BioorganicChemistry

Design, synthesis and molecular docking of novel pyrazolo[1,5-a][1,3,5] triazine derivatives as CDK2 inhibitors

Khulood H. Oudaha,b, Mazin A.A. Najma,b, Nermin Samira,⁎, Rabah A.T. Seryaa,
Khaled A.M Abouzida,c,⁎
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt
b Pharmaceutical Chemistry Department, College of Pharmacy, Al-Ayen University, Ti-Qar, Iraq
c Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Sadat City, Menoufia, Egypt
A R T I C L E I N F O

Keywords:
Pyrazolo[1,5-a][1,3,5]triazine CDK2 inhibitors
Anticancer
Purine bioisostere Docking

A B S T R A C T

Cyclin Dependent Kinases CDKs unpredictable activity has been accounted for a wide assortment of human malignancies, so it might be conceivable to design pharmacologically relevant ligands that go about as specific and potent inhibitors of CDK2 action. In this respect, a series of novel pyrazolo[1,5-a][1,3,5]triazine derivatives were designed, synthesized and evaluated for CDK2 enzyme inhibitory and anticancer activity. Compounds 9f and 10c showed best CDK2 inhibition among the newly synthesized compounds, with percent inhibition at 82.38%, and 81.96% against CDK2 and IC50 of 1.85 and 2.09 µM, respectively. Additionally, the newly syn- thesized compounds were tested for their antiproliferative activity against 60 NCI cell lines. Molecular docking revealed the binding mode of these new compounds into the roscovitine binding site of CDK2 enzyme (PDB code: 3ddq). Conclusively, pyrazolotriazine derivatives represent a talented starting point for further study as antic- ancer drug.

1. Introduction

It is winding up obvious that many sorts of tumors are the con- sequence of irregular signal-transducing proteins that prompt a per- sistent generation of the signal for cell division [1]. Protein Kinases are key controllers of cell work, they constitute one of the biggest and most practically differing gene families. They oversee the catalysis of protein phosphorylation through the exchange of phosphate of ATP to certain residues in the amino acid side chain as indicated by the kind of de- monstrated kinase [2]. CDKs are individuals from serine/threonine ki- nase family and key compounds in cell cycle progression and tran- scription [3] and other major natural procedures including neural

differentiation [4] and metabolism [5]. Binding of a cyclin with asso- ciated kinase to form CDK/cyclin complexes, activates the enzyme and serves to move the cell from one phase to another e.g. CDK2-cyclin A complex controls the G1 to S-stage checkpoint [6]. So CDK/Cyclin complexes are considered an interesting target for therapeutics de- signed to recuperate control of the cell cycle in abnormally dividing cells. CDK2 hyper activation in human tumors is frequently connected with intensification or potentially overexpression of its accomplice cy- clins A and E in a wide assortment of human cancers, yet in particular in breast cancer, ovarian and endometrial carcinomas, lung, thyroid car- cinoma, melanoma and osteosarcoma [7,8,9,10] and clinically, it reg- ularly presents a poor prognosis.

Corresponding authors at: Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt.

E-mail addresses: [email protected] (N. Samir), [email protected] (K.A.M. Abouzid).

https://doi.org/10.1016/j.bioorg.2019.103239

Received 26 June 2019; Received in revised form 19 August 2019; Accepted 30 August 2019
Availableonline04September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

The strong hereditary connection amongst CDK2 and the molecular pathology of the disease has provided the reason for developing small- molecule inhibitors of these kinases. Knowledge of the structure of CDK2 has been a key in driving the outline and in improvement of an expansive number of ATP-competitive inhibitors [11]. Crystallography has uncovered that the ATP-binding site of CDK2 can suit various as- sorted of chemical structures, exploiting different sites of interaction. In addition, residues outside the principle ATP-binding cleft have been recognized that could be targeted to expand specificity and potency [12,13]. The most recent decades, purines heterocycles attracted the researchers and turned into the most utilized scaffolds for the ad- vancement of CDK2 inhibitor that prompts the revelation of many molecules that act as CDK2 inhibitors and potent anticancer [14]. Roscovitine (I) was among the first CDK2 inhibitors that entered clin- ical trials. It induces apoptosis from all phases of the cell cycle in cancer cell line. It inhibits CDKs with various IC50 values: CDK1 (2.7 µM), CDK2 (0.1 µM), CDK7 (0.5 µM), CDK9 (0.8 µM) [15,16]. Due to the
positive feedback of roscovitine and dinaciclib as a potent cytotoXic agents [17], several bioisosteres were synthesized mainly focusing on redistribution of purine heteroatom in order to have similarity with roscovitine in high selectivity and low toXicity and to overcome the disadvantage of its short half-life (i.e. 2–5 h) in human [18]. Thus, the above–mentioned data guided us to synthesize some novel pyrazolo [1,5-a][1,3,5]triazine derivatives (II) as purine bioisosteres and in- vestigate their inhibitory activity against CDK2 enzyme and anticancer activity.

2. Materials and methods

2.1. Chemistry
2.1.1. General
Starting materials and reagents were purchased from Sigma-Aldrich (USA) or Alfa-Aesar Organics and used without further purification.

 

Melting points were recorded on Stuart Scientific apparatus and were uncorrected. The purities of compounds were monitored by analytical TLC, performed on silica gel 60 F254 packed on Aluminium sheets, purchased from Merck, with visualization under U.V. light (254 nm). 1HNMR and 13CNMR spectra were recorded in δ scale given in ppm (on a Bruker 400 MHz for 1H and 101 MHz for 13C spectrophotometer) and referred to TMS as internal reference at Center of Drug Discovery and Research Development, Ain Shams University. FT-IR spectra were re- corded on a Thermo Scientific Nicolet iS10 spectrometer at Ain Shams University. EI-MS spectra and Elemental analyses were performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University.
2.1.2. Synthesis
Compounds 1a-c, 2, 3a, 5 and 6a were prepared according to the previously reported procedure [19–23].
2.1.2.1. General procedure for preparation of compounds (3b and 3c). To the respective isothiocyanates (1b and 1c) (1 equiv., 10 mmol), 5- amino-1H-pyrazole-4-carbonitrile (2) (1 equiv., 10 mmol, 1.08 g) in (10 ml) anhydrous acetone was added, and the reaction miXture was heated under refluX for 3 h to give the thiourea derivatives (3b and c). Ice water was added to the reaction miXture and the resulting solid was collected by filtration, washed with cold water and ethanol, and finally dried at room temperature.
2.1.2.2. 1-(4-Chlorobenzoyl)-3-(4-cyano-1H-pyrazol-5-yl)thiourea
(3b). The titled compound was separated as white solid, crystallization solvent was ethanol (4.50 g, 79.55%); m.p. 166–168 °C; 1H NMR (400 MHz, CDCl3) δ14.02 (s,1H,NH D2O exchangeable), 9.18 (s, 1H,NH D2O exchangeable), 7.73 (d, J = 8.5 Hz, 2H, Ar-H), 7.63 (s, 1H,pyrazole-H), 7.61 (d, J = 8.5 Hz, 2H,Ar-H). 13C NMR (101 MHz, CDCl3) δ 179.82, 166.70, 158.40, 139.45, 137.60, 135.22, 128.80,
127.14, 113.90, 84.60. FT-IR (ὑ max, cm−1): 3250–3120 (NH), 2232
(C^N nitrile), 1680 (C]O amide). Anal. Calcd for C12H8ClN5OS: C, 47.14; H, 2.64; N, 22.91; Found: C, 47.24; H, 2.70; N, 22.97.
2.1.2.3. 1-(4-Bromobenzoyl)-3-(4-cyano-1H-pyrazol-5-yl)thiourea
(3c). The titled compound was separated as pale brown solid, reaction time 3 h, crystallization solvent was ethanol (5.20 g, 80.26%); m.p. 156–158 °C; 1H NMR (400 MHz, CDCl3) δ 14.04 (s, 1H,NH D2O
exchangeable), 9.28 (s, 1H,NH D2O exchangeable), 7.78 (d, J = 8.4 Hz, 2H, Ar-H), 7.67 (s, 1H,pyrazole-H), 7.63 (d, J = 8.4 Hz, 2H, Ar-H).13C NMR (101 MHz, CDCl3) δ 179.72, 166.65, 158.45,
139.32, 136.21, 135.26, 129.15, 126.95, 113.75, 85.22. FT-IR (ὑ
max, cm−1): 3223–3194 (NH), 2225 (C^N nitrile), 1683 (C]O amide). Anal. Calcd for C12H8BrN5OS: C, 41.16; H, 2.30; N, 20.0; Found: C, 41.35; H, 2.35; N, 20.15.
2.1.2.4. General procedure for preparation of compounds (4a-c). A slight excess of sodium hydride (60% suspension in oil, 1 equiv., 0.005 mmol,
0.119 g) was added to a solution of 1-(4-un/substitutedbenzoyl)-3-(4- cyano-1H- pyrazol-5-yl)thiourea (3a-c) (1 equiv., 0.005 mmol) in DMF (5 ml). The miXture was stirred for 10 min at room temperature and

then ethylbromide (1 equiv., 0.005 mmol) was added drop wise to the miXture. The reaction miXture was stirred for further 30–45 min and monitored using TLC (Hexane/ethyl acetate 4:1) to yield the carbamimidothioate derivatives which were not isolated. The reaction miXture was then heated under refluX for 45–60 min and then diluted with cold water. The formed precipitate was collected by filtration and purified by column chromatography on silica gel (200–400 mesh), using a miXture of hexane/ethyl acetate (4:1) as eluent, to give the titled products (4a-c).
2.1.2.5. 2-(Ethylthio)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8- carbonitrile (4a). The product was separated as pale orange crystals, reaction time 45 min, (1.01 g, 71%) m.p.: 160–162 °C; 1H NMR

aliphatic), 1748 (C]O ester), 1672 (C]O amide).
2.1.2.10. Ethyl 5-(3-(4-bromobenzoyl)thioureido)-1H-pyrazole-4-carboxylate (6c). The titled compound was separated as faint yellow crystals (3.1 g, 78.04%); m.p. 203–205 0C; 1H NMR (400 MHz, CDCl3) δ 14.04 (s, 1H, NH D2O exchangeable), 9.23 (s, 1H, NH D2O exchangeable), 7.78 (d, J = 8.6 Hz, 2H, Ar H), 7.66 (s, 1H, pyrazole H), 7.62 (d, J = 8.5 Hz, 2H, Ar H), 4.37 (q, J = 7.1 Hz, 2H, O-CH2 CH3), 1.33 (t, J = 7.1 Hz, 3H, CH2CH3); FT-IR (ὑ max, cm−1): 3338–3100 (NH), 3051 (CH aromatic),
2981 (CH aliphatic), 1745 (C]O ester), 1667 (C]O amide).
2.1.2.11. General procedure for the preparation of 7a-c. To a solution of the ethyl 5-(3-(4-substituted) benzoylthioureido)-1H-pyrazole-4-

(400 MHz, CDCl3) δ 8.67 (d, J = 7.2 Hz, 2H, Ar-H), 8.35 (s, 1H,

carboXylate (6a-c) (1 equiv., 3.5 mmol) in DMF (5 ml), sodium

Pyrazole- H), 7.72–7.59 (m, 3H, Ar-H), 3.32 (q, J = 7.4 Hz, 2H, SCH2), 1.51 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 171.80, 153.32, 152.62, 147.90, 133.06, 132.07, 129.75, 127.39,
112.05, 81.20, 26.09, 13.87. FT-IR (ὑ max, cm−1): 3119 (CH
aromatic), 2854 (CH aliphatic), 2210 (C^N nitrile). MS: (Mwt: 281.07) m/z, 281 [M+, (34.7%)]. Anal. Calcd for C14H11N5S: C,
59.77; H, 3.94; N, 24.89; Found: C, 59.87; H, 3.94; N, 24.99.
2.1.2.6. 4-(4-Chlorophenyl)-2-(ethylthio)pyrazolo[1,5-a][1,3,5]triazine- 8-carbonitrile (4b). The product was separated as pale orange crystals, reaction time 60 min, (1.13 g, 71.57%) m.p.: 185–188 °C; 1H NMR (400 MHz, CDCl3) δ 8.65 (d, J = 8.4 Hz, 2H, Ar-H), 8.35 (s, 1H, Pyrazole-H), 7.76 (d, J = 8.4 Hz, 2H, Ar-H), 3.34 (q, J = 7.2 Hz, 2H, CH2), 1.52 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 171.80, 153.22, 152.61, 147.90, 133.05, 132.07, 129.38, 127.39, 112.06, 81.22, 26.08, 13.85. FT-IR (ὑ max, cm-1): 3110 (CH
aromatic), 2964 (CH aliphatic), 2232 (C^N nitrile). MS: (Mwt: 315.03) m/z, 317 [M++2, (16.3%)], 315 [M+, (38.2%)]. Anal. Calcd
for C14H10ClN5S: C, 53.25; H, 3.19; N, 22.18; Found: C, 53.35; H, 3.19;
N, 22.28.
2.1.2.7. 4-(4-Bromophenyl)-2-(ethylthio)pyrazolo[1,5-a][1,3,5]triazine- 8-carbonitrile (4c). The product was separated as pale orange crystals, reaction time 60 min, (1.25 g, 69.44%) m.p.: 178–180 °C; 1H NMR (400 MHz, CDCl3) δ 8.63 (d, J = 8.6 Hz, 2H, Ar-H), 8.35 (s, 1H, Pyrazole-H), 7.74 (d, J = 8.6 Hz, 2H, Ar-H), 3.31 (q, J = 7.4 Hz, 2H, CH2), 1.51 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 171.80, 153.32, 152.62, 147.90, 133.06, 132.07, 129.75, 127.39, 112.05, 81.20, 26.09, 13.87. FT-IR (ὑ max, cm−1): 3115 (CH
aromatic), 2874 (CH aliphatic), 2239 (C^N nitrile). MS: (Mwt: 360.98) m/z, 362 [M++2, (47.2%)], 360 [M+, (52.8%)]. Anal. Calcd
for C14H10BrN5S: C, 46.68; H, 2.80; N, 19.44; Found: C, 46.78; H, 2.74;
N, 19.67.
2.1.2.8. General procedure for the preparation of 6b and 6c. To a stirred

hydride (60% suspension in oil,1 equiv., 3.5 mmol, 84 mg) was added. The miXture was stirred for 10 min at room temperature and then a solution of ethyl bromide (1 equiv., 3.5 mmol, 0.26 ml) was added dropwise and the stirring was continued for a further 30 min to afford intermediate carbamimidothioate derivatives which were not isolated (checked by TLC). After that, the reaction miXture was heated under refluX for 45–60 min. till completion of reaction (checked by TLC). Then the reaction miXture diluted with cold water, the precipitate solid was collected by filtration, washed with water and ethanol. The product was purified by flash column chromatography on silica gel, using a miXture of hexane:ethyl acetate (7:3) as eluent to afford the titled compounds (7a-c).
2.1.2.12. Ethyl 2-(ethylthio)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8- carboxylate (7a). The titled compound was separated as yellow powder (0.794 mg, 69%); m.p. 121–123 °C; 1H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 7.6 Hz, 2H, Ar H), 8.47 (s, 1H, pyrazole H), 7.62–7.50 (m, 3H, Ar H), 4.34 (q, J = 7.2 Hz, 2H, O-CH2), 3.25 (q, J = 7.3 Hz, 2H, S-CH2), 1.45 (t, J = 7.2 Hz, 3H, CH3), 1.36 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3) δ 171.32, 163.45, 153.85, 152.29, 147.95, 133.92, 132.23, 129.52, 127.42, 99.67, 60.65, 26.72, 14.76, 13.92; FT-IR (ὑ max, cm−1): 3127 (CH aromatic), 2926
(CH aliphatic), 1750 (C]O ester); MS: (Mwt.: 328.39): m/z (% rel. Int.), 328.21 [M+, (28.4%)], 91.06 (100%); Anal. Calcd for
C16H16N4O2S: C, 58.52; H, 4.91; N, 17.06; Found: C, 58.72; H, 4.88;
N, 17.25.
2.1.2.13. Ethyl 4-(4-chlorophenyl)-2-(ethylthio)pyrazolo[1,5-a][1,3,5] triazine-8-carboxylate (7b). The titled compound was separated as yellow powder (0.827 g, 65%); m.p. 122–124 °C; 1H NMR (400 MHz, CDCl3) δ 8.73 (d, J = 8.7 Hz, 2H, Ar H), 8.55 (s, 1H, pyrazole H), 7.58 (d, J = 8.7 Hz, 2H, Ar H), 4.43 (q, J = 7.1 Hz, 2H, O-CH2), 3.34 (q, J = 7.4 Hz, 2H, S-CH2), 1.53 (t, J = 7.4 Hz, 3H, CH3), 1.45 (t, J = 7.1 Hz, 3H, CH3).; 13C NMR (101 MHz, CDCl3) δ 171.37, 163.47, 153.75, 152.27, 147.89, 133.65, 132.64, 129.65, 127.39, 99.85, 60.53,

solution of ethyl 5-amino-1H-pyrazole-4-carboXylate (5) (1 equiv.,

26.76, 14.69, 13.87; FT-IR (ὑ max, cm−1): 3115 (CH aromatic), 2929

10 mmol, 1.55 g) in acetonitrile (15 ml), the solution of the freshly prepared proper substituted benzoyl isothiocyanate (1a-c) (1 equiv., 10 mmol) in acetonitrile (15 ml) was added dropwise. The reaction miXture was then heated under refluX for 1.5–2 h. The miXture was allowed to cool to room temperature. Ice water was added to the reaction miXture and the resulting solid was collected by filtration, washed with cold water and ethanol, recrystallized from ethanol to afford the titled compounds (6b and c).
2.1.2.9. Ethyl 5-(3-(4-chlorobenzoyl)thioureido)-1H-pyrazole-4-carboxylate (6b). The titled compound was separated as white crystals (2.8 g, 79.37%); m.p. 199–201 0C; 1H NMR (400 MHz, CDCl3) δ 14.02 (s, 1H, NH D2O exchangeable), 9.18 (s, 1H, NH D2O exchangeable), 7.73 (d, J = 8.6 Hz, 2H, Ar H), 7.63 (s, 1H, pyrazole H), 7.61 (d, J = 8.5 Hz, 2H, Ar H), 4.43 (q, J = 7.1 Hz, 2H, O-CH2CH3), 1.39 (t, J = 7.1 Hz, 3H, CH2CH3); FT-IR (ὑ max, cm−1): 3289–3120 (NH), 3110 (CH aromatic), 2984 (CH

(CH aliphatic), 1745 (C]O ester); MS: (Mwt.: 362.83): m/z (% rel. Int.), 364.74 [M++2, (13.8%)], 362.43 [M+, (37.2%)], 147.14
(100%); Anal. Calcd for C16H15ClN4O2S: C, 52.96; H, 4.17; N, 15.44;
S, 8.84; Found: C, 52.80; H, 4.35; N, 15.61; S, 8.52.
2.1.2.14. Ethyl 4-(4-bromophenyl)-2-(ethylthio)pyrazolo[1,5-a][1,3,5] triazine-8-carboxylate (7c). The titled compound was separated as yellow powder (0.87 g, 61%); m.p. 124–126 °C; 1H NMR (400 MHz, CDCl3) δ 8.62 (d, J = 8.4 Hz, 2H, Ar H), 8.55 (s, 1H, pyrazole H), 7.74 (d, J = 8.4 Hz, 2H, Ar H), 4.43 (q, J = 7.1 Hz, 2H, O-CH2), 3.33 (q, J = 7.4 Hz, 2H, S-CH2), 1.53 (t, J = 7.4 Hz, 3H, CH3), 1.45 (t, J = 7.1 Hz, 3H, CH3).13C NMR (101 MHz, CDCl3) δ 171.39, 163.42, 153.78, 152.32, 147.92, 133.77, 132.42, 129.57, 127.46, 99.92, 60.59, 26.78, 14.70, 13.95; FT-IR (ὑ max, cm−1): 3130 (CH aromatic), 2935
(CH aliphatic), 1750 (C]O ester); MS: (Mwt.: 407.28): m/z (% rel. Int.), 409.08 [M++2, (36.8%)], 407.06 [M+, (37.2%)], 104.11

(100%); Anal. Calcd for C16H15BrN4O2S: C, 47.18; H, 3.71; N, 13.76; S,
7.87; Found: C, 47.35; H, 3.60; N, 13.86; S, 7.52.

2.1.2.15. General procedure for preparation of compounds (8a-p). A miXture of the appropriate derivative of compounds (4a-c) (1 equiv., 2 mmol) and the selected amine (R) (1 equiv., 2 mmol) were fused in an oil bath at 150 °C for 1–1.5 h. The reaction miXture was then triturated with diethyl ether, and the formed precipitate was collected by filtration and purified by column chromatography on slica gel (200–400 mesh), using a miXture of hexane: ethyl acetate (4:1) as an eluent, to give the target compounds (8a-p).
2.1.2.16. 2-(Benzylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8- carbonitrile (8a). The product was separated as light yellow crystals, reaction time 1 h, (0.33 g, 50.61%) m.p.: 148–150 °C; 1H NMR (400 MHz, CDCl3) δ 8.27 (s,1H,Pyrazole-H), 7.81–7.43 (m, 5H, Ar-H), 7.36–7.28 (m, 5H, Ar-H), 6.66 (s,1H,NH D2O exchangeable), 4.65 (s,

2.1.2.20. 2-(Isobutylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8- carbonitrile (8e). The product was separated as buff crystals, reaction time 1 h, (0.31 g, 53.44%) m.p.: 175–177 °C; 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 7.4 Hz, 2H, Ar-H), 8.13 (s, 1H, Pyrazole-H), 7.67–7.58 (m, 3H, Ar-H), 5.80 (s, 1H, NH D2O exchangeable), 3.43 (d, J = 6.4 Hz, 2H, NHCH2), 2.07–1.94 (m, 1H, CH(CH3)2), 1.05 (d, J = 6.6 Hz, 6H, CH3).13C NMR (101 MHz, CDCl3) δ 156.70, 156.35, 154.72, 147.38, 133.27, 131.10, 129.85, 128.65, 113.20, 100.42, 49.75, 28.15, 20.50. FT-IR (ὑ max, cm−1): 3317 (NH), 3070 (CH aromatic), 2924 (CH
aliphatic), 2229 (C^N nitrile). MS: (Mwt: 292.14) m/z (% rel. Int.), 292 [M+, (32.4%)]. Anal. Calcd for C16H16N6: C, 65.74; H, 5.52; N, 28.75;
Found: C, 65.84; H, 5.52; N, 28.85.
2.1.2.21. 2-(Ethylsulfonyl)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8- carbonitrile (8f). The product was separated as bright yellow crystals, reaction time 1.5 h, (0.32 g, 51.61%) m.p.: 163–165 °C; 1H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 8.2 Hz, 2H Ar-H), 8.11 (s, 1H,
pyrazole-H), 7.67–7.57 (m, 3H, Ar-H), 4.01–3.95 (m, 2H, CH ), 1.75

2H,CH2).13C NMR (101 MHz, CDCl3) δ 156.64, 156.20, 154.30, 147.75,
133.71, 131.10, 129.29, 128.85, 128.33, 127.59, 126.23, 126.52,
(t, J = 7.3 Hz, 3H, CH3).213C NMR (101 MHz, CDCl3) δ 156.02, 154.83,

113.90, 101.95, 46.15. FT-IR (ὑ max, cm−1): 3320 (NH), 3115 (CH
aromatic), 2947 (CH aliphatic), 2225 (C^N nitrile). MS: (Mwt: 326.13): m/z (% rel. Int.), 326 [M+, (38.9%)]; Anal. Calcd for
C19H14N6: C, 69.92; H, 4.32; N, 25.75; Found: C, 69.98; H, 4.30; N,
25.85.2.1.2.17. 2-(Phenethylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8- carbonitrile (8b). The product was separated as pale yellow powder, reaction time 1 h, (0.35 g, 51.47%) m.p.: 138–140 °C; 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 7.6 Hz, 2H, Ar- H), 8.15 (s, 1H, Pyrazole-H), 7.66–7.57 (m, 3H, Ar-H), 7.34–7.21 (m, 5H, Ar-H), 5.88

(s, 1H, NH D O exchangeable), 3.86 (t, J = 6.5 Hz, 2H, NHCH ), 3.02

147.84, 145.7, 133.17, 131.01, 129.92, 128.45, 113.90, 98.13, 45.38,
14.53. FT-IR (ὑ max, cm−1): 3140 (CH aromatic), 2942 (CH aliphatic), 2217 (C^N nitrile), 1257 (SO2). MS: (Mwt: 313.06) m/z (% rel. Int.), 313 [M+, (40.2%)]. Anal. Calcd for C14H11N5O2S: C, 53.66; H, 3.54; N,
22.35; S, 10.23; Found: C, 53.56; H, 3.44; N, 22.35; S, 10.13.
2.1.2.22. 2-(Benzylamino)-4-(4-chlorophenyl)pyrazolo[1,5-a][1,3,5] triazine-8- carbonitrile (8g). The product was separated as light yellow crystals, reaction time 1.5 h, (0.39 g, 54.16%) m.p.: 140–143 °C; 1H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 8.7 Hz, 2H, Ar-H), 8.16 (s, 1H, Pyrazole-H), 7.55 (d, J = 8.7 Hz, 2H, Ar-H), 7.44–7.36 (m, 5H, Ar-H),
6.02 (s, 1H,NH D2O exchangeable), 4.76 (s, 2H, CH2).13C NMR
2 132 (101 MHz, CDCl3) δ 157.58, 156.20, 154.20, 147.56, 139.90, 132.45,
(t, J = 6.5 Hz, 2H, CH2Ar). C NMR (101 MHz, CDCl3) δ 157.46,
128.82, 127.79, 128.45, 128.26, 127.57, 126.32, 113.65, 102.40,
155.02, 154.83, 147.84, 140.17, 138.01, 130.92, 129.45, 128.75,
128.66, 127.33, 126.18, 113.50, 100.95, 42.32, 34.85. FT-IR (ὑ max,
cm−1): 3340 (NH), 3095 (CH aromatic), 2935 (CH aliphatic), 2232 (C^N nitrile). MS: (Mwt: 340.14) m/z (% rel. Int.), 340 [M+, (41.2%)]. Anal. Calcd for C20H16N6: C, 70.57; H, 4.74; N, 24.69; Found: C, 70.67;
H, 4.74; N, 24.75.

2.1.2.18. 2-(3-Isopropoxypropylamino)-4-phenylpyrazolo[1,5-a][1,3,5] triazine-8- carbonitrile (8c). The product was separated as green yellow crystals, reaction time 1 h, (0.35 g, 52.23%) m.p.: 153–157 °C; 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 7.6 Hz, 2H, Ar- H), 8.1–2 (s, 1H, Pyrazole-H), 7.65–7.57 (m, 3H, Ar-H), 6.52 (s, 1H, NH D2O exchangeable), 3.80–3.67 (septet, 1H, OCH(CH3)2), 3.66–3.57 (m, 4H, NHCH2CH2CH2O), 2.01–1.93 (m, 2H, NHCH2CH2CH2O), 1.23 (d, J = 6.0 Hz, 6H, CH3).13C NMR (101 MHz, CDCl3) δ 157.60, 156.20, 154.20, 147.35, 139.25, 132.35, 128.25, 127.30, 113.42, 96.01, 71.76,
67.25, 40.82, 28.85, 22.30. FT-IR (ὑ max, cm−1): 3336 (NH), 3078 (CH
aromatic), 2972 (CH aliphatic), 2228 (C^N nitrile). MS: (Mwt: 336.17)
m/z (% rel. Int.), 336 [M+, (25.2%)]. Anal. Calcd for C18H20N6O: C,
64.27; H, 5.99; N, 24.98; Found: C, 64.37; H, 5.99; N, 25.2.

2.1.2.19. 2-(Butylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8- carbonitrile (8d). The product was separated as pale yellow powder, reaction time 1 h, (0.29 g, 50.0%) m.p.: 168–170 °C; 1H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H, Pyrazole-H), 7.66–7.42 (m, 5H, Ar-H), 5.82 (s, 1H, NH D2O exchangeable), 3.62 (t, J = 3.4 Hz, 2H, NHCH2), 1.70–1.62 (m, 2H, CH2), 1.55–1.47 (m, 2H, CH2), 1.05 (t, J = 7.3 Hz, 3H, CH3).13C NMR (101 MHz, CDCl3) δ 156.46, 156.02, 154.83, 147.89, 133.17, 131.01, 129.92, 128.45, 113.90, 101.76, 42.38, 29.95, 20.24, 14.20. FT-IR (ὑ max, cm−1): 3310 (NH), 3085 (CH aromatic), 2954 (CH
aliphatic), 2231 (C^N nitrile). MS: (Mwt: 292.14) m/z (% rel. Int.), 292 [M+, (28.6%)]. Anal. Calcd for C16H16N6: C, 65.75; H, 5.52; N, 28.75;
Found: C, 65.84; H, 5.52; N, 28.85.

46.16. FT-IR (ὑ max, cm−1): 3335 (NH), 3075 (CH aromatic), 2915 (CH aliphatic), 2227 (C^N nitrile). MS: (Mwt: 360.09) m/z (% rel. Int.), 362 [M++2, (10.8%)], 360 [M+, (32.4%)]. Anal. Calcd for
C19H13ClN6: C, 63.25; H, 3.63; N, 23.29; Found: C, 63.35; H, 3.63; N,
23.34.
2.1.2.23. 4-(4-Chlorophenyl)-2-(phenethylamino)pyrazolo[1,5-a][1,3,5] triazine-8- carbonitrile (8h). The product was separated as light yellow crystals, reaction time 1.5 h, (0.39 g, 52.13%) m.p.: 163–165 °C; 1H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 8.6 Hz, 2H, Ar- H), 8.16 (s, 1H, Pyrazole-H), 7.55 (d, J = 8.6 Hz, 2H, Ar-H), 7.36–7.24 (m, 5H, Ar-H),
5.73 (s, 1H, NH D2O exchangeable), 3.88 (t, J = 6.5 Hz, 2H, NHCH2), 3.03 (t, J = 6.6 Hz, 2H, CH2Ar). 13C NMR (101 MHz, CDCl3) δ 157.86, 155.82, 153.86, 147.92, 140.23, 138.59, 132.93, 129.93, 128.64, 127.67, 126.62, 125.9, 113.37, 100.41, 42.67, 34.68. FT-IR (ὑ max,
cm−1): 3331 (NH), 3103 (CH aromatic), 2923 (CH aliphatic), 2223 (C^N nitrile). MS: (Mwt: 374.10) m/z (% rel. Int.), 376 [M++2, (12.3%)], 374 [M+, (35.7%)]. Anal. Calcd for C20H15ClN6: C, 64.09; H,
4.03; N, 22.42; Found: C, 64.19; H, 4.03; N, 22.48.
2.1.2.24. 4-(4-Chlorophenyl)-2-(3-isopropoxypropylamino)pyrazolo[1,5-a] [1,3,5]triazine-8-carbonitrile (8i). The product was separated as green yellow crystals, reaction time 1 h, (0.39 g, 52.7%) m.p.: 155–158 °C; 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.5 Hz, 2H, ArH), 8.12 (s,1H, Pyrazole H), 7.55 (d, J = 8.5 Hz, 2H, ArH), 6.54 (s, 1H, NH D2O exchangeable), 3.70 (m, 1H, OCH(CH3)2), 3.66–3.59 (m, 4H, NHCH2CH2CH2O), 1.95 (m, 2H, CH2), 1.24 (d, J = 6.0 Hz, 6H, CH3).13C NMR (101 MHz, CDCl3) δ157.58, 156.20, 154.20, 147.56, 139.86, 132.41, 128.82, 127.79, 113.44, 96.0, 71.95, 67.22, 40.91, 28.73, 22.12. FT-IR (ὑ max, cm−1): 3333 (NH), 3102 (CH aromatic), 2969 (CH aliphatic), 2224
(C^N nitrile). MS: (Mwt: 370.13) m/z (% rel. Int.), 372 [M++2, (13.4%)], 370 [M+, (38.9%)]. Anal. Calcd for C18H19ClN6O: C, 58.30;
H, 5.16; N, 22.66; Found: C, 58.40; H, 5.16; N, 22.75.

2.1.2.25. 2-(Butylamino)-4-(4-chlorophenyl)pyrazolo[1,5-a][1,3,5] triazine-8-carbonitrile (8j). The product was separated as pale yellow crystals, reaction time 1.5 h, (0.42 g, 50.73%) m.p.: 174–177 °C; 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 8.7 Hz, 2H, Ar- H), 8.12 (s, 1H, Pyrazole-H), 7.54 (d, J = 8.7 Hz, 2H, Ar-H), 5.75 (s, 1H, NH D2O exchangeable), 3.59 (t, J = 3.7 Hz, 2H, NHCH2), 1.72–1.66 (p, 2H, CH2), 1.51–1.45 (m, 2H, CH2), 1.01 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 157.53, 156.25, 154.25, 147.65, 139.68, 132.14, 128.28, 127.97, 113.26, 104.25, 43.23, 31.06, 20.25, 13.95. FT-IR (ὑ max, cm−1): 3309 (NH), 3080 (CH aromatic), 2945 (CH aliphatic),
2217 (C^N nitrile). MS: (Mwt: 326.10) m/z (% rel. Int.), 328 [M++2, (12.9%)], 326 [M+, (37.7%)]. Anal. Calcd for C16H15ClN5: C, 58.81; H,
4.63; N, 25.72; Found: C, 58.91; H, 4.63; N, 25.82.
2.1.2.26. 4-(4-Chlorophenyl)-2-(isobutylamino)pyrazolo[1,5-a][1,3,5] triazine-8- carbonitrile (8k). The product was separated as buff crystals, reaction time 1 h, (0.43 g, 51.94%) m.p.: 176–178 °C; 1H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H, Pyrazole-H), 7.72 (d, J = 8.5 Hz, 2H, Ar-H), 7.40 (d, J = 8.5 Hz, 2H, Ar-H), 6.32 (s, 1H, NH D2O exchangeable), 3.28 (d, J = 6.4 Hz, 2H, NHCH2), 1.97–1.82 (m, 1H, CH(CH3)2), 0.99 (d, J = 6.7 Hz, 6H, CH3).13C NMR (101 MHz, CDCl3) δ 156.82, 156.12, 153.56, 147.94, 139.83, 132.94, 128.58, 128.72, 113.35, 102.40, 52.15, 27.20, 20.17. FT-IR (ὑ max, cm−1): 3294
(NH), 3070 (CH aromatic), 2950 (CH aliphatic), 2219 (C^N nitrile). MS: (Mwt: 326.10) m/z (% rel. Int.), 328 [M++2, (12.9%)], 326 [M+,
(39.2%)]. Anal. Calcd for C16H15ClN6: C, 58.81; H, 4.63; N, 25.72
Found: C, 58.91; H, 4.63; N, 25.82.
2.1.2.27. 4-(4-Chlorophenyl)-2-(ethylsulfonyl)pyrazolo[1,5-a][1,3,5] triazine-8- carbonitrile (8l). The product was separated as bright yellow crystals, reaction time 1.5 h, (0.35 g, 50.72%) m.p.: 165–167 °C; 1H

2.1.2.30. 4-(4-Bromophenyl)-2-(phenethylamino)pyrazolo[1,5-a][1,3,5] triazine-8- carbonitrile (8o). The product was separated as light yellow crystals, reaction time 1.5 h, (0.44 g, 53.0%) m.p.: 148–151 °C; 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.5 Hz, 2H, Ar-H), 8.16 (s, 1H, Pyrazole-H), 7.57 (d, J = 8.5 Hz, 2H, Ar-H), 7.38–7.16 (m, 5H, Ar-H),
5.77 (s, 1H, NH D2O exchangeable), 3.87 (t, J = 6.6 Hz, 2H, NHCH2), 3.05 (t, J = 6.6 Hz, 2H, CH2Ar). 13C NMR (101 MHz, CDCl3) δ157.82, 155.25, 153.25, 147.19, 140.50, 138.56, 132.95, 129.72, 128.56, 127.20, 126.23, 126.17, 113.20, 100.15, 43.95, 35.70. FT-IR (ὑ max, cm−1): 3328 (NH), 3115 (CH aromatic), 2920 (CH aliphatic), 2222
(C^N nitrile). MS: (Mwt: 418.05) m/z (% rel. Int.), 420 [M++2, (32.7%)], 418 [M+, (28.5%)]. Anal. Calcd for C20H15BrN6: C, 57.29; H,
3.61; N, 20.04; Found: C, 57.39; H, 3.61; N, 20.14.
2.1.2.31. 4-(4-Bromophenyl)-2-(3-isopropoxypropylamino)pyrazolo[1,5- a][1,3,5] triazine-8-carbonitrile (8p). The product was separated as green yellow crystals, reaction time 1.5 h, (0.44 g, 53.14%) m.p.: 162–165 °C; 1H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 8.4 Hz, 2H, Ar- H), 8.12 (s, 1H, Pyrazole-H), 7.53 (d, J = 8.4 Hz, 2H, Ar-H), 6.52 (s, 1H, NH D2O exchangeable), 3.78–3.65 (m, 1H, OCH(CH3)2), 3.64–3.50 (m, 4H, NHCH2CH2CH2O), 2.00–1.92 (m, 2H, CH2), 1.24 (d, J = 6.0 Hz, 6H, CH3). 13C NMR (101 MHz, CDCl3) δ 157.53, 156.35, 154.25, 147.58, 139.85, 132.45, 128.82, 127.82, 113.45, 95.98, 71.96, 67.30, 40.92, 28.94, 22.11. FT-IR (ὑ max, cm−1): 3329 (NH), 3069 (CH
aromatic), 2965 (CH aliphatic), 2220 (C^N nitrile). MS: (Mwt: 414.08)
m/z (% rel. Int.), 416 [M++2, (37.2%)], 414 [M+, (31.8%)]. Anal.
Calcd for C18H19BrN6O: C, 52.06; H, 4.61; N, 20.24; Found: C, 52.16; H,
4.61; N, 20.34.
2.1.2.32. General procedure for the preparation of 9a-o. Compounds ethyl 2-(ethylthio)-4-substituted phenylpyrazolo[1,5-a][1,3,5]triazine-

NMR (400 MHz, CDCl3) δ 8.56 (d, J = 8.2 Hz, 2H, Ar-H), 8.10 (s, 1H,

8-carboXylate (7a-c) (1 equiv., 1 mmol) were fused with the

pyrazole-H), 7.53 (d, J = 8.2 Hz, 2H, Ar-H), 4.00–3.89 (m, 2H, CH2), 1.77 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 156.01,
153.65, 147.91, 145.80, 139.80, 132.49, 128.80, 128.27, 113.75,
96.04, 45.37, 14.50. FT-IR (ὑ max, cm−1): 3145 (CH aromatic), 2941
(CH aliphatic), 2226 (C^N nitrile), 1301 (SO2). MS: (Mwt: 347.02) m/z
(% rel. Int.), 349 [M++2, (13.6%)], 347 [M+, (39.9%)]. Anal. Calcd
for C14H10ClN5O2S: C, 48.35; H, 2.90; N, 20.14; Found: C, 48.25; H,
2.90; N, 20.24.
2.1.2.28. 4-(4-Chlorophenyl)-2-(cyclopropylamino)pyrazolo[1,5-a] [1,3,5]triazine-8- carbonitrile (8m). The product was separated as pale yellow crystals, reaction time 1 h, (0.31 g, 50.0%) m.p.: 178–180 0C; 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.3 Hz, 2H, Ar-H), 8.18 (s, 1H, Pyrazole-H), 7.56 (d, J = 8.3 Hz, 2H, Ar-H), 5.82 (s, 1H ,NH D2O exchangeable), 3.08–2.97 (m, 1H, CH), 0.99 (d, J = 5.8 Hz, 4H, 2CH2). 13C NMR (101 MHz, CDCl3) δ156.25, 155.83, 153.25, 146.25, 139.35, 132.46, 128.38, 127.42, 113.42, 96.35, 28.01, 9.12. FT-IR (ὑ max, cm−1): 3324 (NH), 3115 (CH aromatic), 2922 (CH aliphatic), 2225
(C^N nitrile). MS: (Mwt: 310.07) m/z (% rel. Int.), 312 [M++2, (12.8%)], 310 [M+, (37.9%)]. Anal. Calcd for C15H11ClN6: C, 57.98; H,
3.57; N, 27.05; Found: C, 58.06; H, 3.57; N, 27.15.
2.1.2.29. 2-(Benzylamino)-4-(4-bromophenyl)pyrazolo[1,5-a][1,3,5] triazine-8- carbonitrile (8n). The product was separated as light yellow crystals, reaction time 1.5 h, (0.42 g, 52.5%) m.p.: 163–165 °C; 1H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 8.6 Hz, 2H, Ar- H), 8.16 (s, 1H, Pyrazole-H), 7.55 (d, J = 8.6 Hz, 2H, Ar-H), 7.36–7.24 (m, 5H, ArH),
5.73 (s, 1H, NH D2O exchangeable), 3.88 (s, 2H, NHCH2).13C NMR (101 MHz, CDCl3) δ 157.86, 155.82, 153.86, 147.92, 140.23, 138.59, 132.93, 129.93, 128.64, 127.67, 126.62, 113.37, 100.41, 42.67, 34.68. FT-IR (ὑ max, cm−1): 3331 (NH), 3103 (CH aromatic), 2923 (CH
aliphatic), 2223 (C^N nitrile). MS: (Mwt: 404.04) m/z (% rel. Int.), 406 [M++2, (51.8%)], 404 [M+, (48.2%)]. Anal. Calcd for C19H13BrN6: C,
56.31; H, 3.23; N, 20.74; Found: C, 56.40; H, 3.23; N, 20.80.

appropriate amine (1 equiv., 1 mmol) at 150 °C using oil bath for 1.5–2 h. The reaction miXture was then triturated with diethyl ether and the precipitate was collected by filtration and purified by flash column chromatography on silica gel, using a miXture of hexane/ethyl acetate (8:2) as eluent.
2.1.2.33. Ethyl 2-(benzylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine- 8-carboxylate (9a). The titled compound was separated as yellow powder (0.3 g, 52%); m.p. 143–145 °C; 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 7.6 Hz, 2H, Ar H), 8.39 (s, 1H, H pyrazole), 7.65–7.30 (m, 8H, Ar H), 6.01 (s, 1H, NH D2O exchangeable), 4.82 (s, 2H, N-CH2), 4.41 (q, J = 7.1 Hz, 2H, CH2CH3), 1.44 (t, J = 7.1 Hz, 3H, CH2,CH3) ; 13C NMR (101 MHz, CDCl3) δ 163.25, 157.82, 153.78, 147.32, 145.73, 138.69, 136.81, 133.42, 129.56, 128.92, 128.56, 126.92, 126.48, 98.95, 59.95, 42.65, 14.61; FT-IR (ὑ max, cm−1): 3409 (NH), 3117
(CH aromatic), 2853 (CH aliphatic), 1740 (C]O ester); MS: (Mwt.: 373.41): m/z (% rel. Int.), 373.25 [M+, (35.8%)]., 309.23 (100%);
Anal. Calcd for C21H19N5O2: C, 67.55; H, 5.13; N, 18.76; Found: C, 67.68; H, 5.26; N, 18.92.
2.1.2.34. Ethyl 2-(phenethylamino)-4-phenylpyrazolo[1,5-a][1,3,5] triazine-8-carboxylate (9b). The titled compound was separated as yellow powder (0.31 g, 53%); m.p. 135–137 °C; 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 7.6 Hz, 2H, Ar H), 8.38 (s, 1H, pyrazole H), 7.70–7.55 (m, 3H, Ar H), 7.35–7.26 (m, 5H, Ar H), 5.73 (s, 1H, NH D2O exchangeable), 4.41 (q, J = 7.5 Hz, 2H, CH2CH3), 3.92 (t, J = 6.3 Hz, 2H, CH2), 3.05 (t, J = 6.4 Hz, 2H, CH2), 1.44 (t, J = 7.5 Hz, 3H, CH2CH3); 13C NMR (101 MHz, CDCl3) δ 168.70, 157.87, 152.32, 148.87, 146.59, 139.62, 137.32, 132.29, 129.35, 128.91, 127.54, 126.76, 126.29, 98.79, 60.42, 42.61, 35.23, 14.65; FT-IR (ὑ max,
cm−1): 3368 (NH), 3090 (CH aromatic), 2927 (CH aliphatic), 1745 (C]O ester); MS: (Mwt.: 387.43): m/z(% rel. Int.), 387.23, 194.12 (100%); Anal. Calcd for C22H21N5O2: C, 68.20; H, 5.46; N, 18.08;
Found: C, 68.72; H, 5.22; N, 18.13.

2.1.2.35. Ethyl 4-phenyl-2-(piperidin-1-yl)pyrazolo[1,5-a][1,3,5]triazine- 8-carboxylate (9c). The titled compound was separated as yellow powder (0.35 g, 65%); m.p. 133–135 °C; 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 7.2 Hz, 2H, Ar H), 8.33 (s, 1H, pyrazole H), 7.64–7.54 (m, 3H, Ar H), 4.38 (q, J = 7.1 Hz, 2H, CH2CH3), 4.02 (br s, 4H, piperidine H), 1.72 (br s, 6H, piperidine H), 1.42 (t, J = 7.1 Hz, 3H, CH2CH3). 13C NMR (101 MHz, CDCl3) δ163.35, 157.53, 152.35, 149.32, 143.40, 133.39, 130.89, 129.72, 128.36, 99.20, 60.72, 47.18, 29.30, 24.42, 14.60; FT-IR (ὑ max, cm−1): 3115 (CH aromatic), 2920 (CH aliphatic),
1750 (C]O ester) MS: (Mwt.: 351.40): m/z(% rel. Int.), 351.35, 103.17
(100%); Anal. Calcd for C19H21N5O2: C, 64.94; H, 6.02; N, 19.93;
Found: C, 64.82; H, 6.26; N, 19.85.
2.1.2.36. Ethyl 4-phenyl-2-(pyrrolidin-1-yl)pyrazolo[1,5-a][1,3,5]triazine-8- carboxylate (9d). The titled compound was separated as yellow powder (0.37 g, 72%); m.p. 155–157 °C; 1H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 7.4 Hz, 2H, Ar H), 8.35 (s, 1H, pyrazole H), 7.66–7.55 (m, 3H, Ar H), 4.39 (q, J = 7.1 Hz, 2H, CH2CH3), 3.82 (br s, 4H, N(CH2)2), 2.06 (br s, 4H, pyrroldine (CH2)2), 1.43 (t, J = 7.1 Hz, 3H, CH2CH3); 13C NMR (101 MHz, CDCl3) δ 162.95, 155.72, 153.75, 148.80, 144.65, 132.82, 131.65, 129.42,
127.81, 98.05, 60.25, 47.54, 25.45, 14.70; FT-IR (ὑ max, cm−1): 3098 (CH
aromatic), 2890 (CH aliphatic), 1750 (C]O ester); MS: (Mwt.: 337.38): m/z(% rel. Int.), 337.21, 194.12 (100%); Anal. Calcd for C18H19N5O2: C, 64.08; H, 5.68; N, 20.76; Found: C, 64.15; H, 5.53; N, 20.82.
2.1.2.37. Ethyl 2-(isopropylamino)-4-phenylpyrazolo[1,5-a][1,3,5] triazine-8-carboxylate (9e). The titled compound was separated as yellow powder (0.32 g, 64%); m.p. 159–161 °C; 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 7.3 Hz, 2H, Ar H), 8.35 (s, 1H, pyrazole H), 7.63–7.55 (m, 3H, Ar H), 5.58 (s, 1H, NH D2O exchangeable), 4.38 (q, J = 7.1 Hz, 2H, CH2CH3) 4.1 (m, 1H, CH(CH3)2) 1.41 (t, J = 7.1 Hz, 3H, CH2CH3), 1.34 (d, J = 6.5 Hz, 6H, (CH3)2). 13C NMR (101 MHz, CDCl3) δ 163.36, 157.26, 153.04, 148.85, 144.68, 131.66, 130.91, 128.59, 127.81, 98.82, 59.82, 43.03, 22.78, 14.35; FT-IR (ὑ max, cm−1): 3317 (NH), 3110 (CH aromatic), 2924 (CH aliphatic), 1750
(C]O ester); MS: (Mwt.: 325.37): m/z(% rel. Int.), 325.12, 220.11 (100%); Anal. Calcd for C17H19N5O2: C, 62.75; H, 5.89; N, 21.52;
Found: C, 62.85; H, 5.75; N, 21.46.
2.1.2.38. Ethyl 2-(benzylamino)-4-(4-chlorophenyl)pyrazolo[1,5-a] [1,3,5]triazine-8-carboxylate (9f). The titled compound was separated as yellow powder (0.26 g, 46%); m.p. 133–135 °C; 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.1 Hz, 2H, Ar H), 8.39 (s, 1H, pyrazole), 7.54 (d, J = 8.1 Hz, 2H, Ar H), 7.48–7.28 (m, 5H, Ar H), 5.96 (s, 1H, NH D2O exchangeable), 4.82 (s, 2H, N-CH2), 4.41 (q, J = 7.0 Hz, 2H, CH2CH3), 1.46 (t, J = 7.0 Hz, 3H, CH2CH3). 13C NMR (101 MHz, CDCl3) δ 163.22, 157.78, 153.68, 147.37, 145.68, 138.39, 136.75, 133.40, 129.57, 128.85, 128.63, 126.95, 126,61, 98.93, 59.32, 42.60, 14.58; FT-IR (ὑ max, cm−1): 3346 (NH), 3063 (CH aromatic), 2928 (CH aliphatic),
1745 (C]O ester); MS: (Mwt.: 407.85): m/z(% rel. Int.), 409.36
[M++2, (23.6%)], 407.74 [M+, (69.2%)], 44.10 (100%); Anal. Calcd
for C21H18ClN5O2: C, 61.84; H, 4.45; N, 17.17; Found: C, 61.76; H, 4.61;
N, 17.25.
2.1.2.39. Ethyl 4-(4-chlorophenyl)-2-(phenethylamino)pyrazolo[1,5 a] [1,3,5]triazine −8-carboxylate (9g). The titled compound was separated as faint yellow powder (0.32 g, 55%); m.p. 158–160 °C; 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J = 8.5 Hz, 2H, Ar H), 8.37 (s, 1H, pyrazole H), 7.54 (d, J = 8.5 Hz, 2H, Ar H), 7.35–7.25 (m, 5H, Ar H),
5.69 (s, 1H, NH D2O exchangeable), 4.42 (q, J = 7.4 Hz, 2H, –CH2CH3), 3.92 (t, J = 6.2 Hz, 2H, CH2), 3.04 (t, J = 6.4 Hz, 2H, CH2), 1.44 (t, J = 7.4 Hz, 3H, CH2CH3). 13C NMR (101 MHz, CDCl3) δ 168.78, 157.85, 152.21, 148.89, 146.85, 139.58, 137.22, 132.27, 129.62, 128.95, 127.97, 126.99, 126.31, 98.75, 60.21, 42.66, 35.33, 14.40; FT-IR (ὑ max, cm−1): 3350 (NH), 3078 (CH aromatic), 2920 (CH
aliphatic), 1750 (C]O ester); MS: (Mwt.: 421.88): m/z(% rel. Int.),

423.74 [M++2, (12.8%)], 421.49 [M+, (36.4%)], 194.87 (100%); Anal. Calcd for C22H20ClN5O2: C, 62.63; H, 4.78; N, 16.60; Found: C, 62.71; H, 4.82; N, 16.51.
2.1.2.40. Ethyl 4-(4-chlorophenyl)-2-(piperidin-1-yl)pyrazolo[1,5-a] [1,3,5]triazine-8-carboxylate (9h). The titled compound was separated as faint yellow powder (0.31 g, 58%); m.p. 162–164 °C; 1H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 8.7 Hz, 2H, Ar H), 8.33 (s, 1H, pyrazole H), 7.54 (d, J = 8.7 Hz, 2H, Ar H), 4.38 (q, J = 7.1 Hz, 2H, CH2CH3), 4.02 (br. s, 4H, piperidine H), 1.73 (br. s, 6H, piperidine H),
1.42 (t, J = 7.1 Hz, 3H, CH2CH3).; 13C NMR (101 MHz, CDCl3) δ
163.29, 157.51, 152.30, 149.41, 143.38, 133.35, 130.65, 129.49,
128.29, 99.23, 60.42, 47.20, 29.33, 24.40, 14.40; FT-IR (ὑ max,
cm−1): 3125 (CH aromatic), 2930 (CH aliphatic), 1745 (C]O ester); MS: (Mwt.: 385.85): m/z(% rel. Int.), 387.73 [M++2, (15.7%)], 385.46
[M+, (44.8%)], 308.42 (100%); Anal. Calcd for C19H20ClN5O2: C,
59.14; H, 5.22; N, 18.15; Found: C, 59.22; H, 5.18; N, 18.19.
2.1.2.41. Ethyl 4-(4-chlorophenyl)-2-(pyrrolidin-1-yl)pyrazolo[1,5-a] [1,3,5]triazine-8-carboxylate (9i). The titled compound was separated as faint yellow powder (0.29 g, 56%); m.p. 193–195 °C; 1H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 8.7 Hz, 2H, ArH), 8.35 (s, 1H, pyrazole H), 7.68 (d, J = 8.7 Hz, 2H, Ar H), 4.37 (q, J = 7.1 Hz, 2H, CH2CH3), 3.85 (br s, 4H, N(CH2)2) 2.13 (br s, 4H, pyrrolidine (CH2)2), 1.44 (t, J = 7.1 Hz, 3H, CH2CH3); 13C NMR (101 MHz, CDCl3) δ 162.96, 155.73, 153.76, 148.81, 144.66, 132.80, 131.64, 129.41, 127.80, 98.10, 60.22, 47.53, 25.43, 14.71; FT-IR (ὑ max, cm−1): 3105 (CH
aromatic), 2915 (CH aliphatic), 1750 (C]O ester); MS: (Mwt.: 371.82):
m/z(% rel. Int.), 373.53 [M++2, (12.9%)], 371.68 [M+, (35.4%)],
193.93 (100%); Anal. Calcd for C18H18ClN5O2: C, 58.14; H, 4.88; N,
18.84; Found: C, 58.21; H, 4.92; N, 18.75.
2.1.2.42. Ethyl 4-(4-chlorophenyl)-2-(isopropylamino)pyrazolo[1,5-a] [1,3,5]triazine −8-carboxylate (9j). The titled compound was separated as faint yellow powder (0.26 g, 52%); m.p. 156–158 °C; 1H NMR (400 MHz, CDCl3) 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 8.0 Hz, 2H, Ar H), 8.37 (s, 1H, pyrazole H), 7.56 (d, J = 8.0 Hz, 2H, Ar H), 5.52 (s, 1H, NH D2O exchangeable), 4.46 (q, J = 7.0 Hz, 2H, CH2CH3), 4.2 (m,1H, CH(CH3)2), 1.43 (t, J = 7.0 Hz, 3H, CH2CH3), 1.28 (d, J = 6.3 Hz, 6H, (CH3)2);13C NMR (101 MHz, CDCl3) δ 163.33, 157.22, 153.03, 148.82, 144.64, 131.62, 130.89, 128.56, 127.78,
98.89, 59.75, 43.07, 22.73, 14.30; FT-IR (ὑ max, cm−1): 3317 (NH),
3103 (CH aromatic), 2924 (CH aliphatic), 1750 (C]O ester); MS: (Mwt.: 359.81): m/z(% rel. Int.), 361.74 [M++2, (17.4%)], 359.66
[M+, (52.6%)], 194.12 (100%); Anal. Calcd for C17H18ClN5O2: C,
56.75; H, 5.04; N, 19.46; Found: C, 56.83; H, 5.15; N, 19.72.
2.1.2.43. Ethyl 2-(benzylamino)-4-(4-bromophenyl)pyrazolo[1,5-a] [1,3,5]triazine-8-carboxylate (9k). The titled compound was separated as greenish yellow powder (0.29 g, 52%); m.p. 154–156 °C; 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 8.3 Hz, 2H, Ar H), 8.38 (s, 1H, pyrazole H), 7.70 (d, J = 8.3 Hz, 2H, Ar H), 7.47–7.29 (m, 5H, Ar H),
6.01 (s, 1H, NH D2O exchangeable), 4.82 (s, 2H, N-CH2), 4.41 (q, J = 7.1 Hz, 2H, CH2CH3), 1.43 (t, J = 7.1 Hz, 3H, CH2CH3). 13C NMR (101 MHz, CDCl3) δ163.32, 157.75, 153.72, 147.29, 145.62, 138.71, 136.79, 133.41, 129.72, 128.89, 128.66, 127.23, 126.52, 98.97, 59.89, 42.63, 14.52; FT-IR (ὑ max, cm−1): 3481 (NH), 3093 (CH aromatic),
2977 (CH aliphatic), 1748 (C]O ester); MS: (Mwt.: 452.30): m/z(% rel. Int.), 454.34 [M++2, (33.2%)], 452.16 [M+, (35.4%)], 378.26
(100%); Anal. Calcd for C21H18BrN5O2: C, 55.76; H, 4.01; N, 15.48;
Found: C, 55.82; H, 4.15; N, 15.52.
2.1.2.44. Ethyl 4-(4-bromophenyl)-2-(phenethylamino)pyrazolo[1,5-a] [1,3,5] triazine-8-carboxylate (9l). The titled compound was separated as greenish yellow powder (0.38 g, 66%); m.p. 147–150 °C; 1H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 8.4 Hz, 2H, Ar H), 8.37 (s, 1H,

pyrazole H), 7.54 (d, J = 8.4 Hz, 2H, Ar H), 7.38–7.25 (m, 5H, Ar H),
5.66 (s, 1H, NH D2O exchangeable), 4.40 (q, J = 7.2 Hz, 2H, CH2CH3), 3.92 (t, J = 6.6 Hz, 2H, CH2), 3.05 (t, J = 6.5 Hz, 2H, CH2), 1.41 (t, J = 7.2 Hz, 3H, CH2CH3). 13C NMR (101 MHz, CDCl3) δ 168.73, 157.82, 152.29, 148.82, 146.65, 137.53, 137.35, 132.28, 129.58, 128.87, 127.92, 126.64, 126.25, 98.76, 60.45, 42.65, 35.35, 14.43; FT-IR (ὑ max, cm−1): 3317 (NH), 3065 (CH aromatic), 2918 (CH
aliphatic), 1745 (C]O ester); MS: (Mwt.: 466.33): m/z(% rel. Int.),
468.36 [M++2, (43.8%)], 466.25 [M+, (45.2%)], 309.23 (100%);
Anal. Calcd for C22H20BrN5O2: C, 56.66; H, 4.32; N, 15.02; Found: C, 56.58; H, 4.43; N, 15.18.

2.1.2.45. Ethyl 4-(4-bromophenyl)-2-(piperidin-1-yl)pyrazolo[1,5-a] [1,3,5]triazine-8-carboxylate (9m). The titled compound was separated as greenish yellow powder (0.29 g, 55%); m.p. 168–170 °C; 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 8.6 Hz, 2H, Ar H), 8.33 (s, 1H, pyrazole H), 7.71 (d, J = 8.6 Hz, 2H, Ar H), 4.38 (q, J = 7.1 Hz, 2H, CH2CH3), 4.02 (br s, 4H, piperidine H), 1.73 (br s, 6H, piperidine H), 1.42 (t, J = 7.1 Hz, 3H, CH2CH3); 13C NMR (101 MHz, CDCl3) δ 163.32, 157.52, 152.32, 149.35, 143.52, 133.37, 130.85, 129.53, 128.32, 99.21, 60.45, 47.12, 29.45, 24.38, 14.62; FT-IR (ὑ max, cm−1): 3120
(CH aromatic), 2915 (CH aliphatic), 1750 (C]O ester); MS: (Mwt.: 430.30): m/z(% rel. Int.), 432.24 [M++2, (47.3%)], 430.19 [M+,
(43.9%)], 309.23 (100%); Anal. Calcd for C19H20BrN5O2: C, 53.03; H,
4.68; N, 16.28; Found: C, 53.12; H, 4.57; N, 16.35.

2.1.2.46. Ethyl 4-(4-bromophenyl)-2-(pyrrolidin-1-yl)pyrazolo[1,5-a] [1,3,5]triazine-8-carboxylate (9n). The titled compound was separated as greenish yellow powder (0.23 g, 45%); m.p. 190–192 °C; 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.7 Hz, 2H, Ar H), 8.35 (s, 1H, pyrazole H), 7.72 (d, J = 8.7 Hz, 2H, Ar H), 4.39 (q, J = 7.1 Hz, 2H, CH2CH3), 3.82 (br s, 4H, N(CH2)2) 2.07 (br s, 4H, pyrrolidine (CH2)2), 1.43 (t, J = 7.1 Hz, 3H, CH2CH3). 13C NMR (101 MHz, CDCl3) δ 162.98, 155.75, 153.79, 148.83, 144.68, 132.79, 131.67, 129.40, 127.84, 98.01, 60.21, 47.56, 25.41, 14.72; FT-IR (ὑ max, cm−1): 3108 (CH
aromatic), 2919 (CH aliphatic), 1750 (C]O ester); MS: (Mwt.: 416.27):
m/z(% rel. Int.), 418.28 [M++2, (23.7%)], 416.14 [M+, (27.2%)],
248.22 (100%); Anal. Calcd for C18H18BrN5O2: C, 51.94; H, 4.36; N,
16.82; Found: C, 51.87; H, 3.22; N, 16.77

2.1.2.47. Ethyl 4-(4-bromophenyl)-2-(isopropylamino)pyrazolo[1,5-a] [1,3,5]triazine −8-carboxylate (9o). The titled compound was separated as greenish yellow powder (0.27 g, 54%); m.p. 180–182 °C; 1H NMR (400 MHz, CDCl3); 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 8.0 Hz, 2H, Ar H), 8.36 (s, 1H, pyrazole H), 7.72 (d, J = 8.0 Hz, 2H, Ar H), 5.46 (s,1H, NH D2O exchangeable), 4.43 (q, J = 7.0 Hz, 2H, CH2CH3), 4.15(m, 1H, CH(CH3)2), 1.42 (t, J = 7.0 Hz, 3H, CH2CH3), 1.36 (d, J = 6.3 Hz, 6H, (CH3)2) 13C NMR (101 MHz, CDCl3) δ 163.35, 157.20, 153.06, 148.80, 144.65, 131.63, 130.90, 128.55, 127.75, 98.90, 59.79, 43.05, 22.75, 14.32; FT-IR (ὑ max, cm−1): 3305 (NH),
3125 (CH aromatic), 2947 (CH aliphatic), 1750 (C]O ester); MS: (Mwt.: 404.26): m/z(% rel. Int.), 406.17 [M++2, (62.5%)], 404.21
[M+, (65.1%)], 163.33 (100%); Anal. Calcd for C17H18BrN5O2: C,
50.51; H, 4.49; N, 17.32; Found: C, 50.75; H, 4.61; N, 17.35.

2.1.2.48. General procedure for the preparation of 10a-c. To the compounds ethyl 2-(ethylthio)-4-substituted phenylpyrazolo[1,5-a]

2.1.2.49. N-phenethyl-2-(phenethylamino)-4-phenylpyrazolo[1,5-a] [1,3,5]triazine-8-carboxamide (10a). The titled compound was separated as yellow powder (0.43 g, 61%); m.p. 190–192 °C; 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 7.5 Hz, 2H, Ar H), 8.47 (s, 1H, pyrazole H), 7.82 (s, 1H, NH D2O exchangeable), 7.66–7.20 (mm, 13H, Ar H), 5.85 (s, 1H, NH D2O exchangeable), 3.84 (t, J = 6.3 Hz, 2H, CH2), 3.46 (t, J = 6.2 Hz, 2H, CH2), 2.96 (t, J = 6.6 Hz, 2H, CH2), 2.87 (t, J = 6.7 Hz, 2H, CH2); 13C NMR (101 MHz, CDCl3) δ 162.61, 156.89, 150.03, 147.70, 144.69, 138.13, 133.54, 130.91, 129.02, 128.57, 127.07, 126.69, 126.28, 101.58, 42.21, 39.96, 34.99; FT-IR (ὑ max, cm−1): 3398 (NH), 3175 (NH), 3060 (CH aromatic), 2948 (CH
aliphatic), 1650 (C]O amide); MS: (Mwt.: 462.55): m/z(% rel. Int.),
462.43, 163.07 (100%); Anal. Calcd for C28H26N6O: C, 72.71; H, 5.67;
N, 18.17; Found: C, 72.85; H, 5.83; N, 18.21.
2.1.2.50. N-benzyl-2-(benzylamino)-4-phenylpyrazolo[1,5-a][1,3,5] triazine-8- carboxamide (10b). The titled compound was separated as yellow powder (0.38 g, 58%); m.p. 217–219 0C; 1H NMR (400 MHz, CDCl3)) δ 8.55 (d, J = 7.7 Hz, 2H, Ar H), 8.52 (s, 1H, pyrazole H), 8.05 (s, 1H, NH D2O exchangeable), 7.69–7.57 (m, 3H, Ar H), 7.41–7.22 (m, 10H, Ar H), 6.05 (s, 1H, NH D2O exchangeable), 4.68 (d, J = 5.3 Hz, 2H, CH2), 4.52 (d, J = 5.8 Hz, 2H, CH2); 13C NMR (101 MHz, CDCl3) δ 162.60, 156.82, 150.01, 147.72, 144.63, 138.14, 133.50, 130.85, 129.05, 128.53, 127.02, 126.62, 126.22, 101.55, 42.20, 39.27; FT-IR
(ὑ max, cm−1): 3389 (NH), 3185 (NH), 3065 (CH aromatic), 2945 (CH
aliphatic), 1652 (C]O amide); MS: (Mwt.: 434.49): m/z(% rel. Int.), 434.33, 135.07 (100%); Anal. Calcd for C26H22N6O: C, 71.87; H, 5.10;
N, 19.34; Found: C, 71.75; H, 5.21; N, 19.39.
2.1.2.51. 4-(4-Chlorophenyl)-N-phenethyl-2-(phenethylamino)pyrazolo [1,5-a] [1,3,5] triazine-8-carboxamide (10c). The titled compound was separated as yellow powder (0.38 g, 55%); m.p. 205–207 °C; 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.3 Hz, 2H, Ar H), 8.39 (s, 1H, pyrazole H), 7.79 (s, 1H, NH D2O exchangeable), 7.52 (d, J = 8.3 Hz, 2H, Ar H), 7.36–7.20 (m, 10H, Ar H), 5.71 (s, 1H, NH D2O exchangeable), 3.84 (t, J = 5.5 Hz, 2H, CH2), 3.48 (t, J = 6.0 Hz, 2H, CH2), 2.91 (t, J = 6.1 Hz, 2H, CH2), 2.85(t, J = 5.6 Hz, 2H, CH2); 13C NMR (101 MHz, CDCl3) δ 162.59, 156.85, 150.05, 147.75, 144.65, 138.12, 133.52, 130.95, 129.07, 128.55, 127.05, 126.65, 126.25,
101.53, 42.25, 39.95, 34.90; FT-IR (ὑ max, cm−1): 3420 (NH), 3190
(NH), 3054 (CH aromatic), 2924 (CH aliphatic), 1651 (C]O amide); MS: (Mwt.: 496.99): m/z (% rel. Int.), 498.79 [M++2, (14.7%)], 496.58 [M+, (44.1%)], 91.09 (100%); Anal. Calcd for C28H25ClN6O:
C, 67.67; H, 5.07; N, 16.91; Found: C, 67.65; H, 5.15; N, 16.85.
2.2. Biological evaluation
2.2.1. Antiproliferative activity in-vitro against NCI 60-cell lines
The NCI in-vitro anticancer screening is a two-stage process, begin- ning with the evaluation of all compounds against the full NCI 60 cell line panel representing leukemia, NSCLC, melanoma, colon cancer, CNS cancer, breast cancer, ovarian cancer, renal cancer and prostate cancer at a single dose of 10 µM following the standard NCI protocol [24]. The output from the single dose screen is reported as a mean graph.
2.2.2. In vitro cyclin dependent kinase2 inhibitory activity
The in vitro enzyme inhibition determination for the synthesized compounds was carried out in BPS Bioscience Corporation, San Diego,

[1,3,5]triazine-8-carboXylate (7a, 7c) (1 equiv., 1.5 mmol), an

CA, USA (www.bpsbioscience.com). The CDK2/CyclinA2 activity at

appropriate amine (benzylamine or 2-phenylethylamine) (5 equivalent, 7.5 mmol) was added. The miXture was fused in oil bath at 150 °C for 5–7 h. The reaction miXture was then triturated with diethyl ether and the precipitate was collected by filtration and purified by flash column chromatography on silica gel, using a miXture of hexane/ethyl acetate (4:6) as eluent to give the titled compounds (10a- c).

single dose concentration of 10 µM was performed, where Histone H1 (BPS#52043) served as the enzyme source & Kinase-Glo Plus Luminescence kinase assay kit (Promega#V3772) was used. The assay was performed using Kinase-Glo Plus Luminescence kinase assay kit (Promega). The compounds were diluted in 10% DMSO and 5 µl of the dilution was added to a 50 µl reaction so that the final concentration of DMSO is 1% in all of the reaction. All of the enzymatic reactions were

conducted at 30 °C for 40 min. The 50 µl reaction miXture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT, 10 mM ATP, Kinase substrate and the enzyme (CDK2/CyclinA2). After the enzymatic reaction, 50 µl of Kinase-Glo Plus Luminescence kinase assay solution (Promega) was added to each reaction and incubate the plate for 5 min at room temperature. Luminescence signal was measured using a Bio Tek Synergy 2 microplate reader.
2.3. In silico methods
2.3.1. Molecular docking
Molecular docking study was conducted using C-Docker software in the interface of Accelry’s discovery studio 2.5 (Accelrys Inc., San Diego, C.A, USA) at Faculty of pharmacy, Ain Shams university, drug design laboratory.
In this investigation, docking study and analysis of the binding modes of the target compounds were conducted to interpret the bio- logical results and to gain further vision into binding orientations and interactions. The selected docking pose among the ten retrieved pos- sible ones was chosen based on the similarity of its binding mode to that of the lead compound.
The X-ray crystal structure of CDK2 co-crystallized with Roscovitine (PDB code: 3ddq) was obtained. It is notable that Roscovitine forms the crucial hydrogen bonds with CDK2 through Leu 83 [25].

2.3.2. Validation of docking protocol
Validation of C-Docker protocol used in this study was performed by re-docking of the lead compound roscovitine in the CDK2 active site. This was followed by alignment of the X-ray bioactive conformer of the lead compound roscovitine with the best-fitted pose achieved from the docking run [26]. The alignment showed good coincidence between them with RMSD = 1.624, indicating the ability of the used docking protocol to give valid docking poses

2.3.3. Absorption, distribution, metabolism and excretion (ADME) study
Computer aided ADMET study was performed by using the Accelrys Discovery studio 2.5 software. The ultimate goal of in silico ADMET is to predict disposition behavior of compounds in the whole body by as- sembling all kinetic processes in one global model and hence it is ex- pected to reduce the risk of late-stage attrition of drug development and to optimize screening and testing by looking only at the promising compounds. The study is based on the chemical structure of the mole- cule and involves the calculation of certain descriptors including; aqueous solubility level (Aq Sol), human intestinal absorption level (HIA), blood brain barrier penetration level (BBB), cytochrome P450 2D6 inhibition (CYP2D6), plasma protein binding level (PPB) and he- patotoXicity (Hepa ToX).

Scheme 1. Synthesis of compounds 4a-c and 7a-c. Reagents and conditions: (A) acetone, refluX, 2–3 h, (B) NaH, EtBr, DMF, rt, 30–45 min, (C) refluX, 45–60 min.,
(D) MeCN, refluX, 1.5–2 h, (E) NaH, EtBr, DMF, rt, 30 min, (F) DMF, refluX, 45–60 min.

Scheme 2. Synthesis of compounds 8a-p. Reagents and conditions: (A) Amines, fusion, 1–1.5 h, 150 °C.

3. Results and discussion
3.1. Chemistry
The designed compounds were synthesized according to the che- mical pathways outlined in schemes 1, 2 and 3.
The targeted compounds (4a-c) were synthesized by suitable several steps starting from chlorination of benzoic acids and its derivatives with thionyl chloride to form the corresponding acid chloride. These acid chlorides were treated with ammonium thiocyanate to produce 4- un/ substituted benzoyl isothiocyanates (1). On the other hand, ethoX- ymethylenemalononitrile and hydrazine hydrate 99% were heated under refluX in absolute ethanol to give 5-amino-1H-pyrazole-4-carbo- nitrile (2). Then both benzoyl isothiocyanates derivatives and com- pound 2 were stirred in dry acetone at room temperature then heated under refluX to give the respective thiourea derivatives (3a-c) which were verified by their 1HNMR spectra, where an extra peak of ex- changeable NH of thiourea group appeared at about δ of 14.03 ppm.
The thiourea derivatives (3a-c) were treated with ethyl bromide in the presence of sodium hydride in DMF and stirred at room temperature to generate the ethyl sulfanyl intermediates, which subsequently un- dergoes cyclization by heating under refluX to produce the desired pyrazolotriazine compounds (4a-c). The structures of compounds (4a- c) were confirmed with 1HNMR spectra, where the characteristic peak of NH of the thiourea group disappeared and an extra two peaks of ethyl sulfanyl group appeared in the aliphatic region as quartet and triplet around δ of 3.33 and 1.51 ppm respectively.
The targeted compounds 7a-c were synthesized by the same pro- cedures starting from ethyl 5-amino-1H-pyrazole-4-carboXylate (5).

Compound 5 was prepared as reported by Schmidt et al. [22] using ethyl 2-cyano-3-ethoXyacrylate and hydrazine in ethanol. The product
(5) was separated in high purity and was confirmed by its reported melting point and its spectral and analytical data [22]. Compound (5) was subjected to the reaction with 4-un/substituted benzoyl iso- thiocyanates (1a-c) in acetonitrile to give thiourea derivatives (6a-c) [23]. The synthesized compounds (6a-c) were structurally elucidated by different analytical and spectral data. 1HNMR signals were con- sistent with protons of the targeted compounds. The spectra showed two equally integrated signals at around δ 14.03 and 9.2 ppm re- presenting the D2O exchangeable protons of (NH) thiourea protons, in addition to the signals of the protons of the new aromatic ring system.
FT-IR spectra revealed stretching signal of C]O amide around
1680 cm−1 Then thiourea derivatives (6a-c) were cyclized in the pre-
sence of ethyl bromide and NaH in DMF stirred at rt. for 30 min, to give the carbamimidothioate intermediates which were heated under refluX for 45–60 min to produce the desired compounds (7a-c).
The structures of compounds (7a-c) were confirmed by different analytical and spectral data. 1HNMR signals were consistent with pro- tons of the targeted compounds. The spectra showed additional peak at aliphatic region at around δ 3.30 and 1.49 ppm corresponding to the
newly ethyl group, also disappearance of peaks corresponding to NH of thiourea. FT-IR revealed stretching band of C]O ester around 1750 cm−1 and absence of C]O of amide. The molecular weights of the titled compounds match their calculated ones.
Literature revealed the reaction of pyrazolo[1,5-a][1,3,5]triazine with appropriate amines by fusion and in this study, the 4-(4-un/sub- stitutedphenyl)-2-(ethylthio)pyrazolo[1,5-a][1,3,5]triazine-8-carboni- tril (4a-c) and primary alphatic amines, were fused at 150 °C for 1–1.5 h

Scheme 3. Synthesis of compounds 9a-o and 10a-c. Reagents and conditions: (A) Amine, fusion, 1.5–2 h, 150 °C, (B) Amine, fusion, 5–7 h, 150 °C.to give corresponding pyrazolo[1,5-a] [1,3,5]triazine-8-carbonitrile (8a-p). 1HNMR of the entitled compounds were characterized by the preservation of the characteristic peaks of aromatic pyrazole protons and peak of exchangeable NH at δ 5.5–6.5 ppm. In addition to the ap- pearance of the new peaks of the aryl protons for compounds (8a, 8b, 8g, 8h, 8n and 8o) at δ 7.01–7.92, and the appearance of peaks in the aliphatic region at δ of 3.8 and 1.0 ppm for compounds (8c, 8d, 8e, 8i, 8j, 8k, 8m and 8p).

During the reaction of 3-aminoacetanilide (A) with ethyl sulfanyl pyrazolo[1,5-a][1,3,5]triazine (4a, b), the expected amine derivative
(B) was not obtained upon fusion for more than 1.5 h. Though, the sulfoXide derivative of the ethyl sulfanyl group (8f, 8l) were obtained instead. This could be explained by weak nucleophilicity of aromatic amine compared with aliphatic amine, as shown in Fig. 1.
The formation of this sulfoXide derivative (8f, 8l) was confirmed through 1HNMR spectra which revealed the appearance of the two peaks in the aliphatic protons as quartet and triplet at δ 4.00 and
1.7 ppm respectively, which are more deshielded compared to the starting ethyl sulfanyl derivatives (4a, 4b) that appeared in δ 3.3 and
1.5 ppm due to the presence of the more electron withdrawing SO2 group. Moreover, the absence of the additional aromatic protons, two NH, and acetyl moiety peaks confirmed the failure of the formation of the acetamidophenylamino derivatives of pyrazolo[1,5-a][1,3,5]tria- zine. FT-IR spectrum of compounds (8f and l) showed the appearance of peaks at 1257 and 1301 cm−1 respectively which represent SO2 group, which is not found in the starting ethylsulfanyl compound (4a). Additionally, the mass spectrum of the product (8f and l) revealed a molecular ion peaks of m/z = 313.21 and 347.48 respectively, corre- sponding to their molecular weights, while the starting sulfanylderivative (4a and 4b) have molecular weights of 281.34 and 315.78 respectively.
After that an optimization of compounds 7a-c was performed by nucleophilic substitution of ethylsulfanyl group of pyrazolo[1,5-a] [1,3,5]triazine with different amines to give compounds 9a-o. this re- action was performed by fusion at 150 °C in oil bath. The control of the concentration of amines and time of reaction are required to prevent the reaction of ester group too.
The structures of compounds 9a-o were confirmed by different analytical and spectral data. 1HNMR spectra revealed the characteristic signals of the targeted compounds. First, disappearance of the two ethyl signals of ethylthio group and appearance of signals corresponding to different amines. Regarding compounds 9b, 9g and 9l additional ali- phatic protons showed at around δ 3.05 and 3.92 in addition to the signals of protons of the newly introduced aromatic rings and singlet peak at δ around 5.7 ppm representing the D2O exchangeable protons of NH linker. Compounds 9a, 9f and 9k showed one additional singlet peak at around δ 4.8 ppm corresponding to benzylic protons in addition to protons of the newly introduced aromatic rings and singlet peak at around δ 6.0 ppm representing the D2O exchangeable protons of NH linker. Compounds 9c, 9h and 9m showed two singlet peaks at around δ 4.02 and 1.7 ppm representing the protons of the piperidine rings. Compounds 9d, 9i, and 9n showed two broad singlet peaks equally integrated at around δ 3.8 and 2.08 ppm representing pyrrolidine ring. Finally, compounds 9e, 9j and 9o showed the appearance of signal of the methyl protons which usually overlaps with signal of methyl pro- tons of ester group and the peak of proton near to the amine group appears as multiplet at around δ 4.15 ppm. Mass spectra of the titled compounds revealed that the molecular ion peaks are matched with

Fig. 1. Formation of compounds (8f, 8l).their molecular weights, also compounds (9f-j) showed the presence of M+ and M++2 peaks with relative intensity ratio of 3:1 corresponding to chlorine isotopes In addition, compounds (9k-o) showed the pre- sence of M+ and M++2 peaks with relative intensity ratio of 1:1 cor- responding to bromine isotopes.

Further optimization of ester group occurred by direct fusion of benzylamine or 2-phenylethylamine with compounds (7a, 7c) at 150 °C in oil bath to give compounds (10a-c). However, herein the con- centration of amines is 5 equivalents and the time of reaction is longer so both ethylthio and ester groups reacted, so amines are added on both sides of compounds.
The structures of compounds (10a-c) were confirmed by different analytical and spectral data. 1HNMR showed the disappearance of signals corresponding to ethylthio and ester groups. In addition to the appearance of signals of protons of the newly introduced aromatic rings, compounds (10a, c) showed two singlet peaks at 7.8 and 5.8 ppm corresponding to D2O exchangeable protons of NH linkers, also four equally integrated peaks at aliphatic region representing two ethyl groups. Compound (10b) showed two singlet peaks at around 6.05 and
8.05 ppm corresponding to D2O exchangeable protons of NH linkers and peaks of newly aromatic rings also appeared, in addition to two singlet peaks at δ around 4.62 and 4.52 ppm.

3.2. Biological evaluation
3.2.1. Antiproliferative activity in vitro against NCI 60-cell lines
Twenty of the final compounds were selected by the National Cancer Institute (NCI), NIH Bethesda, Maryland, USA (www.dtp.nci. nih.gov) under the Developmental Therapeutic Program (DTP), namely (7a, 7b, 8c, 8f-h, 8j-m, 9a, 9f-k, 9m, 10a, 10c). The obtained results were presented in Tables 1–3 in the supplementary part.
From the results obtained we can conclude that the 4-chloro phenyl derivative bearing benzyl amine group (9f) showed the highest cell growth inhibition with mean growth inhibition percentage of 41.77%. It exhibited broad spectrum and good anti-proliferative activity against several NCI cell panel as illustrated in Fig. 2.
Moreover, compound (10c), bearing 2-phenylethan-1-amine in one side and phenylethyl amide in the other side together with 4-chloro substituted phenyl group, also exhibited considerable cell growth in- hibition with mean growth inhibition percent of 33.32%. It exhibited broad spectrum and good inhibitory activity against certain cancer cells as illustrated in Fig. 3.
On the other hand, when comparing the results of compounds with their structures we observed that; compounds (9a, 9f, 9k), which bear the unsubstituted analogue, chloro substituted, and the bromo
Fig. 2. %Inhibition of compound (9f) against 26 cell line from 56 NCI cell line that exhibit % inhibition range from 40% to 115%.
Fig. 3. % inhibition of compound 10c against 17 cell line from 56 NCI cell line that exhibit % inhibition range from 43% to 92%.
substituted one respectively if ordered in their antiproliferative activity descendingly, their order will be 9f, 9k then 9a. So the chloro sub- stituted analogue is the most active, then the bromo and the least is the unsubstituted analogue. Likewise, compounds (7b, 8f), bearing chloride group, exhibited more activity than unsubstituted ones (7a, 8l). Accordingly, we can say that the presence of chloride enhanced the activity of compounds (7b, 9g and 10c) on the selected NCI cell lines.
3.2.2. In-vitro cyclin dependent kinase2 inhibitory activity
The percentage inhibition of the enzymatic activity caused by tested compounds against CDK2/CyclinA kinases at a single concentration of 10 µM was investigated. Compounds (8a, 8g, 8h, 8n, 9f, 9g, 9h, 9k, 9m, 10a and 10c) were selected as representatives that exhibited sig- nificant CDK2-cyclin A inhibition percent at 10 µM concentration for further investigation for dose related CDK2-cyclin A enzymatic inhibi- tion at 5 different concentrations (1 nM-10 nM-100 nM- 1 µM- 10 µM) to subsequently calculate their IC50 value. The results were shown in Table 4.
An overview of the results of CDK2 enzymatic activity at 10 µM concentration, compound (9f) bearing 4-chloro substituted phenyl group and benzyl amine and ester side chain has demonstrated good CDK2 inhibition (82.38%). Also compound (10c) bearing 4-chloro substituted and 2-phenylethan-1-amine in both sides exhibited good CDK2 inhibition (81.96%). On the other hand, several other in- vestigated compounds namely (8g, 8h, 9a, 9b, 9c, 9h, 9g, 9k, 9l, 9 m, 10a, and 10b) exhibited significant CDK2 inhibition (above 70%).
From the above studies, structure activity relationship among the newly synthesized pyrazolo[1,5-a][1,3,5]triazine derivatives can be concluded as follows
⁶ In general, the 8-carboXylate compounds have more inhibitory ac- tivity than the 8-carbonitrile congeners (7a compared to 4a, 66.75% and 53.32% respectively, 9a compared to 8a, 70.01% and 68.34% respectively).
⁶ Also, the 2-amino derivatives are more active than the 2-ethyl sul- phide analogues (8a compared to 4a, 68.34% and 53.32% respec- tively, 9a compared to 7a, 70.01% and 66.75% respectively).
⁶ 4 -Chloro substitution at phenyl ring enhanced the CDK2 inhibitory activity as shown by compounds (8g, 8h) (72.25, 74.01%) (9f-j) (61.96–82.38%) respectively when compared to bromo substitution (8n, 8o) (6153, 52.84%) (9k-o) (58.65–78.07%) respectively or unsubstituted compounds (9a-e) (51.93–72.42%) respectively.
⁶ Replacement of 2-ethylthio group of pyrazolo [1,5-a][1,3,5]triazine derivatives with phenyl alkyl amine enhanced the activity compared to ethyl thio and aliphatic or cyclic amines.

⁶ Optimization of the 8-carboXylate ester group into amide group was carried out to increase the hydrophilicity of the side chain oriented in the solvent accessible region, which resulted in increase in CDK2 inhibitory activity, (9a compared to 10a, 70.01% and 75.56%, re- spectively, 9b compared to 10b, 72.42% and 73.89%, respectively and 9g compared to 10c, 74.73% and 81.96%, respectively).

3.3. In silico results
3.3.1. Results of docking of the target compounds into CDK2 active sites
The docking of the synthesized compounds into CDK2 active site revealed that the designed compounds kept the key interactions done by the lead compound roscovitine co-crystallized with CDK2/cyclin A (PDB: code 3ddq) which was reported to be essential for activity and being sandwiched between the side chains of Leu134 and Ile10 [25] (Fig. 4).
Docking of the target compounds showed that synthesized com- pounds have similar binding mode with comparable docking scores to the lead compound.
The performed docking study showed interesting results. Most of the targeted compounds (4b, 4c, 7a-c, 8h, 8j, 8k, 8m, 8o, 9c-f, 9h-k, 9m- o) form a conserved hydrogen bond pattern where the N atom of pyr- azolo ring accepts proton from the backbone Leu 83 residue in the hinge region, which is crucial for CDK2 inhibitory activity. The two compounds (9f and 9k) have significant percentage inhibition of CDK2 (82.38 and 78.07% respectively) may be due to presence of aromatic amine and halide group which appear in all significant active com- pounds. In addition, compound (9f) which has the highest percentage inhibitory activity has the highest docking score among the series (Figs. 5 and 6).
Furthermore, the five compounds (4a, 8f, 8i, 8l and 8p) which showed lower inhibitory activity against CDK2/cyclinA2 (53.32, 27.73, 16, 42.12 and 36.63% respectively), were not bound to Leu83 and that may explain their very poor biological activity against CDK2.
It has been reported that several roscovitine bioisosteres compounds were potent though they had a reversed binding with CDK2 to that of roscovitine. For target compounds (8a, 8g, 8n, 9a, 9b, 6f, 9g, and 10a- c), possessed the reversed binding mode of roscovitine where the amino NH– was bound to Leu 83 through hydrogen bond in the hinge region, their CDK2 inhibitory activity were (68, 72, 61, 72.42, 70.01, 73.49,
74.73, 75.56, 73.89 and 81.96%, respectively). In addition, the activity of these compounds may be due to the presence of another hydrogen bond with backbone Leu83 residue through the ester group in them. On the other hand, compound (10c) has the highest docking score and the most active between them, that may be due to the presence of another

Table 4
Percentage inhibition of CDK2 enzymatic activity of targeted compounds and calculated IC50 of compounds
8a, g, h, n, 9f, g, h, k, m, 10a, c.

Fig. 4. 3D interaction diagram of the top docking pose of the roscovitine in the active site

Fig. 5. 3D interaction diagram of the top docking pose of the compound 9f
Fig. 6. 3D interaction diagram of the top docking pose of the compound 9k.

hydrogen bonding with Leu83 through O-carbonyl group and the pre- sence of chloride group in it which significantly increases the activity. In addition, the presence of hydrophobic groups on both sides of the scaffold is favorable to occupy two deep hydrophobic pockets made up of Phe82, Val64 and His84, Gln85 (Fig. 7).
Fig. 7. 3D interaction diagram of the top docking pose of the compound 10c.

3.3.2. In silico ADMET study
Most of the aqueous solubility of compounds is low except com- pound (8c) which had good aqueous solubility level and compounds 9l, 9k, 9g and 10c which are very low but soluble. All compounds showed HIA level 0 which means that these compounds are expected to be well absorbed, except of 10a and 10c which bear the same amine group. Blood brain barrier penetration level of compounds was found to be 1 or 2 which indicated high to medium BBB penetration; except com- pound (8c) showed low level of BBB penetration and compounds 8f, 8l, 9l, 10a and 10c which showed undefined penetration. The CYP2D6 score predicts the inhibitory and non-inhibitory character of the given chemical structure on Cytochrome P450 2D6 enzyme. Seventeen of the compounds with 0 level were predicted as non-inhibitors; hence no drug-drug interactions would be expected upon administration of these compounds.
The PBB level of half of the compounds was > 95%, thus these drugs would exhibit longer t1/2 and hence less frequent drug adminis- tration. Other compounds were varied between level 0 and 1. The he- patotoXicity level of half of compounds was 0 which indicate non-toXic compound, but Further experimental studies are required to determine the hepatotoXic dose levels for compounds with hepatotoXicity level. The calculated parameters from the ADMET study are illustrated in Table 5 in the supplementary part.
4. Conclusion
Thirty-four novel pyrazolo[1,5-a][1,3,5]triazine derivatives 8a-p, 9a-o, and 10a-c were synthesized by fusion via nucleophilic substitu- tion of ethylsulfanyl group of pyrazolo[1,5-a][1,3,5]triazine (4a-c, and 7a-c) with different amines. Compounds 7a, 7b, 8c, 8f-h, 8j-m, 9a, 9f- k, 9m, 10a, 10c were selected by NCI, USA and screened against 60 cancer cell lines. It was found that, Ethyl 2-(benzylamino)-4-(4-chlor- ophenyl)pyrazolo[1,5-a][1,3,5]triazine-8-carboXylate (9f) showed the highest cell growth inhibition with mean growth inhibition percentage of 41.77%, and compound 4-(4-Chlorophenyl)-N-phenethyl-2-(phe- nethylamino)pyrazolo[1,5-a][1,3,5] triazine-8-carboXamide (10c), also exhibited considerable cell growth inhibition with mean growth in- hibition percent of 33.32%. Furthermore, all compounds (4a-c, 7a-c, 8a-p, 9a-o, and 10a-c) were screened for their In-vitro Cyclin depen- dent kinase2 inhibitory activity. 9f was the most active compound followed by 10c which matches the NCI results. In general, the chloro
substituted analogues are the most active, then the bromo and the least are the unsubstituted analogues. Also, the 8-carboXylate compounds have more inhibitory activity than the 8-carbonitrile congeners. Molecular docking study indicated the significant interactions between

compounds 9f and 10c and CDK2 enzyme. Thereby, it could be de- manded that pyrazolo triazine derivatives represented a talented starting point for further study as anticancer drug.
Acknowledgements
The authors are also thankful to NCI, Division of Cancer Treatment & Diagnosis, Developmental Therapeutics Program (DTP) for per- forming the anti-proliferative activity against 60 cancer cell lines.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bioorg.2019.103239.
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