Ultrasound assisted synthesis of pyrazolo[1,5-a]pyrimidine-antipyrine hybrids and their anti-inflammatory and anti-cancer activities European Journal of Chemistry 11 (1) (2020) 68-79 European Journal of Chemistry View Journal Online View Article Online Ultrasound assisted synthesis of pyrazolo[1,5-a]pyrimidine-antipyrine hybrids and their anti-inflammatory and anti-cancer activities Shunan Kaping 1, Melboureen Sunn 2, Laishram Indira Singha 2 and Jai Narain Vishwakarma 1,* 1 Organic Research Laboratory, Department of Chemical Science, Assam Don Bosco University, Tapesia Gardens, Sonapur 782402, Assam, India shunankaping@yahoo.in (S.K.), jnvishwakarma@rediffmail.com (J.N.V.) 2 Department of Biotechnology, St. Anthony’s College, Shillong 793001, Meghalaya, India melboureensunn@gmail.com (M.S.), laishramsingha@gmail.com (L.I.S.) * Corresponding author at: Organic Research Laboratory, Department of Chemical Science, Assam Don Bosco University, Tapesia Gardens, Sonapur 782402, Assam, India. e-mail: jnvishwakarma@rediffmail.com (J.N. Vishwakarma). 10.5155/eurjchem.11.1.68-79.1942 Received: 20 November 2019 Received in revised form: 11 February 2020 Accepted: 14 February 2020 Published online: 31 March 2020 Printed: 31 March 2020 A series of antipyrinyl-pyrazolo[1,5-a]pyrimidines have been synthesized by reactions of aminopyrazole (4) with various formylated active proton compounds in the presence of KHSO4 (aqueous media), under ultrasound irradiation. The structures of the compounds have been established with the help of spectral and analytical data. N-(1,5-Dimethyl-3-oxo- 2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-7-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6a) was further subjected to X-ray crystallographic studies to avoid any ambiguity of the derived structures. Crystal data for compound 6a, C51H46N12O5 (M =907.00 g/mol): triclinic, space group P-1 (no. 2), a = 9.9554(3) Å, b = 14.0875(4) Å, c = 17.4572(4) Å, α = 79.676(2)°, β = 85.283(2)°, γ = 72.647(2)°, V = 2297.97(11) Å3, Z = 2, T = 296.15 K, μ(MoKα) = 0.088 mm- 1, Dcalc = 1.311 g/cm3, 29732 reflections measured (4.174° ≤ 2Θ ≤ 57.068°), 10681 unique (Rint = 0.0400, Rsigma = 0.0533) which were used in all calculations. The final R1 was 0.0566 (I > 2σ(I)) and wR2 was 0.1663 (all data). The novel compounds were also screened for their biological activities. Aminopyrazole 4-Aminoantipyrine Pyrazolopyrimidines X-ray crystallography Ultrasound irradiation Potassium hydrogen sulphate Cite this: Eur. J. Chem. 2020, 11(1), 68-79 Journal website: www.eurjchem.com 1. Introduction Pyrazolo[1,5-a]pyrimidine, an important pharmacophore and an adjective structure in medicinal chemistry, is well known for its innumerable biological and pharmacological properties [1,2]; like antitumor [3-5], antileukemic [3], anti- neoplastic [6], CNS stimulant [7], antihypertensive [8], adenosine receptors [9], tuberculostatic [10], antibacterial and antifungal [11], anti-inflammatory [12], anti-atherosclerotic [12,13] antischistosomal [14], and antileishmanial activities [15]. A well-known drug Zaleplon [16] which has found a positive role as a sedative for the treatment of insomnia without the risk of dependence consists of the core pyrazolo [1,5-a]pyrimidine ring. Also, 4-aminoantipyrine, containing the functional parent antipyrine has been reported to show myriads of biological activities such as anti-inflammatory [17], analgesic [18], antipyretic [19], antimicrobial [20], and anti- cancer activities [21]. 4-Aminoantipyrine has been used for the protection against oxidative stress as well as prophylactic of some diseases including cancer, and these are found to be important directions in medical applications [22]. In view of these information and in continuation with our research work [23-25] on synthesis of pharmacologically important pyrazolo [1,5-a]pyrimidines, it was found worthy to synthesize novel pyrazolo[1,5-a]pyrimidines incorporating the antipyrine moiety to study their biological properties. Ultrasound (US) irradiation has helped researchers tremendously in accomp- lishing the green goals of chemistry by shortening the reaction time with easy work-up, resulting in high yields of the products and use of readily available solvents and catalysts [26-28]. Ultrasound tool has been advantageously employed to achieve the desired synthesis. 2. Experimental 2.1. Chemistry 2.1.1. Material and methods ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2020 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.11.1.68-79.1942 http://dx.doi.org/10.5155/eurjchem.11.1.68-79.1942 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.11.1.68-79.1942&domain=pdf&date_stamp=2020-03-31 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.11.1.68-79.1942 mailto:shunankaping@yahoo.in mailto:jnvishwakarma@rediffmail.com mailto:melboureensunn@gmail.com mailto:laishramsingha@gmail.com mailto:jnvishwakarma@rediffmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.11.1.68-79.1942&domain=pdf&date_stamp=2020-03-31� Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 69 EtO CN O , 150 oC N N O Ph NH 2 CN O N N O Ph NH CN O NMe2 3 N N O Ph 4 N N O Ph NH2 1 DMF-DMA MWI, 5 min EtOHN2H4 H2O N H O N NH NH2 . Scheme 1 Melting points were recorded by open capillary method and are uncorrected. The IR spectra were recorded on a Perkin-Elmer 983 spectrometer. High-resolution 1H NMR (400 MHz) and 13C NMR (100 MHz) were measured on a DRX-400 Varian spectrometer and Bruker spectrometer, respectively, and CDCl3 and DMSO-d6 was used as the solvent. The chemical shifts (δ, ppm) and the coupling constants (Hz) are reported in the standard fashion with reference to tetramethylsilane (TMS) as internal reference. In the NMR spectral data, the abbreviations s = singlet, t = triplet, m = multiplet, d = doublet, dd = double-doublet are used. The X-ray diffraction data were collected at 296 K with MoKα radiation (λ = 0.71073 Å) using a Bruker Nonius SMART APEX II CCD diffractometer equipped with a graphite monochromator. The structure was solved by direct methods (SHELXS97) and refined by full-matrix least squares based on F2. All calculations were carried out using WinGX system version 1.80.05 [29]. All the non-H atoms were refined in the anisotropic approximation: H-atoms were located at calculated positions. The electron spray mass spectra were recorded on a THERMO Finnigan LCQ Advantage max ion trap mass spectrometer. Elemental analysis was performed on a Vario-EL III instrument. Ultrasound irradiation was carried out in an EQUITRON Digital Ultrasonic Cleaner-2.5 L, model 8425.025.424 at 170 Watt and 50 Hz. Microwave irradiation was carried out in a CEM Discover Benchmate microwave digester at 850 Watt in an open reaction vessel. Formylated active proton compounds were synthesized by our previously reported procedure [30,31]. The aminopyrazole 4 used as a synthon for reactions with formylated active proton compounds were obtained as shown in Scheme 1. Compound 2 was obtained following the procedure reported by Fadda et al. [32] which was then formylated with DMF-DMA using microwave (850 Watt, 5 minutes). The reaction mixture was then without further purification, reacted with hydrazine hydrate in refluxing ethanol [33], yielding the aminopyrazole (4). 