Synthesis and biological evaluation of pyrrolo[2,3-b]pyridine analogues as antiproliferative agents and their interaction with calf thymus DNA

Article abstract of DOI:10.1016/j.ejmech.2016.02.059

A series of thirty two novel pyrrolo[2,3-b]pyridine analogues synthesized, characterized (¹H NMR, ¹³C NMR and MS) and cytotoxic evaluation of these molecules carried out over a panel of three human cancer cell lines including A549 (l

Full text of DOI:10.1016/j.ejmech.2016.02.059

Accepted Manuscript
Synthesis and biological evaluation of pyrrolo[2,3-b]pyridine analogues as
antiproliferative agents and their interaction with calf thymus DNA
Suresh Narva, Surendar Chitti, Bala Bhaskara Rao, Mallika Alvala, Nishant Jain,
Kondapalli Venkata Gowri Chandra Sekhar
PII:
S0223-5234(16)30149-0
10.1016/j.ejmech.2016.02.059
EJMECH 8410
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 November 2015
Revised Date: 23 February 2016
Accepted Date: 24 February 2016
Please cite this article as: S. Narva, S. Chitti, B.B. Rao, M. Alvala, N. Jain, K.V.G. Chandra Sekhar,
Synthesis and biological evaluation of pyrrolo[2,3-b]pyridine analogues as antiproliferative agents and
their interaction with calf thymus DNA, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.02.059.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of pyrrolo[2,3-b]
pyridine analogues as antiproliferative agents and
their interaction with calf thymus DNA
a
a
b
c
b
Suresh Narva , Surendar Chitti , Bala Bhaskara Rao , Mallika Alvala , Nishant Jain , Kondapalli Venkata
a∗
a
Gowri Chandra Sekhar
Department of Chemistry, Birla Institute of Technology and Science, Pilani,
b
Hyderabad Campus, Hyderabad – 500 078, Telangana, India. Centre for Chemical Biology, CSIR-Indian
c
Institute of Chemical Technology, Tarnaka, Hyderabad-500007, Telangana, India. National Institute of
Pharmaceutical Education and Research-Hyderabad, Hyderabad-500037,Telangana, India.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of pyrrolo[2,3-b]pyridine analogues as
antiproliferative agents and their interaction with calf thymus DNA
a
a
b
c
b
Suresh Narva , Surendar Chitti , Bala Bhaskara Rao , Mallika Alvala , Nishant Jain ,
a∗
Kondapalli Venkata Gowri Chandra Sekhar
a
Department of Chemistry, Birla Institute of Technology and Science, Pilani, Hyderabad
Campus, Jawahar Nagar, Hyderabad – 500 078, Telangana, India
b
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad-500007, Telangana, India
c
National Institute of Pharmaceutical Education and Research-Hyderabad, Hyderabad-
5
00037,Telangana, India
Abstract
A series of thirty two novel pyrrolo[2,3-b]pyridine analogues synthesized, characterized
1
13
(
H NMR, C NMR and MS) and cytotoxic evaluation of these molecules carried out over a
panel of three human cancer cell lines including A549 (lung cancer), HeLa (cervical cancer) and
MDA MB-231 (breast cancer), using sulforhodamine B assay method. Few molecules such as 5c,
5
d, 5e, 5h, 5k, 5m, 5n, 5q, 5r, 7f, 7j, 7g and 7k exhibited maximum growth inhibitory action
against the tested cancer cell lines at lower micro molar concentration. Noticeably, compounds
exhibited good growth inhibition in all three cancer cell lines in the range of 0.12 ꢀM to 9.84ꢀM.
Further study exposed that one of the active compound 5d could efficiently intercalate into calf
thymus DNA to form 5d-DNA complex which might block DNA replication to influence their
antiproliferative activity. The molecular interactions of all the synthesized analogs were also
supported by molecular docking simulations. We believe that further optimization of these
compounds will lead to potential anticancer agents.
Keywords: Pyrrolo[2,3-b]pyridine, 1,2,3-triazole, Calf thymus DNA, Antiproliferative activity.
∗
Corresponding author
Tel.:+91 40 66303527 E-mail: kvgc@hyderabad.bits-pilani.ac.in; kvgcs.bits@gmail.com
1

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1
. Introduction
A systematic review of current anti-cancer literature of a variety of small heterocyclic
scaffolds, such as nitrogen heterocyclic compounds, reveals an advantage of antiproliferative
agents as they mimic numerous biomolecules. Among them fused heterocyclic compound such
as 1H-pyrrolo[2,3-b]pyridine (7-aza-indole) is significant scaffold in medicinal chemistry. 1H-
pyrrolo[2,3-b] pyridines are found in numerous natural products such as variolins, isolated from
Antarctic sponge Kirk-patrickiavarialosa [1]. 7-aza-indoles are biologically competent organic
compounds with dissimilar type of actions, such as anti-proliferative [2], protein-kinase
inhibition [3], anti-inflammatory [4], antiviral [5], influenza PB2 inhibition [6], inhibition of
mixed lineage kinase 3 (MLK3) [7] and selective KIT tyrosine kinase inhibition [8].
Many substituent modifications happened at different positions of 7-azindole and their
rd
th
biological activities were evaluated. By changing the substitutions at 3 and 5 positions of 7-
azaindole, [3,5-d]-7-azaindole analogues through fragment-based growing strategy [9] as
phosphatidylinositol-3-kinase alpha (PI3Kα) inhibitors were developed and were found to
exhibit antiangiogenic effect on cancer cells [10]. 7-azaindole containing 4-pyridyl group at the
C-3 position, sulfonamide group at C-5 position analog influenced Tropomyosin-related kinase
A (Trk A) binding affinity (1.67nM) and exhibited good antiproliferative activity against MCF7
cell line. This analog exhibited strong apoptotic and antiangiogenic effects by inhibiting HIF-1α
and vascular endothelial growth factor (VEGF) expression and repressed the angiogenic process
by inhibiting endothelial cell migration and tube formation [11].
7
-azaindole containing rebeccamycin analogues have strong antiproliferative activity. They
are less toxic and arrest the cell cycle in G2+M phase at 0.25 µM than rebeccamycin [12].
Cytotoxicity against various cancer cell lines is considerably enhanced by replacing the amine
ligands of cisplatin by 7-azaindole analogues [13, 14]. The 7-azaindole ring scaffold plays an
important role in controlling the chemical and biological properties like cellular distribution,
cellular accumulation, dissimilar effects at the stage of cell cycle regulation, reduced propensity
for DNA adduct repair and binding to DNA [15].
2

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7
-azaindole–chloro pyridine analogues inhibit the Cdc7 (cell division cycle 7) kinase which
is necessary for activating the DNA replicative complex at the beginning of replication [16-20].
Z)-2-(benzylamino)-5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethylene)-1,3-thiazol-4(5H)-one showed
nM IC50 value and acts as a potent ATP mimetic inhibitor of Cdc7 kinase [21]. 7-azaindole
(
7
core with 6-methyl substitution showed potent in vitro anticancer activity with enhanced
metabolic stability, solubility, and oral bioavailability [22]. 6-substituted pyrrolo[2,3-b]pyridine-
1
-carboxamide analogues, new class of PARP-1 [Poly(ADP-ribose) polymerase] inhibitors, are
involved in maintaining DNA integrity and in regulation of programmed cell death [23, 24] and
showed potent in vitro and in vivo activity when used at a lower dose [25]. 2,5-disubstituted 7-
azaindole analog, methyl 5-(2-chloro-6-methylbenzylamino)-1H-pyrrolo[2,3-b]pyridine-2-
carboxylate potently inhibited Abl and Src kinases with IC50 values 1.4 nM and 3.4 nM
respectively [26]. Hence, substituted 7-azaindoles can be well explored for the synthesis of novel
anticancer agents.
It is very interesting to know that several pharmacophores like naphthofuranones,
methoxyphenyl oximes, and piperazinylindenoquinolinone having oxime (hydroxylamino) as a
functional group display very good anticancer activity. Tseng et.al., synthesized (Z)-4-
(hydroxyimino)naphtho[2,3-b]furan-9(4H)-one
derivative
which
exhibited
potent
antiproliferative activity against selected cell lines. Oximes containing anticancer compounds are
depicted in Figure 1 [27-29].
Insert: Figure 1
1
,2,3-triazoles are imperative class of heterocycles and play a major role in medicinal
chemistry with antifungal [30, 31], antibacterial [32, 33], antiallergic [34], anti-inflammatory
[
35] and anticancer activities [36]. In recent years, copper-catalyzed azide-alkyne cycloaddition
CuAAC) reaction has become a synthetic cornerstone for conjugating building blocks with
(
diverse functionalities [37]. Singh et.al., reported 1,2,3-triazole tethered β-lactam-chalcone
bifunctional hybrids as anticancer agents [38]; Duan et.al., synthesized 1,2,3-triazole-
dithiocarbamate hybrids [39] as well as 1,2,3-triazole-dithiocarbamate-urea hybrids [40] and
evaluated them for the anticancer activity against selected human tumor cell lines. Some of the
compounds exhibited excellent broad spectrum anticancer activity. Ahmed et.al., synthesized
flavone-triazole-tetrahydropyran conjugates and evaluated the compounds for anticancer activity,
3

