Synthesis of novel N-9 substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine derivatives as inducers of apoptosis in MCF-7 breast cancer cells

Article abstract of DOI:10.1039/c5ra23242b

A series of N-9 substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine derivatives (PP05-PP21) were prepared and evaluated for their anticancer activity against a panel of human cancer cell lines. Evaluation of results revealed that some of the synthesized compounds exhibited promising anticancer activity against the examined cancer cell lines. The structure-activity relationship (SAR) studies in the present work revealed that simple N-9 alkyl substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purines are potent anticancer agents. Among all the compounds, PP17 (9-sec-butyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine) showed good inhibitory activity against MCF-7 cells. Cell cycle analysis of the compound suggested that induces G2/M phase arrest. Biochemical experiments showed that PP17 significantly induced MCF-7 cell apoptosis. Therefore, compound PP17 with a potent in vitro anticancer activity can serve as a promising lead compound for further study.

Full text of DOI:10.1039/c5ra23242b

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Synthesis of novel N-9 substituted 6-(4-(4-propoxyphenyl)piperazin-1-
yl)-9H-purine derivatives as inducers of apoptosis in MCF-7 breast
cancer cells †
Manjunath G. Sunagar,a Supreet Gaonkar,a Santosh G. Sunagar,b Narahari Deshapande,a
Ningaraddi S. Belavagi,a Imtiyaz Ahmed M. Khazi,*a
Abstract:
A series of N-9 substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine derivatives (PP05-PP21) were
prepared and evaluated for their anticancer activity against a panel of human cancer cell lines. Evaluation results
revealed that some of the synthesised compounds exhibited promising anticancer activity against the examined
cancer cell lines. The structure-activity relationship (SAR) studies in the present work revealed that simple N-9
alkyl substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purines are potent anticancer agents. Among all the
compounds, PP17 (9-sec-butyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine) showed good inhibitory activity
against MCF-7 cells. Cell cycle analysis of the compound suggested that it is inducing G2/M phase arrest.
Biochemical experiments showed that PP17 significantly induced MCF-7 cell apoptosis. Therefore, compound
PP17 with a potent in vitro anticancer activity can serve as a promising lead compound for further study.
aDepartment of Chemistry, Karnatak University,
Dharwad 580003, Karnataka, India.
Fax: +91-836-2771275,
Tel: +91-836-2215286 Extn 29;
E-mail: drimkorgchem@gmail.com
bBio-X Institutes,
Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education),
Shanghai Jiao Tong University, Shanghai 200240, P.R.China.
E-mail: santosh.sunagar@gmail.com
†Electronic supplementary information (ESI) available.

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Introduction
Purine is the most widely distributed and abundantly available nitrogen heterocyclic system in nature. Both
naturally occurring and synthetic purines have been found to exhibit wide range of biological activities.1 The
diversity of purine libraries against some biological targets has contributed for the development of new therapeutic
agents. In particular, substituted purine derivatives are found in current application as antivirals, interferon inducers,
antimycobacterials, CRH-R1 modulators and inhibitors of Hsp90, leukotriene A4 hydrolase, sulfotransferase,
phosphodiesterases, kinases.2,3 In addition to this specifically functionalised purines have found to treat cancer,
which is one of the most common diseases and a leading cause of human death in the world. Biological target
oriented synthesis has been successful in the case of purine skeleton, in view of the molecular diversity that can be
introduced at C-2, C-6 and C-7. The resulting molecular libraries will bear a close structural resemblance to
adenine, guanine and hypoxanthines. This is reflected in many purines being employed as clinically accepted drugs
in cancer chemotherapy. Some of the purine based anticancer drugs approved by FDA are Idelalisib, Nelarabine,
Clofarabine, Mercaptopurine (Fig.1).4-8
F
CH3
NH2
O
O
N
N
N
N
S
N
N
H
N
N
N
N
Cl
N
NH2
N
HO
HO
F
OH
O
NH
O
N
H
N
N
H
N
OH
OH
N
N
Mercaptopurine
Nelarabine
Clofarabine
Idelalisib
Fig 1. FDA approved purine based anticancer drugs.
Purine based anticancer drugs are nucleoside analogs and they affect the intrinsic structural features of
DNA, leading to stalled replication forks and chain termination.9,10 These drugs act by converting into free
nucleosides resistant to deamination by the enzyme adenosine deaminase (ADA) or by disrupting nucleotide
metabolism by incorporation into DNA or by inhibition of ribonucleotide reductase (RnR).11,12
In substituted purines C-6, N-9 are the most widely exploited anchoring points and they are commercially
available.13,14 Substitution reactions of 6-halogenopurines with different nucleophiles, have been widely exploited
in drug discovery.15 The SNAr reactions between 6-halogenopurine nucleosides with nitrogen, oxygen and sulphur
nucleophiles is well established in the literature.16 Alkylation of purines with different alkyls halides results in the
formation of regioisomeric mixtures of N-7 and N-9 alkylpurines but the desired N-9 compound is normally the
major product. Besides the formation of N-9 as a major isomer, the N-7 isomers and other alkylation products are
also observed.17
Purines are excellent scaffold for the synthesis of significant molecules in biology and the wide diversity of
the chemistries that are possible with this heterocycle. Further, the phenyl piperazine and its analogues are the
important building blocks in drug discovery, across a number of different therapeutic areas including anticancer
therapy.18 In particular, the p-propoxyphenyl piperazine moiety having three rotating bonds and nucleophilic centres

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with conformational flexibility has been a pharmacophore in a number of biologically active compounds. These
phenyl piperazine scaffolds are reported to be used for inhibiting tumor metastasis in cancers like liver cancer,
mammary cancer, ovary cancer, gastric cancer, colon cancer, lung cancer or melanoma etc.19-21 Mechanistic studies
of phenyl piperazine analogues have showed that they inhibit microtubule synthesis, cell cycle progression,
angiogenesis, and destroy the tumor cells through induction of apoptosis.22,23 In the light of this, we have
synthesised novel series of N-9 substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine derivatives. These
molecules were evaluated for their cytotoxicity against selected human cancer cell lines, and were found to exhibit
significant in vitro anticancer activity.
Result and Discussion
Chemistry
The synthesis of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) was carried out as shown in
Scheme 1 & 2. Commercially available 1-(4-(4-hydroxyphenyl)piperazin-1-yl)ethanone (PP01) was treated with 1-
bromo propane in the presence of a base to obtain 1-(4-(4-propoxyphenyl)piperazin-1-yl)ethanone (PP02). This
intermediate (PP02) was de-acetylated by using HCl (4N) to obtain 1-(4-propoxyphenyl)piperazine (PP03). Finally,
we achieved the synthesis of key intermediate 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) by reacting
the intermediate PP03 with 6-chloro purine in presence of a base. The reaction of key intermediate (PP04) with
different alkyl/aryl halides produced N-9 substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purines as shown in
Scheme 2.
OH
O
O
a
b
N
N
N
N
N
N
H
O
O
PP01
PP03
PP02
Scheme 01 Synthesis of 1-(4-propoxyphenyl)piperazine (PP03); reagents and conditions: (a) anhyd. K2CO3, DMF,
120 oC, 12 h; (b) 4N HCl, 80 oC, 3 h.

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O
O
O
N
Cl
N
b
N
a
N
N
N
N
N
N
H
N
N
N
R1
N
H
N
N
6-chloro purine
N
H
N
N
PP03
PP04
R1 =
O
O
O
H3C
O
O
O
PP10
PP17
I
PP09
PP16
PP18
PP05
PP19
CF3
Cl
Scheme 02 Synthesis of 9-substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine; reagents and conditions:
(a) triethyl amine (TEA), n-butanol, 110 0C,12 h; (b) R1-Br, K2CO3, DMF, RT, 10 h.
The key intermediate (PP04) obtained in the previous steps was treated with ethyl 2-bromoacetate in
presence of base to afford ethyl 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetate (PP06), which on
hydrolysis gave the acid 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetic acid (PP07). The
intermediate (PP06) on treating with hydrazine hydrate gave the hydrazide (PP08). Finally, the hydrazide (PP08)
reacted with different substituted aldehydes in ethanol to give the substituted (E)-N'-benzylidene-2-(6-(4-(4-
propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazides as shown in Scheme 3.

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O
O
O
N
N
N
N
N
N
a
b
N
N
N
N
N
N
N
N
H
N
N
N
N
O
O
OH
O
PP07
PP04
PP06
c
O
O
N
N
N
N
PP11; R5= -N02 & R2, R3, R4= H
PP12; R5= -H, R4= -F & R2, R3 =H
PP13; R5= -H, R4= -Cl & R2, R3 =H
PP14; R5= -H, R4= -CF3 & R2, R3 =H
PP15; R5= -H, R4= -OMe & R2, R3 =H
PP20; R2= -OH, R5= -OMe & R3, R4 =H
PP21; R3= -Cl, R4= -F &R2, R5 =H
d
N
N
N
N
N
N
N
N
O
O
NH
R2
NH
N
H2N
R5
R4
PP08
R3
Scheme 03 Synthesis of substituted (E)-N'-benzylidene-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-
yl)acetohydrazide ; reagents and conditions: (a) ethyl 2-bromoacetate, K2CO3, DMF, RT, 12 h; (b) aq. NaOH, 70-80
0 C, 2-3 h; (c) N2H4.H2O, ethanol, reflux, 4 h; (d) substituted aldehydes, ethanol, cat.CH3COOH, 2-3 h.
Biology
In vitro anticancer activity
To evaluate the effects of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine derivatives on the growth of
human cancer cells, the growth inhibitory potential was evaluated using SRB assay in three human cancer cell lines
HeLa (cervix), HCT-15 (colon) and MCF-7 (breast). Initially, all the 17 compounds (PP05-PP21) were screened at
10, 20, 40, 80 µg/mL. Among all the screened compounds, only PP09 and PP17 exhibited significant in vitro
antitumor activity against the tested MCF-7 human cancer cell line at this concentration. Adriamycin (ADR) was
used as the reference drug. The results are summarized in Table 1 & 2. PP09 and PP17 have not shown
considerable percentage control growth in HeLa and HCT-15, whereas both the compounds have shown good
percentage control growth to standard Adriamycin in MCF-7 as shown in Fig. 2, 3 and 4.

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The structure–activity relationship (SAR) studies in the present investigation revealed that 4-
propoxyphenyl piperazine moiety is essential for the optimal anticancer activity. This result prompted us to have
variations at N-9 position keeping 4-propoxyphenyl piperazine constant at the C-6 position with a goal to optimize
anticancer activity. The Schiff bases of acetohydrazides at N-9 position of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-
9H-purine (PP11-15, PP20-21) displayed no activity in terms of both GI50 and % control growth, while the
replacement of Schiff bases at N-9 with one carbon spacer carbonyl attached to aryl and hetero-aryl groups (PP05,
PP10, PP16 & PP18-19) resulted in the moderate activity compared to standard Adriamycin.
Finally, the replacement at N-9 by simple alkyl chains (PP09 & PP17) afforded the molecule with potent
anticancer activity. Interestingly, compound PP17 having iso butyl group at N-9 of purine showed good % control
growth ranging from 29-35 % with GI50 <10 µg/mL. Further introduction of larger alkyl chains such as 2-ethyl
hexyl resulted in the reduction of activity.
Table 1. In vitro cytotoxic effect of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine derivatives (PP05-PP21)
against panel of human cancer cell lines.
% Control Growth (Average Value)*
Compo
-unds
Drug Concentrations (µg/mL)
HCT-15
HeLa
MCF-7
40
10
20
40
80
10
20
40
80
10
20
80
PP05
PP06
PP07
PP08
PP09
PP10
PP11
PP12
PP13
PP14
PP15
PP16
PP17
PP18
PP19
PP20
PP21
ADR
100
96.8
100
100
78.7
100
99.6
100
100
100
100
100
100
99.7
100
100
99.7
-73.4
100
94.5
100
100
70.8
100
99.1
100
100
100
100
100
100
99.1
100
100
98.8
-75.9
100
92.7
100
100
51.9
100
91.7
100
100
100
100
100
100
98.1
100
100
96.7
-79
100
90.7
100
100
38.1
100
80.1
100
100
100
100
100
100
97.0
100
100
94.4
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
99.3
100
100
100
100
100
100
99.6
100
100
100
100
100
100
100
100
100
98.7
100
100
100
100
100
100
96.3
100
100
98.9
100
100
100
100
100
100
98.0
100
100
100
100
100
100
93.8
100
100
99.1
98.6
100
97.4
100
100
100
97.3
97.5
99.1
104
100
58.6
100
95.9
101
102
97.6
97.8
95.0
35.0
95.1
95.6
88.9
85.4
95.7
98.0
99.0
93.7
44.6
100
84.6
92.9
96.4
94.3
93.7
90.2
31.0
92.9
93.8
86.7
82.0
93.6
95.1
95.2
89.9
35.4
100
81.9
92.7
97.8
93.7
96.1
88.1
27.5
90.5
91.5
83.4
79.2
91.9
93.3
96.4
91.6
32.5
100
79.4
98.1
98.4
95.4
100.8
89.1
29.8
88.4
88.3
81.2
75.6
-46.1
-80.6 -21.8 -25.2 -27.6 -32.1 -05.3 -06.3 -15.9
*Average values of triplicate experiments (n=3).

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Table 2. The LC50, TGI and GI50 values of compounds PP05-PP21 as determined by using the SRB Assay.
Compounds
Drug concentrations calculated from graph (µg/mL)
HCT-15
HeLa
TGI
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
<10
MCF-7
TGI
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
<10
LC50
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
36.9
GI50
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
<10
LC50
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
77.4
TGI
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
29.7
GI50
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
<10
LC50
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
89.6
GI50
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
<10
>80
>80
>80
>80
<10
PP05
PP06
PP07
PP08
PP09
PP10
PP11
PP12
PP13
PP14
PP15
PP16
PP17
PP18
PP19
PP20
PP21
ADR
LC50 = Conncentration of drug causing 50% cell kill
GI50 = Concentration of drug causing 50% inhibition of cell growth
TGI = Concentration of drug causing total inhibition of cell growth
ADR = Adriamycin, Positive control
FFig 2. Growth Curve: Human Cervix Cancer Cell Line HeLa Fig 3. Growth Curve: Human Colon Cancer Cell Line HCT-15

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Fig 4. Growth Curve: Human Breast Cancer Cell Line MCF-7
Cell morphology
The effect of PP09 and PP17 on MCF-7 cell morphology was determined by using Microscopy. As shown
in Fig.5 cell morphology remained consistent in control and DMSO treated. However, the concentrations of PP09
up to 40 µg/mL and PP17 up to 10 µg/mL resulted in spherical cells and exhibited reduced adhesion between the
cells. No surviving cells were observed at concentrations exceeding 160 µg/mL in PP09 and 80 µg/mL in PP17.
Fig 5. Effect of PP09 and PP17 on MCF-7 cells morphology.

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Effect on cell cycle arrest
MCF-7 cells were exposed to increasing concentrations of PP09 & PP17 and cell apoptosis was determined using PI
labelled flow cytometry (Fig. 6 & 7). The results of the cell cycle analysis of MCF-7 cells treated with PP09 &
PP17 are summarized in Fig. 8. Compared to the control cells, the percentage of cells in the S and G2/M phase were
increased in the cells incubated with PP09 & PP17. Meanwhile, the proportion of cells in the G0/G1 phase showed
no obvious change. This data suggest that both the compounds may induce G2/M phase arrest in the cell cycle.
Whereas cell cycle arrest was abolished in case of PP17 at 40 µg/mL, as a result less number of cells were observed
in G2/M phase, and more number of cells were observed in G1 phase when compared to other two concentrations.
Thus, doses required for PP09 & PP17 to induce apoptosis were often higher than those that induced growth
inhibition and cell cycle arrest. The ability of certain small molecules to control cell cycle machinery within the S
and G2/M phase has provided stimulating new openings and hopes of developing new types of drugs against cancer.
Fig 6. Flow cytometric analysis in MCF-7 breast cancer cell lines after treatment with compound PP09 at 40 µg/mL,
80 µg/mL & 160 µg/mL concentrations for 24 h. Adriamycin (ADR) was employed as the positive control.

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Fig 7. Flow cytometric analysis in MCF-7 breast cancer cell lines after treatment with compound PP17 at 10 µg/mL,
20 µg/mL & 40 µg/mL concentrations for 24 h. Adriamycin (ADR) was employed as the positive control.
Fig 8. Percentage of cells in different phases was shown using the flow cytometer data on MCF-7 cells treated with
different concentrations of PP09, PP17 and Adriamycin (ADR) for 24 h.
Analysis of apoptosis by Western blotting
It is well studied that the cell cycle arrest at the G2/M phase is shown to induce cellular apoptosis. Hence,
to further confirm that PP09 & PP17 induced apoptosis and cell cycle arrest, the expressions of key apoptosis-
related protein p53 were detected by western blot assay. This protein plays a central role in the execution-phase of
cell apoptosis and cell cycle arrest. As shown in Fig. 9, treatment with PP09 & PP17 for 24 h significantly increased
the level of p53 in a dose-dependent manner compared with the control group. These findings corresponded with the
activation of apoptosis or cell cycle arrest in MCF-7 cells.

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Fig 9. Western blot analysis of compounds PP09, PP17 and Adriamycin in MCF-7 cell lines on p53 protein
expression. Cells were treated with compounds PP09, PP17 and Adriamycin (ADR) for 24 h. p53 protein levels
were examined in western blot analysis.
Conclusions
In summary, we have synthesised a series of novel N-9 substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-
9H-purine derivatives and tested for their in vitro anticancer activities against different human cancer cell lines.
Among all the synthesised compounds only the compounds substituted with simple alkyl groups at N-9 position of
purine moiety (PP09 and PP17) have shown selective anticancer activity in MCF-7 cell line. The SAR
investigations of present work disclosed that simple N-9 alkyl substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-
9H-purines are potent anticancer agents than bulky N-9 substituted groups. Flow cytometric study results revealed
that compounds PP09 and PP17 caused cell cycle arrest in the G2/M phase. Moreover, p53 expression results
suggest that they caused cell death by inducing apoptosis. Based on these results, it is evident that PP17 can serve as
a lead compound for further study.
Experimental
Chemistry
Materials and Methods. All reagents were of analytical grade and were used directly. Solvents were purified and
stored according to standard procedures. Thin-layer chromatography (TLC) was performed on silica gel plates (60
F254; Merck) and visualization was done by UV light or iodine indicators. Column chromatography was performed

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using silica (60–120 mesh size; Merck). Melting points were determined in open capillaries and are uncorrected. 1H
and 13C NMR were recorded on Bruker 400 MHz and 100 MHz FT NMR spectrometer respectively, in CDCl3 and
DMSO-d6 by using TMS as internal standard. Chemical shifts are expressed in parts per million (δ in ppm)
downfield from internal TMS and coupling constants are expressed in Hz. 1H NMR spectroscopic data are reported
in the order: multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet). Liquid Chromatography-Mass Spectrometry
(LC/MS) was carried out on a LC/MS-1290 series Agilent operating in positive and negative ion electro spray
mode, employing a 75 × 4.6 mm C18 column. Cell cycle analysis was carried out in BD FACSCalibur Flow
cytometer using Cell Quest Pro software (BD Bioscience).
Preparation of 1-(4-(4-propoxyphenyl)piperazin-1-yl)ethanone (PP02)
To a solution of 1-(4-(4-hydroxyphenyl)piperazin-1-yl)ethanone (PP01) (5.0 g, 22.6 mmole) in dry DMF
was added anhyd. K2CO3 (6.24 g, 45.2 mmole) under N2 atmosphere and stirred for 10-15 min at room temperature.
Then 1-bromo propane (3.33 g, 27.10 mmole) was added drop wise and stirred at 70 0 C for 15 min. Further, the
reaction mixture was refluxed at 120 0 C for 12 h. After completion of the reaction (checked by TLC), the reaction
mixture was cooled to room temperature. The un-reacted K2CO3 was filtered off and washed with little amount of
DMF. The DMF was evaporated under vacum to obtain crude solid, which on washing with cold water yielded pure
1-(4-(4-propoxyphenyl)piperazin-1-yl)ethanone (PP02).
1
Yield 92 %. Brown solid. m.p. 118-120 0 C, H NMR (400 MHz, CDCl3): δH 1.0 (3H, t, J= 16.0 Hz), 1.65-1.80
(2H, m), 2.12 (3H, s), 3.86 (2H, t, J= 12.0 Hz), 3.06 (4H, s), 3.75 (4H, s), 6.71 (2H, d, J= 7.5 Hz), 6.82 (2H, d, J=
7.5 Hz), 13C NMR (100 MHz, CDCl3): 10.2, 22.1, 23.5, 46.5, 51.0, 72.1, 113.4, 115.8, 145.4, 148.5, 170.1; LC/MS
(ESI): M+1 (C15H22N2O2), found 263.15, calculated 263.16.
Preparation of 1-(4-propoxyphenyl)piperazine (PP03)
1-(4-(4-propoxyphenyl)piperazin-1-yl)ethanone (PP02) (4.0 g, 15.2 mmole) was dissolved in distilled
water (15-20 mL). To this solution HCl (4N) was added under N2 atmosphere. The mixture was stirred for 3h at 80 0
C and progress of reaction was monitored by TLC. The reaction mixture was cooled to 5-10 0 C and neutralised with
aq. NaOH (25%) to obtain solid compound. It was recrystallized from ethyl acetate to offer solid product 1-(4-
propoxyphenyl)piperazine (PP03), which was taken as such for the next step.
Yield 89 %. Pale white solid. m.p. 154-156 0 C, 1H NMR (400 MHz, CDCl3): δH 1.0 (3H, t, J= 16.0 Hz), 1.65-1.80
(2H, m), 2.2 (1H, s), 3.86 (2H, t, J= 12.0 Hz), 3.06 (4H, s), 3.75 (4H, s), 6.77 (2H, d, J= 7.8 Hz), 6.94 (2H, d, J= 7.8
Hz); 13C NMR (100 MHz, CDCl3): 10.47, 22.65, 46.3, 50.88, 69.88, 115.22, 118.90, 144.99, 154.0; LC/MS (ESI):
M+1 (C13H20N2O) found 221.15 , calculated 221.17.
Preparation of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04)
To 1-(4-propoxyphenyl)piperazine (PP03) (4.7 g, 21.35 mmol) obtained in the earlier step was added 6-
chloro purine (3.0 g, 19.40 mmole) in n-butanol and, after 5 minutes triethylamine (5.87 g, 58.20 mmole) was
added drop wise. After the completion of addition, stirring was continued at 110 0 C for 10 h. Upon completion of

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the reaction n-butanol was removed under vacuum and the residual solid was recrystallized from ethanol to obtain
pure 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) in good yields.
Yield 90 %. White solid. m.p.230-232 0 C, 1H NMR (400 MHz, DMSO-d6): δH 0.94 (3H, t, J= 7.6 Hz), 1.63-1.72
(2H, m), 3.10 (4H, s), 3.83 (2H, t, J= 6.4 Hz), 4.35 (4H, s), 6.81 (2H, d, J= 8.0 Hz), 6.92 (2H, d, J= 8.0 Hz), 8.14
(1H, s), 8.22 (1H, s), 13.06 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.8, 22.1, 48.3, 72.1, 115.6, 117.6, 119.3,
140.3, 144.4, 146.7, 152.1, 153.2, 154.5; LC/MS (ESI): M-1 (C18H22N6O) found 337.40, calculated 337.43.
General procedure for the synthesis of 9-substituted-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine
To a solution of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) in dry DMF was added anhyd.
K2CO3 under N2 atmosphere and stirred for 10-15 min at room temperature. Substituted alkyl/aryl bromides were
then added drop wise and stirred at RT for 12 h. After completion of reaction (checked by TLC), the un-reacted
K2CO3 was filtered off. After quenching with ice crude solid was obtained, which on washing with cold water gives
corresponding 9-substituted-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purines. Further the crude compounds were
purified by column chromatography and eluted by methanol and dichloromethane with good yields.
1-Phenyl-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)ethanone (PP05) It was prepared by
following the above method by the addition of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) (300 mg,
0.886 mmol), 2-bromo-1-phenylethanone (212 mg, 1.063 mmol) and K2CO3 (366.9 mg, 2.659 mmol) in dry DMF
1
(10 mL) as a brownish white coloured solid, m.p. 176-178 0 C, (360.21 mg, yield 89.11 %); H NMR (400 MHz,
DMSO-d6): δH 0.94 (3H, t, J= 7.6 Hz), 1.63-1.72 (2H, m), 3.10 (4H, s), 3.83 (2H, t, J= 6.4 Hz), 4.35 (4H, s), 4.8
(2H, s), 6.81 (2H, d, J= 8.0 Hz), 6.92 (2H, d, J= 8.0 Hz), 7.68 (2H, d, J= 8.0 Hz), 7.20-7.29 (3H, m), 8.14 (1H, s),
8.22 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.2, 21.1, 49.3, 62.1, 72.0, 92.3, 114.6, 117.7, 119.8, 127.1, 128.5,
133.0, 137.5, 139.4, 144.4, 146.9, 152.5, 153.7, 155.5, LC/MS (ESI): M+1 (C26H28N6O2) found 456.29, calculated
456.23.
Ethyl 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetate (PP06) It was prepared by following the
above method by the addition of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) (300 mg, 0.886 mmol),
ethyl 2-bromoacetate (177.64 mg, 1.063 mmol) and K2CO3 (366.9 mg, 2.659 mmol) in dry DMF (10 mL) as a white
coloured solid, m.p. 148-150 0 C, (336.71 mg, yield 89.58 %); 1H NMR (400 MHz, DMSO-d6): δH 0.98 (3H, t, J=
3.6), 1.19-1.23 (3H, m), 1.65-1.73 (2H, m), 3.12-3.16 (4H, m), 3.85 (2H, t, J= 4.0 Hz), 4.14-4.20 (2H, m), 4.34-4.36
(4H, m), 5.10 (2H, s), 6.82-6.96 (4H, m), 8.20 (1H, s), 8.26 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.8, 14.6,
22.1, 48.3, 56.9, 61.2, 72.1, 115.6, 117.6, 119.3, 140.3, 144.4, 146.7, 152.1, 153.2, 154.5, 166.9; LC/MS (ESI): M-1
(C22H28N6O3) found 424.26, calculated 424.23.
9-(2-Ethylhexyl)-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP09) It was prepared by following the
above method by the addition of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) (400 mg, 1.181 mmol),
3-(bromomethyl)heptanes (342.11 mg, 1.771 mmol) and K2CO3 (488.93 mg, 3.543 mmol) in dry DMF (10 mL) as a
brown coloured solid, m.p. 90-92 0 C, (457.64 mg, yield 86.05 %); 1H NMR (400 MHz, CDCl3): δH 0.86 (3H, t, J=

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6.45), 0.91 (3H, t, J=6.48 Hz), 0.94 (3H, t, J= 7.6 Hz), 1.10-1.16 (2H, m), 1.20-1.25 (2H, m), 1.27-1.32 (2H, m),
1.34-1.39 (2H, m), 1.50-1.56 (1H, m), 1.63-1.72 (2H, m), 3.10 (4H, s), 3.83 (2H, t, J= 6.4 Hz), 3.90 (2H, d, J=7.24
Hz), 4.35 (4H, s), 6.81 (2H, d, J= 8.0 Hz), 6.92 (2H, d, J= 8.0 Hz), 8.14 (1H, s), 8.22 (1H, s), 13C NMR (100 MHz,
CDCl3): 10.8, 11.6, 14.2, 22.1, 23.5, 26.4, 29.1, 32.3, 36.9, 48.3, 57.5, 72.1, 115.6, 117.6, 119.3, 141.3, 143.4,
148.7, 152.1, 152.2, 154.5; LC/MS (ESI): M-1 (C26H38N6O) found 450.30, calculated 450.32
9-(4-Iodobenzyl)-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP10) It was prepared by following the
above method by the addition of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) (300 mg, 0.886 mmol),
1-(bromomethyl)-4-iodobenzene (315.86 mg, 1.063 mmol) and K2CO3 (366.9 g, 2.659 mmol) in dry DMF (10 mL)
as a brown coloured solid, m.p. 160-162 0 C, (402.30 mg, yield 81.94 %); 1H NMR (400 MHz, DMSO-d6): δH 0.99
(3H, t, J= 7.5 Hz), 1.67-1.77 (2H, m), 3.16 (4H, s), 3.88 (2H, t, J= 6.6 Hz), 4.37 (4H, s), 5.25 (2H, s), 6.78 (2H, d, J=
8.0 Hz), 6.96 (2H, d, J= 8.0 Hz), 7.36 (2H, d, J=5.49 Hz), 7.58 (2H, d, J= 8.29 Hz), 8.26 (1H, s), 8.37 (1H, s), 13
C
NMR (100 MHz, DMSO-d6): 11.1, 21.1, 49.3, 54.6, 74.1, 91.6, 115.2, 118.4, 120.5, 130.5, 135.5, 137.8, 141.3,
143.5, 147.5, 151.1, 153.1, 155.5; LC/MS (ESI): M+1 (C25H27IN6O) found 554.11, calculated 554.14.
7-Methyl-4-((6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)methyl)-2H-chromen-2-one (PP16) It was
prepared by following the above method by the addition of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine
(PP04) (400 mg, 1.182 mmol), 4-(bromomethyl)-7-methyl-2H-chromen-2-one (327.83 mg, 1.301 mmol) and K2CO3
(358.30 g, 3.548 mmol) in dry DMF (10 mL) as a light yellow coloured solid, m.p. 206-208 0 C, (504.0 mg, yield
1
83.07 %); H NMR (400 MHz, CDCl3): δH 0.90 (3H, t, J= 7.6 Hz), 1.63-1.72 (2H, m), 2.36 (3H, s), 3.22 (4H, s),
3.83 (2H, t, J= 6.4 Hz), 3.90 (4H, s), 5.22 (2H, s), 5.93 (1H, s), 6.81 (2H, d, J= 8.0 Hz), 6.92 (2H, d, J= 8.0 Hz), 7.14
(1H, s), 7.58 (1H, d, J=7.8 Hz), 7.66 (1H, d, J=7.8 Hz), 8.14 (1H, s), 8.22 (1H, s), 13C NMR (100 MHz, CDCl3):
10.8, 21.41, 22.1, 48.3, 51.67, 72.1, 113.50, 114.71, 115.6, 116.82, 117.6, 119.3, 128.55, 129.23, 137.0, 139.35,
140.84, 144.4, 146.7, 152.1, 153.2, 154.5, 155.80, 159.65; LC/MS (ESI): M+1 (C29H30N6O3) found 510.26,
calculated 510.24.
9-sec-Butyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP17) It was prepared by following the above
method by the addition of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) (400 mg, 1.1828 mmol), 2-
bromobutane (178.27 mg, 1.3010 mmol) and K2CO3 (358.38 mg, 3.5484 mmol) in dry DMF (10 mL) as a pale
white coloured solid, m.p. 84-86 0 C, (410.26 mg, yield 88.03 %); 1H NMR (400 MHz, CDCl3): δH 0.94 (3H, t, J=
7.6 Hz), 1.08 (3H, t, J= 6.68 Hz), 1.48 (3H, d, J= 7.11 Hz), 1.63-1.72 (2H, m), 1.94-1.99 (2H, m), 3.10 (4H, s), 3.83
(2H, t, J= 6.4 Hz), 4.35 (4H, s), 4.58 (1H, m), 6.81 (2H, d, J= 8.0 Hz), 6.92 (2H, d, J= 8.0 Hz), 8.14 (1H, s), 8.22
(1H, s), 13C NMR (100 MHz,CDCl3): 9.3, 10.7, 22.0, 22.6, 32.5, 48.3, 54.1, 72.5, 115.6, 117.9, 119.7, 141.3, 144.4,
146.7, 152.1, 153.2, 154.5; LC/MS (ESI): M+1 (C22H30N6O) found 394.26, calculated 394.23.
1-(4-Chlorophenyl)-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)ethanone (PP18) It was prepared
by following the above method by the addition of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PP04) (250

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mg, 0.7387 mmol), 2-bromo-1-(4-chlorophenyl)ethanone (205.59 mg, 0.8864 mmol) and K2CO3 (305.82 mg,
2.2161 mmol) in dry DMF (10 mL) as a pale yellow coloured solid, m.p. 196-198 0 C, (305.20 mg, yield 84.28 %);
1H NMR (400 MHz, DMSO-d6): δH 0.97 (3H, t, J= 7.6 Hz), 1.65-1.73 (2H, m), 3.14 (4H, s), 3.86 (2H, t, J= 6.4
Hz), 4.38 (4H, s), 5.18 (2H, s), 6.89 (2H, d, J= 8.0 Hz), 6.98 (2H, d, J= 8.0 Hz), 7.53 (2H, d, J= 8.76 Hz), 7.86 (2H,
d, J= 8.67 Hz), 8.26 (1H, s), 8.37 (1H, s), 13C NMR (100 MHz, DMSO-d6): 11.2 21.1, 48.6, 61.7, 72.6, 115.5,
117.3, 119.5, 128.9, 130.5, 135.3, 138.5, 141.3, 144.4, 146.8, 152.3, 153.1, 154.2, 192.3; LC/MS (ESI): M+1
(C26H27ClN6O2) found 490.14, calculated 490.17.
2-(6-(4-(4-Propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)-1-(4-(trifluoromethoxy)phenyl)ethanone (PP19) It
was prepared by following the above method by the addition of 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine
(PP04) (250 mg, 0.7387 mmol), 2-bromo-1-(4-(trifluoromethoxy)phenyl)ethanone (249.90 mg, 0.8864 mmol) and
K2CO3 (305.82 mg, 2.2161 mmol) in dry DMF (10 mL) as a white coloured solid, m.p. 212-214 0 C, (326.84 mg,
yield 81.90 %); 1H NMR (400 MHz, DMSO-d6): δH 0.96 (3H, t, J= 7.6 Hz), 1.64-1.75 (2H, m), 3.13 (4H, s), 3.87
(2H, t, J= 6.4 Hz), 4.35 (4H, s), 5.24 (2H, s), 6.86 (2H, d, J= 8.0 Hz), 6.95 (2H, d, J= 8.0 Hz), 7.44 (2H, d, J= 8.45
Hz), 7.98 (2H, d, J= 8.45 Hz), 8.26 (1H, s), 8.37 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.6, 21.9, 49.3, 60.9,
71.1, 114.5, 115.8, 117.3, 119.6, 121.6, 129.1, 129.8, 141.3, 144.6, 146.4, 151.9, 153.5, 154.2, 166.4, 190.8;
LC/MS (ESI): M+1 (C27H27F3N6O3) found 540.26, calculated 540.23.
Preparation of 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetic acid (PP07)
To a stirred solution of ethyl 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetate (PP06) (500
mg, 1.177 mmol) in water (5 mL), aq. NaOH (2M) was added and the mixture was stirred at 70-80 0 C for 2-3 h.
The reaction mixture was neutralized with HCl (3M) till the H= 3.0. The resulting solution was extracted with
p
ethyl acetate and the combined organic layers were dried over anhydrous sodium sulfate. After concentration in
vacuo it yielded pure 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetic acid (PP07).
Yield 86 %. Pale white solid. m.p. 240-242 0 C, 1H NMR (400 MHz, CDCl3): δH 0.99 (3H, t, J= 7.6 Hz), 1.67-1.78
(2H, m), 3.16 (4H, s), 3.87 (2H, t, J= 6.4 Hz), 4.38 (4H, s), 4.78 (2H, s), 6.87 (2H, d, J= 8.0 Hz), 6.95 (2H, d, J= 8.0
Hz), 8.12 (1H, s), 8.58 (1H, s), 11.4 (1H, s), 13C NMR (100 MHz, CDCl3): 10.1, 21.7, 49.3, 59.6, 72.9, 115.5, 117.9,
119.8, 141.5, 144.5, 146.9, 152.1, 153.1, 154.5, 176.2; LC/MS (ESI): M-1 (C20H24N6O3) found 396.19, calculated
396.17.
Preparation of 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide (PP08)
To a solution of ethyl 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetate (PP06) (500 mg,
1.178 mmol) in ethanol (10 mL) at RT, hydrazine hydrate was added (176.89 mg, 3.533 mmol) and resulting
solution was stirred at reflux temperature for 4 h. It was then cooled to room temperature and ethanol was removed
under high vacuo and the resulting compound was recrystallized from ethanol to obtain pure 2-(6-(4-(4-
propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide (PP08).

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Yield 91 %. White solid. m.p. 232-234 0 C, 1H NMR (400 MHz, DMSO-d6): δH 0.96 (3H, t, J= 7.2 Hz), 1.66-1.71
(2H, m), 2.0 (2H, s), 3.13 (4H, s), 3.84 (2H, t, J= 6.0 Hz), 4.35 (2H, s), 4.83 (4H, s), 6.82 (2H, d, J= 8.4 Hz), 6.94
(2H, d, J= 8.4 Hz), 8.15 (1H, s), 8.23 (1H, s), 9.46 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.8, 21.7, 40.5, 49.3,
49.8, 71.6, 114.5, 116.3, 119.4, 140.8, 141.5, 147.8, 150.0, 152.6, 154.8, 165.5; LC/MS (ESI): M+1 (C20H26N8O2)
found 410.24, calculated 410.23.
General procedure for the synthesis of substituted (E)-N'-benzylidene-2-(6-(4-(4-propoxyphenyl)piperazin-1-
yl)-9H-purin-9-yl)acetohydrazides
Compound (PP08) was dissolved in absolute ethanol (15 mL). Appropriate aldehydes in absolute ethanol
(10 mL) were added gradually to the mixture, which was refluxed for 2-3 h and cooled. The precipitated product
was filtered off and recrystallized from EtOAc or Methanol to afford pure N-9 substituted (E)-N'-benzylidene-2-(6-
(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazides.
(E)-N'-(3-Nitrobenzylidene)-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide (PP11)
It was prepared by following the above method by the addition of 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-
purin-9-yl)acetohydrazide (PP08) (200 mg, 0.4878 mmol) and 3-nitro benzaldehyde (110.57 mg, 0.7317 mmol) in
1
absolute ethanol (20 mL) as a white coloured solid, m.p. 204-206 0 C, (201.36 mg, yield 75.99 %); H NMR (400
MHz, DMSO-d6): δH 0.96 (3H, t, J= 7.2 Hz), 1.66-1.71 (2H, m), 3.13 (4H, s), 3.84 (2H, t, J= 6.0 Hz), 4.35 (2H, s),
4.83 (4H, s), 6.82 (2H, d, J= 8.4 Hz), 6.94 (2H, d, J= 8.4 Hz), 7.58-7.64 (2H, m), 8.05 (1H, s), 8.15 (1H, s), 8.23
(1H, s), 8.30 (1H, m), 8.65 (1H, s), 9.46 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.3, 22.7, 39.5, 49.1, 49.4, 71.7,
114.5, 115.3, 119.9, 122.9, 124.6, 130.1, 134.3, 136.4, 140.8, 141.9, 143.4, 146.8, 150.4, 152.6, 153.8, 170.5;
LC/MS (ESI): M+1 (C27H29N9O4) found 543.26, calculated 543.24.
(E)-N'-(4-Fluorobenzylidene)-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide (PP12)
It was prepared by following the above method by the addition of 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-
purin-9-yl)acetohydrazide (PP08) (250 mg, 0.6097 mmol) and 4-flouro benzaldehyde (151.36 mg, 1.2195 mmol) in
1
absolute ethanol (20 mL) as a white coloured solid, m.p. 244-246 0 C, (249.80 mg, yield 79.36 %); H NMR (400
MHz, DMSO-d6): δH 0.96 (3H, t, J= 7.2 Hz), 1.66-1.71 (2H, m), 3.13 (4H, s), 3.84 (2H, t, J= 6.0 Hz), 4.35 (2H, s),
4.83 (4H, s), 6.87 (1H, s), 6.90 (2H, d, J= 8.4 Hz), 7.15 (1H, s), 7.80 (2H, d, J= 8.4 Hz), 7.98 (2H, d, J= 8.71 Hz),
8.22 (2H, d, J= 8.71 Hz), 8.50 (1H, s), 9.98 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.4, 22.6, 39.8, 69.8, 114.8,
115.2, 118.9, 129.5, 130.9, 140.7, 141.1, 143.5, 147.7, 150.1, 152.5, 154.8, 165.5, 173.4, LC/MS (ESI): M+1
(C27H29FN8O2) found 516.80, calculated 516.82.
(E)-N'-(4-Chlorobenzylidene)-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide (PP13)
It was prepared by following the above method by the addition of 2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-
purin-9-yl)acetohydrazide (PP08) (250 mg, 0.6097 mmol) and 4-chloro benzaldehyde (128.57 mg, 0.9146 mmol) in
1
absolute ethanol (20 mL) as a white coloured solid, m.p. 238-240 0 C, (270.37 mg, yield 83.32 %); H NMR (400

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MHz, DMSO-d6): δH 0.95 (3H, t, J= 7.3 Hz), 1.65-1.71 (2H, m), 3.14 (4H, s), 3.86 (2H, t, J= 6.0 Hz), 4.37 (2H, s),
4.86 (4H, s), 6.85 (1H, s), 7.12 (1H, s), 7.80 (2H, d, J= 8.4 Hz), 7.90 (2H, d, J= 8.4 Hz), 7.98 (2H, d, J= 8.71 Hz),
8.22 (2H, d, J= 8.71 Hz), 8.42 (1H, s), 9.88 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.6, 22.5, 40.1, 71.9, 114.9,
115.4, 116.0, 119.5, 129.6, 130.8, 140.5, 141.3, 143.6, 147.7, 150.5, 152.6, 154.9, 165.5, 173.6, LC/MS (ESI): M+1
(C27H29ClN8O2) found 532.19, calculated 532.21.
(E)-N'-(4-(Trifluoromethyl)benzylidene)-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-
yl)acetohydrazide (PP14) It was prepared by following the above method by the addition of 2-(6-(4-(4-
propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide (PP08) (220 mg, 0.5365 mmol) and 4- trifluoromethyl
benzaldehyde (140.12 mg, 0.8049 mmol) in absolute ethanol (20 mL) as a light yellow coloured solid, m.p. 250-
1
252 0 C, (248.36 mg, yield 81.75 %); H NMR (400 MHz, DMSO-d6): δH 0.96 (3H, t, J= 7.2 Hz), 1.67-1.72 (2H,
m), 3.22 (4H, s), 3.87 (2H, t, J= 6.4 Hz), 4.39 (2H, s), 5.10 (4H, s), 6.88 (1H, s), 7.05 (1H, s), 7.81 (2H, d, J= 7.6
Hz), 7.91 (2H, d, J= 8.0 Hz), 7.98 (2H, d, J= 8.0 Hz), 8.23 (2H, d, J= 6.0 Hz), 8.40 (1H, s), 9.99 (1H, s), 13C NMR
(100 MHz, DMSO-d6): 10.8, 22.7, 40.5, 72.0, 114.6, 115.5, 116.3, 119.6, 129.7, 131.0, 140.4, 141.6, 143.8, 147.8,
150.4, 152.7, 155.0, 165.7, 173.8, LC/MS (ESI): M-1 (C28H29F3N8O2) found 566.20, calculated 566.20.
(E)-N'-(4-Methoxybenzylidene)-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide
(PP15) It was prepared by following the above method by the addition of 2-(6-(4-(4-propoxyphenyl)piperazin-1-
yl)-9H-purin-9-yl)acetohydrazide (PP08) (250 mg, 0.6097 mmol) and 4-methoxybenzaldehyde (124.43 mg, 0.9146
1
mmol) in absolute ethanol (20 mL) as a white coloured solid, m.p. 256-258 0 C, (263.14 mg, yield 81.80 %); H
NMR (400 MHz, DMSO-d6): ): δH 0.96 (3H, t, J= 7.3 Hz), 1.66-1.72 (2H, m), 3.15 (4H, s), 3.66 (3H, s), 3.87 (2H,
t, J= 6.0 Hz), 4.39 (2H, s), 4.86 (4H, s), 6.87 (1H, s), 7.14 (1H, s), 7.81 (2H, d, J= 8.4 Hz), 7.91 (2H, d, J= 8.71 Hz),
7.99 (2H, d, J= 8.4 Hz), 8.23 (2H, d, J= 8.71 Hz), 8.42 (1H, s), 9.88 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.8,
22.7, 40.6, 55.9, 72.0, 114.8, 115.5, 116.5, 119.4, 129.8, 130.7, 140.6, 141.5, 143.7, 147.5, 150.6, 152.3, 155.0,
165.7, 173.5, LC/MS (ESI): M+1 (C28H32N8O3) found 528.24, calculated 528.26.
(E)-N'-(2-Hydroxy-5-methoxybenzylidene)-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-
yl)acetohydrazide (PP20) It was prepared by following the above method by the addition of 2-(6-(4-(4-
propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide (PP08) (350 mg, 0.8536 mmol) and 2-hydroxy-5-
methoxybenzaldehyde (194.69 mg, 1.2804 mmol) in absolute ethanol (20 mL) as a light yellow coloured solid,
1
m.p. 266-268 0 C, (385.10 mg, yield 82.89 %); H NMR (400 MHz, DMSO-d6): δH 0.95 (3H, t, J= 7.3 Hz), 1.66-
1.72 (2H, m), 3.14 (4H, s), 3.74 (3H, s), 3.86 (2H, t, J= 6.0 Hz), 4.40 (2H, s), 4.87 (4H, s), 5.13 (1H, s), 6.83 (2H, d,
J= 8.4 Hz), 6.92 (2H, d, J= 8.4 Hz), 7.05 (1H, s), 6.98 (2H, d, J= 8.71 Hz), 6.85 (1H, s), 7.15 (1H, s), 8.27 (1H, s),
9.51 (1H, s), 13C NMR (100 MHz, DMSO-d6): 10.6, 22.8, 40.7, 55.9, 72.1, 114.7, 115.6, 116.5, 119.4, 129.7, 130.7,
140.8, 141.5, 143.8, 147.5, 150.8, 152.5, 155.0, 165.7, 173.6, LC/MS (ESI): M+1 (C28H32N8O4) found 544.28,
calculated 544.25.

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(E)-N'-(3-Chloro-4-fluorobenzylidene)-2-(6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purin-9-
yl)acetohydrazide (PP21) It was prepared by following the above method by the addition of 2-(6-(4-(4-
propoxyphenyl)piperazin-1-yl)-9H-purin-9-yl)acetohydrazide (PP08) (350 mg, 0.8536 mmol) and 3-chloro-4-
fluorobenzaldehyde (202.29 mg, 1.2804 mmol) in absolute ethanol (20 mL) as a white coloured solid, m.p. 210-212
1
0 C, (370.10 mg, yield 78.80 %); H NMR (400 MHz, DMSO-d6): δH 0.96 (3H, t, J= 7.2 Hz), 1.66-1.71 (2H, m),
3.15 (4H, s), 3.87 (2H, t, J= 6.0 Hz), 4.37 (2H, s), 4.84 (4H, s), 6.83 (2H, d, J= 8.4 Hz), 6.95 (2H, d, J= 8.4 Hz), 7.14
(2H, d, J= 8.71 Hz), 7.50 (1H, s), 8.12 (1H, s), 8.14 (1H, s), 8.24 (1H, s), 9.47 (1H, s), 13C NMR (100 MHz, DMSO-
d6): 10.5, 22.7, 40.1, 71.7, 114.8, 115.2, 115.9, 119.4, 129.5, 130.9, 140.7, 141.1, 142.1, 147.6, 150.2, 152.5, 154.8,
165.4, 174.1, LC/MS (ESI): M+1 (C27H28ClFN8O2) found 550.23, calculated 550.21.
SRB Assay 24, 25
The cell lines were grown in RPMI 1640 medium containing 10% foetal bovine serum and 2 mM L-
glutamine. Cells were inoculated into 96 well microtiter plates in 100 µL. After cell inoculation, the microtiter
plates were incubated at 37° C in 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of
experimental drugs.
Experimental drugs were initially solubilised in dimethyl sulfoxide at 100mg/mL and diluted to 1mg/mL
using water and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate (1mg/mL)
was thawed and diluted to 100 µg/mL, 200 µg/mL, 400 µg/mL and 800 µg/mL with complete medium containing
test article. Aliquots of 10 µL of these different drug dilutions were added to the appropriate microtiter wells already
containing 90 µL of medium, resulting in the required final drug concentrations i.e.10 µg/mL, 20 µg/mL, 40 µg/mL,
80 µg/mL.
After compound addition, plates were incubated at standard conditions for 48 h and assay was terminated
by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µL of cold 30 % (w/v) TCA (final
o
concentration, 10 % TCA) and incubated for 60 min at 4 C. The supernatant was discarded; the plates were
washed five times with water and air dried. Sulforhodamine B (SRB) solution (50 µL) at 0.4 % (w/v) in 1 % acetic
acid was added to each of the wells, and plates were incubated for 20 min at room temperature. After staining,
unbound dye was recovered and the residual dye was removed by washing five times with 1 % acetic acid. The
plates were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on
plate reader at a wavelength of 540 nm with 690 nm reference wavelength.
Percent growth was calculated on a plate-by-plate basis for test wells relative to control wells. Percent
Growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control
wells X 100. Using the six absorbance measurements the percentage growth was calculated at each of the drug
concentration levels
Cell cycle analysis
Breast cancer cells (MCF-7) were seeded on 35-mm dishes and cultured for 24 hrs, reaching 60–70%
confluency. The cells were incubated for 24 h in the presence or absence of test compounds of PP09 & PP17,

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harvested by trypsinization, resuspended in 200 mL PBS, and fixed in 70% ethanol for 30 min at 4 0 C. Cells were
stained with 1 mL of DNA staining solution (100 µg of RNase A and 40 µg of Propidium iodide (PI)) for 30 min.
The DNA content was measured and assessed on a FACS Calibur Flow cytometer using Cell Quest Pro software
(BD Bioscience).
Western blotting analysis
After the treatment of MCF-7 cells with compounds PP09 & PP17 cells were washed with ice cold
phosphate-buffered saline (PBS) and cells were homogenized and lysed in RIPA buffer [20 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium
pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4] supplemented 1 mM PMSF, and 1 mg/ml of aprotonin,
leupeptin, and pepstatin A. Protein concentration was determined using a Bio-Rad assay. Proteins were resolved by
SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were incubated with
antibodies p53 (Abcam) and b-actin (Santa Cruz Biotechnology) in 5% bovine serum albumin in Tris-buffered
saline with 0.1% Tween-20.
Acknowledgements
The authors gratefully acknowledge the financial assistance from Department of Science & Technology
(DST-SERB), Govt. of India, New Delhi under Major Research Project No. SR/S1/OC-58/2011 and University
Grant Commission (UGC), New Delhi under UPE-FAR-I program, F. No. 14-3/2012 (NS/PE).
References
1. H. Rosemeyer, Chemistry & Biodiversity., 2004, 1, 361-401.
2. M. Legraverend, D. S. Grierson, Bioorg. Med. Chem., 2006, 14, 3987–4006.
3. N. K Anand, C. M. Blazey, O. J. Bowles, J. Bussenius, L. C. Bannen, D. S. Chan, B. Chen, E. Wang Co, S.
Costanzo, S. C. Defina, L. Dubenko, M. Franzini, P. Huang, V. Jammalamadaka, R. G. Khoury, M. H. Kim, R. R.
Klein, D. T. Le, M. B. Mac, J. M. Nuss, J. J. Parks, K. D. Rice, T. H. Tsang, A. L. Tsuhako, Y. Wang, W. Xu, US
Pat, US8076338, 2011.
4. D. S. Sanford, W. G. Wierda, J. A. Burger, M. J. Keating, S. M. O'Brien, Clinical Lymphoma Myeloma and
Leukemia., 2015, 15 (7), 385–391.
5. C. Pui, S. Jeha, P. Kirkpatrick, Nature Reviews Drug Discovery., 2005, 4, 369-370.
6. V. Gandhi, M. J. Keating, G. Bate, P. Kirkpatrick, Nature Reviews Drug Discovery., 2006, 5, 17-18.
7. FDA Orange Book, http://www.accessdata.fda.gov/scripts/cder/ob/docs/queryai.cfm
8. W. B. Parker, Chem Rev., 2009, 109 (7), 2880–2893.
9. D. Sampath, V. A. Rao, W. Plunkett, Oncogene., 2003, 22, 9063–9074.
10. G. B. Elion, Science., 1989, 244 (4900), 41-47.
11. G. R. Goodman, E. Beutler, A. Saven, Best Practice & Research Clinical Haematology., 2003, 16 (1), 101–116.
12. S. Faderl, V. Gandhi, M. J. Keating, S. Jeha, W. Plunkett, H. M. Kantarjian, Cancer., 2005, 103 (10), 1985–1995.

RSC Advances
Page 20 of 21
View Article Online
DOI: 10.1039/C5RA23242B
13. A. R. Kore, B. Yang, B. Srinivasan, Current Organic Chemistry., 2014, 18(16), 2072-2107.
14. D. Bliman, M. Pettersson, M. Bood, M. Grøtli, Tetrahedron Letters., 2014, 55, 2929–2931.
15. M. Legraverend, Tetrahedron., 2008, 64, 8585–8603.
16. J. Liu, M. J. Robins, J. Am. Chem. Soc., 2007, 129, 5962–5968.
17. M. Zhong, M. J. Robins, J. Org. Chem., 2006, 71, 8901–8906.
18. M. Yarim, M. Koksal, I. Durmaz, R. Atalay, Int. J. Mol. Sci., 2012, 13, 8071-8085.
19. J. Fengchao, D. Yulan, J. Jie, WO Pat, WO2012163291, 2012.
20. K. Dong Gyu, S. Dong Su, J. Sin, H. Ji Yeong, L. Myeong Jin, KR Pat, KR2012119229, 2012.
21. W. Matthew A, WO Pat, WO2012061337, 2012.
22. A. Chopra, A. Anderson, C. Giardina, J. Biol. Chem., 2014, 289 (5), 2978–2991.
23. J. Sun, X. Wang, P. Lv, H. Zhu, Sci Rep., 2015, 5, 13934.
24. V. Vichai, K. Kirtikara, Nature Protocols., 2006, 1, 1112 – 1116.
25. P. Skehn, R. Storeng, A. Scudiero, J. Monks, D. McMohan, D. Vistica, T. W. Jonathan, H. Bokesch, S. Kenney, M.
R. Boyd, J. Natl. Cancer Inst., 1990, 82 (13), 1107-1112.

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132x117mm (96 x 96 DPI)