A comprehensive synthesis and antimicrobial evaluation of some fused heterocycles based on coumarin moiety

Chalcones and coumarins represent significant naturally occurring plant constituents which exhibit a wide array of pharmacological and biological activities. Herein, synthesis of coumarin-chalcone hybrid derivatives was achieved in a good yield via Claisen-Schmidt aldolic condensation reaction employing 3-acetylcoumarin as a precursor. The reaction of the new chalcones with malononitrile gave rise to a new substituted pyran ring attached to a coumarin moiety. Subsequently, various C -nucleophiles were allowed to react with 3-(4 H -pyran- 2-yl)coumarin derivative 2a in order to construct novel fused and attached heterocyclic rings bearing different valuable function groups through simple and straightforward reactions. Finally, the antimicrobial activity of the synthesized compounds was evaluated against both Gram-positive and Gram-negative bacteria using Amoxicillin as a standard drug


Introduction
][3][4][5] The name coumarin was coined from Coumarouna odorata Aube which is the Caribbean name of the Tonka bean, from which coumarin was isolated for the first time 200 years ago. 6atural and synthetic coumarins exhibit numerous biological activities such as antibacterial, 7,8 anti-HIV, 9,10 anti-tubercular, 11 antitumoral, 12,13 antioxidant, 14 and anticancer. 15,16Furthermore, they have shown to be valuable as anticoagulant, 17 cytotoxic, 18 anti-inflammatory, 19 antipyretic, 20 antiviral, 21 antileishmanial, 22 and antifungal. 23,24Additionally, they have enzyme inhibition effects of crucial importance. 25][29] In the same context, chalcones are of utmost importance as essential scaffolds for constructing divers rang of different-sized heterocyclic systems of high-biological pertinent. 30s a part of our continuing attempts to synthesize valuable heterocyclic building blocks, 3-acetylcoumarin was exploited as a precursor to construct new chalcones bearing coumarin moiety. 31Subsequently, simple and straightforward reactions were used to synthesize novel coumarin derivatives aiming to promote their synthetic potential and investigate the associated biological activities.
Also, refluxing a mixture of chalcone 1a with N-aminothiosemicarbazide and catalytic amount of piperidine in absolute ethanol afforded 5-(furan-2-yl)-3-(2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazole-1carbothioamide (3) (Scheme 1). 35The spectral data of compound 3 provided sufficient information about its structure.IR spectrum revealed strong absorption bands at 3398, 3352 cm -1 characteristic for NH 2 group, 3218 cm -1 for NH group, 1608 cm -1 for C=N, and a strong absorption band at 1276 cm -1 due to C=S in addition to the appearance of the molecular ion peak at m/z = 354.Moreover, acetylation of compound 2a with acetyl chloride in dry dioxane and few drops of triethylamine yielded N-acetyl derivative 4 (Scheme 2).IR spectrum of the product was devoid from the absorption band of the amino group but exhibited strong absorption bands at 3223 cm -1 due to NH group, at 2212 cm -1 characteristic for nitrile group, and a strong absorption bands at 1688 cm -1 in correspondence with amide carbonyl.Moreover, 1 H NMR data displayed a singlet peak at δ 2.51 ppm integrating for three protons which suggesting the presence of a methyl group that was also detected at 25.91 ppm in 13 C NMR. Additionally, mass spectrum of compound 4 revealed ion peak at m/z = 374 equivalent to molecular formula C 21 H 14 N 2 O 5 .
A new approach to a fused pyrimidine heterocyclic system was achieved via condensation-addition protocol between compound 2a and formamide in refluxing dimethyl formamide to furnish 3-(4-amino-5-(furan-2-yl)-5H-pyrano [2,3-d] pyrimidin-7-yl)-2H-chromen-2-one (6) (Scheme 2).Formation of compound 6 was proved by spectral data.IR spectrum of compound 6 was devoid from the absorption band characteristic for the cyano group.Also, it exhibited two absorption bands at 3244, 3134 cm -1 attributed to NH 2 group and a band at 1620 cm -1 for C=N group.Another piece of evidence was obtained from 1 H NMR spectrum which revealed a singlet signal at 8.40 ppm characteristic for the proton at C2 of pyrimidine and D 2 O exchangeable slightly broad signal at 7.78 ppm corresponding to NH 2 group.The mass spectrum was in adequate agreement with the proposed structure, it revealed a molecular ion peak at m/z = 359 in accordance with the molecular weight of C 20 H 13 N 3 O 4 .
Moreover, compound 2a underwent cyclocondensation reaction with formic acid to form 5-(furan-2-yl)-7-(2-oxo-2H-chromen-3-yl)-3H-pyrano [2,3-d]pyrimidin-4(5H)-one (7) (Scheme 2).IR spectrum of this compound showed the disappearance of nitrile absorption band and the appearance of two new strong absorption bands at 3360, 1689 cm -1 in correspondence with NH group and cyclic amide carbonyl group, respectively.Also, a signal at wavenumber 1607 cm -1 was observed for C=N. 1 H NMR spectrum of compound 7 showed a singlet peak at 8.07 ppm due to the resonance of the proton at C2 in the pyrimidinone ring, on the other hand a resonant frequency was recorded at 7.37 ppm proving the existence of NH group.Furthermore, 13 C NMR spectrum showed a new signal at 159.75 ppm due to carbonyl carbon in pyrimidinone ring.
Interestingly, the structure of compound 7 was also confirmed chemically via exploring an alternative synthetic route of preparation, it included the hydrolysis of the cyano group in compound 2a into amide group utilizing sulfuric acid to yield 2-amino-4-(furan-2-yl)-6-(2-oxo-2H-chromen-3-yl)-4H-pyran-3-carboxamide (8)  followed by the exposure of the later compound 8 to formic acid for 10 h (Scheme 2).The formation of compound 8 was confirmed by its IR spectrum which showed the disappearance of the characteristic nitrile absorption band and the appearance of four absorption bands at 3457, 3385, 3267, 3228 cm -1 corresponding to two NH 2 groups, also a strong band at 1649 cm -1 was reveled due to amide carbonyl group.The mass spectrum of compound 8 disclosed an ion peak at m/z = 350, equivalent to [M + ].
Additionally, aminopyridine derivative 9 was accessible by the cyclocondensation reaction between compound 2a, acetaldehyde, and malononitrile (Scheme 2).The proof of aminopyridine derivative 9 formation was acquired from spectroscopic data such as IR which showed strong absorption bands for NH 2 group at 3340, 3213 cm -1 , aliphatic-CH at 2950 cm -1 , and cyano group at 2208 cm -1 .Moreover, a singlet peak integrating for three protons was recorded in 1 H NMR at 2.51 ppm which suggesting the presence of new methyl group.The methyl group was also observed in 13 C NMR at 24.26 ppm while the mass spectrum showed a molecular ion peak at m/z = 397 equivalent to molecular formula C 23 H 15 N 3 O 4 .
Likewise, condensation between compound 2a and cyclohexanone in the presence of Lewis acid such as anhydrous zinc chloride furnished pyranoquinoline derivative 10 (Scheme 2).The product was asserted by the analytical and spectroscopic data as IR was devoid from cyano group absorption band but exhibited strong absorption bands for amino group at 3410, 3332 cm -1 , aliphatic-CH at 2923 cm -1 , and C=N group at 1616 cm -1 .
A new synthetic approach for the substituted pyridine derivative 7-amino-4-(furan-2-yl)-5-oxo-2-(2-oxo-2H-chromen-3-yl)-5,8-dihydro-4H-pyrano[2,3-b]pyridine-6-carbonitrile (12) was achieved through the interaction between compound 2a and malononitrile in dimethyl formamide containing few drops of piperidine at reflux temperature (Scheme 3).Compound 12 was confirmed by IR spectrum that revealed a strong absorption at 1659 cm -1 for a new carbonyl group along with the ordinary δ-lactone carbonyl group at 1726 cm -1 .Also, bands at 3429, 3372 cm -1 , 3225 cm -1 , and 2212 cm -1 in correspondence with NH 2 , NH, and CN stretching frequencies were observed.Mass spectrum indicated a molecular ion peak at m/z = 399.Additionally, ethyl 5-amino-4-(furan-2-yl)-7-oxo-2-(2-oxo-2H-chromen-3-yl)-7,8-dihydro-4H-pyrano[2,3b]pyridine-6-carboxylate (13) was yielded via the reaction of compound 2a with diethylmalonate in glacial acetic acid (Scheme 3).The structure was confirmed by IR spectrum that revealed no absorption for nitrile group but exhibited strong absorption bands at 3145 cm -1 for NH group, 1688 cm -1 for ester carbonyl group, and at 1668 for amide carbonyl group.Also, 3-cyano-4-(furan-2-yl)-6-(2-oxo-2H-chromen-3-yl)-4H-pyran-2-yl carbamodithioic acid (14) was accessible through the reaction of compound 2a with carbon disulfide in absolute pyridine (Scheme 3).An evidence for the formation of compound 14 was obtained from IR spectrum which was devoid from the characteristic absorption band of amino group but exhibited strong absorption bands for NH group at 3361 cm -1 , SH group at 2352 cm -1 , and C=S group at 1256 cm -1 . 1 H NMR spectrum exhibited singlet signals for NH at 7.30 ppm and at 2.34 ppm for SH.The interaction between compound 2a and phenyl isothiocyanate in pyridine under reflux temperature submitted 1-(3-cyano-4-(furan-2-yl)-6-(2-oxo-2H-chromen-3-yl)-4H-pyran-2-yl)-3-phenylthiourea (15) (Scheme 3).The product structure confirmation was acquired from spectroscopic data and microanalysis, IR showed no absorption for NH 2 group but new absorption bands at 3329 cm -1 corresponding to NH group and 1230 cm -1 corresponding to C=S group were recorded.Mass spectrum of 15 displayed a peak at m/z = 467 in accordance with [M + ].Fusion of 2a with aromatic aldehydes (namely; vanillin, formylchromone, or p-chlorobenzaldehyde) gave rise to Schiff's bases 16a-c (Scheme 3).IR spectrum of compound 16a was devoid from stretching vibrations of amino group, it also showed the appearance of new bands at 3466 and 1608 cm -1 characteristic for OH group and C=N bond, respectively.Moreover, 1 H NMR spectrum exhibited singlet signals at 8.92 ppm for N=CH, 4.11 ppm for OH, and at 3.93 ppm corresponding to CH 3 which was also observed in 13 C NMR at 57.97 ppm.Likewise, IR spectrum of 16b was devoid from stretching vibrations of amino group but revealed an absorption band at 1617 cm -1 characteristic for C=N bond however 16c exhibited the absorption frequency of C=N bond at 1606 cm -1 .Interestingly, Schiff's bases 16a,b underwent a [3+2] cycloaddition reaction with thioglycolic acid in presence of catalytic amount of anhydrous aluminum chloride to give an access to new attached substituted thiazolidinone ring 17a,b (Scheme 3).Spectroscopic data and microanalysis asserted the structure as IR spectrum of 17a showed the appearance of a new C=O group absorption band at 1683 cm -1 whereas 1 H NMR spectrum exhibited singlet at 4.36 ppm integrating for two protons (COCH 2 S of thiazolidinone ).As well, IR spectrum of 17b showed the C=O group absorption band at 1683cm -1 however 1 H NMR spectrum revealed a singlet peak at 4.35 ppm due to resonance of thiazolidinone two protons at C5. Antimicrobial assay.Antibiotics bearing coumarin moiety have attracted a great attention owing to their potent inhibitory effect of bacterial DNA gyrase and topoisomerase. 36n addition, coumarin (2H-1-benzopyran-2-one) exhibited robust antibacterial activity against plentiful Grampositive and Gram-negative bacteria and this mainly attributed to its planar molecular structure and lipophilicity. 37The antimicrobial activity of compounds under investigation was evaluated against Bacillus subtilis, Staphylococcus aureus, and Staphylococcus enteritis as representative examples of Gram-positive bacteria and Escherichia coli as a representative example of Gram-negative bacteria.Antibiotic Amoxicillin was utilized as a control criterion for in vitro antibacterial activity.Antimicrobial activity of newly-synthesized compounds against several pathological strains was expressed as inhibition diameter zones in millimeters (mm) as following in  ; entries 3-6 and 15, respectively.In the same context, all the tested compounds were biologically active against Gramnegative bacteria, namely Escherichia coli, and their activities varied from moderate to good activity except compounds 2, 3, 7, 11, 13 and 15 (Table; entries 1, 2, 6, 10, 12 and 14, respectively) however compound 9 showed the exact activity of the promising compound 17a.

Conclusions
In conclusion, coumarin-chalcone hybrid compounds were used to synthesize new valuable heterocycles aiming to increase their synthetic potential.Moreover, coumarin derivative 2a was used as a valuable scaffold to construct various fused and attached heterocyclic rings via simple and straightforward reactions.Finally, on the screening of newly-synthesized compounds for their antimicrobial activity against selected microbial strains, compound 17a showed an excellent activity against both Gram-positive and Gram-negative bacteria (Table , entry 17).All the new synthesized compounds were well characterized using; elemental analysis, FT-IR, 1 H NMR, 13 C NMR, and ESI-Mass spectrum.

Experimental Section
General.Melting points were determined by an electrothermal melting point apparatus and are uncorrected.The reaction times were determined using the thin-layer chromatography (TLC) technique which was performed with fluorescent silica gel plates HF 245 (Merck) and plates were viewed with iodine.Silica gel (230-400 mesh) was used for flash chromatography separations.The Microanalytical Center of Cairo University performed the microanalyses.IR (KBr) spectra were recorded on a Pye-Unicam infrared spectrophotometer SP 2000 (Faculty of Science, Fayoum University).The mass spectra were run by a Shimadzu-GC-MS-GP 1000 EX using the direct inlet system.Nuclear magnetic resonance spectra were recorded on Varian Mercury 300 MHz spectrometer using TMS as internal standard at National Research Center.Chemical shifts (δ) and coupling constants (J) were recorded in ppm and Hertz units respectively.

Table . Table 1 .
In vitro antimicrobial activity of compounds under investigationThe investigated compounds showed variation in their antibacterial activities (Table).Compounds 8-10, 12, and 14 were active against all examined pathogens (Table; entries 7-9, 11, and 13, respectively).Interestingly, among all the investigated compounds, only 17a revealed the highest activity against all bacterial strains used in this test (Table;entry 17).Compounds 2 * I.Z.Inhibition diameter zones expressed in millimeters (mm); S.D. Standard deviation.a NA : No antimicrobial activity detected.