CDCl ): δ 8.66 (brs, 1H), 8.10 (d, 2H, J = 8.30 Hz), 7.72
s, 2H), 7.62(t, 1H, J = 8.3 Hz), 7.51(s, 2H), 7.54 (t, 1H, J = 8.30
Hz), 7.40 (t, 1H, J = 6.79 Hz), 7.29 (s, 2H), 7.15 (s, 2H), 7.11(s,
3
(
1
2
1
1
1
H), 7.04 (d, 1H, J = 8.30 Hz), 6.82 (t, 1H, J = 8.3 Hz), 5.90 (s,
13
H), 5.25 (s, 2H); C NMR (75 MHz, CDCl ):  157.4, 144.3,
3
43.8, 142.9, 140.9, 138.1, 132.4, 131.0, 129.5, 129.4, 129.1,
28.3, 123.6, 122.1, 122.7, 120.8, 116.8, 113.5, 113.2, 111.9,
o
water:methanol solution. The stock solution was heated to 60 C
to increase the solubility of Harmine. UV-visible spectra was
recorded from 250 nm to 400 nm.
10.5, 105.2 107.9, 72.3, 60.8, 57.1, 56.1; HRMS (ESI): m/z
+
27 4 6
calcd. for C35H O N 544.0539, found 544.05276[M+H]
4.6. Fluorescence emission spectra
3-((4-((Biphenyl-4-yloxy) methyl)-1H-1, 2, 3-triazol-1-yl)
o
methyl)-1-(4-chlorophenyl)-9H-pyrido [3, 4-b] indole (7t).
Fluorescence emission spectra were measured at 25 C using a
0
-1
9
1
8
0% Yield; Mp: 160-163 C; FT-IR (cm ) 3153, 2938, 2362,
Hitachi F4500 spectrofluorimeter (Maryland, USA) using a 1 cm
path length quartz cuvette. Quartz cuvettes was thoroughly
washed with distilled water and dilute nitric acid (approximately
0.1 N) to minimize non-specific binding of the derivative’s
molecules to the surface of the cuvette. Throughout the
fluorescence experiment, concentration of the derivatives were
kept constant (10 µM) and titrated with increasing concentrations
of CT-DNA (multiples of 0.5 µM). Fluorescence spectra were
recorded after each addition of CT-DNA to the fluorescent
cuvette. Emission spectra of 7f, 7n and 7s show prominent peaks
at 512.5 nm, 875 nm and 573 nm respectively. Titration with
Harmine was considered as a control and for 2 µM of Harmine in
the fluorescence cuvette, each time, 0.5 µM of CT-DNA was
added. Harmine was excited at 327 nm and emission spectra were
recorded from 360 nm to 500 nm. Each spectrum was recorded
three times and the average of three scans was taken.
583, 1486, 1465, 1393, 1365, 1326, 1237, 1177, 1127, 1005,
1
31, 745, 724, 692, 434; H NMR (300 MHz, CDCl
3
): δ 8.66
(brs, 1H), 8.10 (d, 2H, J = 8.30 Hz), 7.72 (s, 2H), 7.51(s, 2H),
7
7
5
1
1
1
.54 (t, 1H, J = 8.30 Hz), 7.40 (t, 1H, J = 6.79 Hz), 7.29 (s, 2H),
.15 (s, 2H), 7.11(s, 1H), 7.04 (d, 1H, J = 8.30 Hz), 5.90 (s, 2H),
13
.25 (s, 2H); C NMR (75 MHz, CDCl ):  157.9,157.8, 148.2,
3
42.8, 141.9, 140.9, 139.5,134.0, 133.1, 132.4, 131.0, 129.5,
29.4, 129.1, 127.3, 127.3, 123.6, 122.1, 121.7, 120.8, 116.8,
13.5, 113.2, 111.9, 110.5, 105.2 107.9, 72.3, 60.8, 56.1; HRMS
(
5
ESI): m/z c+alcd. for
C
35
27
H O
4
N
6
542.1746, found
42.1703[M+H]
4
.3. MTT assay
Cytotoxicity of formulations was determined by MTT assay
based on mitochondrial reduction of yellow MTT tetrazolium dye
4
to a highly colored blue Formosan product. 1x10 Cells (counted
4
.7. Viscosity measurements
by Trypan blue exclusion dye method)) in 96 - well plates were
incubated with formulations and standard Erlotinib with series of
Viscosity experiments were conducted on Ostwald’s
o
o
concentrations for 48 hrs at 37 C in MEM with 10% PBS
viscometer, immersed in a water bath maintained at 25 C.
Titrations were performed for each derivative (5µM), while it
