Synthesis, characterization and pharmacological evaluation of 2-aminothiazole incorporated azo dyes

Journal Pre-proof Synthesis, characterization and pharmacological evaluation of 2-aminothiazole incorporated azo dyes B.N. Ravi, Keshavayya J, Mallikarjuna N. M, Vinod Kumar, Shivanand Kandgal PII:

S0022-2860(19)31602-3

DOI:

https://doi.org/10.1016/j.molstruc.2019.127493

Reference:

MOLSTR 127493

To appear in:

Journal of Molecular Structure

Received Date: 9 July 2019 Revised Date:

21 November 2019

Accepted Date: 27 November 2019

Please cite this article as: B.N. Ravi, K. J, M.N. M, V. Kumar, S. Kandgal, Synthesis, characterization and pharmacological evaluation of 2-aminothiazole incorporated azo dyes, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.127493. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Synthesis, Characterization and Pharmacological evaluation of 2-aminothiazole incorporated azo dyes Ravi B Na, Keshavayya Ja*, Mallikarjuna N Ma, Vinod Kumara, Shivanand Kandgalb a

Department of PG Studies and Research in Chemistry, School of Chemical Sciences, Kuvempu University, JnanaSahyadri, Shankaraghatta-577451, Karnataka, India. b

Department of PG Studies and Research in Biotechnology and Bioinformatics, Kuvempu University, JnanaSahyadri, Shankaraghatta-577451, Karnataka, India. (E-mail: [email protected], Tel.: +91-9448446151) Abstract Azo molecules (A1-A3) were prepared by reacting 2-aminothiazole with three different

pyridone

derivatives

as

coupling

components,

viz.,

6-hydroxy-4-methyl-2-oxo-1,2-

dihydropyridine-3-carbonitrile, 6-hydroxy-1,4-dimethyl-2-oxo-1,2-dihydropyridine-3-nitrile and 1-ethyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile

via

diazo-coupling

reactions, under appropriate experimental conditions. The structural characteristics of the synthesized compounds were checked by different spectral techniques. The newly synthesized molecules were tested for their biological activities and the results of biological activity studies of the compounds prepared exhibited potential antimicrobial activity against the respective microorganisms. The synthesized azo dyes also demonstrated promising antimycobacterial activity screened against M. tuberculosis. All the compounds showed considerable cleavage efficiency against supercoiled pBR322 DNA. Further, the in silico molecular docking study was performed against E.coli 24 kDa domain and Cyp51 (cytochrome P450 14α-sterol demethylase) as target receptors. The binding energy of each studied compound was evaluated, and the data revealed that the compound A1 displayed higher binding affinity value and forms hydrogen bonds with the active amino acids.

Keywords: 2-aminothiazole; azo dyes; antimicrobial activity; DNA cleavage; molecular docking.

1. Introduction Azo dyes are the most extensively utilized group of organic compounds and attracted a great deal of interest from the researchers due to their diverse applications in the various fields of chemical science and technology. The usage of these dyes includes as dye stuffs in the pharmaceutical, leather and polymer industry [1-3]. Further, these azo molecules advanced into electronic technology such as non-linear optical devices, liquid crystalline displays, storage devices, dye-sensitized solar cells, and optical communication devices [4-6]. Additionally, azo dyes based on heterocyclic moiety are known for their excellent colouring properties, tinctorial strength, fastness, thermal stability. Further, this class of compounds also exhibits a prominent cell bathochromic effect than the dyes derived from simple aromatic systems [5-16]. Among the heterocyclic azo dyes, those containing thiazole nucleus fascinated the chemists because of its significance into in this field due to their exceptional pharmacological activities [17] such as inhibition of RNA, DNA, protein synthesis, antitumor [18], antihistamines [19-20], anti-inflammatory [21], schistosomicidal agents [22], antituberculosis [23], antibacterial [24], insecticides [25] and production of drugs in chemotherapy [26-27]. Previous reports revealed that the existence of the azo chromophore in the heterocyclic ring enlarges the potential pharmacological activity of that distinct compound [28]. Apart from the pharmacological importance, 2-aminothiazole and its derivatives were utilized worldwide as coupling and diazo components in the dye industry [29]. Nowadays, even though numerous medicines are readily accessible in the market for the treatment of dreadful diseases, still it remains a challenge to prepare the compounds having less toxicity and of more resistance against various pathogens [30]. Therefore, it is required to design potent drug molecules to improve the imminent scientific challenges. Also, thiazole based azo dyes

were biologically more active because of their non-toxicity and specific action against target diseases [31]. From the recent literature, it is reported that the substituted thiazole based azo dyes have exhibited a wide spectrum of biological activities such as antidepressant, antioxidant, cytotoxic, anti-infectious and fungicidal activities [32]. The thiazole and its derivatives having potent biological activities and it may be used as lead drug molecules to cure multiple diseases [33]. Also, pyridone derivatives are comparatively current heterocyclic intermediates for the synthesis of azo molecules [34]. Thus, it is very useful to design new drug molecules containing a thiazole nucleus with numerous remedial properties. In view of the above, the present work explores the synthesis of some novel heterocyclic azo colourants derived from 2-aminothiazole by the conventional diazo-coupling method [3536]. The synthesized azo compounds have been characterized by various physico-chemical techniques. The bio-potency of the synthesized dyes was tested against dissimilar microbial strains. DNA cleavage studies were carried out for prepared azo molecules to examine the potentiality to restrain the relevant pathogens. Further, the in silico molecular docking was also examined to know the mechanism of drug action on a microbial infection. H3C

N N

O

HO

N H

A1

S

H3C

N

CN

CN

N

N

N S

H3C

N

CN

N

O

S

N

O N

N HO A2

CH3

HO

CH3

A3

2. Experimental 2.1 Methods and materials The reagents, solvents, and chemicals used for the current work were purchased from Sigma-Aldrich chemical company. Melting points were measured on the electro-thermal

apparatus using open capillary tubes. Absorption spectra were recorded on the UV-probe-1800 spectrometer in two polar solvents (DMF and DMSO). IR spectra were recorded on a FTIRALPHA BRUKER instrument using potassium bromide pellets in the range of 4000-400 cm-1. The 1H and

13

C NMR spectra were recorded in DMSO-d6 at 400 MHz using an amx500 NMR

spectrometer with tetramethylsilane (TMS) as an internal standard and chemical shifts were indicated as δ values in ppm. The mass spectra of the synthesized compounds were obtained on LCMS, SHIMADZU mass analyzer 2010. 2.2 General procedure for the synthesis of azo molecules A1-A3 Heterocyclic based azo compounds were obtained by the well-known diazotization reaction reported in the literature [37]. The compound A1 is prepared as follows, 2 mmol (0.2 g) of the 1,3-thiazole-2-amine was dissolved in a mixture of propionic and acetic acid (1:2, 6 mL) at room temperature. The resulting solution was stirred by using magnetic stirrer and then cooled to 0-5 0C by using a chilled solution of ice salt-bath. A previously prepared 2 mmol solution of nitrosylsulfuric acid (which was obtained by dissolving 0.138 g of sodium nitrite in 2 mL of sulphuric acid) was added drop by drop to the above chilled amine solution over a period of 20 min with constant stirring, and stirring was continued for 2 h by maintaining the reaction mixture 0-5 0C. After completion of the diazotization reaction, this obtained salt solution was utilized for the coupling reaction. A 2 mmol of the substituted pyridone (R=H) was prepared by dissolving 0.148 g in the aqueous KOH and maintained the temperature at 0-5 0C and then the diazonium chloride solution was poured dropwise into the nucleophilic compound within 15 min in stirred condition for another 2 h. The pH of the whole reaction was adjusted to 5-6 by the simultaneous addition of 10% of the saturated NaHCO3 solution. The completion of the reaction was monitored by TLC, and the resultant product was filtered off, washed a number of times with

distilled water and dried in an oven. The pure azo dyes were collected by recrystallization from ethanol. Similarly the compounds A2 and A3 were prepared by adopting the above method and by using the appropriate coupling components. The schematic route for the synthesis of the above azo dye was shown in Scheme 1. H3C

