Cancer is the most serious health problem and the second major cause of death in the developing countries [1,2]. In spite of significant process in the development of novel chemotherapeutic agents in the last seven decades, success in developing targeted non-toxic drugs with minor side effects has only achieved in the last one [3]. Therefore, the discovery of new selective, potent and safe antitumor agents is a must. Pyrazolo[3,4-d] pyrimidine nucleus is the bio-isostere of purine [4,5]. Hence exhibits promising activity as antitumor by competitive inhibition for ATP kinase enzymes. Many pyrazolo[3,4-d]pyrimidine derivatives were reported as antitumor agents [6-8]. The cytotoxic activity of such compound may attributed to inhibition of several enzymes such as tyrosine kinase [9], Src kinase [10], cyclin dependent kinase (CDK) [11], mammalian target of rapamycin (mTOR) [12] and glycogen synthase kinase (GSK) [13,14]. In addition, the presence of methylsulphanyl group at the 3 position of pyrazolo[3,4-d]pyrimidine nucleus was reported to potentiate the antitumor activity of such nucleus [11,15]. For example, compound 1 and 2 (Figure 1) were exhibited excellent antitumor activity against breast adenocarcinoma cell line MCF 7 with an IC₅₀ values of 12.0 and 7.50 μM respectively [16]. Also, compound 3 displayed superior activity as cytotoxic against A549 cell line with IC₅₀ K_b value of 5.28 μM [4].

Figure 1: ATP, potent antitumor pyrazolo[3,4-d]pyrimidines and designed compounds.

Based on these scientific facts and for further exploration of novel antitumor agents, we supposed that incorporation of these structural features together may results in potent antitumor agents that act on breast adenocarcinoma cell line. In this work, new 3-(methylthio)- 1-phenyl-1H-pyrazolo[3,4-d]pyrimidine derivatives 10-16 were synthesized, incorporating the methylsulphanyl group at the 3 position of pyrazolo[3,4-d]pyrimidine ring system and varying the substituents at the 4 and 5 positions of such ring in order to study the effect of these varying substitutions on the antitumor activity of pyrazolo[3,4-d] pyrimidine nucleus against human breast adenocarcinoma cell line MCF7.

Chemistry

Scheme 1 shows the synthetic pathway of the starting pyrazolo[3,4-d] pyrimidin-4(5H)-one derivatives 8 and 4-chloro-3-(methylthio)-1- phenyl-1H-pyrazolo[3,4-d]pyrimidine (9) which were accomplished via reaction of malononitrile with carbon disulfide in the presence of sodium ethoxide followed by methylation of the product with dimethyl sulphate. The resulting 2(bis(methylthio)methylene)malononitrile was then treated with phenyl hydrazine in absolute ethanol [17]. Cyclization of the 5-amino-3-(methylthio)-1-phenyl-1Hpyrazole-4-carbonitrile (7) by the action of formic acid afforded 3-(methylthio)-1-phenyl-1Hpyrazolo[ 3,4-d]pyrimidin-4(5H)-one [18]. Structure of the latter was confirmed by the disappearance of the C≡N and NH characteristic absorption bands in the IR spectrum of the starting 5-amino- 1Hpyrazole-4-carbonitrile 7. Chlorination of compound 8 with phosphorus oxychloride yielded the 4-chloro derivative 9 [19,20]. The latter was allowed to react with different aliphatic and aromatic amines to afford the target pyrazolo[3,4-d]pyrimidin-4-amine derivatives 10_a-e, 11 and 12 (Scheme 2).

Scheme 1: Synthetic pathway of starting pyrazolo[3,4-d]pyrimidine.

Scheme 2: Synthetic pathway of target compounds 10-12. Reagents: a) CS₂, C₂H₅OH; b) (CH₃)2SO4, C₂H₅OH; c) NH₂NH₂, C₂H₅OH; d) HCOOH; e) POCl3.

Formation of compounds 10-12 was confirmed by spectral data and elemental analyses. The ¹HNMR spectra of these derivatives demonstrated the appearance of a new D₂O exchangeable singlet signals at δ 8.48-8.60 ppm corresponding to the NH protons. Mass spectra of these compounds showed distinctive molecular ion peaks at the right m/z values.

R=2-CH₃, 2-SH, 2,6-Cl₂, 2-COOH, 4-COOC₂H₅

Scheme 3 shows the synthetic pathway of the target compounds 13_a-h through reaction of 3-(methylthio)-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-4(5H)-one (8) with 2-chloro-N-phenylacetamide or 3-chloro-N-phenylpropanamide derivatives. Structures of these amides were confirmed depending on spectral data and elemental analyses. The IR spectra of these compounds showed the characteristic NH stretching bands at the range of 3223-3317 cm^-1. In addition, the ¹HNMR spectra of the same derivatives showed singlet signals corresponding to NH protons at δ 9.72-10.78 ppm.

Scheme 3: Synthetic pathway of target compounds 13-16. Reagents: a) RC₆H₄NH₂, C₂H₅OH/(C₂H₅)3N; b) Cyclohexylamine, C₂H₅OH/(C₂H₅)3N; c) 4-(Aminomethyl)benzoic acid, C₂H₅OH/(C₂H₅)3N.

Ester derivatives 14_a,b were prepared via condensation of compound 8 with alkyl chloroacetate in the presence of potassium carbonate. Structures of these two ester derivatives were confirmed on the basis of their spectral data and elemental analyses. The IR spectra of these esters showed sharp elevation of the wavenumber of the C=O absorption compared to that of the starting amide. Condensation of 14_b with hydrazine hydrate produced the hydrazide 15. The ¹HNMR spectrum of this new compound revealed two D₂O exchangeable signals of the NH and NH₂ protons at 9.41 and 4.30 ppm respectively. Disappearance of the NH₂ signal in the ¹HNMR spectra of compounds 16_a,b confirms their structures.