was added to CT-DNA solution (50 µM) present in the
viscometer. 5µM of Ethidium bromide, Harmine and Hoechst
33258 solution was used as controls in the experiment. DNA
solution was prepared in 100 mM Tris-HCl (pH 7.0). Data were
medium. Then the above media was replaced with 90 µl of fresh
serum free media and 10 µl of MTT reagent (5mg/ml) and plates
o
were incubated at 37 C for 4h, there after the above media was
o
replaced with 200 µl of DMSO and incubated at 37 C for 10
min. Human tumor cell line consisted of PC-3 (Prostate cancer),
MCF-7 (Breast cancer), Hela (Cervical cancer), HT-29 (colon
cancer), HGC (Human gastric carcinoma). In all these
experiments, triplicates were used to determine each point.
1/3
presented as (ŋ/ŋ
hybrid to CT-DNA, where ŋ is the viscosity of CT-DNA in the
presence of hybrid and ŋ is the viscosity of CT-DNA alone.
o
)
versus the ratio of the concentration of the
o
4
.4. Circular dichroism (CD) studies
4.8. Molecular Modelling
Circular dichroism experiments were carried out using JASCO
15 CD spectropolarimeter (Jasco, Tokyo, Japan). CD spectrum
The hybrids 7a–t were constructed using molecular builder of
Maestro 9.5, prepared using Ligprep 2.7 and geometrically
minimized with Macromodel 10.1 based on OPLS-2005 force
field followed by their Macromodel conformational analysis.
Truncated Newton Conjugate Gradient (TNCG) minimization
method was used with 500 iterations and a convergence threshold
8
was recorded from 230 to 310 nm to find out the conformational
changes in the CT DNA after derivatives interaction. For each
‒
6
CD experiment, 15x10 M of CT DNA was used initially.
Further, for the characterization of derivatives ‒DNA interaction
and find the conformational changes in CT DNA. CD spectra
were recorded in 1:1 and 1:2 molar ratios of CT DNA and
derivative respectively. CD titrations were performed in 100 mM
2
of 0.05 kJ/mol was applied. The d(GAAGCTTC) duplex was
prepared and optimized using protein preparation tool. The Glide
XP 6.0 algorithm was employed using a grid box volume of
10x10x10 Å for ligands docking.
o
KBPES buffer (pH 7.0) at 25 C. Each spectrum was recorded
three times and the average of three scans was taken.
Acknowledgments
4
.5. UV-Visible spectral studies
UV-Visible spectroscopic titrations were performed using
ABI Lambda 40 UV-Vis spectrophotometer (Foster City, USA)
o
at 25 C using 1 cm path length quartz cuvette. A stock solution
1
0

Authors C. J., S. N., and S. S. are thankful to DoP, Ministry of
Chemicals and Fertilizers Govt. of India, New Delhi for the
award of Research Fellowship.
A.; Gierlich, J.; Mofid, M. R.; Nir, H.; Tal, S.; Eichen, Y.; Carell,
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PNAS. 2002, 99, 12685.
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1
1

Highlights





A
series of C3-tethered 1,2,3-triazolo-β-
carboline derivatives were synthesized.
Cytotoxicity tested on HeLa, HT-29, MCF, PC-
3
and HGC-27 cell lines.
DNA-binding affinity was evaluated by
spectroscopic studies.
Compounds showed potent DNA electrostatic
bindingand in vitro cytotoxicity.
Viscometric titration and molecular docking
established DNA minor groove binding.
1
2