# Synthesis, Characterization, and Antimicrobial Activity of a Novel Trisazo Dye from 3-Amino-4H-thieno[3,4-c][1]benzopyran-4-one

Authors: Joseph Tsemeugne; Emmanuel Sopbué Fondjo; Jean-de-Dieu Tamokou; Taoufik Rohand; Arnaud Djintchui Ngongang; Jules Roger Kuiate; Beibam Luc Sondengam
Journal: International Journal of Medicinal Chemistry
 (2018)

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## Abstract

A new trisazo dye has been synthesized by coupling the diazonium ion of 3-amino-4H thieno[3,4-c][1]benzopyran-4-one with 2-tert-butyl-4-methoxyphenol. The newly prepared trisazo dye was characterized by its physical, elemental, and spectroscopic data. 2D-NMR (COSY, HSQC, and HMBC) techniques were used to secure the structural assignments. The new trisazo dye (compound7) along with precursors3,4, and6 was screened by microdilution susceptibility assay for antibacterial and antifungal activities towards eight bacterial strains and three yeasts selected on the basis of their relevance as human pathogens. The results showed that compound7 (MIC = 2–128 μg/mL) was the most active as compared with its precursors. The most resistant microorganisms wereV. choleraeNB2 andV. choleraeSG24, whereas the most sensitive microorganism wasC. neoformans. The overall results of this study indicated that compound7 had the greatest potential value against both yeasts and multidrug-resistant bacteria, so further investigation is warranted.


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## Body

## 1. Introduction

The emergence of multidrug microbial resistance and the global warming are among the main challenges that the modern scientists have so far been facing in the recent decades. The fact that many pathogenic microorganisms responsible for several human and animal diseases have developed resistance mechanisms to the classical therapies has stimulated intensive investigations in the fields of natural and synthetic chemistry, with the aim of discovering new drug classes having much better therapeutic profiles. In this respect, the antimicrobial activities of numerous classes of organic substances such as phenols [1–3], coumarins [4–6], thiophenes [7–9], quinones [10, 11], and azo compounds [12–14] are currently the targets of the worldwide drug discovery efforts.Interestingly, we are now seeing the emergence of a new generation of drugs with hybrid molecular architectures combining the biological features of two or more small molecules or even small molecules and biologics. The expectation is that, in the long term, such molecular and/or biologic conjugates could become a dominant form of targeted infectious diseases’ therapies.The idea of combining two or more potentially bioactive substructures to make an integrated new molecular framework with a higher anticipated therapeutic firepower is conceptually simple yet highly successful in the context of infectious diseases that require effective killing of the microbes.The less use of azo compounds in medicinal chemistry is due to the fact that, in the presence of the enzymes of the organism, these compounds are readily reduced to aromatic amines which are reported to be highly mutagenic and carcinogenic [15], justifying therefore their marginal antimicrobial activities [16]. Nevertheless, it has been found that increasing the number of this group in the same compound results in the gradual inhibition of the biodegradation process. Hence, because of the low biodegradation rate of the polyazo compounds due to the important number of the –N=N– fragments in their structures, they should be preferred to their mono and disazo counterparts in the inhibition of the main pathogenic microorganisms of humans [17]. Moreover, previous studies have established the cumulative and synergetic effects of this functional group in the improvement of the biological activities of several hybrid lead compounds [18].In this work, we report for the first time the synthesis of a new trisazo dye from 3-amino-4H-thieno[3,4-c][1]benzopyran-4-one and 2-tert-butyl-4-methoxyphenol, for further investigations in drug development against multidrug-resistant bacteria and fungi. We carried out the antimicrobial screenings of the new compound on eight bacterial strains (among whichV. choleraeNB2,V. choleraeSG24,V. cholerae CO6, andS. flexneri are well known resistant strains) and three pathogen yeasts.


## 2. Experimental

### 2.1. Materials and Methods

#### 2.1.1. General Information

Melting point is corrected and was determined with an Electrothermal Melting Point Apparatus, model 9100. The TLC was carried out on Eastman Chromatogram Silica Gel Sheets (13181; 6060) with a fluorescent indicator. A mixture of ethyl acetate and methylene chloride (7 : 3) was used as an eluent and iodine was used as a revelator for the chromatograms. The IR spectra were measured with a Fourier Transform Infrared spectrometer JASCO FT/IR-4100. The UV spectrum was recorded with a Beckman DU-640 Spectrophotometer. Combustion analyses were carried out with a C, H, N, and S Euro EA from HEKAtech company, and results were found to be in good agreement (±0.3%) with the calculated values. EIMS were measured on Mass Spectrometer LCQ Classic with ESI Source from Thermo Fisher Scientific company.1H-NMR spectrum was recorded in DMSO-d6 with a 250 MHz spectrometer Bruker AV III. 13C-NMR spectrum was recorded in DMSO-d6 with a 62.5 MHz spectrometer Bruker AV III. TMS was used as an internal reference.


#### 2.1.2. Preparation of the Reagents and Starting Materials

All the reagents mentioned in this work were purchased from Aldrich and Fluka and were used without further purification.Starting material4 was prepared according to the procedures mentioned in the literature published earlier [19]. A mixture of 4.65 g of 4-methyl-2-oxo-2H-[1]benzopyran-3-carbonitrile (3) and sulphur (1.22 g, in excess) in ethanol (30 ml) was stirred using a magnetic plate shaker thermostated at 45°C. Ammonia (6 ml) was added dropwise during the first 10 min of the reaction. After 7 h of reaction, the resulting precipitate (5.08 g, 93%) was collected by filtration, washed with distilled water, and recrystallised in benzene to yield a yellow powder. m.p. 195-196°C (Lit. [19], 198-199°C, from benzene). IR (KBr, cm−1) υmax: 3449 (–NH2); 1687 (C=O); 1603 and 1547 (C=C); 1224 (C–O) cm−1. 1H NMR (250 MHz, DMSO-d6, Me4Si,δ, ppm): 7.87 (dd, 1H,J = 8.0 and 1.5 Hz, H-8), 7.78 (br s, 2H, NH2); 7.36 (ddd, 1H,J = 8.0, 7.4 and 1.8 Hz, H-6), 7.22–7.17 (m, 2H, H-7 and H-5), 6.89 (s, 1H, thiophenic H). 13C NMR (62.5 MHz, DMSO-d6, Me4Si,δ, ppm): 166.70 (C-3), 158.98 (C-2), 151 (C-4a), 130.90 (C-8a), 129.26 (C-9), 124.39 (C-6), 123.91 (C-8), 118.05 (C-2a), 117.09 (C-7), 98.07 (C-8b), 97.54 (C-5); m/z (EI) 217 (M+; 100%). Elemental Analysis: C11H7NO2S. Calculated: C: 60.83; H, 3.23; N, 6.45; S, 14.75. Found: C, 61.17; H, 3.36; N, 6.49; S, 14.45.


#### 2.1.3. Preparation of Diazonium Salt Solution

In a similar manner as described in [20], 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. Compound4 was dissolved in DMSO (10 mL) and cooled to 0–5°C. The nitrosylsulfuric acid solution was added to the solution of4 and the temperature was maintained between 0 and 5°C. The clear diazonium salt solution thus obtained consisting of the in situ formed intermediate5 was used immediately in the coupling reaction.


#### 2.1.4. 3-(2-3-(tert-Butyl)-2-hydroxy-5-methoxy-4,6-bis[2-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)diazenyl]phenyldiazenyl)-4H-thieno[3,4-c]chromen-4-one Pentahydrate (7)

The phenol derivative 2-tert-butyl-4-methoxyphenol (540 mg; 3 mmol) was dissolved in DMSO (10 mL) and then cooled in an ice bath at 0–5°C. The diazonium solution of4 previously prepared was added dropwise over 1 hour, and then 15 mL of sodium acetate solution (10%) was added to the mixture. The pH of the mixtures was in the range 9–11. The solid precipitate was collected on a filter and crystallised from methanol to give the title7 (632 mg; 49%) as a green powder. m.p. 200°C. IR (KBr, cm−1) υmax: 3260 (OH), 3073 (Ar. C–H), 1721 (C=O), 1677 (Ar C=C), 1483, 1435 (N=N) cm−1. UV (THF) λmax/nm (logε): 215 (2.95), 240 (4.56), 268 (4.18), 325.5 (4.74), 381 (4.48). 1H NMR (250 MHz, DMSO-d6, Me4Si,δ, ppm): 9.18 (d, 1H,J = 9.75 Hz, 9-H), 9.01 (d, 1H,J = 10.5 Hz, 9′′-H), 8.83 (d, 1H,J = 10.5 Hz, 9′′′-H), 8.59 (dd, 1H,J = 9.0 and 7.8 Hz, 8′′-H), 8.46 (dd, 1H,J = 10.5 and 9.75 Hz, 8′′′-H), 8.32 (dd, 1H,J = 9.75 and 9.75 Hz, 8-H), 8.27 (dd, 1H,J = 9.75 and 9.75 Hz, 7′′-H), 8.15 (dd, 1H,J = 9.75 and 9.0 Hz, 7′′′-H), 8.06 (dd, 1H,J = 11.25 and 9.0 Hz, 7-H), 7.99 (d, 1H,J = 9.0 Hz, 6-H), 7.97 (d, 1H,J = 9.6 Hz, 6′′-H), 7.96 (d, 1H,J = 7.0 Hz, 6′′′-H), 7.67 (s, 1H, 1-H), 7.58 (s, 1H, 1′′′-H), 7.50 (s, 1H, 1′′-H), 3.92 (s, 10H, H2O), 3.90 (s, 1H, OH), 3.50 (s, 3H, OCH3), 1.54 (s, 9H, CH3).13C NMR (62.5 MHz, DMSO-d6, Me4Si,δ, ppm): 164.9 (C-4), 164.2 (C-4′′ and C-4′′′), 157.3 (C-9b′′ and C-9b′′′), 157.2 (C-9b), 155.4 (C-5a′′), 155.3 (C-5a′′′), 154.5 (C-5a), 154.3 (C-3a′′), 154.0 (C-3a′′′), 153.2 (C-3a), 148.3 (C-2′), 138.2 (C-5′), 136.0 (C-3), 135.8 (C-3′′′), 134.9 (C-3′′), 131.6 (C-9a), 130.5 (C-9a′′), 129.7 (C-9a′′′), 129.6 (C-7), 129.4 (C-7′′), 129.1 (C-7′′′), 127.6 (C-9′′ and C-9′′′), 126.8 (C-9), 126.0 (C-8), 125.9 (C-8′′′), 125.8 (C-8′′), 120.5 (C-6′), 118.6 (C-4′), 117.7 (C-6), 117.5 (C-6′′), 117.6 (C-6′′′), 115.4 (C-1′′′), 115.3 (C-1′′), 114.7 (C-1), 113.8 (C-1′), 101.8 (C-3′), 56.7 (OCH3), 25.5 (C(CH3)3), 18.7 (3CH3). m/z (EI): 957(1) [M+∙ + 2H], 525 (1), 516 (2), 459 (21), 393 (3), 338 (5), 279 (1), 247 (19), 230 (6). Elemental Analysis: C44H38N6O13S3 Calculated: C, 55.34; H, 4.01; N, 8.80; S, 10.07. Found: C, 55.36; H, 3.99; N, 8.78; S, 9.99.


### 2.2. Antimicrobial Assay

#### 2.2.1. Microorganisms

A total of eight bacteria and three yeasts were tested for their susceptibility to the studied compounds. The microorganisms used in this study were taken from our laboratory collection. Among the clinical strains ofVibrio choleraeused in this study, strains NB2 and SG24 (1) belonged to O1 and O139 serotypes, respectively. These strains were able to produce cholera toxin and hemolysin [21, 22]. The other microbial species used in this study wereCandida parapsilosis ATCC22019,Candida albicans ATCC9002,Cryptococcus neoformansIP95026,Bacillus subtilis,Escherichia coliS2 (1),Vibrio choleraenon-O1 and non-O139 (strains CO6 and PC2), andShigella flexneri. TheV. choleraenon-O1 and non-O139 strains were positive for hemolysin production but negative for cholera toxin production [22]. The bacterial and fungal species were grown at 37°C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) slants, respectively.


#### 2.2.2. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC)

The antimicrobial activity was investigated by determining the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum fungicidal concentrations (MFCs). MIC of compounds was assessed using the broth microdilution method [23]. Each test compound was dissolved in dimethylsulfoxide (DMSO, Fisher Chemicals) to give a stock solution. This was serially diluted twofold in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi to obtain a concentration range of 512 to 0.25 μg/ml. One hundred microliters of each concentration was introduced into a well (96-well microplate) containing 90 μl of SDB or MHB, and 10 μl of inoculum (1 × 106 CFU/mL for bacteria and 1 × 105 spores/ml for yeasts) was added to obtain a final concentration range of 256 to 0.125 μg/ml. Plates were covered and incubated on the shaker at 37°C for 24 h (bacteria), 48 h (Candida spp.), and 72 h(Cryptococcus). MICs were assessed visually after the corresponding incubation period and were taken as the lowest sample concentration at which there was no growth or virtually no growth.For the minimum microbicidal concentration (MMC) determination, 10μl aliquots from each well that showed no growth of microorganism were plated on Mueller-Hinton Agar or Sabouraud Dextrose Agar and incubated at 37°C for 24 h (bacteria), 48 h (Candida spp.), and 72 h(Cryptococcus). The lowest concentration that yielded no growth after the subculturing was taken as the MBCs or MFCs. Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) for bacteria and nystatin (Sigma-Aldrich, Steinheim, Germany) for yeasts were used as positive controls, while broth with 20 μL of DMSO was used as a negative control. The assay was repeated thrice.


## 2.1. Materials and Methods

### 2.1.1. General Information

Melting point is corrected and was determined with an Electrothermal Melting Point Apparatus, model 9100. The TLC was carried out on Eastman Chromatogram Silica Gel Sheets (13181; 6060) with a fluorescent indicator. A mixture of ethyl acetate and methylene chloride (7 : 3) was used as an eluent and iodine was used as a revelator for the chromatograms. The IR spectra were measured with a Fourier Transform Infrared spectrometer JASCO FT/IR-4100. The UV spectrum was recorded with a Beckman DU-640 Spectrophotometer. Combustion analyses were carried out with a C, H, N, and S Euro EA from HEKAtech company, and results were found to be in good agreement (±0.3%) with the calculated values. EIMS were measured on Mass Spectrometer LCQ Classic with ESI Source from Thermo Fisher Scientific company.1H-NMR spectrum was recorded in DMSO-d6 with a 250 MHz spectrometer Bruker AV III. 13C-NMR spectrum was recorded in DMSO-d6 with a 62.5 MHz spectrometer Bruker AV III. TMS was used as an internal reference.


### 2.1.2. Preparation of the Reagents and Starting Materials

All the reagents mentioned in this work were purchased from Aldrich and Fluka and were used without further purification.Starting material4 was prepared according to the procedures mentioned in the literature published earlier [19]. A mixture of 4.65 g of 4-methyl-2-oxo-2H-[1]benzopyran-3-carbonitrile (3) and sulphur (1.22 g, in excess) in ethanol (30 ml) was stirred using a magnetic plate shaker thermostated at 45°C. Ammonia (6 ml) was added dropwise during the first 10 min of the reaction. After 7 h of reaction, the resulting precipitate (5.08 g, 93%) was collected by filtration, washed with distilled water, and recrystallised in benzene to yield a yellow powder. m.p. 195-196°C (Lit. [19], 198-199°C, from benzene). IR (KBr, cm−1) υmax: 3449 (–NH2); 1687 (C=O); 1603 and 1547 (C=C); 1224 (C–O) cm−1. 1H NMR (250 MHz, DMSO-d6, Me4Si,δ, ppm): 7.87 (dd, 1H,J = 8.0 and 1.5 Hz, H-8), 7.78 (br s, 2H, NH2); 7.36 (ddd, 1H,J = 8.0, 7.4 and 1.8 Hz, H-6), 7.22–7.17 (m, 2H, H-7 and H-5), 6.89 (s, 1H, thiophenic H). 13C NMR (62.5 MHz, DMSO-d6, Me4Si,δ, ppm): 166.70 (C-3), 158.98 (C-2), 151 (C-4a), 130.90 (C-8a), 129.26 (C-9), 124.39 (C-6), 123.91 (C-8), 118.05 (C-2a), 117.09 (C-7), 98.07 (C-8b), 97.54 (C-5); m/z (EI) 217 (M+; 100%). Elemental Analysis: C11H7NO2S. Calculated: C: 60.83; H, 3.23; N, 6.45; S, 14.75. Found: C, 61.17; H, 3.36; N, 6.49; S, 14.45.


### 2.1.3. Preparation of Diazonium Salt Solution

In a similar manner as described in [20], 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. Compound4 was dissolved in DMSO (10 mL) and cooled to 0–5°C. The nitrosylsulfuric acid solution was added to the solution of4 and the temperature was maintained between 0 and 5°C. The clear diazonium salt solution thus obtained consisting of the in situ formed intermediate5 was used immediately in the coupling reaction.


### 2.1.4. 3-(2-3-(tert-Butyl)-2-hydroxy-5-methoxy-4,6-bis[2-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)diazenyl]phenyldiazenyl)-4H-thieno[3,4-c]chromen-4-one Pentahydrate (7)

The phenol derivative 2-tert-butyl-4-methoxyphenol (540 mg; 3 mmol) was dissolved in DMSO (10 mL) and then cooled in an ice bath at 0–5°C. The diazonium solution of4 previously prepared was added dropwise over 1 hour, and then 15 mL of sodium acetate solution (10%) was added to the mixture. The pH of the mixtures was in the range 9–11. The solid precipitate was collected on a filter and crystallised from methanol to give the title7 (632 mg; 49%) as a green powder. m.p. 200°C. IR (KBr, cm−1) υmax: 3260 (OH), 3073 (Ar. C–H), 1721 (C=O), 1677 (Ar C=C), 1483, 1435 (N=N) cm−1. UV (THF) λmax/nm (logε): 215 (2.95), 240 (4.56), 268 (4.18), 325.5 (4.74), 381 (4.48). 1H NMR (250 MHz, DMSO-d6, Me4Si,δ, ppm): 9.18 (d, 1H,J = 9.75 Hz, 9-H), 9.01 (d, 1H,J = 10.5 Hz, 9′′-H), 8.83 (d, 1H,J = 10.5 Hz, 9′′′-H), 8.59 (dd, 1H,J = 9.0 and 7.8 Hz, 8′′-H), 8.46 (dd, 1H,J = 10.5 and 9.75 Hz, 8′′′-H), 8.32 (dd, 1H,J = 9.75 and 9.75 Hz, 8-H), 8.27 (dd, 1H,J = 9.75 and 9.75 Hz, 7′′-H), 8.15 (dd, 1H,J = 9.75 and 9.0 Hz, 7′′′-H), 8.06 (dd, 1H,J = 11.25 and 9.0 Hz, 7-H), 7.99 (d, 1H,J = 9.0 Hz, 6-H), 7.97 (d, 1H,J = 9.6 Hz, 6′′-H), 7.96 (d, 1H,J = 7.0 Hz, 6′′′-H), 7.67 (s, 1H, 1-H), 7.58 (s, 1H, 1′′′-H), 7.50 (s, 1H, 1′′-H), 3.92 (s, 10H, H2O), 3.90 (s, 1H, OH), 3.50 (s, 3H, OCH3), 1.54 (s, 9H, CH3).13C NMR (62.5 MHz, DMSO-d6, Me4Si,δ, ppm): 164.9 (C-4), 164.2 (C-4′′ and C-4′′′), 157.3 (C-9b′′ and C-9b′′′), 157.2 (C-9b), 155.4 (C-5a′′), 155.3 (C-5a′′′), 154.5 (C-5a), 154.3 (C-3a′′), 154.0 (C-3a′′′), 153.2 (C-3a), 148.3 (C-2′), 138.2 (C-5′), 136.0 (C-3), 135.8 (C-3′′′), 134.9 (C-3′′), 131.6 (C-9a), 130.5 (C-9a′′), 129.7 (C-9a′′′), 129.6 (C-7), 129.4 (C-7′′), 129.1 (C-7′′′), 127.6 (C-9′′ and C-9′′′), 126.8 (C-9), 126.0 (C-8), 125.9 (C-8′′′), 125.8 (C-8′′), 120.5 (C-6′), 118.6 (C-4′), 117.7 (C-6), 117.5 (C-6′′), 117.6 (C-6′′′), 115.4 (C-1′′′), 115.3 (C-1′′), 114.7 (C-1), 113.8 (C-1′), 101.8 (C-3′), 56.7 (OCH3), 25.5 (C(CH3)3), 18.7 (3CH3). m/z (EI): 957(1) [M+∙ + 2H], 525 (1), 516 (2), 459 (21), 393 (3), 338 (5), 279 (1), 247 (19), 230 (6). Elemental Analysis: C44H38N6O13S3 Calculated: C, 55.34; H, 4.01; N, 8.80; S, 10.07. Found: C, 55.36; H, 3.99; N, 8.78; S, 9.99.


## 2.1.1. General Information

Melting point is corrected and was determined with an Electrothermal Melting Point Apparatus, model 9100. The TLC was carried out on Eastman Chromatogram Silica Gel Sheets (13181; 6060) with a fluorescent indicator. A mixture of ethyl acetate and methylene chloride (7 : 3) was used as an eluent and iodine was used as a revelator for the chromatograms. The IR spectra were measured with a Fourier Transform Infrared spectrometer JASCO FT/IR-4100. The UV spectrum was recorded with a Beckman DU-640 Spectrophotometer. Combustion analyses were carried out with a C, H, N, and S Euro EA from HEKAtech company, and results were found to be in good agreement (±0.3%) with the calculated values. EIMS were measured on Mass Spectrometer LCQ Classic with ESI Source from Thermo Fisher Scientific company.1H-NMR spectrum was recorded in DMSO-d6 with a 250 MHz spectrometer Bruker AV III. 13C-NMR spectrum was recorded in DMSO-d6 with a 62.5 MHz spectrometer Bruker AV III. TMS was used as an internal reference.


## 2.1.2. Preparation of the Reagents and Starting Materials

All the reagents mentioned in this work were purchased from Aldrich and Fluka and were used without further purification.Starting material4 was prepared according to the procedures mentioned in the literature published earlier [19]. A mixture of 4.65 g of 4-methyl-2-oxo-2H-[1]benzopyran-3-carbonitrile (3) and sulphur (1.22 g, in excess) in ethanol (30 ml) was stirred using a magnetic plate shaker thermostated at 45°C. Ammonia (6 ml) was added dropwise during the first 10 min of the reaction. After 7 h of reaction, the resulting precipitate (5.08 g, 93%) was collected by filtration, washed with distilled water, and recrystallised in benzene to yield a yellow powder. m.p. 195-196°C (Lit. [19], 198-199°C, from benzene). IR (KBr, cm−1) υmax: 3449 (–NH2); 1687 (C=O); 1603 and 1547 (C=C); 1224 (C–O) cm−1. 1H NMR (250 MHz, DMSO-d6, Me4Si,δ, ppm): 7.87 (dd, 1H,J = 8.0 and 1.5 Hz, H-8), 7.78 (br s, 2H, NH2); 7.36 (ddd, 1H,J = 8.0, 7.4 and 1.8 Hz, H-6), 7.22–7.17 (m, 2H, H-7 and H-5), 6.89 (s, 1H, thiophenic H). 13C NMR (62.5 MHz, DMSO-d6, Me4Si,δ, ppm): 166.70 (C-3), 158.98 (C-2), 151 (C-4a), 130.90 (C-8a), 129.26 (C-9), 124.39 (C-6), 123.91 (C-8), 118.05 (C-2a), 117.09 (C-7), 98.07 (C-8b), 97.54 (C-5); m/z (EI) 217 (M+; 100%). Elemental Analysis: C11H7NO2S. Calculated: C: 60.83; H, 3.23; N, 6.45; S, 14.75. Found: C, 61.17; H, 3.36; N, 6.49; S, 14.45.


## 2.1.3. Preparation of Diazonium Salt Solution

In a similar manner as described in [20], 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. Compound4 was dissolved in DMSO (10 mL) and cooled to 0–5°C. The nitrosylsulfuric acid solution was added to the solution of4 and the temperature was maintained between 0 and 5°C. The clear diazonium salt solution thus obtained consisting of the in situ formed intermediate5 was used immediately in the coupling reaction.


## 2.1.4. 3-(2-3-(tert-Butyl)-2-hydroxy-5-methoxy-4,6-bis[2-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)diazenyl]phenyldiazenyl)-4H-thieno[3,4-c]chromen-4-one Pentahydrate (7)

The phenol derivative 2-tert-butyl-4-methoxyphenol (540 mg; 3 mmol) was dissolved in DMSO (10 mL) and then cooled in an ice bath at 0–5°C. The diazonium solution of4 previously prepared was added dropwise over 1 hour, and then 15 mL of sodium acetate solution (10%) was added to the mixture. The pH of the mixtures was in the range 9–11. The solid precipitate was collected on a filter and crystallised from methanol to give the title7 (632 mg; 49%) as a green powder. m.p. 200°C. IR (KBr, cm−1) υmax: 3260 (OH), 3073 (Ar. C–H), 1721 (C=O), 1677 (Ar C=C), 1483, 1435 (N=N) cm−1. UV (THF) λmax/nm (logε): 215 (2.95), 240 (4.56), 268 (4.18), 325.5 (4.74), 381 (4.48). 1H NMR (250 MHz, DMSO-d6, Me4Si,δ, ppm): 9.18 (d, 1H,J = 9.75 Hz, 9-H), 9.01 (d, 1H,J = 10.5 Hz, 9′′-H), 8.83 (d, 1H,J = 10.5 Hz, 9′′′-H), 8.59 (dd, 1H,J = 9.0 and 7.8 Hz, 8′′-H), 8.46 (dd, 1H,J = 10.5 and 9.75 Hz, 8′′′-H), 8.32 (dd, 1H,J = 9.75 and 9.75 Hz, 8-H), 8.27 (dd, 1H,J = 9.75 and 9.75 Hz, 7′′-H), 8.15 (dd, 1H,J = 9.75 and 9.0 Hz, 7′′′-H), 8.06 (dd, 1H,J = 11.25 and 9.0 Hz, 7-H), 7.99 (d, 1H,J = 9.0 Hz, 6-H), 7.97 (d, 1H,J = 9.6 Hz, 6′′-H), 7.96 (d, 1H,J = 7.0 Hz, 6′′′-H), 7.67 (s, 1H, 1-H), 7.58 (s, 1H, 1′′′-H), 7.50 (s, 1H, 1′′-H), 3.92 (s, 10H, H2O), 3.90 (s, 1H, OH), 3.50 (s, 3H, OCH3), 1.54 (s, 9H, CH3).13C NMR (62.5 MHz, DMSO-d6, Me4Si,δ, ppm): 164.9 (C-4), 164.2 (C-4′′ and C-4′′′), 157.3 (C-9b′′ and C-9b′′′), 157.2 (C-9b), 155.4 (C-5a′′), 155.3 (C-5a′′′), 154.5 (C-5a), 154.3 (C-3a′′), 154.0 (C-3a′′′), 153.2 (C-3a), 148.3 (C-2′), 138.2 (C-5′), 136.0 (C-3), 135.8 (C-3′′′), 134.9 (C-3′′), 131.6 (C-9a), 130.5 (C-9a′′), 129.7 (C-9a′′′), 129.6 (C-7), 129.4 (C-7′′), 129.1 (C-7′′′), 127.6 (C-9′′ and C-9′′′), 126.8 (C-9), 126.0 (C-8), 125.9 (C-8′′′), 125.8 (C-8′′), 120.5 (C-6′), 118.6 (C-4′), 117.7 (C-6), 117.5 (C-6′′), 117.6 (C-6′′′), 115.4 (C-1′′′), 115.3 (C-1′′), 114.7 (C-1), 113.8 (C-1′), 101.8 (C-3′), 56.7 (OCH3), 25.5 (C(CH3)3), 18.7 (3CH3). m/z (EI): 957(1) [M+∙ + 2H], 525 (1), 516 (2), 459 (21), 393 (3), 338 (5), 279 (1), 247 (19), 230 (6). Elemental Analysis: C44H38N6O13S3 Calculated: C, 55.34; H, 4.01; N, 8.80; S, 10.07. Found: C, 55.36; H, 3.99; N, 8.78; S, 9.99.


## 2.2. Antimicrobial Assay

### 2.2.1. Microorganisms

A total of eight bacteria and three yeasts were tested for their susceptibility to the studied compounds. The microorganisms used in this study were taken from our laboratory collection. Among the clinical strains ofVibrio choleraeused in this study, strains NB2 and SG24 (1) belonged to O1 and O139 serotypes, respectively. These strains were able to produce cholera toxin and hemolysin [21, 22]. The other microbial species used in this study wereCandida parapsilosis ATCC22019,Candida albicans ATCC9002,Cryptococcus neoformansIP95026,Bacillus subtilis,Escherichia coliS2 (1),Vibrio choleraenon-O1 and non-O139 (strains CO6 and PC2), andShigella flexneri. TheV. choleraenon-O1 and non-O139 strains were positive for hemolysin production but negative for cholera toxin production [22]. The bacterial and fungal species were grown at 37°C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) slants, respectively.


### 2.2.2. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC)

The antimicrobial activity was investigated by determining the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum fungicidal concentrations (MFCs). MIC of compounds was assessed using the broth microdilution method [23]. Each test compound was dissolved in dimethylsulfoxide (DMSO, Fisher Chemicals) to give a stock solution. This was serially diluted twofold in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi to obtain a concentration range of 512 to 0.25 μg/ml. One hundred microliters of each concentration was introduced into a well (96-well microplate) containing 90 μl of SDB or MHB, and 10 μl of inoculum (1 × 106 CFU/mL for bacteria and 1 × 105 spores/ml for yeasts) was added to obtain a final concentration range of 256 to 0.125 μg/ml. Plates were covered and incubated on the shaker at 37°C for 24 h (bacteria), 48 h (Candida spp.), and 72 h(Cryptococcus). MICs were assessed visually after the corresponding incubation period and were taken as the lowest sample concentration at which there was no growth or virtually no growth.For the minimum microbicidal concentration (MMC) determination, 10μl aliquots from each well that showed no growth of microorganism were plated on Mueller-Hinton Agar or Sabouraud Dextrose Agar and incubated at 37°C for 24 h (bacteria), 48 h (Candida spp.), and 72 h(Cryptococcus). The lowest concentration that yielded no growth after the subculturing was taken as the MBCs or MFCs. Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) for bacteria and nystatin (Sigma-Aldrich, Steinheim, Germany) for yeasts were used as positive controls, while broth with 20 μL of DMSO was used as a negative control. The assay was repeated thrice.


## 2.2.1. Microorganisms

A total of eight bacteria and three yeasts were tested for their susceptibility to the studied compounds. The microorganisms used in this study were taken from our laboratory collection. Among the clinical strains ofVibrio choleraeused in this study, strains NB2 and SG24 (1) belonged to O1 and O139 serotypes, respectively. These strains were able to produce cholera toxin and hemolysin [21, 22]. The other microbial species used in this study wereCandida parapsilosis ATCC22019,Candida albicans ATCC9002,Cryptococcus neoformansIP95026,Bacillus subtilis,Escherichia coliS2 (1),Vibrio choleraenon-O1 and non-O139 (strains CO6 and PC2), andShigella flexneri. TheV. choleraenon-O1 and non-O139 strains were positive for hemolysin production but negative for cholera toxin production [22]. The bacterial and fungal species were grown at 37°C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) slants, respectively.


## 2.2.2. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC)

The antimicrobial activity was investigated by determining the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum fungicidal concentrations (MFCs). MIC of compounds was assessed using the broth microdilution method [23]. Each test compound was dissolved in dimethylsulfoxide (DMSO, Fisher Chemicals) to give a stock solution. This was serially diluted twofold in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi to obtain a concentration range of 512 to 0.25 μg/ml. One hundred microliters of each concentration was introduced into a well (96-well microplate) containing 90 μl of SDB or MHB, and 10 μl of inoculum (1 × 106 CFU/mL for bacteria and 1 × 105 spores/ml for yeasts) was added to obtain a final concentration range of 256 to 0.125 μg/ml. Plates were covered and incubated on the shaker at 37°C for 24 h (bacteria), 48 h (Candida spp.), and 72 h(Cryptococcus). MICs were assessed visually after the corresponding incubation period and were taken as the lowest sample concentration at which there was no growth or virtually no growth.For the minimum microbicidal concentration (MMC) determination, 10μl aliquots from each well that showed no growth of microorganism were plated on Mueller-Hinton Agar or Sabouraud Dextrose Agar and incubated at 37°C for 24 h (bacteria), 48 h (Candida spp.), and 72 h(Cryptococcus). The lowest concentration that yielded no growth after the subculturing was taken as the MBCs or MFCs. Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) for bacteria and nystatin (Sigma-Aldrich, Steinheim, Germany) for yeasts were used as positive controls, while broth with 20 μL of DMSO was used as a negative control. The assay was repeated thrice.


## 3. Results and Discussion

### 3.1. Chemistry

The title 2-aminothiophene reagent4 was prepared in excellent yield by applying the third version of the Gewald methodology (Scheme 1) as previously reported [19, 24–26].Scheme 1
Reaction sequences to compound4.The in situ formed intermediate thienyl diazonium sulphate5 (Scheme 2) was generated from the diazotization of compound4 using nitrosylsulfuric acid at a very low temperature (0–5°C). The freshly prepared diazonium solution was then coupled with phenolic reagent 2-tert-butyl-4-methoxyphenol6. The resulting mixture was worked up as usual to yield the azo compound7 (Scheme 2).Scheme 2
Reaction sequences to compound7.The structure of the newly synthesized compound7 was elucidated by IR, UV, NMR, mass spectral studies, and elemental analysis.The IR spectrum of compound7 showed absorption bands ranging between 3260 and 2324 cm−1 due to the stretching vibration of OH of phenol and H2O molecules. Around 1721 cm−1 appeared a band of stretching frequencies of the carbonyl functionality, whereas the characteristic stretching frequencies of the –N=N– linkages were exhibited around 1435 cm−1. The absorption bands at 1677 cm−1 and 763 cm−1 depicted the presence of C=C and Ar–H, respectively. The infrared spectra also showed a distinctive band at 1051 cm−1 due to C–O bond of the methoxy group.The electronic transitions of the UV-visible spectra in DMSO give rise to wavelengthλmax ranging from 215 to 381 nm as a result of π→π∗ transitions of the compound indicating the presence of an extended π-conjugation of the C=C backbone enhanced by the N=N bridges linking the three thienocoumarins moieties to the parent phenolic benzene ring. Five strong absorption bands were observed, whereby those appearing at λmax 215 nm (logε=2.95) and λmax 240 nm (logε=4.56) may be attributed to π→π∗ transition of the benzenoid moiety of the compounds and intraligand π→π∗ transition. The third and fourth bands located at λmax 268 nm (logε=4.18) and λmax 325.5 nm (logε=4.74) were attributed to the electronic n→π∗ transitions of the –N=N– group. The last band appearing in the visible region around λmax 381 nm (logε=4.48) is probably related to a transition involving the whole electronic system of the azo dyes.The structure of compound7 is further strongly supported by its elemental analysis and its HREIMS, which showed a molecular ion peak at m/z 955. The mass spectrum of compound7 exhibited fragment ions at m/z 910, 865, and 847 which could be assigned to [M+∙  − COOH], [M+∙ − 5H2O], and [M+∙  − 6H2O], respectively. The ion fragments at m/z=663, 616, and 230 were assigned as in Scheme 3, confirming the above structural hypothesis.Scheme 3
Significant HRMS fragmentation patterns of compound7.On the1H-NMR spectrum, the singlets observed at δH = 3.50 and 1.54 ppm were, respectively, assigned to the protons of the methoxy group and the protons of the 3 methyl substituents of thetert-butyl group. The thiophenic protons resonated at δH = 7.67, 7.50, and 7.58 ppm as a singlet, while the singlets observed at δH = 9.04 ppm were assigned to the OH group. The 1H-NMR also showed a set of three couples of poorly resolved “ddd”-signals [centered at 8.06 (H-7,J = 11.25 and 9.0 Hz), 8.32 (H-8,J = 9.75 and 9.75 Hz), 8.27 (H-7′′,J = 9.75 and 9.75 Hz), 8.59 (H-8′′,J = 9.0 and 7.8 Hz), 8.15 (H-7′′′,J = 9.75 and 9.0 Hz), and 8.46 (H-8′′′,J = 10.5 and 9.75 Hz), ppm] and a set of three couples of poorly resolved “dd”-signals [centered at 7.99 (H-6,J = 9.0 Hz), 9.18 (H-9,J = 9.75 Hz), 7.97 (H-6′′,J = 9.6 Hz), 9.01 (H-9′′,J = 10.5 Hz), 7.96 (H-6′′′,J = 7.0 Hz), and 8.83 (H-9′′′,J = 10.5 Hz)] overlapping in the range 7.97–9.18 ppm, which were attributed to the protons of the three thienocoumarin substructural units of the coupling product. The 1H-1H-COSY experiment was advantageously used in the accurate assignments of the different 1H-1H connectivities [27].The13C-NMR spectrum of compound7 displayed forty carbon signals instead of forty-four as required by the molecular formula. This could be explained by the overlapping of the signals of the three methyl substituents of thetert-butyl group at δC 18.72 ppm due to their magnetic and chemical equivalence. Furthermore, it was assumed that the signals of two of the three carbonyl functionalities overlapped to give the intense signal at δC 164.17 ppm besides the signal at δC 164.94 ppm assigned to the other carbonyl group. A close inspection of the 3D view (Figure 2) of the optimized structure geometry of compound7 clearly reveals that the electron clouds of the carbonyl groups of the two 3-diazenyl-4H-thieno[3,4-c]chromen-4-one moieties attached at positions −3 and −5 of the central phenolic ring are virtually more involved in the interactions with the electron clouds of the neighboring atoms and groups than the carbonyl group of the 3-diazenyl-4H-thieno[3,4-c]chromen-4-one moiety fixed at position −2 of the core phenolic ring. These observations could justify the slight upfield shifts (Δδ = 166.70–164.17 = 2.53 ppm for the two overlapping carbonyl groups and Δδ = 166.70–164.94 = 1.76 ppm for the other one) that occurred in the resonances of the three carbonyl functions when compared to the chemical shift (δC = 166.70 ppm) of the corresponding carbonyl group in the starting compound4. Based on the DEPT-90/135 experiments, the remaining signals were assigned without ambiguity to the aromatic C atoms bearing hydrogen atoms. The HMBC experiments [27] were used to disclose the two-bond (J2CH), three-bond (J3CH), and occasionally four-bond (J4CH) couplings (Table 1 and Figure 1) between the hydrogen atoms and the tertiary and quaternary carbon atoms, thereby facilitating their identification and their assignments.Table 1
Important HMBC interactions in compound7; 1H and 13C chemical shifts (δ/ppm) in DMSO-d6 as the solvent (25°C).
Compound7
Compound7
C atom
δ
13C
HMBC (H → C)
C atom
δ
13C
HMBC (H → C)
7
129.6
H-8 (8.32)
9a′′
130.5
H-8′′ (8.59), H-7′′ (8.27), H-6′′ (7.97)
9
126.8
H-1 (7.67)
1′′′
115.4
H-9′′′ (8.83), H-1′′′ (7.58)
9a
131.6
H-1 (7.67)
3′′′
135.8
H-9′′′ (8.83), H-7′′′ (8.15), H-1′′′ (7.58)
9b
157.2
H-8 (8.32), H-6 (7.99)
4′′′
164.2
H-7′′′ (8.15)
1′′
115.3
H-1′′ (7.50)
5a′′′
155.3
H-9′′′ (8.83), H-7′′′ (8.15)
5a′′
155.4
H-6′′ (7.97)
6′′′
117.6
H-7′′′ (8.15)
7′′
129.4
H-9′′ (9.01), H-6′′ (7.97)
9′′′
127.6
H-8′′′ (8.46), H-1′′′ (7.58)
8′′
125.8
H-8′′ (8.59)
9a′′′
129.7
H-1′′′ (7.58)
9′′
127.6
H-1′′ (7.50)Figure 1
HMBC interactions in compound 7.Figure 2
Optimized 3D view of compound7. The structure was drawn with the program ACD/3D viewer (freeware) of ACD/Labs.


### 3.2. Antimicrobial Activity

The antimicrobial activity of new trisazo dye along with some of its precursors was examined by microdilution susceptibility assay against eight bacterial strains and three yeasts selected on the basis of their relevance as human pathogens. The results showed that the tested compounds exhibited variable MICs (2 to 256μg/mL) depending on the microbial strains (Table 2). The most resistant microorganisms wereV. choleraeNB2 andV. choleraeSG24, whereas the most sensitive microorganism wasC. neoformans.Compound7 (MIC = 2–128 μg/mL) was more active as compared with its precursors followed in a decreasing order by compound4 (MIC = 4–256 μg/mL), compound6 (MIC = 8–>256 μg/mL), and compound3 (MIC = 32–>256 μg/mL).Table 2
Minimum inhibitory concentrations (MICs) and minimum microbicidal concentrations (MMCs) (µg/ml) of compound 7 and its entire precursors against fungal and bacterial strains.
Microorganisms
Inhibition parameters
3
4
6
7
Referencedrugs∗
Gram-positive bacteria
Bacillus subtilis
MIC
64
16
64
8
2
MBC
256
32
>256
32
2
S. aureus
MIC
64
16
32
2
0.5
MBC
>256
32
64
8
0.5
Gram-negative bacteria
E. coliS2(1)
MIC
64
32
32
8
2
MBC
256
64
>256
32
2
S. flexneri
MIC
128
32
128
16
8
MBC
256
>256
>256
32
8
P. aeruginosa
MIC
128
16
128
8
4
MBC
>256
32
>256
32
4
V. choleraeNB2
MIC
>256
256
256
64
32
MBC
nd
>256
>256
128
32
V. choleraeSG24
MIC
>256
>256
>256
128
64
MBC
nd
nd
nd
256
64
V. cholerae CO6
MIC
256
64
>256
32
8
MBC
>256
128
nd
64
8
Yeasts
C. parapsilosis ATCC22019
MIC
64
4
32
4
2
MFC
256
8
64
4
2
C. albicans ATCC9002
MIC
64
8
64
4
4
MFC
>256
16
128
8
4
C. neoformansIP95026
MIC
32
4
8
4
2
MFC
128
8
16
4
2
C
i
p
r
o
f
l
o
x
a
c
i
n
∗ for bacteria and nystatin for yeasts; nd: not determined; compounds 1, 2, and 5 were not tested.The lowest MIC value of 2μg/mL was recorded onS. aureuswith compound7, whereas the lowest MMC value of 4 μg/mL was obtained onC. parapsilosis andC. neoformans with compound7. However, the highest MIC value of 256 μg/mL was recorded onV. choleraeCO6 with compound3 and onV. cholerae NB2 with compounds4 and6, while the highest MMC value of 256 μg/mL was obtained onV. cholerae SG24 with compound7 and onC. parapsilosis,S. flexneri,E. coli, andB. subtiliswith compound3. A lower MBC/MIC (≤4) value signifies that a minimum amount of compounds is used to kill the microbial species, whereas a higher value signifies the use of comparatively higher amounts of samples for the control of any microorganism [28].The strains ofV. cholerae NB2 and PC2 [21, 22] andShigella flexneri [29] included in the present study were MDR clinical isolates and these were resistant to commonly used drugs such as ampicillin, streptomycin, tetracycline, nalidixic acid, furazolidone, and cotrimoxazole. However, these bacterial strains were found to be sensitive to most of the tested samples, suggesting that their administration may represent an alternative treatment against the diseases caused by these types of MDR pathogenic strains.From the SAR point of view, we can say that compounds4 and6 are not microbicides for the bacterial strainsV. cholerae NB2,S. flexneri, andV. cholerae SG24, with respect to the MBC/MIC data displayed in Table 2. In contrast, compound7is microbicidal for these bacterial strains. The improved antibacterial profiles of the hybrid compound could be due to the synergetic interactions of the primary potent pharmacophores combined in the single molecular platform. The cumulative effects of the three disazo scaffolds certainly contributed to a greater extent to shaping the global biological profile of the new hybrid molecule. These observations that corroborate recent findings from analogous investigation [18].


## 3.1. Chemistry

The title 2-aminothiophene reagent4 was prepared in excellent yield by applying the third version of the Gewald methodology (Scheme 1) as previously reported [19, 24–26].Scheme 1
Reaction sequences to compound4.The in situ formed intermediate thienyl diazonium sulphate5 (Scheme 2) was generated from the diazotization of compound4 using nitrosylsulfuric acid at a very low temperature (0–5°C). The freshly prepared diazonium solution was then coupled with phenolic reagent 2-tert-butyl-4-methoxyphenol6. The resulting mixture was worked up as usual to yield the azo compound7 (Scheme 2).Scheme 2
Reaction sequences to compound7.The structure of the newly synthesized compound7 was elucidated by IR, UV, NMR, mass spectral studies, and elemental analysis.The IR spectrum of compound7 showed absorption bands ranging between 3260 and 2324 cm−1 due to the stretching vibration of OH of phenol and H2O molecules. Around 1721 cm−1 appeared a band of stretching frequencies of the carbonyl functionality, whereas the characteristic stretching frequencies of the –N=N– linkages were exhibited around 1435 cm−1. The absorption bands at 1677 cm−1 and 763 cm−1 depicted the presence of C=C and Ar–H, respectively. The infrared spectra also showed a distinctive band at 1051 cm−1 due to C–O bond of the methoxy group.The electronic transitions of the UV-visible spectra in DMSO give rise to wavelengthλmax ranging from 215 to 381 nm as a result of π→π∗ transitions of the compound indicating the presence of an extended π-conjugation of the C=C backbone enhanced by the N=N bridges linking the three thienocoumarins moieties to the parent phenolic benzene ring. Five strong absorption bands were observed, whereby those appearing at λmax 215 nm (logε=2.95) and λmax 240 nm (logε=4.56) may be attributed to π→π∗ transition of the benzenoid moiety of the compounds and intraligand π→π∗ transition. The third and fourth bands located at λmax 268 nm (logε=4.18) and λmax 325.5 nm (logε=4.74) were attributed to the electronic n→π∗ transitions of the –N=N– group. The last band appearing in the visible region around λmax 381 nm (logε=4.48) is probably related to a transition involving the whole electronic system of the azo dyes.The structure of compound7 is further strongly supported by its elemental analysis and its HREIMS, which showed a molecular ion peak at m/z 955. The mass spectrum of compound7 exhibited fragment ions at m/z 910, 865, and 847 which could be assigned to [M+∙  − COOH], [M+∙ − 5H2O], and [M+∙  − 6H2O], respectively. The ion fragments at m/z=663, 616, and 230 were assigned as in Scheme 3, confirming the above structural hypothesis.Scheme 3
Significant HRMS fragmentation patterns of compound7.On the1H-NMR spectrum, the singlets observed at δH = 3.50 and 1.54 ppm were, respectively, assigned to the protons of the methoxy group and the protons of the 3 methyl substituents of thetert-butyl group. The thiophenic protons resonated at δH = 7.67, 7.50, and 7.58 ppm as a singlet, while the singlets observed at δH = 9.04 ppm were assigned to the OH group. The 1H-NMR also showed a set of three couples of poorly resolved “ddd”-signals [centered at 8.06 (H-7,J = 11.25 and 9.0 Hz), 8.32 (H-8,J = 9.75 and 9.75 Hz), 8.27 (H-7′′,J = 9.75 and 9.75 Hz), 8.59 (H-8′′,J = 9.0 and 7.8 Hz), 8.15 (H-7′′′,J = 9.75 and 9.0 Hz), and 8.46 (H-8′′′,J = 10.5 and 9.75 Hz), ppm] and a set of three couples of poorly resolved “dd”-signals [centered at 7.99 (H-6,J = 9.0 Hz), 9.18 (H-9,J = 9.75 Hz), 7.97 (H-6′′,J = 9.6 Hz), 9.01 (H-9′′,J = 10.5 Hz), 7.96 (H-6′′′,J = 7.0 Hz), and 8.83 (H-9′′′,J = 10.5 Hz)] overlapping in the range 7.97–9.18 ppm, which were attributed to the protons of the three thienocoumarin substructural units of the coupling product. The 1H-1H-COSY experiment was advantageously used in the accurate assignments of the different 1H-1H connectivities [27].The13C-NMR spectrum of compound7 displayed forty carbon signals instead of forty-four as required by the molecular formula. This could be explained by the overlapping of the signals of the three methyl substituents of thetert-butyl group at δC 18.72 ppm due to their magnetic and chemical equivalence. Furthermore, it was assumed that the signals of two of the three carbonyl functionalities overlapped to give the intense signal at δC 164.17 ppm besides the signal at δC 164.94 ppm assigned to the other carbonyl group. A close inspection of the 3D view (Figure 2) of the optimized structure geometry of compound7 clearly reveals that the electron clouds of the carbonyl groups of the two 3-diazenyl-4H-thieno[3,4-c]chromen-4-one moieties attached at positions −3 and −5 of the central phenolic ring are virtually more involved in the interactions with the electron clouds of the neighboring atoms and groups than the carbonyl group of the 3-diazenyl-4H-thieno[3,4-c]chromen-4-one moiety fixed at position −2 of the core phenolic ring. These observations could justify the slight upfield shifts (Δδ = 166.70–164.17 = 2.53 ppm for the two overlapping carbonyl groups and Δδ = 166.70–164.94 = 1.76 ppm for the other one) that occurred in the resonances of the three carbonyl functions when compared to the chemical shift (δC = 166.70 ppm) of the corresponding carbonyl group in the starting compound4. Based on the DEPT-90/135 experiments, the remaining signals were assigned without ambiguity to the aromatic C atoms bearing hydrogen atoms. The HMBC experiments [27] were used to disclose the two-bond (J2CH), three-bond (J3CH), and occasionally four-bond (J4CH) couplings (Table 1 and Figure 1) between the hydrogen atoms and the tertiary and quaternary carbon atoms, thereby facilitating their identification and their assignments.Table 1
Important HMBC interactions in compound7; 1H and 13C chemical shifts (δ/ppm) in DMSO-d6 as the solvent (25°C).
Compound7
Compound7
C atom
δ
13C
HMBC (H → C)
C atom
δ
13C
HMBC (H → C)
7
129.6
H-8 (8.32)
9a′′
130.5
H-8′′ (8.59), H-7′′ (8.27), H-6′′ (7.97)
9
126.8
H-1 (7.67)
1′′′
115.4
H-9′′′ (8.83), H-1′′′ (7.58)
9a
131.6
H-1 (7.67)
3′′′
135.8
H-9′′′ (8.83), H-7′′′ (8.15), H-1′′′ (7.58)
9b
157.2
H-8 (8.32), H-6 (7.99)
4′′′
164.2
H-7′′′ (8.15)
1′′
115.3
H-1′′ (7.50)
5a′′′
155.3
H-9′′′ (8.83), H-7′′′ (8.15)
5a′′
155.4
H-6′′ (7.97)
6′′′
117.6
H-7′′′ (8.15)
7′′
129.4
H-9′′ (9.01), H-6′′ (7.97)
9′′′
127.6
H-8′′′ (8.46), H-1′′′ (7.58)
8′′
125.8
H-8′′ (8.59)
9a′′′
129.7
H-1′′′ (7.58)
9′′
127.6
H-1′′ (7.50)Figure 1
HMBC interactions in compound 7.Figure 2
Optimized 3D view of compound7. The structure was drawn with the program ACD/3D viewer (freeware) of ACD/Labs.


## 3.2. Antimicrobial Activity

The antimicrobial activity of new trisazo dye along with some of its precursors was examined by microdilution susceptibility assay against eight bacterial strains and three yeasts selected on the basis of their relevance as human pathogens. The results showed that the tested compounds exhibited variable MICs (2 to 256μg/mL) depending on the microbial strains (Table 2). The most resistant microorganisms wereV. choleraeNB2 andV. choleraeSG24, whereas the most sensitive microorganism wasC. neoformans.Compound7 (MIC = 2–128 μg/mL) was more active as compared with its precursors followed in a decreasing order by compound4 (MIC = 4–256 μg/mL), compound6 (MIC = 8–>256 μg/mL), and compound3 (MIC = 32–>256 μg/mL).Table 2
Minimum inhibitory concentrations (MICs) and minimum microbicidal concentrations (MMCs) (µg/ml) of compound 7 and its entire precursors against fungal and bacterial strains.
Microorganisms
Inhibition parameters
3
4
6
7
Referencedrugs∗
Gram-positive bacteria
Bacillus subtilis
MIC
64
16
64
8
2
MBC
256
32
>256
32
2
S. aureus
MIC
64
16
32
2
0.5
MBC
>256
32
64
8
0.5
Gram-negative bacteria
E. coliS2(1)
MIC
64
32
32
8
2
MBC
256
64
>256
32
2
S. flexneri
MIC
128
32
128
16
8
MBC
256
>256
>256
32
8
P. aeruginosa
MIC
128
16
128
8
4
MBC
>256
32
>256
32
4
V. choleraeNB2
MIC
>256
256
256
64
32
MBC
nd
>256
>256
128
32
V. choleraeSG24
MIC
>256
>256
>256
128
64
MBC
nd
nd
nd
256
64
V. cholerae CO6
MIC
256
64
>256
32
8
MBC
>256
128
nd
64
8
Yeasts
C. parapsilosis ATCC22019
MIC
64
4
32
4
2
MFC
256
8
64
4
2
C. albicans ATCC9002
MIC
64
8
64
4
4
MFC
>256
16
128
8
4
C. neoformansIP95026
MIC
32
4
8
4
2
MFC
128
8
16
4
2
C
i
p
r
o
f
l
o
x
a
c
i
n
∗ for bacteria and nystatin for yeasts; nd: not determined; compounds 1, 2, and 5 were not tested.The lowest MIC value of 2μg/mL was recorded onS. aureuswith compound7, whereas the lowest MMC value of 4 μg/mL was obtained onC. parapsilosis andC. neoformans with compound7. However, the highest MIC value of 256 μg/mL was recorded onV. choleraeCO6 with compound3 and onV. cholerae NB2 with compounds4 and6, while the highest MMC value of 256 μg/mL was obtained onV. cholerae SG24 with compound7 and onC. parapsilosis,S. flexneri,E. coli, andB. subtiliswith compound3. A lower MBC/MIC (≤4) value signifies that a minimum amount of compounds is used to kill the microbial species, whereas a higher value signifies the use of comparatively higher amounts of samples for the control of any microorganism [28].The strains ofV. cholerae NB2 and PC2 [21, 22] andShigella flexneri [29] included in the present study were MDR clinical isolates and these were resistant to commonly used drugs such as ampicillin, streptomycin, tetracycline, nalidixic acid, furazolidone, and cotrimoxazole. However, these bacterial strains were found to be sensitive to most of the tested samples, suggesting that their administration may represent an alternative treatment against the diseases caused by these types of MDR pathogenic strains.From the SAR point of view, we can say that compounds4 and6 are not microbicides for the bacterial strainsV. cholerae NB2,S. flexneri, andV. cholerae SG24, with respect to the MBC/MIC data displayed in Table 2. In contrast, compound7is microbicidal for these bacterial strains. The improved antibacterial profiles of the hybrid compound could be due to the synergetic interactions of the primary potent pharmacophores combined in the single molecular platform. The cumulative effects of the three disazo scaffolds certainly contributed to a greater extent to shaping the global biological profile of the new hybrid molecule. These observations that corroborate recent findings from analogous investigation [18].


## 4. Conclusion

In summary, the diazotized title 2-aminothiophene derivative5 is a powerful electrophilic reagent which attacks phenolic substrate6 at all its three nonsubstituted ring positions to give with good yield the corresponding trisazo hybrid compound7 whose structural assignment was done on the basis of the available analytic and spectroscopic data. The combination in a single molecular platform of the pharmacophores related to the phenolic, the three thienocoumarin, and the three disazo moieties resulted in a synergy of actions which globally contributed as initially anticipated to enhancing the antimicrobial activities of the reaction product7 when compared with the profiles of the precursors3,4, and6. The results showed that the diazotization followed by the coupling reaction increases the antimicrobial activity of the trisazo compound compared with that of the free 2-aminothiophene and phenol. The overall results of this study indicated that compound7 had the greatest potential value against both yeasts and multidrug-resistant bacteria, so further investigation is warranted.



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*Source: 9197821-2018-02-01.xml*