Synthesis, characterization, antimicrobial activities and electrochemical behavior of new phenolic azo dyes from two thienocoumarin amines

The coupling reactions of diazotized thienocoumarin derivatives with p -acetaminophen, salicylic acid and acetylsalicylic acid gave four products in which the benzene ring of the phenolic reagents were primarily substituted by two 3-diazenyl-4 H -thieno[3,4-c ]chromen-4-one or 3-diazenyl-4-imino-4 H -thieno[3,4-c ]chromene moieties. The newly prepared azo dyes were fully characterized based on their analytical and spectroscopic data. The electrochemical analysis of the novel azo dyes carried out on a glassy carbon electrode unequivocally further confirmed the assigned structures. The synthesized compounds tested for their antimicrobial activity using broth microdilution method, showed good activities.


Introduction
5][6] Nonlinear optical materials (NLO) have several advantages over inorganic materials used for the same reasons.We can thus name the great value of the nonlinear optical coefficient, the easy methods of synthesis and preparation, and the low costs. 7eterocyclic azo dyes are used as better coloring agents in the textile industry. 8The applications of azo compounds in the electronic industry as colorimetric sensors, 9 nonlinear optical (NLO) devices 10,11 and liquid crystalline displays (LCDs) 12 have been intensively investigated.This group of compounds has attracted much attention for their use as potential sensitizers for photodynamic therapy (PDT). 13he azo dyes containing the thiophenic ring have several advantages over other dyes, in particular due to their intense coloration effect. 14,15lectrochemical, photophysical and photochemical properties of azo molecules have been used to predetermine their reactivity or to confirm their structures. 16,17In a recent work, our group reported the electrochemical behavior of azo compounds containing hydroxyl group located at the ortho position of the azo group. 18In the present study, we extend our investigations to diazo dyes possessing different substituents in order to evaluate the influence of the substituent on the progress of the redox reactions.Indeed, another work has shown that the type of substituent on the aromatic ring of some azo compounds has an influence on the oxidation-reduction. 19The purpose of the present work was then to prepare and describe new azo compounds, to determine their degradation under electrochemical conditions, and finally to determine their antimicrobial properties.

Chemistry
The present investigation deals with the synthesis of novel azo compounds using thienocoumarins 2 as starting materials.The general preparation procedure is displayed in Scheme 1. [20][21][22]  The thienyldiazonium ions 3 were readily synthesized by adding thienocoumarin 2 to nitrosyl sulfuric acid at 0-5° C according to literature. 23Diazonium ion 3a was then coupled with three phenol derivatives namely salicylic acid (4a), p-acetaminophen (4b) and acetylsalicylic acid (4c) to yield compounds 5a-c (Scheme 2).

Scheme 2. Reactions' sequences to compounds 5.
The coupling of the in situ formed diazonium ion 3b with 2-acetyl salicylic acid in similar reactions' conditions gave compound 6 (Scheme 3) in good yield.It has to be pointed out, that compound 5d was found in all reactions mentioned as an additional product.This compound resulting from the cyclotrimerization of the intermediate 3-diazenyl-4H-thieno [3,4-c]chromen-4-one ion (3a), was previously reported. 24In the case involving the intermediate ion 3b, the later might undergo oxidation of its imino group into the carbonyl function prior to the cyclotrimerization process.Alternatively 3b might first cyclize before the oxidation of the imino group into the carbonyl function occur.The synthesized compounds 5 and 6 were characterized on the basis of their physical data and spectral analysis.
The infrared spectra of all the synthesized dyes showed intense bands between 3621-3277 cm -1 due to free and associated OH groups and other bands at 1733-1670 cm -1 which were assigned to the carbonyl groups.The FT-IR spectra also showed bands at 1490 cm -1 , assigned to the -N=N-linkage of the azo moieties.
The 13 C NMR spectrum exhibited 22 signals in agreement with the assigned structure 5a.The DEPTspectrum indicates the presence of 12 hydrogenated carbons.3][24] The signal of the carbonyl group of the carboxylic group was found at 185.1 ppm.
As previously mentioned, in the case of compounds 5c we noticed the opening of the coumarin rings. 23,26  also noted that, as earlier described, these products result from multiple copulation of the diazonium moiety on the phenol derivative. 27imilar analysis as above was conducted on the basis of the available elemental and spectroscopic data for the structural assignments of compounds 5b, 5c and 6.

Redox behaviors of the azo dyes
The cyclic voltammograms obtained for compounds 5a-c and 6 showed a quasi-reversible system (peaks I and II).This corresponds to the reduction of the pristine dye's azo functions to hydrazo functions. 28,29Further reduction is observed at peak III.This probably corresponds to the fragmentation of the hydrazo functions to form amine functions.Peak IV corresponds to the further reduction of one of the latter fragmentation products.Based on the foregoing discussion, the electrochemical mechanisms depicting the electrode phenomena are shown in Schemes 4-6.Since one of the hydrazo functions of compound 5c is highly hindered, its fragmentation probably takes place in two steps, with the less hindered function reduced first (scheme 5).

Electrochemical reaction mechanisms of the studied azo dyes
Compound 5a.Three distinct reduction peaks (II, III and IV) were observed for the electroreduction of azo dyes 5a, the first one II due to the formation of the hydrazo product A1 (Scheme 4) which provide quasi reversible oxidation-reduction peaks I during reverse and subsequent forward scans due to the formation of oxidation product 5a and its subsequent reduction to compound A1.The second peak III, is due to the reduction of hydrazo-compound A1 to yield two amines A2 and A3 (Scheme 4).The third peak can therefore be attributed to the reduction of the carbonyl function of compound A2 to yield compound A4.Compound 5c.In the cyclic voltammograms of 5c, four peaks were recorded, of which three cathodic peaks (II, III and IV) in the forward scan and one anodic peak (I) in the reverse scan, indicating the quasi-reversible electrochemical nature of the dye.The anodic peak only appeared in the subsequent scan after the reduction step.

AUTHOR(S)
The first peak can be attributed to the reduction of the two -N=N-bridges (Scheme 5) to yield the hydrazo intermediate B1 which in turn is reduced to give compound 5c during reverse and subsequent forward scan.The second peak III, is due to the reduction of one hydrazo bond of compound B1 particularly the hydrazo bond adjacent to the electron-donating groups at the ortho position to the azo bridge to yield amine B2 and hydrazo-compound B3.The last peak may be attributed to the reduction of the hydrazo-bond of compound B3 to yields two amines B2 and B4.

Antimicrobial activity
The antimicrobial activity of azo compounds and phenol derivatives was performed against bacterial and fungal species and their minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) values are summarized in Table 1.The antimicrobial activities of compounds 1a, 1b, 2a, 2b, 5a, 5b, 5c and 6 have been recorded on 25%(3/12), 16.66%(2/12), 100%(12/12), 100%(12/12), 100%(12/12), 100%(12/12) and 100%(12/12) of the tested microorganisms, respectively.The antibacterial and antifungal activities of the prepared azo compounds 5 and 6 were higher than those of their starting materials as compounds 1a and 1b were not active against all the tested bacterial species (MIC ˃ 256 µg/mL) whereas they displayed weak antifungal activity (MIC = 64 -˃ 256 µg/mL) when compared to that of their derivatives (MIC = 4 -256 µg/mL).Standard drugs ciprofloxacin and nystatin were used as controls for antibacterial and antifungal activities, respectively performed better than most tested compounds (Table 1).Interestingly, compounds 5a, 5b, 5c and 6 were in some cases equipotent with standard drugs against B. subtilis, S. aureus, E. coli, V. cholerae NB2 and SG24 and C. neoformans.The activity of each compound varied depending of the microorganism and compound 5a showed the lowest MIC value of 4 µg/mL against B. subtilis compared to 8 µg/mL for the control ciprofloxacin.Compound 5a and 5b (MIC = 4 -32 µg/mL) were the most potent against all the bacterial and fungal strains followed in decreasing order by compounds 6, 5c, 3a, 3b, 1a, and 1b.Compound 5a with two 3-diazenyl-4Hthieno [3,4-c]chromen-4-one moieties at positions -3 and -5 of the phenolic ring, is equipotent with compound 5b against S. aureus ATCC25923, S. aureus, P. aeruginosa ATCC27853, V. cholerae, C. albicans, C. neoformans and C. tropicalis.In contrast, compound 5c with opened coumarin rings of similar substituents at the same positions on the phenolic ring exhibited similar potency as that of compounds 5a and 5b against C. neoformans and C. tropicalis with MIC values of 4 and 8 µg/mL respectively.A structure-activity-relationship study from the results indicated the necessity of electron withdrawing and releasing groups at different positions on the phenolic ring.So, a combination of electron withdrawing and releasing groups on the phenolic ring can be synthesized and tested with a hope to get promising antimicrobial agents.A lower MMC/MIC (≤4) value signifies that a minimum amount of tested compounds is used to kill the microbial species, whereas, a higher values signifies the use of comparatively higher amount of compound for the control of any microorganism. 30Hence, compounds 1a and 1b showed fungistatic effect on the sensitive fungal strains while compound 2a displayed microbicidal activity against S. aureus ATCC25923, S. aureus, B. subtilis,

Cytotoxic activity
The cytotoxic activity of azo compounds and phenol derivatives against red blood cells (RBCs) was investigated using Triton X-100 as a positive control.The positive control showed about 100% lysis, whereas the phosphate buffer saline (PBS) showed no lysis of RBCs.Interestingly, none of the tested compounds showed cytotoxic activity against RBCs at concentrations up to 256 μg/mL (results not shown).This finding supports the selective toxicity of the tested compounds towards the tested bacteria.

Conclusions
The study of the reactions of the title 2-aminothiophene substrates with the phenolic reagents has confirmed the high reactivity of the intermediate thienyl diazonium ions 3 which coupling degree and regio-orientation on the phenolic rings strongly depend on the nature of the substituents initially present.With salicylic acid the electrophilic attack of the diazonium reagent 3a occurs at substitution free positions -3 and -5, whereas with paraacetaminophen the electrophilic substitutions took place at positions -2 and -3 and with 2-acetylsalicylic acid at positions -3 and -5.In contrast, 2-acetylsalicylic acid undergoes only one electrophilic substitution at position -3 with the intermediate diazonium reagent 3b.All the newly prepared azo dyes where fully characterized by their elemental and spectroscopic data.The electrochemical study on glassy carbon electrode of the new dyestuffs brought further supporting evidences to the structural assignments.The antimicrobial screenings revealed that all the four newly prepared azo dyes 5a, 5b, 5c and 6 have prominent antibacterial and antifungal activities.

Experimental Section
General.All Melting points are corrected and were determined with a STUART SCIENTIFIC Melting Point Apparatus Model SMP3.The T.L.Cs were carried out on Eastman Chromatogram Silica Gel Sheets (13181; 6060) with fluorescent indicator.A mixture of ethyl acetate and methylene chloride (7:3) was used as eluent and iodine was used as revelator for the chromatograms.The IR spectra were measured with a Fourier Transform Infrared spectrometer Bruker Alpha.The UV spectra were recorded with a JENWAY 6715 UV-Vis Spectrophotometer.Combustion analyses were carried out with a C, H, N, S Euro EA from Hekatech company, their results were found to be in good agreement (±0.3%) with the calculated values.EIMS spectra were recorded on a double focusing mass spectrometer (Varian MAT 311A). 1 H-NMR spectra were recorded in DMSO-d6 on a Bruker DRX spectrometer operating at 400 MHz. 13 C-NMR spectra were recorded in DMSO-d6 on a Bruker DRX spectrometer operating at 100 MHz.TMS was used as internal reference.The chemical shifts (δ) and coupling constants (J) are expressed in ppm and hertz respectively.Carbon attribution determined by 13 C, HSQC and HMQC experiments.
All the reagents mentioned in this work were purchased from Aldrich and Fluka and were used without further purification.Starting materials 2a and 2b have been prepared according to literature procedures as published earlier. 22neral procedure for the preparation of coupling products 5a-c and 6.Dry sodium nitrite (2.07 g, 3 mmol) was slowly added over a period of 30 minutes to concentrated sulphuric acid (10 mL) with occasional stirring.The solution was cooled to 0-5 °C.Compounds 2a and 2b was dissolved in DMSO (10 mL) and cooled to 0-5 °C.The nitrosyl sulphuric acid solution was added to the solution of 2a and 2b and the temperature was maintained to 0-5 °C.The clear diazonium salt solutions thus obtained consisting of the in situ formed intermediates 3a and 3b, were used immediately in the coupling reactions.The phenolic coupler (3 mmol) 4a-c was dissolved in DMSO (10 mL) and then cooled in an ice-bath at 0-5 °C.The diazonium solutions of 3a and 3b previously prepared were added drop wise over 1 hour, and then 15 mL of sodium acetate solution (10%) was added to the mixture.The solid precipitate was collected on a filter and crystallised from water-ethanol (50% v/v) mixture to give the corresponding phenolic thienyl azo dyes.Electrochemical procedure.The dyes used were dissolved in dimethylsulfoxide (DMSO).Cyclic voltammograms were recorded in the potential range from -1.5 V to +0.5 V by transferring the GCE into a 0.05 M H2SO4 solution containing an azo dye at a given concentration.The potential scan rate was varied.Tested microorganisms.The antimicrobial activity was performed against nine bacterial and three fungal species.The selected microorganisms were the Gram-positive (Staphylococcus aureus ATCC25923, S. aureus, Bacillus subtilis) and Gram-negative (Pseudomonas aeruginosa ATCC27853, Escherichia coli S2(1), Shigella flexneri SDINT, Vibrio cholerae NB2, V. cholerae SG24, V. cholerae CO6) bacteria and yeast strains of Candida albicans ATCC10231, C. tropicalis PK233 and Cryptococcus neoformans H99.These microorganisms were taken from our laboratory collection.The fungal and bacterial strains were grown at 37 °C and maintained on Sabouraud Dextrose Agar (SDA, Conda, Madrid, Spain) and nutrient agar (NA, Conda) slants respectively.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC).
The antibacterial and antifungal activities were evaluated by determining the MICs and MMCs as previously described. 49MICs of azo compounds and phenol derivatives were determined by broth micro dilution method.Each test sample was dissolved in dimethylsulfoxide (DMSO) to give a stock solution.This was serially diluted two-fold in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for yeasts to obtain a concentration range of 512 to 0.25 μg/mL.Then, 100 µL of each sample concentration was added to respective wells (96-well micro plate) containing 90 µL of SDB/ MHB and 10 µL of inoculum to give final concentration ranges of 256 to 0.125 μg/mL.The final concentrations of microbial suspensions were 2.5x10 5 cells/mL for yeasts and 10 6 CFU/mL for bacteria.Dilutions of nystatin (Sigma-Aldrich, Steinheim, Germany) and ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) were used as positive controls for yeasts and bacteria respectively.Broth with 10 µL of DMSO was used as negative control.MICs were assessed visually and were taken as the lowest sample concentration at which there was no growth or virtually no growth.The lowest concentration that yielded no growth after the sub-culturing was considered as the MMCs. 49All the tests were performed in triplicate.Cytotoxicity assay.Whole blood (10 mL) from albino rats was collected by cardiac puncture in EDTA tubes.The study was conducted according to the ethical guidelines of the Committee for Control and Supervision of Experiments on Animals (Registration no.173/CPCSEA, dated 28 January, 2000), Government of India, on the use of animals for scientific research.Erythrocytes were harvested by centrifugation at room temperature for 10 min at 1000 xg and were washed three times in PBS buffer. 50The cytotoxicity was performed as previously described. 50

Scheme 1 .
Scheme 1. Reactions' sequences to the in situ formed diazonium intermediates.