untitled European Journal of Chemistry 8 (4) (2017) 400‐409 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2017 Atlanta Publishing House LLC ‐ All rights reserved ‐ Printed in the USA http://dx.doi.org/10.5155/eurjchem.8.4.400-409.1645 European Journal of Chemistry Journal webpage: www.eurjchem.com Synthesis and antiproliferative activity of 3‐(substituted)‐4,5,6,7‐tetrahydro‐6‐ (substituted)‐1H‐pyrazolo[3,4‐c]pyridine derivatives Chandrakant Pawar 1,*, Dattatraya Pansare 2 and Devanand Shinde 3 1 Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431004, MS, India 2 Department of Chemistry, Deogiri College, Aurangabad, 431005, MS, India 3 Department of Chemistry, Shivaji University, Vidyanagar, Kolhapur, 416004, MS, India * Corresponding author at: Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431004, MS, India. Tel.: +91.0240.2403308. Fax: +91.0240.2400413. E‐mail address: dbschandrakant13@gmail.com (C. Pawar). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.8.4.400-409.1645 Received: 30 August 2017 Received in revised form: 23 September 2017 Accepted: 23 September 2017 Published online: 31 December 2017 Printed: 31 December 2017   A series of new molecules having 3‐(substituted)‐4,5,6,7‐tetrahydro‐6‐(substituted)‐1H‐ pyrazolo[3,4‐c]pyridine and 3‐(substituted)‐5,6‐dihydro‐6‐(substituted)‐1H‐pyrazolo[3,4‐c] pyridin‐7(4H)‐one derivatives were designed and synthesized in large scale (grams range). The structures of the synthesized compounds were elucidated and confirmed by 1H NMR, 13C NMR, Mass spectra; and purity was also checked through LC/MS and HPLC analysis. The antiproliferative activity of the compounds was checked for lung cancer, cervical cancer, breast cancer and prostate cancer on panel of four cell lines. A few compounds (13c, 13g, 15g and 15h) showed promising antiproliferative activity in the range of 5.12‐17.12 µM which were further tested for their inhibitory activity against panel of 8 human kinases at 10 µM concentrations. The compounds 13c, 13g, 15g and 15h shows prominent inhibitory activity against Aurora‐A, Aurora‐B, CDK5/P25 and mTOR kinases. KEYWORDS Pd/C Antiproliferative Aurora‐A kinases 5,6‐Dihydropyrazole Tetrahydropyridine 4,5,6,7‐Tetrahydropyrazole Cite this: Eur. J. Chem. 2017, 8(4), 400‐409 1. Introduction Cancer treatment is difficult due to plethora of unwanted side effects [1]. In present study, we have chosen 4,5,6,7‐ tetrahydro‐1H‐pyrazolo[3,4‐c]pyridine and 5,6‐dihydro‐1H‐ pyrazolo[3,4‐c]pyridin‐7(4H)‐one as core structure as 4,5,6,7‐ tetrahydro‐1H‐pyrazolo[3,4‐c]pyridine is showing diversified biological activities as anticancer activity [2], 5‐HT6 inhibitors for pain treatment [3], inflammatory disorders [4], GnRH receptor antagonists [5], kinase1 inhibitors [6], cannabinoid receptors [7], inhibitor of blood coagulation factor Xa [8], PDE4 inhibitor [9], COX‐2 inhibitors [10], antimicrobial [11] and P13K inhibitors [12]. The pyrazole is known for adenine mimetic pharmacophore and is useful in inhibitors of several classes of kinases like Aurora, CDK‐2 and MAP kinases as these plays key role in drug discovery [2]. The tetrahydro‐1H‐ pyrazole and their derivatives show diversifying activity. By considering their biological importance herein we report the synthesis of 3‐(substituted)‐4,5,6,7‐tetrahydro‐6‐(substitu‐ ted)‐1H‐pyrazolo[3,4‐c]pyridine and 3‐(substituted)‐5,6‐di hydro‐6‐(substituted)‐1H‐pyrazolo[3, 4‐c]pyridin‐7(4H)‐one and anticancer activity in cell line along with kinase inhibition study. We have optimized routes for their synthesis. The synthetic methods adopted for the preparation of the title compounds 13a‐h and 15a‐h are depicted in Schemes 1 and 2 presented below. 2. Experimental 2.1. Reagent and instrumentation All chemicals, unless otherwise specified, were purchased from commercial sources and were used without further purification. The major chemicals were purchased from Sigma Aldrich and Avra Labs. The development of reactions was monitored by thin layer chromatography (TLC) analysis on Merck pre‐coated silica gel 60 F254 aluminum sheets, visualized by UV light. All reactions were carried out under argon inert atmosphere. Melting points were recorded on SRS OptiMelt. The purity of intermediates was pursued by TLC, NMR, and LC‐ MS. All final compounds and intermediates are characterized by NMR, LC‐MS and purity of final compounds pursued by HPLC and all structures are consistent with proposed struc‐ tures characterization. The 1H NMR spectra were recorded on Varian NMR (400 MHz) spectrometer. The 13C NMR spectra were recorded on Varian NMR (100 MHz) spectrometer. Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 401 N Boc O N Boc O N N Boc N N H N Boc N N Boc HN N N Boc HN N N Boc Br HN N N Boc OH N N N Boc OH N N N Boc O O FF F N N N Boc R1 HN N N Boc R1 N N N Boc R1 R2 N N N H R1 R2 1 2 67 3 4 5 8 9 10a-d 11a-d 13a-h 12a-h g b c d a ef h i j k l Reagents and conditions: (a) DMF‐DMA at 100 °C, 1 h; (b) N2H4.H2O, EtOH, 80 °C, 8 h; (c) (BOC)2O, TEA, DCM, room temperature, 3 h; (d) 2 N aq. HCl, room temperature, 2 h; (e) Pyridine Br2, THF, room temperature, 3 h; (f) 2 N NaOH, 100 °C, 3 h (g) benzyl bromide, 2,6‐lutidine, DMAP, THF, room temperature, 8 h; (h) Triflic anhydride, TEA, DCM, room temperature, 6 h; (i) Aromatic boronic acid, Pd2(dba)3, Ruphos, Cs2CO3, toluene, 100 °C, 6 h, (general procedure); (j) Pd/C, H2, MeOH, 50 psi, room temperature, 3 h (general procedure); (k) Alkyl bromide, 2,6‐lutidine, DMAP, THF, room temperature, 8 h (general procedure); (l) 6 N aq. HCl, room temperature, 6 h (general procedure). Scheme 1 The chemical shifts are reported as NMR spectra δppm units. The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Mass spectra were taken using Varian VG 7070 spectrometer at nominal 5000 resolution. The purity of final compounds was determined by HPLC on an Alltech Alltima C18 column (3.2 × 150 mm, 5 µM) eluting with 5‐80% acetonitrile / 45 nM sodium bicarbonate. 2.2. Synthesis 2.2.1. Synthesis of tert‐butyl‐4‐((dimethylamino)methyl ene)‐3‐oxopiperidine‐1‐carboxylate (2), Step (a) To a stirred solution of N‐tert‐butoxycarbonyl‐3‐piperi‐ done (1) (10.0 g, 50.2 mmol) in N,N‐dimethylformamide dimethylacetal (50 mL). The reaction mixture was heated at 100 °C for 1 h. Progress of reaction was monitored by LC/MS for the consumption of starting material. After completion the reaction, the reaction mixture cooled to room temperature and evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was diluted it with H2O (100 mL) and extracted it with EtOAc (2 × 50 mL). The organic layer was washed with H2O (50 mL) and brine (50 mL), dried it over anhydrous Na2SO4 to obtain yellow solid. The crude material was washed with 10% ethyl acetate:hexane (v:v, 10:90, 100 mL), hexane (100 mL) and diethyl ether (100 mL) to obtain compound 2. Color: Yellow. Yield: 78 % (10.0 g). M.p.: 48‐49 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (sS, 9H, t‐Bu), 2.22 (d, 2H, J = 6.4 Hz, CH2), 2.44 (s, 6H, N‐(CH3)2), 2.81 (m, 2H, CH2), 3.81 (s, 2H, NH‐CH2), 6.89 (s, 1H, N‐CH). 13C NMR (100 MHz, CDCl3, δ, ppm): 28.55, 30.32, 43.47, 47.46, 58.89, 81.2, 101.21, 146.12, 154.42, 192.22. LC‐MS (EI, m/z): 255 (M+H). Anal. calcd. for C13H22N2O3: C, 61.39; H, 8.72; N, 11.01. Found: C, 61.36; H, 8.73; N, 11.03%. 2.2.2. Synthesis of 4,5,6,7‐tetrahydro‐1H‐pyrazolo[4,3‐c] pyridine (3), Step (b) To a stirred solution of compound 2 (10.0 g, 39.4 mmol) was dissolved in ethanol (50 mL) and hydrazine hydrate (3.94 g, 78.7 mmol). The reaction mixture was heated at 80 °C for 8 h and the progress of reaction was monitored by LC‐MS for the consumption of starting material. 