S NH2 N 1,3-thiazol-2-amine

NaNO2/H2SO4 0-5 o C, Stirring

CN

S +

N 2 Cl

-

+

O N

N HO

R

(1-3) Coupling at pH 5-6 Stirr for 2 h

H3C N

S

O

N N

CN

N HO

R

A1-A3 Where R= H, CH 3 , CH 3CH 2

Scheme 1: Synthesis of azo compounds A1-A3 2.2.1

Preparation

of

6-hydroxy-4-methyl-2-oxo-5-[1,3-thiazol-2-yldiazenyl]-1,2-di

hydropyridine-3-carbonitrile (A1): Red crystals (yield: 72%; mp: 175-176 0C). FT-IR (KBr) vmax: OH (3449 cm-1), CH (2983 cm-1), Ali-CH (2821 cm-1), CN (2203 cm-1), C=O (1618 cm-1), N=N (1540 cm-1), C=C (1425 cm-1); 1H NMR (DMSO-d6) δ in ppm 2.4(s, 3H, -CH3), 7.3(d, 1H, ArH, J=3.6 Hz), 7.6(d, 1H, ArH, J=3.6 Hz), 10.4(s, 1H, -NH). 13C NMR (400 MHz, DMSO-d6): δ 15.20, 72.26, 117.34, 118.12, 122.48, 133.20, 141.16, 150.12, 158.79, 172.26. Anal. Calcd (%) for C10H7N5O2S: C (45.97); H (2.70); N (26.81); S (12.27). Found (%): C (45.92); H (2.76); N (26.86); S (12.24). LCMS m/z=262.4 (M+).

2.2.2 Preparation of 6-hydroxy-1,4-dimethyl-2-oxo-5-[1,3-thiazol-2-yldiazenyl]-1,2-di hydro pyridine-3-carbonitrile (A2): Brown crystals (yield: 73%; mp: 180-181 0C). FT-IR (KBr) vmax: OH (3117 cm-1), CH (3081 cm1

), Ali-CH (2827 cm-1), CN (2225 cm-1), C=O (1632 cm-1), N=N (1502 cm-1), C=C (1451 cm-1);

1

H NMR (DMSO-d6) δ in ppm 2.4(s, 3H, -CH3), 3.1(s, 3H, -CH3), 7.4(d, 1H, ArH, J=3.6 Hz),

7.6(d, 1H, ArH, J=3.6 Hz).

13

C NMR (400 MHz, DMSO-d6): δ 15.28, 28.84, 72.20, 117.34,

118.18, 122.58, 133.26, 143.13, 152.12, 158.34, 172.10. Anal. Calcd (%) for C11H9N5O2S: C (47.99); H (3.30); N (25.44); S (11.65). Found (%): C (47.95); H (3.35); N (25.41); S (11.70). LCMS m/z=276.4 (M+). 2.2.3

Preparation

of

1-ethyl-6-hydroxy-4-methyl-2-oxo-5-[1,3-thiazol-2-yldiazenyl]-1,2-

dihydropyridine-3-carbonitrile (A3): Yellowish crystals (yield: 70%; mp: 183-184 0C). FT-IR (KBr) vmax: OH (3107 cm-1), CH (2940 cm-1), Ali-CH (2838 cm-1), CN (2224 cm-1), C=O (1628 cm-1), N=N (1496 cm-1) C=C (1446 cm1

); 1H NMR (DMSO-d6) δ in ppm 1.1(t, 3H, -CH3, J=6.8 Hz), 2.4(s, 3H, -CH3), 3.8(q, 2H, -CH2,

J=7.2 Hz), 7.4(d, 1H, ArH, J=3.6 Hz), 7.6(d, 1H, ArH, J=3.6 Hz). 13C NMR (400 MHz, DMSOd6): δ 15.93, 26.02, 44.99, 72.39, 117.30, 118.26, 122.58, 133.19, 143.12, 152.29, 158.37, 172.13. Anal. Calcd (%) for C12H11N5O2S: C (49.82); H (3.83); N (24.21); S (11.08). Found (%): C (49.77); H (3.81); N (24.24); S (11.10). LCMS m/z=290.4 (M+). 2.3 Pharmacological activity 2.3.1 Antibacterial study The prepared azo dyes (A1-A3) were evaluated for their antibacterial study against two bacterial strains B. subtilis (ATCC 19659) and E. coli (ATCC 25922) by using an agar disc diffusion method [38, 39]. A 48 hours old culture of the above bacterial strains were mixed with