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in which most of the compounds exhibited IC50 in the range of 0.61-1.68 µM [41]. Ma et.al.,
synthesized 1,2,3-triazole-pyrimidine hybrids, which showed IC values ranging from 1.42 to
5
0
6
.52 µM against various cancer cell lines [42]. 1,2,3-triazole based anticancer agents are shown
in Figure 2.
Insert: Figure 2.
Our newly designed scaffold consists of three prime components, i.e., 7-azaindole as a core
rd
st
rd
moiety, oxime at 3 position of 7-azaindole, and 1,2,3-triazole at 1 and 3 position. To explore
the alterations of this conserved hinge region oxime, we modified this region with 1,2,3-triazole.
Herein we report the chemical synthesis of new analogues of 1H-pyrrolo[2,3-b]pyridine, that are
based on some of the major chemotherapeutic pharmacophores in the area of cancer. In this study
st
rd
we designed and synthesized novel 7-azaindole analogues by varying substitutions at 1 and 3
position [scheme 1 and scheme 2] and evaluated them for antiproliferative activity on three
different human cancer cell lines. One such derivative stood out in these screens (Table1) and
was selected as lead molecule for CtDNA binding studies.
2
2
. Results and discussion
.1. Chemistry
The synthesis of pyrrolo[2,3-b]pyridine analogues 5a-u and 7a-k described in this study is
depicted in scheme 1 and 2. 1H-pyrrolo [2,3-b]pyridine-3-carbaldehyde (2) was prepared using
reported procedure of Duff reaction in the presence of hexamethylenetetramine (HMTA), acetic
acid (33%) [43-45]. Formylation occurs at C-3 position of 1H-pyrrolo [2,3-b]pyridine yielding 2
in 75% yield. 1H-pyrrolo [2,3-b]pyridine-3-carbaldehyde (2) was treated with propargyl bromide
in the presence of potassium carbonate (K CO ), dimethyl formamide (DMF) to yield 1-(prop-2-
2
3
st
ynyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (3). This alkylation happened at 1 position of
H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (2). 1-(prop-2-ynyl)-1H-pyrrolo[2,3-b]pyridine-3-
carbaldehyde oxime (4) was synthesized by reacting 1-(prop-2-ynyl)-1H-pyrrolo[2,3-b]pyridine-
-carbaldehyde (3) with hydroxylamine hydrochloride in the presence of ethanol. The title
1
3
compounds (5a-u) were synthesized by click chemistry. 1-(prop-2-ynyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbaldehyde oxime (4) on treatement with various aromatic azides, copper sulfate
pentahydrate and sodium ascorbate in aqueous DMF in one pot yielded 5a-u. 3-bromo-1H-
pyrrolo[2,3-b]pyridine (6) was synthesized by treating pyrrolo[2,3-b]pyridine with N-
4

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bromosuccinimide in presence of DMF at RT [46]. 3-bromo-1H-pyrrolo[2,3-b]pyridine (6) was
treated with various aromatic and aliphatic alkynes in the presence of sodium azide, copper
sulfate pentahydrate and sodium ascorbate, L-proline, sodium carbonate in aq DMSO (9:1) to
yield products 7a-k. All the synthesized compounds were characterized and confirmed by
1
13
HNMR, C NMR and LC-MS.
Insert: Scheme 1 and 2
1
In general H NMR of all the title compounds displayed singlet peak of N-1 proton (7a-k) in
the range of 12.07-12.89 ppm. A sharp peak due to the proton of oxime OH group (5a-u)
resonated in the range 10.73-10.80 ppm. One singlet resonated in the range of 7.18-9.01 due to
the proton of triazole ring. The oxime CH protons, showed singlet in the range δ 7.92-7.97. A
sharp singlet in the range 5.63-5.68 ppm corresponding to the methylene protons was observed.
2
.2. In vitro evaluation of the anticancer activity
In vitro antiproliferative activity of the synthesized compounds 5a-u and 7a-k were evaluated
against three types of human cancer cell lines viz; A549 (Lung cancer), HeLa (Cervical cancer)
and MDA-MB-231 (Breast cancer) employing sulforhodamine B (SRB) assay method [47, 48].
For in vitro chemo sensitivity of tumor cell lines, numerous quick colorimetric assays are
available, while MTT [3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazoliumbromide] assay being
the most extensively used, recently the US National Cancer Institute (NCI) recommended use of
the sulforhodamine B (SRB) protein stain for in vitro chemo sensitivity testing. The SRB assay
appeared to be more responsive than MTT assay, with better linearity with cell number and
higher reproducibility [49, 50]. The growth inhibition data (expressed as GI ) of synthesized
5
0
compounds 5a-u and 7a-k are shown in Table 1.
Insert: Table 1
From the anti-proliferative activity results, it is evident that all the synthesized compounds
have comparable antiproliferative activity with GI50 values ranging from 0.12-9.84 ꢀM. It is
observed that, the majority of the compounds tested displayed significant growth inhibition on
A549 and MDA MB 231 cancer cell lines as compared to HeLa cancer cell line. Compounds 5d
and 5k showed potent anticancer activity against lung cancer cell line (A549) with GI50 values
0
.12 ꢀM and 0.16 ꢀM and compounds 5e and 5m showed potency against breast cancer cell line
with GI50value 0.13 ꢀM while the positive controls, doxorubicin and paclitaxel demonstrated the
5

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GI50 in the range of <0.01-0.09 ꢀM and <0.01-0.023 ꢀM respectively. Compound 5r is potent
against breast cancer cell line with GI value 0.14 ꢀM. Compound 7j inhibited cell growth
5
0
significantly in three human cancer cell lines with GI values 0.18ꢀM against A549, 0.7 ꢀM
5
0
against HeLa and 0.25 ꢀM against MDA MB 231 cell line. The compounds 7a, 7b, 7c, 7d, 7e,
f, 7g, 7h, 7i and 7k had shown comparable values against lung, cervical and breast cancer cell
7
lines with GI50 values ranging from 0.17-9.84 ꢀM as compared to the standard drugs.
Based on the results of antiproliferative activity, the structure activity relationships (SAR) for
these newly synthesized compounds (5a-u and 7a-k) are described as follows. It is interesting to
observe that compound with electron withdrawing chloro substitution at ortho-position (5k)
exhibits good activity (0.16 ꢀM) against A549 cell line compared with other halo substitutions
like fluoro (5j), bromo (5l) and iodo (5m). Compound with electron withdrawing nitro group at
ortho position (5o) showed reduction in activity (0.95 ꢀM) against A549 cell line. Introduction of
electron withdrawing groups like trifluoromethyl (5d), nitro (5h) at para position increased the
activity against A549 cell line compared to electron donating substituents like ethyl (5a) and
methoxy (5g). 3, 4-di substituted compounds exhibited moderate activity against A549 cell line.
st
rd
1
,2,3-triazole ring at either 1 position or 3 position on pyrrolo[2,3-b]pyridine did not have
much effect on activity against A549 cell line. All the synthesized compounds exhibit moderate
activity (0.39-1.86 ꢀM) against HeLa cancer cell line. The compound with iodo substitution
(5m) at ortho position showed better activity (0.13 ꢀM) against MDA MB-231 cancer cell line
compared with fluoro (5j), chloro (5k), bromo (5l) and nitro (5o) groups at ortho positions; 5e
with electron withdrawing chloro substitution at para position showed potent activity (0.13 ꢀM)
than other halo substitutions and also electron donating groups like methoxy (5g). 3,4-
dimethoxy substitution compound showed better activity (0.14 ꢀM) than 3,4-dimethyl, 3,4-
dichloro, 3,4-difluoro compounds against MDA MB-231 cancer cell line. 1,2,3-triazole ring at
rd
3
position on pyrrolo[2,3-b]pyridine analogues from 7a-7k showed good to moderate activity.
Compound with electron withdrawing fluoro at para position (7j) showed good activity against
all three cell lines A549, HeLa, MDA MB-231 respectively with GI50 of 0.18ꢀM, 0.7ꢀM,
0
.25ꢀM compared with electron donating substitutions at para position like methoxy (7k),
methyl (7b), tertiary butyl (7c) and the unsubstituted one (7a). Based on the SAR, we note that
6

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the halo groups like fluoro, chloro, iodo at ortho and para positions play crucial role in
antiproliferative activity.
2
.3. Molecular modeling studies
The molecular docking studies of 5a-u and 7a-k were performed using ALK (Human
anaplastic lymphoma kinase) enzyme using Schrödinger suite 2013. Crystal co-ordinates for
ALK (Human anaplastic lymphoma kinase) were taken from Protein Data Bank (PDB ID:
2
XP2). Docking studies were performed using GLIDE, module of Schrödinger. Docking scores
by standard precision (Glide-SP) docking were shown in Table 1. Molecular docking studies
revealed that these compounds (7f, 7g and 7i) bind to the crizotinib binding site of the human
anaplastic lymphoma kinase with a binding affinity of -8.318, -8.18 and -8.663 respectively,
compared to crizotinib-8.123). The hydroxyl group of 7f, 7g and 7i showed hydrogen bonding
interaction with LYS 1150, ASP 1270 and GLU 1167 amino acids. This orientation is fruitful for
extensive interactions such as hydrophobic interactions. Therefore, substitution with hydroxyl
group in 7f, 7g and 7i resulted in improved docking score, which contributed for the
antiproliferative activity. Amino acid interaction pattern of active compounds 7f, 7g and 7i are
shown in Figure 3 along with crizotinib (PF-02341066) as standard. Crizotinib has shown
docking score of -8.123.
Insert: Figure 3
2
.4. Intercalation with Calf thymus DNA
2
.4.1. UV- Visible spectral studies
UV–visible spectroscopy is frequently used technique to discover the interaction studies
between biological macromolecules and small molecules. We used UV–visible spectroscopy to
investigate the absorbance spectra of 5d-CtDNA interaction (Figure 4). The characteristic peak
of compound 5d alone was observed near 222nm. However, on subsequent addition of CtDNA to
compound 5d, the absorbance of compound gradually decreased, indicating hypochromic effect.
Hypochromic effect interaction of compound 5d with CtDNA indicates strong intermolecular
interaction. This hypochromic effect is due to the overlap of the electron cloud of the compound
5
d with the CtDNA base pairs [51, 52]. Hypochromic effect in UV–visible spectra upon
compound binding to CtDNA is a characteristic of an intercalating binding mode [53, 54].
Insert: Figure 4
7

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b
The intrinsic binding constant K of the compound to CtDNA was determined from following
equation.
[
DNA]/|ɛ - ɛ [DNA]/|ɛ - ɛ + 1/K ɛ - ɛ
a f| = b f| b| b f
Here [DNA] represents the concentration of DNA in base pairs, and ɛ
a f b
, ɛ and ɛ the
apparent extinction coefficient (A /[M]), the extinction coefficient for free complex (M), and
obs
the extinction coefficient for the free complex (M) in the fully bound form, respectively. K
b
is the
–
1
equilibrium binding constant (in M ) of compound binding to DNA. In plots of [DNA]/ɛ - ɛ Vs
a
f
[
DNA], K is obtained by the ratio of slope to intercept (Figure 5). The binding constant K for
b
b
4
-1
compound 5d is 7.16 ×10 M . These results indicate that the binding strength of compound 5d
is good through the intercalate mode.
Insert: Figure 5
2
.4.2. Fluorescence spectral studies
The compound 5d has no fluorescence at room temperature, so the binding of the compound
with CtDNA can’t be predicted directly through the emission spectra. The spectroscopic changes
of Ethidium bromide (EB) on its binding to CtDNA are frequently utilized to study the
interaction between CtDNA and new substances such as synthesized molecule [55, 56]. EB
displays very feeble fluorescence in the aqueous solution, but in the presence of DNA it exhibits
strong fluorescence because of the intercalation to the base pairs in DNA. Intensity of the EB-
DNA adduct allows us to determine the affinity of the binding mode of compound 5d for DNA.
If compound can replace EB from EB-DNA, the fluorescence of the solution will be quenched as
the free EB molecules are readily quenched by the adjacent water molecules [57, 58]. The
fluorescence quenching of EB-CtDNA by the compound 5d is shown in Figure 6. The
quenching of EB-CtDNA by the compound 5d is in good agreement with the linear Stern-Volmer
equation, which provides further evidence that 5d binds to DNA.
Insert: Figure 6
ꢀꢁ
=
1 + ꢂ ꢅ[ꢆ]
ꢃꢄ
ꢀ
In the above equation ꢀ is the emission intensity in the absence of quencher, I is the
ꢁ
emission intensity in the presence of quencher, ꢂ is the Stern-Volmer quenching constant, and
ꢃꢄ
[Q] is the quencher concentration. The shape of Stern-Volmer plot can be used to characterize the
8

ACCEPTED MANUSCRIPT
quenching as being predominantly dynamic or static. Plots of ꢀ ⁄ꢀ versus [Q] appear to be linear.
ꢁ
The linear relationship of ꢀ ⁄ꢀ versus [Q] recommends that the quenching result for this system
ꢁ
is a static type, means non-fluorescence complex is formed between compound 5d and CtDNA.
Ksv is given by the ratio of the slope to the intercept (Figure 7). The Ksv value for the compound
-4
−1
is 5.19 × 10 L M . This data clearly indicates the interaction of 5d with CtDNA.
Insert: Figure 7
2
.5. Activation of p53 protein: Since 5d was demonstrated to intercalate with DNA. Thus
we elucidated whether 5d shows a similar mode of action in cancer cells. To achieve this, we
treated HeLa cells in a dose dependent manner with 5d at 5 and 10 µΜ concentrations for 24 h.
p53 is a tumor suppressor protein that is activated in response to genotoxic stress. Further, we
also observed potent induction of p53 protein levels in 5d treated cells. Tubulin was used as a
loading control, criztonib. Overall, our results show that 5d elicits p53 activation in cancer cells
possibly through intercalating with DNA.
Insert: Figure 8
3
. Conclusion
In summary, a series of pyrrolo[2,3-b]pyridine analogues have been designed and
synthesized, subsequent by easy reaction protocols. All the synthesized compounds were
screened for their growth inhibitory activity against a panel of three different human cancer cell
lines such as A549, HeLa and MDA-MB-231. Most of the tested pyrrolo[2,3-b]pyridine
analogues displayed promising growth inhibitory activity against cancer cell lines. Among all the
synthesized compounds 5c, 5d, 5e, 5h, 5k, 5m, 5n, 5q, 5r, 7f, 7j, 7g and 7k showed maximum
growth inhibitory activity against cancer cell lines at low concentrations. The specific interaction
of compound 5d with calf thymus DNA by intercalate mode, which might further block DNA
replication to exert their antiproliferative activity. Our findings from this work with synthesis,
antiproliferative activity, molecular modeling and DNA binding experiments demonstrate that
this pyrrolo[2,3-b]pyridine analogues could be potential candidates for developing anticancer
agents.
9

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4
4
. Experimental section
.1. Materials and Methods
All reagents were purchased from commercial sources and used with further purification
wherever necessary. 7-Azaindole was purchased from Sigma Aldrich. All reactions were
monitored by analytical thin layer chromatography (TLC) performed on E-Merck 0.25 mm pre
coated silica gel aluminum plates (60 F254) using mixture of pet ether and ethyl acetate.
Visualization of the spots on TLC plates was achieved by exposure to UV light. Column
chromatography was performed using silica gel (Acme, 100-200mesh). Solvents were dried and
purified by distillation prior to use. Solvents for chromatography (Pet ether and ethyl acetate)
were distilled prior to use. Evaporations were carried out under reduced pressure on Heidolph
rotary evaporator. Melting points were obtained using Stuart SMP30 system and are uncorrected.
1
13
1
H and C NMR spectra were recorded on Avance-III 400MHz (400 MHz for H, 100 MHz for
1
3
C), in CDCl or DMSO-d . Chemical shifts have been expressed in parts per million (δ) relative
3
6
to tetramethylsilane (δ = 0.0) as an internal standard and coupling constants (J) in Hertz. Low-
resolution mass spectra (LC-MS) were recorded on LC/MS-2020 Shimadzu. IR spectra were
recorded with an FT-IR spectrophotometer (Jasco FTIR-4200). The UV-visible absorption
spectroscopy was performed on a spectrometer (JASCO model V-650). The fluorescence spectral
titrations were performed on a spectrofluorometer (JASCO model FP-6300).
4
.2. Chemistry
4
.2.1. Synthesis of 1H-pyrrolo [2,3-b]pyridine-3-carbaldehyde (2): To a solution of 1H-
pyrrolo[2,3-b]pyridine (1g, 8.5mmol, 1eq) in acetic acid (33%, 15mL), hexamethylenetetramine
HMTA) (1.79g, 9.35mmol, 1.1eq) was added. The reaction mixture was refluxed at 120° C for
h. Reaction was monitored by TLC and once complete was cooled in an ice bath. The resulting
precipitate was collected and dried to afford the 1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (2).
(
6
4
.2.1.1.
stirred solution of 1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (0.0547 moles, 1eq) in
dimethylformamide (DMF), K CO (0.1094 moles, 2eq) and propargyl bromide (0.0547 moles,
eq) were added. Reaction mixture was stirred at ambient temperature 2h. Reaction was
monitored by TLC and water was added to reaction mixture once complete and was followed by
Synthesis of 1-(prop-2-ynyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (3): To a
2
3
1
1
0

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extraction with ethyl acetate. Combined organic layers were collected and dried over anhydrous
sodium sulphate. Concentrated the organic layer and purified by column chromatography with
-1
pet ether and ethyl acetate (15%) to yield 3. Brown solid; m.p. 122-124° C; IR (KBr, cm ) 3200,
1
2
8
119, 1653, 1524. H NMR (400 MHz, CDCl
3
) δ 10.00 (s, 1H), 8.55 (dd, J = 7.8, 1.6 Hz, 1H),
.43 (dd, J = 4.8, 1.6 Hz, 1H), 8.13 (s, 1H), 7.30 – 7.27 (m, 1H), 5.15 (d, J = 2.6 Hz, 2H), 2.58 (t,
1
3
J = 2.6 Hz, 1H). C NMR (101 MHz, CDCl
3
) δ 184.75, 147.84, 145.26, 137.13, 130.78, 119.28,
1
17.74, 116.97, 76.43, 75.22, 34.49. ESI-MS (m/z): calcd. for C H N O 184.06, found 185.05
11
8
2
+
[
M + H] .
4
.2.1.2.
Synthesis of 1-(prop-2-ynyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde oxime
(4): To a stirred ice cold solution of 1-(prop-2-ynyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde
(0.02173 moles, 1eq) in ethanol, hydroxylamine hydrochloride (0.0260moles, 1.2eq) was added
slowly. Sodium hydroxide solution was added drop wise. Reaction mixture was stirred at room
temperature for 2h and was monitored by TLC. After completion, as indicated by TLC, acetic
acid was added to reaction mixture to neutralize and later filtered to get the title compound.
-1
1
Brown solid; m.p. 184-186° C; IR (KBr, cm ) 3480, 3237, 2124, 1626, 1572, 950. H NMR (400
MHz, DMSO) δ 10.78 (s, 1H), 8.36 (dd, J = 7.8, 1.6 Hz, 1H), 8.34 (dd, J = 4.8, 1.6 Hz, 1H), 8.31
13
(
s, 1H), 7.90 (s, 1H), 7.26 – 7.22 (m, 1H), 5.14 (d, J = 2.6 Hz, 2H), 3.42 (t, J = 2.6 Hz, 1H). C
NMR (101 MHz, DMSO) δ 147.31, 143.70, 130.37, 129.02, 117.30, 116.91, 108.91, 101.95,
+
7
4.49, 71.76, 33.30. ESI-MS (m/z): calcd. for C H N O 199.07, found 200.05 [M + H] .
11
9
3
4
.2.2. General procedure for the synthesis of 1-((1-substituted phenyl-1H-1,2,3-triazol-5-
yl)methyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde oxime (5a-u): To a stirred solution of
t
compound 4 (1.0 mmol) and substituted phenyl azide (1.2 mmol) in butanol-water (1:1) (4 mL),
CuSO .5H O (1 mol %) (0.2 mmol) and sodium ascorbate (5 mol %) (0.2 mmol) were added and
4
2
the reaction mixture was stirred at RT for 2 h. After completion of the reaction, as indicated by
TLC, butanol was removed under reduced pressure. The residue was extracted with ethyl acetate
(3 x10 mL) and combined organic layers were collected and washed with saturated brine
solution, dried over anhydrous MgSO and concentrated in vacuo to get the crude product. The
4
product was further purified by column chromatography using pet ether and ethyl acetae (40%)
to afford the title compounds.
1
1

ACCEPTED MANUSCRIPT
4
.2.2.1.
1-((1-(4-ethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-3-
-1
carbaldehyde oxime (5a). White solid; yield 65%, 0.33g, m.p. 174-176° C; IR (KBr, cm ) 3420,
1
3
4
010, 1660, 1570, 950. H NMR (400 MHz, DMSO) δ 10.79 (s, 1H), 8.77 (s, 1H), 8.37 (dd, J =
.7, 1.4 Hz, 1H), 8.34 – 8.29 (m, 1H), 8.28 (s, 1H), 7.94 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.39
(d, J = 8.6 Hz, 2H), 7.23 (dd, J = 7.8, 4.7 Hz, 1H), 5.65 (s, 2H), 2.64 (q, J = 7.6 Hz, 2H), 1.18 (t,
1
3
J = 7.6 Hz, 3H). C NMR (101 MHz, DMSO) δ 147.99, 145.00, 144.55, 144.44, 144.19, 134.89,
1
31.14, 130.38, 129.42, 122.19, 120.54, 117.50, 117.46, 108.74, 51.59, 28.12, 15.89. ESI-MS
+
(
m/z): calcd. for C H N O 346.17, found 347.23 [M + H] .
1
9
18
6
4
.2.2.2.
1-((1-(4-bromo-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-
pyrrolo[2,3-b] pyridine-3-carbaldehyde oxime (5b). Pale yellow solid; yield 76%, 0.53g, m.p.
-1
1
2
05-207° C; IR (KBr, cm ) 3425, 3015, 1670, 1565, 1130, 950, 575. H NMR (400 MHz,
DMSO) δ 10.79 (s, 1H), 9.01 (s, 1H), 8.36 (d, J = 3.8 Hz, 1H), 8.32-8.30 (m, 2H), 8.28 (s, 1H),
8
1
1
5
.14 (dd, J = 8.7, 2.0 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.94 (s, 1H), 7.23 (dd, J = 7.8, 4.7 Hz,
1
3
H), 5.67 (s, 2H). C NMR (101 MHz, DMSO) δ 147.97, 145.20, 144.37, 144.22, 137.07,
36.34, 131.16, 130.46, 125.70, 122.70, 121.48, 120.06, 120.00, 118.98, 117.53, 117.46, 108.81,
+
1.61. ESI-MS (m/z): calcd. for C H BrF N O 464.02, found 465.09 [M + H] .
1
8
12
3
6
4
3
3
8
.2.2.3.
1-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-
-1
-carbaldehyde oxime (5c). Pale yellow solid; yield 84%, 0.5g, m.p. 166-168° C; IR (KBr, cm )
1
462, 3012, 1658, 1568, 953, 595. H NMR (400 MHz, CDCl ) δ 10.79 (s, 1H), 8.76 (s, 1H),
3
.36 (dd, J = 4.7, 1.4 Hz, 1H), 8.32 – 8.29 (m, 1H), 8.28 (s, 1H), 7.94 (s, 1H), 7.78 (d, J = 8.5
1
3
Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.25 (dd, J = 7.8, 4.7 Hz, 1H), 5.66 (s, 2H). C NMR (100.61
MHz, CDCl ) δ 147.95, 145.03, 144.56, 144.43, 144.15, 134.86, 131.12, 130.36, 129.48, 122.12,
3
1
3
20.58, 117.50, 117.46, 108.75, 51.49. ESI-MS (m/z): calcd. for C H BrN O 396.03, found
17 13 6
+
97.11 [M + H] .
4
.2.2.4.
1-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbaldehyde oxime (5d). Pale yellow solid; yield 86%, 0.5g, m.p. 178-179° C; IR
-1
1
(
KBr, cm ) 3435, 3008, 1648, 1572, 1125, 952. H NMR (400 MHz, CDCl ) δ 10.75 (s, 1H),
3
1
2

ACCEPTED MANUSCRIPT
8
.95 (s, 1H), 8.36 (dd, J = 4.7, 1.4 Hz, 1H), 8.32 – 8.29 (m, 1H), 8.28 (s, 1H), 8.13 (d, J = 8.5
1
3
Hz, 2H), 7.97 (d, J = 8.6 Hz, 2H),7.94 (s, 1H), 7.24 (dd, J = 7.8, 4.7 Hz, 1H), 5.67 (s, 2H). C
NMR (100.61 MHz, CDCl ) δ 147.95, 145.03, 144.56, 144.43, 144.15, 134.86, 131.12, 130.36,
3
1
29.48, 122.12, 121.34, 120.58, 117.50, 117.46, 108.75, 51.48. ESI-MS (m/z): calcd. for
+
C H F N O 386.11, found 387.19 [M + H] .
18
13
3
6
4
3
3
8
.2.2.5.
1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-
-1
-carbaldehyde oxime (5e). Pale yellow solid; yield 76%, 0.4g, m.p. 173-175° C; IR (KBr, cm )
1
398, 3012, 1652, 1574, 956, 792. H NMR (400 MHz, DMSO) δ 10.78 (s, 1H), 8.78 (s, 1H),
.38 (dd, J = 4.7, 1.4 Hz, 1H), 8.34 – 8.29 (m, 1H), 8.28 (s, 1H), 7.93 (s, 1H), 7.77 (d, J = 8.5
13
Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H), 7.26 (dd, J = 7.8, 4.7 Hz, 1H), 5.65 (s, 2H). C NMR (101
MHz, DMSO) δ 147.98, 145.02, 144.54, 144.43, 144.17, 134.88, 131.15, 130.37, 129.45, 122.17,
1
3
20.54, 117.50, 117.46, 108.74, 51.56. ESI-MS (m/z): calcd. for C H ClN O 352.08, found
17 13 6
+
53.16 [M + H] .
4
3
3
8
.2.2.6.
1-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-
-1
-carbaldehyde oxime (5f). Pale yellow solid; yield 79%, 0.4g, m.p. 116-118° C; IR (KBr, cm )
1
430, 3010, 1635, 1565, 1235, 945. H NMR (400 MHz, DMSO) δ 10.79 (s, 1H), 8.81 (s, 1H),
.37 (dd, J = 4.7, 1.4 Hz, 1H), 8.33 – 8.29 (m, 1H), 8.28 (s, 1H), 7.92 (s, 1H), 7.78 (d, J = 8.5
13
Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H), 7.28 (dd, J = 7.8, 4.7 Hz, 1H), 5.67 (s, 2H). C NMR (101
MHz, DMSO) δ 147.98, 145.08, 144.52, 144.42, 144.19, 134.87, 131.16, 130.34, 129.46, 122.18,
1
3
20.53, 117.51, 117.46, 108.74, 51.56. ESI-MS (m/z): calcd. for C H FN O 336.11, found
17 13 6
+
37.19 [M + H] .
4
.2.2.7.
1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]
pyridine-3-carbaldehyde oxime (5g). Brown solid; yield 68%, 0.35g, m.p. 172-173° C; IR (KBr,
-1
1
cm ) 3420, 3005, 1665, 1575, 1210, 960. H NMR (400 MHz, DMSO) δ 10.73 (s, 1H), 8.69 (s,
1
H), 8.37 (dd, J = 4.7, 1.4 Hz, 1H), 8.33 – 8.28 (m, 1H), 8.26 (s, 1H), 7.92 (s, 1H), 7.78 (d, J =
.5 Hz, 2H), 7.28 (dd, J = 7.8, 4.7 Hz, 1H), 7.16 (d, J = 8.6 Hz, 2H), 5.63 (s, 2H), 3.81 (s, 3H).
C NMR (101 MHz, DMSO) δ 147.96, 145.04, 144.54, 144.46, 144.18, 134.86, 131.17, 130.36,
8
1
3
1
3

ACCEPTED MANUSCRIPT
1
29.46, 122.18, 120.53, 117.51, 117.46, 108.74, 57.45, 51.56. ESI-MS (m/z): calcd. for
+
C H N O 348.13, found 349.21 [M + H] .
18
16
6
2
4
.2.2.8.
1-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-3-
-1
carbaldehyde oxime (5h). Yellow solid; yield 90%, 0.49g, m.p. 199-201° C; IR (KBr, cm ) 3440,
1
3
8
002, 1645, 1565, 1520, 1340, 950. H NMR (400 MHz, DMSO) δ 10.76 (s, 1H), 8.76 (s, 1H),
.36 (dd, J = 4.7, 1.4 Hz, 1H), 8.34 – 8.28 (m, 1H), 8.27 (s, 1H), 7.94 (s, 1H), 7.78 (d, J = 8.5
13
Hz, 2H), 7.39 (d, J = 8.6 Hz, 2H), 7.27 (dd, J = 7.8, 4.7 Hz, 1H), 5.66 (s, 2H). C NMR (101
MHz, DMSO) δ 147.96, 145.06, 144.53, 144.44, 144.16, 134.83, 131.16, 130.38, 129.45, 122.17,
1
3
20.54, 117.50, 117.46, 108.74, 51.58. ESI-MS (m/z): calcd. for C H N O 363.11, found
17 13 7 3
+
64.18 [M + H] .
4
.2.2.9.
1-((1-(4-iodophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-3-
-1
carbaldehyde oxime (5i). Brown solid; yield 87%, 0.58g, m.p. 180-182° C; IR (KBr, cm ) 3436,
1
3
009, 1643, 1574, 951, 520. H NMR (400 MHz, DMSO) δ 10.77 (s, 1H), 8.76 (s, 1H), 8.38 (dd,
J = 4.7, 1.4 Hz, 1H), 8.34 – 8.29 (m, 1H), 8.28 (s, 1H), 7.93 (s, 1H), 7.78 (d, J = 8.5 Hz, 2H),
1
3
7
.36 (d, J = 8.6 Hz, 2H), 7.26 (dd, J = 7.8, 4.7 Hz, 1H), 5.67 (s, 2H). C NMR (101 MHz,
DMSO) δ 147.93, 145.11, 144.58, 144.42, 144.18, 134.83, 131.16, 130.38, 129.45, 122.17,
1
4
4
3
3
8
6
20.54, 117.51, 117.46, 108.72, 51.56. ESI-MS (m/z): calcd. for C17H13IN O 444.02, found
+
45.11 [M + H] .
.2.2.10.
1-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-
-1
-carbaldehyde oxime (5j). Pale yellow solid; yield 87%, 0.58g, m.p. 165-166° C; IR (KBr, cm )
1
423, 3005, 1639, 1585, 1310, 945. H NMR (400 MHz, DMSO) δ 10.80 (s, 1H), 8.60 (s, 1H),
.38 (dd, J = 4.7, 1.4 Hz, 1H), 8.32-8.30(m, 2H), 7.97 (s, 1H), 7.76 (dd, J = 7.75 Hz, 1H), 7.68
(dd, J = 7.67 Hz, 1H), 7.61 (td, J = 7.7, 1.8 Hz, 1H), 7.55 (td, J = 7.6, 1.5 Hz, 1H). 7.24 (dd, J =
1
3
7
1
5
3
.24 Hz, 1H), 5.68 (s, 2H). C NMR (100.61 MHz, CDCl ) δ 148.21, 147.32, 142.25, 135.32,
33.82, 132.86, 132.02, 131.27, 130.16, 129.03, 128.87, 126.34, 119.73, 119.04, 118.51, 101.17,
+
2.72. ESI-MS (m/z): calcd. for C H FN O 336.11, found 337.17 [M + H] .
1
7
13
6
4
3
.2.2.11.
1-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-
-1
-carbaldehyde oxime (5k). Brown solid; yield 76%, 0.4g, m.p. 168-169° C; IR (KBr, cm )
1
4

ACCEPTED MANUSCRIPT
1
3
8
395, 3002, 1676, 1581, 955, 810. H NMR (400 MHz, DMSO) δ 10.80 (s, 1H), 8.60 (s, 1H),
.38 (dd, J = 4.7, 1.4 Hz, 1H), 8.32-8.30(m, 2H), 7.97 (s, 1H), 7.76 (dd, J = 7.75 Hz, 1H), 7.68
(dd, J = 7.67 Hz, 1H), 7.61 (td, J = 7.7, 1.8 Hz, 1H), 7.55 (td, J = 7.6, 1.5 Hz, 1H). 7.24 (dd, J =
1
3
7
1
5
3
.24 Hz, 1H), 5.68 (s, 2H). C NMR (100.61 MHz, CDCl ) δ 148.00, 144.44, 144.22, 143.63,
34.84, 132.13, 131.18, 130.98, 130.45, 130.13, 128.91, 128.86, 126.25, 117.52, 117.44, 108.70,
+
1.33. ESI-MS (m/z): calcd. for C17
H13ClN
6
O 352.08, found 353.17 [M + H] .
4
3
3
8
.2.2.12.
1-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-
-1
-carbaldehyde oxime (5l). Brown solid; yield 87%, 0.51g, m.p. 152-154° C; IR (KBr, cm )
1
467, 3043, 1665, 1586, 943, 628. H NMR (400 MHz, DMSO) δ 10.78 (s, 1H), 8.56 (s, 1H),
.38 (dd, J = 4.7, 1.4 Hz, 1H), 8.32-8.30(m, 2H), 7.97 (s, 1H), 7.76 (dd, J = 7.75 Hz, 1H), 7.68
(dd, J = 7.67 Hz, 1H), 7.61 (td, J = 7.7, 1.8 Hz, 1H), 7.55 (td, J = 7.6, 1.5 Hz, 1H). 7.24 (dd, J =
1
3
7
1
5
.24 Hz, 1H), 5.66 (s, 2H). C NMR (100.61 MHz, CDCl ) δ 148.00, 144.44, 144.22, 143.63,
3
34.84, 132.13, 131.18, 130.98, 130.45, 130.13, 128.91, 128.86, 126.25, 117.52, 117.44, 108.70,
+
1.33. ESI-MS (m/z): calcd. for C H BrN O 396.03, found 397.12 [M + H] .
1
7
13
6
4
.2.2.13.
1-((1-(2-iodophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-3-
-1
carbaldehyde oxime (5m). Pale yellow; yield 78%, 0.52g, m.p.154-156° C; IR (KBr, cm ) 3395,
1
3
024, 1654, 1589, 950, 557. H NMR (400 MHz, DMSO) δ 10.79 (s, 1H), 8.56 (s, 1H), 8.36 (dd,
J = 4.7, 1.4 Hz, 1H), 8.32-8.29 (m, 2H), 7.97 (s, 1H), 7.76 (dd, J = 7.75 Hz, 1H,), 7.68 (dd, J =
7
1
1
.67 Hz, 1H), 7.61 (td, J = 7.7, 1.8 Hz, 1H), 7.55 (td, J = 7.6, 1.5 Hz, 1H). 7.24 (dd, J = 7.24 Hz,
1
3
H), 5.68 (s, 2H). C NMR (100.61 MHz, CDCl ) δ 148.00, 144.44, 144.22, 143.63, 134.84,
3
32.13, 131.18, 130.98, 130.45, 130.13, 128.91, 128.86, 126.25, 117.52, 117.44, 108.70, 51.28.
+
ESI-MS (m/z): calcd. for C17
H13IN
6
O 444.02, found 445.11 [M + H] .
4
.2.2.14.
1-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]
pyridine-3-carbaldehyde oxime (5n). Brown solid; yield 91%, 0.47g, m.p. 148-150° C; IR (KBr,
-1
1
cm ) 3425, 3032, 1658, 1570, 1235, 945. H NMR (400 MHz, DMSO) δ 10.78 (s, 1H), 8.58 (s,
1
7
H), 8.38 (dd, J = 4.7, 1.4 Hz, 1H), 8.32-8.28 (m, 2H), 7.96 (s, 1H), 7.76 (s, 1H), 7.71 (s,1H),
.68 (d, J = 7.25 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.24 (d, J = 7.24 Hz, 1H), 5.65 (s, 2H), 3.81
1
3
(
s, 3H). C NMR (101 MHz, DMSO) 148.12, 144.43, 144.21, 143.62, 134.81, 132.72, 131.34,
1
5

ACCEPTED MANUSCRIPT
1
30.21, 130.62, 130.11, 128.91, 128.12, 126.25, 117.52, 117.44, 108.72, 56.23, 51.28. ESI-MS
+
(
m/z): calcd. for C H N O 348.13, found 349.19 [M + H] .
1
8
16
6
2
4
.2.2.15.
1-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]pyridine-3-
-1
carbaldehyde oxime (5o). Yellow solid; yield 67%, 0.36g, m.p. 189-190° C; IR (KBr, cm ) 3420,
1
3
8
081, 1685, 1580, 1512, 1325, 940. H NMR (400 MHz, DMSO) δ 10.75 (s, 1H), 8.69 (s, 1H),
.37 (dd, J = 4.7, 1.4 Hz, 1H), 8.32-8.29 (m, 1H), 8.28 (s,1H), 8.21 (d, 1H), 7.93 (s, 1H), 7.90 (d,
1
3
J = 7.75 Hz, 1H), 7.82 (dd, J = 7.6, 1.5 Hz, 2H), 7.24 (dd, J = 7.24,1.4 Hz, 1H), 5.67 (s, 2H). C
NMR (101 MHz, DMSO) δ 147.99, 144.39, 144.25, 138.01, 134.82, 131.65, 131.14, 130.46,
1
29.44, 128.07, 125.96, 125.39, 119.20, 117.54, 117.44, 108.78, 51.60. ESI-MS (m/z): calcd. for
+
C
17
H
13
N
7
O
3
363.11, found 364.19 [M + H] .
4
.2.2.16.
1-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbaldehyde oxime (5p). Brown solid; yield 87%, 0.48g, m.p. 186-188° C; IR
-1
1
(
KBr, cm ) 3467, 3015, 1651, 1578, 1125, 958, 814. H NMR (400 MHz, DMSO) δ 10.79 (s,
H), 8.83 (s, 1H), 8.36 (dd, J = 4.7, 1.6 Hz, 1H), 8.30 (dd, J = 7.8, 1.6 Hz, 1H), 8.28 (s, 1H), 8.18
dd, J = 6.4, 2.7 Hz, 1H), 7.95 – 7.91 (m, 2H) 7.63 (t J = 6.0 Hz, 1H), 7.23 (dd, J = 7.8, 4.7 Hz,
1
(
1
3
1
1
H), 5.66 (s, 2H). C NMR (101 MHz, DMSO) δ 147.98, 144.98, 144.39, 144.22, 133.95,
31.18, 130.46, 122.83, 122.62, 121.39, 121.08, 118.63, 118.40, 117.52, 117.45, 108.78, 51.62.
+
ESI-MS (m/z): calcd. for C H ClFN O 370.07, found 371.13 [M + H] .
17
12
6
4
.2.2.17.
1-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbaldehyde oxime (5q). Pale yellow solid; yield 81%, 0.47g, m.p. 178-180° C; IR
-1
1
(
KBr, cm ) 3410, 3080, 1660, 1590, 960, 825. H NMR (400 MHz, DMSO) δ 10.76 (s, 1H),
.81 (s, 1H), 8.38 (dd, J = 4.7, 1.6 Hz, 1H), 8.31 (dd, J = 7.8, 1.6 Hz, 1H), 8.26 (s, 1H), 8.18 (dd,
J = 6.4, 2.7 Hz, 1H), 7.95 – 7.91 (m, 2H) 7.62 (t,J = 6.0 Hz, 1H), 7.21 (dd, J = 7.8, 4.7 Hz, 1H),
8
1
3
5
1
.64 (s, 2H). C NMR (101 MHz, DMSO) δ 148.12, 144.91, 144.32, 144.22, 133.94, 131.12,
30.65, 122.75, 122.21, 121.39, 121.08, 118.63, 118.40, 117.52, 117.15, 108.72, 51.64. ESI-MS
+
(m/z): calcd. for C17
H12Cl
2
N
6
O 386.04, found 387.12 [M + H] .
1
6

ACCEPTED MANUSCRIPT
4
.2.2.18.
1-((1-(3,4-difluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbaldehyde oxime (5r). White solid; yield 72%, 0.38g, m.p. 168-169° C; IR
-1
1
(
KBr, cm ) 3395, 3029, 1651, 1583, 1280, 954. H NMR (400 MHz, DMSO) δ 10.78 (s, 1H),
.84 (s, 1H), 8.36 (dd, J = 4.7, 1.6 Hz, 1H), 8.30 (dd, J = 7.8, 1.6 Hz, 1H), 8.27 (s, 1H), 8.19 (dd,
J = 6.4, 2.7 Hz, 1H), 7.95 – 7.91 (m, 2H) 7.62 (t,J = 6.0 Hz, 1H), 7.23 (dd, J = 7.8, 4.7 Hz, 1H),
8
1
3
5
1
.65 (s, 2H). C NMR (101 MHz, DMSO) δ 148.14, 144.92, 144.32, 144.22, 133.94, 131.12,
30.65, 122.75, 122.21, 121.41, 121.08, 118.63, 118.40, 117.54, 117.16, 108.74, 51.68. ESI-MS
+
(
m/z): calcd. for C H F N O 354.11, found 355.19 [M + H] .
1
7
12
2
6
4
.2.2.19.
1-((1-(3,4-dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbaldehyde oxime (5s). Brown solid; yield 86%, 0.49g, m.p. 152-153° C; IR
-1
1
(
KBr, cm ) 3410, 3020, 1650, 1580, 1170, 945. H NMR (400 MHz, DMSO) δ 10.74 (s, 1H),
.71 (s, 1H), 8.37 (dd, J = 4.7, 1.6 Hz, 1H), 8.35 – 8.29 (m, 1H), 8.27 (s,1H), 7.93 (s, 1H), 7.66
d, J = 2.0 Hz, 1H), 7.56 (dd, J = 8.1, 2.3 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H), 7.23 (dd, J = 7.8, 4.7
8
(
1
3
Hz, 1H), 5.63 (s, 2H), 3.29 (s, 3H), 3.26 (s, 3H). C NMR (100.61 MHz, CDCl ) δ 149.69,
3
1
49.32, 147.98, 144.43, 144.37, 144.20, 131.17, 130.46, 130.38, 122.37, 117.51, 117.44, 112.66,
1
3
12.35, 108.72, 105.09, 56.31, 56.22, 51.61. ESI-MS (m/z): calcd. for C H N O 378.14, found
1
9
18
6
3
+
79.22 [M + H] .
4
.2.2.20.
1-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-b]
pyridine-3-carbaldehyde oxime (5t). Brown solid; yield 75%, 0.39g, m.p. 188-190° C; IR (KBr,
-1
1
cm ) 3420, 3038, 1657, 1579, 943. H NMR (400 MHz, DMSO) δ 10.79 (s, 1H), 8.72 (s, 1H),
8
1
.37 (dd, J = 4.7, 1.6 Hz, 1H), 8.32 – 8.30 (m, 1H), 8.28 (s,1H), 7.94 (s, 1H), 7.66 (d, J = 2.0 Hz,
H), 7.56 (dd, J = 8.1, 2.3 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H), 7.23 (dd, J = 7.8, 4.7 Hz, 1H), 5.64
1
3
(
s, 2H), 2.27 (s, 3H), 2.25 (s, 3H). C NMR (101 MHz, DMSO) δ 147.98, 144.53, 144.44,
1
1
44.20, 138.54, 137.52, 134.88, 131.20, 130.95, 130.45, 122.03, 121.30, 117.72, 117.50, 117.45,
08.74, 51.61, 19.83, 19.41. ESI-MS (m/z): calcd. for C H N O 346.15, found 347.23 [M +
19
18
6
+
H] .
4
.2.2.21.
1-((1-(benzo[d][1,3]dioxol-5-yl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbaldehyde oxime (5u). Brown solid; yield 88%, 0.48g, m.p. 208-210° C; IR
1
7

ACCEPTED MANUSCRIPT
-1
1
(
KBr, cm ) 3398 3029, 1654, 1586, 1210, 956. H NMR (400 MHz, DMSO) δ 10.78 (s, 1H),
8
.82 (s, 1H), 8.38 (dd, J = 4.7, 1.6 Hz, 1H), 8.31 (dd, J = 7.8, 1.6 Hz, 1H), 8.26 (s, 1H), 8.18 (dd,
J = 6.4, 2.7 Hz, 1H), 7.95 – 7.91 (m, 2H) 7.62 (t J = 6.0 Hz, 1H), 7.21 (dd, J = 7.8, 4.7 Hz, 1H),
1
3
5
1
5
4
.67 (s, 2H), 3.91 (s, 2H). C NMR (101 MHz, DMSO) δ 148.65, 144.91, 144.37,144.22,
33.99, 131.12, 130.63, 122.75, 122.21, 121.39, 121.08, 118.66, 118.40, 117.52, 117.16, 108.72,
+
8.23, 51.66. ESI-MS (m/z): calcd. for C18
H
14
N
6
O
3
362.11, found 363.21 [M + H] .
.2.2.22.
4
.2.3. Synthesis of 3-bromo-1H-pyrrolo [2, 3-b] pyridine (6): 1H-pyrrolo[2,3-b]pyridine (1) was
dissolved in dimethylformamide (DMF) and added N-bromosuccinamide in DMF solution was
added dropwise at ambient temperature. The reaction mixture was stirred overnight at RT. Once
the reaction is complete as indicated by TLC, water was added to reaction mixture and 3-bromo-
1
H-pyrrolo[2,3-b]pyridine (6) was collected by filtration.
4
.2.4. Synthesis of 3-(4-substituted-1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-b]pyridine (7a-k): 3-
bromo-1H-pyrrolo[2,3-b]pyridine (6) (0.5 mmol, 1 equiv) was mixed with variety of alkynes
0.55mmol, 1.1equiv) 9:1 DMSO/H O in a 20 mL scintillation vial. To this mixture, were added
(
2
L-proline (0.1 mmol, 0.2 equiv), Na CO (0.1 mmol, 0.2 equiv), NaN (0.6 mmol, 1.2 equiv),
2
3
3
sodium ascorbate (0.05 mmol, 0.1 equiv) and CuSO
4
.5H
2
O (0.025 mmol, 0.05 equiv). The
mixture was stirred overnight at 60°C. Upon completion of the reaction (monitoredby TLC), the
crude mixture was poured into water and extracted with ethyl acetate. Combined organic layers
were and dried over anhydrous sodium sulphate to get the crude product. Compound was further
purified by column chromatography with pet ether and ethyl acetate (15%).
4
.2.4.1.
3-(4-phenyl-1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-b]pyridine (7a). White solid;
-1
1
yield 85%, 0.33g, m.p. 161-163° C; IR (KBr, cm ) 3310, 3025, 1655. H NMR (400 MHz,
DMSO) δ 12.89 (s, 1H), 8.71 (s, 1H), 8.27 (dd, J = 4.6, 1.2 Hz, 1H). 8.01 (dd, J = 7.9, 1.5 Hz,
2
1
1
2
H). 7.55 (t, J = 7.2 Hz, 2H). 7.45 (d, J = 7.2 Hz, 2H). 7.15 (dd, J = 7.9, 4.7 Hz, 1H). 6.82 (s,
1
3
H). C NMR (101 MHz, CDCl
3
) δ 148.32, 145.92, 142.86, 132.04, 131.66, 129.38, 128.69,
27.91, 126.64, 125.01, 121.21, 116.82, 101.45. ESI-MS (m/z): calcd. for C H N 261.1, found
1
5
11
5
+
62.17 [M + H] .
1
8

ACCEPTED MANUSCRIPT
4
.2.4.2.
3-(4-p-tolyl-1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-b]pyridine (7b). Pale yellow
-1
1
solid; yield 84%, 0.35g, m.p. 117-119° C; IR (KBr, cm ) 3322, 3032, 1663. H NMR (400 MHz,
DMSO) δ 12.10 (s, 1H), 8.30 (dd, J = 4.6, 1.2 Hz, 1H). 8.21 (s, 1H), 7.84 (dd, J = 7.9, 1.5 Hz,
1
7
1
H). 7.75 (d, J = 7.2 Hz, 2H). 7.72 (d, J = 7.2 Hz, 1H). 7.25 (d, J = 4.3 Hz, 2H). 7.17 (dd, J =
1
3
3
.9, 4.7 Hz, 1H). 2.32 (s, 3H). C NMR (101 MHz, CDCl ) δ 147.62, 144.33, 142.86, 129.92,
28.48, 127.42, 126.89, 126.50, 126.04, 119.15, 116.82, 100.22, 87.99, 21.30. ESI-MS (m/z):
+
calcd. for C H N 275.12, found 276.19 [M + H] .
1
6
13
5
4
.2.4.3.
3-(4-(4-tert-butylphenyl)-1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-b]pyridine (7c).
-1
1
Pale yellow solid; yield 83%, 0.40g, m.p. 121-123° C; IR (KBr, cm ) 3328, 3028, 1665. H
NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.30 (dd, J = 4.6, 1.2 Hz, 1H), 8.19 (s, 1H), 7.85 (dd,
J = 7.9, 1.5 Hz, 1H), 7.77 (d, J = 7.2 Hz, 2H), 7.71 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 4.3 Hz, 2H),
1
3
7
1
3
.18 (dd, J = 7.9, 4.7 Hz, 1H), 1.30 (s, 9H). C NMR (101 MHz, DMSO) δ 149.26, 147.63,
44.23, 131.14, 131.01, 128.51, 126.89, 126.36, 126.04, 125.86, 119.16, 116.82, 100.15, 34.82,
+
1.50. ESI-MS (m/z): calcd. for C19
H
19
N
5
317.16, found 317.24 [M + H] .
4
.2.4.4.
3-(4-butyl-1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-b]pyridine (7d). Brown solid;
-
1
1
yield 79%, 0.29g, m.p. 156-157° C; IR (KBr, cm ) 3325, 3056, 1667, 1472. H NMR (400 MHz,
DMSO) δ 12.09 (s, 1H), 8.30 (dd, J = 4.6, 1.2 Hz, 1H), 7.84 (dd, J = 7.9, 1.5 Hz, 1H), 7.76 (s,
1
1
H), 7.19 (dd, J = 7.9, 4.7 Hz, 1H), 7.08 (dd, J = 7.9, 4.7 Hz, 1H), 2.01 (t, J = 6.8 Hz, 3H), 1.55-
1
3
.45 (m, 6H). C NMR (100.61 MHz, CDCl ) δ 148.76, 147.36, 144.72, 142.74, 128.60, 126.89,
3
119.14, 116.87, 101.22, 10.25, 9.29, 8.86, 8.12. ESI-MS (m/z): calcd. for C H N 241.13, found
13 15 5
+
2
42.21 [M + H] .
4
.2.4.5.
3-(4-cyclopropyl-1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-b]pyridine (7e). Pale
-1
1
yellow solid; yield 69%, 0.23g, m.p. 148-149° C; IR (KBr, cm ) 3342, 3024, 1651, 1425. H
NMR (400 MHz, DMSO) δ 12.11 (s, 1H), 8.31 (dd, J = 4.6, 1.2 Hz, 1H). 7.85 (dd, J = 7.9, 1.5
Hz, 1H), 7.74 (s, 1H), 7.18 (dd, J = 7.9, 4.7 Hz, 1H), 7.06 (dd, J = 7.9, 4.7 Hz, 1H), 2.00 (tt, J =
1
3
7
.2 Hz, J = 6.9 Hz, 1H), 0.92 (t, J = 7.2 Hz, 2H), 0.72 (t, J = 6.8 Hz, 2H). C NMR (100.61
1
9

ACCEPTED MANUSCRIPT
MHz, CDCl
3
) δ 148.72, 147.62, 144.32, 142.74, 128.60, 126.89, 126.04, 119.16, 116.81, 100.26,
+
8
.90, 8.37. ESI-MS (m/z): calcd. for C H N 225.1, found 226.19 [M + H] .
1
2
11
5
4
.2.4.6.
2-(1-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-4-yl)propan-2-ol (7f). Pale
-1
yellow solid; yield 87%, 0.32g, m.p. 149-151° C; IR (KBr, cm ) 3420, 3336, 3054, 1676, 1450.
1
H NMR (400 MHz, DMSO) δ 12.08 (s, 1H), 8.30 (dd, J = 4.6, 1.2 Hz, 1H). 7.93 (dd, J = 7.9,
4
1
1
2
.7 Hz, 1H), 7.83 (dd, J = 7.9, 4.7 Hz, 1H), 7.72 (s, 1H), 7.06 (dd, J = 7.9, 4.7 Hz, 1H), 6.44 (s,
1
3
H), 1.35 (s, 6H). C NMR (101 MHz, DMSO) δ 148.86, 147.62, 144.33, 128.48, 126.89,
13 5
26.04, 119.15, 116.82, 100.22, 87.59. 32.04. ESI-MS (m/z): calcd. for C12H N O 243.11, found
+
44.21 [M + H] .
4
.2.4.7.
3-(1-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-4-yl)propan-1-ol
(7g).
-1
White solid; yield 73%, 0.27g, m.p. 150-152° C; IR (KBr, cm ) 3425, 3327, 3055, 1675, 1446.
1
H NMR (400 MHz, DMSO) δ 12.10 (s, 1H), 8.31 (dd, J = 4.6, 1.2 Hz, 1H), 7.86 (dd, J = 7.9,
1
1
1
2
.5 Hz, 1H), 7.78 (s, 1H), 7.18 (dd, J = 7.9, 4.7 Hz, 1H), 7.09 (dd, J = 7.9, 4.7 Hz, 1H), 4.01 (s,
1
3
H), 1.61-1.48 (m, 6H). C NMR (100.61 MHz, CDCl
3
) δ 148.32, 147.12, 144.62, 142.74,
28.65, 126.82, 119.12, 116.87, 101.22, 11.02, 10.24, 9.31. ESI-MS (m/z): calcd. for C H N O
1
2
13
5
+
43.11, found 244.18 [M + H] .
4
.2.4.8.
Ethyl1-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazole-4-carboxylate
(7h).
-1
Pale yellow solid; yield 78%, 0.30g, m.p. 165-167° C; IR (KBr, cm ) 3317, 3062, 1672, 1424.
1
H NMR (400 MHz, DMSO) δ 12.08 (s, 1H), 8.30 (dd, J = 4.6, 1.2 Hz, 1H), 7.84 (dd, J = 7.9,
1
.5 Hz, 1H), 7.79 (s, 1H), 7.17 (dd, J = 7.9, 4.7 Hz, 1H), 7.06 (dd, J = 7.9, 4.7 Hz, 1H), 3.78 (q,J
1
3
=
3
6.4 Hz, 2H), 2.41(t, J = 6.2 Hz 3H). C NMR (100.61 MHz, CDCl ) δ 152.21, 148.31, 147.32,
1
44.48, 142.52, 128.61, 126.82, 119.13, 116.82, 101.81, 62.32, 15.32. ESI-MS (m/z): calcd. for
+
C
12
H
11
N
5
O
2
257.09, found 258.17 [M + H] .
4
.2.4.9.
(1-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-4-yl)methanol (7i). White
-1
1
solid; yield 83%, 0.27g, m.p. 120-121° C; IR (KBr, cm ) 3435, 3325, 3020, 1670. H NMR (400
MHz, DMSO) δ 12.11 (s, 1H), 8.28 (dd, J = 4.6, 1.2 Hz, 1H), 7.84 (dd, J = 7.9, 1.5 Hz, 1H), 7.77
(s, 1H), 7.19 (dd, J = 7.9, 4.7 Hz, 1H), 7.08 (dd, J = 7.9, 4.7 Hz, 1H), 4.12 (s, 1H), 3.12 (s, 2H).
2
0

ACCEPTED MANUSCRIPT
1
3
C NMR (100.61 MHz, CDCl
3
) δ 148.32, 147.12, 144.62, 142.74, 128.65, 126.82, 119.12,
+
1
16.87, 101.22, 56.43. ESI-MS (m/z): calcd. for C H N O 215.08, found 216.17 [M + H] .
10
9
5
4
.2.4.10.
3-(4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-b]pyridine
(7j).
-1
1
Brownsolid; yield 69%, 0.29g, m.p. 127-128° C; IR (KBr, cm ) 3342, 3050, 1671, 1260. H
NMR (400 MHz, DMSO) δ 12.10 (s, 1H), 8.31 (dd, J = 4.6, 1.2 Hz, 1H). 8.20 (s, 1H), 7.86 (dd,
J = 7.9, 1.5 Hz, 1H). 7.73 (d, J = 7.2 Hz, 2H). 7.71 (d, J = 7.2 Hz, 1H). 7.23 (d, J = 4.3 Hz, 2H).
1
3
7
1
.12 (dd, J = 7.9, 4.7 Hz, 1H). C NMR (101 MHz, CDCl ) δ 148.52, 144.32, 142.84, 129.97,
3
28.28, 127.12, 126.89, 126.50, 126.16, 119.15, 116.86, 101.22, 100.16. ESI-MS (m/z): calcd.
+
for C H FN 279.09, found 280.15 [M + H] .
1
5
10
5
4
.2.4.11.
3-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-b]pyridine (7k).
-1
Pale yellow solid; yield 76%, 0.33g, m.p. 165-166° C; IR (KBr, cm ) 3325, 3050, 1670, 1175.
1
H NMR (400 MHz, DMSO) δ 12.08 (s, 1H), 8.29 (dd, J = 4.6, 1.2 Hz, 1H), 8.20 (s, 1H), 7.81
(dd, J = 7.9, 1.5 Hz, 1H), 7.79 (d, J = 7.2 Hz, 2H), 7.71 (d, J = 7.2 Hz, 1H), 7.22 (d, J = 4.3 Hz,
1
3
2
1
3
H), 7.18 (dd, J = 7.9, 4.7 Hz, 1H), 3.82 (s, 3H). C NMR (101 MHz, CDCl ) δ 147.59, 144.31,
42.82, 129.54, 128.67, 127.40, 126.83, 126.71, 126.04, 119.15, 116.81, 101.21, 100.43, 56.38.
+
ESI-MS (m/z): calcd. for C H N O 291.11, found 292.18 [M + H] .
16
13
5
4
.3. Biology
The cell lines, A549-lung (doubling time: 22 h), HeLa-cervix (doubling time: 19 h) and
MDA MB 231-breast cancer (doubling time: 27 h) which were used in this study were procured
from American Type Culture Collection (ATCC), United States. In addition, WPMY-1 was
employed as a normal prostatic myofibroblast cell line. The synthesized test compounds were
evaluated for their in vitro antiproliferative activity in these three different human cancer cell
lines. A protocol of 48 h continuous drug exposure was used, and a SRB cell proliferation assay
was used to estimate cell viability or growth. All the cell lines were grown in Dulbecco's
modified Eagle's medium (containing 10% FBS in a humidified atmosphere of 5% CO at 37
2
°
C). Cells were trypsinized when sub-confluent from T25 flasks/60 mm dishes and seeded in 96-
well plates in 100 ꢀL aliquots at plating densities depending on the doubling time of individual
cell lines. The microtiter plates were incubated at 37 °C, 5% CO , 95% air, and 100% relative
2
2
1

ACCEPTED MANUSCRIPT
humidity for 24 h prior to addition of experimental drugs and were incubated for 48 h with
different doses (0.01, 0.1, 1, 10 and 100µM) of prepared derivatives. After 48 h incubation at 37
°
C, cell monolayers were fixed by the addition of 10% (wt/vol) cold trichloroacetic acid and
incubated at 4 °C for 1 h and were then stained with 0.057% SRB dissolved in 1% acetic acid
for 30 min at room temperature. Unbound SRB was washed with 1% acetic acid. The protein –
bound dye was dissolved in 10 mM Tris base solution for OD was measured at 510 nm using a
microplate reader (Enspire, Perkin Elmer, USA). Using the seven absorbance measurements
[time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five
concentration levels (Ti)], the percentage growth was calculated at each of the drug
concentrations levels. Percentage growth inhibition was calculated as:
[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz
[(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz.
The dose response parameter, GI was calculated for each experimental agent. Growth
5
0
inhibition of 50 % (GI50) was calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug
concentration resulting in a 50% reduction in the net protein increase (as measured by SRB
staining) in control cells during the drug incubation. Values were calculated for this parameter if
the level of activity is reached; however, if the effect is not reached or is exceeded, the value for
that parameter was expressed as greater or less than the maximum or minimum concentration
tested.
4
.4. Western blot
Hela cells were seeded in 6-well plates at 0.2 million cells per well in complete growth
medium. Following treatment of cells with 5d and cirtzonib for duration of 24 h, cells were
washed with PBS and subsequently whole cell lystes were prepared in Laemmli’s sample buffer.
Samples were immediately heated to 95 °C for 3 min. Equal volumes of samples were run on an
SDS-10% polyacrylamide gel and were transferred to a nitrocellulose membrane employing
semidry transfer at 50 mA for 1 h. Blots were probed with mouse anti-human p53 and α-tubulin,
diluted to 1 : 5000 ml (Sigma) and stained with the rabbit anti-mouse secondary antibody
coupled with horseradish peroxidase diluted to 1 : 5000 ml (Sigma). Bands were visualized using
an enhanced chemiluminescence protocol (Pierce) and radiographic film (Kodak).
2
2

ACCEPTED MANUSCRIPT
Acknowledgments
We acknowledge the financial support from Department of Biotechnology (DBT)
BT/PR4801/MED/29/370/2012), Government of India, New Delhi. N.S. thanks UGC, New
(
Delhi for the award of senior research fellowship.
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