Novel 1,5-diphenyl-6-substituted 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones induced apoptosis in RKO colon cancer cells

Article abstract of DOI:10.3109/14756366.2015.1118686

Novel 1,5-diphenyl-6-substituted-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones were synthesized and characterized. All compounds were screened for their anti-proliferative activities in five different cancer cell lines. The results showed that compounds 7a and 7b comprising aminoguanidino or guanidino moiety at position 6 inhibited proliferation of RKO colon cancer cells with IC₅₀ of 8 and 4 μM, respectively. Compounds 7a and 7b induced apoptosis in RKO cells, which was confirmed by TUNEL and annexin V-FITC assays. Flow cytometric analysis indicated that compounds 7a and 7b arrested RKO cells in the G1 phase and the most active compound 7b increased levels of p53, p21, Bax, ERK1/2 and reduced levels of Bcl2 and Akt. Compound 7b also activates release of cytochrome c, which is consistent with activation of caspase-9. Additionally, compound 7b increased caspase-3 activity and cleaved PARP-1 in RKO cells. Collectively, these findings could establish a molecular basis for the development of new anti-cancer agents.

Full text of DOI:10.3109/14756366.2015.1118686

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Novel 1,5-diphenyl-6-substituted 1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-ones induced apoptosis in RKO
colon cancer cells
Ahmed Malki, Hayam M. A. Ashour, Rasha Y. Elbayaa, Doaa A. E. Issa, Hassan
A. Aziz & Xiaozhuo Chen
To cite this article: Ahmed Malki, Hayam M. A. Ashour, Rasha Y. Elbayaa, Doaa A. E. Issa,
Hassan A. Aziz & Xiaozhuo Chen (2015): Novel 1,5-diphenyl-6-substituted 1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-ones induced apoptosis in RKO colon cancer cells, Journal of Enzyme
Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2015.1118686
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1118686
Published online: 18 Dec 2015.
Submit your article to this journal
Article views: 12
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [University of California, San Diego]
Date: 01 January 2016, At: 22:09

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–14
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1118686
!
RESEARCH ARTICLE
Novel 1,5-diphenyl-6-substituted 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
ones induced apoptosis in RKO colon cancer cells
Ahmed Malki1*, Hayam M. A. Ashour2, Rasha Y. Elbayaa2,3, Doaa A. E. Issa2,4, Hassan A. Aziz1, and Xiaozhuo Chen5
1Biomedical Science Program, Department of Health Sciences, College of Art and Sciences, Qatar University, Doha, Qatar,
3
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt, Department of Analytical &
Pharmaceutical Chemistry, Faculty of Pharmacy & Drug Manufacturing, Pharos University, Alexandria, Egypt, and 4Department of Pharmaceutical
5
Sciences, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon, and 5Department of Biomedical Sciences, Edison Biotechnology Institute,
Molecular and Cellular Biology Program, Ohio University, Athens, Ohio, USA
Abstract
Keywords
Novel 1,5-diphenyl-6-substituted-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones were synthesized and
characterized. All compounds were screened for their anti-proliferative activities in five different
cancer cell lines. The results showed that compounds 7a and 7b comprising aminoguanidino or
guanidino moiety at position 6 inhibited proliferation of RKO colon cancer cells with IC50 of 8
and 4 mM, respectively. Compounds 7a and 7b induced apoptosis in RKO cells, which was
confirmed by TUNEL and annexin V-FITC assays. Flow cytometric analysis indicated that
compounds 7a and 7b arrested RKO cells in the G1 phase and the most active compound 7b
increased levels of p53, p21, Bax, ERK1/2 and reduced levels of Bcl2 and Akt. Compound 7b also
activates release of cytochrome c, which is consistent with activation of caspase-9. Additionally,
compound 7b increased caspase-3 activity and cleaved PARP-1 in RKO cells. Collectively, these
findings could establish a molecular basis for the development of new anti-cancer agents.
Apoptosis, caspase-3, PARP-1, pyrazolo[3,4-
d]pyrimidin-4(5H)-ones, p53, RKO
History
Received 20 September 2015
Revised 26 October 2015
Accepted 4 November 2015
Published online 8 December 2015
Introduction
of pathological conditions such as cancer, neurodegenerative
disorders and autoimmune diseases4. Therefore, inhibition of cell
cycle progression and induction of apoptotic cancer cell death
represents a key strategy for the prevention and treatment of
cancer. Apoptosis is triggered by two major apoptosis-initiating
pathways: the extrinsic and intrinsic pathways. The extrinsic
pathway is regulated by a sub-group of tumor necrosis factor
receptors (TNFR) superfamily that includes TNFR, FAS and
TRAIL. The intrinsic pathway is initiated from within the cell and
depends on the balance of the pro- and anti-apoptotic members
of Bcl-2 family proteins5–7. The most widely studied form of
intrinsic apoptosis is initiated by the stress-mediated release of
cytochrome c from the mitochondria that results in the release
of apoptogenic proteins into the cytosol. This is followed by
activation of caspase-9 and caspase-3 and apoptosis can be
executed through protease activity targeting substrate proteins8.
The pyrazolo[3,4-d]pyrimidine nucleus is considered as an
isostere to the purine nucleus and hence exhibits promising anti-
proliferative activities. Several members of this family were found
to induce apoptosis and/or reduce cell proliferation in many
Colorectal cancer (CRC) is the fourth most common form of
cancer and the second leading cause for cancer-related deaths1.
The prevalence of CRC is still increasing despite enhanced
understanding of the pathogenesis of this disease and establish-
ment of improved screening strategies for this malignancy.
Current data suggest that epidemiological risk factors for colon
cancer, other than genetic risk factors, include smoking, physical
inactivity, obesity and food with high fat and carbohydrates that
could promote tumor growth2. Although several drugs have been
developed for the management of cancer, the high systemic
toxicity and drug resistance remain significant obstacles to their
success in many cancers3. Thus, the discovery of new therapeutic
targets for cancer and development of novel, more effective
therapeutic agents remain major challenges in the field of cancer
chemotherapy.
Programmed cell death (PCD), or apoptosis, is an essential
physiological process that leads to cellular self-destruction and
plays essential roles in development and maintenance of tissue
homeostasis. Deregulation of apoptosis is implicated in a variety
cancer cell lines such as breast, colon, lung and liver cell lines9–17
.
*Permanent address: Biochemistry Department, Faculty of Science,
Alexandria University, Alexandria 21521, Egypt
Different mechanisms account for the cytotoxic effect of this class
of compounds, where they have been reported to act as inhibitors
of different enzymes such as CDK 214, epidermal growth factor15,
Src or dual Src/AbI inhibitors16–19, mammalian target of
rapamycin inhibitors20 or modulators of oxygen stress in cancer
cells9. Over the past two decades, considerable attention has been
devoted to pyrazolo[3,4-d]pyrimidin-4-one as a prime target for
Address for correspondence: Dr Ahmed Malki, Health Sciences
Department, College of Art and Sciences, Qatar University, Doha,
Qatar. Tel: 974-44036557. E-mail: ahmed.malki@qu.edu.qa,
Rasha Y. Elbayaa, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Alexandria University, Alexandria, 21521, Egypt. Tel: +2 (03)
4871317. Fax: +2 (03) 4873273. E-mail: rashabayaa72@gmail.com

2
A. Malki et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Figure 1. Chemical structure of previously
reported pyrazolo[3,4-d]pyrimidines
endowed with anti-cancer and apoptosis
inducing activities (A–D) and the synthesized
compounds (E).
the synthesis of anti-tumor agents. In this context, several capillaries using Stuart capillary melting point apparatus (Stuart
pyrazolo[3,4-d]pyrimidin-4-ones were reported to possess anti- Scientific Stone, Staffordshire, UK) and were uncorrected.
proliferative activity against human colon cancer cell Infrared (IR) spectra were recorded on Perkin Elmer 1430
line HCT116 and other cell lines (e.g. compounds A–D; infrared spectrophotometer (Perkin Elmer, Beaconsfield, UK) and
Figure 1)12,21,22
1
.
measured by ꢀꢀ cmꢀ1 scale using KBr cell. H NMR spectra were
Encouraged by the above-mentioned findings and in continu- scanned on Varian Mercury VX-300 MHz (San Diego, CA) or
ation of our efforts linked with discovering and exploring novel Bru¨ker avance 400 MHz (Bruker, UK), using tetramethylsilane as
lead structures as potent chemotherapeutic agents23–26, it was an internal standard and DMSO-d6 as the solvent (chemical shifts
attempted to synthesize new pyrazolo[3,4-d]pyrimidin-4-ones are given in ꢁ ppm). Splitting patterns were designated as follows:
comprising a variety of substituents at position 6 (E; Figure 1) s: singlet; br s: broad singlet; d: doublet; t: triplet; q: quartet; m:
to be evaluated for their anti-cancer activity. The substituents at multiplet. 13C NMR spectra were recorded on a Varian Mercury
position 6 include various pharmacophores and functionalities VX-300 MHz or Bru¨ker 400 MHz spectrometer in DMSO-d6 and
that are believed to be responsible for the biological significance measured in ꢁ scale. Mass spectra were run on a gas chromato-
of some relevant anti-cancer agents such as alkylamino22,27 graph/mass spectrometer Shimadzu GCMS-QP 2010 Plus (70 eV)
together with other pharmacophoric groups that would confer (Kyoto, Japan). Microanalyses were performed at the regional
different electronic and lipophilic environment, which would Center for Mycology and Biotechnology, Alazhar University and
influence the targeted anti-cancer activity. Since many guanidines the found values were within 0.4% of the theoretical values.
were reported to possess cytotoxic and apoptosis inducing Follow-up of the reactions and checking the purity of the
activities28–30, we deemed it interesting to explore the effect of compounds was performed by thin-layer chromatography on silica
introducing guanidino and aminoguanidino moieties at position 6. gel-precoated aluminum sheets (Type 60 GF254; Merck,
Moreover, due to the reported anti-tumor and apoptosis inducing Germany) and the spots were detected by exposure to UV lamp
activities of some hydrazones12,31–35 it was considered worth- at l 254 nm for few seconds.
while to extend substitution at position 6 to include the structural
Ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate (1)36 and
elements of hydrazones. These combinations were suggested in an 1,5-diphenyl-6-thioxo-6,7-dihydro-1H-pyrazolo[3,4-d]pyrimidin-
attempt to investigate the possible synergistic influence of such 4(5H)-one (2)37 were prepared according to previously published
structure hybridizations on the anticipated biological activity, reaction conditions.
hoping to discover and develop apoptosis inducers as potential
new anti-cancer agents.
6-(Methylsulfanyl)-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (3)
To a stirred solution of the thione 2 (6.4 g, 20 mmol) in dry
dimethyl formamide (20 ml) containing anhydrous potassium
carbonate (2.8 g, 20 mmol), methyl iodide (3.4 g, 1.5 ml, 24 mmol)
was slowly added. The reaction mixture was left stirred at room
temperature for 6 h then poured onto crushed ice. The separated
solid product was filtered, washed with water, dried and
Methods and materials
Chemistry
All reagents and solvents were purchased from commercial
suppliers and were dried and purified when necessary by standard
techniques. Melting points were determined in open glass

DOI: 10.3109/14756366.2015.1118686
Apoptosis in RKO colon cancer cells
3
crystallized from ethanol. Yield: 5.21 g, 78%; m.p.: 170–172 ꢁC. water 4:1). IR (KBr, cmꢀ1): 3280 (NH), 3107, 2925 (CH), 1700
IR (KBr, cmꢀ1): 3055, 2930 (CH), 1706 (C ¼ O), 1594 (C ¼ N), (C ¼ O), 1600 (C ¼ N), 1551, 1492, 1458 (C ¼ C). 1H NMR
1529, 1503, 1477 (C ¼ C), 1276, 1064 (C–S–C). 1H NMR (300 MHz, DMSO-d6) ꢁ 4.45 (d, J ¼ 6.9 Hz, 2H, CH2), 7.01–7.64
(300 MHz, DMSO-d6) ꢁ 2.45 (s, 3H, SCH3), 7.32–7.40 (m, 3H, (m, 13H, aromatic-H and NH), 7.93 (d, J ¼ 7.2 Hz, 2H, phenyl
phenyl C3,4,5–H), 7.50–7.60 (m, 5H, phenyl-H), 8.1 (d, J ¼ 7.8 Hz, C2,6–H), 8.08 (s, 1H, pyrazolopyrimidine C3–H). 13C NMR
2H, phenyl C2,6–H), 8.28 (s, 1H, pyrazolopyrimidine C3–H). 13C (DMSO-d6) ꢁ 45.71 (CH2), 101.29 (pyrazolopyrimidine C3a),
NMR (DMSO-d6) ꢁ 16.05 (CH3), 104.58 (pyrazolopyrimidine 120.75, 126.32, 127.18, 127.44, 128.36, 128.92, 129.37, 129.65,
C3a), 121.76, 127.31, 129.72, 129.92, 130.04, 130.41, 136.26, 130.71, 136.95, 139.31, 139.77 (phenyl and benzyl-C), 140.07
136.98 (phenyl-C), 138.76 (pyrazolopyrimidine C3), 150.76 (pyrazolopyrimidine C3), 153.10 (pyrazolopyrimidine C7a),
(pyrazolopyrimidine C7a), 157.29 (C ¼ O), 164.34 (pyrazolopyr- 153.91 (C ¼ O), 157.93 (pyrazolopyrimidine C6). MS (m/z,
imidine C6). Anal. Calcd. for C18H14N4OS (334.39): C, 64.65; H, %): 393 (M+, 56.9), 314 (100). Anal. Calcd. for C24H19N5O
4.22; N, 16.75. Found: C, 64.48; H, 4.36; N, 16.59.
(393.44): C, 73.27; H, 4.87; N, 17.80. Found: C, 73.51; H, 4.92;
N, 18.02.
6-(Methylsulfonyl)-1,5-diphenyl-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (4)
6-(2-(3,4-dimethoxyphenyl)ethylamino)-1,5-diphenyl-1H-pyra-
zolo[3,4-d]pyrimidin-4(5H)-one (5c). Yield: 0.5 g, 54%; m.p.: 84–
86 ꢁC (methylene chloride/petroleum ether 60/80) (2:1). IR (KBr,
cmꢀ1): 3384 (NH), 3061, 2997, 2932, 2834 (CH), 1703 (C ¼ O),
1597 (C ¼ N), 1554, 1498, 1458 (C ¼ C), 1260, 1236, 1063, 1026
To an ice-cold well-stirred solution of the methylsulfanyl
derivative 3 (6.7 g, 20 mmol) in dimethyl formamide (50 ml),
potassium hydrogenpersulfate (36.9 g, 60 mmol) dissolved in
water (140 ml) was slowly added over a period of 2 h. The
reaction mixture was further stirred at room temperature for 16 h
then filtered to remove inorganic material. The filterate was
poured into ice-cold water and the obtained solid product was
filtered, dried and crystallized from dioxane. Yield: 5.43 g, 74%;
m.p.: 215–217 ꢁC. IR (KBr, cmꢀ1): 3062, 2962 (CH), 1715
(C ¼ O), 1594 (C ¼ N), 1563, 1527, 1504, 1488, 1458 (C ¼ C),
1346, 1162 (SO2), 1291, 1093 (C–S–C). 1H NMR (400 MHz,
DMSO-d6) ꢁ 2.76 (s, 3H, SO2CH3), 7.40–7.73 (m, 8H, phenyl-H),
8.19 (d, J ¼ 7.8 Hz, 2H, phenyl C2,6–H), 8.53 (s, 1H, pyrazolo
pyrimidine C3–H). 13C NMR (DMSO-d6) ꢁ 31.12 (CH3), 103.74
(pyrazolopyrimidine C3a), 121.44, 127.21, 129.09, 129.48,
129.75, 130.42, 135.51, 137.11 (phenyl-C), 138.86 (pyrazolopyr-
imidine C3), 151.11 (pyrazolopyrimidine C7a), 156.51 (C ¼ O),
157.66 (pyrazolopyrimidine C6). MS (m/z, %): 366 (M+, 5.1), 287
(100). Anal. Calcd. for C18H14N4O3S (366.39): C, 59.01; H, 3.85;
N, 15.29. Found: C, 58.88; H, 4.16; N, 15.39.
1
(C–O–C). H NMR (400 MHz, DMSO-d6) ꢁ 2.78 (t, J ¼ 6.8 Hz,
2H, C-CH2), 3.48 (t, J ¼ 6.8 Hz, 2H, N-CH2), 3.70, 3.73 (2s, each
3H, 2OCH3), 6.22 (t, J ¼ 6.8 Hz, 1H, NH, D2O exchangeable),
6.68 (d, J ¼ 8.1 Hz, 1H, dimethoxyphenyl C6-H), 6.72 (s, 1H,
dimethoxy phenyl C2-H), 6.87 (d, J ¼ 8.1 Hz, 1H, dimethoxyphe-
nyl C5-H), 7.27–7.61 (m, 8H, phenyl-H), 8.14 (s, 1H, pyrazolo-
pyrimidine C3-H), 8.24 (d, J ¼ 8.0 Hz, 2H, phenyl C2,6-H).
13C NMR (DMSO-d6) ꢁ 34.66 (C–CH2), 44.05 (NCH2), 55.91,
56.06 (OCH3), 101.23 (pyrazolopyrimidine C3a), 112.57, 113.05,
120.25, 120.86, 126.38, 128.76, 129.43, 129.84, 130.56, 132.10,
135.03, 136.98, 139.59, 147.89, 149.22 (phenyl-C, dimethox-
yphenyl-C and pyrazolopyrimidine C3), 153.39 (pyrazolopyrimi-
dine C7a), 153.87 (C ¼ O), 157.93 (pyrazolopyrimidine C6).
MS (m/z, %): 467 (M+, 51), 151 (100). Anal. Calcd. for
C27H25N5O3 (467.52): C, 69.36; H, 5.39; N, 14.98. Found: C,
69.72; H, 5.41; N, 15.16.
1,5-Diphenyl-6-(propylamino)-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (5d). Yield: 0.44 g, 64%; m.p.: 144–146 ꢁC (methylene
chloride/petroleum ether 60/80) (2:1). IR (KBr, cm ꢀ 1): 3280
(NH), 3062, 2925 (CH), 1700 (C ¼ O), 1594 (C ¼ N), 1551, 1504,
General procedure for the synthesis of 1,5-diphenyl-6-(substituted
amino)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones (5a–i)
1
1492, 1458 (C ¼ C). H NMR (400 MHz, DMSO-d6) ꢁ 0.84 (t,
A mixture of 4 (0.73 g, 2 mmol) and the selected substituted J ¼ 6.9 Hz, 3H, CH3), 1.53–1.62 (m, 2H, C-CH2), 3.26 (q,
amine (2 mmol) in dry dioxane (10 ml) was heated under reflux J ¼ 6.9 Hz, 2H, N–CH2), 6.29 (t, J ¼ 6.9 Hz, 1H, NH, D2O
for 6–8 h. The reaction mixture was concentrated under reduced exchangeable), 7.30–7.62 (m, 8H, phenyl-H), 8.15 (s,
pressure and the remaining residue was triturated with diethyl 1H, pyrazolopyrimidine C3-H), 8.22 (d, J ¼ 8.0 Hz, 2H, phenyl
ether whereupon a white solid was separated. It was filtered, C2,6-H). 13C NMR (DMSO-d6) ꢁ 11.63 (CH3), 21.97 (CH2), 43.86
washed with diethyl ether and crystallized from the proper (N–CH2), 100.96 (pyrazolopyrimidine C3a), 120.66, 126.65,
solvent.
6-(Cyclohexylamino)-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimi-
129.46, 129.81, 130.09, 130.67, 134.95, 136.92 (phenyl-C),
139.41 (pyrazolopyrimidine C3), 154.01 (pyrazolopyrimidine
din-4(5H)-one (5a). Yield: 0.57 g, 74%; m.p.: 180–182 ꢁC C7a), 157.46 (C ¼ O), 158.16 (pyrazolopyrimidine C6). MS (m/z,
(methylene chloride/petroleum ether 60/80) (2:1). IR (KBr, %): 345 (M+, 52.8), 317 (100). Anal. Calcd. for C20H19N5O
cmꢀ1): 3332 (NH), 3064, 2930, 2849 (CH), 1700 (C ¼ O), 1597 (345.4): C, 69.55; H, 5.54; N, 20.28. Found: C, 69.48; H, 5.76;
1
(C ¼ N), 1556, 1492, 1454 (C ¼ C). H NMR (400 MHz, DMSO- N, 20.39.
d6) ꢁ 1.06–1.30 (m, 6H, cyclohexyl C3,4,5–H2), 1.53–1.82 (m, 4H,
6-(2-Morpholinoethylamino)-1,5-diphenyl-1H-pyrazolo[3,4-
cyclohexyl C2,6–H2), 3.83–3.85 (m, 1H, cyclohexyl C1–H), 5.43 d]pyrimidin-4(5H)-one (5e). Yield: 0.43 g, 52%; m.p.: 187–
(d, J ¼ 7.2 Hz, 1H, NH, D2O exchangeable), 7.17–7.63 (m, 8H, 189 ꢁC (ethanol). IR (KBr, cmꢀ1): 3288 (NH), 3063, 2946,
phenyl-H), 8.10 (s, 1H, pyrazolopyrimidine C3–H), 8.20 (d, 2902, 2845 (CH), 1702 (C ¼ O), 1596 (C ¼ N), 1553, 1474
J ¼ 7.6 Hz, 2H, phenyl C2,6-H). 13C NMR (DMSO-d6) ꢁ 25.10 (C ¼ C). 1H NMR (400 MHz, DMSO-d6) ꢁ 2.24–2.30 (m, 4H,
(cyclohexyl C3,5), 25.54 (cyclohexyl C4), 31.66 (cyclohexyl C2,6), morpholine C3,5-H2), 2.43 (t, J ¼ 6.6 Hz, 2H, NCH2), 3.38–3.47
51.83 (cyclohexyl C1), 101.13 (pyrazolopyrimidine C3a), 120.62, (m, 6H, morpholine C2,6–H2 and NCH2), 5.97 (s, 1H, NH, D2O
124.88, 127.36, 128.73, 129.43, 130.67, 134.85, 136.94 (phenyl- exchangeable), 7.31–7.71 (m, 8H, phenyl-H), 8.12 (s, 1H,
C), 139.43 (pyrazolopyrimidine C3), 153.07 (pyrazolopyrimidine pyrazolopyrimidine C3-H), 8.20 (d, J ¼ 8.0 Hz, 2H, phenyl C2,6
-
C7a), 153.19 (C ¼ O), 157.96 (pyrazolopyrimidine C6). MS (m/z, H). 13C NMR (DMSO-d6) ꢁ 38.65 (NCH2), 53.22 (morpholine
%): 385 (M+, 55), 288 (100). Anal. Calcd. for C23H23N5O C3,5), 55.90 (NCH2), 66.61 (morpholine C2,6), 101.31 (pyrazolo-
(385.46): C, 71.67; H, 6.01; N, 18.17. Found: C, 71.83; H, 6.09; pyrimidine C3a), 120.88, 126.43, 129.47, 129.81, 129.92, 130.71,
N, 18.35.
6-(Benzylamino)-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-
135.20, 137.00 (phenyl-C), 139.51 (pyrazolopyrimidine C3),
153.33 (pyrazolopyrimidine C7a), 153.86 (C ¼ O), 157.80 (pyr-
4(5H)-one (5b). Yield: 0.58 g, 74%; m.p.: 201–203 ꢁC (dioxane/ azolopyrimidine C6). MS (m/z, %): 416 (M+, 0.1), 100 (100).

4
A. Malki et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Anal. Calcd. for C23H24N6O2 (416.48): C, 66.33; H, 5.81; N, 107 (100). Anal. Calcd. for C21H21N5O4 (407.42): C, 61.91; H,
20.18. Found: C, 66.59; H, 5.86; N, 20.43.
5.20; N, 17.19. Found: C, 61.68; H, 5.46; N, 17.39.
6-(2-(Pyrrolidin-1-yl)ethylamino)-1,5-diphenyl-1H-pyra-
6-(2-Aminoethylamino)-1,5-diphenyl-1H-pyrazolo[3,4-d]pyri-
zolo[3,4-d]pyrimidin-4(5H)-one (5f). Yield: 0.41 g, 52%; m.p.: midin-4(5H)-one (5j). Yield: 0.36 g, 52%; m.p.: 199–201 ꢁC
106–107 ꢁC (methylene chloride/petroleum ether 60/80) (2:1). IR (methylene chloride/petroleum ether 60/80) (2:1). IR (KBr,
(KBr, cmꢀ1): 3318 (NH), 3062, 2950, 2924, 2850 (CH), 1721 cmꢀ1): 3357, 3302 (NH), 3063, 2957, 2917, 2858 (CH), 1694
1
1
(C ¼ O), 1597 (C ¼ N), 1557, 1493 (C ¼ C). H NMR (400 MHz, (C ¼ O), 1596 (C ¼ N), 1557, 1495 (C ¼ C). H NMR (400 MHz,
DMSO-d6) ꢁ 1.56–1.60 (m, 4H, pyrrolidine C3,4-H2), 2.36–2.40 DMSO-d6) ꢁ 2.72 (t, J ¼ 6.2 Hz, 2H, NCH2), 3.32 (t, J ¼ 6.2 Hz,
(m, 4H, pyrrolidine C2,5–H2), 2.57 (t, J ¼ 6.8 Hz, 2H, NCH2), 3.39 2H, NCH2), 6.30 (br s, 2H, NH2, D2O exchangeable), 6.40 (br s,
(m, 2H, NCH2), 6.13 (t, J ¼ 6.8 Hz, 1H, NH, D2O exchangeable), 1H, NH, D2O exchangeable), 7.31–7.62 (m, 8H, aromatic-H),
7.32–7.60 (m, 8H, phenyl-H), 8.15 (d, J ¼ 7.4 Hz, 2H, phenyl 8.15 (s, 1H, pyrazolopyrimidine C3–H), 8.20 (d, J ¼ 8.4 Hz, 2H,
C2,6-H), 8.32 (s, 1H, pyrazolopyrimidine C3-H). 13C NMR phenyl C2,6–H). 13C NMR (DMSO-d6) ꢁ 44.58, 45.96, (NCH2),
(DMSO-d6) ꢁ 22.56 (pyrrolidine C3,4), 34.63, 51.03 (NCH2), 101.15 (pyrazolopyrimidine C3a), 120.79, 126.43, 129.45, 129.61,
53.64 (pyrrolidine C2,5), 104.48 (pyrazolopyrimidine C3a), 129.93, 130.65, 135.0, 136.95 (phenyl-C), 139.40 (pyrazolopyr-
121.78, 128.50, 129.15, 129.71, 129.89, 130.46, 136.14, 136.90 imidine C3), 154.08 (pyrazolopyrimidine C7a), 158.09 (C ¼ O),
(phenyl-C), 138.66 (pyrazolopyrimidine C3), 150.71 (pyrazolo- 162.06 (pyrazolopyrimidine C6). MS (m/z, %): 346 (M+, 5.75),
pyrimidine C7a), 157.37 (C ¼ O), 164.32 (pyrazolopyrimidine 304 (100). Anal. Calcd. for C19H18N6O (346.39): C, 65.88; H,
C6). MS (m/z, %): 400 (M+, 48), 85 (100). Anal. Calcd. for 5.24; N, 24.26. Found: C, 66.02; H, 5.27; N, 24.53.
C23H24N6O (400.48): C, 68.98; H, 6.04; N, 20.99. Found: C,
69.07; H, 6.11; N, 21.24.
6-(2-Hydroxyethylamino)-1,5-diphenyl-1H-pyrazolo[3,4-
6-Ethoxy-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (6). Yield: 0.54 g, 82%; m.p.: 143–144 ꢁC (ethanol). IR (KBr,
cmꢀ1): 3052, 2979, 2930 (CH), 1714 (C ¼ O), 1571 (C ¼ N),
1
d]pyrimidin-4(5H)-one (5g). Yield: 0.44 g, 64%; m.p.: 172– 1550, 1531, 1493, 1421 (C ¼ C), 1265, 1056 (C–O–C). H NMR
173 ꢁC (dioxane/diethyl ether) (1:1). IR (KBr, cmꢀ1): 3436 (400 MHz, DMSO-d6) ꢁ 1.17 (t, J ¼ 6.9 Hz, 3H, CH3), 4.39 (q,
(OH), 3291 (NH), 3098, 3044, 2935, 2868 (CH), 1687 (C ¼ O), J ¼ 6.9 Hz, 2H, CH2), 7.30–7.70 (m, 8H, phenyl-H), 8.10 (d,
1
1596 (C ¼ N), 1554, 1495 (C ¼ C). H NMR (400 MHz, DMSO- J ¼ 7.8 Hz, 2H, phenyl C2,6–H), 8.35 (s, 1H, pyrazolopyrimidine
d6) ꢁ 3.40 (t, J ¼ 6.9 Hz, 2H, NCH2), 3.43 (t, J ¼ 6.9 Hz, 2H, C3–H). 13C NMR (DMSO-d6) ꢁ 14.22 (CH3), 66.0 (CH2), 103.75
OCH2), 4.70 (s, 1H, OH, D2O exchangeable), 6.01 (s, 1H, NH, (pyrazolopyrimidine C3a), 121.58, 127.21, 129.05, 129.43,
D2O exchangeable), 7.14–7.61 (m, 8H, phenyl-H), 8.01 (s, 1H, 129.93, 130.04, 135.51, 137.12 (phenyl-C), 138.36 (pyrazolopyr-
pyrazolopyrimidine C3-H), 8.21 (d, J ¼ 7.4 Hz, 2H, phenyl imidine C3), 151.11 (pyrazolopyrimidine C7a), 156.50 (C ¼ O),
C2,6–H). 13C NMR (DMSO-d6) ꢁ 44.40 (NCH2), 59.10 (OCH2), 157.66 (pyrazolopyrimidine C6). MS (m/z, %): 332 (M+, 87), 287
101.14 (pyrazolopyrimidine C3a), 120.53, 126.48, 129.71, (100). Anal. Calcd. for C19H16N4O2 (332.36): C, 68.66; H, 4.85;
130.01, 130.48, 130.70, 134.84, 136.92 (phenyl-C), 139.34 N, 16.86. Found: C, 68.38; H, 5.06; N, 17.09.
(pyrazolopyrimidine C3), 153.22 (pyrazolopyrimidine C7a),
153.98 (C ¼ O), 158.12 (pyrazolopyrimidine C6). MS (m/z, %):
General procedure for the synthesis of compounds 7a, b
347 (M+, 36.67), 302 (100). Anal. Calcd. for C19H17N5O2
Method A. A mixture of 4 (0.36 g, 1.0 mmol), aminoguanidine or
guanidine HCl (1.2 mmol) and anhydrous sodium acetate (0.1 g,
1.2 mmol) in dry dioxane/dimethyl sulfoxide (5:1) (25 ml) was
heated under reflux for 8 h. The reaction mixture was then
concentrated under vacuum then poured into cold water. The
separated solid was filtered, dried and crystallized from ethanol.
Method B. A mixture of 4 (0.36 g, 1.0 mmol), aminoguanidine
or guanidine bicarbonate (1.2 mmol) in dry dioxane/dimethyl
sulfoxide (5:1) (15 ml) was heated under reflux for 8 h. The
reaction mixture was then concentrated under vacuum then
poured into cold water. The separated solid was filtered, dried and
crystallized from ethanol.
(347.37): C, 65.69; H, 4.93; N, 20.16. Found: C, 65.87; H, 5.01;
N, 20.43.
6-(1-Hydroxy-2-methylpropan-2-ylamino)-1,5-diphenyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (5h). Yield: 0.46 g, 62%;
m.p.: 286–287 ꢁC (ethanol). IR (KBr, cmꢀ1): 3405 (OH), 3329
(NH), 3066, 3037, 2965, 2862 (CH), 1667 (C ¼ O), 1604 (C ¼ N),
1556, 1494, 1456 (C ¼ C). 1H NMR (400 MHz, DMSO-d6) ꢁ 1.60
(s, 6H, 2CH3), 3.60 (s, 2H, CH2), 4.50 (s, 1H, OH, D2O
exchangeable), 6.13 (s, 1H, NH, D2O exchangeable), 7.13–7.50
(m, 8H, phenyl-H), 8.28 (d, J ¼ 7.2 Hz, 2H, phenyl C2,6-H), 7.95
(s, 1H, pyrazolopyrimidine C3-H). 13C NMR (DMSO-d6) ꢁ 29.12
(CH3), 50.37 (C(CH3)2), 66.80 (OCH2), 99.71 (pyrazolopyrimi-
dine C3a), 120.34, 124.98, 126.84, 128.72, 129.11, 130.10,
136.73, 139.87 (phenyl-C), 140.60 (pyrazolopyrimidine C3),
157.71 (pyrazolopyrimidine C7a), 158.68 (C ¼ O), 160.95 (pyr-
azolopyrimidine C6). MS (m/z, %): 375 (M+, 31.8), 69 (100).
Anal. Calcd. for C21H21N5O2 (375.42): C, 67.18; H, 5.64; N,
18.65. Found: C, 67.35; H, 5.73; N, 18.91.
(4-Oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimi-
din-6-ylamino)guanidine (7a). Yield: 0.17 g, 48%, method A;
0.25 g, 70% method B, m.p.: 265–266 ꢁC (ethanol). IR (KBr,
cmꢀ1): 3280 (NH), 3062, 2925 (CH), 1700 (C ¼ O), 1594
1
(C ¼ N), 1551, 1504, 1492, 1458 (C ¼ C). H NMR (400 MHz,
DMSO-d6) ꢁ 4.46 (s, 2H, NH2, D2O exchangeable), 7.14–7.58
(m, 11H, 8 phenyl-H and 3NH), 7.93 (d, J ¼ 7.6 Hz, 2H, phenyl
C2,6–H), 8.07 (s, 1H, pyrazolopyrimidine C3–H). 13C NMR
(DMSO-d6) ꢁ 101.92 (pyrazolopyrimidine C3a), 121.80, 126.82,
127.04, 128.93, 129.33, 129.81, 129.90, 135.87 (phenyl-C),
138.80 (pyrazolopyrimidine C3), 152.49 (pyrazolopyrimidine
C7a), 158.31 (C ¼ O), 159.04 (pyrazolopyrimidine C6), 160.90
(C ¼ NH). MS (m/z, %): 360 (M+, 56), 288 (100). Anal. Calcd. for
C18H16N8O (360.37): C, 59.99; H, 4.48; N, 31.09. Found: C,
59.68; H, 4.46; N, 31.19.
6-(1,3-Dihydroxy-2-(hydroxymethyl)propan-2-ylamino)-1,5-
diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (5i). Yield:
0.34 g, 42%; m.p.:4300 ꢁC (ethanol). IR (KBr, cmꢀ1): 3280
(NH), 3062, 2925 (CH), 1700 (C ¼ O), 1594 (C ¼ N), 1551, 1504,
1
1492, 1458 (C ¼ C). H NMR (400 MHz, DMSO-d6) ꢁ 3.50 (s,
6H, 3CH2), 4.40 (s, 1H, OH, D2O exchangeable), 6.3 (s, 1H, NH,
D2O exchangeable), 7.30–7.78 (m, 8H, phenyl-H), 8.08–8.22 (m,
2H, phenyl C2,6–H), 8.4 (s, 1H, pyrazolopyrimidine C3–H). 13C
NMR (DMSO-d6) ꢁ 60.12 (C(CH2OH)3), 65.98 (OCH2), 103.73
(pyrazolopyrimidine C3a), 121.42, 127.13, 129.07, 129.46,
129.72, 129.93, 135.51, 137.08 (phenyl-C), 138.36 (pyrazolopyr-
imidine C3), 151.09 (pyrazolopyrimidine C7a), 156.46 (C ¼ O),
157.64 (pyrazolopyrimidine C6). MS (m/z, %): 407 (M+, 96.8),
(4-Oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)guanidine (7b). Yield: 0.17g, 50%, method A; 0.25g, 73%
method B, m.p.: 275–277 ꢁC. IR (KBr, cmꢀ1): 3401, 3350, 3190
(NH), 3057, 2893, 2836 (CH), 1676 (C ¼ O), 1611 (C ¼ N), 1574,

DOI: 10.3109/14756366.2015.1118686
Apoptosis in RKO colon cancer cells
5
1493 (C ¼ C). 1H NMR (400 MHz, DMSO-d6) ꢁ 7.08–7.76 13H, phenyl-H), 7.94 (d, J ¼ 7.6 Hz, 2H, phenyl C2,6–H), 8.42 (s,
(m, 12H, 8 phenyl-H and 4NH), 7.94 (d, J ¼ 7.6 Hz, 2H, phenyl 1H, pyrazolopyrimidine C3–H), 8.91, 9.77 (2s, each 1H, 2NH,
C2,6-H), 8.10 (s, 1H, pyrazolopyrimidine C3–H). 13C NMR D2O exchangeable). 13C NMR (DMSO-d6) ꢁ 101.76 (pyrazolo-
(DMSO-d6) ꢁ 102.15 (pyrazolopyrimidine C3a), 121.97, 127.52, pyrimidine C3a), 121.42, 121.68, 121.69, 122.57, 127.65, 127.84,
128.03, 128.78, 128.93, 129.88, 129.94, 136.93 (phenyl-C), 139.21 129.90, 129.33, 132.44, 133.94, 134.11, 138.90 (phenyl-C),
(pyrazolopyrimidine C3), 152.33 (pyrazolopyrimidine C7a), 157.76 139.22 (pyrazolopyrimidine C3), 152.65 (pyrazolopyrimidine
(C ¼ O), 158.0 (pyrazolopyrimidine C6), 159.92 (C ¼ NH). MS (m/ C7a), 153.40 (C ¼ O), 157.67 (pyrazolopyrimidine C6), 164.0
z, %): 345 (M+, 100). Anal. Calcd. for C18H15N7O (345.36): C, (C ¼ O). MS (m/z, %): 422 (M+, 75), 289 (100). Anal. Calcd. for
62.60; H, 4.38; N, 28.39. Found: C, 62.87; H, 4.33; N, 28.62.
C24H18N6O2 (422.44): C, 68.24; H, 4.29; N, 19.89. Found: C,
68.45; H, 4.13; N, 19.55.
6-Hydrazino-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (8)
N0-(4-Oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyri-
midin-6-yl)-4-methoxybenzohydrazide (10b). Yield: 0.7 g, 70%;
m.p.: 245–247 ꢁC (dioxane/water). IR (KBr, cmꢀ1): 3270, 3164
(NH), 3058, 2949 (CH), 1677, 1705 (C ¼ O), 1590 (C ¼ N), 1554,
1490, 1450, 1415 (C ¼ C), 1260, 1025 (C–O–C). 1H NMR
(400 MHz, DMSO-d6) ꢁ 3.70 (s, 3H, OCH3), 6.90 (d, J ¼ 8.4 Hz,
2H, methoxyphenyl C3,5–H), 7.06–7.68 (m, 10H, phenyl-H),
7.88 (d, J ¼ 8.4 Hz, 2H, methoxyphenyl C2,6–H), 8.10 (s, 1H,
pyrazolopyrimidine C3–H), 8.82, 9.76 (2s, each 1H, 2NH, D2O
exchangeable). 13C NMR (DMSO-d6) ꢁ 55.61 (OCH3), 101.84
(pyrazolopyrimidine C3a), 114.24 (methoxyphenyl C3,5), 121.12,
121.69, 126.22, 126.73, 128.83, 128.74, 129.74, 130.45, 134.12,
136.45, 143.36, 151.52, 156.22 (phenyl-C, methoxyphenyl C1,2,4,6
A mixture of 4 (7.3 g, 20 mmol) and hydrazine hydrate 98% (2.0 g,
1.94 ml, 40 mmol) in ethanol (40 ml) was stirred at room
temperature for 6 h. The separated white solid was filtered,
washed with ethanol, dried and crystallized from dioxane. Yield:
4.95 g, 78% (reported 38%); m.p.: 268–270 ꢁC (as reported)38.
General procedure for the synthesis of N0-(4-oxo-1,5-diphenyl-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)substituted sulfo-
hydrazides 9a, b and N0-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-6-yl)substituted hydrazides (10a, b)
To a suspension of 8 (0.7 g, 2.2 mmol) in dry pyridine (7 ml), the and pyrazolopyrimidine C3,7a), 156.44 (C ¼ O), 158.41 (pyrazo-
appropriate aryl sulfonyl chloride or aroyl chloride (2.6 mmol) lopyrimidine C6), 163.5 (C ¼ O). MS (m/z, %): 452 (M+, 33), 289
was slowly added at ice-cold temperature. The reaction mixture (100). Anal. Calcd. for C25H20N6O3 (452.46): C, 66.36; H, 4.46;
was stirred at room temperature over night then poured into ice- N, 18.57. Found: C, 66.18; H, 4.30; N, 18.22.
cold water. The obtained precipitate was filtered, washed with
water, dried and crystallized from the proper solvent.
N0-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyri-
midin-6-yl)benzenesulfohydrazide (9a). Yield: 0.54 g, 54%; m.p.: pyrimidin-4(5H)-ones (11a–c)
General procedure for the synthesis of 6-(2-(substituted
benzylidene)hydrazino)-1,5-diphenyl-1H-pyrazolo[3,4-d]
216–218 ꢁC (ethanol). IR (KBr, cmꢀ1): 3380, 3244 (NH), 3099,
A mixture of 6 (0.7 g, 2.2 mmol) and the appropriate aldehyde
3052, 2917 (CH), 1704 (C ¼ O), 1566 (C ¼ N), 1525, 1495, 1459,
1426 (C ¼ C), 1341, 1167 (SO2). 1H NMR (400 MHz, DMSO-d6)
ꢁ 7.30–7.95 (m, 13H, phenyl-H), 8.10 (d, J ¼ 7.6 Hz, 2H, phenyl
C2,6–H), 8.45 (s, 1H, pyrazolopyrimidine C3–H), 8.90, 9.98 (2s,
each 1H, 2NH, D2O exchangeable). 13C NMR (DMSO-d6)
ꢁ 101.79 (pyrazolopyrimidine C3a), 121.37, 121.61, 121.70,
122.53, 127.04, 129.36, 129.50, 129.93, 132.30, 134.05, 138.93,
139.10 (phenyl-C), 139.19 (pyrazolopyrimidine C3), 152.69
(pyrazolopyrimidine C7a), 153.33 (C ¼ O), 157.65 (pyrazolopyr-
imidine C6). MS (m/z, %): 458 (M+, 94), 289 (100). Anal. Calcd.
for C23H18N6O3S (458.49): C, 60.25; H, 3.96; N, 18.33. Found: C,
60.41; H, 4.02; N, 18.47.
(2.2 mmol) in absolute ethanol (20 ml) containing two drops
glacial acetic acid was heated under reflux for 4 h. The reaction
mixture was allowed to attain room temperature and the separated
solid product was filtered, washed with ethanol, dried and
crystallized from the proper solvent.
6-(2-(4-Methoxybenzylidene)hydrazino)-1,5-diphenyl-1H-pyr-
azolo[3,4-d]pyrimidin-4(5H)-one (11a). Yield: 0.79 g, 82%; m.p.:
238–239 ꢁC (ethanol) (reported m.p.: 239–240 ꢁC)38.
6-(2-(3,4-Dimethoxybenzylidene)hydrazino)-1,5-diphenyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (11b). Yield: 0.86 g, 84%;
m.p.: 225–226 ꢁC (dioxane) (reported m.p.: 215–216 ꢁC)38.
6-(2-(3,4,5-Trimethoxybenzylidene)hydrazino)-1,5-diphenyl-
1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (11c). Yield: 0.92 g,
84%; m.p.: 236–237 ꢁC (ethanol) (reported m.p.: 240–241 ꢁC)38.
N0-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyri-
midin-6-yl)-4-methoxy benzenesulfohydrazide (9b). Yield: 0.56 g,
54%; m.p.: 238–240 ꢁC (ethanol). IR (KBr, cm ꢀ 1): 3271, 3158
(NH), 3061, 2951 (CH), 1707 (C ¼ O), 1594 (C ¼ N), 1556, 1493,
1
Biological screening
1458, 1416 (C ¼ C), 1342, 1162 (SO2), 1262, 1024 (C–O–C). H
NMR (400 MHz, DMSO-d6) ꢁ 3.75 (s, 3H, CH3), 7.05–7.93 (m,
12H, phenyl-H and methoxyphenyl C3,5–H), 8.0 (d, J ¼ 7.6 Hz,
2H, methoxyphenyl C2,6–H), 8.07 (s, 1H, pyrazolopyrimidine C3-
H), 8.80, 9.84 (2s, each 1H, 2NH, D2O exchangeable). 13C NMR
(DMSO-d6) ꢁ 56.50 (OCH3), 101.80 (pyrazolopyrimidine C3a),
114.63 (methoxyphenyl C3,5), 121.66, 126.76, 128.02, 128.34,
129.75, 129.92, 130.43, 132.45, 134.05, 136.40, 143.34, 151.48,
154.12 (phenyl-C, methoxyphenyl C1,2,4,6 and pyrazolopyrimidine
C3,7a), 155.24 (C ¼ O), 157.44 (pyrazolopyrimidine C6). MS (m/z,
%): 488 (M+, 34.19), 185 (100). Anal. Calcd. for C24H20N6O4S
(488.52): C,59.01; H, 4.13; N, 17.20. Found: C, 59.19; H, 4.31; N,
17.02..
In vitro anti-tumor activity
MTT bromide mitochondrial activity assay. Human cancer cell
lines, lung cancer cells (H1299), liver cancer cells (HepG2), colon
cancer cells (RKO), human vulvar epidermoid carcinoma cells
(A431) and breast cancer cells (MCF-7) were obtained from
ATCC (Manassas, VA). Cells were grown in RPMI 1640 medium
containing 10% fetal bovine serum at 37 ꢁC and 5% CO2. FHC
cells were cultured in DMEM-F12 containing 25 mM HEPES,
10 ng/ml cholera toxin, 5 mg/ml insulin, 5 mg/ml transferrin,
100 ng/ml hydrocortisone and 10% fetal bovine serum. The
effect of compounds on the proliferation of cancer cells was
determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
liumbromide (MTT; Sigma, St. Louis, MO) assay. Cancer cells
were seeded into 96-well plates at a concentration of 5000 cells
per well with a culture medium exposed to each derivative at
different concentrations. After 24 h incubation, MTT was added
N0-(4-Oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyri-
midin-6-yl)benzohydrazide (10a). Yield: 0.7 g, 75%; m.p.: 228–
230 ꢁC (dioxane). IR (KBr, cm ꢀ 1): 3374, 3246 (NH), 3098, 3047,
2916 (CH), 1680, 1702 (C ¼ O), 1565 (C ¼ N), 1528, 1490, 1465,
1
1429 (C ¼ C). H NMR (400 MHz, DMSO-d6) ꢁ 7.03–7.74 (m,

6
A. Malki et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
into each well. The absorbance of formazan product of MTT was iodide (PI) and will remain unstained by the Annexin V-FITC
measured at 570 nm using a microtiter plate reader. The probe.
percentage of cell viability was determined as the ratio of the
Flow cytometric analysis. Cells were seeded at a density of 3–
absorbance of the sample versus the control. Each assay was 5 ꢂ 105/10 cm plate and incubated for 24 h before treatment.
performed in triplicate and standard deviation was determined. Media were changed to media containing compounds 7a and 7b.
The IC50 of each derivative was calculated as the concentration After 24 h, cells were harvested by trypsinization. The cells were
showing 50% cell growth inhibition during 24 h of compound washed with PBS and fixed with ice-cold 70% ethanol while
treatment.
vortexing. Finally, the cells were washed and resuspended in PBS
Enzyme-linked immunosorbent apoptosis assay. Cells were containing 5 mg/ml RNase A (Sigma) and 50 mg/ml propidium
seeded at a density of 2 ꢂ 104/well in a 96-well plate and iodide (Sigma) for analysis. Cell cycle analysis was performed
incubated for 24 h. Media were changed to media containing using FACScan Flow Cytometer (Becton Dickson, Franklin
compounds 7a and 7b (8 and 4 mM, respectively). Cells were then Lakes, NJ) according to the manufacturer’s protocol. Windows
incubated for 24 h. An ELISA assay was performed using Cell multiple document interfaces (WinMDI, Scripps, CA) software
Death Detection ELISA PLUS kit (Roche-Applied Science, was used to calculate the cell-cycle phase distribution from the
Indianapolis, IN) for the quantitative in vitro determination of resultant DNA histogram, and data were expressed as a percent-
cytoplasmic histone-associated DNA fragments (mono- and age of cells in the G0/G1 and G2/M phases. The apoptotic cells
oligonucleosomes) in apoptosing cells. Briefly, cells were lysed were identified on the DNA histogram as a subdiploid peak.
with 200-ml lysis buffer for 30 min at room temperature. The
Western blot analysis. Total protein was extracted from treated
lysate was centrifuged at 200g for 10 min. One hundred and fifty and untreated cells using lysis buffer (10 mM Tris–HCl (pH 7.5),
microliters of supernatant was collected, of which 20 ml was 1 mM EDTA, 1% triton X-100, 150 mM NaCl, 1 mM dithiothretol,
incubated with anti-histone biotin and anti-DNA peroxidase at 10% glycerol, 0.2 mM phenylmethysulphonyl fluoride and prote-
room temperature for 2 h. After washing with incubation buffer ase inhibitors) for 30–50 min on ice. The extracts were
three times, 100 ml of substrate solution (2,20-azino-di(3-ethyl- centrifuged at 13 000g for 15 min at 4 ꢁC to remove cell debris.
benzthiazolin-sulphuric acid) was added to each well and Folin Lowry (Pierce, Grand Island, NY) protein assay was used to
incubated for 15–20 min at room temperature. The absorbance figure the protein concentration in the cell lysates. Proteins were
was measured using Spectra Max Plus reader (Sunnyvale, CA) at resolved by electrophoresis on 8–10% sodium dodecyl sulfate
405 nm. Cells without compounds treatment were used as control polyacrylamide gel loading equal amount of proteins per lane.
group. Each assay was done in triplicate and the standard The resolved proteins were transferred onto polyvinylidene
deviation was determined.
difluoride membrane and then probed with primary antibody
Detection of apoptosis by TUNEL method. For in situ against the protein of interest prepared in 5% milk/PBS-T. The
detection of apoptotic cells, TUNEL (terminal-deoxynucleotidyl membrane was washed using PBS with Tween 20 (PBS-T) and
transferase meditated nick end labeling) assay was performed then appropriate secondary antibody conjugated to horseradish
using DeadEndTM fluorimetric tunnel system (Promega , Radnor, peroxidase was used for visualization of the bands using ECL
PA). Cells were cultured on four-chamber slides (VWR, Radnor, chemiluminescence kit (GE, Pittsburgh, PA). Anti-(p21), anti-
PA) at a density of 2 ꢂ 104 cells/chamber. After treatment with (p53), anti-Bax, anti-ERK1/2, anti-Akt, and anti-Bcl-2, anti-
compounds 7a and 7b (8 and 4 mM, respectively), cells were cytochrome-c, anti-caspase-9, anti-caspase-3, anti-PARP1, were
washed with phosphate buffer saline (PBS) and fixed by purchased from SantaCruz, Dollas, TX. Pixel density of the
incubation in 4% paraformaldehyde for 20 min at 4 ꢁC, then proteins studied was calculated using Image J, version 1.41o, NIH
permeabilized with 0.05% triton X-100 for 5 min at 4 ꢁC. The (Scripps, North Torrey, CA). The values obtained were first
fixed cells were then incubated with digoxigenin-conjugated normalized to loading control b-actin and the fold change was
dUTP in terminal deoxynucleotide transferase recombinant measured by normalizing to that of the control (0 h) value. At
(rTdT)-catalyzed reaction and nucleotide mixture for 60 min at least two independent experiments were performed.
37 ꢁC in a humidified atmosphere and then immersed in stop/wash
Caspase-3 activation assay. Caspase-3 activity was assayed
buffer for 15 min at room temperature. Cells were then washed according to the manufacturer’s protocol. Briefly, 5 ꢂ 106 cells
with PBS to remove unincorporated fluorescein-12-dUTP. After were lysed in 100 ml lysis buffer containing 10 mM HEPES (4-
washing with PBS, cells were incubated in 1 mg/ml 2-(4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 2 mM
amidinophenyl)-6-indole carbamidine dihydrochloride (DAPI) EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propa-
and fluorescin isothiocyanate (FITC) solution for 15 min in dark nesulfonate (CHAPS), 350 mg/ml phenylmethylsulfonyl fluoride
(data not shown). Cells were observed with fluorescence micros- and 5 mM dithiothreitol. Cells were homogenized by three
copy (RT slider Spot, Diagnostic Instruments, Inc.) and photo- cycles of freezing and thawing and then centrifuged to remove
graphed at 100 ꢂ magnification.
the cellular debris. Each sample was then incubated in buffer
Annexin V staining assay. The assay is used to measure and containing 10 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS,
compare changes in apoptosis, 1 ꢂ 106 cells were seeded in 100- 5 mM EDTA supplemented with Ac-DEVD-AFC (acetyl-Asp-
mm dishes and treated as described above, immediately Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) for
1 h at
trypsinized, counted for trypan blue exclusion, washed in PBS, room temperature and then reaction was stopped with 1 N
and resuspended in 100 ml binding buffer containing Annexin V HCl. The optical density at 405 nm (OD 405) was measured
(Annexin V-Fluos kit, Roche, Switzerland). Cells were analyzed using a spectrophotometer (Spectra Max Plus, Sunnyvale, CA).
by flow cytometry using FACScan Flow Cytometer (Becton
Caspase-9 activation assay. The assay is based on the ability of
Dickson, Franklin Lakes, NJ) according to the manufacturer’s the active enzyme to cleave the chromophore from the enzyme
protocol after the addition of propidium iodide. Cells in the early substrate LEHD-pNA (Ac-Leu-Glu-His-Asp-pNA). Cell lysates
stages of apoptosis with intact cell membranes and surface were incubated with peptide substrate in assay buffer (100 mM
exposed phosphatidylserine will stain positive for Annexin V- NaCl, 50 mM HEPES, 10 mM dithiothreitol, 1 mM EDTA, 10%
FITC. Late apoptotic and necrotic cells, which have lost plasma glycerol and 0.1% CHAPS, pH 7.4) for 2 h at 37 ꢁC. The release of
membrane integrity, will become dually labeled with Annexin V- p-nitroaniline was monitored at 405 nm. Results are represented
FITC and Propidium Iodide (green and red fluorescence). Live as the percentage of change of activity compared with the
cells with intact plasma membranes will exclude propidium untreated control. The assay was repeated with and without

DOI: 10.3109/14756366.2015.1118686
Apoptosis in RKO colon cancer cells
7
Scheme 1. Synthetic pathways for the target
compounds 5–7.
O
EtOOC
O
N
N
N
N
H N
N
2
N
N
ii
i
N
H CS
N
S
N
H
3
2
3
1
iii
O
N
O
O
N
N
N
N
N
N
vi
iv
N
N
1
N
H CO S
R
N
3
2
N
RHN
7a,b
4
5a-j
v
O
N
N
N
N
H CH CO
3
2
6
For 5:a, R= cyclohexyl; b, R= benzyl; c, R= 3,4-dimethoxyphenethyl
d, R = -CH CH CH ; e, R = morpholinoethyl; f, R= pyrrolidinoethyl;
2
2
3
g, R = -CH CH OH; h, R= -C(CH ) (CH OH); i, R= -C(CH OH) ; j, = -CH CH NH
2
2
3 2
2
2
3
2
2
2
1
1
For 7:a, R = -NHNHC(=NH)-NH ; b, R = -NHC(=NH)-NH
2
2
Reagents and Conditions: i) methyl N-phenyldithiocarbamate / 1N NaOH / DMF /
reflux; ii) methyl iodide / anhydrous K CO / DMF / r.t.; iii) potassium
2
3
hydrogenpersulfate / DMF / 0°C; iv) the appropriate amine / dry dioxane / reflux; v)
aminoguanidine or guanidine HCl / anhydrous NaOAC / absolute EtOH/reflux; vi)
aminoguanidine or guanidine HCl / anhydrous NaOAC / dioxane, DMSO mixture / reflux
(method A); aminoguanidine or guanidine bicarbonate / dioxane, DMSO mixture / reflux
(methodB)
caspase-9 inhibitor (LEDH-CHO, Ac-Ala-Ala-Val-Ala-Leu-Leu- methylsulfanyl derivative 3. At this stage, it was decided to
Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-His-Asp- oxidize the methylsulfanyl moiety to the corresponding methyl-
CHO). For inhibition studies, cells were pre-incubated with sulfonyl group to be utilized in the subsequent nucleophilic
20 mM LEHD-CHO for1 h before the addition of 4 mM 7b for an substitution reactions for preparation of the target compounds.
additional 24 h. Each value was expressed as mean SD of three Oxidation of 3 to the corresponding methylsulfonyl derivative 4
determinations.
was achieved by reaction with potassium hydrogenpersulfate
(oxone) following reaction conditions previously reported for the
synthesis of analogous compounds39. IR spectrum of the latter
compound displayed absorption bands of SO2 at 1346 and
1162 cmꢀ1, while its 1H NMR spectrum showed a relatively
deshielded singlet at 2.76 ppm attributed to SO2CH3 protons.
Moreover, the mass spectrum showed a molecular ion peak at m/
z ¼ 366, which was in accordance with its molecular formula.
Refluxing the methylsulfonyl derivative 4 with cycloalkyl, aryl,
aralkyl or alkyl amines in dry dioxane resulted in the target 6-
substituted amino-1H pyrazolo[3,4-d]pyrimidin-4(5H)ones 5a–j
in a good yield. In the first trial to prepare the aminoguanidino
and guanidino derivatives (7a, b) the methylsulfonyl derivative 4
was reacted with the corresponding aminoguanidine or guanidine
HCl salts in ethanol in presence of anhydrous sodium acetate but
the 6-ethoxy derivative 6 was separated as a major product
indicating nucleophilic replacement of the methylsulfonyl moiety
by the solvent. However, when a mixture of dry dioxane/dimethyl
sulfoxide (5:1) was used as solvent (method A) instead of ethanol
Statistical analysis
The quantitative ratios of different groups were compared using
Student’s t-test. Probability values of p50.05 were regarded as
statistically significant. All statistical tests were two sided.
Results and discussion
Chemistry
The synthetic strategies adopted for the synthesis of the
intermediate and final compounds are depicted in Schemes 1
and 2. In Scheme 1, the amino ester 136 was readily cyclized to the
corresponding 6-thioxo-6,7-dihydro-1H-pyrazolo[3,4-d]pyrimi-
din-4(5H)-one 237 through reaction with methyl N-phenyldithio-
carbamate in DMF containing 1 N NaOH adopting previously
reported procedure. Stirring 2 with methyl iodide in dry DMF
containing anhydrous potassium carbonate gave rise to the

8
A. Malki et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
O
N
Scheme 2. Synthetic pathways for the target
compounds 9–11.
N
N
N
H CO S
3
2
4
i
O
N
N
N
N
H NHN
2
8
ii
iv
O
N
O
N
iii
N
N
H
N
N
N
1
R
N
N
N
RO S
N
N
H
2
H
9a, b
11a-c
O
N
N
H
N
R
N
N
N
H
O
10a, b
For 9, 10:a, R= phenyl; b, R= 4-methoxyphenyl
For 11: a, R = 4-methoxyphenyl; b, R = 3,4-dimethoxyphenyl; c, R = 3,4,5-trimethoxyphenyl
1
1
1
Reagents and Conditions: i)NH NH .xH O 98% / absoluteEtOH / r.t. ii)benzene or p-methoxy
2
2
2
benzene sulfonylchloride / dry pyridine / r.t.; iii) benzoyl or p-methoxybenzoyl chloride /
dry pyridine /r.t.; iv) aryl aldehyde / EtOH / reflux
Table 1. IC50 (mM)* of the synthesized 6-substituted 1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-ones against five cancer cell lines.
it was possible to isolate compounds 7a, b in a moderate yield. In
another trial to prepare these compounds, the bicarbonate salts of
aminoguanidine and guanidine were refluxed with the methyl-
sulfonyl derivative 4 in a mixture of dry dioxane/dimethyl
sulfoxide (5:1) (method B), and it was possible to isolate
compounds 7a, b in a better yield. IR spectra of compounds 7a,
Compound No.
H1299
HepG2
RKO
A431
MCF-7
5a
5b
5c
5d
5e
5f
5g
5h
5i
26
32
36
35
26
19
38
28
16
12
24
18
49
63
44
47
54
52
47
36
64
75
66
92
48
22
44
64
88
75
89
b displayed NH absorption bands in the range 3401–3190 cmꢀ1
,
4100
52
15
while 1H NMR spectra revealed D2O exchangeable singlets
characteristic for aminoguanidine and guanidine NH at their
expected chemical shifts. Moreover, 13C NMR showed resonance
for the aminoguanidino and guanidino carbon at 160.90 and
159.92 ppm, respectively.
62
4100
69
4100
70
88
4100
77
22
95
35
25
18
14
19
77
28
48
18
11
8
4
78
95
28
5j
24
35
23
22
In Scheme 2, the methylsulfonyl derivative 4 was utilized for the 7a
4100
4100
4100
4100
66
7b
9a
9b
10a
10b
11a
11b
11c
Cisplatin
preparation of the starting hydrazino compound 8. Thus, stirring an
ethanolic suspension of 4 with hydrazine hydrate at room
temperature afforded the hydrazine derivative 8 in an excellent
yield. It should be noted here that compound 8 was differently
prepared in a low yield38 from the corresponding thione 2.
Reacting 8 with benzene or p-methoxybenzene sulfonylchloride in
dry pyridine resulted in the arylsulfonyl derivatives 9a, b in a good
yield. IR spectra of these compounds showed the characteristic SO2
absorption bands at 1341, 1342 and 1162, 1167 cmꢀ1, while their
1H NMR spectra revealed two D2O-exchangeable signals at 8.80,
8.90 and 9.84, 9.98 ppm attributed to two NH groups. Similarly,
4100
4100
37
4100
40
30
4100
24
10
4100
95
12
28
27
22
29
52
90
88
86
20
4100
66
25
88
4100
65
20
4
12
17
13
H1299, human Lung carcinoma cell line; HepG2, liver cancer cell line;
RKO, human colon cancer cell line; A431, human vulvar epidermoid
carcinoma cell line; MCF-7, human breast cancer cell line.
reacting 8 with benzoyl or p-methoxybenzoyl chloride in dry *The values are means of three independent experiments.

DOI: 10.3109/14756366.2015.1118686
Apoptosis in RKO colon cancer cells
9
Figure 2. Compounds 7a and 7b reduced cell viability and induced apoptosis in RKO cells. (A) A dose–response curve for compounds 7a and 7b on
FHC normal colon cancer cells was constructed. Each data point was an average of results from three independent experiments performed in triplicate
and presented as mean SD. (B) Comparison of 7a and 7b cytotoxicity in FHC and RKO cells. Both cells were treated with 8 and 4 mM of compounds
7a and 7b, respectively. Untreated cells were used as control. (C) Comparison of 7a and 7b induced apoptosis in FHC and RKO cells. FHC and RKO
cells were treated with 8 and 4 mM of compounds 7a and 7b, respectively, for 24 h. Untreated cells were used as control. ELISA assay was applied for
apoptotic cell detection. Each experiment was performed in triplicate. Data are presented as mean SD (n ¼ 3). Significant differences between the
control and test compounds are indicated by * (*p50.05). (D) TUNEL assay was used to confirm induction of apoptosis in RKO cells. Cells were
treated with 8 and 4 mM of 7a and 7b, respectively, for 24 h. Untreated cells act as control. Lack of staining in control (untreated) cells, represents that
the cells are actively proliferating, i.e. without apoptotic cell death and induction of apoptosis in treated cells was confirmed by appearance of TUNEL
positive cells. Images were acquired for each fluorescence channel, using suitable filters at 200 ꢂ magnification.
pyridine resulted in the aroyl derivatives 10a, b. Finally, it was least sensitive cell line toward the test compounds, whereas the
planned to synthesize some arylhydrazones incorporating methoxy RKO cell line was the most sensitive. Compounds 7a and 7b
groups in order to study the effect of magnification of compounds’ emerged with IC50 values against RKO cells (IC50 ¼8 and 4 mM,
lipophilicity40,41 on the anticipated biological activity. respectively) lower than cisplatin (IC50 ¼17 mM), whereas they
Consequently, condensation of 8 with methoxylated aromatic showed IC50 values against A431 (IC50 ¼17 and 20 mM, respect-
aldehydes in refluxing ethanol furnished the target azomethine ively) comparable to that of cisplatin (IC50 ¼20 mM). Moreover,
derivatives 11a–c in a good yield as previously reported38.
compounds 10b and 11c exhibited the lowest IC50 values in this
study against MCF-7 cells (IC50 ¼10 and 12 mM, respectively)
compared to the reference standard (IC50 ¼13 mM). Since com-
pounds 7a and 7b showed the lowest IC50 values in this study
against RKO colon cancer cells, they were further investigated for
Biological screening
Cytotoxicity test
Cytotoxicity of the test compounds was assayed in five cancer cell their cytotoxicity against normal colon cells (FHC). The MTT cell
lines, namely lung cancer cells (H1299), liver cancer cells viability assay indicated that compounds 7a and 7b were less
(HepG2), colon cancer cells (RKO), human vulvar epidermoid cytotoxic to FHC cells (Figure 2A) and they reduced cell viability
carcinoma cells (A431) and breast cancer cells (MCF-7) by a to approximately 50% in RKO colon cancer cells compared to
tetrazolium based (MTT) colorimetric assay and utilizing cis- approximately 76% and 87% in FHC normal colon cancer cells
platin as a reference standard. The results of in vitro cytotoxic after treatment of cells with 8 and 4 mM of 7a and 7b, respectively
activity are expressed as IC50, which is the concentration of (Figure 2B).
compound (in mM) that inhibits survival of the cells by 50%
(Table 1). The effect of the synthesized compounds on the growth structure–activity relationships could be tentatively deduced.
of the five cancer cell lines revealed that HepG2 cell line was the Compounds comprising an amino group at position
Based on the results in Table 1, the following preliminary
6

10 A. Malki et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Figure 3. Impact of 7a and 7b on cell cycle. (A and B) Flow cytometric analysis of RKO cells stained with Annexin V-FITC and PI. (A) Untreated
RKO cells were used as control, (B) RKO cells treated with 8 mM 7a and 4 mM 7b for 24 h. (C) Comparison of 7a and 7b induced apoptosis in RKO
colon cancer cells using Annexin V/FITC staining assay. Each bar represents the mean SD (n ¼ 3). (D) Percent of distribution of cell cycle phases of
RKO colon cancer cells after treatment with 7b for 24 h. (E) A representative image of flow cytometric analysis showing G1 arrest. (F) Total protein
was extracted from RKO cells and used for Western blotting analysis to determine the differences in p21 between untreated cells (control; C) and cells
treated with 7b for 24 h. b-Actin was used as loading control.

DOI: 10.3109/14756366.2015.1118686
Apoptosis in RKO colon cancer cells 11
Figure 4. Effect of compound 7b on apoptotic
signaling in RKO cells. (A) Cells were
treated with 4 mM of compound 7b for 24 h.
The cell lysates were collected and the levels
of p53, Akt, Bcl-2, ERK1/2 were studied by
Western blotting analysis using specific
antibodies. b-Actin was used as loading
control. C: control (untreated cells). (B) Total
protein and mitochondrial fraction were
exacted and Western blotting analysis was
applied to determine the protein levels of Bax
in treated and non-treated cells. b-Actin was
used as loading control and IV-Cox was used
as internal mitochondrial control.
substituted with cyclohexyl, aralkyl, alkyl or substituted alkyl
Finally, it could be concluded that presence of substituents
moieties (5a–g) exhibited mild activity against the five cell such as aminoguanidino or guanidino at position 6 of the
lines except for compound 5b (benzyl derivative) that displayed pyrazolo[3,4-d]pyrimidinone scaffold (compounds 7a and 7b)
significant activity against A431 (IC50 ¼15 mM) and 5f potentiated the cytotoxic activity especially against RKO colon
(pyrrolidinoethyl derivative), which demonstrated moderate cancer cell line. Accordingly, compounds 7a and 7b were chosen
activity against H1299 and A431 (IC50 ¼19 and 15 mM, for further investigations.
respectively). Substitution at C-1 of 5g (2-hydroxyethyl deriva-
tive) by two methyl groups or two hydroxymethyl groups
Compounds 7a and 7b induced apoptosis in RKO cells
resulted in compounds 5h and 5i with improved anti-prolifera-
tive activity toward H1299 (IC50 ¼28 and 16 mM, respectively),
Enzyme linked immunosorbent apoptosis assay. Compounds 7a
HepG2 (IC50 ¼36 and 28 mM, respectively), RKO (IC50 ¼22
and 7b were further investigated for their induction of apoptosis in
both FHC and RKO cells using Cell Death Detection ELISAPLUS
,
and 18 mM, respectively), and A431 (IC50 ¼35 and 25 mM,
respectively). However, substitution of the hydroxy group in 5g
by an amino group (compound 5j) or replacement of the 2-
hydroxyethylamino group in 5g by aminoguanidino or guani-
dino moieties (compounds 7a and 7b) potentiated the anti-
proliferative activity toward H1299, RKO, A431 and MCF-7
cell lines. In this context, compounds 7a and 7b emerged with
the highest activity in this study toward RKO cells as they
showed IC50 values of 8 and 4 mM, respectively. Replacement
of the substituted amino group by phenyl; or 4-methoxyphenyl
sulfonylhydrazino (compounds 9a and 9b), benzo; or 4-
methoxybenzohydrazide (10a and 10b) or azomethine moieties
(11a–c) did not potentiate the activity against the five cancer
cell lines except for the moderate activity of 9a, 10b and 11c
against MCF7 cell line (IC50 ¼22, 10 and 12 mM, respectively).
which is used for the quantitative in vitro determination of
cytoplasmic histone-associated DNA fragments (mono- and
oligonucleosomes) in apoptosing cells. The results revealed that
8 mM of 7a and 4 mM of 7b increased apoptosis in RKO cells by
approximately 8- and 11-folds, respectively, compared to non-
treated control (p50.05) (Figure 2C). The results also showed
that compounds 7a and 7b induced apoptosis in RKO cells more
than FHC cells and this was more pronounced in case of
compound 7b.
TUNEL assay. To ascertain induction of apoptosis, the results
were confirmed by immune histochemical analysis using TUNEL
assay. TUNEL assay involves labeling of the 30-hydroxyl DNA
ends generated during DNA fragmentation by means of rTdT and
labeled dUTP. The localized fluorescence of apoptotic cells

12 A. Malki et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Figure 5. Compound 7b induced apoptosis through initiation of the mitochondrial pathway. (A) Western blot analysis was used to determine the release
of cytochrome c in the cytosol and mitochondria of 7b-treated RKO cells. b-Actin was used as loading control and IV-Cox was used as internal
mitochondrial control. C: control (untreated cells). (B) Effect of 7b on the level of caspase-9 in RKO cells after treatment with 4 mM 7b for 24 h. (C)
Effect of 7b and its combination with caspase-9 inhibitor on 7b-induced apoptosis. Cells were preincubated with 20 mM LEHD-CHO for 1 h before the
addition of 4 mM of 7b for an additional 24 h. Induction of apoptosis was determined at 405 nm and compared to control (untreated cells). (D)
Activation of caspase-3 in RKO cells after treatment with 4 mM 7b for 24 h. (For B, C and D; each data point is the mean of three independent
experiments and expressed as M SD. The asterisk indicates a significant difference between control (C; untreated cells) and 7b-treated cells;
p50.05). (E) Western blot analysis was used to determine the expression levels of caspase-3 and cleaved PARP1 after treating cells with 7b for 24 h.
b-Actin was used as loading control.
(labeled-dUTP) in a blue background (DAPI) for nuclei staining results suggest that these two compounds induce apoptosis in
or green background (FITC) was visualized using fluorescence RKO cells.
microscope. The assay results (Figure 2D) revealed the pres-
ence of nuclear condensation and TUNEL-positive cells
(stained cells).
Flow cytometric analysis. Flow cytometry is regarded as one of
the most powerful and specific methods for the integrated study of
molecular and morphological events occurring during cell death
and cell proliferation43. Generally, anti-cancer drugs inhibit the
proliferation of cancer cell either by induction of cell cycle arrest
or by apoptosis, or by combination of both these modes. However,
by measuring the DNA content, it becomes possible to identify
apoptotic cells; to recognize the cell cycle phase specificity and to
quantitate apoptosis. Therefore, the effect of 7a and 7b on cell
cycle progression was studied by flow cytometry in PI stained
cells. The percent of cells in G1, S, G2, M phases and apoptosis
were determined after treating cells with 8 and 4 mM of 7a and 7b,
respectively, for 24 h (Figure 3D and E). The results revealed a
significant increase in the percent of G1 population (55% and
57.5%, respectively) compared to control group (40%); however,
Impact of compounds 7a and 7b on cell cycle
Annexin V FITC dual staining assay. The apoptotic effect of
compounds 7a and 7b was finally confirmed by Annexin V. FITC/
PI (AV/PI) dual staining assay42. In this assay, RKO cells were
treated with 8 mM 7a and 4 mM 7b for 24 h to study the apoptotic
effect. Data in Figure 3(A) showed representative image for
untreated group, where the percentage of viable cells was 97%,
whereas data in Figure 3(B) revealed that RKO cells treated with
7a and 7b showed 20% and 28% apoptosis, respectively.
Figure 3(C) reveals that there is a threefold increase in induction
of apoptosis in RKO cells treated with 7b compared to control
untreated cells under the same experimental conditions. These

DOI: 10.3109/14756366.2015.1118686
Apoptosis in RKO colon cancer cells 13
no significant difference was found in the percent of cells in the S significantly reduced 7b induced apoptosis (Figure 5C).
phase. Moreover, the apoptotic cells increased to 20% and 26.5% Additionally, the treatment of cells with 7b increased caspase-3
after treating RKO cells with 7a and 7b, respectively, compared to activity (Figure 5D). Finally, monitoring of the cleavage of PARP-
control cells (9%). Thus, it can be concluded that compounds 7a 1 poly (ADP-ribose) polymerase, a target of caspase-3, further
and 7b inhibit the proliferation of RKO cells and the growth confirmed the above results. As shown in Figure 5(E), cleaved
inhibition is associated with induction of cycle G1 phase arrest PARP1 (89 kDa fragment) was detected after 24 h exposure to 7b.
and apoptosis.
Conclusion
Effect of compound 7b on the expression level of signaling
proteins
In the present investigation, new 1,5-diphenyl-6-substituted 1H-
pyrazolo[3,4-d]pyrimidine-4(5H)-ones were synthesized and
screened for their anti-tumor activity in five cancer cell lines.
Results revealed that compounds 7a and 7b were the most
active derivatives in this study against RKO colon cancer cells
To further investigate the underlying pathways of 7b induced G1
phase arrest and apoptosis, the expression of some apoptotic and
survival proteins involved in apoptosis such as p21, p53, Bax and
and they showed less cytotoxic effect on normal colon cells (FHC)
anti-apoptotic proteins such as Bcl-2 in addition to protein
than colon cancer cells (RKO). The results of apoptosis assays and
cell cycle analysis demonstrated that compounds 7a and 7b
obviously inhibit the proliferation of RKO cancer cells by
inducing apoptosis and arresting the cell cycle at G1 phase. The
kinases, e.g. ERK1/2, AKT was evaluated. p21WAF1 is con-
sidered as an important regulator of cell cycle44.To determine
whether compound 7b-induced G1 arrest was associated with
increased p21/WAF1 level, cells were analyzed for the expression
most active compound 7b increased levels of p53, p21, Bax,
of this protein after treatment with 4 mM of 7b for 24 h and the
ERK1/2 and decreased the level of Bcl-2 and AKT. The
expression of this protein was quantified using Imagine J software
regulatory effects of derivative 7b on the Bcl-2 family are
correlated with the release of cytochrome c from the mitochondria
into the cytoplasm and the activation of caspase-9. Compound 7b
also increased caspase-3 and cleaved PARP-1 in RKO colon
cancer cells. Finally, it could be concluded that compound 7b was
found to induce apoptosis in RKO colon cancer cells via
(Scripps, North Torrey Pines, CA). The results showed that, in
comparison to the non-treated control, 7b increased p21 level by
twofold (Figure 3F). The tumor suppressor protein p53 plays a
critical role in inducing cell growth arrest or apoptosis through its
transcriptional activity45. Activation of p53 leads to activation of
the intrinsic pathway through initiation of the apoptotic cascade
modulating mitochondrial signaling pathway. Accordingly, com-
that leads to activation of many pro-apoptotic proteins such as
pound 7b was identified as a new lead compound that merits
further optimization and development as an anti-cancer candidate.
Bax and Bid. Bax, a Bcl-2 family protein, induces cell death
through disruption of mitochondrial permeability and subsequent
release of cytochrome c, where it engages in a cascade of
Declaration of interest
interactions that leads to the execution stage of apoptosis. The
obtained data displayed that 7b increased level of p53 by threefold
(Figure 4A) and increased level of Bax in both mitochondrial and
total fractions by four- and threefold, respectively (Figure 4B). It
is known that interactions between the pro- and anti-apoptotic
members of Bcl-2 family integrate diverse upstream signals to
determine the cellular response. Apoptosis occurs through
competitive dimerization between the two protein groups46.
Accordingly, an increased ratio of Bax to Bcl-2 leads to
programmed cell death. Therefore, we analyzed the level of
Bcl-2 protein following treatment with 4 mM 7b for 24 h. The
obtained data portrayed that the level of Bcl-2 protein was
decreased by 2.5-fold. Finally, we analyzed the influence of 7b on
functional status of ERK1/2 and AKT, two pivotal protein kinases
involved in mitogenic signal transduction to the cell cycle control
system. The results displayed that 7b upregulated ERK1/2 and
downregulated Akt by 4.5- and 3-fold, respectively (Figure 4A).
The authors report no conflicts of interest. The authors alone are
responsible for the contents and writing the paper. The authors
would like to thank the Department of Health Sciences, Qatar
University for financial support of the cancer biology part of this
work.
References
1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics 2014. CA Cancer J
Clin 2014;64:9–29.
2. Haggar FA, Boushey RP. Colorectal cancer epidemiology: inci-
dence, mortality, survival, and risk factors. Clin Colon Rectal Surg
2009;22:191–7.
3. Borowski E, Bontemps-Gracz MM, Piwkowska A. Strategies for
overcoming ABC-transporters-mediated multidrug resistance
(MDR) of tumor cells. Acta Biochim Pol 2005;52:609–27.
4. Meier P, Vousden KH. Lucifer’s labyrinth – ten years of path finding
in cell death. Mol Cell 2007;28:746–54.
5. Gupta S, Kass GE, Szegezdi E, Joseph B. The mitochondrial death
pathway: a promising therapeutic target in diseases. J Cell Mol Med
2009;13:1004–33.
6. Brunelle JK, Letai A. Control of mitochondrial apoptosis by the Bcl-
2 family. J Cell Sci 2009;122:437–41.
7. Galluzzi L, Morselli E, Kepp O, et al. Mitochondrial gateways to
cancer. Mol Aspects Med 2010;31:1–20.
8. Portt L, Norman G, Clapp C, et al. Anti-apoptosis and cell survival:
a review. Biochim Biophys Acta 2011;1813:238–59.
9. Rashad AE, Mahmoud AE, Ali MM. Synthesis and anticancer
effects of some novel pyrazolo[3,4-d]pyrimidine derivatives by
generating reactive oxygen species in human breast adenocarcinoma
cells. Eur J Med Chem 2011;46:1019–26.
10. Ghorab MM, Ragab FA, Alqasoumi SI, et al. Synthesis of some new
pyrazolo[3,4-d]pyrimidine derivatives of expected anticancer and
radioprotective activity. Eur J Med Chem 2010;45:171–8.
11. Kumar A, Ahmad I, Chhikara BS, et al. Synthesis of 3-
phenylpyrazolopyrimidine-1,2,3-triazole conjugates and evaluation
of their Src kinase inhibitory and anticancer activities. Bioorg Med
Chem Lett 2011;21:1342–6.
Compound 7b induced apoptosis through release of cytochrome c
and activation of caspases
Cytochrome c, a component of the mitochondrial electron transfer
chain, is released into the cytosol during the early phases of
apoptosis47. The mitochondrial cytochrome c release in the
cytosol was determined by immunoblotting of the cell lysate of 7b
treated cells. Data from Western blot analysis (Figure 5A) showed
that the treatment of RKO cells with 4 mM 7b for 24 h increased
the expression of cytochrome c in cytoplasm and reduced its
expression in mitochondria (Figure 5A). To determine whether
caspase-9 and -3 activation is involved in 7b induced apoptosis,
caspase-9 and -3 were investigated in RKO cells. The obtained
data (Figure 5B) revealed that the treatment of cells with 7b
increased significantly caspase-9 activity (Figure 5B).
Furthermore, pretreatment of RKO cells with the specific
caspase-9 inhibitor LEHD-CHO prior to treatment with 7b

14 A. Malki et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
12. Hassan GS, Kadry HH, Abou-Seri SM, et al. Synthesis and in vitro
cytotoxic activity of novel pyrazolo[3,4-d]pyrimidines and related
pyrazole hydrazones toward breast adenocarcinoma MCF-7 cell line.
Bioorg Med Chem Lett 2011;19:6808–17.
13. Abd El Hamid MK, Mihovilovic MD, El-Nassan HB. Synthesis of
novel pyrazolo[3,4-d]pyrimidine derivatives as potential anti-breast
cancer agents. Eur J Med Chem 2012;57:323–8.
14. Kim DC, Lee YR, Yang B, et al. Synthesis and biological
evaluations of pyrazolo[3,4-d]pyrimidines as cyclin-dependent
kinase 2 inhibitors. Eur J Med Chem 2003;38:525–32.
imidazo[2,1-b]thiazoles and from the new heterocyclic system
thiazolo[20,30:2,3]imidazo[4,5-c]quinoline. J Med Chem 2005;48:
3085–9.
30. Andreani A, Burnelli S, Granaiola M, et al. New antitumor
imidazo[2,1-b]thiazole guanylhydrazones and analogues. J Med
Chem 2008;51:809–16.
31. Zhang H, Drewe J, Tseng B, et al. Discovery and SAR of indole-2-
carboxylic acid benzylidenehydrazides as a new series of potent
apoptosis inducers using a cell based HTS assay. Bioorg Med Chem
2004;12:3649–55.
15. Traxler P, Bold G, Frei J, et al. Use of a pharmacophore model for
the design of EGFR tyrosine kinase inhibitors: 4-(phenylamino)pyr-
azolo[3,4-d]pyrimidines. J Med Chem 1997;40:3601–16.
16. Indovina P, Giorgi F, Rizzo V, et al. New pyrazolo[3,4-d]pyrimidine
Src inhibitors induce apoptosis in mesothelioma cell lines through
p27 nuclear stabilization. Oncogene 2012;31:929–38.
17. Carraro F, Naldini A, Pucci A, et al. Pyrazolo[3,4-d]pyrimidines as
potent antiproliferative and proapoptotic agents toward A431 and
8701-BC cells in culture via inhibition of c-Src phosphorylation. J
Med Chem 2006;49:1549–61.
18. Santucci MA, Corradi V, Mancini M, et al. C6-unsubstituted
pyrazolo[3,4-d]pyrimidines are dual Src/Abl inhibitors effective
against imatinib mesylate resistant chronic myeloid leukemia cell
lines. Chem Med Chem 2009;4:118–126.
19. Radi M, Dreassi E, Brullo C, et al. Design, synthesis, biological
activity, and ADME properties of pyrazolo[3,4-d]pyrimidines active
in hypoxic human leukemia cells: a lead optimization study. . J
Med Chem 2011;54:2610–26.
20. Richard DJ, Verheijen JC, Curran K, et al. Incorporation of water-
solubilizing groups in pyrazolopyrimidine mTOR inhibitors: dis-
covery of highly potent and selective analogs with improved human
microsomal stability. Bioorg Med Chem Lett 2009;19:6830–5.
21. Markwalder JA, Arnone MR, Benfield PA, et al. Synthesis and
biological evaluation of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyr-
imidin-4-one inhibitors of cyclindependent kinases. J Med Chem
2004;47:5894–911.
22. El-Enany MM, Kamel MM, Khalil OM, El-Nassan HB. Synthesis
and antitumor activity of novel 6-aryl and 6-alkylpyrazolo[3,4-
d]pyrimidin-4-one derivatives. Eur J Med Chem 2010;45:5286–91.
23. Rostom SAF, Ashour HMA, Abd ElRazik HA. Synthesis and
biological evaluation of some novel polysubstituted pyrimidine
derivatives as potential antimicrobial and anticancer agents. Arch
Pharm Chem Life Sci 2009;342:299–310.
24. Ashour HMA, Abdel Wahab AE. Synthesis and biological evalu-
ation of novel pyrazoles and pyrazolo[3,4-d]pyrimidines incorporat-
ing a benzenesulfonamide moiety. Arch Pharm Chem Life Sci 2009;
342:238–52.
25. Rostom SAF, Badr MH, Abd ElRazik HA, et al. Synthesis of some
pyrazolines and pyrimidines derived from polymethoxy chalcones as
anticancer and antimicrobial agents. Arch Pharm Chem Life Sci
2011;344:572–87.
32. Cocco MT, Congiu C, Lilliu V, Onnis V. Synthesis and in vitro
antitumoral activity of new hydrazinopyrimidine-5-carbonitrile
derivatives. Bioorg Med Chem 2005;14:366–72.
33. Rollas S, Ku¨c¸u¨kgu¨zel G. Biological activities of hydrazone deriva-
tives. Molecules 2007;12:1910–39.
34. Nasr T, Bondock S, Youns M. Anticancer activity of new coumarin
substituted hydrazide-hydrazone derivatives. Eur J Med Chem 2014;
76:539–48.
35. Baviskar AT, Banerjee UC, Gupta M, et al. Synthesis of imine-
pyrazolopyrimidinones and their mechanistic interventions on
anticancer activity. Bioorg Med Chem 2013;21:5782–93.
36. Peet NP, Lentz NL, Sunder S, et al. Conformationally restrained,
chiral (phenylisopropyl)amino-substituted pyrazolo[3,4-d]pyrimi-
dines and purines with selectivity for adenosine A1 and A2
receptors. J Med Chem 1992;35:3263–9.
37. Garin J, Loscertales MP, Melendez E, et al. Methyl N-aryldithio-
carbamates: useful reagents for the annelation of pyrimidines and
1,3-oxazines to five-membered heterocyclic rings. Heterocycles
1987;26:1303–12.
38. Shaaban MA, Abou Sier AH, El-Ansary AK, et al. Synthesis and
pharmacological activities of certain pyrazolotriazolopyrimidine
and pyrazolotriazepine derivatives. Bull Facul Pharm (Cairo Univ)
2001;39:67–77.
39. Trost BM, Curran DP. Chemoselective oxidation of sulfides to
sulfones using potassium hydrogenpersulfate. Tetr Lett 1981;22:
1287–90.
40. Lin CF, Yang JS, Chang CY, et al. Synthesis and anticancer activity
of benzyloxybenzaldehyde derivatives against HL-60 cells. Bioorg
Med Chem 2005;13:1537–44.
41. Barboni L, Giarlo G, Ballini R, Fontana G. 14Beta-hydroxy-10-
deacetylbaccatin III as a convenient, alternative substrate for the
improved synthesis of methoxylated second-generation taxanes.
Bioorg Med Chem Lett 2006;16:5389–91.
42. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A
novel assay for apoptosis flow cytometric detection of phosphati-
dylserine expression on early apoptotic cells using fluorescein
labelled Annexin V. J Immunol Methods 1995;184:39–51.
43. Shrivastava HY, Ravikumar T, Shanmugasundaram N, et al.
Cytotoxicity studies of chromium(III) complexes on human dermal
fibroblasts. . Free Radic Biol Med 2005;38:58–69.
44. Haupt S, Berger M, Goldberg Z, Haupt Y. Apoptosis – the p53
network. J Cell Sci 2003;116:4077–85.
26. Malki A, Elbayaa RY, Ashour HMA, et al. Novel thiosemicarbazides
induced apoptosis in human MCF-7 breast cancer cells via JNK
signaling. J Enzyme Inhib Med Chem 2015;30:786–95.
27. Ibrahim DA, El-Metwally AM. Design, synthesis, and biological
evaluation of novel pyrimidine derivatives as CDK2 inhibitors. Eur J
Med Chem 2010;45:1158–66.
28. Zhang H-Z, Crogan-Grundy C, May C, et al. Discovery and
structure–activity relationships of (2-(arylthio)benzylideneamino)-
guanidines as a novel series of potent apoptosis inducers. Bioorg
Med Chem Lett 2009;17:2852–8.
29. Andreani A, Granaiola M, Leoni A, et al. Potential antitumor agents.
37.1 Synthesis and antitumor activity of guanylhydrazones from
45. Taguchi T, Kato Y, Baba Y, et al. Protein levels of p21, p27, cyclin E
and Bax predict sensitivity to cisplatin and paclitaxel in head and
neck squamous cell carcinomas. Oncol Rep 2004;11: 421–46.
46. Pellecchia M, Reed JC. Inhibition of anti-apoptotic Bcl-2 fam-
ily proteins by natural polyphenols: new avenues for cancer
chemoprevention and chemotherapy. Curr Pharm Des 2004;10:
1387–98.
47. Liu X, Kim CN, Yang J, et al. Induction of apoptotic program in
cell-free extracts: requirement for dATP and cytochrome c. Cell
1996;86:147–57.