sterile physiological saline (0.9%), and the turbidity was adjusted to the standard inoculum of McFarland scale 0.5 (106 colony forming units (CFU) per mL). The mixture of Muller Hinton Agar (20 mL) and Sabouraud dextrose agar was used for the antibacterial activity. The inoculum was spread on the surface of the solidified media and Whatman no. 1 filter paper discs (5 mm in diameter) impregnated with the target compounds (20 µL/disc) were placed on the plates. Streptomycin and DMSO were used as positive and negative controls respectively. The plates inoculated with the bacteria were incubated for 24 hours at 37°C. The diameters of the zone of inhibition were calculated in millimeters (mm). All the tests were achieved in triplicate, and the average value was taken as the final reading. 2.3.2 Antifungal study The synthesized azo dyes (A1-A3) were subjected to antifungal activity against two fungal strains; Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 627) by Food Poison assay [40]. The fungal strains were sub-cultured using Potato Dextrose Agar (PDA) medium. The PDA medium was sterilized by autoclave at 121°C (15 lb/sq. inch), for 15 minutes. The 30 mL each of molten PDA was inoculated with selected fungus (5 mm disc of the fungus grown) was transferred to a sterilized Petri-plates. The diameter of the zone of inhibition was read with the help of an ‘antibiotic zone reader’. The trials were carried out in triplicate and the percentage of inhibition (IP) of the mycelial growth was determined by using the equation, IP = 

 

 x100

(1)

Where C and T represent the mycelial growth (cm) of three replicates of control and treated Petri dishes correspondingly. Fluconazole and DMSO were used as a positive and negative control for the study.

2.3.3 Antimycobacterial studies In vitro antimycobacterial activities of the newly synthesized azo molecules were evaluated against Mycobacterium tuberculosis (H37RV strain) by Microplate Almar Blue Assay (MABA) according to the reported procedure as described by Mangalam et al [41]. This is an effective method that uses thermally steady reagent and displays a good association with proportional to the BACTEC radiometric technique [42]. Previously sterilized 96-well plates are used for the present study. The test samples were prepared in dry DMSO and serially diluted in 100 µL of the Middlebrook 7H9 broth to obtain final concentration ranges from 100 to 0.8 µg/mL. Plates were covered and sealed with parafilm and incubated at 37 ºC for five days. After the completion of the incubation period, the tested samples were treated with freshly prepared 25 mL of Almar Blue reagent and 10% tween 80 in 1:1 ratio and again the plates were incubated for 24 h. A blue and pink colour change was noticed in the wells, and in the experiment, the blue colour signifies the absence of bacterial growth and pink colour specifies that of the development of bacteria. Then, the minimum inhibitory concentration (MIC) of the compounds was recognized by monitoring the colour change in the plates. The screened azo dyes were compared with the reference drugs pyrazinamide, streptomycin and ciprofloxacin with MIC values 3.125, 6.25 and 3.125 µg/mL correspondingly. 2.3.4 DNA cleavage studies The DNA cleavage activity of the prepared azo compounds was studied by gel electrophoresis technique [43]. The previously cultured pBR-322 was used for the present experiment. A solution (10 mg/mL) of the synthesized azo dyes was prepared in freshly distilled DMSO and was added separately to the DNA sample, and the mixtures were incubated at 37 oC for 2h. For the gel electrophoresis technique, agarose gel was prepared by mixing 250 mg of

agarose in 25 mL of TAE (Tris-acetate-EDTA) buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 ltr) and it was boiled. When the gel attains 55 oC, it was added to the gel cassette fitted with a comb and then the gel was left for solidification. The comb was cautiously removed from the gel and loaded to the electrophoresis chamber flooded with TAE buffer. The mixture of DNA sample with bromophenol (1:1 ratio) was cautiously filled into the wells, along with the reference DNA marker and a current of 50 V was circulated into the electrophoretic chamber for about half an hour. After completion of electrophoresis, the gel was separated carefully from the chamber and marked with an ethidium bromide solution for about 10-15 min. The bands appeared on the plate were observed under UV trans-illuminator, and the photographs were taken to interpret the cleavage efficiency of the synthesized compounds. 2.3.5 Molecular docking studies The molecular docking is one of the effective tools to understand the mechanism of action of the drug with the biological receptors. The chemical structures of the prepared azo molecules were obtained by using ACD/ChemSketch 2015.2.5 with proper 2D orientation and energy of minimization was carried out using the Dundee PRODRG2 server and saved in PDBQT file format for docking analysis. The 3D structures of the target proteins were downloaded from the Brookhaven Protein Data Bank (PDB). The binding affinities of the ligands into the active sites of protein were studied using the Auto Dock Vina programme. The crystal structure of antibacterial and antifungal target proteins, E.coli 24kDa domain (PDB ID: 1KZN) and, Cyp51 (cytochrome P450 14α-sterol demethylase) (PDB ID: 1EA1) were chosen for the docking studies. The output results were analyzed by Ligplot and Pymol software [44, 45].

3. Results and discussion The 2-aminothiazole based azo dyes (A1-A3) were prepared by the diazo-coupling reaction, and the synthetic route adopted for the preparation was depicted in Scheme 1. The structures of the azo dyes were accomplished by elemental analysis, UV-Visible, FTIR, (1H and

13

C) NMR and

mass spectrometric techniques. The prepared azo compounds were found to be in good agreement with the proposed molecular structures. The physical and analytical data of azo dyes were tabulated in Table 1. Table 1: Analytical and physical data of the synthesized azo dyes A1-A3 Compounds

A1

A2

A3

Mol. formula

C10H7N5O2S

C11H9N5O2S

C12H11N5O2S

Mol.Wt.

261.25

275.28

289.31

M. P. (oC)

175-176

180-181

183-184

Colour

Red

Brown

Yellow

Elemental analysis (%) Calcd. (Found) C

H

N

S

45.97

2.70

26.81

12.27

(45.92)

(2.76)

(26.86)

(12.24)

47.99

3.30

25.44

11.65

(47.95)

(3.35)

(25.41)

(11.70)

49.82

3.83

24.21

11.08

(49.77)

(3.81)

(24.24)

(11.10)

3.1 Electronic spectra The prepared heterocyclic azo compounds (A1-A3) were investigated for their electronic spectral studies in the range of 200-800 nm at the concentration of 10-6 M in DMF and DMSO solvent. The results of the electronic spectral studies were presented in Table 2 and Figs. 1-3. From the results, it is revealed that the absorption maxima of all the dyes exhibited two distinct absorption bands at 322-324 and 439-443 nm, attributed to the corresponding π→π* and n→π* transitions. The above transitions are due to the interaction of azo-aromatic chromophore and

intramolecular charge transfer. The bathochromic shift was examined in all the colouring compounds due to the basic solvent interaction between the nonbonding electrons on the nitrogen atom/atoms of the azo chromophore and the pi-electrons of the aromatic system [46]. The involvement of a cyano group influences the blue shift towards the lower wavelength and it can be illustrated by substitution effect. On the other hand, the number of electron donating groups present on the diazo component results in the bathochromic shift. This is due to the interaction between the H atom of dye with the solvent molecule. Table 2: UV-Visible and molar absorptivity data of the synthesized compounds A1-A3 Compounds

λmax nm

Log ε

DMF

DMSO

DMF

DMSO

A1

441, 324

443, 323

4.74, 4.38

4.99, 4.17

A2

441, 323

439, 323

4.85, 4.13

4.95, 4.22

A3

440, 323

439, 322

5.02, 4.33

4.89, 4.05

Fig. 1: UV-Visible spectra of azo dye A1

Fig.2: UV-Visible spectra of azo dye A2

Fig.3: UV-Visible spectra of azo dye A3

3.2 Infrared spectral data Infrared spectroscopy is an important tool for the identification of functional groups present in molecules. The IR spectra (Figs. S1-S3 which are provided in the Supplementary material) of the newly prepared azo compounds (A1-A3) were recorded, and the results were tabulated in Table 3. A strong absorption band was observed in the region 1618 cm-1 may be attributed due to the presence of carbonyl group in the compound A1 and a broad band for hydroxyl group appeared at 3449 cm-1. The remaining bands at 2983, 2821, 2203, 1540 and 1425 cm-1 observed were assigned for Ar-CH, Ali-CH, -CN-, -N=N- and –C=C- groups respectively. For the compound A2, a sharp absorption peak with high intensity appeared in the range 1632 cm-1 corresponding to the presence of carbonyl function group and a medium intensity broad band observed at 3117 cm-1 was assigned to -OH stretching vibrations. The remaining groups like Ar-CH, Ali-CH, -CN-, -N=N- and –C=C- are observed at 3081, 2827, 2225, 1502 and 1451 cm-1 respectively. Similarly, for the compound, A3 IR spectral data are tabulated in Table 3. Table 3: Important IR absorption bands for azo dyes A1-A3 (cm-1)

Compounds

ʋOH

ʋAr-CH

ʋAli-CH

ʋC=C

ʋC=O

ʋCN

ʋN=N

A1

3449

2983

2821

1425

1618

2203

1540

A2

3117

3081

2827

1451

1632

2225

1502

A3

3107

2940

2838

1446

1628

2224

1496

3.3 1H and 13C NMR Spectral data Proton NMR of the azo dyes (A1-A3) was recorded in the DMSO-d6 solvent at ambient temperature and the spectra were displayed in Figs. S4-S6 which are provided in the Supplementary material. In general, the synthesized compounds were composed of 1,3thiazole and pyridone moieties via diazo linkage. In the 1H NMR spectra, the NH proton of dye A1 appeared as a singlet at 10.46 ppm. In all the azo dyes A1-A3, the common 1,3-thiazole aromatic protons were resonated as multiplet in the range 7.69-7.31 ppm. Wherein for the pyridone derivatives (A1-A3), the common methyl proton was observed as a singlet in the range 2.45-2.46 ppm. The methyl proton of the compound A2 attached to the ring nitrogen in pyridone moiety was obtained as a singlet in the range 3.18 ppm. A triplet and quartet at 1.10-1.13 and 3.83-3.88 ppm corresponding to -CH3 and -CH2 protons of the compound A3 attached to ring nitrogen. In the case of all the synthesized compounds, the hydroxyl group was not observed due to interaction with the DMSO solvent [47]. The 13C NMR spectra of the synthesized azo compounds were recorded in a DMSOd6 solvent using Bruker spectrophotometer at 25 oC and are shown in Figs. S7-S9 which are provided in the Supplementary material. The

13

C NMR spectra of the compounds A1-A3

exhibited signals at δ 118.12-118.26, 133.19-133.26 and 150.12-152.29 ppm are corresponding to carbon atoms of the thiazole ring. The peaks between at δ 15.20-15.93 ppm are due to carbon atoms of the methyl group connected to pyridone ring, at δ 72.20-72.39 ppm are due to carbon atoms of the coupling compound attached to the azo group. The peaks at δ 117.30-117.34 ppm are because carbon of nitrile group attached carbon atom and the peaks at δ 122.48-122.58 ppm are attributed to ring carbon atoms attached to the nitrile group. The peaks were observed at δ 158.34-158.79 ppm are due to the carbon atoms of carbonyl carbon, the signals at δ 141.16-

143.13 ppm are related to the carbon atoms of the methyl group attached to the pyridone ring. 172.10-172.26 ppm is due to the carbon atoms of pyridone moiety attached to the OH group. In the case of compounds A2 and A3, the peaks were observed at δ 26.84 ppm resultant to a carbon atom of the methyl group linked to a nitrogen atom of the pyridone nucleus, the signals at δ 26.02, 44.99 ppm are due to the carbon atoms of the methyl and methylene groups attached to the nitrogen atom of pyridone core. 3.4 Mass spectral studies The mass spectra of the newly synthesized azo dyes A1-A3 were recorded, and the spectra were presented in Figs. S10-S12 which are provided in the Supplementary material. The mass spectra of the azo molecules showed [M+1] peak corresponding to their molecular mass by showing the peaks at m/z 262.4, 276.4, 290.4 for compounds A1, A2 and A3 respectively. The obtained results are consistent with the formula weight, and this confirms the molecular structure of the compounds. 3.5 Antimicrobial activity The results of the antimicrobial activity of prepared azo dyes on bacterial growth are displayed in Table 4. The antimicrobial results of the newly synthesized dyes displayed a broad spectrum of activity against screened pathogens (E. coli, B. subtilis, A. flavus, and C. albicans) and showed relatively potential activity against B. subtilis and C. albicans. Further, among the studied compounds, the azo dye A3 showed higher activity compared to the other two dyes A2 and A1 owing to the existence of electron donating atoms [48]. In common, the synthesized compounds are potential bacteriostatic and fungistatic, as they possess electron rich groups like H, -CH3, -CH3CH2 that facilitates the antimicrobial activity [49].

Table 4: The MIC (mg/mL) value for antimicrobial activity of the azo compounds (A1-A3) Compounds

Bacteria

Fungi

E. coli

B. subtilis

A. flavus

C. albicans

25

50

25

50

25

50

25

50

mg/mL

mg/mL

mg/mL

mg/mL

mg/mL

mg/mL

mg/mL

mg/mL

A1

2.0±0.28

1.5±0.5

1.8±0.32

1.5±0.24

33

65

25

66

A2

2.1±0.25

1.5±0.21

2.4±0.21

2.3±0.12

44

62

25

55

A3

2.1±0.30

1.6±0.23

2.4±0.30

2.3±0.40

44

70

55

75

Streptomycin

2.2±0.26

2.4±0.28

2.5±0.30

2.6±0.35

-

-

-

-

Fluconazole

-

-

-

-

46

82

35

70

3.6 Antimycobacterial activity In the recent years, tuberculosis has become a one of the most dangerous infectious disease, and finally, it leads to the cause of death worldwide [50]. Still, it is a challenge for the chemists and researchers to design effective anti-TB drugs. Therefore, in the present study, the azo dyes A1-A3 were screened for their antimycobacterial activity against M. tuberculosis and, the results are represented in Fig. 4. From the outcome of the results, it is revealed that compound A3 displayed a significant inhibitory effect with MIC equal to 12.5 µg/mL which is almost nearer with the reference drug streptomycin. Further, dyes A1 and A2 demonstrated the moderate activity with MIC equal to 50 and 25 µg/mL respectively. In common, all the studied compounds displayed considerable activity, and this effect could be attributed to their charge density distribution.

Fig. 4: Antimycobacterial results of the synthesized azo dyes A1-A3

3.7 DNA cleavage studies All the synthesized compounds (A1-A3) were evaluated for their DNA cleavage efficiency on pBR 322 DNA by gel electrophoresis technique. The gel picture presenting the cleavage activity of the synthesized azo dyes was presented in Fig. 5. From the close observation of the gel picture, it was noticed that the concentration of the DNA molecule was decreased after electrophoresis which might be attributed because of the breaking of DNA by the title compounds. This may be due to the interaction between the dye molecules and the DNA. These findings indicated that compound A2 has shown the complete cleavage of all forms of DNA. Whereas A1 and A3 partially cleaved Form I DNA and Form II DNA compared to control DNA. Therefore, the tested compounds were shown appreciable cleavage properties and hence they can be considered as effective drugs to inhibit the growth of a pathogenic organism by cleaving the genome.

Fig. 5: DNA cleavage activity on supercoiled pBR 322 DNA: 1: Standard DNA, 2: Control DNA (untreated pBR 322 DNA), A: A1, B: A2, C: A3. 3.8 Docking studies The in silico molecular docking studies are extensively used to achieve an optimized conformation for both the target receptors and the drug with a relative orientation between them [51]. Therefore, the free energy of the whole system gets decreased. To investigate the interaction of the synthesized compounds with the target receptors E. coli 24kDa domain and Cyp51, the molecular docking was done and the results obtained were displayed in Figs. (6a and 6b) and Tables (5 and 6). The tested compounds (A1-A3) exhibited a well-established H-bond with active pocket amino acids in the enzyme active sites. All the synthesized compounds displayed good binding energy and exhibited the H-bonding with one or the other active pocket amino acids. From Tables 5 and 6, the compound A1 showed the highest binding energy -7.4 and -7.0 kcal/mol with a strong affinity towards Asp73 and Pro386 respectively.

Table 5: Antibacterial docking scores of the prepared azo compounds (A1-A3) Compounds

Affinity

H-bonds

(kcal/mol) A1

-7.4

H-bond length

H-bond with

Hydrophobic interactions

Asp73

Glu50, Ile78, Gly77, Pro79, Asn46,

(Å) 3

3.05, 3.13, 3.14

Val167, Thr165, Val143, Val71 A2

A3

-6.4

-6.3

2

2

3.32, 2.81

3.08, 3.19

Asn46

Ala47, Asp73, Asp49, Val120, Ile90,

Glu50

Ile78, Met91, Val143

Arg136

Ile90, Ile78, Ala47, Thr165, Glu50,

Asn46

Pro79, Arg76

Table 6: Antifungal docking scores of the synthesized azo molecules (A1-A3) Compounds

Affinity

H-bonds

(kcal/mol) A1

-7.0

1

H-bond length

H-bond

(Å)

with

2.87

Pro386

Hydrophobic interactions

Gly388, Leu324, Cys394, Ala400, Leu315, Thr260, Thr264, Phe387, Pro320, Leu321

A2

-6.8

-

-

-

Pro320, Pro386, Leu315, Phe387, Leu321, Gly388, Leu324, His392, Ala256, Gly396, Cys394, Thr260, Ala400

A3

-6.8

4

2.84, 3.28, 2.99,

Arg96

Cys394, His392, Leu324, Arg393, Tyr76,

3.08

Gln72

Leu321, Met79

Figure 6a: Interaction of azo dyes A1-A3 with E.coli 24kDa domain

Figure 6b: Interaction of azo dyes A1-A3 with cytochrome P450 14α-sterol demethylase

4. Conclusion In this study, we have synthesized three new derivatives (A1-A3) of 2-aminothiazole based azo dyes and were characterized by various analytical techniques. The prepared azo dyes showed brilliant colour and brightness properties due to the existence of chromophores in their structures. The synthesized azo dyes were explored for their biological properties, specifically antimicrobial activity against two bacterial (E. coli and Bacillus subtilis) and two fungal (A. flavus and Candida albicans) strains respectively. The antimicrobial results showed significant activity against tested pathogenic strains. The azo dye A3 exhibited the highest activity compared to A1 and A2 due to the presence of electron rich species. All the prepared dyes illustrated potential antimycobacterial activity against M. tuberculosis. We have also studied the DNA cleavage properties of the azo dyes against supercoiled pBR322, and the results indicated that of the tested compounds A2 cleaved the DNA completely whereas A1 and A3 partially. Further, the molecular docking investigations were carried out to find the possible binding between synthesized azo dyes with the receptor E.coli 24 kDa domain and Cyp51. The results showed a well-established bonding with favorable binding energy. Besides, all the azo dyes were proficient to form H-bond with the different amino acids in the active enzyme sites. Acknowledgment One of the authors, Mr. Ravi B N is grateful to the UGC, New Delhi, India for providing BSR Fellowship to carry out the research work and we also express our sincere thanks to the Department of PG Studies and Research in Chemistry, Kuvempu University, Shimoga for providing laboratory facilities. We are also thankful to Manipal University, Manipal for providing spectral data and Biogenics, Hubli for providing DNA cleavage studies.

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Highlights
2-Aminothiazole based azo dyes were synthesized by using diazo-coupling reaction at lower temperature.
The prepared azo molecules were characterized by different physical and spectral techniques.
The

title

compounds

were

screened

for

their

antimicrobial,

antimycobacterial and DNA cleavage studies and all the compounds exhibited significant biological properties.
The in silico molecular docking was also studied to know the mechanism of drug action on a microbial infection.

Authors Contributions Section Authors Name

Credit Roles

Mr. Ravi B N

Methodology, Validation and Writing - Original Draft

Prof. J Keshavayya

Supervision and Project administration (Corresponding author)

Mr. Mallikarjuna N M

Writing - Review & Editing

Mr. Vinod Kumar

Resources

Dr. Shivanand Kandgal

Software

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: