Synthesis, Reactions and Antimicrobial Activities of 8-Ethoxycoumarin Derivatives

Condensation of 3-acetyl-8-ethoxycoumarin (3) with thiosemicarbazide gave ethylidenehydrazinecarbothioamide 5, which was transformed into the thiazolidin-4-one derivatives 6,7. Interaction of 3 with DMF/POCl3 gave β-chloroacroline derivative 8. Treatment of 3 with malononitrile gave benzo[c]chromone and 2-aminobenzonitrile derivatives 9 and 10, respectively with respect to the reaction conditions. Condensation of 3-(2-bromoacetyl)-8-ethoxycoumarin (4) with o-phenylenediamine gave 3-(quioxaline-2-yl)-8-ethoxycoumarin hydrobromide (11), while 4 reacted with 2-aminopyridine to give chromenopyridopyrimidine derivative 12. Condensation of 4 with potassium thio-cyanate/methanol gave an unexpected derivative, 2H-chromeno-3-carboxy(methyl-carbonimidic)thioanhydride 16, which upon treatment with (NH2)2·H2O gave 3-ethoxy-2-hydroxybenzaldehyde azine 19. Interaction of 4 with thiourea derivatives gave thiazole derivatives 20a–c. The structures of the newly synthesized compounds were confirmed by their spectra data. The newly synthesized compounds were also screened for their antimicrobial activity.

Treatment of 3 with malononitrile in boiling methanolic piperidine solution instead of triethyl-amine, gave a product which formulated as 2-amino-4,6-bis(8-ethoxycouarin-3-yl)benzonitrile (10) (Scheme 5). The formation of 10 could be explained by condensation of the intermediate (A) with malononitrile to give another intermediate (C) which underges intramolecular cyclization through the nucleophilc addition of active methylene group to one of the carbonitrile functions followed by aromatization [32] (Scheme 5).
The formation of 12 can be explained by a nucleophilic addition of the more nucleophilic cyclic secondary nitrogen of 2-aminopyridine to the electrophilic carbonyl carbon of 3-(2-bromoacetyl)-8ethoxycoumarin (4) instead of nucleophilic replacement of bromine; the addition intermediate undergoes a subsequent cyclodehydration with the addition of the exonucleophilic nitrogen atom of 2-aminopyridine at position 2 to the active site of the coumarin ring (C-4), and the intermediate then acts as a nucleophile which attacks a second molecule of 4 at C-4 followed by aromatization and elimination of a bromoacetaldehyde molecule to give the final product 12. Previously Ramanna et al. [35]  In the present research, treatment of 4 with KSCN in boiling methanol gave 2-bromoethylene-8ethoxy-2H-chromene-3-carboxylic(methylcarbonimidic)thioanhydride (16) rather than the corresponding 3-thiocyanatoacetylcoumarin derivative 17 (Scheme 9). Scheme 9. Synthesis of compounds 16, 18 and 19 with MS fragmentaion patterns.
The structure was supported by the following evidence: (i) in the IR spectrum, the absence of SCN and the presence of NH at 3,141 cm −1 ; (ii) the 1 H-NMR spectrum exhibited a characteristic singlet for OCH 3  The structures of 20-22 were confirmed by their spectral data. The IR spectra showed NH 2 bands at υ 3,387, 3,302, 3,148 cm −1 and a CO band at υ 1,705 cm −1 for 20a; NH bands at υ 3,302, 3,148 cm −1 and CO band at υ 1,697 cm −1 for 20b, and CN band at υ 2,262 cm −1 and CO band at υ 1,722 cm −1 for 20c; C-H (aliphatic) at υ 2,978 cm −1 , CN band at υ 2,222 cm −1 a CO band at υ 1,728 cm −1 for 21 and a CN band at υ 2,191 cm −1 , a CO band at υ 1,720 cm Condensation of 20c with various aromatic aldehydes 23 afforded the corresponding dicoumarin-3-ylthiazole derivatives 24, 25 and iminocoumarin derivative 26, while condensation of 20c with aromatic aldehydes in methanolic piperidine solution give 27a,b. Compound 20c was also readily coupled with p-methoxybenzenediazonium chloride to afford 28 (Scheme 11). The structure of 28 was supported by its independent synthesis from 4 and (4-methoxyphenylazo)-2cyanoethanethioamide (29) (Scheme 11).

Reaction of 20c with Aromatic Aldehydes
A solution of 20c (5 mmol) in absolute methanol (40 mL) was refluxed with 4-methoxybenzaldehyde and/or 4-chlorobenzaldehyde (5 mmol) in the presence of piperidine for 1 hour. The solid formed was filtered off, washed with ethanol and dried under reduced pressure. The crude product was recrystallized from a suitable solvent to give 27a,b. The physical and spectral data of compounds 27a,b are as follows:

Antibacterial Activity
The newly synthesized compounds were screened for their antimicrobial activities in vitro against two Gram-negative Bordetella bronchiseptica (ATCC 4617) and Escherichia coli (ATCC 14169) and four Gram-positive Bacillus pumilus (ATCC 14884), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29737) and Staphylococcus epidermidis (ATCC 12228) pathogenic bacteria and two fungi Candida albicans (ATCC 10231) and Saccharomyces cervesia (ATCC 9080). The activities of these compounds were tested using the disc diffusion method [40] for bacteria and the paper disk diffusion method [41] for fungi. The area of zone of inhibition was measured using Ampicillin (25 μg mL −1 ) as standard antibiotic Micostatin (25 μg mL −1 ) was used as a reference antifungal. The tested compounds were dissolved in N,N-dimethylformamide (DMF) to give a solution of 1 mg mL −1 . The inhibition zones were measured in millimeters at the end of an incubation period of 48 hours at 28 °C. N,N-dimethylformamide (DMF) showed no inhibition zone. Test results are shown in Table 1.

Conclusions
Our interest in the synthesis of the title compounds was to focus on their study as antimicrobial agents as a part of our program which is aimed at the development of new heterocyclic compounds as more potent antimicrobial agents. In this paper we reported the synthesis of some 8-ethoxycoumarin derivatives bearing side chains, thiazole derivatives and the antimicrobial evaluation of all the novel compounds. The structures of the novel compounds were elucidated on the basis of IR, 1 H-NMR, 13 C-NMR and MS data. The screening results demonstrated that replacing the hydrogen atom attached to the coumarin nucleus at C-3 with a side chain as in compound 5 and thiazoles 7 and 28 results in wide spectrum antimicrobial activity against all tested bacteria and fungi compared to ampicillin and mycostatin, while the other compounds with other side chains showed moderate to weak activity.