Novel pyrazolopyrimidines: Synthesis, in?vitro cytotoxic activity and mechanistic investigation

Article abstract of DOI:10.1016/j.ejmech.2017.07.003

A series of novel pyrazolo[3,4-d]pyrimidines bearing benzenesulfonamide moiety 5a-f, 6 and 7 were synthesized. Cytotoxic screening was conducted against MCF-7 and HepG2. 6-(4-Methoxyphenyl)-4-oxopyrazolopyrimidine derivative 5e and 4-imino-6-oxopyrazolopyrimidine derivative 6 revealed potent cytotoxic activity with IC₅₀ 1.4 μM (MCF-7) and 0.4 μM (HepG2), respectively compared to that of doxorubicin, (IC₅₀ = 1.02 μM and 0.9 μM, respectively). Compounds 5e and 6 were subjected to cell cycle analysis and apoptosis assay after 24 h and 48 h treatment. Compound 5e arrested cell at G1 phase, while 6 arrested cell at S and G2/M phases, respectively. The apoptotic effect of both compounds were evidenced by pre G1 apoptosis as its percentage increased by time (7.38%, 11.61%) and (13.92%, 16.71%), respectively. Apoptosis induction capability was confirmed by the effect on early and late apoptosis and augmentation of caspase-3 level. Furthermore, compound 6 inhibited CDK2 enzyme with IC₅₀ = 0.19 μM and increased levels of its regulators, P21 and P27 by 10.06% and 8.5%, respectively. Moreover, a molecular docking study of compound 6 on CDK2 enzyme was adopted to explore binding interaction with amino acid residues of its active site.

Full text of DOI:10.1016/j.ejmech.2017.07.003

Accepted Manuscript
Novel pyrazolopyrimidines: Synthesis, in vitro cytotoxic activity and mechanistic
investigation
Ghaneya S. Hassan, Doaa E. Abdel Rahman, Yassin M. Nissan, Esraa A.
Abdelmajeed, Tamer M. Abdelghany
PII:
S0223-5234(17)30525-1
10.1016/j.ejmech.2017.07.003
EJMECH 9566
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 May 2017
Revised Date: 30 June 2017
Accepted Date: 2 July 2017
Please cite this article as: G.S. Hassan, D.E. Abdel Rahman, Y.M. Nissan, E.A. Abdelmajeed,
T.M. Abdelghany, Novel pyrazolopyrimidines: Synthesis, in vitro cytotoxic activity and mechanistic
investigation, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.003.
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Graphical abstract
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Novel pyrazolopyrimidines: Synthesis, in vitro cytotoxic activity and mechanistic
investigation
Ghaneya S. Hassana, Doaa E. Abdel Rahmana, Yassin M. Nissana*, Esraa A. Abdelmajeedb and
Tamer M. Abdelghanyc.
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini St.,
Cairo 11562, Egypt.
bNational Cancer Institute, Cairo University, Fom Elkhalig, Kasr Elaini St., Cairo 11796, Egypt
cPharmacology and Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Nasr
City, Cairo 11787, Egypt.
*Corresponding author: Y. M. Nissan, Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Cairo University, Kasr Elini Street., Cairo 11562, Egypt.
Tel.: +201000848844; E-mail address: yassin.nissan@hotmail.com
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Abstract
A series of novel pyrazolo[3,4-d]pyrimidines bearing benzenesulfonamide moiety 5a-f, 6 and 7
were synthesized. Cytotoxic screening was conducted against MCF-7 and HepG2. 6-(4-
Methoxyphenyl)-4-oxopyrazolopyrimidine derivative 5e and 4-imino-6-oxopyrazolopyrimidine
derivative 6 revealed potent cytotoxic activity with IC50 1.4 µM (MCF-7) and 0.4 µM (HepG2),
respectively compared to that of doxorubicin, (IC50 = 1.02 µM and 0.9 µM, respectively).
Compounds 5e and 6 were subjected to cell cycle analysis and apoptosis assay after 24h and 48h
treatment. Compound 5e arrested cell at G1 phase, while 6 arrested cell at S and G2/M phases,
respectively. The apoptotic effect of both compounds were evidenced by pre G1 apoptosis as its
percentage increased by time (7.38%, 11.61%) and (13.92%, 16.71%), respectively. Apoptosis
induction capability was confirmed by the effect on early and late apoptosis and augmentation of
caspase-3 level. Furthermore, compound 6 inhibited CDK2 enzyme with IC50 = 0.19 µM and
increased levels of its regulators, P21 and P27 by 10.06% and 8.5%, respectively. Moreover, a
molecular docking study of compound 6 on CDK2 enzyme was adopted to explore binding
interaction with amino acid residues of its active site.
Key words: pyrazolopyrimidines; cytotoxicity; CDK2 inhibitor; apoptosis.
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1. Introduction
Cancer, according to World Health Organization (WHO), is the second leading cause of death
globally and accounted for 8.8 million deaths in 2015. That is nearly 1 in 6 of all global deaths
[1]. Higher proliferation rate and evasion of apoptosis are considered two of common cancer
hallmarks [2]. Cell division and programmed cell death "apoptosis" are intimately coupled [3].
The linkage of cell cycle and apoptosis has been influenced by some proteins like tumor protein
(
p53), Protein Kinase C (PKC), B-cell lymphoma 2 (Bcl-2), Nuclear Factor kappa-light-chain-
enhancer of activated B cells (NF-κB), Cyclin Dependant Kinase (CDK), cyclins and Casein
Kinase I (CKI) [4]. Cyclin dependent kinases (CDKs) are the master regulators that drive the cell
cycle engine [5]. Their activities are down regulated by inhibitors (CDKIs) [6]. Deregulation of
CDKs can activate CDKs inappropriately and as a consequence, promote proliferation of cancer
[7]. CDK2, the S-phase CDK, control the G1/S transition. Also, regulate the G2/M transition that
prevents cells with damaged DNA from initiating mitosis [8]. Also CDK2 participate in subset of
apoptosis cascade [9]. Inhibition of CDK2 activity causes cell cycle arrest at G1/S and G2/M
phases and induction of apoptosis [10]. Consequently, the development of CDK inhibitors might
offer selective and tolerable treatment for cancer.
During the past decade many CDK inhibitors have been developed and characterized. The first
CDK inhibitor, purine derivative, Roscovitine I is considered as a pan-selective CDK inhibitor
with multiple effects on cell proliferation, cell cycle progression and induction of apoptosis in
cancer cells [11]. However, Roscovitine exhibited toxicity and low efficacy [7]. Dinaciclib II is a
pyrazolo[1,5-a]pyrimidine derivative that inhibits (CDKs). It is being evaluated in clinical trials
for various cancer indications [12], (Figure 1).
Literature survey revealed the biological activity of pyrazolo[3,4-d]pyrimidine, an isoster of
purine, as CDK2 inhibitors and apoptotic inducers [7,11,13]. It was reported that, presence of
benzenesulfonamide group on pyrazolo[3,4-d]pyrimidine scaffold strengthens its anticancer
activity [14].
Inspired by the above findings, we aimed to design and synthesize CDK2 inhibitors with
pyrazolo[3,4-d]pyrimidine scaffold bearing benzenesulfonamide moiety 5-7. In order to explore
the activity of the synthesized compounds, in vitro cytotoxic screening against liver cancer
(HepG2) and breast cancer (MCF-7) cell lines was conducted. Moreover, to elucidate the
mechanism of action for cytotoxic activity, the most active compounds were subjected to cell
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cycle analysis and apoptotic assay. In addition, the CDK2 enzyme inhibition assay was
conducted. Finally, simulation docking study was carried out to gain insight into the possible
binding mode of the tested compound in the CDK2 enzyme binding site.
2. Results and Discussion
2.1. Chemistry
Sulfanilamide 1 was diazotized in hydrochloric acid with sodium nitrite at 5 °C. The diazonium
salt was then reduced by sodium sulfite to give the 4-hydrazinobenzenesulfonamide 2 [15]. The
key intermediate, 4-(5-amino-4-cyano-1H-pyrazol-1-yl)benzenesulfonamide 3 was prepared by
reacting compound 2 with ethoxymethylene malononitrile in absolute ethanol and anhydrous
sodium acetate [16]. The reaction proceeded via SN2 mechanism in two steps leading to
intramolecular pyrazole cyclization [17,18].
Pyrazole-4-carboxamide derivative 4 was prepared by stirring amino compound 3 with sulfuric
acid for 16 h at room temperature. Its IR spectrum revealed presence of bands at 3460, 3452,
3344, 3278 and 3170 cm-1 which correspond to 3 NH2 groups. Also a new band at 1662 cm-1 was
attributed to C=O; in addition to disappearance of band at 2216 cm-1 for CN group. Mass
spectrum showed its molecular ion peak at m/z 281.
Pyrazolo[3,4-d]pyrimidin-4-one derivatives 5a-f were achieved by reacting pyrazole-4-
carboxamide 4 with the appropriate aromatic aldehyde using DMF as a solvent in the presence of
1
iodine as mild Lewis acid and oxidant. H NMR spectra of compounds 5a-f showed the
appearance of singlet signal at δ: 12.57-12.79 ppm attributed to pyrimidine NH proton in
addition to increased number of aromatic protons. Compound 5b showed a triplet signal at δ:
7.44 ppm and a doublet of doublet signal at δ: 8.31 ppm assigned to H-3´,5´ and H-2´,6´ protons
of p-fluoro substituted phenyl moiety, respectively. This was explained on the basis of proton
coupling with fluorine atom [19]. 1H NMR spectrum of compound 5e showed singlet signal at δ:
3.87 ppm attributed to OCH3 protons while that of compound 5f had two singlet signals at δ:
3.87 and 3.91 ppm attributed to two OCH3 protons. 13C NMR spectrum of 5d showed signals of
aromatic carbons. Mass spectra of 5a-f showed their molecular ion peaks (Scheme 1).
Fusion of 5-aminopyrazole-4-carbonitrile, 3 with urea yielded pyrazolopyrimidine derivative, 6.
IR spectrum of compound 6 showed the appearance of a band at 1685 cm-1 assigned to C=O
1
group, while its H NMR spectrum showed appearance of three singlet signals at δ: 7.48, 8.77
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and 11.05 ppm attributed to three NH protons in addition to a singlet signal at δ: 7.37 ppm
attributed to SO2NH2 protons. 13C NMR spectrum of compound 6 showed signals corresponding
to aromatic protons and mass spectrum showed its molecular ion peak at m/z 306.
4-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzenesulfonamide 7 was achieved by refluxing
amino derivative 3 with formamide for 16 h. 1H NMR of compound 7 showed the appearance of
the singlet signal at δ: 8.36 ppm which corresponds to H-3 pyrazolopyrimidine, and mass
spectrum showed its molecular ion peak at m/z 290 (Scheme 1).
2.2. Biological Evaluation
2.2.1. Cytotoxic Screening
All newly synthesized compounds were tested against the human breast adenocarcinoma cell line
(MCF-7), and human hepatocellular carcinoma cell line (HepG2) at the department of
Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University by the method of
MTT assay.
The newly synthesized compounds 3, 4, 5a-f, 6 and 7 were evaluated for their in vitro cytotoxic
activity against two cell lines using Doxorubicin as reference standard; all the results are
displayed in Table 1.
Results showed that the synthesized pyrazole derivatives 3 and 4 were inactive against both
MCF-7 and HepG2 cell lines, while the pyrazolo[3,4-d]pyrimidines 5-7 demonstrated variable
activities against both cell lines. Substitutions at position 4 and 6 were proven crucial for
pyrazolo[3,4-d]pyrimidines activity. 6-Substituted pyrazolo[3,4-d]pyrimidin-4-one derivatives
with monosubstituted phenyl 5b-e were very active, while unsubstituted 5a and disubstituted 5f
were inactive against the both cell lines. Compound 5e showed promising activity against MCF-
7 with IC50 1.40 µM comparable to the standard used doxorubicin with IC50 1.02 µM, while it
was inactive against HepG2. Also, compound 5c showed good activity with IC50 2.50 µM
against MCF-7 and moderate activity with IC50 23.90 µM against HepG2. Compound 5b and 5d
exhibited mild activity against MCF-7 with IC50 38 µM and 14.46 µM, respectively. However,
against HepG2 compound 5b showed good activity with IC50 2.66 µM and compound 5d showed
mild activity with IC50 42.6 µM. On the other hand, compound 6 showed promising activity
against HepG2 cell line with IC50 0.40 µM even more active than doxorubicin with IC50 0.90
µM.
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4-Substituted pyrazolo[3,4-d]pyrimidin-4-one with amino group, compound 7, showed mild
activity against MCF-7 with IC50 40.70 µM.
Therefore, compounds 5e and 6 were subjected to further investigations to explore the possible
mechanism of action on MCF-7 and HepG2, respectively.
2.2.2. Cell cycle analysis and apoptosis assay
Compounds 5e and 6 were subjected to cell cycle analysis, apoptotic assay and enzyme assays at
confirmatory diagnostic unit, VACSERA-EGYPT.
2.2.2.1. Cell cycle analysis and apoptosis detection
Cell cycle is the series of growth and development steps that lead to cell division and DNA
replication. The cell cycle consists of four distinct phases: G1 phase, S phase (synthesis), G2
phase and M phase. During G1, preparation of energy and DNA replication blocks occurs. The S
phase is the stage when DNA replicates. During G2, the new DNA is checked and repaired. The
M (mitosis) stage where nuclear and cytoplasmic division take place.
Effect of compounds 5e and 6 on cell cycle progression and induction of apoptosis in the MCF-7
and HepG2 cell lines was studied. MCF7 and HepG2 cell lines were incubated with IC50
concentration of compound 5e and 6 for 24 h and 48 h. The cell lines were stained with PI /
Annexin V and analyzed by flow cytometry using BD FASCC alibur. Quantification of the
results (Table 2, Figure 2) revealed that percentage of pre G1 apoptosis induced by compound
5e on MCF-7 after 24h incubation was 7.38%. This percent was increased to 11.61% after 48h.
A high percent of cell accumulation was observed in G1 and S phase in MCF-7 treated with
compound 5e after 24h and 48h incubation indicating arrest of cell cycle at G1/S transition.
Compound 6 on HepG2 induced pre G1 apoptosis by 13.92% after 24h incubation that increased
to 16.71% after 48h incubation. Also, HepG2 treated with compound 6 showed cell
accumulation at S phase and G2/M phase after 24h incubation. It showed high cell accumulation
at G2/M after 48h incubation. This indicated that it arrested cell cycle at G2/M phase.
2.2.2.2. Apoptosis assay
Cell cycle analysis of MCF-7 and HepG2 after treatment with compound 5e and 6, respectively
showed presence of pre-G1 peak which is an indication of apoptosis. To confirm the ability of
both compounds to induce apoptosis, cells were stained with Annexin V/ PI, incubated for 24h
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and 48h and analyzed. Analyses of early and late apoptosis showed that, compounds 5e and 6
were indeed able to induce significant levels of apoptosis (Table 3, Figure 3).
2.2.2.3. Caspase-3 activity (Key executor of apoptosis)
Caspases (cysteinyl aspartate specific proteinases) are essential in the regulation of apoptosis.
There are two types of apoptotic caspases: Initiator (apical) caspases and effector (executioner)
caspases. The best recognized biochemical hallmark of apoptosis is the activation of caspases.
Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced
by a wide variety of apoptotic signals. Sensitive and reproducible detection of active caspase-3 is
important to advance the understanding of cellular functions and multiple pathologies of
etiologies [20,21]. Compounds 5e and 6 were subjected to caspase-3 activation assay using
ELISA technique. Results were expressed in term of Caspase-3 concentration Pg/ml (Table 4).
From the table of the result, it was found that compound 5e increased caspase-3 concentration in
MCF-7 by 32 fold, while compound 6 induced activation caspase-3 in HepG2 by 36 fold
compared to its control. In light of these results, both compounds may be considered as
promising apoptotic inducers.
2.2.2.4. Cyclin dependent Kinase 2 (in vitro) inhibition assay (IC50 determination)
Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases where their catalytic
activities are modulated by interactions with cyclins and Cdk inhibitors (CKIs). The regulation of
the cell cycle is a well orchestrated pairing of different members of the cyclin dependent kinases
(CDKs) and its regulatory subunits (cyclin). For instance, binding of CDK2 with cyclin E is
necessary for cells to make the transition from G1 into S phase [8]. Association of CDK2 with
cyclin A is responsible for DNA synthesis [9] and activation of M-cyclin/CDK complex [8].
The activity of CDKs is restrained by CDK inhibitors (CKIs). CKIs are subdivided into two
classes based on their structure and CDK specificity. One of them are Cip/Kip family members
[p21Cip1 (Cdkn1a), p27Kip1 (Cdkn1b) and p57Kip2 (Cdkn1c)] are more promiscuous and interfere
with the activities of cyclin E-, A- and B-dependent kinase complexes [5].
To elucidate the S phase and G2/M phase arrest caused by compound 6, the kinase inhibitory
effect of 6 was evaluated against CDK2 and its IC50 was determined. After treatment with
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different concentration of compound 6, IC50 was calculated from concentration–response curve
to be 0.19 µM (Table 5).
2.2.2.5. P21 and P27 expression
Compound 6 was subjected to P21 and P27 (CDKs inhibitors) expression assay to evaluate its
effect on both. Results were expressed in term of residual concentration Pg/mL and % activation
(Table 6). Compound 6 was found to activate P21 by 10.06% and P27 by 8.5% which will be
reflected on CDK2 inhibition.
2.3. Molecular docking
CDK2 includes primarily the conserved kinase core with no extension at the N terminus and a
short extension of 12 residues at the C terminus with small insert within the core. This catalytic
core made up of ATP binding pocket, PSTAIRE-like cyclin-binding site and protein substrate
binding site [22]. The backbone structure of CDK2 can be divided into two lobes. The smaller N-
terminal lobe of CDK2 consists of a sheet of five antiparallel β-strands (β1-β5) and a single large
helix (α1). The larger C-terminal lobe contains a pseudo-4-helical bundle (α2-α4, α6), a small β-
ribbon (β6-β8) and two additional helices (α5, α7) [22]. CDK2 regulation, both positive and
negative, depends on complexion with cyclins and its phosphorylation state. The ATP-binding
pocket is a CDK2-Mg2+ATP binary complex. Its site is found in the cleft between the two lobes
[23]. Thr14 and Tyr15 are both in the middle of the glycine- rich loop that serves as a phosphate
anchor in ATP binding. Also, it is considered as CDK2 inhibitory phosphorylation site. Thr160
is found at the apex of the T-loop, is considered as CDK2 activating phosphorylation site. The
hydroxyl group of Thr160 is oriented toward the glycine rich loop of the ATP binding site.
The ATP phosphates are held in position by ionic and hydrogen bonding interactions with
several residues including Lys33, Asp145 and the backbone amides of the glycine rich loop
between β1 and β2. Oxygen from each of the three phosphates of ATP contributes to the
octahedral co-ordination of the Mg2+ ion. The three other residues involved in the co-ordination
of Mg2+ are Asn132, Asp145 and a water molecule [23].
In order to explore the binding mode of compound 6 on the active site of CDK2, molecular
docking of compound 6 was performed using protein data bank file (PDB: 1y8y) [24] by
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Molecular Operating Environment (MOE) 2008.10 release of Chemical Computing Group,
Canada.
The binding affinity of the ligand was evaluated with energy score (S, Kcal/mol). Low dock
score indicates good affinity. Hydrogen bond, arene arene and arene cation interaction were also
used to assess the binding models.
The inhibitor CT7-CDK2 complex was precisely reproduced by the docking procedure as
demonstrated by low root mean square deviation, rmsd (0.2341) and dock score (-18.0923
kcal/mol), i.e. the docking protocol was valid. As shown in (Figure 4) the inhibitor CT7 (IC50 =
2.0 µM) [24] nearly fits in the active site forming eight hydrogen bonding interactions with the
active site residues. N of pyrimidine binds with Lys33 (3.17 Å). Leu83 interacts by two
hydrogen bonds with NH (2.70 Å) and N pyrazole (3.10 Å). SO2 of ligand interacts with Asp86
(2.90 Å) and Lys89 (3.18 Å) and also through water molecule with His84, Gln85 and Lys89
(3.11 Å).
Compound 6 (IC50 0.19 µM) with dock score (-18.6608 Kcal/mol), binds with active site forming
eight hydrogen bonds in addition to one arene cation interaction. The amino group of SO2NH2
binds with Thr14 (2.55 Å) and Asp145 (1.29 Å). N of pyrazole interacts with Lys33 (2.93 Å) and
the imino NH interacts with Leu83 (2.50 Å). In addition, the oxygen atom of SO2NH2 binds with
Thr14 and Lys123 through water molecule (2.24 Å) while NH of pyrimidine binds with Asp86
and Gln131 through water molecule (1.94 Å) in addition to arene cation interaction between
benzene ring and Lys33, reflecting its action as CDK-2 inhibitor (Figure 5).
3. Conclusion
In this study, a series of pyrazolopyrimidine derivatives were synthesized and their cytotoxicity
against MCF-7 and HepG2 were evaluated. It was found that; compound 5e was potent against
MCF-7 with IC50 comparable to standard used (Doxorubicin). However, pyrazolopyrimidine 6
demonstrated high potency against HepG2 with IC50 less than standard used doxorubicin.
Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds 5e and
6 on MCF-7 and HepG2 cell lines were investigated. Cell cycle analysis showed that
pyrazolopyrimidine derivative 5e arrested cell cycle of MCF-7 at G1 phase. While
pyrazolopyrimidine derivative 6 arrested cell cycle of HepG2 at S and G2/M phases. Also, both
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compounds exhibited apoptotic induction capability. This was evidenced by presence of pre G1
peak and confirmed by apoptotic induction assay and augmenting caspase-3 activation. In the
light of cell cycle results, compound 6 subjected to CDK2 inhibition assay with IC50 0.19 µM.
Furthermore, it increased the expression of CDK2 inhibitors P21 and P27. Molecular docking of
compound 6 on CDK2 enzyme (PDB: 1y8y) confirmed the biological results.
4. Experimental
4.1. Chemistry
Melting points were determined on Stuart melting point apparatus (Stuart Scientific, Redhil, UK)
and Peak Find-Memory-9 and were uncorrected. IR spectra were recorded as potassium bromide
disc on Shimadzu FT-IR Affinity-1 spectrometer and Bruker FT-IR spectrophotometer. They
were expressed in wave number (cm-1). The NMR spectra were recorded on a Varian Mercury
1
VX-300 and Bruker AVANCE III400 MHz FT-NMR spectrometer. H spectra were run at 400
MHz and 13C spectra were run at 100 MHz in deuterated dimethylsulphoxide (DMSO-d6).
Chemical Shifts are quoted in δ as parts per million (ppm) downfield from tetramethylsilane
(TMS) as internal standard. Mass spectra were recorded using Single Quadruple mass
spectrometer ISQ LT. Microanalyses were carried out at The Regional Center for Mycology and
Biotechnology, Al-Azhar University. TLC were carried out using Kieselgel 60 F254 sheets
(Merck, Darmstadt, Germany), the developing solvents were dichloromethane/methanol (9:1)
and the spots were visualized at 366, 254 nm by UV Vilber Lourmat 77202 (Vilber, Marne La
Vallee, France).
4-Hydrazinobenzenesulfonamide HCl,
2
[15] and 4-(5-amino-4-cyano-1H-pyrazol-1-
yl)benzenesulfonamide 3 [16] were synthesized according to the reported methods.
4.1.1. 5-Amino-1-(4-sulfamoylphenyl)-1H-pyrazole-4-carboxamide 4
The 5-aminopyrazole-4-carbonitrile 3 (0.26 g, 1 mmol) was stirred in sulfuric acid (8.2 mL) at
room temperature for 16 h. The reaction mixture was poured on crushed ice (50 mL) and was
neutralized with saturated sodium bicarbonate solution. The crude solid was filtered, washed
with water, dried and crystallized from ethanol.
Rf=0.12; yield: 88%; mp 161 °C (decomp.); 1H NMR (400 MHz, DMSO-d6) δ ppm: 6.58 (s, 2H,
NH2, exch. D2O), 7.50 (s, 2H, SO2NH2 exch. D2O), 7.78 (d, J = 8.7 Hz, 2H, H-2,6 Ar), 7.94 (d, J
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= 8.6 Hz, 3H, H-3 pyrazole, H-3,5 Ar), 7.97 (s, 2H, CONH2, exch. D2O); IR (KBr) υmax\cm-1:
3460, 3452, 3344, 3278, 3170 (3NH2), 3076 (CH Ar), 1662 (C=O), 1612, 1593, 1566, 1543
(C=N, NH, C=C), 1327, 1168 (SO2); MS (70 eV): m/z (%): 281 (3.53) [M+]; elemental analysis
calcd (%) for C10H11N5O3S: C 42.70, H 3.94, N 24.90; Found: C 42.94, H 3.75, N 25.12.
4.1.2. General Procedure for synthesis of compounds 5a-f
A mixture of compound 4 (0.28 g, 1 mmol), appropriate aromatic aldehyde (1 mmol) and
molecular iodine (0.285 g, 2 mmol) was heated at 80 °C in dimethylformamide (5 mL) for 12-16
h (reaction monitored by TLC). The reaction mixture was cooled, poured on crushed ice and
aqueous solution of 5% sodium thiosulfate (15 mL) was added. The precipitate was filtered,
washed and dried. The crude product was crystallized from methanol.
4.1.2.1.4-(4-Oxo-6-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzenesulfonamide
5a
1
Reaction time 16 h; Rf=0.45; yield: 80 %; mp 250 °C (decomp.); H NMR (400 MHz, DMSO-
d6) δ ppm: 7.47 (s, 2H, SO2NH2 exch. D2O), 7.59 (t, J = 7.6 Hz, 2H, H-3´,5´Ar ), 7.65 (d, J = 7.3
Hz, 1H, H-4´Ar), 8.05 (d, J = 8.5 Hz, 2H, H-2,6 Ar), 8.23 (d, J = 7.3 Hz, 2H, H-2´,6´ Ar), 8.40
(d, J = 8.7 Hz, 2H, H-3,5 Ar), 8.44 (s, 1H, H-3 pyrazolopyrimidine), 12.74 (s, 1H, NH exch.
D2O); IR (KBr) υmax\cm-1: 3363, 3240, 3178 (NH2, NH), 3066 (CH Ar), 1670 (C=O), 1624,
1600, 1550, 1523, 1500 (C=N, NH, C=C), 1346, 1157 (SO2); MS (70 eV): m/z (%): 367 (54.90)
[M+]; elemental analysis calcd (%) for C17H13N5O3S: C 55.58, H 3.57, N 19.06; Found: C 55.83,
H 3.63, N 19.42.
4.1.2.2.4-[6-(4-Fluorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl]
benzenesulfonamide 5b
1
Reaction time 16 h; Rf=0.47; yield: 75%; mp >300 °C; H NMR (400 MHz, DMSO-d6) δ ppm:
7.44 (t, J = 8.8 Hz, 2H, H-3´,5´Ar), 7.47 (s, 2H, SO2NH2 exch. D2O), 8.04 (d, J = 8.7 Hz, 2H, H-
2,6 Ar), 8.31 (dd, J = 8.7, 5.4 Hz, 2H, H-2´,6´Ar), 8.39 (d, J = 8.7 Hz, 2H, H-3,5 Ar), 8.43 (s,
1H, H-3 pyrazolopyrimidine), 12.76 (s, 1H, NH exch. D2O); IR (KBr) υmax\cm-1: 3367, 3240,
3209 (NH2, NH), 3089 (CH Ar), 1662 (C=O), 1600, 1597, 1558, 1527 (C=N, NH, C=C), 1350,
1165 (SO2); MS (70 eV): m/z (%): 385 (1.41) [M+]; elemental analysis calcd (%) for
C17H12FN5O3S: C 52.98, H 3.14, N 18.17; Found: C 53.17, H 3.26, N 18.43.
4.1.2.3.4-[6-(4-Chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-
yl]benzenesulfonamide 5c
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1
Reaction time 12 h; Rf=0.64; yield: 80%; mp >300 °C; H NMR (400 MHz, DMSO-d6) δ ppm:
7.47 (s, 2H, SO2NH2 exch. D2O), 7.66 (d, J = 8.5 Hz, 2H, H-3´,5´Ar), 8.04 (d, J = 8.7 Hz, 2H, H-
2,6 Ar), 8.24 (d, J = 8.6 Hz, 2H, H-2´,6´ Ar), 8.38 (d, J = 8.7 Hz, 2H, H-3,5 Ar), 8.43 (s, 1H, H-3
pyrazolopyrimidine), 12.76 (s, 1H, NH exch. D2O); IR (KBr) υmax\cm-1: 3325, 3224, 3167 (NH2,
NH), 3080 (CH Ar), 1693 (C=O), 1612, 1597, 1543 (C=N, NH, C=C), 1346, 1161 (SO2); MS
(70 eV): m/z (%): 401 (0.79) [M+]; elemental analysis calcd (%) for C17H12ClN5O3S: C 50.81, H
3.01, N 17.43; Found: C 51.12, H 3.04, N 17.78.
4.1.2.4.4-[6-(4-Bromophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-
yl]benzenesulfonamide 5d
1
Reaction time 12 h; Rf=0.51; yield: 80%; mp >300 °C; H NMR (400 MHz, DMSO-d6) δ ppm:
7.48 (s, 2H, SO2NH2 exch. D2O), 7.79 (d, J = 8.4 Hz, 2H, H-3´,5´Ar), 8.04 (d, J = 8.7 Hz, 2H, H-
2,6 Ar), 8.15 (d, J = 8.6 Hz, 2H, H-2´,6´ Ar), 8.38 (d, J=8.7 Hz, 2H, H-3,5 Ar), 8.43 (s, 1H, H-3
pyrazolopyrimidine), 12.79 (s, 1H, NH exch. D2O); 13C NMR (100 MHz, DMSO-d6) δ ppm:
106.94 (C3a pyrazolopyrimidine), 121.94 (C3,5), 126.58 (C4´), 127.53 (C2,6), 130.85 (C3´,5´),
131.53 (C1´), 132.28 (C2´,6´), 137.38 (C1), 141.10 (C4), 142.50 (C3 pyrazolopyrimidine),
153.12 (C7a pyrazolopyrimidine), 156.09 (C6 pyrazolopyrimidine), 158.50 (C4
pyrazolopyrimidine); IR (KBr) υmax\cm-1: 3332, 3232, 3167 (NH2, NH), 3109 (CH Ar), 1693
(C=O), 1600, 1590, 1543, 1500 (C=N, NH, C=C), 1346, 1161 (SO2); MS (70 eV): m/z (%): 446
(0.95) [M+], 448 (1.60) [M++2]; elemental analysis calcd (%) for C17H12BrN5O3S: C 45.75, H
2.71, N 15.69; Found: C 46.02, H 2.74, N 15.87.
4.1.2.5.4-[6-(4-Methoxyphenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-
yl]benzenesulfonamide 5e
1
Reaction time 16 h; Rf=0.49; yield: 80%; mp >300 °C; H NMR (400 MHz, DMSO-d6) δ ppm:
3.87 (s, 3H, OCH3), 7.13 (d, J = 8.8 Hz, 2H, H-3´,5´Ar), 7.46 (s, 2H, SO2NH2 exch. D2O), 8.04
(d, J = 8.7 Hz, 2H, H-2,6 Ar), 8.24 (d, J = 8.4 Hz, 2H, H-2´,6´ Ar), 8.40 (d, J = 8.6 Hz, 3H, H-3
pyrazolopyrimidine, H-3,5 Ar), 12.57 (s, 1H, NH exch. D2O); IR (KBr) υmax\cm-1: 3356, 3244,
3197 (NH2, NH), 3028 (CH Ar), 1685 (C=O), 1612, 1593, 1558 (C=N, NH, C=C), 1330, 1157
(SO2); MS (70 eV): m/z (%): 397 (4.58) [M+]; elemental analysis calcd (%) for C18H15N5O4S: C
54.40, H 3.80, N 17.62; Found: C 54.69, H 4.06, N 17.27.
4.1.2.6.4-[6-(3,4-Dimethoxyphenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-
yl]benzenesulfonamide 5f
12

ACCEPTED MANUSCRIPT
Reaction time 16 h; Rf=0.46; yield: 80%; mp 263 °C (decomp.); 1H NMR (400 MHz, DMSO-d6)
δ ppm: 3.87 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 7.17 (d, J = 8.5 Hz, 1H, H-5´Ar), 7.47 (s, 2H,
SO2NH2 exch. D2O), 7.85 (s, 1H, H-2´Ar), 7.94 (d, J = 8.5 Hz, 1H, H-6´Ar), 8.04 (d, J = 8.7 Hz,
2H, H-2,6 Ar), 8.41 (s, 1H, H-3 pyrazolopyrimidine), 8.43 (d, J = 8.7 Hz, 2H, H-3,5 Ar), 12.57
(s,1H, NH exch. D2O); IR (KBr) υmax\cm-1: 3356, 3259, 3201 (NH2, NH), 3093 (CH Ar), 1685
(C=O), 1600, 1597, 1560, 1531 (C=N, NH, C=C), 1342, 1161 (SO2); MS (70 eV): m/z (%): 427
(1.18) [M+]; elemental analysis calcd (%) for C19H17N5O5S: C 53.39, H 4.01, N 16.38; Found: C
53.69, H 4.06, N 16.42.
4.1.3.4-(4-Imino-6-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)benzenesulfonamide 6
A mixture of aminocyanopyrazole compound 3 (0.55 g, 2 mmol) and urea (0.5 g, 8 mmol) was
heated at 200 °C for 20 min. (till the melted solution became a solid mass). The reaction mixture
was cooled and the yellowish brown precipitate was solubilized in 2N sodium hydroxide and
heated till boil (clear solution) then acidified with dilute hydrochloric acid. The obtained solid,
re-precipitated twice, was filtered, washed with water and dried. The crude product was
crystallized from ethanol.
Rf=0.2; yield: 88%; mp >300 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.37 (s, 2H, SO2NH2
exch. D2O), 7.48 (s, 1H, NH exch. D2O), 7.92 (d, J = 8.7 Hz, 2H, H-2,6 Ar), 8.21 (s, 1H, H-3
pyrazolopyrimidine), 8.41 (d, J = 8.7 Hz, 2H, H-3,5 Ar), 8.77 (s, 1H, NH exch. D2O), 11.05 (s,
1H, NH exch. D2O); 13C NMR (100 MHz, DMSO-d6) δ ppm: 93.22 (C3a pyrazolopyrimidine),
119.97 (C3,5), 127.16 (C2,6), 136.98 (C7a pyrazolopyrimidine), 140.71 (C1), 142.15 (C3
pyrazolopyrimidine), 153.97 (C4), 156.83 (C4 pyrazolopyrimidine), 159.12 (C6
pyrazolopyrimidine); IR (KBr) υmax\cm-1: 3402, 3317 (NH2, 3NH), 3086 (CH Ar), 1685 (C=O),
1612, 1593, 1563, 1516 (C=N, NH, C=C), 1307, 1153 (SO2); MS (70 eV): m/z (%): 306 (1.46)
[M+]; elemental analysis calcd (%) for C11H10N6O3S: C 43.13, H 3.29, N 27.44; Found: C 43.28,
H 3.40, N 27.78.
4.1.4. 4-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzenesulfonamide 7
A mixture of compound 3 (0.55 g, 2 mmol) and formamide (3 mmol) was heated under reflux for
16 h. The reaction mixture was cool, poured onto ice-water, filtered and dried. The crude product
was crystallized from methanol.
13

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Rf=0.38; yield: 88%; mp >300°C; 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.42 (s, 4H, NH2, SO2
NH2 exch. D2O), 7.99 (d, J = 8.0 Hz, 2H, H-2,6 Ar), 8.36 (s, 1H, H-6 pyrazolopyrimidine), 8.43
(s, 1H, H-3 pyrazolopyrimidine), 8.47 (d, J = 8.0 Hz, 2H, H-3,5 Ar); IR (KBr) υmax\cm-1: 3414,
3398, 3242, (2NH2), 3080 (CH Ar), 1660, 1587, 1562, 1504 (C=N, NH, C=C), 1327, 1155
(SO2); MS (70 eV): m/z (%): 290 (9.03) [M+]; elemental analysis calcd (%) for C11H10N6O2S: C
45.51, H 3.47, N 28.95. Found: C 45.67, H 3.14, N 29.15.
4.2. Biological Evaluation
4.2.1. Cytotoxicity screening
Exponentially growing cells from two cancer cell lines were trypsinized, counted and seeded at
the appropriate densities (2000-1000 cells/0.33 cm2 well) into 96-well microtiter plates. Cells
then were incubated in a humidified atmosphere at 37 ˚C for 24 h. Then, cells were exposed to
different concentrations of compounds (0.1, 10, 100, 1000 µM) for 72 h. Then the viability of
treated cells were determined using MTT technique as follow. Media were removed; cells were
incubated with 200 µL of 5% MTT solution/well (Sigma Aldrich, MO) and were allowed to
metabolize the dye into colored-insoluble formazan crystals for 2 h. The remaining MTT
solution were discarded from the wells and the formazan crystals were dissolved in 200 µL/well
acidified isopropanol for 30 min., covered with aluminum foil and with continuous shaking using
a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc, MI) at room temperature. Absorbance
was measured at 570 nm using a Stat FaxR 4200 plate reader (Awareness Technology, Inc., FL).
The cell viability were expressed as percentage of control and the concentration that induces
50% of maximum inhibition of cell proliferation (IC50) were determined using Graph Pad Prism
version 5 software (Graph Pad software Inc, CA) [25,26].
4.2.2.1. Cell cycle analysis and apoptosis detection
The MCF-7 and HepG2 cell lines were treated with 1.4 µM of compound 5e and 0.4 µM of
compound 6, respectively for 24 or 48 h. After treatment, the cells were suspended in 0.5 mL of
PBS, collected by centrifugation, and fixed in ice-cold 70% (v/v) ethanol washed with PBS, re-
suspended with 0.1 mg/mL RNase, stained with 40 mg/ml PI, and analyzed by flow cytometry
using FACScalibur (Becton Dickinson). The cell cycle distributions were calculated using
Phoenix Flow Systems and Verity Software House [27].
14

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4.2.2.2. Apoptosis Assay
The MCF-7 and HepG2 were treated with 1.4 µ M of compound 5e and 0.4 µM of compound 6
respectively for 24 or 48 h. After treatment, the cells were suspended in 0.5 mL of PBS,
collected by centrifugation, and fixed in ice-cold 70% (v/v) ethanol, centrifuged the ethanol-
suspended cells for 5 min, suspended in 5 mL PBS and centrifuged for 5 min., re-suspended with
1 mL PI staining solution (0.1 mg/ml RNase) + PE Annexin V (component no. 51-65875X) and
kept in dark at 37 °C for 10 min., finally analyzed by flow cytometry using FACScalibur (Becton
Dickinson). The cell cycle distributions were calculated using Phoenix Flow Systems and Verity
Software House [28].
4.2.2.3. Caspase-3 activity (Key executor of apoptosis)
The Invitrogen Caspase-3 (active) Human ELISA is designed to detect and quantify the level of
human active caspase-3 protein when cleaved at Asp175/Ser176. Briefly, MCF-7 and HepG2
cells were treated with 5e (1.4 µM) and 6 (0.4 µM). The cells were harvested by trypsinization
and rinsed with PBS. After centrifugation, the pellet (105- 106 cells) was suspended in 1 mL of
PBS. Cells were freezed at < -20 oC and thawed with gentle mixing. Freeze/thaw cycle repeated
for 3 times, then centrifuged at 1500×g for 10 min. at 2-8oC to remove cellular debris. Human eia
kit is a solid phase sandwich Enzyme Linked Immuno-Sorbent Assay (ELISA). A monoclonal
specific antibody for human target protein has been coated onto the wells of the microtiter strips
provided. Samples, including a standard containing the human target protein control specimens,
and unknowns, are pipette into these wells and then a rabbit antibody specific for human active
target protein is added to the wells. During the first incubation, the human target protein binds to
the immobilized (capture) antibody and the specific active protein antibody serves as a detection
antibody by binding to the immobilized active protein. After the first incubation step and
washing to remove excess protein and detection antibody, a horseradish peroxidase-labeled Anti-
Rabbit IgG (Anti-Rabbit IgG HRP) is added. This binds to the detection antibody to complete the
four-member sandwich. After a third incubation and washing to remove all the excess Anti-
Rabbit IgG HRP, a substrate solution is added, which is acted upon by the bound enzyme to
produce color. The color intensity is measured using ROBONIK P2000
SPECTROPHOTOMETER at a wavelength of 450 nm. The intensity of this colored product is
15

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directly proportional to the concentration of human active protein present in the original
specimen [29].
4.2.2.4. Cyclin dependent Kinase 2 in vitro inhibition assay
This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody
specific for CDKN2A has been pre-coated onto a microplate. Standards and samples are pipette
into the wells and any CDKN2A present is bound by the immobilized antibody. After removing
any unbound substances, a biotin-conjugated antibody specific for CDKN2A is added to the
wells. After washing, avidin conjugated horseradish peroxidase (HRP) is added to the wells.
Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added
to the wells and color develops in proportion to the amount of CDKN2A bound in the initial step.
The color development is stopped and the intensity of the color is measured using ROBONIK
P2000 SPECTROPHOTOMETER at a wavelength of 450 nm. To determine IC50 different
concentrations of compound 6 (0.004, 0.02, 0.1, 0.4 and 0.5 ug/mL) was performed. The IC50
value was calculated according to the equation for Boltzman sigmoidal concentration–response
curve using the nonlinear regression fitting models (Graph Pad, Prism Version 5).
4.2.2.5. P21 and P27 expression
This assay employs the quantitative sandwich enzyme immunoassay technique. The kit used for
P21Cip1 is ab136945 –p21 Human ELISA kit while that used for P27Kip1DRG® Human p27
ELISA (EIA-4491). Samples and standards are added to wells coated with a monoclonal
antibody specific for P27Kip1. The plate is then incubated, washed, leaving only bound p27Kip1 the
plate. A yellow solution of polyclonal antibody to humanp27Kip1 is then added. This binds the
p27Kip1 captured on the plate. The plate is then incubated, washed to remove excess antibody. A
blue solution of HRP conjugate is added to each well, binding to the p27Kip1 polyclonal. The
plate is again incubated, washed to remove excess HRP conjugate. TMB Substrate solution is
added. The substrate generates a blue color when catalyzed by the HRP. Stop solution is added to
stop the substrate reaction. The resulting yellow color intensity is measured using ROBONIK
P2000 SPECTROPHOTOMETER at a wavelength of 450 nm.
*Same procedure is applied for P21Cip
16

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4.3. Molecular Docking
Docking study of the synthesized compound was performed by Molecular Operating
Environment (MOE) 2008.10 release of Chemical Computing Group, Canada. The program
operated under “Window XP” operating system installed on an Intel Pentium IV PC with a 2.8
MHz processor and 512 RAM. All minimizations were performed with MOE until a RMSD
gradient of 0.05 Kcal mol-1 Å-1 with MMFF94 force field and the partial charges were
automatically calculated. The score function, dock function (S, Kcal/mol) developed by MOE
program was used for the evaluation of the binding affinity of the ligand. The X-ray crystal
structure of the enzyme (PDB code: 1y8y) [24] was obtained from the protein data bank in PDB
format. The enzyme was prepared for docking studies: i) 3D protonation for the amino acid side
chain and ligand. ii) Isolation of the active site, fixation to be dealt with as rigid structure and
recognition of the amino acids. iii) Creation of dummies around the active site. iv) Studying the
interaction of the ligand with the amino acids of the active site.
The 3D structure of the synthesized compound were built using MOE and subjected to the
following procedure: i) 3D protonation of the structure. ii) Running conformational analysis
using systemic search. iii) Selecting the least energetic conformer. iv) Applying the same
docking protocol used with ligand.
Prior to the docking of the pyrazolopyrimidine derivative 6, redocking of the native ligand bound
in the CDK2 active site was performed to validate the docking protocol. The generated most
stable conformer of the compound was virtually docked into the predefined active site of the
CDK2. The developed docked models were energetically minimized and then used to predict the
interaction of the ligand with the amino acids in the active site of the enzyme.
Acknowledgment
Authors thank Dr. Essam Rashwan, Head of confirmatory diagnostic unit, VACSERA-EGYPT
for carrying out the cell cycle analysis, apoptosis and enzyme assays.
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[19] R. M. Silverstein, F. X. Webster, D. G. Kiemle, Spectrophotometric identification of
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Figures Captions
Figure 1: Chemical structure of some CDK2 inhibitors.
Figure 2: Cell cycle analysis of compound 5e and 6 on MCF-7 and HepG2, respectively.
Figure 3: Apoptosis induction analysis using Annexin V/PI of compound 5e and 6 on MCF-7
and HepG2, respectively.
Figure 4: 2D (above) and 3D (below) interaction of ligand CT7 on the active site of CDK2.
Figure 5: 2D (above) and 3D (below) interaction of compound 6 on the active site of CDK2.
Table 1: In vitro cytotoxic activity (IC50 µM) of compounds 3-7 against breast cancer cell line
(MCF-7) and liver cancer cell line (HepG2).
Table 2: Cell Cycle analysis results for compounds 5e and 6.
Table 3: Apoptosis induction analysis using Annexin V/PI for compounds 5e and 6.
Table 4: Results of caspase-3 activation for compounds 5e and 6.
Table 5: Results of in vitro cyclin dependent kinase 2 inhibition (IC50 determination) for
compound 6.
Table 6: Results of P21 and P27 expression for compound 6.
Scheme 1. Synthesis of compounds 1-7.
19

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OH
H
N
N
N
N
N
N
OH
N
N
HN
N
HN
I
N
O
II
Figure 1: Chemical structure of some CDK2 inhibitors.
Figure 2: Cell cycle analysis of compound 5e and
6 on MCF-7 and HepG2, respectively.
20

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Figure 3: Apoptosis induction analysis using Annexin V/PI of compound 5e and
and HepG2, respectively.
6 on MCF-7
21

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Figure 4: 2D (above) and 3D (below) interaction of ligand CT7 on the active site of CDK2.
22

ACCEPTED MANUSCRIPT
Figure 5: 2D (above) and 3D (below) interaction of compound
6 on the active site of CDK2.
23

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Table 6: In vitro cytotoxic activity (IC50 µM) of compounds 3-7 against breast cancer cell line
(MCF-7) and liver cancer cell line (HepG2).
IC50 µM
Compounds Breast cancer cell line
(MCF-7)
Liver cancer cell line
(HepG2)
>100
>100
3
>100
>100
38.00
2.50
>100
77.60
2.66
4
5a
5b
5c
23.90
42.60
>100
>100
0.40
14.46
1.40
5d
5e
>100
>100
40.70
1.02
5f
6
95.50
0.90
7
DOX.
Table 7: Cell Cycle analysis results for compounds 5e and 6.
Cell line Sample code
Control
% G0-G1
71.70
64.70
72.30
73.11
36.55
9.62
%S
%G2-M
5.23
%Apoptosis
0.62
22.45
27.92
16.09
21.69
25.66
24.67
MCF-7
5e / 24h
5e / 48h
Control
0.00
7.38
0.00
11.61
0.84
4.36
HepG2
6
6
/ 24h
/ 48h
23.78
48.91
13.92
16.71
24

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Table 8: Apoptosis induction analysis using Annexin V/PI for compounds 5e and 6.
Cell line Sample code % Early apoptosis % Late apoptosis
Control
5e / 24h
5e / 48h
Control
0.39
4.39
6.49
0.61
7.64
9.76
0.18
2.58
3.64
0.14
4.79
5.92
MCF-7
HepG2
6
6
/ 24h
/ 48h
Table 9: Results of caspase-3 activation for compounds 5e and 6.
Sample Conc. Caspase-3 conc.
Cell line
MCF-7
code
µM
(Pg/ml)
13.9
Control
1.40
440.9
17.2
5e
Control
HepG2
0.4
615.4
6
Table 10: Results of in vitro cyclin dependent kinase 2 inhibition (IC50 determination) for
compound 6.
Assay conc. µM
% Inhibition
0.004
0.02
0.1
0.4
0.5
12.591523 39.032691 53.129834 68.79842
81.808807
Table 6: Results of P21 and P27 expression for compound
6.
Sample Assay conc. Residual conc.
% Activation
code
Control
µM
Pg/mL
228.6
0.00
10.06
0.00
8.5
P21
P27
0.40
251.6
316.6
343.5
6
Control
0.40
6
25

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H2N
.HCl
NH
NH2
(a)
SO2NH2
SO2NH2
1
2
(b)
H2N
O
CN
N
NH2
NH2
N
N
N
N
NH2
N
N
N
(f)
(c)
SO2NH2
SO2NH2
SO2NH2
7
3
4
(e)
(d)
R
O
HN
NH
N
NH
R'
O
N
N
N
N
N
5a R = H, R' = H
5b R = H, R' = F
H
5c R = H, R' =Cl
5d R = H, R' =Br
5e R = H, R' = OCH3
5f R = OCH3, R' = OCH3
SO2NH2
SO2NH2
6
5
Scheme 1. Synthesis of compounds 1-7
Reagents and conditions: (a) i- NaNO2, HCl, 5 oC; ii- Na2SO3, HCl; (b) Ethoxy methylene
malononitrile, anhyd. sodium acetate, absolute ethanol, reflux, 8h; (c) H2SO4, stir at room
temperature, 16 h; (d) i- Aromatic aldehyde, DMF, 80 oC, 12-16 h; ii- 5% Na2S2O3 ; (e) Urea,
fusion, 20 min.; (f) Formamide, reflux, 16 h.
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ACCEPTED MANUSCRIPT
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