Efficient synthesis of diversely substituted pyrazolo[1,5-a]pyrimidine derivatives promoted by ultrasound irradiation in water and their antibacterial activities European Journal of Chemistry 11 (4) (2020) 304-313 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.11.4.304-313.2033 European Journal of Chemistry View Journal Online View Article Online Efficient synthesis of diversely substituted pyrazolo[1,5-a]pyrimidine derivatives promoted by ultrasound irradiation in water and their antibacterial activities Susma Das 1, Shilpika Khanikar 1, Shunan Kaping 1, Jayanti Datta Roy 2, Arnab Sen 2, Philippe Helissey 3 and Jai Narain Vishwakarma 1 1 Organic Research Laboratory, Department of Chemical Science, Assam Don Bosco University, Tapesia Gardens, Sonapur 782402, Assam, India susmadas91@gmail.com (S.D.), shilpikakhanikar11@gmail.com (S.K.), shunankaping@yahoo.in (S.K.), jnvishwakarma@rediffmail.com (J.N.V.) 2 Indian Council of Agricultural Research Complex for North Eastern Hill Region, Umiam 793103, Meghalaya, India jayntidr@gmail.com (J.D.R.), arnabsen123@gmail.com (A.S.) 3 Laboratoire de Chimie Thérapeutique, UMR CNRS 8638, Faculte des Sciences Pharmaceutiques, Université Paris Descartes, Sorbonne Paris Cité 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France philippe.helissey@parisdescartes.fr (P.H.) * Corresponding author at: Organic Research Laboratory, Department of Chemical Science, Assam Don Bosco University, Tapesia Gardens, Sonapur 782402, Assam, India. e-mail: jnvishwakarma@dbuniversity.ac.in (J.N. Vishwakarma). 10.5155/eurjchem.11.4.304-313.2033 Received: 25 August 2020 Received in revised form: 30 September 2020 Accepted: 02 October 2020 Published online: 31 December 2020 Printed: 31 December 2020 A green synthetic route leading to the discovery of a series of diversely substituted pyrazolo[1,5-a]pyrimidines, having CO2Et group embedded at position-2 has been unraveled in this article. A series of formylated active proton compounds that were chosen to react with a carboxylate substituted-3-aminopyrazole under ultrasonic irradiation in the presence of a mild acid as a catalyst and aqueous ethanol medium afforded the desired products. The molecular structures of all these synthesized compounds were established by their spectral and analytical data. A model molecule 3d, subjected to single-crystal X-ray crystallography analysis further confirms their molecular structure. The crystal crystallized to a monoclinic cell with P21/c space group, a = 7.468 (5) Å, b = 27.908 (17) Å, c = 7.232 (4) Å, β = 104.291 (7)o, V =1460.7(15) Å3, Z = 4, μ(MoKα) = 0.096 mm-1, Dcalc = 1.352 Mg/m3 16667 measured reflection (5.63 ≤ 2Θ ≤ 57.57°), 3720 unique (Rint = 0.0965, Rsigma = 0.0945) which were used in all calculations. The final R1 was 0.0750 (I > 2σ(I)) and wR2 was 0.2226 (all data). These compounds were further explored for their antibacterial potential, and a few of them have exhibited encouraging results. Enaminone Single crystal 3-Aminopyrazole Ultrasonic irradiation X-ray crystallography Antibacterial properties Cite this: Eur. J. Chem. 2020, 11(4), 304-313 Journal website: www.eurjchem.com 1. Introduction Synthesis of pyrazolo[1,5-a]pyrimidines has always been a hotbed for organic and pharmacological studies due to its analogy to purine and thus has occupied a unique position in the design and synthesis of biologically active agents, thereby providing a very interesting core for drug developments [1-3]. The drugs like Zaleplon (1) and Indiplon (2) which are very effectively used for the treatment of sleep disorders or Ocinaplon (3) used as anxiolytic agents, all contain pyrazolo [1,5-a]pyrimidine as their structural core. They have been reported to have the advantages of rapid absorption, rapid onset, adequate duration of action, and no residual effect on daytime performance [4-8]. Inspired by this, our group has reported various pyrazolo[1,5-a]pyrimidine hybrids (4a-b, 5, 6, 7, 8(a-c)) all of which exhibit various bio-efficiencies (Figure 1) [9-13]. Motivated by this notable significance of pyrazolo[1,5- a]pyrimidine and in continuation with our research activities, we settled to further add on to this class of compounds. We settled to synthesize pyrazolo[1,5-a]pyrimidines with a carboethoxy group (CO2Et) in position 2 of the pyrazolo[1,5- a]pyrimidine ring system. Our group anticipated that the presence of CO2Et group could act as a bridge or connectors to various other nucleophilic molecules, which themselves have significant biological implications. This alteration could bulge on to novel compounds with unique properties or can supplement to the individual parent properties. In this article, we have reported a successful synthesis of pyrazolo[1,5-a]pyrimidines with the desired CO2Et group at position 2. The novel pyrazolo[1,5-a]pyrimidine derivatives and some known pyrazolo[1,5-a]pyrimidine analogues were accomplished using one of our previously reported methods ABSTRACT RESEARCH ARTICLE KEYWORDS http://dx.doi.org/10.5155/eurjchem.11.4.304-313.2033 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.11.4.304-313.2033 mailto:susmadas91@gmail.com mailto:shilpikakhanikar11@gmail.com mailto:shunankaping@yahoo.in mailto:jnvishwakarma@rediffmail.com mailto:jayntidr@gmail.com mailto:arnabsen123@gmail.com mailto:philippe.helissey@parisdescartes.fr mailto:jnvishwakarma@dbuniversity.ac.in http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.11.4.304-313.2033&domain=pdf&date_stamp=2020-12-31 Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 305 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 Figure 1. Pyrazolo[1,5-a]pyrimidine derivatives are marketed as drugs (1, 2, and 3), and a few of our previously reported pyrazolo[1,5-a]pyrimidine derivatives 4a-8c. wherein we have used ultrasonic irradiation in aqueous media assisted by a mild acid [10]. Some of the compounds although already reported in the literature [14-18], our method has the preference of relatively lower reaction time and higher yield using eco-friendly solvents compared to those used in literature. Thus, our method is extrapolated in synthesizing a quite different range of azolopyrimidine derivatives adding to its other advantages of being a green, short reaction time, use of aqueous media and high yield synthetic route. Enaminones are an important class of synthons for the synthesis of pyrimidine rings from aminopyrazoles. A wide range of precursor enaminones required were synthesized via the reported procedure from our laboratory [19]. These active methylene compounds were then irradiated with 3-amino- pyrazole as mentioned above, yielding a series of target pyrazolo[1,5-a]pyrimidines analogues. Furthermore, these compounds were screened for their antibacterial activities. 2. Experimental 2.1. Instrumentations The melting points of each of the compounds were recorded by the open capillary method and are uncorrected. 1H and 13C NMR spectra were recorded on a Bruker AV-II 400 and 300 MHz, using (CH3)4Si as the internal standard in CDCl3. In the NMR spectral data, the abbreviations s, d, dd, t, and m stand for singlet, doublet, double-doublet, triplet, and multiplet, respect- tively. Chemical shift (δ, ppm) and coupling constant (Hz) are reported in a standard fashion. The electron spray mass spectra were recorded on a THERMO Finnigan LCQ Advantage max ion trap mass spectrometer. The FT-IR spectra were recorded on a Perkin-Elmer Spectrum Two spectrometer. The X-ray diffract- tion data were collected at 293 K with MoKα radiation (λ = 0.71073 Å) using a Bruker APEX-II CCD (Charge Coupled Device) [20] diffractometer equipped with a graphite mono- chromator. The structures were solved using SHELXT [21] and refined with SHELXL [22] by full-matrix least-squares based on F2. All non-H-atoms were refined in the anisotropic approxi- mation: H-atoms were located at the calculated positions. The details of the structure of compound 3d have been deposited with the Cambridge Crystallographic Data Centre No. CCDC- 1886698. 2.2. Synthesis 2.2.1. Synthesis of ethyl 7-(substituted-phenyl)pyrazolo[1,5- a]pyrimidine-3-carboxylate (3a-ii) A mixture of ethyl-3-amino-1H-pyrazole-4-carboxylate (1) (0.5 mmol) and enaminones 2a-f (0.5 mmol) was dissolved in 2 mL ethanol and to this was added a 2 mL aqueous solution of KHSO4 (1 mmol). 306 Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 Scheme 1. Optimization of reaction conditions. Scheme 2. Synthesis of compounds 3b-3i, 3ii. The resulting mixture was then subjected to ultrasound irradiation at room temperature (60-65 °C for 2g-i). The progress of the reaction was monitored by thin-layer chroma- tography and on completion (6-12 minutes), the reaction mixture was cooled to room temperature and the precipitated product was collected by filtration, washed repeatedly with water to ensure complete removal of the acid and finally dried to give practically pure products 3a-ii in 78-96 % yields (Schemes 1 and 2). Further purification of the products was achieved by column chromatography (silica gel, 20 % Ethyl acetate:Hexane). Ethyl 7-phenylpyrazolo[1, 5-a]pyrimidine-3-carboxylate (3a): Color: Milky white crystals. Yield: 95 %. M.p.: 133-134 °C. FT-IR (KBr, ν, cm-1): 1677 (C=O) (ester), 1613 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.43 (t, J = 7.2 Hz, 3H, CH3), 4.46 (q, J = 7.2 Hz, 2H, CH2), 7.08 (d, J = 4.4 Hz, 1H, C6-H), 7.57- 7.60 (m, 3H, aromatic), 7.99-8.01 (m, 2H, aromatic), 8.60 (s, 1H, C2-H), 8.81 (d, J = 4.4 Hz, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.7, 60.5, 103.3, 109.2, 128.9, 129.6, 130.3, 131.7, 147.5, 148.1, 1 49.0, 152.4, 162.7. MS (EI, m/z (%)): 268 (MH)+. Ethyl 7-(4-chlorophenyl)pyrazolo[1, 5-a]pyrimidine-3-car boxylate (3b): Color: White solid. Yield: 87 %. M.p.:158-159 °C (159-160 °C) [14]. FT-IR (KBr, ν, cm-1): 1720 (C=O) (ester), 1617 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.43 (t, 3H, J = 7.2 Hz, CH3), 4.46 (q, 2H, J = 7.2 Hz, CH2), 7.07 (d, 1H, J = 4.4 Hz, C6-H), 7.56 (d, J = 8.4 Hz, 2H, aromatic), 7.98 (d, J = 8.4 Hz, 2H, aromatic), 8.60 (s, 1H, C2-H), 8.81 (d, J = 4.4 Hz, 1H, C5- H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.7, 60.6, 103.5, 109.0, 128.73, 129.3, 131.0, 138.1, 146.9, 147.6, 149.1, 152.4, 162.6. MS (EI, m/z (%)): 302 (MH)+. Ethyl 7-(4-methylphenyl)pyrazolo[1, 5-a]pyrimidine-3- car boxylate (3c): Color: Milky white solid. Yield: 94 %. M.p.: 115 °C (112-114 °C) [14]. FT-IR (KBr, ν, cm-1): 1689 (C=O) (ester), 1608 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.43 ( t, 3H, J = 7.2 Hz, CH3), 2.46 (s, 3H, CH3), 4.46 (q, 2H, J = 7.2 Hz, CH2), 7.06 (d, J = 4.4 Hz, 1H, C6-H), 7.38 (d, J = 8.0 Hz, 2H, aromatic), 7.91 (d, J = 8.0 Hz, 2H, aromatic), 8.59 (s, 1H, C2- H), 8.79 (d, J = 4.4 Hz, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.7, 21.7, 60.5, 103.1, 108.8, 127.4, 129.5, 129.6, 142.3, 147.5, 148.2, 149.1, 152.4, 162.7. MS (EI, m/z (%)): 282 (MH)+. Ethyl 7-(4-methoxyphenyl)pyrazolo[1, 5-a]pyrimidine-3- car boxylate (3d): Color: Pale yellow crystals. Yield: 93 %. M.p.:133-134 °C (131-133 °C) [14]. FT-IR (KBr, ν, cm-1): 1689 (C=O) (ester), 1611 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm):1.43 (t, J = 7.2 Hz, 3H, CH3), 3.90 (s, 3H, OCH3), 4.46 (q, J = 7.2 Hz, 2H, CH2), 7.05 (d, J = 4.4 Hz, 1H, C6-H), 7.07-7.10 (m, 2H, aromatic), 8.03-8.06 (m, 2H, aromatic), 8.60 (s, 1H, C2-H), 8.76 (d, J = 4.4 Hz, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.7, 55.6, 60.4, 103.0, 108.4, 114.4, 122.4, 131.4, 147.4, 147.8, 149.2, 152.3, 162.3, 162.7. MS (EI, m/z (%)): 298 (MH)+. Ethyl 7-(4-bromophenyl)pyrazolo[1, 5-a]pyrimidine-3- car boxylate (3e): Color: Yellow solid. Yield: 79 %. M.p.: 162- 163 °C. FT-IR (KBr, ν, cm-1): 1719 (C=O) (ester), 1616 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.43 (t, J = 6.8 Hz, 3H, CH3), 4.46 (q, J = 6.8 Hz, 2H, CH2), 7.08 (s, 1H, C6-H), 7.73 (d, J = 8 Hz, 2H, aromatic), 7.91 (d, J = 8 Hz, 2H, aromatic), 8.60 (s, 1H, C2-H), 8.82 (s, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.5, 60.4, 103.3, 108.8, 126.3, 129.0, 130.9, 132.1, 146.7, 147.4, 148.9, 152.3, 162.4. MS (EI, m/z (%)): 346 (M)+. Ethyl 7-([1,1'-biphenyl]-4-yl)pyrazolo[1, 5-a]pyrimidine-3- carboxylate (3f): Color: Pale Yellow crystals. Yield: 78 %. M.p.: 172-174 °C. FT-IR (KBr, ν, cm-1): 1693 (C=O) (ester), 1629 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.43 (t, J = 9.6 Hz, 3H, CH3), 4.47 (q, J = 9.6 Hz, 2H, CH2), 7.12 (d, J = 4.2 Hz, 1H, C6-H), 7.37‒7.50 (m, 3H, aromatic), 7.64-7.67 (m, 2H, aromatic), 7.79 (d, J = 11.2 Hz, 2H, aromatic), 8.10 (d, J = 11.2 Hz, 2H, aromatic), 8.63 (s, 1H, C2-H), 8.81 (d, J = 4.2 Hz, 1H, C5- H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.7, 60.5, 103.3, 109, 127.3, 127.6, 128.3, 129.0, 129.1, 130.1, 139.9, 144.6, 147.5, 147.7, 149.1, 152.4, 162.7. MS (EI, m/z (%)): 344 (MH)+. Ethyl 7-(4-nitrophenyl)pyrazolo[1, 5-a]pyrimidine-3-car boxylate (3g): Color: Yellow solids. Yield: 91 %. M.p.: ˃235 °C. FT-IR (KBr, ν, cm-1): 1717 (C=O) (ester), 1598 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.44 (t, 7.2 Hz, 3H, CH3), 4.47 (q, J = 7.2 Hz, 2H, CH2), 7.14 (d, J = 4 Hz, 1H, C6-H), 8.22 (d, J = 8.8 Hz, 2H, aromatic), 8.44 (d, J = 8.8 Hz, 2H, aromatic), 8.62 (s, 1H, C2-H), 8.88 (d, J = 4.4 Hz, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 13C NMR (100 MHz, CDCl3): 14.7, 60.8, 104.1, 109.7, 124.1, 130.9, 136.2, 145.5, 147.8, 149.0, 149.5, 152.4, 162.4. MS (EI, m/z (%)): 313 (MH)+. Ethyl 7-(pyridin-3-yl)pyrazolo[1, 5-a]pyrimidine-3-car boxylate (3h): Color: White solid. Yield: 82 %. M.p.: 190-192 °C (177-178 °C) [16]. FT-IR (KBr, ν, cm-1): 1690 (C=O) (ester), 1546 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.41 (t, J = 7.2 Hz, 3H, CH3), 4.44 (q, J = 7.2 Hz, 2H, CH2), 7.14 (d, J = 4.4 Hz, 1H, C6-H), 7.51-7.55 (dd, J = 8 Hz, 1H, 3.6 Hz, aromatic), 8.48 (d, J = 8 Hz, 1H, aromatic), 8.59 (s, 1H, C2-H), 8.80 (d, J = 3.6 Hz, 1H, aromatic), 8.86 ( d, J = 4.4 Hz, 1H, C5-H), 9.15 (s, 1H, aromatic). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.5, 60.4, 103.6, 109.0, 123.2, 126.5, 137.1, 144.8, 147.4, 148.7, 149.6, 152.1, 152.2, 162.2. MS (EI, m/z (%)): 268 (M)+. Ethyl 5-(pyridin-2-yl)pyrazolo[1, 5-a]pyrimidine-3-car boxylate (3i): Color: Milky White. Yield: 31 %. M.p.: 139-140 °C. FT-IR (KBr, ν, cm-1): 1701 (C=O) (ester), 1622 (C=C) (aromatic). Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 307 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 Scheme 3. Synthesis of compounds 3j-3l. 1H NMR (400 MHz, CDCl3, δ, ppm): 1.46 (t, J = 7.2 Hz, 3H, CH3), 4.44 (q, J = 7.2 Hz, 2H, CH2), 7.39-7.43 (m, 1H, aromatic), 7.85-7.90 (m, 1H, aromatic), 8.23 (d, J = 7.6 Hz, 1H, C6-H), 8.57 (s, 1H, aromatic), 8.70 (s, 1H, C2-H), 8.71 (s, 1H, aromatic), 8.79 (d, J = 7.2 Hz, 1H, C7-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.5, 60.2, 103.3, 107.1, 122.4, 125.7, 136.0, 137.1, 147.3, 148.1, 149.2, 153.2, 158, 162.5. MS (EI, m/z (%)): 269 (MH)+. Ethyl 7-(pyridin-2-yl)pyrazolo[1, 5-a]pyrimidine-3-carboxy late (3ii): Color: White solid. Yield: 66 %. M.p.: 143-144 °C (145 °C) [16,18]. FT-IR (KBr, ν, cm-1): 1701 (C=O) (ester), 1622 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.44 (t, J = 6.8 Hz, 3H, CH3), 4.47 (q, J = 6.8 Hz, 2H, CH2), 7.48-7.51 (m, 1H, aromatic), 7.86 (d, J = 3.6 Hz, 1H, C6-H), 7.95 (t, J = 7.2 Hz, 1H, aromatic), 8.65 (s, 1H, C2-H), 8.82-8.83 (m, 1H, aromatic), 8.89 (d, J = 3.6 Hz, 1H,C5-H), 8.98 (d, J = 7.6 Hz,1H, aromatic). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.5, 60.4, 103.1, 109.6, 125.8, 126.4, 136.7, 145.2, 147.2, 147.6, 149.1, 150.1, 152.4, 162.5. MS (EI, m/z (%)): 269 (MH)+. 2.2.2. Synthesis of ethyl 7-methylpyrazolo[1,5-a]pyrimidine- 3-carboxylate (3j-l) To a solution of ethyl-3-amino-1H-pyrazole-4-carboxylate (1) (0.5 mmol) and enaminones (2j-l) (0.5 mmol) in 2 mL ethanol was added KHSO4 (1 mmol) dissolved in 2 mL of water. The mixture thus obtained was irradiated in an ultrasonic bath at 60-65 °C for compound 2j and at room temperature for compounds 2k and 2l. On completion of the reaction (12-18 minutes, monitored by TLC), the reaction mixture was allowed to stand for 1 hour. The precipitated product was collected by filtration, washed repeatedly with water to remove traces of acid, and finally dried to give practically pure product 3j-l in 83- 96 % yields (Scheme 3). The products were further purified to give analytically pure products by column chromatography (silica gel, 20 % Ethyl acetate:Hexane). Ethyl 6-acetyl-7-methylpyrazolo[1,5-a]pyrimidine-3-carboxy late (3j) [15]: Color: Pale brown crystals. Yield: 87 %. M.p.:147- 148 °C. FT-IR (KBr, ν, cm-1): 1726 (C=O) (ester), 1603(C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.40 (t, J = 7.2 Hz, 3H, CH3), 2.55-2.75 (m, 3H, CH3), 3.15 (s, 3H, CH3), 4.43 (q, J = 7.2 Hz, 2H, CH2), 8.63 (s, 1H, C2-H), 9.07 (s, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.4, 15.2, 29.6, 60.3, 104.0, 119.3, 147.1, 149.0, 151.4, 151.8, 162.0, 196.0. MS (EI, m/z (%)):248 (MH)+. Diethyl 7-methylpyrazolo[1,5-a]pyrimidine-3,6-dicarboxy late (3k) [15]: Color: Milky white solid. Yield: 76 %. M.p.: 140- 141 °C. FT-IR (KBr, ν, cm-1): 1730 (C=O) (ester), 1610 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.40-1.46 (m, 6H, 2CH3), 3.23 (s, 3H, CH3), 4.41-4.48 (m, 4H, 2CH2), 8.63 (s, 1H, C2- H), 9.18 (s, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.2, 14.4, 15.1, 60.5, 61.9, 104.1, 112.5, 147.9, 148.6, 152.3, 152.9, 162.1, 164.0. MS (EI, m/z (%)): 278 (MH)+. 3-Ethyl-6-methyl 7-methylpyrazolo[1,5-a]pyrimidine-3,6- dicarboxylate (3l) [15]: Color: Milky white crystals. Yield: 84 %. M.p.: 131-132 °C. FT-IR (KBr, ν, cm-1): 1726 (C=O) (ester), 1706 (C=O) (ester). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.41 (t, J = 6.4 Hz, 3H, CH3), 3.23 (s, 3H, CH3), 3.99 (s, 3H, OCH3), 4.43 (q, J = 6.4 Hz, 2H, CH2), 8.62 (s, 1H, C2-H), 9.17 (s, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.4, 15.2, 52.7, 60.5, 104.2, 112.2, 147.9, 148.7, 152.5, 152.8, 162.0, 164.4. MS (EI, m/z (%)): 264 (MH)+. 2.2.3. Attempted synthesis of ethyl 8,8-dimethyl-6-oxo- 6,7,8,9-tetrahydropyrazolo[1,5-a]quinazoline-3- carboxylate (3m) A mixture of 0.5 mmol of dimedone and 1 mmol of N,N- dimethylformamide dimethylacetal taken in a round bottom flask was subjected to microwave irradiation for 10 minutes to obtain 2-((dimethylamino)methylene)-5,5-dimethylcyclohexa- ne-1,3-dione (2m). The solvent was distilled off under reduced pressure to give a viscous mass. To this flask was added ethyl 3- amino-1H-pyrazole-4-carboxylate (1) (0.5 mmol) and the mixture was dissolved in 2 mL ethanol. Subsequently, KHSO4 (1 mmol) dissolved in 2 mL water was added and the resultant mixture was irradiated in an ultrasound cleaner for 10 minutes at 60-65 °C. The initial dark yellow solution gradually turned light to give a milky white mixture. At the end of the reaction, two product spots were seen at Rf = 0.6 and Rf = 0.4 as monitored by TLC (50 % ethyl acetate:hexane). The reaction mixture was allowed to cool to room temperature. The precipitated product obtained at this stage was collected by filtration, washed repeatedly with water to remove traces of acid, and finally dried to give a solid in 98 % yield. The two products could be isolated by column chromatography (silica gel, 20 % ethyl acetate: hexane) to give compounds 3m and 3mm in 60 % and 23 % yields, respectively (Scheme 4). The spectral and analytical data of compounds 3m and 3mm are presented here. Ethyl 8, 8-dimethyl-6-oxo-6, 7, 8, 9-tetrahydropyrazolo[1, 5- a]quinazoline-3-carboxylate (3m): Color: Milky white crystals. Yield: 60 %. M.p.: 154-155 °C. FT-IR (KBr, ν, cm-1): 1688 (C=O) (ester), 1610 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.23 (s, 6H, 2CH3), 1.41 (t, J = 6.8 Hz, 3H, CH3), 2.61 (s, 2H, cyclic CH), 3.39 (s, 2H, cyclic CH), 4.43 (q, J = 6.8 Hz, 2H, CH2), 8.65 (s, 1H, C2-H), 9.20 (s, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.4, 28.5, 29.6, 32.7, 37.3, 50.8, 60.7, 105.1, 114.1, 148.4, 149.4, 150.1, 153.0, 161.9, 194.0. MS (EI, m/z (%)):288 (MH)+. Ethyl 3-(((4, 4-dimethyl-2, 6-dioxocyclohexylidene)methyl) amino)-1H-pyrazole-4-carboxylate (3mm): Color: White solid. Yield: 23 %. M.p.: 163-164 °C. FT-IR (KBr, ν, cm-1): 3205 (N-H), 1743 (C=O) (ester), 1690 (C=O) (ketone). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.25 (s, 6H, 2CH3), 1.37 (t, J = 6.8 Hz, 3H, CH3), 2.48 (s, 2H, cyclic H), 2.52 (s, 2H, cyclic H), 4.31 (m, 1H, aliphatic NH), 4.47 (q, J = 6.8 Hz, 2H, CH2), 8.09 (s, 1H, C5-H), 9.09 (d, J = 13.6 Hz, 1H, aliphatic NCH), 13.86 (d, J = 13.6 Hz, 1H, aromatic NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.4, 28.5, 31.1, 37.4, 51.6, 51.7, 60.9, 102.9, 109.6, 133.2, 148.1, 149.3, 162.9, 198.1, 199.9. MS (EI, m/z (%)): 306 (MH)+. 2.2.4. Synthesis of ethyl 7-(naphthalen-2-yl)pyrazolo[1,5- a]pyrimidine-3-carboxylate (3n) Firstly, the (E)-3-(dimethylamino)-1-(naphthalen-2-yl) prop-2-en-1-one (2n) was obtained by reacting 2-acetyl napthalene (0.5 mmol) with DMF-DMA (1 mmol) in a round bottom flask under microwave irradiation for 9 min. 308 Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 Scheme 4. Synthesis of compounds 3m and 3mm. Scheme 5. Synthesis of compound 3n. The flask was sucked dry under reduced pressure to give a brown product 2n. To this flask was added compound 1 (0.5 mmol) and the mixture was dissolved in 2 mL ethanol. Subsequently, KHSO4 (1 mmol) dissolved in 2 mL water was added and the resulting mixture was heated in an ultrasonic bath at 60-65 °C. At the end of the reaction (18 minutes), the product precipitated out which was collected by filtration, washed repeatedly with cold water to remove traces of acid, and finally dried to give a crude mass. The product was further purified by column chromatography (silica gel column, 30 % ethyl acetate:hexane) to obtain the analytically pure product 3n (Scheme 5). Ethyl 7-(naphthalen-2-yl)pyrazolo[1, 5-a]pyrimidine-3-car boxylate (3n): Color: Brown crystals. Yield: 87 %. M.p.:134-135 °C. FT-IR (KBr, ν, cm-1): 1717 (C=O) (ester), 1612 (C=C) (aromatic). 1H NMR (400 MHz, CDCl3, δ, ppm):1.45 (t, J = 6.8 Hz, 3H, CH3), 4.48 (d, J = 6.8 Hz, 2H, CH2), 7.19 (s, 1H, C6-H) 7.59- 7.62 (m, 2H, aromatic), 7.88-8.03 (m, 4H, aromatic), 8.57 (s, 1H, aromatic), 8.65 (s, 1H, C2-H), 8.85 (s, 1H, C5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 14.5, 60.4, 103.1, 109.3, 125.4, 127.0, 127.5, 127.8, 128.2, 128.5, 130.1, 130.3, 132.7, 134.5, 147.4, 147.9, 149.0, 152.3, 162.6. MS (EI, m/z(%)): 318 (MH)+. 2.3. Antibacterial assay The antibacterial activity of the synthesized compounds was studied by 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (p-INT) dye assay followed by well- diffusion assay. The standard strain of bacteria (two Gram- positive and two Gram-negative) was used for the study viz Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 25923), Staphylococcus aureus (ATCC 25923) and one laboratory isolate of Listeria monocytogenes and maintained in the laboratory of Animal Health Division, ICAR-RC for NEH region, Umiam, Meghalaya, India. All bacterial cultures were revived on nutrient agar and checked for their purity by Gram staining and identified after revival in the automated Integrated DNA Technologies (IDT) and antibiotic susceptibility testing (AST) system (BD Phoenix 100) for proper identification. Initially, evaluation of the synthetic compounds for antibac- terial activity was done by p-INT dye assay. The bacteria were inoculated in nutrient broth and kept overnight at 37 °C. On the subsequent day, the compounds were diluted two-fold from the stock solution of 2000 µg/mL and a concentration of 15.6 to 1000 µg/mL was used. For comparison, non-culture control and non-compound control were also taken. The compounds were compared with the standard drug, Ampicillin (39-5000 µg/mL). The plates were incubated at 37 °C for 8 hours and bacterial growth of the compounds was assessed in which p-INT changed from yellow to purple where the bacterial growth occurred (Figure 2). The minimum inhibition concentration (MIC) was also noted [23]. In order to further observe the bacterial growth at the concentration which was showing prominent antibac- terial activity, 10 µL of samples from the well were further pipetted on nutrient agar by spot inoculation and kept for 24 hours at 37 °C. The concentrations of compounds exhibiting prominent antibacterial activity were punched with a sterile cork borer of 4 mm size and 100 μL of each sample was pipetted in the wells. The plates were incubated at 37 °C for 24 hours and the diameter of growth inhibition zone around the wells was measured [24]. The following control agents were used: positive control agent: Ampicillin (100 mg/mL) and negative control agent: 5 % DMSO. 3. Results and discussion 3.1. Chemistry Sequentially, to synthesize the class of pyrazolo[1,5- a]pyrimidines (3a-n) with CO2Et functionality, readily available ethyl 3-amino-1H-pyrazole-4-carboxylate (1) was chosen and reacted with a series of formylated active proton compounds (2a-n). The structural configurations of all these compounds were confirmed by their spectral and analytical data and compared with those already reported [14-18]. Firstly, to optimize the reaction conditions and to obtain the best reaction strategy, an equimolar mixture of 3-amino- pyrazole 1 was reacted with enaminone 2a derived from acetophenone in the presence of two equivalents of KHSO4. Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 309 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 Table 1. Reaction optimization under various conditions No Reaction condition Solvent Yield (%) Reaction time Melting point (°C) 1 Room temperature H2O 60 7h (incomplete) 133-134 2 Room temperature H2O:EtOH (1:1, v:v) 72 7h (incomplete) 133-134 3 Conventional heating at 60 °C H2O 63 2.5 h 133-134 4 Conventional heating at 60 °C H2O:EtOH (1:1, v:v) 73 2 h 133-134 5 US at room temperature H2O 71 9 min 134-135 6 US at room temperature H2O:EtOH (1:1, v:v) 94 6 min 133-134 7 US at 60 °C H2O 71 9 min 133-134 8 US at 60 °C H2O:EtOH (1:1, v:v) 90 6 min 134-135 Table 2. Summary of all synthesized pyrazolo[1,5-a]pyrimidine derivatives. Product Reaction condition Yield (%) Reaction time (min) Melting point (°C) 3a Room temperature 95 6 134-135 (133-134) 3b Room temperature 87 5 158-159 (158.5-160) 3c Room temperature 94 9 115-116 (112-114) 3d Room temperature 93 8 133-134 (131-133) 3e Room temperature 79 8 162-163 (162-162.5) 3f Room temperature 78 10 172-174 3g 60-65 °C 91 12 162-163 3h 60-65 °C 82 10 190-192 (177-178) 3i 60-65 °C 31 8 139-140 3ii 60-65 °C 66 8 143-144 (145) 3j 60-65 °C 78 10 147-148 3k Room temperature 76 6 140-141 3l Room temperature 84 12 131-132 3m 60-65 °C 60 10 154-155 3mm 60-65 °C 23 10 163-164 3n 60-65 °C 87 12 134-135 8 hours 18 hours 3a 3b 3c 3d 3e Amp 3a 3b 3c 3d 3e Amp Figure 2. Microlitre plate showing the effect of compounds 3a-e at 8 hours and 18 hours against the standard culture of Escherichia coli (ATCC 25922) in p-INT dye and well diffusion assays (Amp: Ampicillin). The reaction was carried out in different solvent systems and reaction conditions. It was found that ethanol:water (1:1, v:v) combination at room temperature under ultrasonic irradiation gave the best outcome in terms of yield and reaction rate. Towards the end of the reaction, as monitored by thin layer chromatography (TLC), the product precipitated out and was isolated in 95 % yield. The structure of the product was assigned to be ethyl 7-phenylpyrazolo[1,5-a]pyrimidine-3- carboxylate (3a, Scheme 1) on the basis of its spectral and analytical data. The detailed optimization results are presented in Table 1. Encouraged by the above results, this reaction condition was adopted and extrapolated to achieve the desired pyrazolo [1,5-a]pyrimidine series 3b-ii. The reaction of 3-aminopyrazole (1) with enaminones 2b-i, under the established reaction conditions occurred effortlessly with an overall yield of 78-97 % yields in 5-12 minutes. However, the reaction of compound 1 with compound 2i yielded two regioisomeric products 3i and 3ii, whose structures are confirmed by their spectral and analytical data (Scheme 2). After satisfactory results obtained in the first few reactions, it was decided to expand the series. For this, formylated active methylene compounds 2j-l were chosen (prepared in situ by reacting the active proton compound with DMF-DMA as reported in [11,12]) and irradiated with 1 under similar conditions as mentioned in Scheme 3 to obtain the products 3j- l in 76-84 % yields in 6-12 minutes. The structures of compounds 3j-l have been confirmed by their spectral and analytical data. Furthermore, when 2-((dimethylamino)methylene)-5,5- dimethylcyclohexane-1,3-dione (2m) derived from dimedone was reacted with compound 1 under irradiation in an ultra- sonic bath at 60-65 °C for 10 minutes, two products were isolated in 60 and 23 % yields (Scheme 4). The structures of these compounds were established to be compounds 3m (cyclized) and 3mm (uncyclized) on the basis of their spectral and analytical data. At last, when (E)-3-(dimethylamino)-1-(naphthalen-2-yl) prop-2-en-1-one (2n was reacted with compound 1 following the same methodology, product 3n precipitated in 87 % yield (Scheme 5). Its structure has also been confirmed by its spectral and analytical data. All products along with their reaction conditions, yields, melting point, and reaction time are summarized in Table 2. The structures of the above compounds were confirmed by their spectral and analytical data (1H NMR, 13C NMR, FT-IR, and Mass spectrometry). 310 Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 Table 3. Crystal data and structure refinement for compound 3d. Parameters Obtained specification CCDC No. 1886698 Empirical formula C16H15N3O3 Formulae weight 297.31 Temperature (K) 296 (2) Crystal system Monoclinic Space group P21/c a (Å) 7.468 (5) b (Å) 27.908 (17) c (Å) 7.232 (4) β (o) 104.291 (7) Volume (Å3) 1460.7 (15) Z 4 Dcalc(Mg/m3) 1.352 μ (mm-1) 0.096 F (000) 624.0 Radiation MoKα (λ = 0.71073 Å) 2θ range for data collection 5.63 to 57.57 Index range -10≤h≤10, -36≤k≤37, -9≤l≤9 Reflection collected 16667 Independent reflections 3720 [Rint=0.0965, Rsigma=0.0945] Data/restraints/parameters 3720/0/202 Goodness of fit on F2 1.018 Final R indexes [I≥2σ(I)] R1=0.0750, wR2=0.1775 Final R indexes [all data] R1=0.1645, wR2=0.2226 Largest diff peak/hole (e.Å-3) 0.63/-0.42 For further confirmation of their structures, the selected compound was subjected to X-ray crystallography. Thus, in the 1H NMR spectra, the CH3 and CH2 protons of the CO2Et group of compounds 3a-d, 3f-i, 3j resonated as triplets at δ 1.40-1.43 ppm and as quartets at δ 4.43-4.47 ppm, respectively, with a coupling constant of 7.2 Hz, whereas, in the case of compound 3k, the signals of the two methyl groups merged to give multiplet in the range δ 1.40-1.46 ppm. The CH3 and CH2 protons of CO2Et group of compounds 3e, 3ii, 3l, 3m, 3mm, and 3n gave a triplet close to δ 1.43 ppm and a quartet at around δ 4.44 ppm, respectively, with a coupling constant at around 6.4 Hz. Furthermore, the spectra of compound 3l showed multiplet at δ 4.41-4.48 ppm due to the two methylene groups of the two diethyl groups. The C6-H and C5-H protons of compounds 3a-d and 3f-h gave doublets at around δ 7.14 and 8.81 ppm, respectively, with a coupling constant of around 4.4 Hz. In compound 3e, the C6-H and C5-H protons showed singlets at δ 7.08 and 8.82 ppm, respectively. The formation of regioisomers 3i and 3ii was distinguished with the coupling constant on the basis of a report by Stanislav Radl et al. [25]. Compound 3ii showed doublets for C6-H and C5-H protons at δ 7.86 and 8.89 ppm, respectively, with a coupling constant of 4 Hz, whereas compound 3i, reflected substitution at C5 with the disap- pearance of the signals due to C5-H proton and in lieu showed doublets at δ 8.23 and 8.79 ppm for C6-H and C7-H protons with coupling constant of 7.4 Hz. The C2-H proton resonated as a singlet close to δ 8.60 ppm for compound 3a-j, 3ii. In compound 3e, unlike in others, the C6-H and C5-H protons gave singlets at δ 7.08 and 8.82 ppm, respectively. For compounds 3j-l, the C2-H proton resonated as a singlet at δ 8.63 ppm and the C6-H proton appeared as a singlet close to δ 9.12 ppm. The C2-H, C5-H protons of compound 3m resonated as singlets at δ 8.65 and 9.20 ppm, respectively. In the case of uncyclized product, 3mm, C5-H showed signal as a singlet at δ 8.09 ppm. The aliphatic -NH- gave a doublet splitting pattern at δ 4.31 ppm with coupling constant 6.8 Hz while -NHC- showed doublet splitting at δ 9.09 with coupling constant 13.6 Hz. For compound 3n, the C2-H, C5-H, and C6-H protons appeared as singlets at δ 8.65, 8.85, and 7.19 ppm, respectively. The other aromatic protons of compounds 3a-n and 3ii appeared in their usual range. Furthermore, 13C NMR, FT-IR, and mass spectroscopy were in support of the structure. 3.2. X-ray crystallography of compound 3d The structure confirmations of the compounds were also supported by X-ray crystallography. Ethyl 7-(4-methoxy phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (3d), taken as a model was analyzed for its crystal structure. X-ray data of the crystal 3d were recorded using a Bruker APEX-II CCD area detector diffractometer. Cosco colored needle-shaped crystals of the compound were obtained by dissolving the compound in a mixture of 1:4 hexane:dichloromethane and then leaving undisturbed to recrystallize slowly. The compound 3d (C16H15 N3O3, Dx = 1.352 Mg m-3) crystallized in a monoclinic cell (space group P21/c) with, MoKα radiation, λ = 0.71073 Å, 16667 measured, 3720 independent, 1681 observed reflection, Rint = 0.097, phi and ω scans, refinement on F2, R[F2>2σ(F2)] = 0.077, wR(F2) = 0.208, 202 parameters, 0 restraints, ∆ρ max = 0.63Å-3, ∆ρ min = -0.42 e.Å-3 (Table 3). The molecular graphic was performed using ORTEP-3 and displacement ellipsoids are drawn at 50 % probability level (Figure 3). Short contacts or hydrogen bonds were absent in this molecule (Figure 4). The selected bond lengths and bond angles of the crystal structures are mentioned in Tables 4 and 5, respectively. The C- C bond distance in the phenyl ring and pyrazolopyrimidine ring ranged between 1.376-1.387 and 1.369-1.399 Å, respectively. The single bond lengths between C13-C12, C10-C9, N3-N1, C11- N2, and N1-C8 are almost equivalent to those of supposedly double bonds C12-C11, N2-C1O, C9-C8, and C13-N3 [26]. This may be due to the delocalization in the ring system. 3.3. Biological activities The activities of these compounds were studied as a zone of inhibition in millimeters. Ampicillin (39-5000 µg/mL) was used as an agent for positive control and 5 % DMSO was used as the negative control. Among all 15 compounds, compounds 3a-e was found to be the most effective against Escherichia coli (ATCC 25922) and least active against other standard cultures. In the p-INT dye assay test (Figure 5), compounds 3a-e exhibited prominent antibacterial activity as there was no appearance of color from 15.6-1000 µg/mL, 7-8 hours. When the plates were further incubated for 18 hrs and then the dye added, the appearance of dye color was observed, thereby confirming the growth of the bacteria. Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 311 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 Table 4. Selected bond length of compound 3d. Atom Atom Length (Å) Atom Atom Length (Å) N1 C8 1.370(4) C5 C4 1.388(4) N1 N3 1.375(3) C5 C8 1.479(4) N1 C11 1.393(4) C7 C2 1.378(4) O3 C2 1.372(4) C7 C6 1.385(4) O3 C1 1.424(4) C11 C12 1.389(4) N3 C13 1.327(4) C8 C9 1.369(4) N2 C10 1.317(4) C2 C3 1.387(4) N2 C11 1.352(4) C12 C13 1.399(4) O1 C14 1.218(4) C12 C14 1.455(5) O2 C14 1.346(4) C4 C3 1.376(4) O2 C15 1.523(5) C15 C16 1.401(6) C5 C6 1.383(4) Table 5. Selected bond angle of compound 3d. Atom Atom Atom Angle (°) Atom Atom Atom Angle (°) C8 N1 N3 124.7(2) N1 C8 C5 120.7(3) C8 N1 C11 122.9(2) C5 C6 C7 121.4(3) N3 N1 C11 112.4(2) O3 C2 C7 124.6(3) C2 O3 C1 117.5(3) O3 C2 C3 115.3(3) C13 N1 N1 103.3(2) C7 C2 C3 120.0(3) C10 N2 C11 115.9(3) C12 C12 C13 104.9(3) C12 O2 C15 151.6(3) C12 C12 C14 123.9(3) C6 C5 C4 118.6(3) C3 C4 C5 120.5(3) C6 C5 C8 122.1(3) C8 C9 C10 120.6(3) C4 C5 C8 119.2(3) N2 C10 C9 124.6(3) C2 C7 C6 119.3(3) N3 C13 C12 114.0(3) N2 C11 C12 133.2(3) C4 C3 C2 120.2(3) N2 C11 N1 121.4(3) O1 C14 O2 124.1(3) C12 C11 N1 105.4(3) O1 C14 C12 122.9(4) C9 C8 N1 114.7(3) O2 C14 C12 113.0(3) C9 C8 C5 124.6(3) C16 C15 O2 104.5(4) Figure 3. ORTEP diagram of the single crystal structure of compound 3d as determined by X-ray crystallography. Figure 4. Packing diagram of compound 3d. Furthermore, the well content showed antibacterial activity within 7-8 hrs, when inoculated on nutrient agar plate exhibited growth on the agar plates on incubation for 24 hrs. Thus, the findings clearly indicate that the compounds 3a-e have bacteriostatic effect but not bactericidal property. Likewise, the well diffusion assay also showed zone of inhibition of compounds 3a: 12.33 mm, 3b: 11.33 mm, 3c: 11.33 mm, 3d: 11.33 mm, 3e: 10.66 mm) against E. coli (Table 6, Figure 6). The rest of the compounds failed to show any effect against E. coli. The test compounds 3a-e were found to be unproductive against Klebsiella pneumoniae which is a multidrug-resistant standard strain and hence the compounds may not be able to work against the multidrug resistant mechanism of the isolate. 312 Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 Table 6. Minimum inhibitory concentrations of pyrazolo[1,5-a]pyrimidine compounds (3a-3n) and standard drug against Escherichia coli (ATCC 25922) in p- INT dye and well diffusion assays. Compound MIC values (µg/mL) (p-INT dye assay) Diameter (mm) (well diffusion assay) 2000 µg/mL 1000 µg/mL 500 µg/mL 250 µg/mL 125 µg/mL 3a 15.62±3.285 12.33±0.623 10.33±0.577 <10 mm <10 mm <10 mm 3b 15.62±7.724 11.33±0.235 10.66±0.235 <10 mm <10 mm <10 mm 3c 15.62±3.285 11.33±0.471 11.00±0.408 <10 mm <10 mm <10 mm 3d 15.62±3.285 11.33±0.471 10.66±0.471 <10 mm <10 mm <10 mm 3e 15.62±7.724 10.66±0.471 11.00±0.408 <10 mm <10 mm <10 mm 3f >2000 - 3g >2000 - 3h >2000 - 3i >2000 - 3ii >2000 - 3j >2000 - 3k >2000 - 3l >2000 - 3m >2000 - 3n >2000 - Ampicillin 15.62±3.285 - Figure 5. Minimum Inhibitory Concentration (MIC) of the investigated compounds against Escherichia coli. Figure 6. Zones of inhibition (mm) showing the antimicrobial activity of the investigated compounds against Escherichia coli. All compounds were also found to be ineffective against gram-positive organisms which may be explained by the fact that the nature of the cell wall is different for Gram-positive as compared to Gram-negative. In all studies, the effect was compared with the standard drug ampicillin. Similar studies were carried out by Omasa et. al. [27] in which forty-eight compounds belonging to anthraquinones, naphthoquinones, benzoquinones, flavonoids (chalcones and polymethoxylated flavones) and diterpenoids (clerodanes and kauranes) were tested for antimicrobial potential against a panel of sensitive and multi-drug resistant bacteria. Tsemeugne et. al. [28] reported the antimicrobial activity of a synthesized novel trisazo dye from 3-amino-4H-thieno[3,4-c][1]benzopyran-4- one and Bin et. al. [29] reported the antimicrobial activity of indole diketopiperazine alkaloids. Pyrazolo[1,5-a]pyrimidine derivatives are widely known to be used as antibacterial, antifungal and antiviral agents in biological systems [30,31]. Pyrimidine based systems have been also found to be active surface antimicrobial agents [32]. 4. Conclusion In this article, we have reported the synthesis of a few known and a few novel pyrazolopyrimidine derivatives using an efficient, eco-friendly, rapid, high yielding method. This general method can be applied to further expand the library of pyrazolo[1,5-a]pyrimidines. The most important aspect of the reported procedure lies in the fact that the products precipitate out and could be isolated by simple filtration in a practically pure state. There has been no ambiguity in structural assignment and was additionally supported by the X-ray crystallographic studies of one of the synthesized compounds. Compounds 3a-e showed antibacterial activities against Escherichia coli (ATCC 25922) in p-INT dye and well diffusion 0 5 10 15 20 25 M IC v al ue s (µ g/ m L) Compound ID 0 2 4 6 8 10 12 14 16 2000 µg/mL 1000 µg/mL Di am et er o f i nh ib iti on z on e (m m ) Concentration of compounds (µg/mL) 3a 3b 3c 3d 3e Das et al. / European Journal of Chemistry 11 (4) (2020) 304-313 313 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.4.304-313.2033 assays. However, these compounds were found to be bacteriostatic in nature and hence they can play a role in controlling the growth of bacteria in the biological system and can also act as surface cleaning agents. Acknowledgements We thank Rev. Fr. Dr. Stephen Mavely, Vice-Chancellor and Rev. Fr. Joseph Nellanant, Assam Don Bosco University for providing infrastructure for the execution of this work. We also wish to express our gratitude to Sophisticated Analytical Instrument Facility-Central Drug Research Institute (SAIF- CDRI), Lucknow, Sophisticated Analytical Instrumentation Centre (SAIC), Tezpur University, Tezpur for providing spectral and analytical data, X-ray analysis data and biological analysis of our compounds. We are thankful to Farlando Diengdoh, Asst. Prof., St. Anthony’s College, Shillong for helping us in solving the X-ray structure of the crystals. We are also grateful to the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, New Delhi and Indian Council of Agricultural Research (ICAR)-Barapani, Shillong for a research grant. Supporting information CCDC-1886698 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 interest: 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 and Indian Council of Agricultural Research (ICAR)-Barapani, Shillong, India. 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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). https://www.ccdc.cam.ac.uk/structures/ mailto:data_request@ccdc.cam.ac.uk http://orcid.org/0000-0002-2391-8792 http://orcid.org/0000-0001-9845-2002 http://orcid.org/0000-0001-7898-5039 http://orcid.org/0000-0002-7240-7606 http://orcid.org/0000-0003-1629-2231 http://orcid.org/0000-0003-4558-0961 http://orcid.org/0000-0001-9068-4554 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. Instrumentations 2.2. Synthesis 2.2.1. Synthesis of ethyl 7-(substituted-phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (3a-ii) 2.2.2. Synthesis of ethyl 7-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate (3j-l) 2.2.3. Attempted synthesis of ethyl 8,8-dimethyl-6-oxo-6,7,8,9-tetrahydropyrazolo[1,5-a]quinazoline-3-carboxylate (3m) 2.2.4. Synthesis of ethyl 7-(naphthalen-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (3n) 2.3. Antibacterial assay 3. Results and discussion 3.1. Chemistry 3.2. X-ray crystallography of compound 3d 3.3. Biological activities 4. Conclusion Acknowledgements Supporting information Disclosure statement Funding ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: