untitled European Journal of Chemistry 5 (3) (2014) 457‐462 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2014 Eurjchem Publishing ‐ Printed in the USA http://dx.doi.org/10.5155/eurjchem.5.3.457‐462.1040 European Journal of Chemistry Journal homepage: www.eurjchem.com Synthesis of new 3,4‐dihydropyrano[c]chromene derivatives and their evaluation as acetyl cholinesterase inhibitors Younes Bouazizi a,b,*, Anis Romdhane b, and Hichem Ben Jannet b a Chemistry Department, Faculty of Science, Jazan University, 2097, Saudi Arabia b Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Bioorganic Chemistry and Natural Products, Faculty of Sciences, University of Monastir, Monastir, 5000, Tunisia *Corresponding author at: Chemistry Department, Faculty of Science, Jazan University, 2097, Saudi Arabia. Tel.: +96.653.7135063. Fax: +96.653.7135065. E‐mail address: bouazizi_younes@yahoo.fr (Y. Bouazizi). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.5.3.457‐462.1040 Received: 02 March 2014 Received in revised form: 26 April 2014 Accepted: 27 April 2014 Online: 30 September 2014 KEYWORDS 2‐Amino‐4‐phenyl‐4,5‐dihydro‐5‐oxopyrano[2,3‐c]chromen‐3‐carbonitrile derivatives (8a‐d) have been isolated in good yields by the reaction of corresponding 4‐hydroxycoumarin (1) with substituted aldehydes (2a‐d) and malononitrile (3) under reflux conditions. The reactivity of α‐functionalized iminoethers (9a‐d) with hydrazine, hydroxylamine and piperidine was studied. The synthesized compounds were characterized by various techniques including spectroscopy. Compounds 8‐11 were also evaluated as potential acetylcholinesterase inhibitors. Chromene Iminoethers Pyrrolochromene Pyridinochromene Dihydropyrano[c]chromene Anti‐acetylcholinesterase activity 1. Introduction 4‐Hydroxycoumarins (2H‐1‐benzopyran‐2‐ones) have evoked a great deal of interest due to their biological properties and characteristic conjugated molecular architecture. Many of them display important pharmacological effects, including analgesic [1], anti‐arthritis [2], anti‐inflammatory [3], anti‐ pyretic [4], anti‐bacterial [5], anti‐viral [6], and anti‐cancer [7] properties. 4‐Hydroxycoumarin and its derivatives have been effectively used as anticoagulants for the treatment of disorders in which there is excessive or undesirable clotting, such as thrombophlebitis [8], pulmonary embolism [9], and certain cardiac conditions [10]. A number of comparative pharmacological investigations of the 4‐hydroxycoumarin derivatives have shown good anticoagulant activity combined with low side effects and little toxicity [11]. Our research has been devoted to the development of several heterocyclic systems derived from 4H‐pyrans (chromenes) as starting material a new class of heterocyclic systems with the hope that they may be biologically active. We report here, facile syntheses approaches to several heterocyclic systems derived from 4H‐pyrans (chromenes) as starting material, for which we have evaluated their anti‐acetylcholin‐ esterase activity. 2. Experimental 2.1. Instrumentation 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded in deuterated CDCl3 and DMSO‐d6 on a Bruker AC‐300 using non‐deuterated solvents as internal reference. All chemical shifts were reported as δ values (ppm) and coupling constants (J) were expressed in Hz. All reactions were monitored by TLC using aluminium sheets of SDS silica gel 60 F254, 0.2 mm. 2.2. Biological properties Acetylcholinesterase enzymatic activity was measured by the Ellman test [12], 98 μL (50 mM) Tris‐HCl buffer pH = 8. 30 μL sample and 7.5 μL acetylcholinesterase solutions containing 0.26 U/mL were mixed in an ELISA plate well and left to incubate for 15 min. Subsequently, 22.5 μL of AtchI (Acetyl thiocholine iodide, substrate concentration = 0.023 mg/mL) and 142 μL of DTNB (5,5‐Dithio‐bis(2‐nitrobenzoic acid), chromogen concentration = 3 mM) were added. 458 Bouazizi et al. / European Journal of Chemistry 5 (3) (2014) 457‐462 Scheme 1 The absorbance at 405 nm was read when there action reached the equilibrium. A control action was carried out using water instead of compound. The absorbance value obtained was considered 100% activity. Inhibition (%) was calculated with Equation (1). I % = 100‐(Asample/Acontrol) × 100 (1) where, Asample is the absorbance of the reaction containing the extract and Acontrol the absorbance of the reaction control. Tests were carried out in triplicate and a blank with Tris‐HCl buffer instead of enzyme solution was done. Sample concentration providing 50% inhibition (IC50) was obtained plotting the inhibition percentage against compound solution concentra‐ tions. 2.3. Synthesis Starting materials were prepared using standard methods [13,14]. 2.3.1. Reaction of 4‐hydroxycoumarin (1) with compounds 2a‐d General procedure: To a stirred mixture of 4‐hydroxy coumarin (1) (3 mmol) and benzaldyde (2a‐d) (3 mmol) and malononitrile (3) (3 mmol) in absolute ethanol (30 mL) was added anhydrous sodium carbonate (32.68 mg, 0.308 mmol) and the mixture was heated under reflux. A TLC control showed that the reaction was completed after an hour. After cooling, the mixture diluted with cold ethanol when a solid formed which collected by filtration, washed several times with cold ethanol and dried and recrystallized from ethanol to afford the chromenes 8a‐d (Scheme 1). 2‐Amino‐5‐oxo‐4‐phenyl‐4,5‐dihydropyrano[3,2‐c]chromene‐ 3‐carbonitrile (8a): Color: White. Yield: 80%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 4.45 (s, 1H, CH‐Ph), 7.28‐7.38 (m, 5H, NH2, Ar‐H), 7.47‐7.53 (m, 4H, Ar‐H), 7.79 (t, 1H, J = 8.1 Hz, Ar‐ H), 7.90 (d, 1H, J = 7.8 Hz, Ar‐H). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 159.9 (1C, CO), 158.3 (1C, Ar‐C), 153.7 (1C, Ar‐C), 152.5 (1C, Ar‐C), 143.7 (1C, Ar‐C), 133.3 (1C, Ar‐C), 128.8 (2C, Ar‐C), 127.9 (2C, Ar‐C), 127.4 (1C, Ar‐C), 125.0 (1C, Ar‐C), 122.8 (1C, Ar‐C), 119.6 (1C, Ar‐C), 116.9 (1C, Ar‐C), 113.3 (1C, CN), 104.3 (1C, Ar‐C), 58.2 (1C, Ar‐C), 37.3 (1C, CH‐Ph). Anal. calcd. for C19H12N2O3: C, 72.15; H, 3.82; N, 8.86. Found: C, 72.20; H, 3.85; N, 8.90%. 2‐Amino‐4‐(4‐chlorophenyl)‐5‐oxo‐4,5‐dihydropyrano[3,2‐c] chromene‐3‐carbonitrile (8b): Color: White. Yield: 75%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 4.72 (s, 1H, CH‐Ph), 7.40‐8.13 (m, 10H, NH2, Ar‐H). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 160.4 (1C, CO), 158.9 (1C, Ar‐C), 154.4 (1C, C‐NH2), 153.2 (1C, Ar‐C), 143.1 (1C, Ar‐C), 133.8 (1C, C‐Cl), 130.6 (2C, Ar‐C), 128.8 (2C, Ar‐C), 127.9 (1C, Ar‐C), 127.4 (1C, Ar‐C), 125.0 (1C, Ar‐C), 122.8 (1C, Ar‐C), 119.6 (1C, Ar‐C), 113.3 (1C, CN), 104.4 (1C, Ar‐C), 58.6 (1C, Ar‐C), 38.3 (1C, CH‐Ph). 2‐Amino‐5‐oxo‐4‐(p‐tolyl)‐4,5‐dihydropyrano[3,2‐c]chrome ne‐3‐carbonitrile (8c): Color: White. Yield: 80%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.25 (s, 3H, CH3), 4.40 (s 1H, CH‐Ph‐ CH3), 7.12‐7.93 (m, 10H, NH2, Ar‐H). 13C NMR (75 MHz, DMSO‐ d6, δ, ppm): 159.8 (1C, CO), 158.4 (1C, Ar‐C), 153.7 (1C, C‐NH2), 152.5 (1C, Ar‐C), 140.8 (1C, Ar‐C), 136.7 (1C, Ar‐C), 133.2 (1C, Ar‐C), 129.5 (2C, Ar‐C), 128.9 (2C. Ar‐C), 127.9 (1C, Ar‐C), 122.8 (1C, Ar‐C), 119.7 (1C, Ar‐C), 116.9 (1C, Ar‐C), 113.4 (1C, CN), 104.6 (1C, Ar‐C), 58.6 (1C, Ar‐C), 37.0 (1C, CH‐Ph‐Me), 21.0 (1C, CH3). Anal. calcd. for C20H14N2O3: C, 72.12; H, 4.27; N, 8.48. Found: C, 72.16; H, 4.30; N, 8.50%. 2‐Amino‐5‐oxo‐4‐(4‐methoxyphenyl)‐4,5‐dihydropyrano[3,2‐ c]chromene‐3‐carbonitrile (8d): Color: White. Yield: 85%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 3.71 (s, 3H, CH3O), 4.39 (s, 1H, CH), 6.80‐7.85 (m, 10H, NH2, Ar‐H). Bouazizi et al. / European Journal of Chemistry 5 (3) (2014) 457‐462 459 Scheme 2 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 161.2 (1C, CO), 158.8 (1C, Ar‐C), 158.5 (1C, C‐NH2), 154.5 (1C, C‐O‐Me), 151.7 (1C, Ar‐ C), 135.9 (1C, Ar‐C), 133.1 (2C, Ar‐C), 129.0 (1C, Ar‐C), 125.1 (1C, Ar‐C), 121.4 (1C, Ar‐C), 119.8 (1C, Ar‐C), 117.2 (1C, Ar‐C), 115.0 (2C, Ar‐C), 111.7 (1C, CN), 106.4 (1C, Ar‐C), 59.1 (1C, Ar‐ C), 56.1 (1C, CH3‐O), 36.7 (1C, CH‐Ph‐Me). 2.3.2. Reaction of compounds 8a‐d with triethylortho formate General procedure: A mixture of compounds 8a‐d (0.01 mmol), triethylorthoformate (0.01 mmol) and Ac2O (30 mL) was refluxed for 6 h. The solid product that precipitated during the reflux was filtered off, dried and recrystallized from ethanol to give compounds 9a‐d (Scheme 2). Ethyl‐N‐(3‐cyano‐5‐oxo‐4‐phenyl‐4,5‐dihydropyrano[3,2‐c] chromen‐2‐yl)formimidate (9a): Color: White solid. Yield: 80%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.34 (t, 3H, J = 9 Hz, CH3‐ CH2‐O), 4.38 (q, 2H, J = 9 Hz, CH3‐CH2‐O), 4.76 (s, 1H, CH‐Ph), 7.27‐8.22 (m, 9H, Ar‐H), 8.92 (s, 1H, N=CH). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 162.4 (1C, C‐N=CH), 159.4 (1C, CO), 158.6 (1C, Ar‐C), 155.0 (1C, ‐N=CH‐O), 153.4 (1C, Ar‐C), 152.1 (1C, Ar‐ C), 133.5 (2C, Ar‐C), 133.0 (1C, Ar‐C), 132.0 (1C, Ar‐C), 129.3 (2C, Ar‐C), 124.6 (1C, Ar‐C), 123.4 (1C, Ar‐C), 116.8 (1C, Ar‐C), 116.3 (1C, Ar‐C), 113.9 (1C, CN), 103.1 (1C, Ar‐C), 82.9 (1C, Ar‐ C), 64.1 (1C, ‐O‐CH2‐CH3), 37.5 (1C, CH‐Ph), 13.8 (1C, CH3‐CH2‐ O). Ethyl‐N‐(4‐(4‐chlorophenyl)‐3‐cyano‐5‐oxo‐4,5‐dihydropyra no[3,2‐c]chromen‐2‐yl)formimidate (9b): Color: White. Yield: 75%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.35 (t, 3H, J = 9 Hz, CH3‐CH2‐O), 4.39 (q, 2H, J = 9 Hz, CH3‐CH2‐O), 4.76 (s, 1H, CH‐ Ph), 7.42‐8.22 (m, 8H, Ar‐H), 8.93 (s, 1H, N=CH). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 162.7 (1C, Ar‐C), 159.4 (1C, CO), 155.0 (1C, O‐CH=N), 153.9 (1C, Ar‐C), 152.2 (1C, Ar‐C), 140.5 (1C, Ar‐ C), 133.2 (1C, Ar‐C), 132.3 (1C, Ar‐C), 130.2 (2C, Ar‐C), 128.6 (2C, Ar‐C), 124.7 (1c, Ar‐C), 123.6 (1C, Ar‐C), 116.6 (1C, Ar‐C), 116.4 (1C, Ar‐C), 112.8 (1C, CN), 102.5 (1C, Ar‐C), 82.2 (1C, Ar‐ C), 64.2 (1C, O‐CH2‐CH3), 37.7 (1C, CH‐Ph), 13.8 (1C, CH3‐CH2‐ O). Ethyl‐N‐(3‐cyano‐5‐oxo‐4‐(p‐tolyl)‐4, 5‐dihydropyrano [3,2‐ c]chromen‐2‐yl)formimidate (9c): Color: White. Yield: 70%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.35 (t, 3H, J = 9 Hz, CH3‐ CH2‐O), 3.32 (s, 3H, CH3), 4.38 (q, 2H, J = 9 Hz, O‐CH2‐CH3), 4.65 (s, 1H, CH‐Ph), 7.18‐8.20 (m, 8H, Ar‐H), 8.92 (s, 1H, N=CH‐O). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 162.4 (1C, Ar‐C), 159.4 (1C, CO), 155.2 (1C, O‐CH=N), 153.6 (1C, Ar‐C), 152.1 (1C, Ar‐C), 138.6 (1C, Ar‐C), 136.9 (1C, Ar‐C), 133.0 (1C, Ar‐C), 129.2 (2C, Ar‐C), 128.0 (2C, Ar‐C), 124.7 (1c, Ar‐C), 123.5 (1C, Ar‐C), 116.7 (1C, Ar‐C), 116.4 (1C, Ar‐C), 112.9 (1C, CN), 103.0 (1C, Ar‐C), 82.8 (1C, Ar‐C), 64.2 (1C, O‐CH2‐CH3), 37.9 (1C, CH‐Ph), 20.6 (1C, CH3), 13.8 (1C, CH3‐CH2‐O). Ethyl‐N‐(3‐cyano‐4‐(4‐methoxyphenyl)‐5‐oxo‐4,5‐dihydro pyrano[3,2‐c]chromen‐2‐yl)formimidate (9d): Color: White. Yield: 78 %. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.35 (t, 3H, J = 9 Hz, CH3‐CH2‐O), 3.33 (s, 3H, OCH3), 4.38 (q, 2H, J = 9 Hz, O‐ CH2‐CH3), 4.63 (s, 1H, CH‐Ph), 6.83‐8.21 (m, 8H, Ar‐H), 8.91 (s, 1H, N=CH‐O). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 162.4 (1C, Ar‐C), 159.4 (1C, CO), 158.7 (1C, O‐CH=N), 155.1 (1C, Ar‐C), 153.4 (1C, Ar‐C), 152.1 (1C, Ar‐C), 133.6 (1C, Ar‐C), 133.0 (1C, Ar‐C), 129.4 (2C, Ar‐C), 124.7 (1C, Ar‐C), 123.5 (1c, Ar‐C), 116.8 (1C, Ar‐C), 116.4 (1C, Ar‐C), 113.9 (2C, Ar‐C), 112.9 (1C, CN), 103.2 (1C, Ar‐C), 82.9 (1C, Ar‐C), 64.2 (1C, O‐CH2‐CH3), 55.1 (1C, O‐CH3), 37.5 (1C, CH3), 13.8 (1C, CH3‐CH2‐O). 2.3.3. Reaction of compound 8a with formic acid A mixture of the chromene 8a (0.01 mmol) and formic acid (20 mL) was refluxed for 5 h. The mixture cooled it as a solid started to form and the precipitate filtered off, then washed with water and diethyl ether. The solid recrystallized from ethanol and afforded the 4‐phenyl‐3,4‐dihydropyrano[3,2‐c] chromene‐2,5‐dione, 10 (Scheme 2). 4‐Phenyl‐3,4‐dihydropyrano[3,2‐c]chromene‐2,5‐dione (10): Color: White. Yield: 80%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.99 (dd, 1H, J = 1.2 Hz, CH2), 3.57 (dd, 1H, J = 7.8 Hz, CH2), 4.45 (d, 1H, J = 7.8 Hz, CH‐Ph), 7.22‐7.87 (m, 9H, Ar‐H). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 165.6 (1C, O‐CO‐CH2), 160.7 (1C, O‐CO‐ C), 157.7 (1C, Ar‐C), 153.1 (1C, Ar‐C), 140.5 (1C, Ar‐C), 133.6 (1C, Ar‐C), 129.6 (1C, Ar‐C), 128.0 (2C, Ar‐C), 127.1 (2C, Ar‐C), 125.4 (1C, Ar‐C), 123.0 (1C, Ar‐C), 117.2 (1C, Ar‐C), 113.8 (1C, Ar‐C), 106.6 (1C, Ar‐C), 36.7 (1C, CH‐Ph), 35.8 (1C, CH2). 2.3.4. Reaction of compounds 9a‐d with hydrazine hydrate General procedure: A solution of compounds 9a‐d (0.01 mmol) and hydrazine hydrate (5 mL) in EtOH (50 mL) was sttired at room temperature for 1 h. The solid product was collected by filtration and recrystallized from ethanol to give compounds 11a‐d (Scheme 3). 9‐Amino‐8‐imino‐7‐phenyl‐8,9‐dihydrochromeno[3’,4’,5,6] pyrano[2,3‐d]pyrimidin‐6(7H)‐one (11a): Color: White. Yield: 72%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 4.99 (s, 1H, CH‐Ph), 5.73 (s, 2H, NH2), 7.14‐7.94 (m, 10H, NH, Ar‐H), 8.17 (s, 1H, CH=N). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 163.1 (1C, CO), 161.0 (1C, Ar‐C), 160.3 (1C, Ar‐C), 157.2 (1C, C=NH), 155.0 (1C, Ar‐C), 152.5 (1C, Ar‐C), 142.1 (1C, N=C‐N‐NH2), 133.3 (1C, Ar‐ C), 128.9 (2C, Ar‐C), 128.7 (1C, Ar‐C), 127.7 (2C, Ar‐C), 125.3 (1C, Ar‐C), 123.1 (1C,Ar‐C), 117.0 (1C, Ar‐C), 113.9 (1C, Ar‐C), 106.2 (1C, Ar‐C), 96.6 (1C, Ar‐C), 34.0 (1C, CH). 9‐Amino‐7‐(4‐chlorophenyl)‐8‐imino‐8,9‐dihydrochromeno [3’,4’,5,6]pyrano[2,3‐d]pyrimidin‐6(7H)‐one (11b): Color: White. Yield: 70%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 4.99 (s, 1H, CH‐Ph), 5.76 (s, 2H, NH2), 7.12‐7.93 (m, 9H, NH, Ar‐H), 8.17 (s, 1H, CH=N). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 164.0 (1C, C=NH), 163.1 (1C, CH=N), 159.8 (1C, CO), 154.2 (1C, Ar‐C), 460 Bouazizi et al. / European Journal of Chemistry 5 (3) (2014) 457‐462 O O O CN HN Ar O O O CN Ar N ONH PiperidineNH4OH NH2NH2.H2O 9a-d O O NN O NH NH2 Ar 13a12a-d 11a-d 1 2 3 4 7a 5 4a 5a 6 78 9 10 11 11a 11b 12 12a O O CN N N O Scheme 3 152.0 (1C, Ar‐C), 151.3 (1C, Ar‐C), 140.8 (1C, Ar‐C), 132.8 (1C, Ar‐C), 131.6 (1C, Ar‐C), 130.7 (2C, Ar‐C), 127.9 (2C, Ar‐C), 124.8 (1C, Ar‐C), 122.5 (1C, Ar‐C), 116.5 (1C, Ar‐C), 113.2 (1C, Ar‐C), 104.3 (1C, Ar‐C), 99.6 (1C, Ar‐C), 34.8 (1C, CH). 9‐Amino‐8‐imino‐7(p‐tolyl)‐8,9‐dihydrochromeno[3’,4’,5,6] pyrano[2,3‐d]pyrimidin‐6(7H)‐one (11c): Color: White. Yield: 78%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.50 (s, 3H, CH3), 4.97 (s, 1H, CH‐Ph), 5.77 (s, 2H, NH2), 7.15‐7.91 (m, 9H, NH, Ar‐ H), 8.16 (s, 1H, CH=N). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 159.8 (1C, CO), 154.2 (1C, Ar‐C), 152.0 (1C, Ar‐C), 151.3 (1C, C=NH), 148.4 (1C, Ar‐C), 147.2 (1C, Ar‐C), 140.9 (1C, CH=N), 132.8 (1C, Ar‐C), 131.6 (1C, Ar‐C), 130.7 (2C, Ar‐C), 127.9 (2C, Ar‐C), 124.7 (1C, Ar‐C), 122.5 (1C, Ar‐C), 116.5 (1C, Ar‐C), 113.1 (1C, Ar‐C), 104.3 (1C, Ar‐C), 99.8 (1C, Ar‐C), 54.9 (1C, CH), 34.8 (1C, CH3). 9‐Amino‐8‐imino‐7(4‐methoxyphenyl)‐8,9‐dihydrochromeno [3’,4’,5,6]pyrano[2,3‐d]pyrimidin‐6(7H)‐one (11d): Color: White. Yield: 78%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.50 (s, 3H, OCH3), 4.90 (s, 1H, CH‐Ph), 5.76 (s, 2H, NH2), 6.79‐7.92 (m, 9H, NH, Ar‐H), 8.15 (s, 1H, CH=N). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 159.8 (1C, CO), 158.2 (1C, Ar‐C), 154.0 (1C, Ar‐C), 153.8 (1C, C=NH), 151.9 (1C, C‐OCH3), 150.8 (1C, Ar‐C), 148.0 (1C, Ar‐ C), 146.8 (1C, Ar‐C), 133.9 (1C, Ar‐C), 132.6 (1C, Ar‐C), 129.8 (2C, Ar‐C), 124.7 (1C, Ar‐C), 122.4 (1C, Ar‐C), 116.5 (1C, Ar‐C), 113.5 (2C, Ar‐C), 113.2 (1C, Ar‐C), 104.9 (1C, Ar‐C), 54.9 (1C, OCH3), 34.8 (1C, CH). 2.3.5. Reaction of compounds 9a‐d with hydroxylamine General procedure: A mixture of compounds 9a‐d (0.01 mmol) and hydroxylamine (10 mL) in methanol:THF (v:v, 14:6) (20 mL) was stirred at room temperature for 1 h. The solid product was collected and recrystallized from ethanol to give compounds 12a‐d (Scheme 3). N‐(3‐Cyano‐5‐oxo‐4‐phenyl‐4,5‐dihydropyrano[3,2‐c]chro men‐2‐yl)formimidamide (12a): Color: White. Yield: 85%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 4.55 (s, 1H, CH‐Ph), 7.26‐ 8.06 (m, 10H, NH, Ar‐H), 8.44 (d, 1H, J = 13.8 Hz, ‐NH), 8.56 (dd, 1H, J = 9.6 Hz, CH=NH). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 160.0 (1C, CO), 158.4 (1C, Ar‐C), 156.0 (1C, Ar‐C), 154.2 (1C, C=NH), 152.5 (1C, C‐OCH3), 143.0 (1C, Ar‐C), 133.2 (1C, Ar‐C), 128.9 (2C, Ar‐C), 128.3 (1C, Ar‐C), 127.6 (2C, Ar‐C), 125.0(1C, Ar‐C), 123.3 (1C, Ar‐C), 119.3 (1C, Ar‐C), 116.8 (1C, Ar‐C), 113.6 (1C, CN), 103.7 (1C, Ar‐C), 74.4 (1C, Ar‐C), 34.8 (1C, CH). N‐(4‐(4‐Chlorophenyl)‐3‐cyano‐5‐oxo‐4,5‐dihydropyrano [3, 2‐c]chromen‐2‐yl)formimidamide (12b): Color: White. Yield: 85%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 4.55 (s, 1H, CH‐Ph), 7.25‐8.04 (m, 9H, NH, Ar‐H), 8.44 (d, 1H, J = 13.8 Hz, ‐NH), 8.56 (dd, 1H, J = 9.6 Hz, CH=NH). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 162.6 (1C, CO), 160.5 (1C, Ar‐C), 159.8 (1C, Ar‐C), 156.8 (1C, C=NH), 154.6 (1C, C‐OCH3), 152.1 (1C, Ar‐C), 140.5 (1C, Ar‐ C), 132.9 (1C, Ar‐C), 131.8 (1C, C‐Cl), 130.3 (2C, Ar‐C), 128.1 (2C, Ar‐C), 124.8 (1C, Ar‐C), 122.6 (1C, Ar‐C), 116.5 (1C, Ar‐C), 113.3 (1C, CN), 105.2 (1C, Ar‐C), 95.6 (1C, Ar‐C), 33.0 (1C, CH). N‐(3‐Cyano‐5‐oxo‐4‐(p‐totyl)‐4,5‐dihydropyrano[3,2‐c]chro men‐2‐yl)formimidamide (12c): Color: White. Yield: 80%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.50 (s, 3H, CH3), 5.1 (s, 1H, CH‐Ph), 7.25‐8.04 (m, 9H, NH, Ar‐H), 8.44 (d, 1H, J = 13.8 Hz, ‐ NH), 8.56 (dd, 1H, J = 9.6 Hz, CH=NH). 13C NMR (75 MHz, DMSO‐ d6, δ, ppm): 162.5 (1C, CO), 160.4 (1C, Ar‐C), 159.7 (1C, Ar‐C), 156.6 (1C, C=NH), 154.3 (1C, C‐OCH3), 152.0 (1C, Ar‐C), 138.6 (1C, Ar‐C), 136.3 (1C, Ar‐C), 132.7 (1C, Ar‐C), 128.8 (2C, Ar‐C), 128.2 (2C, Ar‐C), 124.8 (1C, Ar‐C), 122.5 (1C, Ar‐C), 116.5 (1C, Ar‐C), 113.3 (1C, CN), 105.7 (1C, Ar‐C), 96.1 (1C, Ar‐C), 33.1 (1C, CH), 20.5(1C, CH3). N‐(3‐Cyano‐4‐(4‐methoxyphenyl)‐5‐oxo‐4,5‐dihydropyrano [3,2‐c]chromen‐2‐yl)formimidamide (12d): Color: White. Yield: 85%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 3.80 (s, 3H, OCH3), 4.80 (s, 1H, CH‐Ph), 7.34‐8.02 (m, 9H, NH, Ar‐H), 8.40 (d, 1H, J = 13.8 Hz, ‐NH), 8.50 (dd, 1H, J = 9.6 Hz, CH=NH). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 160.5 (1C, CO), 159.4 (1C, Ar‐C), 158.7 (1C, Ar‐C), 155.6 (1C, C=NH), 153.2 (1C, C‐OCH3), 151.5 (1C, Ar‐ C), 138.5 (1C, Ar‐C), 136.6 (1C, Ar‐C), 130.7 (2C, Ar‐C), 128.3 (1C, Ar‐C), 125.0 (1C, Ar‐C), 123.7 (1C, Ar‐C), 115.1 (1C, Ar‐C), 116.0 (1C, Ar‐C), 113.9 (2C, Ar‐C), 113.2 (1C, CN), 104.7 (1C, Ar‐ C), 96.1 (1C, Ar‐C), 38.7(1C, CH). Bouazizi et al. / European Journal of Chemistry 5 (3) (2014) 457‐462 461 2.3.6. Reaction of compound 9a with piperidine Compound 9a (1 mmol), piperidine (3 mL) and toluene (20 mL) was refluxed for 3 h. The solvent was removed under reduced pressure and the resulting solid was recrystallized from ethanol:petrol ether (v:v, 14:6) (20 mL) to give compound 13a (Scheme 3). 5‐Oxo‐4‐phenyl‐2‐((piperidin‐1‐ylmethylene)amino)‐4,5‐di hydropyrano[3,2‐c]chromene‐3‐carbonitrile (13a): Color: White. Yield: 80%. 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.56‐1.64 (m, 6H, CH2‐CH2‐CH2), 3.65‐3.71 (m, 4H, CH2‐N‐CH2), 4.55 (s, 1H, CH‐Ph), 7.22‐8.32 (m, 9H, Ar‐H), 8.57 (s, 1H, N=CH‐N). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 160.1 (1C, Ar‐C), 157.7 (1C, CO), 154.3 (1C, Ar‐C), 153.2 (1C, Ar‐C), 152.5 (1C, Ar‐C), 143.1 (1C, Ar‐C), 133.2 (1C, Ar‐C), 128.9 (2C, Ar‐C), 128.2 (1C, Ar‐C), 127.6 (2C, Ar‐C), 124.9 (1C, Ar‐C), 123.9 (1C, Ar‐C), 119.5 (1C, Ar‐C), 116,7 (1C, Ar‐C), 113,6 (1C, Ar‐C), 103,7 (1C, Ar‐C), 74,1 (1C, Ar‐C), 50.8 (1C, CH2), 43.3 (1C, CH2), 38.7 (1C, Ar‐C), 26.5 (1C, CH2), 25.1 (1C, CH2), 24.0 (1C, CH2). 3. Results and discussion 3.1. Synthesis Treatment of 4‐hydroxycoumarin (1) with aryl aldehydes (2a‐d) and malononitrile (3) in the boiling ethanol during several hours in the presence of anhydrous sodium bicarbonate as a catalyst gave 2‐amino‐5‐oxo‐4‐phenyl‐4,5‐ dihydropyrano[3,2‐c]chromene‐3‐carbonitrile derivatives (8a‐ d) in high yields (75‐85%) (Scheme 1). The 1H NMR spectra of compound 8a displays a signal at δ 4.45 ppm that ascribable to the proton H4. In addition, the aromatic protons are observed between δ 7.28 and 7.90 ppm (see experimental), and the expected singlet for the proton H8 is observed at δ 7.79 ppm (Scheme 1). The observed high regioselectivity is most probably associated with the reaction sequence outlined in Scheme 1. Initial Knoevenagal reaction between aldehydes substitutes 2a‐ d and malonitrile (3) produces the unsaturated nitrile 4, which, undergoes a Michael reaction with the base derived coumarin anion, 5. The resulting Michael adduct 6 then undergoes intra molecular cyclization producing the annelatediminopyran 7. Subsequent tautomeric[1,3] sigmatropic shift gives compounds 8a‐d. Under these conditions, the reaction proceeds sufficiently rapidly and smoothly to afford the target chromenes 8a‐d in high yields without Michael adducts 6 being detected. However, the proposed mechanism is supported to some degree by isolation of analogous Michael adducts in the previously studied reaction of 4‐hydroxycoumarin with arylidenecyano acetamides [3]. Refluxing compounds 8a‐d with triethylorthoformate in acetic anhydride at reflux afforded the corresponding ethyl‐N‐ (3‐cyano‐5‐oxo‐phenyl‐4,5‐dihydropyrano[3,2‐c]chromen‐ 2‐yl)formimidate (9a‐d) while with formic acid, chromene‐2,5‐ one‐2,5‐dione (10) were formed, Scheme 2. Hydrazinolysis of compound 9a‐d in ethanol at room temperature afforded the 9‐amino‐8‐imino‐7‐phenyl‐8,9‐di hydrochromeno [3’,4’,5,6]pyrano[2,3‐d]pyrimidin‐6(7H)‐one derivatives, 11a‐d. Reaction of compounds 9a‐d with hydroxylamine in MeOH‐ THF at room temperature yielded the N‐(3‐cyano‐5‐oxo‐4‐ phenyl‐4,5‐dihydropyrano[3,2‐c]chromen‐2‐ yl)formimidamide deriva‐tives, 12a‐d. Interaction of compounds 9a‐d with piperidine in toluene afforded the chromen 5‐oxo‐4‐phenyl‐2‐((piperidin‐1‐ylmethylene) amino)‐4,5‐dihydropyrano[3,2‐c]chromene‐3‐carbonitrile, 13a, Scheme 3. The structures of products are characterized by 1H NMR along with 13C NMR are in agreement with the proposed structures. 3.2. Biological properties (Acetylcholinesterase inhibition) Inhibition of acetylcholinesterase (AChE), the key enzyme in the breakdown of acetylcholine, is considered one of the treatment strategies against several neurological disorders such as Alzheimer’s disease, senile dementia, ataxia, and myasthenia gravis [15,16]. The acetylcholinesterase (AChE) inhibition was determined using an adaptation of the method described in the literature [17]. All compounds were analyzed on what concerns their acetylcholinesterase inhibition activity (Table 1). Values oscillating between 0.010 and 0.130 mg/mL were obtained. Compared to those given in the literature for crude pure products [17], we can say that the synthesized compounds 8, 9, 10 and 11 are considered good inhibitors of acetylcholinesterase. The greatest inhibitory activity was exhibited by compound 11a (Ar = Ph) (IC50 = 0.110 μg/mL). It has been shown that the activity of these derivatives depends in general on the nature of Ar. In compound 10, the acetylcholinesterase inhibition decreases from Ar = Ph (9a) to Ar = p‐MeOPh (9d). The same phenomena have been observed with compounds 9a‐d and 8a‐d. It has been also shown that the activity decreases considerably when Ar varied in the order Ph, p‐ClPh, p‐MePh and p‐MeOPh. The substitution of both the phenyl seems to affect the activity of the chromene skeleton. Table 1. Acetylcholinesterase inhibition capacity represented by IC50 (mg/mL) of compounds 8, 9, 10 and 11. Compound Acetylcholiinesterase inhibition capacity represented by IC50 (mg/mL) 8a 0.091 8b 0.065 8c 0.064 8d 0.035 9a 0.130 9b 0.048 9c 0.030 9d 0.021 10 0.077 11a 0.110 11b 0.033 11c 0.025 11d 0.010 4. Conclusion In conclusion, this work reports the synthesis of 3,4‐ dihydropyrano[c]chromene derivatives and their evaluation as acetyl cholinesterase inhibitors, via the simple and useful 4‐hydroxycoumarin (1) with substituted aldehydes 2a‐d and malonitrile (3) under reflux reaction conditions. 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