402 Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 Reagents and conditions: (a) KMnO4, 18‐Crown‐6, DCM, room temperature, 6 h, (general procedure); (b) 6 N HCl, room temperature, 6 h (general procedure). Scheme 2 The reaction mixture was cooled to room temperature and evaporated under reduced pressure to obtain yellow gummy material. The obtained crude material was purified by silica gel (100‐200 mesh) column chromatography by using 10‐40% ethyl acetate:hexane (v:v, 10‐40:90‐60). The obtained compound was washed with diethyl ether (100 mL) to obtain compound 3 [13]. Color: Yellow. Yield: 68.3%, 6 g. M.p.: 74‐75 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.46 (s, 9H, t‐Bu), 2.22 (d, 2H, J = 6.4 Hz, CH2), 2.80 (m, 2H, CH2), 3.84 (s, 2H, NH‐ CH2), 7.31 (s, 1H, Ar‐H), 12.6 (br, 1H, Ar‐NH). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 19.21, 28.88, 38.41, 47.11, 80.2, 114.12, 133.57, 142.26, 152.88. LC‐MS (EI, m/z): 225 (M+H). Anal. calcd. for C11H17N3O2: C, 59.17; H, 7.67; N, 18.82. Found: C, 59.14; H, 7.68; N, 18.80%. 2.2.3. Synthesis of di‐tert‐butyl 4,5‐dihydro‐7H‐pyrazolo [3,4‐c]pyridine‐1,6‐dicarboxylate (4), Step (c) To a stirred solution of compound 3 (5.00 g, 22.4 mmol) in DCM (50 mL), triethylamine (6.00 ml, 44.8 mmol) was added BOC anhydride (7.33 g, 33.6 mmol) and stirred reaction mixture to room temperature for 3 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After completion the reaction, the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was washed with 10% ethyl acetate:hexane (v:v, 10:90) (25 mL), hexane (50 mL) and diethyl ether (50 mL) to obtain compound 4. Color: Yellow. Yield: 91 %, 6.6 g. M.p.: 55‐56 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.56 (s, 18H, N‐(t‐Bu)2), 2.21 (d, 2H, J = 6.2 Hz, CH2), 2.79 (m, 2H, CH2), 3.83 (s, 2H, N‐CH2), 7.31 (s, 1H, Ar‐H). 13C NMR (100 MHz, CDCl3, δ, ppm): 21.92, 28.41, 31.32, 44.38, 77.2, 116.6, 135.23, 143.84, 148.66. LC‐MS (EI, m/z): 325 (M+H). Anal. calcd. for C16H25N3O4: C, 59.42; H, 7.79; N, 12.99. Found: C, 59.40; H, 7.80; N, 12.97%. 2.2.4. Synthesis tert‐butyl 4,5,6,7‐tetrahydropyrazolo[3,4‐c] pyridine‐1‐carboxylate (5), Step (d) To a stirred solution compound 4 (6.50 g, 20.1 mmol) was dissolved in 2 N HCl (65 mL) and stirred reaction mixture to room temperature for 2 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After completion the reaction, the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was washed with 10% ethyl acetate:hexane (v:v,10:90, 25 mL), hexane (50 mL) and diethyl ether (50 mL) to obtain compound 5. Color: Yellow. Yield: 89.3 %, 4 g. M.p.: 61‐62 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.56 (s, 9H, N‐ t‐Bu), 2.21 (d, 2H, J = 6.2 Hz, CH2), 2.78 (m, 2H, CH2), 3.83 (s, 2H, N‐CH2), 7.31 (s, 1H, Ar‐H). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.26, 28.85, 31.52, 44.28, 78.84, 98.62, 134.36, 144.18, 148.77. LC‐MS (EI, m/z): 225 (M+H). Anal. calcd. for C11H17N3O2: C, 59.17; H, 7.67; N, 18.82. Found: C, 59.19; H, 7.64; N, 18.80%. 2.2.5. Synthesis tert‐butyl 3‐bromo‐4,5,6,7‐tetrahydro pyrazolo[3,4‐c]pyridine‐1‐carboxylate (6), Step (e) To a stirred solution compound 5 (4.00 g, 17.9 mmol) in THF (40 mL) was added pyridine hydrobromide (5.74 g, 35.8 mmol) drop wise at 0 °C. The reaction mixture was stirred at room temperature for 3 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After comp‐ letion the reaction, the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was washed with 10% ethyl acetate:hexane (v:v, 10: 90, 25 mL), hexane (50 mL), and diethyl ether (50 mL), to obtain compound 6. Color: Yellow. Yield: 92 %, 5 g. M.p.: 145‐146 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.39 (s, 9H, N‐t‐Bu), 2.49 (d, 2H, J = 6.2 Hz, CH2), 2.88 (m, 2H, CH2), 3.79 (s, 2H, N‐CH2). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.33, 28.41, 30.76, 4312, 78.81, 115.61, 123.10, 139.18, 148.88. LC‐ MS (EI, m/z): 303 (M+H). Anal. calcd. for C11H16BrN3O2: C, 47.72; H, 5.34; N, 13.91. Found: C, 47.73; H, 5.32; N, 13.90%. 2.2.6. Synthesis tert‐butyl 4,5,6,7‐tetrahydro‐3‐hydroxy pyrazolo[3,4‐c]pyridine‐1‐carboxylate (7), Step (f) To a stirred solution compound 6 (5.00 g, 16.5 mmol) in 2 N NaOH (50 mL) and heat reaction mixture to 100 °C for 3 h. Progress of reaction was monitored by LC‐MS for the con‐ sumption of starting material. After completion the reaction, the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was washed with 10% ethyl acetate:hexane (v:v, 10:90, 50 mL), hexane (50 mL) and diethyl ether (50 mL) to obtain compound 7. Color: Yellow. Yield: 88.4 %, 3.5 g. M.p.: 87‐88 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.54 (s, 9H, N‐t‐Bu), 2.20 (d, 2H, J = 6.2 Hz, CH2), 2.78 (m, 2H, CH2), 3.79 (s, 2H, N‐CH2). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.23, 28.41, 31.76, 44.36, 78.88, 99.10, 134.17, 144.16, 147.46. LC‐MS (EI, m/z): 240 (M+H). Anal. calcd. for C11H17N3O3: C, 55.22; H, 7.16; N, 17.56. Found: C, 55.20; H, 7.18; N, 17.58%. 2.2.7. Synthesis of tert‐butyl 6‐benzyl‐4,5,6,7‐tetrahydro‐3‐ hydroxypyrazolo[3,4‐c]pyridine‐1‐carboxylate (8), Step (g) To a stirred solution of compound 7 (3.50 g, 14.6 mmol) in THF (35 mL) was added 2,6‐lutidine (3.14 g, 29.3 mmol), DMAP (0.36 g, 2.93 mmol) and benzyl bromide ( 2.98 g, 16.1 mmol). The reaction mixture was stirred at room temperature for 6 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After completion the reaction, the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was washed with 10% ethyl acetate:hexane (v:v, 10:90, 50 mL), hexane ( 50 mL), and diethyl ether (50 mL), to obtain compound 8. Color: Yellow. Yield: 83 %, 4.5 g. M.p.: 133‐134 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.42 (s, 9H, t‐Bu), 2.34 (d, 2H, J = 6.4 Hz, CH2), 2.68 (m, 2H, CH2), 3.60 (s, 2H, CH2), 3.70 (s, 2H, N‐CH2), 7.38‐7.28 (m, 5H, Ar‐H). 13C NMR (100 MHz, CDCl3, δ, ppm): 9.88, 28.41, 43.44, 52.12, 60.18, Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 403 78.81, 98.67, 127.62, 128.21, 128.34, 128.46, 128.58, 135.11, 135.21, 144.66, 148.78. LC‐MS (EI, m/z): 330 (M+H). Anal. calcd. for C18H23N3O3: C, 65.63; H, 7.04; N, 12.76. Found: C, 65.65; H, 7.01; N, 12.78%. 2.2.8. Synthesis of tert‐butyl 3‐(2,2,2‐trifluoroacetoyloxy)‐6‐ benzyl‐4,5,6,7‐tetrahydropyrazolo[3,4‐c]pyridine‐1‐ carboxylate (9), Step (h) To a stirred solution of synthesis of compound 8 (3.50 g, 10.6 mmol) in DCM (35 mL), triethylamine (2.12 ml, 15.9 mmol) was added triflouromethanesulfonicanhydride (3.60 g, 12.8 mmol) drop wise at 0 °C. The reaction mixture was stirred at room temperature for 12 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After completion the reaction, the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was washed with 10% ethyl acetate:hexane (v:v, 10:90, 50 mL), hexane (50 mL), and diethyl ether (50 mL), to obtain compound 9. Color: Yellow. Yield: 88.5 %, 4 g. M.p.: 173‐174 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.40 (s, 9H, t‐Bu), 2.34 (d, 2H, J = 6.4 Hz, CH2), 2.68 (m, 2H, CH2), 3.60 (s, 2H, CH2), 3.70 (s, 2H, N‐CH2), 7.38‐7.28 (m, 5H, Ar‐H). 13C NMR (100 MHz, CDCl3, δ, ppm): 9.86, 28.41, 43.44, 52.12, 60.18, 78.80, 98.68, 112.13, 127.72, 128.22, 128.32, 128.46, 128.58, 135.13, 135.22, 144.66, 148.78, 168.34. LC‐MS (EI, m/z): 426 (M+H). Anal. calcd. for C20H22F3N3O4: C, 56.47; H, 5.21; N, 9.88. Found: C, 56.44; H, 5.20; N, 9.90%. 2.2.9. General procedure for synthesis of compounds 10a‐d, Step (i) To a stirred solution of compound 9 (1 mmol) in toluene (5 mL) was substituted aromatic boronic acid (2 mmol), 2‐ dicyclohexylphosphino‐2,6‐diisopropoxybiphenyl (0.2 mmol), cesium carbonate (3 mmol) and tris(dibenzylideneacetone) dipalladium(0) (0.2 mmol). The reaction mixture was purged with argon for 10 min and heat reaction mixture to 100 °C for 6 h. Progress of reaction was monitored by LC‐MS. After completion the reaction, the reaction mixture was filtered through a pad of celite, washed with EtOAc (10 mL) and saturated cold sodium chloride solution (2×5 mL) and organic layer was evaporated under reduced pressure to obtain crude gummy material (10a‐d). The obtained crude was purified by silica gel (230‐400 mesh) by using ethyl acetate:heptane (15:85, v:v) to obtain compound 10a‐d. Tert‐butyl 3‐(benzofuran‐2‐yl)‐6‐benzyl‐4, 5, 6, 7‐tetrahydro pyrazolo[3,4‐c]pyridine‐1‐carboxylate (10a): Color: Brown. Yield: 83%. M.p.: 131‐132 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.38 (s, 9 H, t‐Bu), 2.33 (d, 2H, J = 6.4 Hz, CH2), 2.67 (m, 2H, CH2), 3.60 (s, 2H, CH2), 3.71 (s, 2H, N‐CH2), 6.65 (s, 1H, Ar‐ H), 7.13 (m, 1H, Ar‐H), 7.18 (m, 1H, Ar‐H), 7.38‐7.28 (m, 7H, Ar‐H). LC‐MS (EI, m/z): 430 (M+H). Anal. calcd. for C26H27N3O3: C, 72.71; H, 6.34; N, 9.78. Found: C, 72.70; H, 6.36; N, 9.75%. Tert‐butyl 3‐(benzo[b]thiophen‐2‐yl)‐6‐benzyl‐4, 5, 6,7‐tetra hydropyrazolo[3,4‐c]pyridine‐1‐carboxylate (10b): Color: Brown. Yield: 81%. M.p.: 145‐146 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.39 (s, 9H, t‐Bu), 2.33 (d, 2H, J = 6.4 Hz, CH2), 2.68 (m, 2H, CH2), 3.61 (s, 2H, CH2), 3.7 (s, 2H, N‐CH2), 7.25 (s, 1H, Ar‐H), 7.38‐7.28 (m, 7H, Ar‐H), 7.68‐7.81 (m, 2H, Ar‐H). LC‐MS (EI, m/z): 446 (M+H). Anal. calcd. for C26H27 N3O2S: C, 70.09; H, 6.11; N, 9.43. Found: C, 70.06; H, 6.13; N, 9.41%. Tert‐butyl 6‐benzyl‐4,5,6,7‐tetrahydro‐3‐(quinolin‐3‐yl)pyra zolo[3,4‐c]pyridine‐1‐carboxylate (10c): Color: Brown. Yield: 63%. M.p.: 154‐155 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.39 (s, 9H, t‐Bu), 2.35 (d, 2H, J = 6.4 Hz, CH2), 2.67 (m, 2H, CH2), 3.61 (s, 2H, CH2), 3.70 (s, 2H, N‐CH2), 7.38‐7.28 (m, 5H, Ar‐H), 7.66‐7.45 (m, 3H, Ar‐H), 8.19‐8.02 (m, 2H, Ar‐H), 8.78 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 441 (M+H). Anal. calcd. for C27H28N4O2: C, 73.61; H, 6.41; N, 12.72. Found: C, 73.64; H, 6.39; N, 12.74%. Tert‐butyl 6‐benzyl‐4,5,6,7‐tetrahydro‐3‐(quinolin‐5‐yl)pyra zolo[3,4‐c]pyridine‐1‐carboxylate (10d): Color: Brown. Yield: 66%. M.p.: 161‐162 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.39 (s, 9H, t‐Bu), 2.33 (d, 2H, J = 6.4 Hz, CH2), 2.68 (m, 2H, CH2), 3.61 (s, 2H, CH2), 3.70 (s, 2H, N‐CH2), 7.38‐7.21 (m, 5H, Ar‐H), 7.66‐7.45 (m, 3H, Ar‐H), 8.19‐8.02 (m, 2H, Ar‐H), 8.78 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 441 (M+H). Anal. calcd. for C27H28N4O2: C, 73.61; H, 6.41; N, 12.72. Found: C, 73.63; H, 6.42; N, 12.73%. 2.2.10. General procedure for synthesis of compounds (11a‐ d), Step (j) To a stirred solution of compounds 10a‐d (1 mmol) in methanol (10 mL) was added palladium on carbon (10 mol%) and keep the reaction in Parr Shaker apparatus by applying hydrogen gas pressure of 50 psi for 3 h at room temperature. Progress of reaction was monitored by LC‐MS for the con‐ sumption of starting material. After completion the reaction, the reaction mixtures filtered through a pad of celite and obtain filtrate evaporated under reduced pressure to obtain crude semisolid material for compound 11a‐h. The obtained crude was washed with cold pentane and cold diethyl ether to obtain solid compounds 11a‐h. Tert‐butyl 3‐(benzofuran‐2‐yl)‐4, 5, 6, 7‐tetrahydropyrazolo [3,4‐c]pyridine‐1‐carboxylate (11a): Color: Brown. Yield: 87%. M.p.: 115‐116 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.39 (s, 9H, t‐Bu), 2.33 (d, 2H, J = 6.4 Hz, CH2), 2.67 (m, 2H, CH2), 3.60 (s, 2H, N‐CH2), 6.65 (s, 1H, Ar‐H), 7.13 (m, 1H, Ar‐H), 7.18 (m, 1H, Ar‐H), 7.38‐7.28 (m, 2H, Ar‐H). LC‐MS (EI, m/z): 340 (M+H). Anal. calcd. for C19H21N3O3: C, 67.24; H, 6.24; N, 12.38. Found: C, 67.22; H, 6.21; N, 12.39%. Tert‐butyl 3‐(benzo[b]thiophen‐2‐yl)‐4, 5, 6, 7‐tetrahydro pyrazolo[3,4‐c]pyridine‐1‐carboxylate (11b): Color: Yellow. Yield: 90%. M.p.: 121‐122 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.39 (s, 9H, t‐Bu), 2.33 (d, 2H, J = 6.4 Hz, CH2), 2.68 (m, 2H, CH2), 3.61 (s, 2H, N‐CH2), 7.25 (s, 1H, Ar‐H), 7.38‐7.24 (m, 2H, Ar‐H), 7.68‐7.81 (m, 2H, Ar‐H). LC‐MS (EI, m/z): 356 (M+H). Anal. calcd. for C19H21N3O2S: C, 64.20; H, 5.95; N, 11.82. Found: C, 64.18; H, 5.96; N, 11.84%. Tert‐butyl 4,5,6,7‐tetrahydro‐3‐(quinolin‐3‐yl)pyrazolo[3, 4‐ c]pyridine‐1‐carboxylate (11c): Color: Yellow. Yield: 86%. M.p.: 138‐139 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.39 (s, 9H, t‐Bu), 2.35 (2H, d, J = 6.4 Hz, CH2), 2.66 (m, 2H, CH2), 3.61 (s, 2H, N‐CH2), 7.66‐7.45 (m, 3H, Ar‐H), 8.19‐8.02 (m, 2H, Ar‐H), 8.78 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 351 (M+H). Anal. calcd. for C20H22N4O2: C, 68.55; H, 6.33; N, 15.99. Found: C, 68.56; H, 6.34; N, 15.97%. Tert‐butyl 4,5,6,7‐tetrahydro‐3‐(quinolin‐5‐yl)pyrazolo[3, 4‐ c]pyridine‐1‐carboxylate (11d): Color: Yellow. Yield: 84%. M.p.: 140‐141 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.39 (s, 9H, t‐Bu), 2.33 (d, 2H, J = 6.4 Hz, CH2), 2.68 (m, 2H, CH2), 3.61 (s, 2H, N‐CH2), 7.66‐7.45 (m, 3H, Ar‐H), 8.19‐8.02 (m, 2H, Ar‐H), 8.78 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 351 (M+H). Anal. calcd. for C20H22N4O2: C, 68.55; H, 6.33; N, 15.99. Found: C, 68.57; H, 6.32; N, 15.98%. 2.2.11. General procedure for synthesis of compounds (12a‐ h), Step (k) To a stirred solution of compounds 11a‐d (1 mmol) was dissolved in THF (10 mL). Then, added 2,6‐lutidine (2 equiv.), DMAP (0.2 equiv.) and benzyl bromide (1.2 equiv.) and stirred reaction mixture to room temperature for 8 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After completion the reaction the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was washed with 20% ethyl acetate:hexane (v:v, 20:80), hexane and diethyl ether to 404 Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 obtain yellow semisolid compound. Crystallization of crude was done by using pentane and diethyl ether to obtain solid compounds 12a‐h. Tert‐butyl 3‐(benzofuran‐2‐yl)‐4,5, 6, 7‐tetrahydro‐6‐(3‐met hoxyphenyl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (12a): Color: Yellow. Yield: 85 %. M.p.: 134‐135 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.38 (s, 9H, t‐Bu), 2.84 (d, 2H, J = 8Hz, CH2), 3.62 (s, 2H, CH2), 3.78 (s, 3H, O‐CH3), 4.40 (d, 2H, J =7.8 Hz, N‐ CH2), 6.97 (d, 1H, J =7.8 Hz, Ar‐H), 7.18‐7.08 (m, 2H, Ar‐H), 7.41‐7.21 (m, 3H, Ar‐H), 7.46 (t, 1H, J = 7.6 Hz, Ar‐H), 7.74‐7.61 (m, 2H, Ar‐H). LC‐MS (EI, m/z): 446 (M+H). Anal. calcd. for C26H27N3O4: C, 70.09; H, 6.11; N, 9.43. Found: C, 70.07; H, 6.12; N, 9.44%. Tert‐butyl 3‐(benzo[b]thiophen‐2‐yl)‐4, 5, 6, 7‐tetrahydro‐6‐ (3‐methoxyphenyl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (12b): Color: Yellow. Yield: 88 %. M.p.: 144‐145 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.38 (s, 9H, t‐Bu), 2.97‐2.81 (m, 2H, CH2), 3.63 (s, 2H, CH2), 3.76 (s, 3H, O‐CH3), 4.36 (d, 2H, J = 8.4 Hz, N‐CH2), 6.41 (t, 1H, J = 7.6 Hz, Ar‐H), 6.54 (s, 1H, Ar‐H), 6.62 (m, 1H, Ar‐H), 7.18‐7.04 (m, 2H, Ar‐H), 7.32‐7.18 (m, 2H, Ar‐H), 7.73‐7.68 (m, 2H, Ar‐H). LC‐MS (EI, m/z): 462 (M+H). Anal. calcd. for C26H27N3O3S: C, 67.66; H, 5.90; N, 9.10. Found: C, 67.63; H, 5.92; N, 9.12%. Tert‐butyl 4,5,6,7‐tetrahydro‐6‐(3‐methoxyphenyl)‐3‐(quino lin‐3‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (12c): Color: White. Yield: 91%. M.p.: 147‐148 °C. 1H NMR (400 MHz, DMSO‐ d6, δ, ppm): 1.38 (s, 9H, t‐Bu), 2.92 (d, 2H, J = 8.2 Hz, CH2), 3.61 (t, 2H, J = 8.2 Hz, CH2), 3.72 (s, 3H, O‐CH3), 4.42 (s, 2H, N‐CH2), 6.44 (s, 1H, Ar‐H), 6.56 (s, 1H, Ar‐H), 6.64 (d, 1H, J = 7.6 Hz, Ar‐ H), 7.18 (t, 1H, J = 7.8 Hz, Ar‐H), 7.81‐7.55 (dd, 2H, J = 8.4 Hz, 16.8 Hz, Ar‐H), 8.08 (t, 2H, J = 8.2 Hz, Ar‐H), 8.58 (s, 1H, Ar‐H), 9.38 (s, 1H, Ar‐H), 9.20 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 457 (M+H). Anal. calcd. for C27H28N4O3: C, 71.03; H, 6.18; N, 12.27. Found: C, 71.01; H, 6.20; N, 12.26%. Tert‐butyl 4,5,6,7‐tetrahydro‐6‐(3‐methoxyphenyl)‐3‐(quino lin‐5‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (12d): Color: White. Yield: 89%. M.p.: 148‐149 °C. 1H NMR (400 MHz, DMSO‐ d6, δ, ppm): 1.38 (s, 9H, t‐Bu), 3.01 (s, 2H, CH2), 3.61 (s, 2H, CH2), 3.76 (s, 3H, O‐CH3), 4.38 (d, 2H, J = 8 Hz, N‐CH2), 6.38 (t, 1H, J = 7.6 Hz, Ar‐H), 6.60 (s, 1H, Ar‐H), 6.66 (t, 1H, J = 7.6 Hz, Ar‐H), 7.16 (d, 1H, J = 8.8 Hz, Ar‐H), 7.78‐7.58 (m, 2H, Ar‐H), 8.18‐8.01 (m, 2H, Ar‐H), 8.42 (s, 1H, Ar‐H), 9.38 (s, 1H, Ar‐H), 9.20 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 457 (M+H). Anal. calcd. for C27H28N4O3: C, 71.03; H, 6.18; N, 12.27. Found: C, 71.02; H, 6.19; N, 12.26%. Tert‐butyl 3‐(benzofuran‐2‐yl)‐4,5,6,7‐tetrahydro‐6‐(pyrimi din‐2‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (12e): Color: White. Yield: 86%. M.p.: 155‐156 °C. 1H NMR (400 MHz, DMSO‐ d6, δ, ppm): 1.38 (s, 9H, t‐Bu), 2.86 (d, 2H, J = 8Hz, CH2), 4.08 (s, 2H, CH2), 4.96 (d, 2H, J = 7.8 Hz, N‐CH2), 6.60 (s, 1H, Ar‐H), 7.18‐7.02 (d, 1H, Ar‐H), 7.49‐7.20 (m, 2H, Ar‐H), 7.76‐7.10 (m, 2H, Ar‐H), 8.42 (s, 2H, Ar‐H). LC‐MS (EI, m/z): 418 (M+H). Anal. calcd. for C23H23N5O3: C, 66.17; H, 5.55; N, 16.78. Found: C, 66.15; H, 5.53; N, 16.79%. Tert‐butyl 3‐(benzo[b]thiophen‐2‐yl)‐4,5,6,7‐tetrahydro‐6‐ (pyrimidin‐2‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (12f): Color: White. Yield: 84%. M.p.: 154‐155 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.38 (s, 9H, t‐Bu), 3.06 (d, 2H, J = 8.2 Hz, CH2), 4.18 (s, 2H, CH2), 4.96 (d, 2H, J = 7.6 Hz, N‐CH2), 6.56 (s, 1H, Ar‐H), 7.16‐7.02 (d, 1H, Ar‐H), 7.49‐7.14 (m, 2H, Ar‐H), 7.86‐7.54 (m, 2H, Ar‐H), 8.54 (s, 2H, Ar‐H). LC‐MS (EI, m/z): 434 (M+H). Anal. calcd. for C23H23N5O2S: C, 63.72; H, 5.35; N, 16.15. Found: C, 63.71; H, 5.37; N, 16.16%. Tert‐butyl 4,5,6,7‐tetrahydro‐6‐(pyrimidin‐2‐yl)‐3‐(quinolin‐ 3‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (12g): Color: White. Yield: 84%. M.p.: 165‐166 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.38 (s, 9H, t‐Bu), 2.92 (d, 2H, J = 8.4 Hz, CH2), 4.12 (q, 2H, J = 8.4 Hz, CH2), 4.94 (s, 2H, N‐CH2), 6.72 (t, 1H, J = 7.6 Hz, Ar‐H), 7.74 (t, 1H, J = 8 Hz, Ar‐H), 7.84 (t, 1H, J = 8 Hz, Ar‐H), 8.12 (d, 1H, J = 7.8 Hz, Ar‐H), 8.22 (d, 1H, J = 7.8 Hz, Ar‐H), 8.42 (d, 1H, J = 7.6 Hz, Ar‐H), 8.56 (s, 1H, Ar‐H), 9.38‐9.22 (br, 2H, Ar‐H). LC‐MS (EI, m/z): 429 (M+H). Anal. calcd. for C24H24N4O2: C, 67.27; H, 5.65; N, 19.61. Found: C, 67.25; H, 5.64; N, 19.63%. Tert‐butyl 4,5,6,7‐tetrahydro‐6‐(pyrimidin‐2‐yl)‐3‐(quinolin‐ 5‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (12h): Color: Yel‐ low. Yield: 93%. M.p.: 161‐162 °C. 1H NMR (400 MHz, DMSO‐ d6, δ, ppm): 1.38 (s, 9H, t‐Bu), 2.62 (t, 2H, J = 8.2 Hz, CH2), 4.04 (q, 2H, J = 8.2 Hz, CH2), 4.98 (s, 2H, N‐CH2), 6.66 (s, 1H, Ar‐H), 7.44 (br, 1H, Ar‐H), 7.58 (d, 1H, J = 8 Hz, Ar‐H), 7.83‐7.68 (m, 2H, Ar‐H), 8.23‐8.01 (m, 2H, Ar‐H), 8.28 (s, 1H, Ar‐H), 8.92 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 429 (M+H). Anal. calcd. for C24H24N4O2: C, 67.27; H, 5.65; N, 19.61. Found: C, 67.24; H, 5.66; N, 19.64%. 2.2.12. General procedure for synthesis of compound 13a‐h, Step (l) To a stirred solution of compounds 12a‐h (1 mmol) was dissolved in 2 N dioxane in HCl (10 mL) and stirred reaction mixture to room temperature for 6 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After completion the reaction, the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The crude obtained was washed with 10% ethyl acetate:hexane, hexane and diethyl ether to obtain crude 13a‐h as yellow solid material. The crude was purified by column chromatography (silica gel, 230‐400 mesh) by using 25‐75% ethyl acetate and hexane to obtain desired compounds 13a‐h as solid materials. 3‐(Benzofuran‐2‐yl)‐4, 5, 6, 7‐tetrahydro‐6‐(3‐methoxyphen yl)‐1H‐pyrazolo[3,4‐c]pyridine (13a): Color: White. Yield: 91%. M.p.: 155‐156 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.84 (d, 2H, J = 8 Hz, CH2), 3.62 (s, 2H, CH2), 3.78 (s, 3H, O‐CH3), 4.39 (d, 2H, J = 7.8 Hz, N‐CH2), 6.96 (d, 1H, J = 7.8 Hz, Ar‐H), 7.2‐7.12 (m, 2H, Ar‐H), 7.36‐7.21 (m, 3H, Ar‐H), 7.46 (t, 1H, J = 7.6 Hz, Ar‐H), 7.74‐7.63 (m, 2H, Ar‐H), 13.38 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 10.34, 48.55, 51.72, 55.85, 97.56, 103.11, 103.21, 106.81, 111.12, 121.18, 122.34, 123.76, 124.44, 130.84, 135.67, 150.42, 150.67, 155.67, 161.82. LC‐MS (EI, m/z): 345 (M+H). Anal. calcd. for C21H19N3O2: C, 73.03; H, 5.54; N, 12.17. Found: C, 73.05; H, 5.53; N, 12.15%. HPLC: r.t. = 5.68 min, purity = 98.3%. 3‐(Benzo[b]thiophen‐2‐yl)‐4,5, 6, 7‐tetrahydro‐6‐(3‐methoxy phenyl)‐1H‐pyrazolo[3,4‐c]pyridine (13b): Color: Yellow. Yield: 89%. M.p.: 161‐162 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.97‐2.81 (m, 2H, CH2), 3.63 (s, 2H, CH2), 3.76 (s, 3H, O‐CH3), 4.36 (d, 2H, J = 8.4 Hz, N‐CH2), 6.38 (t, 1H, J = 7.6 Hz, Ar‐H), 6.55 (s, 1H, Ar‐H), 6.63 (m, 1H, Ar‐H), 7.18‐7.04 (m, 2H, Ar‐H), 7.32‐7.2 (m, 2H, Ar‐H), 7.73‐7.68 (m, 2H, Ar‐H), 13.4 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 10.24, 48.45, 51.62, 55.85, 97.66, 102.11, 102.21, 106.81, 111.2, 120.18, 122.34, 123.66, 124.44, 130.84, 135.66, 150.32, 150.67, 154.67, 161.72. LC‐MS (EI, m/z): 361 (M+H). Anal. calcd. for C21H19N3OS: C, 69.78; H, 5.30; N, 11.63. Found: C, 69.77; H, 5.32; N, 11.61%. HPLC: r.t. = 9.21 min, purity = 99.5%. 3‐(4, 5, 6,7‐Tetrahydro‐6‐(3‐methoxyphenyl)‐1H‐pyrazolo[3, 4‐c]pyridin‐3‐yl)quinolone (13c): Color: Yellow. Yield: 80%. M.p.: 179‐180 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.92 (d, 2H, J = 8.2 Hz, CH2), 3.61 (t, 2H, J = 8.2 Hz, CH2), 3.72 (s, 3H, O‐CH3), 4.42 (s, 2H, N‐CH2), 6.34 (s, 1H, Ar‐H), 6.56 (s, 1H, Ar‐ H), 6.64 (d, 1H, J = 7.6 Hz, Ar‐H), 7.16 (t, 1H, J = 7.8 Hz, Ar‐H), 7.81‐7.59 (dd, 2H, J = 8.4 Hz, 16.8 Hz, Ar‐H), 8.08 (t, 2H, J = 8.2 Hz, Ar‐H), 8.56 (s, 1H, Ar‐H), 9.38 (s, 1H, Ar‐H), 13.26 (br. s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.72, 49.24, 52.46, 55.46, 97.8, 103.21, 106.67, 127.34, 127.84, 128.22, 128.42, 128.88, 129.42, 129.41, 130.71, 130.85, 145.21, 148.73, 150.65, 162.10. LC‐MS (EI, m/z): 357 (M+H). Anal. calcd. for C22H20N4O: C, 74.14; H, 5.66; N, 15.72. Found: C, 74.17; H, 5.65; N, 15.71%. HPLC: r.t. = 4.89 min, purity = 96.6%. 5‐(4,5,6,7‐Tetrahydro‐6‐(3‐methoxyphenyl)‐1H‐pyrazolo [3, 4‐c]pyridin‐3‐yl)quinolone (13d): Color: Off white. Yield: 86%. M.p.: 184‐185 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.01 (s, 2H, CH2), 3.61 (s, 2H, CH2), 3.76 (s, 3H, O‐CH3), 4.39 (d, 2H, J Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 405 = 8 Hz, N‐CH2), 6.38 (t, 1H, J = 7.6 Hz, Ar‐H), 6.58 (s, 1H, Ar‐H), 6.65 (t, 1H, J = 7.6 Hz, Ar‐H), 7.16 (d, 1H, J = 8.8 Hz, Ar‐H), 7.8‐ 7.58 (m, 2H, Ar‐H), 8.18‐8.01 (m, 2H, Ar‐H), 8.43 (s, 1H, Ar‐H), 9.38 & 9.20 (s, 1H, Ar‐H), 13.2 (br, 1H, NH, 1H). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.62, 49.34, 52.56, 54.46, 97.86, 102.21, 106.77, 127.44, 127.84, 128.32, 128.42, 128.88, 129.42, 129.40, 130.60, 130.85, 145.31, 148.63, 151.14, 162.60. LC‐MS (EI, m/z): 357 (M+H). Anal. calcd. for C22H20N4O: C, 74.14; H, 5.66; N, 15.72. Found: C, 74.11; H, 5.69; N, 15.71%. HPLC: r.t. = 5.09 min, purity = 98.0%. 3‐(Benzofuran‐2‐yl)‐4, 5, 6, 7‐tetrahydro‐6‐(pyrimidin‐2‐yl)‐ 1H‐pyrazolo[3,4‐c]pyridine (13e): Color: Yellow. Yield: 88%. M.p.: 167‐168 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.86 (d, 2H, J = 8Hz, CH2), 4.08 (s, 2H, CH2), 4.96 (d, 2H, J = 7.8 Hz, N‐ CH2), 6.65 (s, 1H, Ar‐H), 7.16‐7.02 (d, 1H, Ar‐H), 7.49‐7.21 (m, 2H, Ar‐H), 7.76‐7.6 (m, 2H, Ar‐H), 8.42 (s, 2H, Ar‐H), 13.4 & 12.9 (br. s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 10.41, 48.67, 51.72, 102.82, 103.03, 110.62, 111.67, 121.12, 123.46, 123.86, 135.40, 144.20, 150.66, 162.84, 175.88. LC‐MS (EI, m/z): 318 (M+H). Anal. calcd. for C18H15N5O: C, 68.13; H, 4.46; N, 22.07. Found: C, 68.11; H, 4.48; N, 22.09%. HPLC: r.t. = 7.58 min, purity = 93.7%. 3‐(Benzo[b]thiophen‐2‐yl)‐4, 5, 6,7‐tetrahydro‐6‐(pyrimidin‐ 2‐yl)‐1H‐pyrazolo[3,4‐c]pyridine (13f): Color: Yellow. Yield: 91%. M.p.: 197‐198 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.06 (d, 2H, J = 8.2 Hz, CH2), 4.18 (s, 2H, CH2), 4.96 (d, 2H, J = 7.6 Hz, N‐CH2), 6.66 (s, 1H, Ar‐H), 7.16‐7.04 (d, 1H, Ar‐H), 7.49‐ 7.17 (m, 2H, Ar‐H), 7.86‐7.59 (m, 2H, Ar‐H), 8.52 (s, 2H, Ar‐H), 13.4 & 12.0 (br. s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 10.40, 48.66, 51.72, 102.72, 103.30, 110.62, 111.67, 121.22, 123.46, 123.72, 135.48, 144.20, 148.66, 162.84, 174.78. LC‐MS (EI, m/z): 334 (M+H). Anal. calcd. for C18H15N5S: C, 64.84; H, 4.53; N, 21.01. Found: C, 64.86; H, 4.51; N, 21.03%. HPLC: r.t. = 5.84 min, purity = 97.5%. 3‐(4,5,6,7‐Tetrahydro‐6‐(pyrimidin‐2‐yl)‐1H‐pyrazolo[3, 4‐ c]pyridin‐3‐yl)quinolone (13g): Color: White. Yield: 76 %. M.p.: 185‐186 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.92 (d, 2H, J = 8.4 Hz, CH2), 4.12 (q, 2H, J = 8.4 Hz, CH2), 4.94 (s, 2H, N‐ CH2), 6.7 (t, 1H, J = 7.6 Hz, Ar‐H), 7.74 (t, 1H, J = 8 Hz, Ar‐H), 7.84 (t, 1H, J = 8 Hz, Ar‐H), 8.14 (d, 1H, J = 7.8 Hz, Ar‐H), 8.22 (d, 1H, J = 7.8 Hz, Ar‐H), 8.42 (d, 1H, J = 7.6 Hz, Ar‐H), 8.58 (s, 1H, Ar‐H), 9.38‐9.22 (br, 2H, Ar‐H),13.2 & 12.8 (br. s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.76, 49.12, 52.46, 103.24, 110.36, 128.62, 128.88, 129.42, 129.51, 129.62, 130.41, 134.83, 144.42, 145.12, 148.80, 157.90, 162.78. LC‐MS (EI, m/z): 329 (M+H). Anal. calcd. for C19H16N6: C, 69.50; H, 4.91; N, 25.59. Found: C, 69.52; H, 4.93; N, 25.60%. HPLC: r.t. = 6.59 min, purity = 95.8%. 5‐(4,5,6,7‐Tetrahydro‐6‐(pyrimidin‐2‐yl)‐1H‐pyrazolo[3, 4‐ c]pyridin‐3‐yl)quinolone (13h): Color: White. Yield: 88 %. M.p.: 188‐189 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.62 (t, 2H, J = 8.2 Hz, CH2), 4.04 (q, 2H, J = 8.2 Hz, CH2), 4.98 (s, 2H, N‐CH2), 6.63 (s, 1H, Ar‐H), 7.44 (br, 1H, Ar‐H), 7.68 (d, 1H, J = 8 Hz, Ar‐ H), 7.83‐7.68 (m, 2H, Ar‐H), 8.23‐8.01 (m, 2H, Ar‐H), 8.40 (s, 1H, Ar‐H), 8.92 (s, 1H, Ar‐H), 13.2 (br. s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 15.55, 49.33, 53.66, 110.21, 116.68, 119.7, 121.22, 125.58, 127.79, 129.10, 130.00, 130.42, 136.46, 137.12, 147.45, 150.51, 159.12, 162.84. LC‐MS (EI, m/z): 329 (M+H). Anal. calcd. for C19H16N6: C, 69.50; H, 4.91; N, 25.59. Found: C, 69.51; H, 4.90; N, 25.59%. HPLC: r.t. = 4,78 min, purity = 99.2%. 2.2.13. General procedure for synthesis of compounds 14a‐ h, Step (a) To a stirred solution of compounds 12a‐h (1 mmol) was dissolved in DCM (10 mL). Then added KMnO4 (2 equiv.) and 18‐crown‐6 (0.5 equiv.) and stirred reaction mixture to room temperature for 6 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After comp‐ letion the reaction, the reaction mixture was diluted with DCM (10 mL) and washed it with water (3×5 mL). Separated and collected the organic layer, washed it with 5 mL of brine and dried organic layer over anhydrous Na2SO4 and evaporated it under reduced pressure to obtain crude compounds 14a‐h as semisolid compound. The crude obtained was washed with 5% ethyl acetate: hexane, hexane and diethyl ether to obtain yellow semisolid compound. The obtained compound was crystallized by using cold pentane and cold diethyl ether to obtain solid compounds 14a‐h. Tert‐butyl 3‐(benzofuran‐2‐yl)‐4,5,6,7‐tetrahydro‐6‐(3‐met hoxyphenyl)‐7‐oxopyrazolo[3,4‐c]pyridine‐1‐carboxylate (14a): Color: Brown. Yield: 75 %. M.p.: 165‐166 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (s, 9H, t‐Bu), 3.21 (t, 2H, J = 7.6 Hz, 2H, CH2), 3.31 (s, 3H, O‐CH3), 4.18 (d, 2H, J = 7.6 Hz, N‐CH2), 6.8 (d, 1H, J = 7.6 Hz, Ar‐H), 6.87‐6.79 (m, 2H, Ar‐H), 7.44‐7.21 (m 4H, Ar‐H), 7.76‐7.6 (m, 2H, Ar‐H). LC‐MS (EI, m/z): 460 (M+H). Anal. calcd. for C26H25N3O5: C, 67.96; H, 5.48; N, 9.14. Found: C, 67.98; H, 5.47; N, 9.15%. Tert‐butyl 3‐(benzo[b]thiophen‐2‐yl)‐4, 5, 6, 7‐tetrahydro‐6‐ (3‐methoxyphenyl)‐7‐oxopyrazolo[3,4‐c]pyridine‐1‐carboxylate (14b): Color: Brown. Yield: 77%. M.p.: 171‐172 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (s, 9H, t‐Bu), 3.40 (t, 2H, J = 8.4 Hz, CH2), 3.74 (s, 3H, O‐CH3), 4.24 (d, 2H, J = 6.2 Hz, N‐CH2), 6.38 (d, 1H, J = 7.6 Hz, Ar‐H), 6.54 (s, 1H, Ar‐H), 6.63‐6.57 (m, 2H, Ar‐H), 7.2‐7.11 (m, 3H, Ar‐H), 7.56‐7.44 (dd, 2H, J = 8.2 Hz, 4.1 Hz, Ar‐H). LC‐MS (EI, m/z): 476 (M+H). Anal. calcd. for C26H25N3O4S: C, 65.67; H, 5.30; N, 8.84. Found: C, 65.65; H, 5.31; N, 8.83%. Tert‐butyl 4, 5, 6, 7‐tetrahydro‐6‐(3‐methoxyphenyl)‐7‐oxo‐ 3‐(quinolin‐3‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (14c): Color: Brown. Yield: 78%. M.p.: 181‐182 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (s, 9H, t‐Bu), 3.21 (br s, 2H, CH2), 3.83 (s, 3H, O‐CH3), 4.16 (s, 2H, N‐CH2), 6.93 (d, 1H, J = 7.8 Hz, Ar‐ H), 7.12 (d, 1H, J = 8 Hz, Ar‐H), 7.18 (s, 1H, Ar‐H), 7.42 (m, 1H, Ar‐H), 7.58 (t, 1H, J = 7.2 Hz, Ar‐H), 7.72 (t, 1H, J = 7.2 Hz, Ar‐ H), 7.84 (d, 1H, J = 7.6 Hz, Ar‐H), 8.14 (d, 1H, J = 7.6 Hz, Ar‐H), 8.56 (s, 1H, Ar‐H), 9.41 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 471 (M+H). Anal. calcd. for C27H26N4O4: C, 68.92; H, 5.57; N, 11.91. Found: C, 68.91; H, 5.55; N, 11.92%. Tert‐butyl 4, 5, 6, 7‐tetrahydro‐6‐(3‐methoxyphenyl)‐7‐oxo‐ 3‐(quinolin‐5‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (14d): Color: Brown. Yield: 76%. M.p.: 188‐189 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (s, 9H, t‐Bu), 3.75 (s, 3H, O‐CH3), 3.80 (d, 2H, J = 7.6 Hz, CH2), 4.38 (d, 2H, J = 7.8 Hz, N‐CH2), 6.34 (t, 1H, J = 7.6 Hz, Ar‐H), 6.68 (s, 1H, Ar‐H), 6.75 (t, 1H, J = 7.8 Hz, Ar‐H), 7.14 (m, 1H, Ar‐H), 7.88 (m, 1H, Ar‐H), 8.14‐7.98 (m, 3H, Ar‐H), 8.37‐8.33 (d, 1H, J = 7.8 Hz, Ar‐H), 9.16 (s, 1H, Ar‐H). LC‐ MS (EI, m/z): 471 (M+H). Anal. calcd. for C27H26N4O4: C, 68.92; H, 5.57; N, 11.91. Found: C, 68.93; H, 5.55; N, 11.92%. Tert‐butyl 3‐(benzofuran‐2‐yl)‐4, 5, 6, 7‐tetrahydro‐7‐oxo‐6‐ (pyrimidin‐2‐yl)pyrazolo[3,4‐c]pyridine‐1‐carboxylate (14e): Color: White. Yield: 77%. M.p.: 163‐164 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (s, 9H, t‐Bu), 3.22 (s, 2H, CH2), 4.24 (d, 2H, J = 8.2 Hz, N‐CH2), 7.41‐7.16 (m 4H, Ar‐H), 7.68‐7.54 (m, 2H, Ar‐H), 8.84 (d, 2H, J = 7.8 Hz, Ar‐H). LC‐MS (EI, m/z): 431 (M+H). Anal. calcd. for C23H21N5O4: C, 64.03; H, 4.91; N, 16.23. Found: C, 64.02; H, 4.94; N, 16.25%. Tert‐butyl 3‐(benzo[b]thiophen‐2‐yl)‐4, 5, 6, 7‐tetrahydro‐7‐ oxo‐6‐(pyrimidin‐2‐yl)pyrazolo[3, 4‐c]pyridine‐1‐carboxylate (14f): Color: Brown. Yield: 71 %. M.p.: 155‐156 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (s, 9H, t‐Bu), 3.48 (s, 2H, CH2), 4.44 (d, 2H, J = 8.2 Hz, N‐CH2), 7.48‐7.16 (m 4H, Ar‐H), 7.78‐7.58 (m, 2H, Ar‐H), 8.84 (d, 2H, J = 7.6 Hz, Ar‐H). LC‐MS (EI, m/z): 448 (M+H). Anal. calcd. for C23H21N5O3S: C, 61.73; H, 4.73; N, 15.65. Found: C, 61.72; H, 4.75; N, 15.63%. Tert‐butyl 4, 5, 6, 7‐tetrahydro‐7‐oxo‐6‐(pyrimidin‐2‐yl)‐3‐ (quinolin‐3‐yl)pyrazolo[3, 4‐c]pyridine‐1‐carboxylate (14g): Color: Brown. Yield: 76 %. M.p.: 161‐162 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (s, 9H, t‐Bu), 3.26 (q, 2H, J = 7.6 Hz, CH2), 4.26 (t, 2H, J = 7.6 Hz, N‐CH2), 7.28 (t, 1H, J = 7.6 Hz, Ar‐H), 7.56 (q, 1H, J = 8.2 Hz, Ar‐H), 7.82 (t, 1H, J = 8.2 Hz, Ar‐ 406 Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 H), 8.18‐8.02 (q, 2H, Ar‐H), 8.60 (s, 1H, Ar‐H), 8.83 (d, 2H, Ar‐ H), 9.38 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 443 (M+H). Anal. calcd. for C24H22N6O3: C, 65.15; H, 5.01; N, 18.99. Found: C, 65.14; H, 5.03; N, 18.98%. Tert‐butyl 4, 5, 6, 7‐tetrahydro‐7‐oxo‐6‐(pyrimidin‐2‐yl)‐3‐ (quinolin‐5‐yl)pyrazolo[3, 4‐c]pyridine‐1‐carboxylate (14h): Color: Brown. Yield: 73%. M.p.: 164‐165 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.41 (s, 9H, t‐Bu), 2.8 (q, 2H, CH2), 4.2 (q, 2H, N‐CH2), 7.26 (t, 1H, J = 7.2 Hz, Ar‐H), 7.42 (d, 1H, J = 7.4 Hz, Ar‐H), 7.64 (d, 1H, J = 8 Hz, Ar‐H), 7.82 (t, 1H, J = 7.6 Hz, Ar‐H), 8.12 (d, 2H, J = 7.4 Hz, Ar‐H), 8.8 (d, 2H, J = 7.6 Hz, Ar‐H), 8.96 (s, 1H, Ar‐H). LC‐MS (EI, m/z): 443 (M+H). Anal. calcd. for C24H22N6O3: C, 65.15; H, 5.01; N, 18.99. Found: C, 65.13; H, 5.02; N, 18.97%. 2.2.14. General procedure for synthesis of compounds 15a‐ h, Step (b) To a stirred solution of compounds 14a‐h (1 mmol) was dissolved in 2 N dioxane in HCl (10 mL) and stirred reaction mixture to room temperature for 6 h. Progress of reaction was monitored by LC‐MS for the consumption of starting material. After completion the reaction, the reaction mixture evaporated under reduced pressure to obtain yellow gummy material. The obtained crude was washed with 10% ethyl acetate: hexane, hexane and diethyl ether to obtain crude 15a‐h as yellow solid material. The obtained compound was purified by column chromatography by using silica gel (230‐400 mesh) by using 25‐75% ethyl acetate and hexane to obtain desired compound 15a‐h as solid materials. 3‐(Benzofuran‐2‐yl)‐5,6‐dihydro‐6‐(3‐methoxyphenyl)‐1H‐ pyrazolo[3,4‐c]pyridin‐7(4H)‐one (15a): Color: White. Yield: 87%. M.p.: 191‐192 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.21 (t, 2H, J = 7.6 Hz, CH2), 3.31 (s, 3H, O‐CH3), 4.18 (d, 2H, J = 7.6 Hz, N‐CH2), 6.82 (d, 1H, J = 7.6 Hz, Ar‐H), 6.89‐6.79 (m, 2H, Ar‐H), 7.40‐7.21 (m 4H, Ar‐H), 7.76‐7.60 (m, 2H, Ar‐H), 14.4 (br, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 10.64, 51.82, 55.85, 97.46, 103.31, 103.61, 106.61, 111.02, 121.18, 122.34, 123.76, 124.44, 128.84, 134.67, 150.42, 150.67, 156.61, 156.88, 161.72. LC‐MS (EI, m/z): 360 (M+H). Anal. calcd. for C21H17N3O3: C, 70.18; H, 4.77; N, 11.69. Found: C, 70.16; H, 4.74; N, 11.70%. HPLC: r.t. = 6.18 min, purity = 99.3%. 3‐(Benzo[b]thiophen‐2‐yl)‐5, 6‐dihydro‐6‐(3‐methoxyphen yl)‐1H‐pyrazolo[3,4‐c]pyridin‐7(4H)‐one (15b): Color: Yellow. Yield: 88%. M.p.: 188‐189 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.40 (t, 2H, J = 8.4 Hz, CH2), 3.74 (s, 3H, O‐CH3), 4.24 (d, 2H, J = 6.2 Hz, N‐CH2), 6.38 (d, 1H, J = 7.6 Hz, Ar‐H), 6.50 (s, 1H, Ar‐H), 6.63‐6.57 (m, 2H, Ar‐H), 7.21‐7.11 (m, 3H, Ar‐H), 7.56‐ 7.44 (dd, 2H, J = 8.2 Hz, 4.1Hz, Ar‐H) 14.4 (br, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 13.54, 52.82, 55.65, 98.46, 104.31, 104.61, 108.66, 111.12, 121.18, 122.34, 123.76, 124.44, 128.84, 134.67, 150.42, 150.67, 156.8, 157.00, 161.73. LC‐MS (EI, m/z): 375 (M+H). Anal. calcd. for C21H17N3O2S: C, 67.18; H, 4.56; N, 11.19. Found: C, 67.16; H, 4.58; N, 11.18%. HPLC: r.t. = 7.63 min, purity = 99.7%. 5,6‐Dihydro‐6‐(3‐methoxyphenyl)‐3‐(quinolin‐3‐yl)‐1H‐pyra zolo[3,4‐c]pyridin‐7(4H)‐one (15c): Color: Off white. Yield: 91%. M.p.: 193‐194 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.21 (br s, 2H, CH2), 3.83 (s, 3H, O‐CH3), 4.16 (s, 2H, N‐CH2), 6.93 (d, 1H, J = 7.8 Hz, Ar‐H), 7.12 (d, 1H, J = 8 Hz, Ar‐H), 7.18 (s, 1H, Ar‐H), 7.40 (m, 1H, Ar‐H), 7.58 (t, 1H, J = 7.2 Hz, Ar‐H), 7.73 (t, 1H, J = 7.2 Hz, Ar‐H), 7.84 (d, 1H, J = 7.6 Hz, Ar‐H), 8.15 (d, 1H, J = 7.6 Hz, Ar‐H), 8.56 (s, 1H, Ar‐H), 9.43 (s, 1H, Ar‐H), 14.8 (br, 1H, NH). 13C NMR (CDCl3, 100 MHZ): 12.22, 49.44, 52.46, 55.46, 97.80, 102.21, 106.60, 127.34, 127.84, 128.22, 128.42, 128.88, 129.62, 129.41, 130.71, 130.85, 145.21, 148.63, 150.60, 168.16. LC‐MS (EI, m/z): 371 (M+H). Anal. calcd. for C22H18N4O2: C, 71.34; H, 4.90; N, 15.13. Found: C, 71.32; H, 4.91; N, 15.14%. HPLC: r.t. = 7.20 min, purity = 99.3%. 5, 6‐Dihydro‐6‐(3‐methoxyphenyl)‐3‐(quinolin‐5‐yl)‐1H‐ pyrazolo[3,4‐c]pyridin‐7(4H)‐one (15d): Color: Off white. Yield: 92%. M.p.: 206‐207 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.75 (s, 3H, O‐CH3), 3.80 (d, 2H, J = 7.6 Hz, CH2), 4.38 (d, 2H, J = 7.8 Hz, N‐CH2), 6.38 (t, 1H, J = 7.6 Hz, Ar‐H), 6.58 (s, 1H, Ar‐H), 6.65 (t, 1H, J = 7.8 Hz, Ar‐H), 7.14 (m, 1H, Ar‐H), 7.88 (m, 1H, Ar‐H), 8.14‐7.98 (m, 3H, Ar‐H), 8.37‐8.33 (d, 1H, J = 7.8 Hz, Ar‐ H), 9.16 (s, 1H, Ar‐H), 12.9 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.62, 49.44, 52.56, 54.46, 97.76, 102.31, 106.76, 127.44, 127.84, 128.32, 128.42, 128.88, 129.42, 129.40, 130.60, 130.85, 145.31, 148.63, 152.14, 168.60. LC‐MS (EI, m/z): 371 (M+H). Anal. calcd. for C22H18N4O2: C, 71.34; H, 4.90; N, 15.13. Found: C, 71.31; H, 4.92; N, 15.11%. HPLC: r.t. = 7.81 min, purity = 96.1%. 3‐(Benzofuran‐2‐yl)‐5,6‐dihydro‐6‐(pyrimidin‐2‐yl)‐1H‐pyra zolo[3,4‐c]pyridin‐7(4H)‐one (15e): Color: Yellow. Yield: 79%. M.p.: 176‐177 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.22 (q, 2H, CH2), 4.24 (d, 2H, J = 8.2 Hz, N‐CH2), 7.41‐7.16 (m 4H, Ar‐H), 7.78‐7.61 (m, 2H, Ar‐H), 8.84 (d, 2H, J = 7.8 Hz, Ar‐H), 14.4 (br, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 10.24, 43.92, 102.84, 110.36, 111.62, 116.21, 121.26, 123.36, 124.82, 133.11, 133.26, 150.45, 156.46, 158.10, 169.32. LC‐MS (EI, m/z): 332 (M+H). Anal. calcd. for C18H13N5O2: C, 65.25; H, 3.95; N, 21.14. Found: C, 65.23; H, 3.97; N, 21.15%. HPLC: r.t. = 5.17 min, purity = 99.6%. 3‐(Benzo[b]thiophen‐2‐yl)‐5, 6‐dihydro‐6‐(pyrimidin‐2‐yl)‐ 1H‐pyrazolo[3,4‐c]pyridin‐7(4H)‐one (15f): Color: Yellow. Yield: 84%. M.p.: 202‐203 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.48 (q, 2H, CH2), 4.44 (d, 2H, J = 8.2 Hz, N‐CH2), 7.48‐ 7.16 (m 4H, Ar‐H), 7.78‐7.38 (m, 2H, Ar‐H), 8.84 (d, 2H, J = 7.6 Hz, Ar‐H), 14.3 (br, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 10.24, 43.92, 102.84, 110.36, 111.62, 116.21, 121.26, 123.36, 124.82, 133.11, 133.26, 150.45, 156.46, 158.10, 169.32. LC‐MS (EI, m/z): 347 (M+H). Anal. calcd. for C18H13N5OS: C, 62.23; H, 3.77; N, 20.16. Found: C, 62.21; H, 3.76; N, 20.18%. HPLC: r.t. = 8.12 min, purity = 98.1%. 5, 6‐Dihydro‐6‐(pyrimidin‐2‐yl)‐3‐(quinolin‐3‐yl)‐1H‐pyra zolo[3,4‐c]pyridin‐7(4H)‐one (15g): Color: White. Yield: 85%. M.p.: 211‐212 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.26 (q, 2H, J= 7.6 Hz, CH2), 4.26 (t, 2H, J = 7.6 Hz, N‐CH2), 7.38 (t, 1H, J = 7.6 Hz, Ar‐H), 7.6 (q, 1H, J = 8.2 Hz, Ar‐H), 7.80 (t, 1H, J = 8.2 Hz, Ar‐H), 8.18‐8.02 (q, 2H, Ar‐H), 8.6(s, 1H, Ar‐H), 8.83 (d, 2H, J = Hz, Ar‐H), 9.38 (s, 1H, Ar‐H), 14.1 (br. s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.53, 44.57, 69.36, 110.31, 116.12, 127, 127.11, 128.45, 128.85, 129.11, 129.34, 130.17, 132.23, 134.68, 157.88, 158.12. LC‐MS (EI, m/z): 343 (M+H). Anal. calcd. for C19H14N6O: C, 66.66; H, 4.12; N, 24.55. Found: C, 66.67; H, 4.10; N, 24.57%. HPLC: r.t. = 8.42 min, purity = 98.0%. 5, 6‐Dihydro‐6‐(pyrimidin‐2‐yl)‐3‐(quinolin‐5‐yl)‐1H‐pyra zolo[3,4‐c]pyridin‐7(4H)‐one (15h): Color: White. Yield: 80%. M.p.: 209‐210 °C. 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.80 (q, 2H, CH2), 4.20 (q, 2H, N‐CH2), 7.36 (t, 1H, J = 7.2 Hz, Ar‐H), 7.40 (d, 1H, J = 7.4 Hz, Ar‐H), 7.64 (d, 2H, J = 8 Hz, Ar‐H), 7.81 (t, 1H, J = 7.6 Hz, Ar‐H), 8.12 (d, 1H, J = 7.4 Hz, Ar‐H), 8.80 (d, 2H, J = 7.6 Hz, Ar‐H), 8.96 (s, 1H, Ar‐H), 13.98‐14.2 (br. s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 15.35, 45.33, 110.20, 116.78, 119.47, 121.20, 125.38, 127.29, 129.31, 130.00, 130.12, 136.46, 137.12, 147.45, 150.51, 156.60, 159.12, 162.84. LC‐MS (EI, m/z): 343 (M+H). Anal. calcd. for C19H14 N6O: C, 66.66; H, 4.12; N, 24.55. Found: C, 66.67; H, 4.11; N, 24.54%. HPLC: r.t. = 6.37 min, purity = 99.3%. 2.3. Biological evaluation All the synthesized compounds were tested for their in vitro anticancer activity against various cancer cell lines. Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 407 Table 1. In vitro anticancer screening of the synthesized compounds against five cell lines. Compound A‐549 a Si f HeLa b Si f MCF‐7 c Si f DU‐145 d Si f HUVEC e 13a 22.72±0.11 4.04 23.87±0.08 3.84 24.12±0.06 3.80 28.86±0.22 3.18 91.8±0.28 13b 15.81±0.11 5.13 14.32±0.04 6.74 26.32±0.06 3.67 33.73±0.12 2.86 96.6±0.14 13c 6.81±0.11 12.73 11.32±0.04 7.65 17.32±0.06 5.00 10.73±0.12 8.07 86.6±0.28 13d 10.65±0.11 8.41 18.79±0.22 4.76 16.86±0.12 5.31 20.82±0.11 4.30 89.6±0.28 13e 13.86±0.08 6.39 24.38±0.06 3.63 13.63±0.12 6.50 11.52±0.22 7.69 88.7±0.12 13f 15.72±0.11 6.08 26.87±0.08 3.55 24.12±0.06 3.96 38.86±0.22 2.46 95.6±0.28 13g 5.12±0.11 17.42 9.12±0.22 9.78 9.36±0.12 9.52 13.52±0.11 6.59 89.2±0.28 13h 13.25±0.14 6.53 17.78±0.08 4.87 13.82±0.08 6.26 11.72±0.06 7.38 86.6±0.19 15a 10.82±0.11 8.69 13.39±0.22 7.02 11.36±0.12 8.28 9.52±0.11 9.88 94.1±0.26 15b 14.13±0.12 6.18 15.16±0.08 5.76 16.12±0.12 5.42 11.62±0.11 7.52 87.4±0.22 15c 23.86±0.08 3.24 14.38±0.06 6.50 20.63±0.12 4.53 11.52±0.22 8.12 93.6±0.12 15d 11.72±0.11 7.81 8.87±0.08 10.32 13.12±0.06 6.98 18.86±0.22 4.85 91.6±0.28 15e 23.82±0.11 3.55 20.99±0.22 4.03 19.36±0.12 4.36 12.52±0.11 6.75 84.6±0.28 15f 10.78±0.14 8.12 18.78±0.08 4.66 14.82±0.08 5.91 18.72±0.06 4.67 87.6±0.19 15g 10.82±0.11 8.78 8.59±0.22 11.07 8.36±0.12 11.37 17.52±0.11 5.42 95.1±0.26 15h 9.13±0.12 9.81 14.16±0.08 6.32 6.12±0.12 14.17 11.62±0.11 7.71 89.6±0.22 Doxil 1.71±0.11 51.57 1.82±0.13 48.46 1.91±0.08 46.17 1.62±0.08 54.44 88.2±0.18 a A‐549: Human lung cancer cell line. b HeLa: Human cervical cancer cell line (ATCC CCL‐2). c MCF‐7: Human breast cancer cell line. d DU‐145: Human prostate cancer cell line. e HUVEC: Human umbilical vein endothelial cell line (ATCC CRL‐1730). f Selectivity Index (SI) = IC50 of pure compound in normal cell line/IC50 of same compound in cancer cell line. IC50 ‐ The concentration required to inhibit 50% of cell population. The anticancer activity test is performed according to the proce‐dure developed by the National Cancer Institute (NCI, USA) in the ‘In vitro Anticancer Drug Discovery Screen’ that uses the protein‐binding dye Sulforhodamine B (SRB) to assess cell growth [14,15]. Briefly, cells are grown in 96‐well plates in suspension and then exposed for 48 hours to four serial concentrations of 1×10‐7, 1×10‐6, 1×10‐5, 1×10‐4 and 1×10‐3 M of each compound. Cells were fixed and stained with protein binding SRB stain. Excess stain is washed and bound stain was solubilized, and the absorbance was measured at 492 nm in a plate reader. Concentration of the compounds that inhibited 50% of the net cell growth, growth inhibition of 50% (GI50), was calculated from the dose response curve obtained for each test compound and cell line. GI50 values were presen‐ ted in micro molar (μM) concentration. Doxorubicin was used as positive control for the comparison of cytotoxicity of synthesized compounds. Assays were performed in triplicate on three independent experiments and their mean values are taken as a final reading. The result of this study indicates that compound 13c, 13g, 15g and 15h shows prominent anti‐ cancer activity in all cell lines, having growth inhibition of 50 (GI50) values of 5.12 to 17.52 µM (Table 1). All experiments were performed in duplicate and repeated three times. 3. Results and discussion 3.1. Chemistry In Scheme 1, Step (a) is enamine formation which is done by reacting compound 1 with DMF‐DMA heating at 100 °C for obtaining compound 2 with 78% yield. The compound 2 is reacted with N2H4.H2O in EtOH at 80 °C for 8 h to obtain compound 3 with having 68.3% yield. The structure of 4,5,6,7‐ tetrahydro‐1H‐pyrazolo[4,3‐c]pyridine is confirmed by singlet at δ 7.31 ppm in 1H NMR [16]. Purification of the compound 3 required purification by using column chromatography. The overall yield obtained by this method is greater than earlier reports [13]. 4,5,6,7‐Tetrahydro‐1H‐pyrazolo[4,3‐c]pyridine (3) is reacted with di‐tert‐butyl dicarbonate (Boc anhydride) using triethylamine as base to obtain di‐tert‐butyl‐4,5‐di hydro‐7H‐pyrazolo[3,4‐c]pyridine‐1,6‐dicarboxylate (4) with yield 91%. The compound 4 is having BOC protection on both nitrogen’s confirmed by 1H NMR showing singlet for 18 H at δ 1.56 ppm. The Step (d) is deportation of aliphatic N‐BOC which is achieved by treating compound 4 with 2 N HCl for 2 h to obtain compound 5, confirmed by 1H NMR showing singlet for 9 H at δ 1.56 ppm. The compound 5 was treated with pyridine Br2 at room temperature for 3 h to obtain compound 6 with 92% yield. The structure of tert‐butyl‐3‐bromo‐4,5,6,7‐tetra hydropyrazolo[3,4‐c]pyridine‐1‐carboxylate (6) was confir‐ med by disappearance of singlet at δ 7.31 ppm in 1H NMR. The compound 6 reacted with aqueous NaOH in heating for 3 h. There is formation of compound 7 in 88.4% yield which is confirmed by desired mass in LC‐MS. The compound 7 is protected by using benzylbromide in THF by using mixture of bases as 2,6‐leutidine and DMAP at room temperature for compound 6h to obtain compound 8 with 83% yield, with 1H NMR signals at δ 7.38‐7.28 ppm (m, 5H). The compound 8 having free hydroxyl group which is protected by using triflic anhydride at room temperature for 12 h to obtain compound 9 with 88.5% yield. t‐Butyl 3‐(2,2,2‐trifluoroacetoyloxy)‐6‐ benzyl‐4,5,6,7‐tetrahydropyrazolo[3, 4‐c]pyridine‐1‐carboxy‐ late (9) is key intermediate for the synthesis of final com‐ pounds 13a‐h and 15a‐h. The compound 9 was treated with different aromatic boranic acids at 100 °C for 6 h to obtain compounds 10a‐d with yields in the range from 63 to 83% yields after purifications by silica gel (100‐200 mesh) column chromatography. Debenzylation of compounds 10a‐d was done by using Pd/C in EtOH for 50 psi of hydrogen for 3 h at room temperature to obtain compounds 11a‐d with 84 to 90% yields. Alkylation of compounds 11a‐d was done by using substituted aromatic bromides in THF by using mixed bases 2,6‐leutidine and DMAP for 3 h at room temperature to obtain compounds 12a‐h with yields 84 to 93%. The cleavage of protecting group of compounds 12a‐h was done by using aqueous 6 N HCl at room temperature for 6 h to obtain compounds 13a‐h with yields 76 to 91%. The mixed bases used in Steps (g) and (k) to enhance the reactivity of secondary amine used for reaction. In Scheme 2, compounds 12a‐h are treated with KMnO4 in DCM and 18‐crown‐6 used as phase transfer catalyst at room temperature for 6 h to give compounds 14a‐h with yields 71 to 78%, which is confirmed by vanishing singlet at 3.78 in 1H NMR. The compound 14a‐h are converted to compounds 15a‐ h by using aqueous 6 N HCl at room temperature for 6 h with 79 to 92% yields. The final compounds 13a‐h and 15a‐h are obtained with reaction yields 76 to 92%. Purity of all final compounds and key intermediates is >95% which are further used for biological activity studies. 408 Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 Table 2. Inhibitory activity of compound 13c, 13g, 15g and 15h against panel of eight human kinases. Kinase % Inhibition Compound 13c Compound 13g Compound 15g Compound 15h Aurora‐A 73 64 51 57 Aurora‐B 41 70 77 73 CDK2/cyclinA 28 37 23 33 CDK2/cyclinE 17 22 21 23 CDK5/P25 66 59 70 56 EGFR 14 21 15 18 mTOR 44 68 46 48 PDK1 23 28 19 33 3.2. Biological studies All the newly synthesized compounds 13a‐h and 15a‐h were evaluated for their antiproliferative activities against a panel of four different human cancer cell lines. The IC50 for each synthesized compounds are calculated with respect to one human normal cell line Human umbilical vein endothelial cell line (ATCC CRL‐1730) and results are summarized in Table 1. These values represent the concentration required to inhibit 50% cell population compared with the control cells treated with DMSO and positive control Doxorubicin under similar conditions. From substituted tetra‐hydro‐6‐(substituted)‐1H‐pyra‐ zolo[3,4‐c]pyridine derivatives (13a‐h and 14a‐h), the IC50 value ranges from 5.12 to 38.86 µM all four cell lines. For cell line A‐549, compound 13g is most active with IC50 value of 5.12 µM; and compound 13c is also active with IC50 value of 6.81 µM along with compounds 13b, 13e, 13f, 13h, 15b and 15d are moderately active with IC50 value of 15.81, 13.86, 15.72, 13.25, 14.13 and 11.72 µM, respectively. The com‐ pounds 13a, 15c and 15e are most inactive compounds in the series. For cell line HeLa, it’s have IC50 values are in between 8.59 to 26.87 µM. The compounds 15g is most active with IC50 value of 8.59 µM along with compound 15d and 13g with IC50 values of 8.87 and 9.12 µM, respectively. The compounds 13b, 13c, 15a, 15c and 15h are moderately active with IC50 values ranging in between 11.32 to 14.38 µM. Remaining compounds are less active with IC50 value in between 15.16 to 26.87 µM. For cell line MCF‐7, the IC50 values are in the range of 6.12 and 26.32 µM. The compounds 15h, 15g and 13g are most active IC50 values of 6.12, 8.63 and 9.36 µM, respectively, total five compounds are moderately active with IC50 values in the range of 11.36 to 14.82 µM and eight compounds are less active with IC50 values are in the range of 16.12 to 26.32 µM. For cell line DU‐145, the IC50 value ranges from 9.52 to 38.56 µM. The compounds 15a is most active with IC50 value of 9.52 µM along with compound 13c having IC50 value of 10.37 µM are most active. The compounds 13e, 13h, 15b, 15c, 15d, 15h and 15e are also active compounds in DU‐145 cell line with IC50 values in the range of 10‐12 µM total seven compounds are less active with IC50 values in the range of 13.52 to 38.56 µM. Compound 13a having 3‐methoxy phenyl and benzofuran‐ 2‐yl groups is inactive compared with standard with IC50 values in the range of 22.72 to 28.87 µM in all cell lines. Compound 13b is moderately active with IC50 value of 14.33 µM of HeLa cell line and in remaining all cell lines it is inactive. The compound 13c is active in A‐549 cell line and DU‐145 and is moderately active in HeLa and it is most inactive in MCF‐7 due to presence of 3‐methoxy phenyl and 3‐yl quinolone groups. The compound 13d is moderately active in A‐549 and in remaining cell lines, it is inactive. Compound 13e is inactive in cell lines HeLa and in remaining cell lines, it is moderately active with IC50 values 11.82 to 13.86 µM, its having pyrimidine and benzo‐furan group. Compound 13f is mostly inactive in all cell lines because the presence of pyrimidine and benzo‐thiophene group. The compound 13g is active com‐ pound in all cell lines with IC50 values in the range of 5.12 to 9.36 µM and for DU‐145, it is moderately active with IC50 values of 13.52 µM due to the presence of pyrimidine and 3‐yl quinolone group. Compound 13h, for HeLa cell line, is inactive with IC50 value of 17.78 µM and, for remaining cell lines, it is moderately active with IC50 values in the range of 11.72 to 13.82 µM due to presence of pyrimidine and 5‐yl‐quinolone group. The compound 15a having 3‐methoxy phenyl and benzofuran‐2‐yl groups are mostly active in all cell lines with IC50 values in the range of 10.82 to 13.39 µM and is active in DU‐145 with IC50 value of 9.52 µM. The compound 15b is moderately active in A‐549 and DU‐145 cell lines and it is inactive in HeLa and MCF‐7 cell line. The compound 15c is moderately active in HeLa and DU‐145 (IC50 value of 11.52 µM) cell lines and it is inactive in A‐549 and MCF‐7 cell line 23.86 and 20.63 µM, respectively. The compound 15d is active for HeLa cell line with IC50 value of 8.87 µM and it is also moderately active for A‐549 with IC50 value of 11.72 µM, for MCF‐7 cell line with IC50 value of 13.12 µM with moderate active interestingly it is in active for DU‐145 cell line with IC50 value of 18.86 µM. The compound 15e having pyrimidine and benzo‐furan group is moderately active for DU‐145 cell line with IC50 value of 12.52 µM and for remaining cell lines, it is inactive. The compound 15f having IC50 values of 10.78 µM is active for A‐549 cell line and it is inactive for remaining all cell lines with IC50 values of 14.82 to 18.78 µM. The compound 15g is active for A‐549, HeLa and MCF‐7 cell lines with IC50 values of 10.82, 8.59 and 8.36 µM, respectively. It is inactive with DU‐ 145 cell line with IC50 value of 17.52 µM with pyrimidine and 3‐yl‐quinolone groups. The compound 15h having pyrimidine and 5‐yl‐quinolone groups is most active in MCF‐7 cell line with IC50 value of 6.12 µM and A‐549 with IC50 value of 9.13 µM also it is moderately active in HeLa and DU‐145 cell lines with IC50 values of 14.16 and 11.62 µM, respectively. From cell line data compounds 13c, 13g, 15g and 15h are most active which are having pyrimidine‐2‐yl group and quinoline3/5‐yl groups, compared with compounds having 3‐methoxy phenyl, benzofuran and benzothiophene groups. The compound 13c is more active than compound 13d as both of these compounds are separated by position of nitrogen in the qunioline ring, the 3‐methoxy compounds with benzofuran and benzothiophene are less active than compounds having pyrimidine‐2‐yl substitutions. Interestingly compounds having substituted 4,5,6,7‐tetrahydro group and substituted 5,6‐dihydro groups are moderate to active on all four cell lines and that substituted 5,6‐dihydro groups are more active than that of substituted 4,5,6,7‐tetrahydro group. These are results from both series of compounds. There is not much difference in their inhibitions in all four cancer cell lines. Further we have studied the most active compounds 13c, 13g, 15g and 15h on human kinases. The compounds 13c, 13g, 15g and 15h are most active in cell line studies, so further we have tested for its activity against a panel of eight human kinase at 10 µM concentrations. For Aurora‐A kinase compounds, they shows 73, 64, 51 and 57% inhibitions, respectively. The results are summarized in Table 2. For Aurora‐B kinase, compound 13c shows 41% inhi‐ bitions and for remaining compounds 13g (70%), 15g (77%) and 15h (73%) inhibitions. For CDK/cyclinA, CDK/cyclinE, EGFR and PDK1, the inhibition is in the range of 17 to 37%. CDK5/P25 kinase and mTOR kinase the inhibitions are in the range of 44 to 70%. For Aurora‐A, Aurora‐B, CDK5/P25 and Pawar et al. / European Journal of Chemistry 8 (4) (2017) 400‐409 409 mTOR kinase, all the compounds shows promising inhibitions to great extent. The inhibition results shows compound 13c is active for aurora‐A kinase and CDK5/P25 kinase and it shows less inhibition for remaining kinases. Compounds 13g, 15g and 15h shows >50% inhibitions. For EGFR, PDK1, CDK2/ cyclinE and CDK2/cyclinA kinases, most of compounds shows <40% inhibitions. 4. Conclusion We have synthesized 3‐(substituted)‐4,5,6,7‐tetrahydro‐6‐ (substituted)‐1H‐pyrazolo[3, 4‐c]pyridine (13a‐h) and 3‐( substituted)‐5, 6‐dihydro‐6‐(substituted)‐1H‐pyrazolo[3, 4‐c] pyridin‐7(4H)‐one (15a‐h). The synthesis mainly required protection, deportation, N‐alkylation and Suzuki coupling reactions. 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