2.1.2. Synthesis 2.1.2.1. Synthesis of antipyrinyl-7-arylpyrazolo[1,5-a] pyrimidines (6a-e) Aminopyrazole (4) (1 mmol), enaminones 5 (1 mmol), and KHSO4 (2 mmol) were suspended in 5 mL of ethanol:water (1:1, v:v) system and the resulting mixture was irradiated under the influence of ultrasound waves for 3-6 minutes resulting in the formation of a precipitated product (Scheme 2). After the completion of reaction monitored by thin layer chromatography (TLC), the precipitate was collected by filtration, washed repeatedly with water to ensure complete removal of acid and dried to give practically pure pyrazolo pyrimidines (6) in 93-96 % yields. Further, purification was achieved by column chromatography using silica gel and 100 % EtOAc. N-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol- 4-yl)-7-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6a): Color: Yellow solid. Yield: 95 %. M.p.: 191-192 °C. FT-IR (KBr, ν, cm-1): 3448 (NH), 1645 (CO), 1637 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.43 (s, 3H, CH3), 3.17 (s, 3H, NCH3), 7.10 (d, 1H, C6-pyrimidine, J = 4.5 Hz), 7.30-7.34 (m, 1H, ArH), 7.45- 7.50 (m, 4H, ArH), 7.57-7.62 (m, 3H, ArH), 8.03-8.06 (m, 2H, ArH), 8.72 (d, 1H, C5-pyrimidine, J = 4.5 Hz), 8.75 (s, 1H, C2- pyrazole), 9.57 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.7, 36.0, 105.5, 108.8, 108.9, 124.4, 127.1, 128.9, 129.3, 129.5, 130.0, 131.8, 134.5, 146.7, 147.5, 148.4, 149.0, 151.1, 160.9, 161.1. MS (EI, m/z (%)): 425 (MH)+. Anal. calcd. for C24H20N6O2: C, 67.91; H, 4.75; N, 19.80. Found: C, 67.98; H, 4.72; N, 19.75 %. N-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol- 4-yl)-7-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (6b): Color: Yellow solid. Yield: 94 %. M.p.: 252-253 °C. FT-IR (KBr, ν, cm-1): 3448 (NH), 1675 (CO), 1624 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.42 (s, 3H, CH3), 2.47 (s, 3H, CH3), 3.17 (s, 3H, NCH3), 7.07 (d, 1H, C6-pyrimidine, J = 4.5 Hz), 7.28-7.32 (m, 1H, ArH), 7.39 (d, 2H, ArH, J = 7.8 Hz), 7.44-7.49 (m, 4H, ArH), 7.96 (d, 2H, ArH, J = 7.8 Hz), 8.68 (d, 1H, C5-pyrimidine, J = 4.5 Hz), 8.73 (s, 1H, C2-pyrazole), 9.56 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm):12.8, 21.7, 36.4, 105.4, 108.4, 109.1, 124.2, 126.9, 127.1, 129.2, 129.5, 129.6, 134.7, 142.5, 146.6, 147.5, 148.4, 149.1, 151.0, 160.9, 161.3. MS (EI, m/z (%)): 439 (MH)+. Anal. calcd. for C25H22N6O2: C, 68.48; H, 5.06; N, 19.17. Found: C, 68.59; H, 5.04; N, 19.12 %. 7-(4-Chlorophenyl)-N-(1, 5-dimethyl-3-oxo-2-phenyl-2, 3- dihydro-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carbox amide (6c): Color: Yellow solid. Yield: 95 %. M.p.: 261-262 °C. FT-IR (KBr, ν, cm-1): 3448 (NH), 1675 (CO), 1624 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.42 (s, 3H, CH3), 3.16 (s, 3H, CH3), 7.09 (d, 1H, C6-pyrimidine, J = 4.5 Hz), 7.30-7.33 (m, 1H, ArH), 7.44-7.49 (m, 4H, ArH), 7.57 (d, 2H, ArH, J = 8.7 Hz), 8.03 (d, 2H, ArH, J = 8.7 Hz), 8.71-8.75 (m, 2H, C2-pyrazole, C5- pyrimidine), 9.51 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.8, 36.4, 105.7, 108.6, 109.0, 124.2, 127.0, 128.3, 129.2, 130.9, 134.2, 134.6, 138.1, 146.7, 147.0, 147.3, 149.1, 151.1, 160.7, 161.3. MS [ESI]: m/z 459 (MH)+. Anal. calcd. for C24H19ClN6O2: C, 62.82; H, 4.17; N, 18.31. Found: C, 62.70; H, 4.15; N, 18.35 %. N-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol- 4-yl)-7-(4-methoxyphenyl)pyrazolo[1, 5-a]pyrimidine-3-carbox amide (6d): Color: Light yellow solid. Yield: 93 %. M.p.: 247- 248 °C. FT-IR (KBr, ν, cm-1): 3449 (NH), 1655 (CO), 1643 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.41 (s, 3H, CH3), 3.12 (s, 3H, NCH3), 3.90 (s, 3H, OCH3), 7.07-7.10 (m, 3H, C6-pyrimi 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 70 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 Ar1 CN NMe2 N H O An N H N NH2 N N Ph O An = US, 60 oC KHSO4 EtOH, H2O Ar O NMe2 Ar = C6H5, 4-CH3C6H4, 4-ClC6H4, 4-MeOC6H4, 4-NO2C6H4; Ar1 = C6H5, 4- CH3OC6H4 N H OAn N N N Ar 5 4 6a-e 7 N H O An N N N NH2 8a,b Ar1 US, 60 oC KHSO4 EtOH, H2O Scheme 2 dine, ArH), 7.26-7.28 (m, 1H, ArH), 7.45-7.47 (m, 4H, ArH), 8.09 (d, 2H, ArH, J = 8.7 Hz), 8.65 (d, 1H, C5-pyrimidine, J = 4.5 Hz), 8.73 (s, 1H, C2-pyrazole), 9.53 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.9, 36.6, 55.6, 87.2, 105.4, 107.8, 109.5, 114.3, 122.1, 123.8, 126.5, 129.2, 131.4, 135.1, 146.6, 147.6, 148.0, 149.4, 150.9, 160.8, 162.1. MS (EI, m/z (%)): 455.2 (MH)+. Anal. calcd. for C25H22N6O3: C, 66.07; H, 4.88; N, 18.49. Found: C, 66.14; H, 4.84; N, 18.44 %. N-(1,5-Dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazol- 4-yl)-7-(4-nitrophenyl)pyrazolo[1,5-a]pyrimidine-3-carbox amide (6e): Color: Yellow solid. Yield: 96 %. M.p.: 223-224 °C. FT-IR (KBr, ν, cm-1): 3418 (NH), 1656 (CO), 1624 (CO). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.69 (s, 3H, CH3), 3.53 (s, 3H, CH3), 7.74-7.83 (m, 3H, ArH), 7.92-7.96 (t, 2H, ArH), 8.06 (d, 1H, C6-pyrimidine, J = 4.5 Hz), 8.82 (d, 2H, ArH, J = 9.1 Hz), 8.89 (d, 2H, ArH, J = 9.1 Hz), 9.16 (s, 1H, C2-pyrazole), 9.39 (d, 1H, C5-pyrimidine, J = 4.5 Hz), 9.56 (s, 1H, NH). MS (EI, m/z (%)): 470.3 (MH)+. Anal. calcd. for C24H19N7O4: C, 61.40; H, 4.08; N, 20.89. Found: C, 61.47; H, 4.10; N, 20.93 %. 2.1.2.2. Synthesis of 7-amino-(1,5-dimethyl-3-oxo-2-phenyl- 2,3-dihydro-1H-pyrazol-4-yl)-6-arylpyrazolo[1,5-a] pyrimidine-3-carboxamide (8a,b) A mixture of aminopyrazole (4) (1 mmol) and enamino nitriles 7 (1 mmol) in the presence of KHSO4 (2 mmol) in 5 mL of ethanol:water (1:1, v:v) was US irradiated for 3-10 minutes to give a precipitate (Scheme 2). After the completion of reaction (monitored by TLC), the precipitate was collected by filtration, washed repeatedly with ethanol-water (1:1) ensuring complete removal of acid, and then dried to give practically pure pyrazolopyrimidines (8) in 87-92 % yields. Further, purification was carried out by column chromate- graphy (silica gel, 100 % EtOAc-hexane). 7-Amino-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H- pyrazol-4-yl)-6-phenylpyrazolo[1, 5-a]pyrimidine-3-carbox amide (8a): Color: Pale White. Yield: 87 %. M.p.: 184-186 °C. FT-IR (KBr, ν, cm-1): 3640 (NH2), 3199 (NH), 1659 (CO), 1635 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.35 (s, 3H, CH3), 3.11 (s, 3H, NCH3), 6.90 (s, 2H, NH2), 7.38-7.49 (m, 10H, ArH), 8.49- 8.52 (m, 1H, C5-pyrimidine), 9.19 (s, 1H, C2-pyrazole), 9.35 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 11.0, 35.8, 85.0, 106.3, 117.2, 123.6, 123.8, 126.5, 127.0, 127.8, 129.1, 129.5, 129.6, 130.4, 132.7, 135.0, 139.4, 152.9, 162.0, 163.4. MS (EI, m/z (%)): 440.4 (MH)+. Anal. calcd. for C24H21N7O2: C, 65.59; H, 4.82; N, 22.31. Found: C, 65.65; H, 4.78; N, 22.26 %. 7-Amino-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H- pyrazol-4-yl)-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine- 3- carboxamide (8b): Color: Pale White. Yield: 92 %. M.p.: 275- 276 °C. FT-IR (KBr, ν, cm-1): 3394 (NH2), 3297 (NH), 1678 (CO), 1662. 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.36 (s, 3H, CH3), 3.11 (s, 3H, NCH3), 3.85 (s, 3H, OCH3), 7.11 (m, 2H, NH2), 7.26-7.46 (m, 9H, ArH), 8.22 (s, 1H, C5-pyrimidine), 8.53 (s, 1H, C2-pyrazole), 9.31 (s, 1H, NH). 13C NMR (100 MHz, DMSO- d6, δ, ppm): 11.0, 35.8, 55.2, 102.6, 104.4, 107.3, 114.6, 115.0, 123.5, 125.0, 126.4, 129.1, 130.8, 135.0, 145.1, 146.1, 146.6, 151.4, 159.0, 160.5, 161.7. MS (EI, m/z (%)): 532.1 (M+Na+ K+H)+. Anal. calcd. for C25H23N7O3: C, 63.96; H, 4.94; N, 20.88. Found: C, 63.79; H, 4.90; N, 20.75 %. 2.1.2.3. Synthesis of antipyrinyl-7-hetarylpyrazolo[1,5-a] pyrimidines (9,10) Aminopyrazole (4) (1 mmol), enaminones I/II (1 mmol), and KHSO4 (2 mmol) were suspended in 5 mL of ethanol:water (1:1, v:v) and the mixture was irradiated under the influence of US waves for 5-6 minutes giving a precipitated product (Scheme 3). After the completion of reaction monitored by TLC, the precipitate was collected by filtration, washed repeatedly with water ensuring complete removal of acid and dried to produce practically pure pyrazolopyrimidines (9, 10) in 90-94 % yields. Further, purification was achieved by column chromatography using silica gel and 100 % EtOAc. N-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol- 4-yl)-7-(pyridin-4-yl)pyrazolo[1, 5-a]pyrimidine-3-carbox amide (9): Color: Yellow solid. Yield: 94 %. M.p.: 212-213 °C. FT-IR (KBr, ν, cm-1): 3414 (NH), 1664 (CO), 1638 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.40 (s, 3H, CH3), 3.14 (s, 3H, NCH3), 7.21 (d, 1H, C6-pyrimidine, J = 4.12 Hz), 7.30-7.32 (m, 1H, ArH), 7.46-7.47 (m, 4H, ArH) 8.02 (d, 2H, 3',5'-H, J = 5.5 Hz), 8.74 (s, 1H, C2-pyrazole), 8.78 (d, 1H, C5-pyrimidine, J = 4.12 Hz), 8.89 (d, 2H, 2',6'-H, J = 5.5 Hz), 9.36 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.8, 36.5, 106.3, 108.9, 109.2, 123.4, 124.0, 126.8, 129.2, 134.9, 138.0, 145.1, 146.9, 147.1, 149.5, 150.1, 151.2, 160.4, 162.0. MS (EI, m/z (%)): 425.4 (M)+. Anal. calcd. for C23H19N7O2: C, 64.93; H, 4.50; N, 23.05. Found: C, 64.98; H, 4.51; N, 23.00 %. N-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol- 4-yl)-7-(pyridin-3-yl)pyrazolo[1, 5-a]pyrimidine-3-carbox amide (10): Color: Yellow solid. Yield: 90 %. M.p.: 238-239 °C. FT-IR (KBr, ν, cm-1): 3356 (NH), 1679 (CO), 1613 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.41 (s, 3H, CH3), 3.13 (s, 3H, NCH3), 7.16 (d, 1H, C6-pyrimidine, J = 4.12 Hz), 7.27-7.28 (m, 1H, ArH), 7.46-7.47 (m, 4H, ArH), 7.55-7.56 (m, 1H, 5'-H), 8.55 (d, 1H, 4'-H), 8.75-8.76 (m, 2H, 6'H, C5-pyrimidine), 8.84 (d, 1H, 2'-H, J = 5.0 Hz), 9.18 (s, 1H, C2-pyrazole), 9.42 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.8, 36.5, 87.2, 106.2, 108.6, 109.2, 123.9, 126.4, 126.6, 129.2, 135.0, 137.2, 145.2, 146.9, 147.2, 149.5, 149.8, 151.1, 152.4, 160.5, 162.0. MS (EI, m/z (%)): 426.2 (MH)+. Anal. calcd. for C23H19N7O2: C, 64.93; H, 4.50; N, 23.05. Found: C, 65.01.; H, 4.49; N, 23.00 %. 2.1.2.4. Synthesis of 6-acetyl/carboalkoxy-N-(1,5-dimethyl- 3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-7-methyl pyrazolo[1,5-a]pyrimidine-3-carboxamide (13a, b) 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 71 O NMe2 N O NMe2 N An N H O N H N NH2 4 An N H O N N N N An N H O N N N N i, I i, II i: US, 60 °C, KHSO4, EtOH:H2O 9 10 I: II: An: N N O Scheme 3 N H O N N N Me N N Me O Me O R CH3 OO R NMe2 13a,b 12 US, 60 °C R: CH3, OCH3 CH3 OO R DMF-DMA MWI, 850 W 11 KHSO4 EtOH:H2O + 4 Scheme 4 In order to synthesize the target pyrazolo[1,5-a] pyrimidine (13), formylated active proton compounds of type 12 were required. This was accomplished by the microwave irradiation of acyclic active proton compounds (11) (1 mmol) with DMF-DMA in a microwave digester for 5 minutes. The reaction mixture (monitored by TLC) was evaporated to dryness under reduced pressure. To the resulting residue, aminopyrazole (4) (1 mmol) was added, and the content was dissolved in 5 mL of ethanol:water mixture (1:1, v:v). KHSO4 (2 mmol) was then added, and the solution was subjected to ultrasound (US) irradiation for 4-6 min giving a precipitate (Scheme 4). After the completion of reaction (monitored by TLC), the precipitate was collected by filtration, washed repeatedly with ethanol: water (1:1, v:v), and dried over anhydrous CaCl2 to give practically pure products (13a, b) in 92-96 % yields. Further, purification was achieved by column chromatography (silica gel, 100 % EtOAc). 6-Acetyl-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H- pyrazol-4-yl)-7-methylpyrazolo[1, 5-a]pyrimidine-3-carbox amide (13a): Color: Light yellow solid. Yield: 92 %. M.p.: 211- 212 °C. FT-IR (KBr, ν, cm-1): 3328 (NH), 1692 (CO), 1680 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.41 (s, 3H, CH3), 2.72 (s, 3H, CH3), 3.12 (s, 3H, NCH3), 3.21 (s, 3H, CH3), 7.27-7.29 (m, 1H, ArH), 7.45-7.46 (m, 4H, ArH), 8.80 (s, 1H, C5-pyrimidine), 9.03 (s, 1H, C2-pyrazole), 9.30 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.9, 15.4, 30.0, 36.6, 106.6, 109.2, 119.0, 123.9, 126.6, 129.2, 135.0, 145.8, 148.6, 149.2, 150.9, 152.2, 160.1, 161.9, 195.9. MS (EI, m/z (%)): 405.3 (MH)+. Anal. calcd. for C21H20N6O3: C, 62.37; H, 4.98; N, 20.78. Found: C, 62.44; H, 4.95; N, 20.73 %. Methyl 3-((1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H- pyrazol-4-yl)carbamoyl)-7-methylpyrazolo[1, 5-a]pyrimidine- 6-carboxylate (13b): Color: Light yellow solid. Yield: 96 %. M.p.: 214-215 °C. FT-IR (KBr, ν, cm-1): 3313 (NH), 1736 (CO), 1690 (CO). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.39 (s, 3H, CH3), 3.12 (s, 3H, NCH3), 3.28 (s, 3H, CH3), 4.00 (s, 3H, OCH3), 7.27-7.28 (m, 1H, ArH), 7.45-7.46 (m, 4H, ArH), 8.79 (s, 1H, C5- pyrimidine), 9.10 (s, 1H, C2-pyrazole), 9.25 (s, 1H, NH). 13C NMR (400 MHz, CDCl3, δ, ppm): 12.8, 15.3, 36.5, 52.9, 106.8, 109.0, 112.0, 123.9, 126.6, 129.2, 135.0, 146.2, 148.3, 149.6, 151.8, 153.2, 160.2, 161.9, 164.5. MS (EI, m/z (%)): 422.0 (M)+. Anal. calcd. for C21H20N6O4: C, 59.99; H, 4.79; N, 19.99. Found: C, 59.81; H, 4.81; N, 19.94 %. 2.2. Biology 2.2.1. Material and methods 2.2.1.1. Anti-inflammatory test Griess reagent system (naphthylethylenediamine dihydro chloride, sulphanilamide and nitrite standard) from Promega (USA), Freund’s Complete Adjuvant (FCA) from GeNei (India), Wright stain, sodium chloride, disodium hydrogen phosphate dihydrate, potassium dihydrogen orthophosphate, dimethyl sulfoxide (DMSO) were procured from HiMEDIA (India), methanol was purchased from MERCK (India). Swiss Albino mice of both sexes were purchased from Pasteur institute, Shillong. The animals were kept in a tempe- rature controlled room with a 12 hours light and dark cycle. Cell lines CHO K1 (Chinese Hamster Ovary) were procured from National Centre for Cell Science (NCCS), Pune. 2.2.1.1.1. Measurement of paw edema This experiment was performed according to Lai et al. [34] with certain modifications. Swiss Albino male and female mice aged between 10-12 weeks (3 per group) were injected with about 50 µL of FCA into left hind paw of the mice to induce inflammation. The test compounds (50 mg/kg body weight in DMSO) were then administered 1 hour after FCA injection and the diameter of paw edema was then measured at different time intervals such as 0, 1, 2, 3 and 24 hours using a caliper. After 24 hours, blood was collected by retro-orbital bleeding and stored for nitric oxide assay and differential WBC count. The paws were then excised, weighed and kept in ice- cold normal saline. These were then homogenized in 10 % ice- cold normal saline, centrifuged at 12,000 rpm and the supernatant was collected and stored at -20 ᵒC for nitric oxide assay. 2.2.1.1.2. Nitric oxide assay The amount of nitric oxide produced was calculated using Griess reaction. The sulfanilamide and naphthylethylene diamine dihyrochloride (NED) solutions were made to equilibrate to room temperature for 15 to 30 minutes. 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 72 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 Table 1. Optimization of reaction conditions. 4 C6H5 NMe2 O 6a + 5a N N O Ph N H O N NH NH2 N H O N N NN N O Ph Entry Mode Temperature (°C) Solvent Reaction time (min) Yield (%) 1 Silent 25 Water 80 51 2 Silent 25 Water-Ethanol 65 60 3 Silent 60 Water 60 40 4 Silent 60 Water-Ethanol 50 50 5 Sonication 25 Water 6 58 6 Sonication 25 Water-Ethanol 5 67 7 Sonication 60 Water 5 62 8 Sonication 60 Water-Ethanol 4 94 To a 96 well microtiter plate, 50 µL of the sample was added in triplicate. To each of these wells, 50 µL of the sulfanilamide solution was then added and incubated for 5 to 10 minutes at room temperature protected from light. After the incubation period, 50 µL of the NED solution was added to each of the wells and incubated again in dark at room temperature. A purple colour developed which was further quantified at 520 nm. Three columns in the 96 wells plate were used for the nitrite standard reference curve in which a six serial fold dilution of 100 µM nitrite solution (50 µL/well) in triplicate was performed to generate the nitrite standard reference curve. Differential WBCs count. Differential WBCs counts were performed according to the method described by Houwen [35]. In brief, blood film was prepared on glass slides till it dried. The film was fixed in absolute methanol for 30 seconds. The slides were then stained with Wright’s stain for 2 minutes in a horizontal position after which Sorensen’s buffer (KH2PO4, Na2HPO4, pH = 6.4) was added and mixed. This was allowed to stand for 3 minutes and then rinsed with distilled water and dried. The slides were then observed under a microscope and differential WBC counting was done. 2.2.1.2. Anti-cancer assay CHO K1 cell lines were procured from NCCS (Pune), Dulbecco’s modified Eagle medium/Nutrient mixture F-12, dimethyl sulfoxide, fetal bovine serum (FBS), sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, trypsin phosphate versene glucose (TPVG) solution, 3-[4,5-dimethylthiazoyl-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) were purchased from HiMEDIA. Antibiotic antimycotic solution was purchased from Sigma. The cytotoxicity of the test compounds was assessed using MTT assay, according to Freshney [36] with certain modify- cations. CHO K1 cells at a concentration of 0.5 × 10 to 103 in 100 µL growth medium (Dulbecco’s modified Eagle medium/ Nutrient mixture F-12 with 10 % FBS) per well were seeded in a 96 well-microtiter plate. The cells were then incubated in the CO2 incubator at 37 °C. After 24 hours, the medium was removed and 80 µL of the fresh medium was added. To six wells, 20 µL (10 mg/mL in 10 % DMSO) of test compounds (in triplicate) was added, while three wells were loaded with 20 µL of 10 % DMSO to serve as control. The plate was again incubated for 24 hours and at the end of incubation period, the spent medium was replaced with 100 µL of the fresh medium and 20 µL of MTT (5 mg/mL) reagent prepared in phosphate buffer saline (PBS) was added to all the wells. The plates were then wrapped in aluminium foil and returned to the incubator for 2 hours until an insoluble purple formazan product was formed. After 2 hours, the medium was removed and 200 µL of 10 % DMSO was added to all the wells to dissolve the formazan product. Contents were mixed and the absorbance was recorded at 570 nm. 3. Results and discussion 3.1. Chemistry To start with, 4-aminoantipyrine was fused with ethyl cyanoacetate at 150 °C for the formation of compound 2 [32]. This was then formylated with DMF-DMA using microwave (850 W, 5 min). The reaction mixture was subsequently treated with hydrazine hydrate in refluxing ethanol [33], yielding practically pure aminopyrazole 4 (Scheme 1). The aminopyrazole 4 thus obtained was used as a synthon for subsequent reactions without further purification. In order to optimize the conditions for the reaction between pyrazole 4 and enaminones, the reaction of compounds 4 and 5a was taken as a model experiment and a series of experiments was conducted under various conditions as presented in Table 1. Sonication at 60 °C in water-ethanol gave the desired product 6a within 4 minutes in 94 % yield. The most remarkable aspect of this condition is that the product precipitated out and could be isolated by simple filtration in practically pure form. Therefore, it was decided to carry out the rest of the reactions of the series in water- ethanol at 60 °C under US irradiation. Thus, 3-Aminopyrazole 4 was irradiated for 3-4 minutes with enaminones 5 and KHSO4 in an aqueous medium in an ultrasonic bath at 60 °C (Scheme 2) to give products 6a-e in 93-96 % yields. The structures of these compounds were well established on the basis of their spectral and analytical data. These reaction conditions could well be applied for the reactions of 4 with enaminonitriles (7) giving the 7-amino- pyrazolopyrimidines 8a, b in 87-92 % overall yields (Scheme 2). The synthetic protocol was applied for the synthesis of antipyrinyl-pyrazolo[1,5-a]pyrimidines with hetaryl group at C-7 position of the pyrimidine ring. This was achieved by ultrasound irradiation of 3-aminopyrazole 4 with hetarylen aminones I or II and KHSO4 in water-ethanol mixture 60 °C (Scheme 3). The products were obtained in 90-96 % yields in 5-6 minutes. Further, the reaction of aminopyrazole 4 with formylated active proton compounds 12 (Scheme 4) derived from 1,3-diketones and prepared in situ was carried out under similar reaction conditions. The reaction proceeded smoothly with the product 13a, b precipitating out in 92-96 % yields within 4-6 minutes. 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 73 Table 2. Synthesis of antipyrinyl-pyrazolo[1,5-a]pyrimidines. Pyrazolo[1,5-a]pyrimidines Isolated yield (%) Time (min) Melting point (°C) 6a 95 3 191-192 6b 94 4.5 252-253 6c 95 4 261-262 6d 93 4 247-248 6e 96 4 223-224 8a 87 10 184-186 8b 92 3 275-276 9 94 6 212-213 10 90 5 238-239 13a 92 6 211-212 13b 96 4 214-215 Figure 1. The molecular structure of compound 6a. The infrared spectra of compounds 6a-e and 8a, b showed NH stretching peak in the region 3199-3449 cm-1 and the NH2 spectra of compounds 8a,b gave peaks at around δ 3394-3640 ppm. The carbonyl groups gave characteristic bands at around 1645-1678 and 1624-1662 cm-1. In the 1H NMR spectra of compounds 6a-d, the methyl group gave sharp singlet at around δ 2.42 ppm and in compounds 8a,b it showed at δ 23.5 and 23.6 ppm, respectively. The N-methyl group gave singlet at about δ 3.16 ppm for compounds 6a-c, and at δ 3.12 and 3.11 ppm for compound 6d and compounds 8a,b, respectively. The C6-H and C5-H protons of the pyrimidine ring for compounds 6a, 6b resonated as doublets (J = 4.5 Hz) at about δ 7.09 and 8.70 ppm, respectively, whereas for compound 6c the C5-H and C2-H protons signals overlapped and appeared as multiplet in the range δ 8.71-8.75 ppm. In compound 6d the C6-H proton signal was obscured by the aromatic protons while the C5-H signal appeared as doublet at δ 8.65 ppm. The C2-H protons for compound 6a appeared as singlet at δ 8.75 ppm and for compound 6b and 6d it resonated as singlet at δ 8.73 ppm. The methyl groups of compound 6e resonated as singlet at δ 2.69 and 3.53 ppm. The C6-H and C5-H protons of the pyrimidine ring of compound 6e resonated as doublet (J = 4.5 Hz) at δ 8.06 and 9.39 ppm, respectively, while the C2-H proton appeared as singlet at δ 9.16 ppm. The NH proton for compounds 6a-e gave sharp singlet at around δ 9.55 ppm. The methoxy group protons of compound 8b resonated as singlet in their usual range at δ 3.85 ppm. The amino protons appeared as singlet at around δ 6.90-7.11 ppm. The C5-H proton of compound 8a appeared as multiplet at δ 8.49-8.52 ppm, while that in compound 8b resonated as singlet at δ 8.22 ppm. The C2-H proton gave signal at δ 9.19 ppm in compound 8a and at δ 8.53 ppm in compound 8b. The -NH proton resonated as singlet at around δ 9.35 ppm. The spectral data of compounds 9, 10, 13a and 13b (experimental section) were in full agreement with the assigned structures. The 13C NMR spectra, of all the compounds showed signals at about δ 160.4-160.8 and δ 161.3-162.0 ppm indicating the presence of carbonyl groups. Mass spectrometry results were in full support of the assigned structures. The synthesized antipyrinyl-pyrazolo[1,5-a]pyrimidines are mentioned in Table 2. 3.2. X-ray crystallography The structure of N-(1,5-dimethyl-3-oxo-2-phenyl-2,3- dihydro-1H-pyrazol-4-yl)-7-phenylpyrazolo[1,5-a]pyrimidine- 3-carboxamide (6a) was further supported by using X-ray crystallography. The crystal data and structure refinement [37] values of 6a are mentioned in Table 3. The molecular structure of compound 6a is depicted in Figure 1. The details of the structure of compound 6a have been deposited with the Cambridge Crystallographic Data Centre No. CCDC-1401935. The bond length and angles are within the normal ranges [38]. Selected bond lengths and bond angles are given in Tables 4 and 5. The compound exists as a dimer in an asymmetric unit arranged in an opposite manner, with a molecule of acetone trapped. This could be probably due to the existence of short contacts between N5 and H21A at a distance of 2.744 Å, C42 and H20 at a distance of 2.828 Å and C34 and C11 at a distance of 3.391 Å which are definitely shorter than the sum of the corresponding van der Waal radii of N (1.55 Å) and H (1.2 Å); C (1.7 Å) and H (1.2 Å); and C (1.7 Å) and C (1.7 Å) respectively. 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 74 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 Table 3. Crystal data and structure refinement for N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-7-phenylpyrazolo[1,5-a]pyrimidine-3- carboxamide (6a). Parameter Value Empirical formula C51H46N12O5 Formula weight 907.00 Temperature (K ) 296.15 Crystal system triclinic Space group P-1 a (Å) 9.9554(3) b (Å) 14.0875(4) c (Å) 17.4572(4) α (°) 79.676(2) β (°) 85.283(2) γ (°) 72.647(2) Volume (Å3) 2297.97(11) Z 2 ρcalc (g/cm3) 1.311 μ (mm-1) 0.088 F(000) 952.0 Crystal size (mm3 ) 0.30 × 0.14 × 0.08 Radiation MoKα (λ = 0.71073) 2Θ range for data collection (°) 4.174 to 57.068 Index ranges -13 ≤ h ≤ 13, -18 ≤ k ≤ 18, -23 ≤ l ≤ 22 Reflections collected 29732 Independent reflections 10681 [Rint = 0.0400, Rsigma = 0.0533] Data/restraints/parameters 10681/0/627 Goodness-of-fit on F2 1.058 Final R indexes [I≥2σ (I)] R1 = 0.0566, wR2 = 0.1433 Final R indexes [all data] R1 = 0.1032, wR2 = 0.1663 Largest diff. peak/hole / (e Å-3) 0.47/-0.30 Table 4. Selected bond lengths for compound 6a (Å). Atom Atom Length Atom Atom Length Atom Atom Length N3 C14 1.408(2) C1 C2 1.394(3) C35 C36 1.388(3) N3 C13 1.366(2) C1 C6 1.387(3) C35 C37 1.470(2) N1 C10 1.353(2) C2 C3 1.381(3) C37 O3 1.223(2) N1 C9 1.320(2) C6 C5 1.385(3) C30 C29 1.390(3) N2 N8 1.3713(19) C3 C4 1.371(3) C30 C25 1.397(3) N2 C12 1.329(2) C4 C5 1.371(3) C38 C39 1.431(3) N5 C34 1.351(2) N10 N9 1.418(2) C38 C47 1.355(2) N5 C33 1.321(2) N10 C23 1.397(2) C32 C33 1.390(3) N6 N7 1.374(2) N10 C22 1.463(3) C39 O4 1.232(2) N6 C36 1.327(2) N9 C24 1.381(2) C39 N12 1.391(2) N4 C37 1.362(2) N9 C15 1.417(2) C41 C46 1.381(3) N4 C38 1.407(2) C24 O2 1.231(2) C41 C42 1.379(3) N7 C34 1.392(2) C23 C21 1.488(3) C41 N12 1.426(2) N7 C31 1.373(2) C15 C16 1.374(3) C29 C28 1.382(3) N8 C10 1.391(2) C15 C20 1.384(3) C25 C26 1.380(3) N8 C7 1.374(2) C16 C17 1.387(3) C26 C27 1.370(3) C10 C11 1.393(2) C20 C19 1.390(3) C28 C27 1.386(4) C11 C12 1.397(2) C18 C17 1.351(3) C46 C45 1.388(3) C11 C13 1.467(2) C18 C19 1.372(4) C42 C43 1.388(3) C14 C24 1.448(3) C34 C35 1.399(3) C43 C44 1.375(4) C14 C23 1.346(3) C31 C30 1.475(2) C45 C44 1.376(4) C7 C8 1.368(3) C31 C32 1.377(3) C47 C48 1.486(3) C7 C1 1.478(3) N13 N12 1.415(2) O5 C50 1.168(3) C13 O1 1.231(2) N13 C47 1.396(2) C52 C50 1.428(4) C8 C9 1.402(3) N13 C49 1.480(2) C51 C50 1.447(4) Also, the π-π stacking of the pyrazole rings could be the contributing factor. In both molecules, the pyrazolo[1,5- a]pyrimidine rings are planar. The bond length and angles are within the normal ranges [38]. The bond distances for C35- C36, C33-C32, C9-C8 and C11-C12 are 1.388, 1.390, 1.402 and 1.397 Å, respectively, which are much shorter than that expected for C-C single bonds. Also the bond lengths of C34- C35, C31-C32, C10-C11 and C7-C8 are 1.399, 1.377, 1.393 and 1.368 Å, respectively, which are longer than C=C bond and close to that of C-C single bond. This indicates a considerable degree of delocalization of the aromatic 10-pi-electrons around the heterocyclic pyrazolopyrimidine ring system [38]. 3.3. Biological activities 3.3.1. Anti-inflammatory assay 3.3.1.1. Percentage Inhibition of Paw Diameter Untreated paw edema bearing mice served as control group. The mice (except the control) were then treated with the compounds (50 mg/kg body weight) via intraperitoneal (i.p) injection. The ability of the test compound to reduce the edema caused by Freund’s Complete Adjuvant (FCA) was taken as a parameter. When induced with injection of FCA into the plantar side of left hind paw of the mice, all the groups of mice showed an increase in the paw diameter. In our study, we found that when these mice were treated with the test compounds (except the control which is untreated) some of the compounds showed a decrease in the paw diameter, which indirectly means that the edema has reduced. The ability of these test compounds to reduce the paw edema was calculated as percentage inhibition of paw diameter by using the formula ((a-b)/a)×100 [39] where ‘a’ and ‘b’ denote the mean increase in paw diameter of the control and drug treated mice respectively. The decrease/increase in the paw diameter was monitored at different intervals of zero, one, two, three and 24 h, respectively. Values are expressed as ±SEM. 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 75 Table 5. Selected bond angles for compound 6a (°) Atom Atom Atom Angle Atom Atom Atom Angle Atom Atom Atom Angle C13 N3 C14 124.55(16) C4 C5 C6 121.1(2) N4 C37 C35 114.44(17) C9 N1 C10 115.84(16) N9 N10 C22 113.35(17) O3 C37 N4 123.36(18) C12 N2 N8 103.34(14) C23 N10 N9 105.25(14) O3 C37 C35 122.16(18) C33 N5 C34 115.53(16) C23 N10 C22 118.16(16) C29 C30 C31 118.25(19) C36 N6 N7 103.22(15) C24 N9 N10 110.77(15) C29 C30 C25 118.40(18) C37 N4 C38 126.10(17) C24 N9 C15 127.45(16) C25 C30 C31 123.33(17) N6 N7 C34 111.76(14) C15 N9 N10 120.53(15) N4 C38 C39 120.26(17) C31 N7 N6 125.94(15) N9 C24 C14 104.21(16) C47 C38 N4 130.70(18) C31 N7 C34 122.24(15) O2 C24 C14 130.40(18) C47 C38 C39 109.03(16) N2 N8 C10 112.15(14) O2 C24 N9 125.28(18) C31 C32 C33 120.61(18) N2 N8 C7 125.92(15) C14 C23 N10 109.90(17) O4 C39 C38 129.87(18) C7 N8 C10 121.92(15) C14 C23 C21 130.48(18) O4 C39 N12 124.77(18) N1 C10 N8 122.37(15) N10 C23 C21 119.60(16) N12 C39 C38 105.33(16) N1 C10 C11 131.74(16) C16 C15 N9 118.75(18) N5 C33 C32 124.65(18) N8 C10 C11 105.88(15) C16 C15 C20 120.4(2) C46 C41 N12 120.91(19) C10 C11 C12 104.28(15) C20 C15 N9 120.9(2) C42 C41 C46 120.6(2) C10 C11 C13 128.06(16) C15 C20 C19 119.0(2) C42 C41 N12 118.5(2) C12 C11 C13 127.66(16) C17 C18 C19 119.6(2) C28 C29 C30 120.8(2) N3 C14 C24 121.04(16) C18 C17 C16 121.5(3) C26 C25 C30 120.1(2) C23 C14 N3 129.44(18) C18 C19 C20 120.5(2) C27 C26 C25 121.3(2) C23 C14 C24 109.41(16) N5 C34 N7 122.14(16) C29 C28 C27 120.3(2) N2 C12 C11 114.34(16) N5 C34 C35 131.75(17) C41 C46 C45 119.8(2) N8 C7 C1 120.75(16) N7 C34 C35 106.11(15) C41 C42 C43 119.3(2) C8 C7 N8 115.04(16) N7 C31 C30 121.13(16) C26 C27 C28 119.2(2) C8 C7 C1 124.21(17) N7 C31 C32 114.81(16) C44 C43 C42 120.1(3) N3 C13 C11 114.37(16) C32 C31 C30 124.05(17) C44 C45 C46 119.5(3) O1 C13 N3 123.06(17) N12 N13 C49 113.85(15) C43 C44 C45 120.7(2) O1 C13 C11 122.56(16) C47 N13 N12 105.77(14) N13 N12 C41 119.13(15) C7 C8 C9 121.05(17) C47 N13 C49 118.22(15) C39 N12 N13 109.40(14) C2 C1 C7 117.81(18) C34 C35 C37 130.17(17) C39 N12 C41 124.40(16) C6 C1 C7 123.48(17) C36 C35 C34 103.82(16) N13 C47 C48 119.71(16) C6 C1 C2 118.67(19) C36 C35 C37 125.82(17) C38 C47 N13 109.46(16) N1 C9 C8 123.77(17) N6 C36 C35 115.08(18) C38 C47 C48 130.75(18) C3 C2 C1 120.5(2) C4 C3 C2 120.4(2) O5 C50 C52 122.5(3) C5 C6 C1 119.9(2) C5 C4 C3 119.5(2) O5 C50 C51 119.9(4) From the Table 6, it shows that compound 8b led to a percentage inhibition in the paw diameter of the mice. Table 6 reveals the percentage inhibition of paw edema of the different test groups. In this study, it was found that the standard drug ibuprofen could produce the highest percentage inhibition of up to 66.67 % at four hours but this inhibition was found to be absent at 24 hours. The highest percentage inhibition of the edema at 24 hours after intraperitoneal (i.p) drug administration was seen in case of test compound 8b (25 %) and 13a (16.57 %), respectively, while at four hours, this effect was seen to be highest in case of test compound 8b, followed by test compounds 9 and 13a, 13b, 6a, 6c, 6e and 10, respectively. 3.3.1.2. Concentration of nitric oxide (NO) This assay was used to assess the potentiality of the test compounds as anti-inflammatory agents. As nitric oxide is produced by inducible nitric oxide synthase (iNOS) in activated macrophages during inflammation, this compound therefore can serve as a good biological marker for inflammation. This means that the agents that can inhibit the over production of nitric oxide may have anti-inflammatory activities [36,40]. The concentration of nitric oxide was assessed in paw exudates and whole blood. Of the different compounds tested, compound 13b was found to exert marked reduction in the concentration of NO in paw exudates (Figure 2) while in blood, compound 6a showed some magnitude of reduction (Figure 3). 3.3.1.3. Differential WBC count in the blood During inflammation, a variety of cells such as neutrophils, eosinophils and basophils mediate an effective immune response [41]. Differential leukocyte count in blood smear was performed for the test compounds (Table 7) to study its anti- inflammatory properties. When compared with the untreated blood sample count, the blood samples that showed a reduc- tion in the percentage count of the neutrophils, eosinophils and basophils might have anti-inflammatory effect. Most of the compounds tested resulted in lower counts of both neutrophils and eosinophils as compared to control mice. The percentage count of neutrophils was found to be significantly reduced in case of 8b and 13b (p < 0.0005***), the effects of which are higher than that of ibuprofen (p < 0.001**) treated mice. The test compound 13a showed comparable effects to ibuprofen in the reduction of neutrophils (p < 0.001**). In the reduction of eosinophils, test compounds 10 and 8a (p < 0.001**) showed comparable activities to ibuprofen (p < 0.001**), while test compound 8b showed lower effect (p < 0.005*). It is clearly understood that the compounds with the highest ability to reduce the marked indicators of inflammation, neutrophils and eosinophils are test compounds 8b and 13b. 3.3.2. Anti-cancer assay 3.3.2.1. MTT-based cytotoxic assay The anti-cancer screening of the synthesized compounds were performed in cultured Chinese hamster ovary K1 (CHO K1) cell lines. The ability of the enzyme succinate tetrazolium reductase to metabolize 3-[4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide to purple formazan product after treatment with the test compounds in comparison to the untreated (control) cells was assessed. It was found that the test compounds 6a, 6b, 8a, 8b, 13a, and 13b, resulted in a decrease in metabolism of MTT by CHO K1 cells (Table 8). In particular, test compound 6a produced the lowest metabolism of MTT by these cells (Figure 4), followed by test compounds 8b, 13a. This assay is commonly used to monitor cell viability and proliferation and hence can also be used for assessing cytotoxicity [36,42]. 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 76 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 Table 6. Percentage inhibition of paw edema in two different test groups, one administered with test compounds and the other with ibuprofen. Test groups Mean paw diameter ± SEM (mm) Before FCA injection 1 h 2 h 3 h 4 h 24 h Control 2±0.00 3.5±0.00 3.7±0.17 3.3±0.17 3.5±0.29 3.2±0.57 Ibuprofen 2±0.00 4.2±0.17 3.8±0.17 3.6±0.28 3.0±0.28 3.8±0.17 6a 2±0.00 4.3±0.29 3.9±0.06 3.3±0.35 3.2±0.17 3.8±0.46 6b 2±0.00 4.0±0.00 4.0±0.00 3.7±0.23 3.8±0.17 4.3±0.46 6c 2±0.00 4.0±0.00 3.1±0.05 3.2±0.17 3.3±0.17 3.5±0.29 6d 2±0.00 4.0±0.00 3.7±0.34 3.5±0.29 3.5±0.35 3.9±0.57 6e 2±0.00 4.0±0.00 3.9±0.06 3.5±0.17 3.3±0.00 3.2±0.17 8a 2±0.00 3.8±0.17 3.9±0.06 3.6±0.17 3.5±0.17 3.2±0.17 8b 2±0.00 3.7±0.17 3.3±0.12 2.9±0.06 2.8±0.00 2.9±0.06 9 2±0.00 3.9±0.00 3.5±0.00 3.3±0.00 2.9±0.06 3.2±0.17 10 2±0.00 4.0±0.00 3.6±0.12 3.1±0.06 3.3±0.12 4.0±0.00 13a 2±0.00 3.7±0.17 3.5±0.17 3.3±0.23 2.9±0.06 3.0±0.12 13b 2±0.00 4.0±0.00 3.3±0.17 3.1±0.17 3.0±0.12 3.2±0.17 Test groups Mean increase in paw diameter (mm) 1 h 2 h 3 h 4 h 24 h Control 1.5 1.7 1.3 1.5 1.2 Ibuprofen 2.0 1.2 1.0 0.5 1.8 6a 2.3 1.9 1.3 1.2 1.8 6b 2.0 2.0 1.7 1.8 2.3 6c 2.0 1.1 1.2 1.3 1.5 6d 2.0 1.7 1.5 1.5 1.9 6e 2.0 1.9 1.5 1.3 1.2 8a 1.8 1.9 1.6 1.5 1.2 8b 1.7 1.3 0.9 0.8 0.9 9 1.9 1.5 1.3 0.9 1.2 10 2.0 1.6 1.1 1.3 2.0 13a 1.7 1.5 1.3 0.9 1.0 13b 2.0 1.3 1.1 1.0 1.2 Test groups Percentage inhibition in paw diameter 1 h 2 h 3 h 4 h 24 h Control Ibuprofen -33.33 29.41 23.07 66.67 -50.00 6a -53.33 -11.76 0.00 20.00 -50.00 6b -33.33 -17.65 -30.77 -20.00 -91.67 6c -33.33 35.29 7.69 13.33 -25.00 6d -33.33 0.00 -15.38 0.00 -58.33 6e -33.33 -11.76 -15.38 13.33 0.00 8a -20.00 -11.76 -23.08 0.00 0.00 8b -13.33 23.53 30.77 46.67 25.00 9 -26.67 11.76 0.00 40.00 0.00 10 -33.33 5.88 15.38 13.33 -66.67 13a -13.33 11.76 0.00 40.00 16.57 13b -33.33 25.53 15.38 33.33 0.00 Table 7. Differential WBC count in three different test groups: untreated paw edema bearing mice (control), ibuprofen treated paw edema bearing mice and test compounds treated paw edema bearing mice. The values are ±SEM (* denote significance against paw edema bearing control mice at p < 0.0005***, p < 0.001**, p < 0.005*). Test groups Lymphocytes Neutrophils Eosinophils Basophils Monocytes Control 62.67 14.00 6.00 8.67 8.67 Ibuprofen 86.67 4.00 2.00 6.67 0.33 6a 68.00 16.00 6.00 7.30 2.70 6b 77.33 8.67 6.00 6.00 2.00 6c 69.33 19.33 7.33 3.33 0.67 6d 64.67 19.33 9.33 4.67 2.00 6e 78.67 10.00 8.00 2.67 0.67 8a 82.67 6.33 2.00 9.00 0.00 8b 93.00 1.67 3.00 3.00 0.00 9 76.00 10.00 8.67 4.67 0.67 10 71.33 27.33 0.67 0.67 0.00 13a 79.67 6.67 5.00 7.00 1.67 13b 88.00 3.00 4.00 3.00 2.00 Table 8. MTT assay in CHO K1 cell lines after exposure to 20 µL of 10 mg/mL test compounds (in 10 % DMSO). Absorbance was recorded at 570 nm. Cells exposed to 10 % DMSO served as control. Values are mean ± SD of three readings. Test compounds Absorbance at 570 nm Standard deviation Control 0.730 ±0.3400 6a 0.020 ±0.0145 6b 0.445 ±0.0488 6c 0.726 ±0.3530 6d 0.705 ±0.1321 6e 0.687 ±0.6026 8a 0.394 ±0.2100 8b 0.197 ±0.0600 9 0.654 ±0.0770 10 0.646 ±0.0682 13a 0.201 ±0.0505 13b 0.370 ±0.0912 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 77 ** *** 0 1 2 3 4 5 6 7 8 9 C on ce nt ra tio n of N O in p aw e xu da te s (µ M ) Test groups Control Ibuprofen 6a 6b 6c 6d 6e 8a 8b 9 10 13a Figure 2. Concentration of nitric oxide in paw exudates of different test groups (treated with test compounds and ibuprofen) in comparison to the control (untreated) paw edema bearing mice. The edema was induced by injection of FCA into the plantar side of left hind paw of the mice. The mice (except the control) were then treated with the compounds (50 mg/kg body weight) via i.p injection. After 24 h, the paws were excised, weighed, homogenized and centrifuged at 12,000 rpm in 10 per cent ice-cold normal saline. The supernatant was collected and used for NO assay. Absorbance was recorded at 520 nm and the concentration of NO was calculated using the standard nitrite curve. Values are expressed as mean ±SEM (*denotes significance against paw edema bearing control mice at p < 0.0005***, p < 0.001**, p < *0.005). *** * 0 5 10 15 20 25 30 35 40 45 50 C on ce nt ra tio n of N O in b lo od (µ M ) Test groups Control Ibuprofen 6a 6b 6c 6d 6e 8a 8b Figure 3. Concentration of nitric oxide (NO) in blood groups of treated and untreated paw edema bearing mice. Untreated groups served as control. The edema was induced by injection of FCA into the plantar side of the left hind paw of the mice. These mice (except the control) were then treated with the compounds (50 mg/kg body weight) via i.p injection. After 24 h, blood was collected by retro-orbital bleeding and used for nitric oxide assay. Absorbance was recorded at 520 nm and the concentration of nitric oxide was quantified using the standard nitrite curve. Values are expressed as mean ±SEM (*denotes significance against paw edema bearing control mice at p < 0.0005***, p < 0.001**, p < *0.005). ±0.34 ±0.0145 ±0.0488 ±0.353 ±0.1321 ±0.6026 ±0.21 ±0.06 ±0.077 ±0.0682 ±0.0505 ±0.0912 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Ab so rb an ce a t 5 70 nm Test groups Figure 4. The cytotoxic effects of the test compounds on CHO K1 cell lines were compared against the control using the MTT based assay. CHO K1 cells were grown in microtiter plates and treated with 20 µL of 10 mg/mL test compounds (in 10 % DMSO). Their ability to metabolise MTT was measured by taking the absorbance at 570 nm. Cells exposed to 10 % DMSO were used as control. This means that the lower the production of purple formazan in cells treated with the test compounds, the lower is the number of viable cells, the higher is their cytotoxic effects and hence the greater is their potential as anticancer compounds. Hence, these compounds 6a, 6b, 8a, 8b, 13a, 13b may be concluded to have cytotoxic effect on CHO K1 cell lines. 4. Conclusion We were able to successfully combine the bio-labile rings together in a molecular framework. The synthetic protocol features a new route that utilizes ultrasound irradiation for the diversification of pyrazolopyrimidine derivatives. The 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 78 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 starting materials used were developed by methods reported from our group from the commercially available active proton compounds suggesting that further modification of this component may be a viable strategy for future development of bioactive compound libraries. From the results obtained, the test compound 13b can be selected as a good candidate for anti-inflammatory effects. This test compound showed an ability to decrease the paw edema up to 33.33 % at four hours and also show a potential to decrease the concentration of nitric oxide to a level that is at par with the known drug ibuprofen. On the basis of the anti-cancer assay, test compound 6a was found to show the maximum cytotoxic effect, followed by compounds 8b, 13a, 13b, 8a and 6b, respectively. The percentage inhibition of these test compounds was found to be compounds 6a (97.10 %), 8b (72.96 %), 13a (72.46 %), 13b (49.24 %) and 8a (45.75 %). Further investigation on these compounds are therefore crucial in order to elucidate the exact mechanism of action on target cells and hence to therefore add them in the list of potent anti-inflammatory and anti-cancer drugs. Acknowledgements Authors wish to thank Rev. Fr. Dr. Stephen Mavely, Vice Chancellor, Assam Don Bosco University for providing infrastructure for the execution of this work. Authors also wish to express their gratitude to IIT, Guwahati, Tezpur University, Tezpur, SAIF-NEHU, Shillong and SAIF-CDRI, Lucknow. Our thanks are also due to the Department of Biotechnology (DBT), Government of India for a research grant and the Department of Biotechnology (DBT), St. Anthony’s College, Shillong for carrying out the biological assays. SK thanks NER-BPMC-DBT, New Delhi for a research fellowship. This manuscript is a part of the PhD thesis of Dr. Shunan Kaping, Assam Don Bosco University, India, 2016 [www.shodhganga.inflibnet.ac.in] Supporting information CCDC-1401935 contains the supplementary crystallo- graphic data for this paper. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/, or by e- mailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. Funding Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi-110 003, India. ORCID Shunan Kaping http://orcid.org/0000-0001-7898-5039 Melboureen Sunn http://orcid.org/0000-0002-9377-4544 Laishram Indira Singha http://orcid.org/0000-0002-0360-2168 Jai Narain Vishwakarma http://orcid.org/0000-0001-9068-4554 References [1]. Elkhawaga, A. M.; El-Dean, A. M. K.; Radwan, S. M.; Ahmed, M. M. Bull. Korean Chem. Soc. 2009, 30, 561-566. [2]. Dreassi, E.; Zizzari, A. T.; Mori, M.; Fillipi, I.; Belfiore, A.; Naldini, A.; Carraro, F.; Santucci, A.; Shenone, S.; Botta, M. Eur. J. Med. Chem. 2010, 45, 5958-5964. [3]. Earl, R. A.; Pugmire, R. J.; Revanker, G. R.; Townsend, L. B. J. Org. Chem. 1975, 40, 1822-1828. [4]. Curran, K. J.; Verheijen, J. C.; Kaplan, J.; Richard, D. J.; Toral-Barza, L.; Hollander, I.; Lucas, J.; Ayral-Kaloustian, S.; Yu, K.; Zask, A. Bioorg. Med. Chem. Lett. 2010, 20, 1440-1444. [5]. Hassan, A. S.; Hafez, T. S.; Osman, S. A. Sci. Pharm. 2015, 83, 27-39. [6]. Ferrari, S. M.; Motta, C. 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C 2006, 62, 186-189. 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 http://www.shodhganga.inflibnet.ac.in/ https://www.ccdc.cam.ac.uk/structures/ mailto:data_request@ccdc.cam.ac.uk http://orcid.org/0000-0001-7898-5039 http://orcid.org/0000-0002-9377-4544 http://orcid.org/0000-0002-0360-2168 http://orcid.org/0000-0001-9068-4554 Kaping et al. / European Journal of Chemistry 11 (1) (2020) 68-79 79 [39]. Ahmad, F.; Khan, R.; Rasheed, S. J. Islamic Acad. Sci. 1992, 5, 111-114. [40]. Kang, O. H.; Chae, H. S.; Oh, Y. C.; Choi, J. G.; Lee, Y. S.; Jang, H. J.; Kim, J. H.; Kim, Y. C.; Sohn, D. H.; Park, H.; Kwon, D. Y. Am. J. Chin. Med. 2008, 36, 913-928. [41]. Khan, F. H. The Elements of Immunology, 1st edition, Pearson Education India, 2009. [42]. Alam, A.; Imliwati, L.; Rapthap, C.; Singh, V. Ind. J. Exp. Biol. 2001, 39, 201-208. Copyright © 2020 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.1.68-79.1942 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Chemistry 2.1.1. Material and methods 2.1.2. Synthesis 2.1.2.1. Synthesis of antipyrinyl-7-arylpyrazolo[1,5-a] pyrimidines (6a-e) 2.1.2.2. Synthesis of 7-amino-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-6-arylpyrazolo[1,5-a] pyrimidine-3-carboxamide (8a,b) 2.1.2.3. Synthesis of antipyrinyl-7-hetarylpyrazolo[1,5-a] pyrimidines (9,10) 2.1.2.4. Synthesis of 6-acetyl/carboalkoxy-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-7-methyl pyrazolo[1,5-a]pyrimidine-3-carboxamide (13a, b) 2.2. Biology 2.2.1. Material and methods 2.2.1.1. Anti-inflammatory test 2.2.1.1.1. Measurement of paw edema 2.2.1.1.2. Nitric oxide assay 2.2.1.2. Anti-cancer assay 3. Results and discussion 3.1. Chemistry 3.2. X-ray crystallography 3.3. Biological activities 3.3.1. Anti-inflammatory assay 3.3.1.1. Percentage Inhibition of Paw Diameter 3.3.1.2. Concentration of nitric oxide (NO) 3.3.1.3. Differential WBC count in the blood 3.3.2. Anti-cancer assay 3.3.2.1. MTT-based cytotoxic assay 4. Conclusion Acknowledgements Supporting information Disclosure statement Funding ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField110: PrintField111: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: PrintField210: PrintField211: