Novel pyrazolo[3,4-d]pyrimidine with 4-(1H-benzimidazol-2-yl)-phenylamine as broad spectrum anticancer agents: Synthesis, cell based assay, topoisomerase inhibition, DNA intercalation and bovine serum albumin studies

Article abstract of DOI:10.1016/j.ejmech.2016.09.093

A series of new pyrazolo[3,4-d]pyrimidine possessing 4-(1H-benzimidazol-2-yl)-phenylamine moiety at C4 position and primary as well as secondary amines at C6 position has been designed and synthesized. Their antitumor activities were evaluated against a panel of 60 human cancer cell lines at National Cancer Institute (NCI). Six compounds displayed potent and broad spectrum anticancer activities at 10?μM. Compounds 8, 12, 14 and 17 proved to be the most active and efficacious candidate in this series, with mean GI₅₀values of 1.30?μM, 1.43?μM, 2.38?μM and 2.18?μM, respectively against several cancer cell lines. Further biological evaluation of these compounds suggested that these compounds induce apoptosis and inhibit human topoisomerase (Topo) IIα as a possible intracellular target. UV-visible and fluorescence studies of these compounds revealed strong interaction with ct-DNA and bovine serum albumin (BSA).

Full text of DOI:10.1016/j.ejmech.2016.09.093

Accepted Manuscript
Novel pyrazolo[3,4-d]pyrimidine with 4-(1H-benzimidazol-2-yl)-phenylamine as broad
spectrum anticancer agents: Synthesis, cell based assay, topoisomerase inhibition,
DNA intercalation and bovine serum albumin studies
Prinka Singla, Vijay Luxami, Raja Singh, Vibha Tandon, Kamaldeep Paul
PII:
S0223-5234(16)30856-X
10.1016/j.ejmech.2016.09.093
EJMECH 8959
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 May 2016
Revised Date: 3 September 2016
Accepted Date: 28 September 2016
Please cite this article as: P. Singla, V. Luxami, R. Singh, V. Tandon, K. Paul, Novel
pyrazolo[3,4-d]pyrimidine with 4-(1H-benzimidazol-2-yl)-phenylamine as broad spectrum anticancer
agents: Synthesis, cell based assay, topoisomerase inhibition, DNA intercalation and bovine serum
albumin studies, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.093.
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Graphical Abstract
Novel pyrazolo[3,4-d]pyrimidine with 4-(1H-benzimidazol-2-yl)-phenylamine
as broad spectrum anticancer agents: Synthesis, cell based assay,
topoisomerase inhibition, DNA intercalation
and bovine serum albumin studies
Prinka Singla1, Vijay Luxami1*, Raja Singh2, Vibha Tandon2 and Kamaldeep Paul1*
1School of Chemistry and Biochemistry, Thapar University, Patiala – 147004, INDIA
2Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi-110 067,
INDIA
Email: kpaul@thapar.edu

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Novel pyrazolo[3,4-d]pyrimidine with 4-(1H-benzimidazol-2-yl)-phenylamine as
broad spectrum anticancer agents: Synthesis, cell based assay, topoisomerase
inhibition, DNA intercalation and bovine serum albumin studies
Prinka Singla1, Vijay Luxami1* Raja Singh2, Vibha Tandon2, and Kamaldeep Paul1*
1School of Chemistry and Biochemistry, Thapar University, Patiala – 147004, INDIA
2Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi-110 067, INDIA
E-mail: kpaul@thapar.edu, vluxami@thapar.edu
5
Abstract: A series of new pyrazolo[3,4-d]pyrimidine possessing 4-(1H-benzimidazol-2-yl)-
10 phenylamine moiety at C4 position and primary as well as secondary amines at C6 position has been
designed and synthesized. Their antitumor activities were evaluated against a panel of 60 human
cancer cell lines at National Cancer Institute (NCI). Six compounds displayed potent and broad
spectrum anticancer activities at 10 µM. Compounds 8, 12, 14 and 17 proved to be the most active and
efficacious candidate in this series, with mean GI50 values of 1.30 µM, 1.43 µM, 2.38 µM and 2.18
15 µM, respectively against several cancer cell lines. Further biological evaluation of these compounds
suggested that these compounds induce apoptosis and inhibit human topoisomerase (Topo) IIα as a
possible intracellular target. UV-visible and fluorescence studies of these compounds revealed strong
interaction with ct-DNA and bovine serum albumin (BSA).
Keywords: Pyrazolo[3,4-d]pyrimidine, Benzimidazole, Antitumor activity, ct-DNA, Bovine serum
20 albumin.
1. Introduction
Cancer is a multifaceted disease that represents one of the leading causes of mortality, exceeded
only by heart disease [1]. Although, chemotherapy is the main remedy for cancer treatment, but the use
of available chemotherapeutics is often limited due to undesirable side effects and a limited choice of
25 available anticancer drugs [2]. It is becoming important to investigate new agents and targets for the
treatment of cancer. Thus, a considerable research for new anticancer agents has been fuelled by
various industries and academics to unveil new targets and mechanisms based on the lead candidates
of different classes of compounds [3].
Nitrogen heterocycles have received a great deal of attention in the literature due to their role as
30 active pharmacophores. Roscovitine, belongs to the family of purine, is used for the treatment of non-

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small cell lung cancer (NSCLC), leukemia, herpes simplex infection, Cushing's disease, HIV infection
etc [4]. Roscovitine has been tested in several phase I and II clinical trials both as monotherapy and
combination therapy in several human cancers. Roscovitine induces apoptosis in many cancer cell
lines at all phases of the cell cycle [5]. In addition, roscovitine is also shown as synergistic effect with
other anti-cancer agents, such as doxorubicin [6,7], taxol [7], 5-fluorouracil [7], vinblastine [7],
alemtuzumab [8], paclitaxel [9], cisplatin [10], irinotecan [11], etoposide (cytotoxic anticancer drug,
belongs to topoisomerase II enzyme inhibitor drug class [12] and tamoxifen [13]. Despite many
successful preclinical studies with roscovitine, clinical trials are not very encouraging. Therefore, there
is need for the contemporary medicinal chemists to further design and identify new potent purine
5
10 analogue with improve anticancer activity [14].
Among the favorable bioisosteres to purine ring is pyrazolo[3,4-d]pyrimidine. Designing of pyrazolo
[3,4-d]pyrimidine ring system has been taken to provide new compounds related with various
biologically active purines, in the hope that new anti-tumor agents might be discovered. Literature
survey revealed the interesting anticancer activities of certain pyrazolo[3,4-d]pyrimidine derivatives
15 over various cancer cell lines [15]. Encouraging by the affirmative anticancer activity of several
pyrazolo[3,4-d]pyrimidine members, and as a continuation of our ongoing efforts to identify new
potent anticancer agents [16], a series of novel 6-substituted-2-benzimidazoanilino pyrazolo[3,4-
d]pyrimidine has been synthesized. Our design is principally based on isosteric replacement of purine
scaffold as well as installment of more lipophilic aromatic and aliphatic primary amines at C4 and C6
20 positions of pyrazolo[3,4-d]pyrimidine, respectively (Figure 1). We investigate the impact of replacing
primary amines at C6 position with secondary amines on the antineoplastic activity. To the best of our
knowledge, this substitution pattern of pyrazolo[3,4-d]pyrimidine is not explored so far, which
motivated us to synthesize this type of compounds to explore their anticancer activity. Furthermore,
the ability of these compounds to inhibit the relaxation activity of supercoiled pHOT1 DNA mediated
25 by human topoisomerase IIα, suggest that nuclear enzyme is a potential intracellular target.
Figure 1
Since, it is commonly accepted that deoxyribonucleic acid (DNA) and proteins are considered as the
main molecular targets in the action of drugs. Many compounds exert their drug effects through
binding to DNA or proteins, which is the basis of designing and discovering new and more efficient
30 drugs [17]. The studies on syntheses and interactions of small molecules with DNA and proteins have
been an active field of research. The interaction of a small molecule with DNA initially causes

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conformational changes in the double helix of DNA, subsequently interrupts replication, transcription,
and repair, and finally kills the fast growing cells [18]. In the design of small molecules with DNA
recognizing capacity, various pyrazolo[3,4-d]pyrimidine [19] have attracted much interest which
damage DNA and cause cell death. Thus, pyrazolo[3,4-d]pyrimidine has attracted considerable
attention owing to their capability of interacting with DNA.
5
Because of the high cost of using human serum albumin (HSA), bovine serum albumin (BSA) with
lower cost is widely used as an important model protein concerning the interaction between
compounds and serum albumins [20]. Compared with the number of organic molecules [21], relatively
few pyrazolo[3,4-d]pyrimidine derivatives have been investigated for their protein, BSA-binding
10 activity [22]. Therefore, the reactivity of pyrazolo[3,4-d]pyrimidine towards DNA and protein BSA is
useful in the design and synthesis of anticancer therapeutics.
2. Results and Discussion
2.1. Chemistry
With a view to prepare the target [4-(1H-benzimidazol-2-yl)-phenyl]-(6-substituted-1H-pyrazolo[3,4-
15 d]pyrimidin-4-yl)-amine, the key intermediate 4,6-dichloro-pyrazolo[3,4-d]pyrimidine (4) was
synthesized from 5-aminopyrazole-4-carbonitrile 1, which was prepared from 2-(ethoxymethylene)
malononitrile. Condensation of commercially available 2-(ethoxymethylene)malononitrile with
hydrazine afforded 5-aminopyrazole-4-carbonitrile (1) which underwent partial hydrolysis with
sulphuric acid to produce 5-amino-1H-pyrazole-4-carboxamide (2) in good yield (89%). Treatment of
0
20 carboxamide (2) with molten urea at 190 C gave the intermediate product 1H-pyrazolo[3,4-
d]pyrimidine-4,6(5H,7H)-dione (3), that was easily chlorinated with excess phosphorous oxychloride,
furnished 4,6-dichloropyrazolo[3,4-d]pyrimidine (4) in good yield (87%). As illustrated in Scheme 1,
the substitution of 4-(1H-benzimidazol-2-yl)-phenylamine moiety (5) at C4 position of pyrazolo[3,4-
d]pyrimidine was the next step for synthesis of the target compounds 7-18. Nucleophilic displacement
25 of 4,6-dichloropyrazolo[3,4-d]pyrimidine (4) with benzimidazole (5) was successfully accomplished
under room temperature for 24 h in moderate yield (79%). 1H NMR spectrum of 6, showed the signals
at δ 9.75 (s, 1H, ArH), 8.13-8.08 (m, 2H, ArH), 7.92 (d, 1H, ArH), 7.72 (d, 1H, ArH), 7.62 (bs, 1H,
NH, D2O exchangeable), 7.15-7.13 (m, 2H, ArH), 6.66 (d, 2H, ArH), 5.02 (bs, 1H, NH, D2O
exchangeable), while broad singlet of NH2 protons at δ 4.45 of 4-(1H-benzimidazol-2-yl)-phenylamine
1
30 disappeared in the H NMR of compound 6. The appendage of the second functionality, primary and
secondary amines, at C6 position of pyrazolo[3,4-d]pyrimidine scaffold was achieved by refluxing of

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amines in the presence of potassium carbonate in n-butanol, afforded the final compounds 7-18 (66-
78%). The synthesized compounds were well characterized by NMR and mass spectrometry.
Scheme 1
2.2. In vitro evaluation for antitumor activity
5
The structure of the newly synthesized compounds were submitted to National Cancer Institute (NCI),
Bethesda, Maryland, USA and six derivatives (7, 8, 11, 12, 14 and 17), were selected based on the
degree of structural variation and computer modelling techniques for investigation of their anticancer
activities. According to the NCI protocol, the selected compounds were first pre-screened at a single
dose concentration of 10 µM towards the full panel of approximately 60 human cancer cell lines
10 derived from 9 different cancer types: leukaemia, melanoma, lung, colon, central nervous system,
ovarian, renal, prostate and breast cancers. The mean growth percentage (GP) for the full panel, as well
as a range of GP with the lowest and the highest values among individual cancer cell lines are
presented in Table 1.
As revealed from the results, all of the tested compounds, except 7 and 11, displayed strong growth
15 inhibitory activity (mean GP range of -77.92 to 57.44) over the examined cell lines at 10 µM
concentration. The benzylamine derivative 14 demonstrated superior cytostatic activity while
compound 12 (2-hydroxyethylpiperazine) showed better cytotoxic activities rather than pyrrolidine (8)
and 4-fluororaniline (17). Such finding denotes that primary amine moiety is more favourable for
cytostatic and secondary amine for cytotoxic anticancer activity. This superiority in the activity may be
20 attributed to the corresponding increase in compound lipophilicity, and therefore permeability and
penetration into cancer cells. By referring to the total number of sensitive cells for each tested
compound, it was found that most of the target compounds exhibited broad spectrum antitumor
activity covering different cancer cell lines. Among the responsive cell lines, U251 (CNS cancer) was
noticed to be highly sensitive (minus GP values, lethal effect) for derivatives 11 and 12. Melanoma
25 cell lines SK-MEL-5 and LOX-IMVI were found to be the most susceptible cell lines for derivatives
14 and 17, respectively. Moreover, the renal cancer cell line (786-0) was the most responsive cells to
compound 8. Compounds 8, 12 and 14 showed pronounced cytotoxic activity over the majority of
tested cell lines (> 50 cell lines), with mean GP values of -32.52, -77.92 and -8.30, respectively. Their
detailed antiproliferative activities on the growth percentage of the NCI-60 cell lines, at single dose of
30 10 µM, are depicted in Figure S29-S34 (Supporting Information).

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Table 1
Compounds 8, 12, 14 and 17 exhibited interesting growth inhibitory results in the preliminary
single dose screen and were passed for the advanced 5-dose testing mode against the full panel. Three
response parameters were calculated for each compound against each cell line: GI50 (the concentration
producing 50% growth inhibition); TGI (the concentration leading to 100% growth inhibition); LC50
(the concentration causing 50% lethality). Moreover, the mean graph midpoint (MG-MID) were
calculated for each of those parameters, producing an average activity parameter over all cell lines for
the tested compounds. The subpanel and full panel MG-MID GI50 values of tested nine compounds
5
10 along with roscovitine [23] as a reference compound are shown in Table 2.
Table 2
Investigation of the data listed in Table 2 clearly indicated remarkable potency of all compounds.
All of the active compounds showed lower full panel MG-MID GI50 values (1.30-2.38 µM) than
roscovitine (MG-MID GI50 value of 24.5 µM). Moreover, their subpanel MG-MID GI50 values over
15 the majority of cancer types are better than those of the reference compound. The effect of substitution
pattern of amine moiety at C6 position of pyrazolo[3,4-d]pyrimidine was examined. It has been
observed that secondary amine derivatives 8 and 12 displayed superior antiproliferative potencies
(MG-MID GI50 = 1.30 µM and 1.43 µM, respectively) rather the primary amine compounds 14 and 17
(MG-MID GI50 = 2.38 µM and 2.18 µM, respectively). Regarding the secondary amine, compound 8
20 with pyrrolidine moiety was slightly more potent than the corresponding 2-hydroxyethylpiperazine 12.
This may be owed to the differences in the electronic and/or steric effects of 2-hydroxyethylpiperazine
in respect to pyrrolidine. Similarly, among the primary amine derivatives, compound 14 with benzyl
amine showed reduced activity than 4-fluoroaniline member 17. Therefore, it could be concluded that
fluorine is preferential for achieving good antiproliferative activity. On the other hand, while
25 considering the subpanel MG-MID GI50, it was noticed that compounds 8 and 12 exhibited the best
subpanel with GI50, in most cases, are less than 2 µM. Since roscovitine is mainly active for non-small
lung cancer (NSLC) and leukaemia, it is noteworthy mentioning that all compounds are highly potent
than roscovitine over NSLC and leukaemia. In view of the efficacy relevant parameters (MG-MID TGI
and MG-MID LC50 values) of the most potent compounds 8, 12, 14 and 17, it was evident that all of
30 them are highly effective than roscovitine. The pyrazolo[3,4-d]pyrimidines 8 and 12 exerted
remarkable efficacies (MG-MID TGI < 5.00 µM and MG-MID LC50 < 11.0 µM) towards most of the

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tested subpanel cancer cells. Interestingly, compounds 8 and 12 proved to be the most efficacious
derivatives in this new series with full panel MG-MID TGI and LC50 values of 3.32 µM and 2.99 µM,
and 10.5 µM and 6.06 µM, respectively (Figures S35-S46).
2.3. Topoisomerase IIα inhibitory activity
5
Based upon the antitumor activity, we investigated the ability of the active derivatives to inhibit
relaxation activity catalysed by topoisomerase IIα. In these experiments, supercoiled plasmid DNA
was incubated with Topo IIα at increasing concentrations of the compounds (10, 25, 50, 75, and 100
µM) and DNA relaxation products were then resolved by gel electrophoresis on 1% agarose gel.
Figure 2 showed the effect of the test compounds on the relaxation of supercoiled DNA mediated by
10 topoisomerase IIα. Etoposide, a well-known DNA intercalating and DNA binding topoisomerase IIα
inhibitor was used as a positive control at 25 µM concentration. In the presence of compounds 7, 8, 11,
12, 14 and 17, the appearance of band corresponding to supercoiled DNA can be observed at 50 µM
concentration, indicating inhibition of the topoisomerase IIα activity. Further investigation showed that
these compounds also inhibit Topo IIα activity at both 75 µM and 100 µM concentrations. This data
15 suggested that pyrazolo[3,4-d]pyrimidine compounds served as Topo IIα specific inhibitors at lower
concentration. To check whether these compounds targets topoisomerase I enzyme, relaxation of
supercoiled assay was performed, using camptothecin at 20 µM concentrations as positive control. As
per our observations, these six compounds 7, 8, 11, 12, 14 and 17 did not show significant inhibition of
Topo I activity even up to 100 µM concentration, suggesting that these pyrazolo[3,4-d]pyrimidine
20 derivatives do not inhibit topoisomerase I.
Figure 2
2.4. DNA binding studies
UV-visible and fluorescence spectroscopy are frequently used techniques to studies the interactions
between biological macromolecules and small molecules. We used UV-visible spectroscopy to
25 investigate the absorbance spectra of compounds 7, 8, 11, 12, 14 and 17 with ct-DNA interaction in
phosphate buffer (10 mM, pH 7.4) (Figure 3). The characteristic peaks of compounds were observed in
the range of near 324-335 nm. However, on subsequent addition of ct-DNA to these compounds, the
absorbance was gradually decreased, indicating hypochromic effect along with concomitant increase in
absorption intensity at another λmax = 257-264 nm (compound 7; λmax = 260 nm, compound 8; λmax
=
30 259 nm, compound 11; λmax = 259 nm, compound 12; λmax = 259 nm, compound 14; λmax = 264 nm,

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compound 17; λmax = 257 nm) (Table S1). Hypochromic effect of these compounds with ct-DNA
indicates strong intermolecular interaction. This is due to the overlapping of electron cloud of
pyrazolo[3,4-d]pyrimidine-benzimidazoles (compounds 7, 8, 11, 12, 14 and 17) with ct-DNA base
pairs [24]. Hypochromic effect in UV-visible spectra upon compounds, binding to ct-DNA is a
characteristic of an intercalating binding mode [25].
5
Figure 3
Fluorescence experiments were also undertaken to investigate the interaction of synthesized
compounds (7, 8, 11, 12, 14 and 17) with DNA. The binding of compounds with DNA, by maintaining
the concentration of compounds constant (i.e., 10 µM), and varying the concentration of DNA from 0
10 to 20 µM, was studied by fluorescence spectroscopy. On excitation at 300 nm, these compounds
showed intense band between 382 nm (compounds 7 and 14) and 466 nm (compounds 8, 11, 12 and
17). On increasing the concentration of ct-DNA to compounds, the fluorescence intensities were
regularly decreasing while the maximum emission wavelength was slightly shifted in case of
compounds 12 and 17 (Figure 4).
15
Figure 4
Subsequently, stabilities of pyrazolo[3,4-d]pyrimidine benzimidazoles–DNA complexes were
determined by calculating the binding constant value from Benesi–Hildebrand equation [26].
20
1/(Af − Aobs) = 1/(Af − Afc) + {1/[K(Af − Afc)]}[ligand] ------ 1
where K is the binding constant, Af is the absorbance of the free host, Aobs is the absorbance observed,
and Afc is the absorbance at saturation.
The plot of 1/(Af − Aobs) versus 1/[DNA] was constructed using the titration data and linear fitting,
yielding the binding constant, in the order of 104 M–1 through absorption and emission titrations (Table
25 S1). These calculated binding constant values showed moderate bindings of pyrazolo[3,4-
d]pyrimidine-benzimidazoles with DNA. The results on stability agree with other DNA intercalating
drugs, viz., 5-fluorouracil and mesalamine, which show 104 M-1 order of binding constant values [27].
This constant value might be sufficient to interfere with DNA replication and thus, could provide drug
efficiency.
30 Further, ethidium bromide displacement studies have been performed with pyrazolo[3,4-
d]pyrimidine-benzimidazoles for determination of binding mode. Ethidium bromide (EB) is one of the

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sensitive fluorescent dye that binds to DNA via intercalative mode. In EB displacement assay, any
molecule that binds to DNA via the same mode as EB will replace EB from the DNA helix and results
in a decrease in the fluorescent intensity of EB.DNA system [28]. The EB.DNA complex showed the
emission band at 606 nm. However, on subsequent addition of compounds 8, 12, 14 and 17 to the
EB.DNA complex caused the decrease in emission band at 606 nm. There were significant changes in
emission intensity of EB.DNA complex, suggesting that these compounds bind to DNA in
intercalative mode (Figure S47).
5
2.5. Fluorescence quenching with bovine serum albumin (BSA).
Fluorescence quenching experiment is an efficient approach for exploring the binding mechanism of
10 protein with ligand at molecular level [29]. The fluorescence measurements offer necessary
information about the environment around fluorophore. Generally, the quenching process occurs due
to a variety of molecular interactions viz., excited state reactions, molecular relocations, energy
transfer, ground state complex development and collision [30]. Moreover, the binding of drug to BSA
proteins may direct to loss or improvement of the biological properties of the original drug, or
15 necessitate the paths for drug transportation [31]. Hence, the interaction of BSA with pyrazolo[3,4-
d]pyrimidine-benzimidazole conjugates was studied by the fluorescence emission spectrum in the
presence of different concentrations of compounds (0–30 µM). The emission spectra of BSA were
recorded and the emission profile obtained at 298 K is represented in Figure 5. Bovine serum albumin
demonstrates a strong fluorescence emission peak at ~352 nm on excitation at 280 nm. The result
20 specifies that with increasing concentration of compounds 7, 8, 11, 12, 14 and 17, the fluorescence
intensity of BSA decreased regularly with an apparent quenching process. Moreover, upon the addition
of these compounds, there were slightly λem blue shifts (4 nm at 20 µM pyrazolo[3,4-d]pyrimidine-
benzimidazoles) of maximum emission wavelength, which signified non-covalent interactions via π–π
stacking between the aromatic rings of compounds 7, 8, 11, 12, 14, 17 and BSA with changes in
25 protein conformation [32].
Figure 5
3. Conclusion
In summary, a series of new 6-substituted-pyrazolo[3,4-d]pyrimidine with 4-(1H-benzimidazol-2-yl)-
phenylamine side chain at C4 position has been designed and synthesized as roscovitine analogues. A
30 selected group of 6 compounds was assessed for its anticancer activity over a panel of 60 human
cancer cell lines at 10 µΜ concentration. Most of the examined compounds, especially 8, 12, 14 and 17

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showed superior antitumor activities rather roscovitine over the majority of cancer cell lines with low
micromolar GI50 values. A SAR study has been made and revealed the crucial role of both 4-(1H-
benzimidazol-2-yl)-phenylamine and amines for attaining the best antineoplastic activity. Compound
8, with pyrrolidine group, was noticed to be the most potent and efficacious member. Compounds 7, 8,
11, 12, 14 and 17 showed significant inhibition of human topoisomerase IIα relaxation activity at 50
µM concentration. To explore the possible binding capability of the newly synthesized analogs
towards DNA, UV–vis and fluorescence techniques were used. Results from both studies predicted
that pyrazolo[3,4-d]pyrimidine interacted with ct-DNA through intercalation mode. In the current
study, the binding interaction of pyrazolo[3,4-d]pyrimidine with BSA has been studied by fluoresence
5
10 spectroscopic method. It has been observed that pyrazolo[3,4-d]pyrimidine derivatives quench
fluorescence of BSA with blue shift in maximum emission wavelength which signify the π-π
interactions between the compounds and BSA. These findings are valuable in understanding the
anticancer activity of this class of compounds as well as laying a foundation for the rational design of
novel, powerful agents for probing and targeting nucleic acids and proteins, which are expected to
15 provide an important insight into the field of DNA and protein interactions.
4. Experimental Section
4.1. Chemistry
All materials were obtained from commercial suppliers and used without further purification unless
otherwise noted. Melting points were determined in open capillaries and were uncorrected. Reactions
20 were monitored by thin-layer chromatography (TLC) carried out on silica gel plates (GF 254) using
UV light as visualizing agent. Column chromatography was performed with silica gel mesh size 60-
1
120. H NMR and 13C NMR spectra were recorded on Jeol-400 (1H, 400 MHz; 13C, 100 MHz)
spectrometer at ambient temperature, using CDCl3, and DMSO-d6 as solvents. Chemical shifts are
reported in parts per million (ppm) with TMS as an internal reference and J values are given in Hz.
25 Mass spectrometric data were recorded at Waters Micromass Q-Tof Micro. Elemental analysis was
done with Thermo Scientific (Flash 2000) analyzer. Hexane: ethylacetate and chloroform: methanol
were the adopted solvent systems. UV-vis studies were carried out on a Specord PC machines using
slit width of 1.0 nm and matched quartz cells. The fluorescence spectra were determined on a Varian
Cary Eclipse fluorescence spectrometer.
30 4.2. 5-Amino-1H-pyrazole-4-carbonitrile (1)

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Hydrazine (5 g, 0.156 mol) was carefully added into 2-(ethoxymethylene) malononitrile (8.64 g, 0.070
mol) in ethanol and heated on the steam-bath for 30 minutes. After completion of the reaction, white
precipitate was gradually appeared that kept in a refrigerator overnight. The product was then filtered
and washed with a small amount of cold ethanol to afford the desired product 5-amino-1H-pyrazole-4-
carbonitrile (1) as white solid; (3.05 g, 86%); mp 168-170 0C (Lit. mp 169-170 0C) [33].
5
4.3. 5-Amino-1H-pyrazole-4-carboxylic acid amide (2)
5-Amino-1H-pyrazole-4-carbonitrile (1) (5g, 0.046 mol)) was added to cooled concentrated sulphuric
0
acid (10 ml) with stirring so that the temperature did not rise above 20 C. The addition took about
one-half hour and solution was stirred at room temperature for 4 h. The acid solution was then poured
10 with stirring into ice and the solution was kept overnight in the refrigerator. The solid was then filtered
and washed with water to free of excess sulphuric acid to afford the desired product 5-amino-1H-
0
pyrazole-4-carboxylic acid amide (2) as white solid; (5.19 g, 89%); mp 220-222 C (Lit. mp 222-225
0C) [33].
4.4. 1,7-Dihydro-pyrazolo[3,4-d]pyrimidine-4,6-dione (3)
15 5-Amino-1H-pyrazole-4-carboxylic acid amide (2) (5g, 0.0396 mol) and urea (10 g, 0.1666 mol) were
heated together at 190 0C for 20 minutes. The clear solution went mushy and heating was continued for
0
another 20 minutes at 190 C until the mushy melt became too solid to stir. The resulting solid was
dissolved in hot dilute sodium hydroxide and the boiling basic solution was then carefully acidified
with hydrochloric acid. The solution was allowed to stand approximately ten minutes and was then
20 filtered to afford the desired product 1,7-dihydro-pyrazolo[3,4-d]pyrimidine-4,6-dione (3) as white
solid; (3.73 g, 62%); mp > 300 0C [33].
4.5. 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (4)
To 1,7-dihydro-pyrazolo[3,4-d]pyrimidine-4,6-dione (3) (1.233 mol), phosphorous oxychloride (2.84
mol) was added with stirring at 20 °C. After stirring for 15 min, the mixture was heated at 55 °C, and
25 triethylamine (2.53 mol) was added over a period of 1 h at a rate that maintains the internal
temperature below 65 °C, then mixture was slowly heated to an internal temperature of 85 °C for 10
min and then heated at 108-110 °C for 4 h to obtain a clear brown-yellow solution. The reaction
mixture was cooled to an internal temperature of 40 °C and warm water was added over a period of

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30-40 min. The solid was collected by filtration to afford pure 4,6-dichloro-1H-pyrazolo[3,4-
d]pyrimidine (4) as light yellow solid; (5.35 g, 87 %); mp: 173-175 oC.
4.6. 4-(1H-Benzimidazol-2-yl)-phenylamine (5)
A mixture of 4-aminobenzoic acid (5 g, 5.78 mmol) and o-phenylenediamine (3.9 g, 3.68 mmol) were
0
5
stirred in polyphosphoric acid (12.5 gm) at 200 C for 5 h. The reaction mixture was cooled and
poured into crushed ice. The precipitate was then stirred in cold water. Ammonium hydroxide solution
was added until the pH 7 was achieved. The resulting solid was filtered and washed several times with
methanol and column chromatographed on silica gel in ethylacetate: hexane (4:1) to afford the desired
o
1
product 4-(1H-benzimidazol-2-yl)-phenylamine (5) as white solid; yield: 82%; mp: 207-209 C; H
10 NMR (400 MHz, DMSO-d6): δ = 7.96 (d, 2H, J = 8.72 Hz, ArH), 7.55-7.51 (m, 2H, ArH), 7.16-7.14
13
(m, 2H, ArH), 6.75 (d, 2H, J = 8.24 Hz, ArH), 4.45 (bs, 2H, NH2); C NMR (100 MHz, CDCl3 +
DMSO-d6): δ = 152.2, 148.4, 127.5, 120.9, 118.6, 113.8 (ArC); MS (ESI), m/z: 210.2 (M++1).
4.7. [4-(1H-Benzimidazol-2-yl)-phenyl]-(6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amine (6)
To a solution of 4-(1H-benzimidazol-2-yl)-phenylamine (5) (1 g, 0.005 mol) in isopropyl alcohol (25
15 ml), 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (4) (0.99 g, 0.005 mol) was added and stirred at room
temperature for 24 h. After washing the crude solid with isopropyl alcohol, dried to obtain pure [4-
(1H-benzimidazol-2-yl)-phenyl]-(6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amine (6) as white
o
1
solid; yield: 79%; mp: > 300 C; H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 9.75 (s, 1H, ArH),
8.13-8.08 (m, 2H, ArH), 7.92 (d, 1H, J = 8.60 Hz, ArH), 7.72 (d, 1H, J = 8.28 Hz, ArH), 7.62 (bs, 1H,
20 NH), 7.15-7.13 (m, 2H, ArH), 6.66 (d, 2H, J = 8.72 Hz, ArH), 5.02 (s, 1H, NH); 13C NMR (100 MHz,
CDCl3 + DMSO-d6): δ = 147.6, 142.5, 132.2, 130.2, 129.3, 129.2, 124.0, 120.8 (ArC); MS (ESI), m/z:
362.7 (M++1); Anal. Calcd for C18H12ClN7: C, 59.76; H, 3.34; N, 27.10, Found: C, 59.83; H, 3.32; N,
27.14.
4.8. General procedure for the preparation of compounds 7-18
25 [4-(1H-Benzimidazol-2-yl)-phenyl]-(6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amine (6) (0.100 g,
0.2 mmol) was refluxed with various amines (0.7 mol) in the presence of potassium carbonate (0.057
g, 0.4 mol) in n-butanol (20 ml) for 24 h and reaction was monitored by TLC; crude solid was obtained
with evaporation of the solvent under vacuum that was purified by column chromatography using
chloroform: methanol as eluents to give pure compounds 7-18.

ACCEPTED MANUSCRIPT
4.8.1. [4-(1H-Benzimidazol-2-yl)-phenyl]-(6-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amine
(7)
White powder; yield: 77%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.08 (d, 2H, J
= 8.72 Hz, ArH), 7.85 (s, 1H, ArH), 7.82 (d, 2H, J = 8.72 Hz, ArH), 7.76 (d, 2H, J = 8.68 Hz, ArH),
7.50 (bs, 1H, NH), 6.75 (d, 2H, J = 8.72 Hz, ArH), 3.86 (t, 6H, J = 5.04 Hz, mor-CH2), 3.73-3.71 (m,
5
13
2H, mor-CH2); C NMR (100 MHz, CDCl3 + DMSO-d6): δ = 164.6, 163.8, 150.8, 142.6, 137.3,
127.1, 122.3, 120.8, 119.1, 114.1 (ArC), 66.0 (O-CH2), 63.0 (N-CH2); MS (ESI), m/z: 413.3 (M++1);
Anal. Calcd for C22H20N8O: C, 64.07; H, 4.89; N, 27.17, Found: C, 64.45; H, 4.65; N, 27.45.
4.8.2. [4-(1H-Benzimidazol-2-yl)-phenyl]-(6-pyrrolidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amine
10 (8)
White powder; yield: 69%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.01 (d, 1H, J
= 9.16 Hz, ArH), 7.98 (s, 1H, ArH), 7.88 (bs, 1H, NH), 7.86 (d, 1H, J = 8.68 Hz, ArH), 7.69 (d, 1H, J
= 5.04 Hz, ArH), 7.67 (d, 1H, J = 5.04 Hz, ArH), 7.18 (d, 1H, J = 3.20 Hz, ArH), 7.17 (d, 1H, J = 3.20
Hz, ArH), 7.00 (t, 2H, J = 8.72 Hz, ArH), 3.61-3.59 (m, 4H, pyrr-CH2), 1.99-1.94 (m, 4H, pyrr-CH2);
15 13C NMR (100 MHz, CDCl3 + DMSO-d6): δ = 163.9, 151.6, 138.7, 135.6, 135.2, 134.3, 133.2, 128.6,
127.5, 125.9, 122.7, 118.1, 116.4, 114.9, 114.7, 101.8 (ArC), 46.4 (N-CH2), 29.2 (CH2); MS (ESI),
m/z: 397.4 (M++1); Anal. Calcd for C22H20N8: C, 66.65; H, 5.08; N, 28.26, Found: C, 66.51; H, 5.36;
N, 28.31.
4.8.3. [4-(1H-Benzimidazol-2-yl)-phenyl]-(6-piperidin-1-yl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amine
20 (9)
o
1
White powder; yield: 72%; mp: > 300 C; H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.46 (s, 1H,
ArH), 8.31 (s, 1H, ArH), 8.12 (d, 1H, J = 8.72 Hz, ArH), 7.89 (d, 1H, J = 8.24 Hz, ArH), 7.70-7.66 (m,
1H, ArH), 7.61-7.59 (m, 2H, ArH), 7.22-7.19 (m, 1H, ArH), 7.03 (t, 1H, J = 8.24 Hz, ArH), 3.83-3.80
13
(m, 4H, pip-CH2), 1.71-1.62 (m, 6H, pip-CH2); C NMR (100 MHz, CDCl3 + DMSO-d6): δ = 151.6,
25 142.2, 135.7, 135.5, 128.4, 127.3, 125.8, 120.8, 114.4, 114.2, 108.5 (ArC), 46.5 (N-CH2), 26.2 (CH2),
25.2 (CH2); MS (ESI), m/z: 411.4 (M++1); Anal. Calcd. for C23H22N8: C, 67.30; H, 5.40; N, 27.30,
Found: C, 67.07; H, 5.65; N, 27.18.
4.8.4. [4-(1H-Benzimidazol-2-yl)-phenyl]-[6-(4-methyl-piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-
4-yl]-amine (10).
30 White powder; yield: 74%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.14 (d, 1H, J
= 8.68 Hz, ArH), 8.04 (s, 1H, ArH), 7.95 (bs, 1H, NH), 7.81 (d, 1H, J = 8.68 Hz, ArH), 7.64-7.60 (m,

ACCEPTED MANUSCRIPT
2H, ArH), 7.22 (d, 1H, J = 3.20 Hz, ArH), 7.20 (d, 1H, J = 3.20 Hz, ArH), 7.04 (t, 2H, J = 8.68 Hz,
ArH), 3.88 (t, 4H, J = 5.04 Hz, pip-CH2), 2.53 (t, 2H, J = 4.56 Hz, pip-CH2), 2.48 (t, 2H, J = 5.04 Hz,
13
pip-CH2), 2.30 (s, 3H, N-CH3); C NMR (100 MHz, CDCl3 + DMSO-d6): δ = 165.8, 158.5, 156.1,
151.6, 142.1, 135.6, 128.4, 127.3, 125.8, 114.4, 114.2, 108.5 (ArC), 46.5 (N-CH2), 40.9 (N-CH2), 13.4
(N-CH3); MS (ESI), m/z: 426.5 (M++1); Anal. Calcd for C23H23N9: C, 64.92; H, 5.45; N, 29.63,
Found: C, 64.74; H, 5.41; N, 29.75.
5
4.8.5. N6-(2-Amino-ethyl)-N4-[4-(1H-benzimidazol-2-yl)-phenyl]-1H-pyrazolo[3,4-d]pyrimidine-4,6-
diamine (11)
White powder; yield: 73%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.12 (d, 1H, J
10 = 8.68 Hz, ArH), 7.85 (bs, 1H, NH), 7.82-7.79 (m, 2H, ArH), 7.62-7.60 (m, 2H, ArH), 7.22-7.20 (m,
2H, ArH), 7.03 (t, 2H, J = 8.72 Hz, ArH), 3.64 (bs, 1H, NH), 3.53 (t, 2H, J = 4.12 Hz, N-CH2), 2.99 (t,
13
2H, J = 5.52 Hz, N-CH2); C NMR (100 MHz, CDCl3 + DMSO-d6): δ = 148.7, 144.7, 135.1, 131.5,
128.3, 125.2, 119.5, 114.7, 114.5, 113.3 (ArC), 52.3 (N-CH2), 42.6 (N-CH2); MS (ESI), m/z: 386.1
(M++1); Anal. Calcd for C20H19N9: C, 62.32; H, 4.97; N, 32.71, Found: C, 62.45; H, 4.93; N, 32.65.
15 4.8.6. 2-(4-{4-[4-(1H-Benzimidazol-2-yl)-phenylamino]-1H-pyrazolo[3,4-d]pyrimidin-6-yl}-piperazin-
1-yl)-ethanol (12)
White powder; yield: 72%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.14 (d, 1H, J
= 9.16 Hz, ArH), 7.95 (s, 1H, ArH), 7.85 (bs, 1H, NH), 7.80 (d, 1H, J = 8.68 Hz, ArH), 7.63-7.59 (m,
3H, ArH), 7.23-7.19 (m, 2H, ArH), 7.04 (t, 1H, J = 8.92 Hz, ArH), 3.88 (t, 4H, J = 4.56 Hz, N-CH2),
13
20 3.72 (t, 2H, J = 5.48 Hz, N-CH2), 3.67 (t, 2H, J = 5.48 Hz, N-CH2), 2.62-2.57 (m, 4H, N-CH2); C
NMR (100 MHz, CDCl3 + DMSO-d6): δ = 164.4, 163.5, 163.1, 151.4, 142.2, 135.7, 134.8, 130.3,
128.3, 127.2, 125.8, 115.2, 109.2, 108.5 (ArC), 66.1 (O-CH2), 46.4 (N-CH2), 45.4 (N-CH2), 43.0 (N-
CH2); MS (ESI), m/z: 456.1 (M++1); Anal. Calcd for C24H25N9O: C, 63.28; H, 5.53; N, 27.67, Found:
C, 63.49; H, 5.49; N, 27.75.
25 4.8.7.N4-[4-(1H-Benzimidazol-2-yl)-phenyl]-N6-(2-morpholin-4-yl-ethyl)-1H-pyrazolo[3,4-
d]pyrimidine-4,6-diamine (13).
White powder; yield: 70%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.43-8.23 (m,
1H, ArH), 8.10 (d, 2H, J = 8.68 Hz, ArH), 7.86 (s, 1H, NH), 7.66-7.58 (m, 3H, ArH), 7.20 (d, 1H, J =
3.20 Hz, ArH), 7.19 (d, 1H, J = 3.20 Hz, ArH), 7.04 (t, 1H, J = 8.24 Hz, ArH), 5.93 (bs, 1H, NH),

ACCEPTED MANUSCRIPT
13
3.71-3.55 (m, 8H, mor-CH2), 2.62 (t, 2H, J = 5.96 Hz, N-CH2), 2.57-2.52 (m, 2H, N-CH2); C NMR
(100 MHz, CDCl3 + DMSO-d6): δ = 165.3, 164.5, 163.7, 150.6, 137.2, 135.7, 135.1, 135.0, 128.4,
127.2, 125.3, 117.7, 114.8, 100.5 (ArC), 66.1 (O-CH2), 52.9 (N-CH2), 42.9 (N-CH2), 36.5 (N-CH2);
MS (ESI), m/z: 456.1 (M++1); Anal. Calcd for C24H25N9O: C, 63.28; H, 5.53; N, 27.67, Found: C,
63.29; H, 5.49; N, 27.69.
5
4.8.8.
N4-[4-(1H-Benzimidazol-2-yl)-phenyl]-N6-benzyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine
(14)
White powder; yield: 66%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.57 (bs, 1H,
NH), 8.03 (d, 1H, J = 1.84 Hz, ArH), 7.32 (d, 1H, J = 8.72 Hz, ArH), 7.26 (d, 3H, J = 7.32 Hz, ArH),
10 7.21-7.16 (m, 3H, ArH), 7.13 (d, 3H, J = 8.72 Hz, ArH), 7.01 (d, 3H, J = 5.92 Hz, ArH), 5.27 (s, 2H,
13
N-CH2); C NMR (100 MHz, CDCl3 + DMSO-d6): δ = 164.0, 163.8, 158.8, 156.4, 151.0, 137.9,
135.7, 135.3, 135.1, 134.2, 128.3, 127.2, 125.9, 121.8, 117.7, 116.1, 114.4, 114.2, 101.6 (ArC), 46.2
(N-CH2); MS (ESI), m/z: 433.1 (M++1); Anal. Calcd for C25H20N8: C, 69.43; H, 4.66; N, 25.91,
Found: C, 69.54; H, 4.62; N, 25.98.
15 4.8.9.2-{4-[4-(1H-Benzimidazol-2-yl)-phenylamino]-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino}-ethanol
(15)
White powder; yield: 78%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.12 (t, 3H, J
= Hz, ArH, NH), 8.02 (s, 1H, ArH), 7.80 (s, 2H, ArH), 7.63-7.61 (m, 1H, ArH), 7.50-7.45 (m, 1H,
ArH), 7.19-7.16 (m, 1H, ArH), 7.00 (d, 1H, J = 7.32 Hz, ArH), 6.31 (bs, 1H, NH), 3.75 (t, 2H, J = 4.56
13
20 Hz, O-CH2), 3.57 (t, 2H, J = 3.20 Hz, N-CH2); C NMR (100 MHz, CDCl3 + DMSO-d6): δ = 165.4,
163.6, 163.3, 151.3, 141.6, 135.1, 128.1, 127.0, 125.5, 121.0, 120.7, 114.1, 113.9, 108.2 (ArC), 60.5
(O-CH2), 46.2 (N-CH2); MS (ESI), m/z: 387.1 (M++1); Anal. Calcd for C20H18N8O: C, 62.17; H, 4.70;
N, 29.00, Found: C, 62.24; H, 4.66; N, 29.20.
4.8.10.N6-Allyl-N4-[4-(1H-benzimidazol-2-yl)-phenyl]-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (16)
25 White powder; yield: 69%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.13 (d, 1H, J
= 8.68 Hz, ArH), 8.01 (d, 1H, J = 8.72 Hz, ArH), 7.76 (t, 1H, J = 8.72 Hz, ArH), 7.64-7.61 (m, 2H,
ArH), 7.29-7.23 (m, 2H, ArH), 7.02 (d, 2H, J = 8.92 Hz, ArH), 6.62 (bs, 1H, NH), 6.02-5.93 (m, 1H,
13
allyl-CH), 5.29 (d, 2H, J = 18.32 Hz, allyl-CH2), 5.15 (d, 2H, J = 10.08 Hz, N-CH2); C NMR (100
MHz, CDCl3 + DMSO-d6): δ = 164.4, 150.5, 126.5, 121.8, 121.7, 119.2, 114.2, 114.0 (ArC), 53.9 (N-

ACCEPTED MANUSCRIPT
CH2); MS (ESI), m/z: 383.1 (M++1); Anal. Calcd for C21H18N8: C, 65.95; H, 4.74; N, 29.30, Found: C,
65.76; H, 4.71; N, 29.39.
4.8.11.
N4-[4-(1H-Benzimidazol-2-yl)-phenyl]-N6-(4-fluoro-phenyl)-1H-pyrazolo[3,4-d]pyrimidine-
4,6-diamine (17)
5
White powder; yield: 67%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.47 (bs, 1H,
NH), 8.27 (s, 2H, ArH), 8.22 (d, 1H, J = 8.72 Hz, ArH), 7.92 (d, 1H, J = 8.68 Hz, ArH), 7.87 (d, 1H, J
= 7.80 Hz, ArH), 7.69-7.66 (m, 4H, ArH), 7.29-7.27 (m, 1H, ArH), 7.09 (s, 1H, ArH), 7.05 (d, 2H, J =
13
8.68 Hz, ArH); C NMR (100 MHz, DMSO-d6): δ = 150.6, 139.8, 138.3, 138.1, 127.3, 124.4, 122.7,
122.5, 120.6, 120.5, 114.6, 114.8 (ArC); MS (ESI), m/z: 437.1 (M++1); Anal. Calcd for C24H17FN8: C,
10 66.05; H, 3.93; N, 25.67, Found: C, 66.41; H, 3.89; N, 25.61.
4.8.12.
N4-[4-(1H-Benzimidazol-2-yl)-phenyl]-N6-cyclohexyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-
diamine (18)
White powder; yield: 68%; mp: > 300 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 8.27 (bs, 1H,
NH), 8.16 (s, 1H, ArH), 8.11 (d, 1H, J = 8.72 Hz, ArH), 7.90 (d, 1H, J = 6.88 Hz, ArH), 7.84 (s, 1H,
15 ArH), 7.68-7.65 (m, 3H, ArH), 7.20-7.17 (m, 1H, ArH), 7.01 (t, 1H, J = 8.68 Hz, ArH), 5.54 (bs, 1H,
NH), 3.87-3.84 (m, 1H, CH), 2.06-2.04 (m, 2H, CH2), 1.86-1.60 (m, 4H, CH2), 1.39-1.08 (m, 4H,
13
CH2); C NMR (100 MHz, CDCl3 + DMSO-d6): δ = 151.6, 142.2, 135.3, 133.8, 130.8, 128.3, 127.2,
125.6, 115.7, 110.2, 108.5 (ArC), 52.8 (N-CH2), 46.5 (CH2), 43.0 (CH2), 36.4 (CH2); MS (ESI), m/z:
425.2 (M++1); Anal. Calcd for C24H24N8: C, 67.90; H, 5.70; N, 26.40, Found: C, 67.72; H, 5.50; N,
20 26.21.
4.9. Procedure for in vitro anticancer screening
The human tumor cell lines of cancer screening were grown in RPMI 1640 medium containing 5%
fetal bovine serum and 2 mM L-glutamine. Cells were inoculated in 96 well plates in 100 µL per well
at plating densities ranging from 5,000 to 40,000 cells/well that depends upon the doubling time of
°
25 individual cell lines. The microtiter plates were then incubated at 37 C, 95% air, 5% CO2, and 100%
relative humidity for 24 h. Two plates of each cell line were then fixed in situ with TCA. Experimental
drugs were dissolved in DMSO at 400-fold the desired final maximum test concentration and stored
frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was used and diluted
to two times the desired final maximum test concentration with complete medium containing 50 µg/ml
30 gentamicin. Additional four, 10-fold or ½ log serial dilutions were made to give total of five drug

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concentrations plus control. Aliquots of 100 µL of these drug dilutions were added to the appropriate
°
microtiter wells. Following the addition, plates were incubated at 37 C, 5% CO2, 95% air, and 100%
relative humidity for 48 h. For adherent cells, the assay was terminated by the addition of cold TCA.
Cells were fixed in situ by the addition of 50 µL of cold 50% (w/v) TCA and incubated at 4 °C for 60
min. The plates were washed five times with tap water by discarding the supernatant and air dried.
Sulphorhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well
followed by incubation of plates for 10 min at room temperature. After staining, unbound dye was
removed by washing five times with 1% acetic acid and the plates were air dried before subsequent
solubilization with 10 mM trizma base. The absorbance was read at a wavelength of 515 nm on an
5
10 automated plate reader. With seven absorbance measurements [time zero (Tz), control growth (C), and
test growth in the presence of drug at five concentration levels (Ti)], percentage growth was calculated
at each of the drug concentration levels. Percentage growth inhibition was calculated as:
[(Ti -Tz)/(C - Tz)] × 100 for concentrations for which Ti ≥ Tz; [(Ti -Tz)/Tz] × 100 for
concentrations for which Ti < Tz.
15 Three dose response parameters were calculated for each experimental agent. Growth inhibition of
50% (GI50) was calculated from [(Ti -Tz)/(C - Tz)] × 100 = 50. Drug concentration resulting in total
growth inhibition (TGI) was calculated from Ti = Tz. LC50 was calculated from [(Ti-Tz)/Tz]×100 = 50.
4.10. Relaxation Assay of Human Topoisomerase IIα.
Relaxation of negatively supercoiled plasmid DNA by human topoisomerase IIα was assayed in 20 µL
20 of reaction buffer (50 mM Tris-Cl, pH 8.0, 120 mM KCl, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM
dithiothreitol) containing 500 ng of supercoiled pHOT1 plasmid DNA and 1 unit of Topo IIα enzyme.
After incubation at 37 °C for 30 min, the reactions were stopped by the addition of 5 ml of 50%
glycerol, 50 mM EDTA, and 0.5% (v/v) bromophenol blue. After electrophoresis in 1 % agarose gel
with 1XTBE buffer (1 litre of 5X stock solution-54 g of tris base, 27.5 g of boric acid, 20 ml of 0.5 M
25 EDTA pH 8.1), the DNA was stained with ethidium bromide and photographed over UV light.
4.11. Procedure for DNA binding studies
4.11.1. Materials and methods
The stock solution of ct-DNA (Sigma Chemical Co., USA) was prepared by dissolving an appropriate
30 amount of ct-DNA in Millipore water. The purity of ct-DNA was verified by monitoring the ratio of

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the absorbance at 260 and 280 nm, and the ratio of A260/A280 was 1.82, indicating that ct-DNA was
sufficiently free of protein contamination. The concentration of ct-DNA in terms of the nucleotide
phosphate (i.e. nucleotide) was determined to be 2.56 × 10-3 molL-1 by UV-vis absorption at 260 nm
using a molar absorption coefficient of ε260 = 6600 L mol-1cm-1(expressed as the molarity of phosphate
groups). All stock solutions were diluted to the required concentrations with phosphate buffer (pH
7.4). All other reagents were of analytical reagent grade, and ultrapure water was used throughout the
experiment. All stock solutions were stored at 0–4 0C.
5
4.11.2. UV-vis absorption spectra
All the spectra were recorded at ambient temperature (300 K). UV-vis spectra were recorded on
10 Shimadzu-2400 PC spectrometer with 1 cm cuvette. Any background buffer signal was electronically
subtracted. Absorption titrations were performed with constant ligand concentration of 20 µM and
increasing ct-DNA concentration (up to 15 µM). The control experiment was done by adding equal
volumes of buffer solution instead of DNA solution to the same concentration of compound. The
control experiment shows no significant change in the absorption spectra of the compound.
15 4.11.3. Fluorescence spectra
Emission spectra were recorded with Varian Cary Eclipse fluorescence spectrometer. Fluorescence
titration spectra were obtained with a constant ligand concentration (20 µM) and increasing amount of
ct-DNA (up to 17 µM).
4.11.4. Binding constants
20 Binding constant K for the ligand–DNA complex was estimated from absorption and fluorescence
titration data using the Benesi–Hildebrand equation-1 that suggested strong interactions with ct-DNA.
1/(Af − Aobs) = 1/(Af − Afc) + {1/[K(Af − Afc)]}[ligand] ------ 1
where K is the binding constant, Af is the absorbance of the free host, Aobs is the absorbance observed,
and Afc is the absorbance at saturation.
25 K was determined from the ratio of intercept to slope obtained from the linear fit of the plot of 1/(Af −
Aobs) versus 1/[DNA], respectively. The control experiment was done by adding equal volumes of
buffer solution instead of DNA solution to the same concentration of the compound. No significant
change in the emission spectra was observed in control experiment.

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4.11.5. Competitive binding study of compounds with ct-DNA in the presence of ethidium bromide
Competitive binding of compounds with DNA in the presence of intercalator ethidium bromide were
studied. Ethidium bromide was dissolved in Milli-Q water to prepare a stock solution with 1 mM
strength and diluted immediately before use. Fluorescence spectra were taken in the presence of
ethidium bromide while increasing the amount of ct-DNA (0 to 20 µM) and using an excitation
wavelength of 480 nm. Compounds were added with increasing concentration of 2 µM (up to 50 µM),
mixed thoroughly after each addition and the emission spectrum was recorded with λex = 480 nm.
5
4.12. Procedure for BSA screening
4.12.1. Materials and methods
10 The stock solution of BSA (Sigma Chemical Co., USA) was prepared by dissolving an appropriate
amount of solid BSA in 0.1 M phosphate buffer at pH 7.4 and stored at 0−4 °C in the dark. Stock
solutions of compounds 7-18 (10−3 mol L−1) were prepared in DMSO. All stock solutions were diluted
to the required concentrations with phosphate buffer (pH 7.4). All other reagents were of analytical
reagent grade.
15 4.12.2. Fluorescence spectra
Emission spectra were recorded with Varian Cary Eclipse fluorescence spectrometer. The excitation
and emission wavelengths for BSA were 280 nm and 351 nm with a slit width of 10 nm. Very dilute
solutions of BSA and compounds 7-18 were used to avoid inner filter effect. The titration experiments
were performed by varying the concentration of compounds 7-18 and keeping the BSA concentration
20 (10 µM).
ACKNOWLEDGMENTS
KP thanks the Department of Science and Technology, New Delhi (EMR/2014/000669). PS is grateful
for a DST/Inspire Fellowship (Fellow Code-IF110542). NIH, Bethesda, USA for anticancer activities
and SAI labs, Thapar University are also acknowledged.
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Figures Captions
Figure 1. Rational design of the target compounds
Figure 2. Inhibition of relaxation activity of Topo IIα in the presence of compounds 7, 8, 11, 12, 14
and 17. Ethidium bromide stained agarose gel showing: lane 1, pHOT1 plasmid DNA (C); lane 2,
relaxation of plasmid DNA by Topo IIα (EC); lanes 3–7 for compound 7 (A), lanes 8-12 for compound
8 (A) and lanes 13-17 for compound 11 (A); lanes 3–7 for compound 12 (B), lanes 8-12 for compound
14 (B) and lanes 13-17 for compound 17 (B) inhibition of relaxation of plasmid DNA by Topo IIα in
the presence of 10, 25, 50, 75 and 100 µM of compounds, respectively. Lane 18 indicated the
etoposide at 25 µM as positive control (PC).
5
10 Figure 3. UV-Vis spectral changes of compounds 7, 8, 11, 12, 14 and 17 at the concentration of 10
µM upon addition of ct-DNA (0 µΜ-20 µΜ) in phosphate buffer (10 mΜ, pH 7.4).
Figure 4. Fluorescence spectral changes of compounds 7, 8, 11, 12, 14 and 17 (λex = 300 nm) at the
concentration of 10 µM upon addition of ct-DNA (0 µΜ - 20 µΜ) in phosphate buffer (10 mΜ, pH
7.4).
15 Figure 5. Emission spectral changes of BSA at concentration of 10 µM upon addition of compounds 7,
8, 11, 12, 14 and 17 (0 µΜ-30 µΜ) in phosphate buffer (10 mΜ, pH 7.4).
Table 1. Overview of the preliminary anticancer assay at single dose concentration of 10 µM
Table 2. Compounds 8, 12, 14, 17 and roscovitine having median growth inhibitory (GI50, µM), total
growth inhibitory (TGI, µM) and median lethal concentrations (LC50, µM) of in vitro subpanel tumor
20 cell lines.
Scheme 1. Synthetic route for the preparation of target compounds 7-18.
Fragment Replacement
N
(Increase in conjugation)
N
HN
H
N
N
HN
N
HO
N
N
N
N
H
Isosteric replacement
N
R2R1N
N
H
lipophilic group insertion
lipophilic group insertion
(Primary and secondary amines)
Figure 1. Rational design of the target compounds
25
A

ACCEPTED MANUSCRIPT
B
5
Figure 2. Inhibition of relaxation activity of Topo IIα in the presence of compounds 7, 8, 11, 12, 14
and 17. Ethidium bromide stained agarose gel showing: lane 1, pHOT1 plasmid DNA (C); lane 2,
relaxation of plasmid DNA by Topo IIα (EC); lanes 3–7 for compound 7 (A), lanes 8-12 for compound
8 (A) and lanes 13-17 for compound 11 (A); lanes 3–7 for compound 12 (B), lanes 8-12 for compound
14 (B) and lanes 13-17 for compound 17 (B) inhibition of relaxation of plasmid DNA by Topo IIα in
10 the presence of 10, 25, 50, 75 and 100 µM of compounds, respectively. Lane 18 indicated the
etoposide at 25 µM as positive control (PC).

ACCEPTED MANUSCRIPT
Figure 3. UV-vis spectral changes of compounds 7, 8, 11, 12, 14 and 17 at concentration of 10 µM
upon addition of ct-DNA (0 µΜ-20 µΜ) in phosphate buffer (10 mΜ, pH 7.4).

ACCEPTED MANUSCRIPT
Figure 4. Fluorescence spectral changes of compounds 7, 8, 11, 12, 14 and 17 (λex = 300 nm) at the
concentration of 10 µM upon addition of ct-DNA (0 µΜ - 20 µΜ) in phosphate buffer (10 mΜ, pH
7.4).

ACCEPTED MANUSCRIPT
Figure 5. Emission spectral changes of BSA at concentration of 10 µM upon addition of compounds 7,
8, 11, 12, 14 and 17 (0 µΜ-30 µΜ) in phosphate buffer (10 mΜ, pH 7.4).
Table 1. Overview of the preliminary anticancer assay at single dose concentration of 10 µM
60 cell lines assay in single dose (10 µM)
Compound
No.
Mean
Growth %
Range of growth The most sensitive
cell line
Positive
cytostatic
effecta
Positive
cytotoxic
effectb
No. of
sensitive cell
lines
82.24
50.10 to104.00
CCRF-CEM
(Leukaemia)
1/54
0/54
1/54
7
-32.52
61.83
-77.92
-8.30
-92.36 to 98.93
786.0 (renal cancer)
8/55
11/54
1/54
43/55
6/54
51/55
17/54
54/54
59/59
8
-55.40 to 124.82 U251 (CNS cancer)
-99.72 to 106.08 U251 (CNS cancer)
11
12
14
53/54
31/59
-96.36 to 41.08
SK-MEL-5
(Melanoma)
LOX-IMVI
(Melanoma)
28/59
57.44
-64.57 to 107.39
11/55
6/55
17/55
17

ACCEPTED MANUSCRIPT
a The ratio between number of cell lines with percent growth from 0-50 and total number of cell lines. b The ratio between
number of cell lines with percent growth of <0 and total number of cell lines.
Table 2. Compounds 8, 12, 14, 17 and roscovitine having median growth inhibitory (GI50, µM), total
growth inhibitory (TGI, µM) and median lethal concentrations (LC50, µM) of in vitro subpanel tumor
cell lines.
5
Compounds
Activity
I
II
III
IV
V
VI
VII
VIII
IX
MG-
MIDa
1.30
3.32
10.5
1.43
2.99
6.06
2.38
7.87
37.3
2.18
5.27
17.7
24.5
81.8
96.0
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
2.38 1.08 2.32 0.86 0.67 1.10 1.22 1.37 0.73
6.70 3.17 5.88 2.01 1.53 2.73 2.67 3.14 2.08
31.8 11.5 2.23 4.17 4.32 6.85 7.04 20.6 6.35
0.44 1.57 0.72 1.47 1.81 1.84 1.76 1.49 1.80
1.22 3.23 2.12 3.26 3.41 3.59 3.26 2.96 3.83
8
12
b
6.26 5.71 5.72 5.83 6.09 5.85 5.97 7.02
2.67 2.54 2.00 2.96 1.85 2.52 2.30 2.56 2.04
6.43 8.99 5.12 16.7 4.29 7.83 6.16 9.43 5.85
14
17
b
55.3 34.7 28.1 19.3 41.8 31.2 41.8 46.3
2.62 2.11 1.78 2.06 2.80 2.49 1.84 1.95 1.99
6.33 5.37 3.83 5.08 3.74 6.77 7.35 4.14 4.80
b
32.5 6.80 20.4 6.92 28.9 18.2 9.22 19.0
37.5 23.6 24.8 24.7 14.8 29.3 24.9 17.9 22.7
b
Roscovitine
84.8 84.9 82.2 57.6 93.8 97.0 81.5 73.0
b
b
b
b
b
b
b
97.1
94.9
I, leukaemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal
cancer; VIII, prostate cancer; IX, breast cancer. a Full panel mean-graph midpoint (µM). b Compounds showed values >100
µM.
10