For 13_a-d n=1, R=H, 2-Cl, 3-CH₃, 4-COOC₂H₅; For 13_e-h: n=2, R=H, 2-Cl, 3-CH₃, 4-COOC₂H₅; For 14_a-b: R=CH₃, C₂H₅; For 16_a-b: R=H, OH

Reagents: For 13_a-d: a) ClCH₂CONHC₆H₄R, K₂CO₃, C₂H₅OH; For 13eh : a) ClCH₂CH₂CONHC₆H₄R, K₂CO₃, C₂H₅OH; b) ClCH₂CH₂COOR, K₂CO₃, C₂H₅OH; c) NH₂NH₂; d) RC₆H₄CHO, C₂H₅OH

In vitro antitumor screening

All of the newly synthesized derivatives were evaluated for antitumor activity by measuring the inhibitory effect of such compounds against human breast adenocarcinoma cell line MCF7 using MITT technique [21,22]. The MTT Cell Proliferation Assay measures the reduction in cancer cell viability due to apoptosis or necrosis as a response to external factor. The yellow colored tetrazolium salt of MTT is reducible by the action of metabolically active cells, through dehydrogenase enzymes that leads to generation of NADH and NADPH reducing equivalents. The produced intracellular purple formazan can be solubilized and spectrophotometrically quantified [23]. The results of in vitro antitumor activity were compared with doxorubicin as a reference antitumor agent. The parameter used herein is the IC₅₀, which represents the concentration needed for 50% inhibition of the cell viability. A relation between the IC₅₀ values of the new compounds that showed more than 50% inhibition against MCF-7 and that of the reference antitumor agent are shown in Table 1 and represented graphically in Figure 2.

Table 1: Results of in vitro cytotoxic activity of compounds showed more than 50% inhibition of MCF 7 adenocarcinoma cell line.

Figure 2: IC₅₀ in µM of the synthesized compounds and doxorubicin against MCF7 adenocarcinoma cell line.

General

All melting points were taken on electro thermal (LA9000 SERIS) digital melting point apparatus and are uncorrected. IR spectra were recorded on PyeUnicam Sp 1000 spectrophotometer and were carried out at the Pharmaceutical Analytical Unit, Faculty of Pharmacy, Al- Azhar University, Egypt. The ¹HNMR and ¹³CNMR spectra were recorded in DMSO-D₆ either on Varian Mercury VXR-300 NMR spectrophotometer at the Microanalytical Unit of Cairo University or BURKER 400 MHZ spectrophotometer at the Nuclear Magnetic Resonance Lab, Faculty of Pharmacy, Zagazig University, Egypt. Chemical shifts were related to that of the solvent. TMS was used a standard. Mass spectra were recorded on Hewlett Packard 5988 spectrometer at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Progress of the reactions were monitored by TLC pre-coated with UV fluorescent silica gel and was visualized using UV lamp and different solvent systems as mobile phases. 5-Amino-3-(methylthio)-1-phenyl-1H-pyrazole-4-carbonitrile (7) and 3-(methylthio)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)- one (8) were prepared according to published method¹⁷. Compound 9 was obtained following reported procedure [19]. 2-chloro-Narylactamide and 3-chloro-N-arylpropanamide derivatives were prepared as reported [24].

General procedure for synthesis of 3-(methylthio)-1-phenyl- N-aryl-1H-pyrazolo[3,4-d]pyrimidin-4-amines 10_a-e: A mixture of compound 9 (10 mmol) and the appropriate aniline derivative (10 mmol) in absolute ethanol (35 ml) containing trimethylamine (15 mmol) was heated under reflux for 6 hours. The reaction mixture was cooled, and the separated solid was filtered, dried and finally recrystallized from ethanol.

3-(Methylthio)-1-phenyl-N-o-tolyl-1H-pyrazolo[3,4-d] pyrimidin-4-amine (10a): White solid; Yield: 85%; m. p. 130-131°C. IR (KBr) cm^-1: 3372 (NH), 3047 (CH aromatic), 2924 (CH aliphatic). ¹HNMR (DMSO-d6) δ ppm: 8.45 (s, 1H, NH, D₂O exchangeable), 8.42 (s, 1H, pyrimidine-H2), 8.19 (d, 2H, J=1.80 Hz, phenylpyrazole-H2, H6), 7.80 (t, 2H, J=7.80 Hz, phenylpyrazole-H3, H5), 7.59 (t, 1H, J=2.10 Hz, phenylpyrazole-H4), 7.56 (t, 1H, J=6.90 Hz, phenyl-H5), 7.38 (d, 1H, J=1.50 Hz, phenyl-H3),7.35 (d, 1H, J=1.50 Hz, phenyl-H6), 7.29 (t, 1H, J=6.00 Hz, phenyl-H4), 2.50 (s, 3H, SCH₃), 2.30 (s, 3H, Ar-CH₃). MS (m/z): 347 (C₁₉H₁₇N₅S, 53.48%, M⁺), 332 (C₁₈H₁₅N₅S, M-CH₃, 74.57%), 77 (C₆H₅, 100%). Analytical Calculated for: (C₁₉H₁₇N₅S) (M.W.=347): C, 65.68; H, 4.39; N, 20.16%; Found: C, 65.81; H, 4.89; N, 20.31%.

2-(3-(Methylthio)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4- ylamino)benzenethiol (10_b): White solid; Yield: 75%; m. p. 141-142°C. IR (KBr) cm^-1: 3291 (NH), 3053 (CH aromatic), 2918 (CH aliphatic). ¹HNMR (DMSO-d6) δ ppm: 12.42 (s, 1H, SH, D₂O exchangeable), 8.07 (s, 1H, pyrimidine-H2), 8.06 (d, 2H, J=4.80 Hz, phenylpyrazole-H2, H6), 7.93 (t, 2H, J=10.00 Hz, phenylpyrazole-H3, H5), 7.58 (t, 1H, J=3.00 HZ, phenylpyrazole-H4), 7.49 (t, 1H, J=10.00 HZ, phenyl-H5), 7.44 (d, 1H, J=2.80 Hz, phenyl-H3),7.35 (d, 1H, J=4.00 Hz, phenyl-H6), 7.32 (t, 1H, J=3.60 Hz, phenyl-H4), 2.60 (s, 3H, SCH₃). MS (m/z): 365 (C₁₈H₁₅N₅S₂, 2.33%, M⁺). Analytical Calculated for: (C₁₈H₁₅N₅S₂) (M.W.=365): C, 59.16; H, 4.14; N, 19.16%; Found: C, 59.21; H, 3.83; N, 19.47%.

N-(2,6-dichlorophenyl)-3-(methylthio)-1-phenyl-1Hpyrazolo[ 3,4-d]pyrimidin-4-amine (10_c): White solid; Yield: 63%; m. p. 231-232°C. IR (KBr) cm^-1: 3455 (NH), 3050 (CH aromatic), 2938 (CH aliphatic). ¹HNMR (DMSO-d6) δ ppm: 10.40 (s, 1H, NH, D₂O exchangeable), 8.66 (s, 1H, pyrimidine-H2), 8.18 (d, 2H, J=4.80 Hz, phenylpyrazole-H2, H6), 8.04 (t, 2H, J=8.10 Hz, phenylpyrazole-H3, H5), 7.59 (t, 1H, J=6.90 Hz, phenylpyrazole-H4), 7.54 (d, 2H, J=7.20 Hz, phenyl-H3,H5), 7.39 (t, 1H, J=6.60 Hz, phenyl-H4), 2.60 (s, 3H, SCH₃). MS (m/z): 403 (C₁₈H₁₃Cl₂N₅S, 0.41%, M+2), 401 (C₁₈H₁₃Cl₂N₅S, 1.46%, M⁺), 366 (C₁₈H₁₃ClN₅S, 2.42%), 331 (C₁₈H₁₃N₅S, 1.47%), 256 (C₁₂H₁₀N₅S, 3.01%), 241 (C₁₂H₉N₄S, 4.62%). Analytical Calculated for: (C₁₈H₁₃Cl₂N₅S) (M.W.=401): C, 53.74; H, 3.26; N, 17.41%; Found: C, 53.92; H, 3.23; N, 17.68%.

4-(3-(Methylthio)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4- ylamino)benzoic acid (10_d): White solid; Yield: 85%; m. p. 162-164°C. IR (KBr) cm^-1: 3397 (OH), 3360 (NH), 3047 (CH aromatic), 2924 (CH aliphatic), 1689 (C=O). ¹HNMR (DMSO-d6) δ ppm: 13.80 (s, 1H, OH, D₂O exchangeable), 11.25 (s, 1H, NH, D₂O exchangeable), 8.52 (s, 1H, pyrimidine-H2), 8.50-7.30 (m, 9H, Ar-H), 2.62 (s, 3H, SCH₃). MS (m/z): 377 (C₁₉H₁₅N₅O₂S, 1.19%, M⁺), 333 (C₁₈H₁₅N₅S, 2.4%). Analytical Calculated for: (C₁₉H₁₅N₅O₂S) (M.W.=377): C, 60.47; H, 4.01; N, 18.56%; Found: C, 60.64; H, 4.09; N, 18.73%.

Ethyl-4-((3-(methylthio)-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-4-yl)amino)benzoate (10_e): White solid; Yield: 85%; m. p. 123-124°C. IR (KBr) cm^-1: 3372 (NH), 3033 (CH aromatic), 2940 (CH aliphatic), 1710 (C=O). ¹HNMR (DMSO-d6) δ ppm: 8.79 (s, 1H, NH, D₂O exchangeable), 8.60 (s, 1H, pyrimidine-H2), 8.17 (d, 2H, J=1.80 Hz, phenylpyrazole-H2, H6), 7.97 (t, 2H, J=8.70 Hz, phenylpyrazole-H3, H5), 7.56 (d, 2H, J=8.10 Hz, phenyl-H2,H6), 7.37 (t, 1H, J=6.00 Hz, phenylpyrazole-H4), 7.33 (t, 2H, J=6.00 Hz, phenyl H2,H6), 4.23 (q, 2H, J=7.20 Hz, CH₂CH₃), 2.62 (s, 3H, S-CH₃), 1.3 (t, 3H, J=6.6 Hz, CH₂CH₃). MS (m/z): 405 (C₂₁H₁₉N₅O₂S, 6.85%, M⁺), 376 (C₁₉H₁₄N₅O₂S, 3.72%), 256 (C₁₂H₁₀N₅S, 2.27%), 241 (C₁₂H₉N₄S, 12.52%). Analytical Calculated for: (C₂₁H₁₉N₅O₂S) (M.W.=405): C, 62.21; H, 4.72; N, 17.27%; Found: C, 62.47; H, 4.81; N, 17.49%.

N-Cyclohexyl-3-(methylthio)-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-4-amine (11): Into a solution of equimolar amounts of compound 9 and cyclohexylamine (10 mmol each) in ethanol (30 ml), trimethylamine (15 mmol) was added. The reaction mixture was heated under reflux for 6 hours then allowed to cool. The crude product was filtered out, dried and finally recrystallized from ethanol. White solid; Yield: 74%; m. p. 114-116 ºC. IR (KBr) cm^-1: 3397 (NH), 3031 (CH aromatic), 2925 (CH aliphatic). ¹HNMR (DMSO-d6) δ ppm: 8.36 (s, 1H, pyrimidine-H2), 8.16 (d, 2H, H6, J=8.10, phenyl-H2), 7.55 (t, 2H, H₅, J=8.40, phenyl-H3), 7.33 (t, 1H, J=7.20, phenyl-H4), 6.32 (s, 1H, NH, D₂Oexchangeable), 2.71 (s, 3H, SCH₃) 1.97-1.36 (m, 11H, cyclohexyl). MS (m/z): 339 (C₁₈H₂₁N₅S, 23.10%, M⁺), 257 (C₁₂H₁₁N₅S, M-C₆H₁₁, 100%). Analytical Calculated for: (C₁₈H₂₁N₅S) (M.W.=339): C, 63.69; H, 6.24; N, 20.63%; Found: C, 63.85; H, 6.32; N, 20.86%.

4-((3-(Methylthio)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin- 4-ylamino)methyl)benzoic acid (12): Into a solution of equimolar amounts of compound 9 and 4-(aminomethyl)benzoic acid (10 mmol each) in ethanol (30 ml), trimethylamine (15 mmol) was added. The reaction mixture was heated under reflux for 6 hours then allowed to cool. The crude product was filtered out, dried and finally recrystallized from ethanol. White solid; Yield: 70%; m. p. 170-171°C. IR (KBr) cm^-1: 3382 (broad OH), 3027 (CH aromatic), 2985 (CH aliphatic), 1699 (C=O). ¹HNMR (DMSO-d6) δ ppm: 10.82 (s, 1H, OH), 8.39 (s, 1H, pyrimidine-H2), 7.88 (d, 2H, phenyl-H2, H6, J=7.2), 7.30 (m, 5H, phenylpyrazole), 6.87 (d, 2H, phenyl-H3, H5, J=7.2), 6.28 (s, 1H, NH), 4.32 (s, 2H, CH₂), 2.68 (s, 3H, S-CH₃). MS (m/z): 391 (C₂₀H₁₇N₅O₂S, M⁺, 100%), 270 (C₁₉H₁₄N₅O₂S, M-CH₃, 46.20%), 256 (C₁₂H₁₁N₅S, M-COOH-C₆H₄CH₂, 43.23%). Analytical Calculated for: (C₂₀H₁₇N₅O₂S) (M.W.=391): C, 61.37; H, 4.38; N, 17.89%; Found: C, 61.59; H, 4.43; N, 18.15%.

General procedure for synthesis of 2-(3-(methylthio)-4-oxo-1- phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl)-N-arylacetamide and N-arylpropanamide 13_a-h: Into a solution of compound 8 (10 mmol each) in DMF (30 ml) containing potassium carbonate (0.5 g), the appropriate 2-chloro N-arylacetamide or 3-chloro- N-arylpropanamide (10 mmol) was added. The reaction mixture was heated under reflux for 3 hours. After complete reaction, the reaction mixture was filtered while hot, concentrated, cooled and the resulting solid product was dried and finally recrystallized from ethanol.

2-(3-(Methylthio)-4-oxo-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-5(4H)-yl)-N-phenylacetamide (13_a): White solid; Yield: 85%; m. p. 164-165°C. IR (KBr) cm^-1: 3307 (NH), 3053 (CH aromatic), 2925 (CH aliphatic), 1672 (C=O). ¹HNMR (DMSO-d6) δ ppm: 10.42 (s, 1H, NH, D₂O exchangeable), 8.47 (s, 1H, pyrimidine-H2), 8.04 (t, 2H, J=7.20 Hz, Aniline-H3,H5), 7.59 (d, 2H, J=2.00 Hz, Aniline-H2,H6), 7.57 (d, 2H, J=8.40 Hz, phenylpyrazole-H3,H5), 7.41 (t, 1H, J=7.20 Hz, 7.34 (t, 2H, J=7.60 Hz, phenylpyrazole-H2,H6), 7.07 (t, 1H, J=7.20 Hz, phenylpyrazole-H4), 4.87 (s, 2H, CH₂C=O), 2.63 (s, 3H, S-CH₃). ¹³CNMR (DMSO-d6 400 MHz) δ ppm: 13.26,48.59, (Aliphatic CH₃ and CH₂), 104.94, 119.53, 121.76, 124.07, 127.39, 129.36, 129.72, 138.45, 139.04, 146.08, 152.96, 153.18, 156.51 (Aromatic carbons), 165.74 (C=O). MS (m/z): 391 (C₂₀H₁₇N₅O₂S, M, 42.33%), 299 (C₁₄H₁₁N₄O₂S, M-NHC₆H₅, 100%), 271 (C₁₃H₁₁N₄OS, 51.75%), 257 (C₁₂H₉N₄OS, 8.2%). Analytical Calculated for: (C₂₀H₁₇N₅O₂S) (M.W.=391): C, 61.37; H, 4.38; N, 17.89%; Found: C, 61.48; H, 4.43; N, 18.12%.

N-(2-chlorophenyl)-2-(3-(methylthio)-4-oxo-1-phenyl-1Hpyrazolo[ 3,4-d]pyrimidin-5(4H)-yl)acetamide (13_b): White solid; Yield: 78%; m. p. 146-147°C. IR (KBr) cm^-1: 3258 (NH), 3072 (CH aromatic), 2932 (CH aliphatic), 1695 (C=O). ¹HNMR (DMSO-d6) δ ppm: 10.09 (s, 1H, NH, D₂O exchangeable), 8.48 (s, 1H, pyrimidine-H2), 8.04 (d, 2H, J=7.80 Hz, Phenylpyrazole-H2, H6), 7.75 (d, 1H, J=8.10 Hz, Aniline-H6), 7.57 (d, 1H, J=8.10 Hz, Aniline-H3), 7.55 (t, 2H, J=8.10 Hz, Phenylpyrazole-H3,H5), 7.53(t, 1H, J=8.10 Hz, phenylpyrazole-H4), 7.4 (t, 1H, J=7.60 Hz, Aniline-H5), 7.2 (t, 1H, J=7.60 Hz, Aniline-H4), 4.87 (s, 2H, CH₂C=O), 2.63 (s, 3H, S-CH₃). MS (m/z): 427 (C₂₀H₁₆ClN₅O₂S, M+2, 1.52%), 4.82%,425 (C₂₀H₁₆ClN₅O₂S, M⁺, 4.82%), 299 (C₁₄H₁₁N₄O₂S, M-NHC₆H₄Cl, 84.7%), 271 (C₁₃H₁₁N₄OS, 100%). Analytical Calculated for: (C₂₀H₁₆ClN₅O₂S) (M.W.=425): C, 56.40; H, 3.79; N, 16.44%; Found: C, 56.61; H, 3.76; N, 16.58%.

2-(3-(Methylthio)-4-oxo-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-5(4H)-yl)-N-m-tolylacetamide (13_c): White solid; Yield: 82%; m. p. 152-153°C. IR (KBr) cm^-1: 3299 (NH), 3038 (CH aromatic), 2925 (CH aliphatic), 1672 (C=O). ¹HNMR (DMSO-d6) δ ppm: 10.37 (s,1H, NH, D₂O exchangeable), 8.46 (s, 1H, pyrimidine-H2), 8.06 (d, 2H, J=7.60 Hz, Phenylpyrazole-H2H6), 7.59 (d, 1H, J=7.60 Hz, Aniline-H6), 7.43 (t, 2H, J=5.60 Hz, phenylpyrazole-H3,H5), 7.39 (t, 1H, J=7.60 Hz, phenylpyrazole-H4), 7.36 (s, 1H, Aniline-H2), 7.22 (t, 1H, J=8.00 Hz, Aniline-H5), 6.90 (d, 1H, J=7.60 Hz, Aniline-H4), 4.87 (s, 2H, CH₂C=O), 2.63 (s, 3H, S-CH₃), 2.27 (s, 3H, Ar-CH₃). MS (m/z): 405 (C₂₁H₁₉N₅O₂S, M, 52.08%), 299 (C₁₄H₁₁N₄O₂S, M-NHC₆H₄CH₃, 100%), 271 (C₁₃H₁₁N₄OS, 67.81%). Analytical Calculated for: (C₂₁H₁₉N₅O₂S) (M.W.=405): C, 62.21; H, 4.72; N, 17.27%; Found: C, 62.38; H, 4.76; N, 17.49%.

Ethyl 4-(2-(3-(methylthio)-4-oxo-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-5(4H)-yl)acetamido)benzoate (13_d): White solid; Yield: 65%; m. p. 72-73°C. IR (KBr) cm^-1: 3287 (NH), 3058 (CH aromatic), 2988 (CH aliphatic), 1695 (C=O). ¹HNMR (DMSO-d6) δ ppm: 10.78 (s, 1H, NH), 8.4 (s, 1H, pyrimidine-H2), 8.70 (d, 2H, J=8.80 Hz, Aniline-H3,H5), 7.80 (d, 2H, J=6.80 Hz, Aniline-H2,H6), 7.80 (t, 2H, J=8.40 Hz, Phenylpyrazole-H3,H5), 7.51 (t, 2H, J=8.10 Hz, Phenylpyrazole-H2,H6), 7.42 (t, 1H, J=7.20 Hz, phenylpyrazole-H4), 4.90 (s, 2H, CH₂C=O), 4.30 (q, 2H, J=6.80 Hz, CH₂CH₃), 2.60 (s, 3H, S-CH₃), 1.29 (t, 3H, J=7.20 Hz, CH₂CH₃). MS (m/z): 463 (C₂₃H₂₁N₅O4S, M, 5.65%), 299 (C₁₄H₁₁N₄O₂S, M-NHC₆H₄COOC₂H₅, 19.63%), 271 (C₁₃H₁₁N₄OS, 18.52%), 174 (C₉H₅N₂S, 100%). Analytical Calculated for: (C₂₃H₂₁N₅O4S) (M.W.=463): C, 59.60; H, 4.57; N, 15.11%; Found: C, 59.70; H, 4.63; N, 15.15%.

3-(3-(Methylthio)-4-oxo-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-5(4H)-yl)-N-phenylpropanamide (13_e): White solid; Yield: 80%; m. p. 154-155°C. IR (KBr) cm^-1: 3317 (NH), 3010 (CH aromatic), 2924 (CH aliphatic), 1674 (C=O). ¹HNMR (DMSO-d6) δ ppm: 12.44 (s, 1H, NH, D₂O exchangeable), 8.44 (s, 1H, pyrimidine-H2), 8.02 (t, 2H, J=7.20 Hz, Phenylpyrazole-H2,H6), 7.54 (d, 2H, J=2.00 Hz, Aniline-H2,H6), 7.50 (d, 2H, J=8.40 Hz, phenylpyrazole-H3,H5), 7.37 (t, 1H, J=7.20 Hz), 7.33 (t, 2H, J=7.60 Hz, phenylpyrazole-H4), 7.28 (t, 1H, J=7.20 Hz, Aniline-H3,H5), 7.25 (t, 1H, J=7.20 Hz, Aniline-H4), 4.26 (t, 2H, J=7.20 Hz NCH₂), 2.87 (t, 2H, J=7.20 Hz CH₂C=O), 2.61 (s, 3H, S-CH₃). ¹³CNMR (DMSO-d6 400 MHz) δ ppm: 13.27, 31.12, 31.22 (Aliphatic carbons), 105.86, 119.62, 121.66, 122,79, 129.74, 138.60, 145.89, 150.40, 152.79, 153.48, 156.58, 157.42, 162.74 (Aromatic carbons), 169.09 (C=O). MS (m/z): 405 (C₂₁H₁₉N₅O₂S, M, 45.17%), 313 (C₁₅H₁₃N₄O₂S, M- NHC₆H₅, 48.33%), 285 (C₁₄H₁₃N₄OS, 7.07%), 259 (C₁₂H₉N₄OS, 100%). Analytical Calculated for: (C₂₁H₁₉N₅O₂S) (M.W.=405): C, 62.21; H, 4.72; N, 17.27%; Found: C, 62.45; H, 4.75; N, 17.53%.

N-(2-chlorophenyl)-3-(3-(methylthio)-4-oxo-1-phenyl-1,4- dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)propanamide (13_f): White solid; Yield: 70%; m. p. 149-150°C. IR (KBr) cm^-1: 3301 (NH), 3083(CH aromatic), 2931 (CH aliphatic), 1680 (C=O). ¹HNMR (DMSO-d6) δ ppm: 9.72 (s, 1H, NH D₂O exchangeable), 8.44 (s, 1H, pyrimidine-H2), 8.03 (d, 2H, J=7.80 Hz, Phenylpyrazole-H2,H6), 7.61 (d, 1H, J=8.10 Hz, Aniline-H6), 7.57 (d, 1H, J=8.10 Hz, Aniline-H3), 7.46 (t, 2H, J=8.10 Hz, Phenylpyrazole-H3H5), 7.39 (t, 1H, J=8.10 Hz, phenylpyrazole-H4), 7.34 (t, 1H, J=7.50 Hz, Aniline-H5), 7.20 (t, 1H, J=7.60 Hz, Aniline-H4), 4.29 (t, 2H, J=80 Hz, CH₂CH₂), 2.8 (t, 2H, J=10.00 Hz, CH₂-C=O), 2.50 (s, 3H, S-CH₃). MS (m/z): 441 (C₂₁H₁₈ClN₅O₂S, M+2, 3.78%, M⁺), 439 (C₂₁H₁₈ClN₅O₂S, M, 11.49%), 313 (C₁₅H₁₃N₄O₂S, 35.54%), 285 (C₁₄H₁₃N₄OS, 5.89%), 257 (C₁₂H₉N₄OS, 8.2%). Analytical Calculated for: (C₂₁H₁₈ClN₅O₂S) (M.W.=439): C, 57.34; H, 4.12; N, 15.92%; Found: C, 57.49; H, 4.19; N, 16.08%.

3-(3-(methylthio)-4-oxo-1-phenyl-1,4-dihydro-5Hpyrazolo[ 3,4-d]pyrimidin-5-yl)-N-(m-tolyl)propanamide (13_g): White solid. Yield: 55%; m. p. 138-141°C. IR (KBr) cm^-1: 3223 (NH), 3049(CH aromatic), 2980 (CH aliphatic), 1693 (C=O). 1H NMR (DMSO-d6) δ ppm: 9.92 (s, 1H, NH D₂O exchangeable), 8.45 (s, 1H, pyrimidine-H2), 8.02 (d, 2H, J=7.8 Hz, Phenylpyrazole-H2H6), 7.56 (d, 1H, J=7.80 Hz, Aniline-H6), 7.38 (t, 2H, J=5.70 Hz, phenylpyrazole- H3,H5), 7.32 (t, 1H, J=8.40 Hz, phenylpyrazole-H4), 7.17 (s, 1H, Aniline-H2), 7.14 (t, 1H, J=8 Hz, Aniline-H5), 6.85 (d, 1H, J=7.50 Hz, Aniline-H4), 4.2 (t, 2H, SCH₂), 2.8 (t, 2H, CH₂-C=O), 2.6 (s, 3H, Ar- CH₃), 2.2 (s, 3H, S-CH₃). MS (m/z): 419 (C₂₂H₂₁N₅O₂S, M, 2.06%,), 314 (C₁₅H₁₄N₄O₂S, 10.23%), 257 (C₁₂H₉N₄OS, 8.2%), 105 (C₇H₆N, 100%). Analytical Calculated for: (C₂₂H₂₁N₅O₂S) (M.W.=419): C, 62.99; H, 5.05; N, 16.69%; Found: C, 63.21; H, 5.11; N, 16.87%.

Ethyl 4-(3-(3-(methylthio)-4-oxo-1-phenyl-1,4-dihydro-5Hpyrazolo[ 3,4-d]pyrimidin-5-yl)propanamido)benzoate (13_h): White solid. Yield; 85%; m. p. 160 °C. IR (KBr) cm^-1: 3287 (NH), 3058 (CH aromatic), 2988 (CH aliphatic), 1695 (C=O). ¹HNMR (DMSO-d6) δ ppm: 10.78 (s, 1H, NH), 8.40 (s, 1H, pyrimidine-H2), 8.71 (d, 2H, J=8.80 Hz, Aniline-H3,H5), 7.81 (d, 2H, J=6.80 Hz, Aniline-H2H6), 7.81 (t, 2H, J=8.40 Hz, Phenylpyrazole-H3H5), 7.53 (t, 2H, J=8.10 Hz, Phenylpyrazole-H2H6), 7.42 (t, 1H, J=7.20 Hz, phenylpyrazole-H4), 3.90 (t, 2H, J=7.20, SCH₂), 4.35 (t, 2H, J=7.20, CH₂C=O), 4.3 (q, 2H, J=6.80 Hz, CH₂CH₃), 2.6 (s, 3H, S-CH₃), 1.29 (t, 3H, J=7.20 Hz, CH₂CH₃). MS (m/z): 477 (C₂₄H₂₃N₅O4S, 27.54%, M⁺), 432 (C₂₂H₁₈N₅O₃S, 2.51%), 313 (C₁₅H₁₃N₄O₂S, 55.20%), 285 (C₁₄H₁₃N₄OS, 8.39%). Analytical Calculated for: (C₂₄H₂₃N₅O4S) (M.W.=477): C, 60.36; H, 4.85; N, 14.65%; Found: C, 60.64; H, 4.93; N, 14.85%.

General procedure for synthesis of alkyl 2-(3-(methylthio)-4- oxo-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl)acetate 14_a-b: Into a solution of compound 9 (10 mmol) in DMF (30 ml) containing potassium carbonate (0.5 gm), the appropriate alkyl-2-chloroacetate (10 mmol) was added. The reaction mixture was heated under reflux for 4 hours. After complete reaction (as indicated by TLC), the reaction mixture was filtered while hot, concentrated, cooled and the resulting solid product was recrystallized from ethanol.

Methyl -2-(3-(methylthio)-4-oxo-1-phenyl-1,4-dihydro-5Hpyrazolo[ 3,4-d]pyrimidin-5-yl)acetate (14_a): White solid; Yield: 70%; m. p. 78-79°C. IR (KBr) cm^-1: 3044 (CH aromatic), 2948 (CH aliphatic), 1747 (C=O). ¹HNMR (DMSO-d6) δ ppm: 8.47 (s, 1H, pyrimidine-H2), 8.02 (d, 2H, J=7.80 Hz, phenyl-H2,H6), 7.57 (t, 2H, J=8.10 Hz phenyl-H3, H5), 7.41 (t, 1H, J=7.20 Hz, phenyl-H4), 4.85 (s, 2H, CH₂C=O), 3.73 (s, 3H, O-CH₃), 2.63 (s, 3H, S-CH₃). ¹³C NMR (DMSO-d6. 400 MHz) δ (ppm): 13.25,47.03,52.95 (Aliphatic carbons), 104.77, 121.85, 127.46, 129.68, 138.33, 146.12, 152.52, 152.79, 156.30 (Aromatic carbons), 168.81 (C=O). MS (m/z): 330 (C₁₅H₁₄N₄O₃S, M, 100%), 315 (C₁₄H₁₁N₄O₃S, M-CH₃, 1.07%). Analytical Calculated for: (C₁₅H₁₄N₄O₃S) (M.W.=330): C, 54.54; H, 4.27; N, 16.96%; Found: C, 54.71; H, 4.36; N, 17.21%.

Ethyl 2-(3-(methylthio)-4-oxo-1-phenyl-1,4-dihydro-5Hpyrazolo[ 3,4-d]pyrimidin-5-yl)acetate (14_b): White solid; Yield: 85%; m. p. 82-83°C. IR (KBr) cm^-1: IR (KBr)cm^-1: 3044 (CH aromatic), 2948 (CH aliphatic), 1747 (C=O). ¹HNMR (DMSO-d6) δ ppm: 8.47 (s, 1H, pyrimidine-H2), 8.02 (d, 2H, J=7.80 Hz, phenyl-H2,H6), 7.57 (t, 2H, J=8.00 Hz phenyl-H3, H5), 7.41 (t, 1H, J=7.20 Hz, phenyl-H4), 4.85 (s, 2H, CH₂C=O), 3.73 (s, 3H, O-CH₃), 2.63 (s, 3H, S-CH₃). ¹³C NMR (DMSO-d6. 400 MHz) δ (ppm): 13.25,47.03,52.95 (Aliphatic carbons), 104.77, 121.85, 127.46, 129.68, 138.33, 146.12, 152.52, 156.30 (Aromatic carbons), 168.81 (C=O). MS (m/z): 330 (C₁₅H₁₄N₄O₃S, M, 100%), 314 (C₁₄H₁₀N₄O₃S, M-CH₃, 100%), 299 (C₁₄H₁₁N₄O₂S, 4.76%), 257 (C₁₂H₉N₄OS, 2.35%). Analytical Calculated for: (C₁₅H₁₄N₄O₃S) (M.W.=330): C, 54.54; H, 4.27; N, 16.96%; Found: C, 54.71; H, 4.36; N, 17.21%.

Synthesis of 2-(3-(methylthio)-4-oxo-1-phenyl-1,4-dihydro-5Hpyrazolo[ 3,4-d]pyrimidin-5-yl)acetohydrazide (15): Into a solution of 14_b (10 mmol) in ethanol (30 ml), hydrazine- hydrate (20 mmol) was added. The reaction mixture was heated under reflux for 6 hours. After complete reaction, the reaction allowed to cool. The separated solid was and filtered out, recrystallized from ethanol. White solid; Yield: 65%; m. p. 122-123°C. IR (KBr) cm^-1: 3307 (NHNH₂), 3017 (CH aromatic), 2984 (CH aliphatic), 1673 (C=O). ¹HNMR (DMSO-d6) δ ppm: 9.41 (s, 1H, NH D₂O exchangeable), 8.40 (s, 1H, pyrimidine-H2), 8.05 (d, 2H, J=8.10 Hz, phenyl-H2, H6), 7.58 (t, 2H, J=7.20 Hz, phenyl-H3, H5), 7.39 (t, 1H, J=1.20 Hz, phenyl-H4), 4.6 (s, 2H, CH₂C=O), 4.30 (s, 2H, NH₂ D₂O exchangeable), 2.5 (s, 3H, S-CH₃). MS (m/z): 330 (C₁₄H₁₄N₆O₂S, 13.32%, M⁺), 299 (C₁₄H₁₁N₄O₂S, 100%), 271 (C₁₃H₁₁N₄OS, 65.87%), 257 (C₁₂H₉N₄OS, 1.79%). Analytical Calculated for: (C₁₄H₁₄N₆O₂S (M.W.=330): C, 50.90; H, 4.27; N, 25.44%; Found: C, 51.23; H, 4.34; N, 25.61%.

General procedure for synthesis of N’-benzylidene derivatives- 2-(3-(methylthio)-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d] pyrimidin-5-yl)acetohydrazide 16_a-b: Into a solution of 15 (10 mmol) in glacial acetic acid (20 ml), benzaldehyde derivatives (10 mmol) was added. The mixture was then heated under reflux for 5 hours. The reaction mixture was concentrated and allowed to cool. The separated solid was filtered and finally recrystallized from ethanol.

(E)-N’-benzylidene-2-(3-(methylthio)-4-oxo-1-phenyl-1,4- dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)acetohydrazide (16_a): White solid; Yield: 73%; m. p. 189-190°C. IR (KBr) cm^-1: 3196 (NH), 3044 (CH aromatic), 2929 (CH aliphatic), 1680 (C=O). ¹HNMR (DMSO-d6) δ ppm: 11.88 (s, 1H, NH D₂O exchangeable), 8.49 (s, 1H, pyrimidine-H2), 8.07 (t, 3H, J=8.00 Hz, phenyl-H3,H4,H5), 7.75 (d, 2H, J=8.00 Hz, phenyl-H2,H6), 7.58 (d, 2H, J=8.00 Hz, phenylpyrazole- H2,H6), 7.46 (t, 2H, J=6.00 Hz, Phenylpyrazole-H3,H5), 7.44 (t, 1H, J=7.60 Hz, phenylpyrazole-H4), 7.39 (s,1H, CH=N), 5.24 (s, 2H, CH₂C=O), 2.63 (s, 3H, S-CH₃). ¹³C NMR (DMSO-d6. 400 MHZ) δ (ppm): 13.26, 46.88, 47.44 (Aliphatic carbons), 104.96 121.73, 127.63, 129.93, 130.67, 134.46, 138.46, 146.06, 147.88, 152.94, 153.12, 156.51, 163.76, (Aromatic carbons), 168.64 (C=O). MS (m/z): 418 (C₂₁H₁₈N₆O₂S, 2.30%, M⁺), 299 (C₁₄H₁₁N₄O₂S, 100%), 271 (C₁₃H₁₁N₄OS, 65.87%). Analytical Calculated for: (C₂₁H₁₈N₆O₂S (M.W.=418): C, 60.27; H, 4.34; N, 20.08%; Found: C, 51.23; H, 4.34; N, 25.61%.

(E)-N’-(4-hydroxybenzylidene)-2-(3-(methylthio)-4-oxo- 1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl) acetohydrazide (16_b):White solid; Yield: 78%; m. p. 205-206 C. IR (KBr) cm^-1: 3017 (CH aromatic), 2984 (CH aliphatic), 1673 (C=O), 3307 (NH). ¹HNMR (DMSO-d6) δ ppm: 11.67 (s, 1H, NH D₂O exchangeable), 9.97 (s, 1H, OH D₂O exchangeable), 8.47 (s, 1H, pyrimidine-H2), 8.05 (d, 2H, J=8.00 Hz, phenyl-H2,H6), 7.97 (s, 2H, CH=N), 7.58 (t, 4H, J=8.00 Hz, phenylpyrazole-H3,H5, phenyl-H3,H5), 7.40 (t, 1H, J=8.00 Hz, phenylpyrazole-H4), 6.86 (d, 2H, J=8.00 Hz, Phenylpyrazole- H2,H6), 5.19 (s, 2H, CH₂C=O), 2.63 (s, 3H, S-CH₃). ¹³C NMR (DMSO-d6. 400 MHZ) δ (ppm): 13.26,20.89,47.37 (Aliphatic carbons), 104.96, 116.20, 121.72, 125.42, 127.33, 129.68, 138.47, 145.13, 148.16, 152.94, 156.52, 159.90, 163.33, (Aromatic carbons), 168.24 (C=O). MS (m/z): 434 (C₂₁H₁₈N₆O₃S, 2.66%, M⁺), 328 (C₁₄H₁₂N₆O₂S, 19.44%), 271 (C₁₃H₁₁N₄OS, 4.40%), 257 (C₁₂H₉N₄OS, 1.79%). Analytical Calculated for: (C₂₁H₁₈N₆O₃S (M.W.=434): C, 58.08; H, 4.18; N, 19.34%; Found: C, 51.23; H, 4.34; N, 25.61%.

Materials and methods

Human breast adenocarcinoma cell line MCF7, were purchased from the American Type Cell Culture Collection (ATCC, Manassas, USA) and grown on Roswell Park Memorial Institute Medium (RPMI 1640) supplemented with 100 g/ml of streptomycin, 100 units/ ml of penicillin and 10% of heat inactivated fetal bovine serum in a humidified, 5% (v/v) CO₂ atmosphere at 37°C.

Measurement of potential antitumor

The antitumor activity of newly synthesized pyrazolo[3,4-d] pyrimidines were measured in vitro on human breast adenocarcinoma cell line MCF7 using SulfoRhodamine-B stain (SRB) assay applying the method of 3-[4,5-dimethylthiazole-2-yl]-2,5-dimethyltetrazolium bromide (MTT) technique [21,22]. Exponentially grown cells from the selected cancer cell line were trypsinized, counted and seeded at the appropriate densities (2000-1000 cells/0.33 cm²). Cells were then incubated in a humidified atmosphere at 37°C. for 24 hours. Then, cells were exposed to different concentrations of the test compounds (0.1, 1, 10, 100, 1000 πM) for 72 hours. After that, the viability of treated cells was determined according to MTT technique. The viability of cells was expressed as percentage of control and the concentration that induces 50% inhibition of cell proliferation (IC₅₀). The relation between the surviving fraction and the compound concentration was plotted and the IC₅₀ was calculated for each compound. Results are given in Table 1.

A series of novel 1-phenyl-3-methylsulphanylpyrazolo[3,4-d] pyrimidines 10-16 was synthesized. The antitumor activity of this new series was investigated against human breast adenocarcinoma cell line MCF7. Ten of the test compounds showed moderate activity relative to that of doxorubicin. The N-arylacetamide derivatives (13_a-h) exhibited better antitumor activity than all other series. Among this series, compound 13a displayed the highest activity with IC₅₀ equal to 23 μM. As it obvious from the results in Table 1 and Figure 2, increasing the linker length by one more CH₂ unit in compounds 13_a-h results in dramatic fall in the activity. Presence of hydrogen bond donor at the para position of the aromatic ring in the new derivatives 16_{a, b} is essential for the activity. This becomes clear upon comparing the MIC values of 16_a (above 326 μM) with that of 16_b which is only 61 μM. Further studies are required in order to determine the mechanism of the antitumor action and to identify the SAR of other positions of pyrazolo[3,4-d]pyrimidine nucleus.

The collaborative support of members of the Cancer Biology Department, National Institute of Cancer, Egypt in determining the antitumor activity of new compounds is highly appreciated.