Novel pyrazolo[3,4-d]pyrimidines: design, synthesis, anticancer activity, dual EGFR/ErbB2 receptor tyrosine kinases inhibitory activity, effects on cell cycle profile and caspase-3-mediated apoptosis

Abstract A series of novel pyrazolo[3,4-d]pyrimidines was synthesised. Twelve synthesised compounds were evaluated for their anticancer activity against 60 human tumour cell lines by NCI (USA). Compound 7d proved prominent anticancer activity. It showed 1.6-fold more potent anti-proliferative activity against OVCAR-4 cell line with IC50 = 1.74 μM. It also exhibited promising potent anticancer activity against ACHN cell line with IC50 value 5.53 μM, representing 2.2-fold more potency than Erlotinib. Regarding NCI-H460 cell line, compound 7d (IC50 = 4.44 μM) was 1.9-fold more potent than Erlotinib. It inhibited EGFR and ErbB2 kinases at sub-micromolar level (IC50 = 0.18 and 0.25 µM, respectively). Dual inhibition of EGFR and ErbB2 caused induction of apoptosis which was confirmed by a significant increase in the level of active caspase-3 (11-fold). It showed accumulation of cells in pre-G1 phase and cell cycle arrest at G2/M phase.


Introduction
The EGFR family comprises four distinct membrane tyrosine kinase receptors: (EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ ErbB4) located in the plasma membrane and activated in response to ligand binding 1,2 . The interaction of EGFRs with the ligands causes receptor homo-and hetero-dimerisation, this interaction initiates a cascade of events that control diverse biological processes such as proliferation, differentiation, migration and apoptosis. Thus, deregulation of this transduction pathway has been implicated in many types of human cancers and is associated with poor clinical prognosis [2][3][4][5] . Moreover, members of EGFR family have been shown to be oncogenic. High expression levels of EGFR and ErbB2 has been implicated in the development of various types of cancers including breast, lung, colorectal, ovarian, prostate, head and neck [6][7][8] . EGFR and ErbB2 pathways are interconnected since both receptors have the highest homology among the EGFR family members in their kinase catalytic domains and share many similar biochemical and kinetic properties 9 . Additionally, the use of multiple ErbB receptors for cell proliferation and survival in tumour cells and the synergistic transforming effects of EGFR and ErbB2 lead to the hypothesis that targeting both the EGFR and ErbB2 catalytic domains simultaneously through dual EGFR/ErbB2 inhibition would have superior therapeutic effects relative to single-agent treatment for cancer 10 . Furthermore, a multi-targeted approach may improve the outcome of anti-EGFR therapies since the blockade of EGFR by EGFR tyrosine kinase inhibitors is insufficient to eradicate established tumors because of independently activated survival pathways 11 . Erlotinib (TarcevaTM) 12 , Gefitinib (IressaTM) 13 and Lapatinib (Tykerb TM ) 14 (Figure 1) are low-molecular-weight dual inhibitors of EGFR/ErbB2 tyrosine kinases that compete with adenosine triphosphate (ATP) to block the catalytic domain of these receptors. They had been approved for the chemotherapeutic treatment of cancer patients. The main structural features of these drugs are quinazoline scaffold containing a substituted phenyl amino pyrimidine moiety. Purine is a heterocyclic nucleus which exists in the chemical architecture of various bioactive compounds. It is an important pharmacophore, which is widely used in the development of protein kinase inhibitors via introduction of substituents on the 2, 6 and 9 positions 15 . Pyrazolo [3,4d]pyrimidine as a bioisostere of purine has drawn a considerable attention as a privileged scaffold for the design and discovery of novel anticancer agents 16,17 . In addition, pyrazolo [3,4-d]pyrimidine or its bioisostere pyrrolo [2,3-d]pyrimidine are common structural motifs of potent dual EGFR/ErbB2 inhibitors such as PKI166 18, I (AEE788) 19 , II 20 and III 21 ( Figure 1). Motivated by all these findings, we have designed and synthesised a series of pyrazolo [3,4-d]pyrimidines as novel small molecules targeting both EGFR and ErbB2 tyrosine kinases to be useful for treatment of cancer via inhibition of cell growth and induction of apoptosis. Our strategy was directed towards carrying out the chemical modifications on the general features of anilinoquinazoline scaffold to substantiate the effect of such modifications on the anticancer activity and to identify potent anticancer agents ( Figure  2). Initially, we aimed to replace the benzene moiety in the quinazoline skeleton by an isostere (pyrazole one). This respected nucleus is always bearing a 4-fluorophenyl group at N1 since fluorinated compounds are one of the research hotspots in modern medicinal chemistry 22 . Moreover, incorporation of a fluorine atom provides compounds with enhanced both pharmacokinetic and physicochemical properties as compared to their non-fluorinated analogs 23,24 . The second modification focused on introducing various phenyl amino groups on the pyrimidine moiety. We have introduced unsubstituted phenyl amino group, phenyl amino group substituted with electron donating groups or phenyl amino group substituted with electron withdrawing groups. The third modification included incorporating the phenyl amino group to the pyrimidine nucleus through a spacer such as azomethine group or piperazinyl linker. In the fourth modification, we have focused on replacement of the phenyl amino group by small pharmacophoric moieties as carbonyl, amino, morpholine, 4-methylpiperazine or hydrazinyl groups. These groups at such position are well acknowledged for the anticancer activity of the fused pyrimidine rings 25,26 . Finally, additional amino group was introduced at C-6 position of pyrazolopyrimidine core. Twelve of the newly synthesised pyrazolopyrimidines were subjected to in vitro anticancer screening by the National Cancer Institute (USA) against 60 different human cell lines. The most potent compound was selected to be further studied through determination of its half maximal inhibitory concentration (IC 50 ) values against ovarian cancer OVCAR-4, lung cancer NCI-H460, NCI-H226 and renal cancer ACHN cell lines. In order to explore the mechanistic pathways of the anticancer activity of 7d, it was evaluated in EGFR, ErbB2 and active caspase-3 assays. Moreover, we also investigated its effect on the normal cell cycle profile and induction of apoptosis in the OVCAR-4 cell line.     General procedure for the preparation of compounds (7a-d) A mixture of the chloro derivative 6 (0.3 g, 0.001 mol) and the selected aromatic amine (0.001 mol) was dispersed in isopropanol (2 ml). The reaction mixture was heated under reflux for 6 h. The reaction mixture was cooled. The obtained solid was filtered, dried and recrystallised from ethanol to afford 7a-d.

Anticancer activity
Measurement of anticancer activity against a panel of 60 cell lines Anticancer activity screening of the newly synthesised compounds was measured in vitro utilising 60 different human tumour cell lines provided by US National Cancer Institute according to previously reported standard procedure [27][28][29] as follows: Cells are inoculated into 96-well microtitre plates in 100 ml. After cell inoculation, the microtitre plates are incubated at 37 C, 5% CO 2 , 95% air and 100% relative humidity for 24 h prior to addition of experimental compounds. After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental compounds are solubilised in dimethyl sulphoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of compound addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 mg/mL gentamicin. Aliquots of 100 ml of the compounds dilutions are added to the appropriate microtitre wells already containing 100 ml of medium, resulting in the required final compound concentration. Following compound addition, the plates are incubated for an additional 48 h at 37 C, 5% CO 2 , 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold trichloroacetic acid (TCA). Cells are fixed in situ by the gentle addition of 50 ml of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 C. The supernatant is discarded, and the plates are washed five times with tap water and air-dried. Sulphorhodamine B (SRB) solution (100 ml) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 min at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air-dried. Bound stain is subsequently solubilised with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 ml of 80% TCA (final concentration, 16% TCA). Using the absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of compound (Ti)], the percentage growth is calculated for each compound. Percentage growth inhibition is calculated as:

MTT assay protocol
The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method 30 of monitoring in vitro cytotoxicity is well suited for use with multiwell plates. The assessment of cell population growth is based on the capability of living cells to reduce the yellow product MTT to a blue product, formazan, by a reduction reaction occurring in the mitochondria. The five cell lines were incubated for 24 h in 96-microwell plates. The number of living cells in the presence or absence (control) of the various test compounds is directly proportional to the intensity of the blue colour, measured by spectrophotometry using (ROBONIK P2000 Spectrophotometer) at a wavelength of 570 nm. Measure the background absorbance of multiwell plates at 690 nm and subtract from the 570 nm measurement. Five concentrations ranging from 0.01 lM to 100 lM (with semi-log decrease in concentration) were tested for each of the compounds under study. Each experiment was carried out in triplicate. The IC 50 values [the concentration required for 50% inhibition of cell viability] were calculated using sigmoidal dose response curve-fitting models.

Measurement of inhibitory activity against EGFR
Compound 7d was selected to be evaluated against EGFR enzyme using EnzyChrom TM Kinase Assay Kit (EKIN-400) according to manufacturer's instructions. In brief, set up 20 lL reaction mixture containing the EGFR kinase, ATP and substrate in the provided assay buffer. Set up a blank control that contains ATP and substrate but no enzyme. Incubate at desired temperature for 30 min. Prepare 900 lL 10 lM (adenosine diphosphate) ADP premix by mixing 3 lL 3 mM standard and 897 lL distilled water. Transfer 20 lL standards into separate wells of the plate. Prepare enough working reagent for each well. Add 40 lL working reagent to each assay well. Tap plate to mix. Incubate at room temperature for 10 min. Read fluorescence intensity at k exc ¼ 530 nm and k em ¼ 590 nm.Calculate kinase activity.

Measurement of inhibitory activity against ErbB2
Compound 7d was evaluated against ErbB2 enzyme using HTScanV R HER2/ErbB2 Kinase Assay Kit according to manufacturer's instructions. In brief, add 10 lL of 10 mM ATP to 1.25 ml of 6 lM substrate peptide. Dilute the mixture with dH 2 0 to 2.5 ml. Transfer enzyme from À80 C to ice. Allow enzyme to thaw on ice. Microcentrifuge briefly at 4 0 C. Return immediately to ice. Add

Measurement of the effect of compound 7d on the level of caspase-3 protein (Marker of apoptosis):
The level of human active caspase-3 protein was evaluated using Invitrogen (Catalog KHO1091) ELISA kit. The manufacturer's instructions were followed in the following procedures. Add 100 lL of the standard diluent buffer to the zero standard wells. Add 100 lL of standards and controls or diluted samples to the appropriate microtitre wells. Cover wells with and incubate for 2 h at room temperature. Thoroughly aspirate or decant solution from wells and discard the liquid. Pipette 100 lL of caspase-3 (active) detection antibody solution into each well. Cover plate and incubate for 1 h at room temperature. Add 100 lL Anti-Rabbit IgG HRP working solution to each well. Cover wells with the plate cover and incubate for 30 min at room temperature. Add 100 lL of stabilised chromogen to each well. The liquid in the wells will begin to turn blue. Incubate for 30 min at room temperature. Stop solution has been added to each well. The solution in the wells should change from blue to yellow. Read the plate within 2 h after adding the stop solution. Use a curve fitting software to generate the standard curve.

Cell cycle analysis of compound 7d
The OVCAR-4 cells were treated with compound 7d at its IC 50 concentration for 24 h. After treatment, the cells were washed twice with ice-cold 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 propidium iodide (PI), and analysed by flow cytometry using FACS Calibur (Becton Dickinson) 31 . The cell cycle distributions were calculated using Cell-Quest software (Becton Dickinson). Exposure of OVCAR-4 cells to compound 7d resulted in an interference with the normal cell cycle distribution as indicated.

Chemistry
The synthesis of the new compounds is illustrated in Schemes 1 and 2. The preparation of the starting material 1 was achieved via heating triethyl orthoacetate and malononitrile in acetic anhydride as previously reported 32    Bold values signifies the growth inhibition parentage is higher than 50%. spectra of 7a-d showed the presence of an absorption band at 3444 cm À1 corresponding to NH group. The 1 H NMR spectra supported the formation of 7a-d through the appearance of the D 2 O exchangeable singlet signal at d 8.75 -8.90 ppm. The appearance of extra aromatic protons corresponding to substitution on the 4-amino moiety was another significant proof of the success of the reaction. The synthesis of the target compounds 8a-d was accomplished through the reaction of the 4-chloropyrazolo [3,4d]pyrimidine derivative 6 with the selected secondary amine in ethanol in the presence of a catalytic amount of triethylamine. The 1 H NMR spectra of these compounds showed the presence of the 4 CH 2 protons of the morpholinyl or piperazinyl moiety at d 3.20 -3.78 ppm, while the 13C NMR spectra indicated the presence of 4 CH 2 carbons at d 45.6 -66.4 ppm. The hydrazinyl derivative 9 was obtained in a good yield via reacting the 4-chloropyrazolo [3,4-d]pyrimidine 6 with hydrazine hydrate in ethanol. The IR spectrum of compound 9 showed two absorption bands at the range 3309 -3120 cm À1 indicating the presence of both NH and NH 2 groups. The 1 H NMR spectrum revealed the presence of two D 2 O exchangeable singlet signals at d 4.80 and 8.90 ppm with proper integration, corresponding to NH 2 and NH protons, sequentially. Condensation of the 4-hydrazinyl derivative 9 with the selected aromatic aldehyde in ethanol in the presence of glacial acetic acid yielded compounds 10a-d in good yields. The 1 H NMR of the target compounds revealed the appearance of a singlet signal at the range of d 8.19 -8.31 ppm corresponding to N¼CH proton, in addition to additional characteristic aromatic protons denoting the success of the condensation reaction.

Growth inhibition against a panel of 60 human tumor cell lines
In this study, 12 of the newly synthesised pyrazolopyrimidines were subjected to in vitro anticancer screening by the National Cancer Institute (USA) against 60 different human cell lines including (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer). The selected compounds were evaluated at single dose (1 0 À5 M). The growth inhibition percentages obtained from the single dose test for the selected compounds are shown in Tables 1 and 2. The analysis of the obtained in vitro data revealed that two lung cell lines NCI-H226 and NCI-H460 were sensitive to compound 7d with growth inhibition percentages 62.91 and 58.46, sequentially. Compound 7d also displayed potent cytotoxic activity against renal cell line ACHN with growth inhibition percentage 60.41 and against ovarian cancer cell line OVCAR-4 with growth inhibition percentage 53.12. Compound 6 showed promising selectivity against lung cancer cell line NCI-H522 with growth inhibition percentage 58.95. Other test compounds exhibited no activity against most investigated cell lines.     Detection of IC 50 of compound 7d against lung cancer NCI-H460, NCI-H226, renal cancer ACHN and ovarian cancer OVCAR-4 cell lines Compounds 7d was selected to be further studied through determination of its half maximal inhibitory concentration (IC 50 ) values against the most sensitive cancer cell lines compared to Erlotinib as a reference anticancer drug. The results of the mean values of experiments performed in triplicate were summarised in Table 3 and represented graphically in Figure 3. The in vitro results showed that the most sensitive cell line for compound 7d was ovarian (OVCAR-4) with IC 50 ¼ 1.74 lM, which was 1.6 times more potent than Erlotinib. It also showed promising potent anticancer activity against renal (ACHN) cell line with IC 50 value 5.53 lM representing 2.2 folds more potency than Erlotinib. Regarding lung cancer NCI-H460 cell line, compound 7d (IC 50 ¼ 4.44 lM) was 1.9 folds more potent than Erlotinib. It is worthy mention that compound 7d (IC 50 ¼ 18.73 lM) was almost 3.5 folds more potent than Elrlotinib against NCI-H460 EGFR-WT cell line. It possessed moderate anticancer activity against NCI-N226 cell line with IC 50 ¼ 17.36 lM. Structural activity relationship analysis revealed that anti-proliferative activity of the newly synthesised pyrazolo [3,4d]pyrimidines correlates well with substitution pattern on position 4. It is worth noting that compound 7d, a highly potent anticancer agent was among pyrazolopyrimidines 7a-d having a phenyl amino group directly attached to the pyrimidine nucleus. Further analysis of these compounds clearly revealed the substitution pattern on this phenyl amino moiety had a notable effect on the anticancer activity. Grafting 3-Cl and 4-F substituents to the 4-anilino moiety enhanced the anti-proliferative activity against several tumor cell lines, otherwise 4-F and 4-CH 3 substituents resulted in inactive members. Furthermore, the incorporation of the phenyl amino group through azomethine or piperazinyl linker regardless of the substitution pattern on the phenyl group abolished activity. Finally, replacement of the 4-anilino group with pharmacophoric moieties as carbonyl, amino, morpholine, 4-methylpiperazine or hydrazinyl groups or introducing additional    amino group at C-6 position resulted in a dramatic loss in the anti-proliferative activity.

Measurement of the effect of 7d on EGFR enzyme
We have investigated the inhibitory effect of compound 7d on EGFR enzyme in vitro. The anti-proliferative activity of this compound appeared to correlate well with its ability to inhibit EGFR at submicromolar range with IC 50 value 0.18 mM (Table 4 and Figure 4).

Measurement of the effect of 7d on ErbB2 enzyme
Since EGFR and ErbB2 receptors have the highest homology among the EGFR family members in their kinase catalytic domains and share many similar biochemical and kinetic properties. We have measured the inhibitory effect of compound 7d on ErbB2 tyrosine kinase in vitro. Compound 7d showed excellent inhibitory activity, at the submicromolar level with IC 50 value 0.25 mM (Table 4 and Figure 4).
Measurement of the effect of compound 7d on caspase-3 level EGFR and ErbB2 tyrosine kinase inhibitors are well known to potentiate the intrinsic apoptotic pathway via increase in caspase-3 enzyme activity 34,35 . To evaluate the effect of compound 7d on the level of active caspase-3, OVCAR-4 cells were treated with compound 7d at its IC 50 value for 24 h, before the enzyme assay. Compound 7d resulted in a significant increase (almost 11 folds) in the level of active caspase-3 compared to control (Table 5 and Figure 5).

Cell cycle analysis and detection of apoptosis
The effect of the most active compound 7d on the cell cycle progression and induction of apoptosis in OVCAR-4 cells was studied. OVCAR-4 cells were exposed to compound 7d at its IC 50 values for 24 h and its effect on the normal cell cycle profile and induction of apoptosis was analysed. Exposure of OVCAR-4 cells to compound 7d resulted in an interference with the normal cell cycle distribution of this cell line. Interestingly, compound 7d resulted in an increase in the cells accumulated in pre-G1 phase by almost 11-folds compared to control, the apoptotic activity was affirmed by the presence of a sub-G1 peak which may result from degradation or fragmentation of the genetic materials. Also, it showed significant increase in the percentage of cells at G2/M phases by 5 folds, compared to control (Table 6 and Figures 6, 7). These results suggest that compound 7d exerted its cytotoxic activity by promoting cycle arrest at G2/M phase and apoptotic induction.

Apoptosis determination by Annexin-V assay
The apoptotic activity of compound 7d was further studied using Annexin V-FITC assay, which includes dual staining using Annexin V, a Ca 2þ -dependent protein, and Propidium iodide (PI). Annexin V binds to phosphatidylserine (PS) expressed only on the surface of the apoptotic cells and fluoresces green after interacting with the fluorochrome labelled annexin-V. On the other hand, PI stains DNA and enters only dead cells. This assay can give a differential analysis to the percentages of living, early apoptotic, late apoptotic and necrotic cells 36 . As shown in Figures 8, 9 and Table 7,  after 24 h of treatment of OVCAR-4 cells with compound 7d at its IC 50 concentration a decrease in the percentage of the survived cells was observed. Moreover, a significant increase in the percentage of Annexin-V positive cells (almost 9 folds more than control) occurred indicating an early apoptosis (lower right quadrant). In addition, an increase by 7 folds more than control in the percentage of PI positive cells (upper left quadrant) indicating necrotic cells. Some treated cells were in a late apoptotic stage (upper right quadrant), this was indicated by the significant increase in the percentage of Annexin V positive, PI positive cells (16 folds more than control).

Conclusion
A series of novel pyrazolo [3,4-d]pyrimidines was synthesised. Twelve synthesised compounds were evaluated for their anticancer activity by NCI (USA). Compound 7d exhibited potent anticancer activity at low concentrations. Pyrazolopyrimidine 7d proved marked anticancer activity higher than Erlotinib. It showed 1.6-fold more potent anti-proliferative activity against ovarian (OVCAR-4) cell line with IC 50 ¼ 1.74 lM. It also exhibited promising potent anticancer activity against renal (ACHN) cell line with IC 50 value 5.53 lM representing 2.2-fold more potency than Erlotinib. Regarding lung cancer NCI-H460 cell line, compound 7d (IC 50 ¼ 4.44 lM) was 1.9 folds more potent than Erlotinib. It is worthy mention that compound 7d was almost 3.5-fold more potent than Elrlotinib against NCI-H460 EGFR-WT cell line. It possessed moderate anticancer activity against NCI-N226 cell line with IC 50 ¼ 17.36 lM. The anti-proliferative activity of pyrazolopyrimidine 7d appeared to correlate well with its ability to inhibit both EGFR and ErbB2 tyrosine kinases at sub-micromolar level with IC 50 values 0.18 and 0.25 mM, sequentially. Dual inhibition of EGFR and ErbB2 enzymes leads to induction of the intrinsic pathway of apoptosis. This mechanistic pathway was confirmed by a significant increase in the level of active caspase-3, which is the key executer of apoptosis compared to the control (10.8 folds). Moreover, compound 7d showed accumulation of cells in pre-G1 phase and annexin-V and propidium iodide staining in addition to cell cycle arrest at G2/M phase. Pyrazolo [3,4-d]pyrimidine 7d is a privileged multi-targeted scaffold for the design and discovery of novel anticancer agents.