Synthesis, characterization and biological investigations of potentially bioactive heterocyclic compounds containing 4-hydroxy coumarin.

In this paper, we have reported the synthesis of a series of heterocyclic azo dyes containing 4-hydroxy coumarin by diazo-coupling reaction. The structural aspect of the newly synthesized compounds was accomplished by various physico-chemical techniques like UV-Visible, FT-IR, NMR, and mass spectrometry. The computational calculations and geometrical optimization of the newly synthesized azo dyes were investigated by using Gaussian software with the help of Density functional theory (DFT)/B3LYP method using 6-31G(d,p) basis set at gaseous phase. Also, the quantum chemical parameters were evaluated to understand the structural activity concept of the dyes. The pharmacological efficacy of the azo dyes was investigated by antimicrobial, antitubercular, DNA cleavage and in silico molecular docking studies. All the newly synthesized compounds were able to exhibit significant inhibitory activity against tested microbes. Further, the in silico molecular docking showed effective binding properties of the compounds against RpsA target receptor.

Introduction

The coumarin derivatives were found to be having innumerable pharmacological properties and exhibit various biochemical and therapeutic applications depending upon the pattern of the substitution [1, 2, 3]. Among the family of the coumarins, 4-hydroxy coumarin have been extensively studied for a wide range of biological activities including anticoagulant, insecticidal, anthelminthic, hypnotic, antifungal, phytoalexin, and HIV protease inhibition activities [4, 5, 6, 7]. These special applications of 4-hydroxycoumarin have motivated considerable interest in this class of compounds for number of researchers across the globe [8, 9, 10, 11].

The recent development in the synthesis of heterocyclic compounds containing azo chromophore has become more important as they exhibit different properties based on effective conjugation and substituent's electronic effect. The coumarin which exhibits brilliant physical, thermal, optical and biological properties can be used as dyes for colouring of fabrics, fluorophores and optical brightening agents [12, 13, 14]. Some of the work reported on the azo group incorporated in the mesogenic core of coumarin has increased the dipole moments and stability of coumarin azo-ester series as related to the coumarin esters [15]. Alternatively, a molecule which has azo linkage is generally exhibited reversible isomerization transformations upon irradiation with ultraviolet (~365 nm) and visible (~450 nm) light. The molecules containing extended conjugation and more number of electron releasing substituents on the diazo group relatively enhances the electron density of the molecule which in turn intense optical absorption and related optical properties [16, 17, 18]. Also, the azo benzene derivatives found more applications in liquid crystal studies due to its rod-like shape, photosensitive ability and photo induced alignment with reversible cis-trans transformation, dimerization in cross linking material and irreversible photo degradation [19]. The azo dyes containing coumarin nucleus has potential biological, optical and electrochemical properties and therefore it has been extensively studied by number of researchers [20, 21, 22, 23].

From the above observations and extensive findings on the coumarin analogues, in the present work we have described the synthesis of coumarin based azo dyes and their structural characterization. The quantum chemical technique was used to optimize the molecular geometry of the newly synthesized compounds and the biological activities were also carried out to check the inhibitory effect against the organisms.

Experimental

Materials and methods

All the chemicals and reagents used are of analytical grade and purchased from Sigma Aldrich Chemical Company and used as such received. Melting points of the compounds were recorded on an electro thermal apparatus and are uncorrected. The electronic absorption spectra were recorded on UV-1800 Shimadzu spectrophotometer in the range of 200–800 nm using 10−6 M solution of dimethylsulphoxide (DMSO), N,N-dimethylformamide (DMF), tetrahydrofuran (THF) and dichloromethane (DCM). The FT-IR spectra were obtained using Perkin Elmer- RX-FTIR spectrophotometer using KBr pellets. The 1H and 13C-NMR spectra were recorded with the aid of Bruker spectrometer 400 MHz and 100 MHz respectively; chemical shifts (δ) were recorded in parts per million (ppm) with respect to tetramethylsilane (TMS). The mass spectra were recorded on LC-MS 2010, SHIMADZU mass spectrometer. The computational studies were carried out by Density functional theory (DFT)/B3LYP method using Gaussian 09Wsoftware using 6-31G(d,p) basis set at gas phase. Geometry optimization, vibrational analysis and quantum chemical parameters were analyzed at same basis set level [24].

General procedure for the synthesis of coumarin based azo dyes (F21–F24)

A well-stirred and ice-cold solution of heterocyclic amines (0.002 mmol) in hydrochloric acid (2 ml of HCl in 3 ml of H2O) were added drop wise with constant stirring to the solution of sodium nitrite in sulphuric acid (2 mL) at 0–5 °C and stirred for 2 h to get the diazonium salt solution. Then, this diazonium salt solution was added to the ice-cold solution of 4-hydroxy coumarin in aqueous KOH solution and stirred for another 1h. The obtained coloured precipitates were filtered, washed with distilled water until it is free from impurities and recrystallized in ethanol to afford coloured azo dyes (F–F).

4-Hydroxy-3-[(4-nitrophenyl) diazenyl]-2H-chromen-2-one (F21)

Yellow coloured solid, 80% yield, m.p. 182–184 °C. FT-IR (KBr, ʋmax, cm−1):3421 (OH), 3072(Ar-CH), 1660 (C=O), 1461 (N=N), 1421 (C–N). 1H NMR (DMSO-d, δ ppm): 16.22 (s, 1H, OH), 8.34 (d, J=8 Hz, 1H, Ar–H), 8.24 (d, J=8 Hz, 1H, Ar–H), 8.08 (d, J=7.6 Hz, 1H, Ar–H), 7.99–7.80 (m, 2H, Ar–H), 7.54 (t, J=8 Hz, 1H, Ar–H), 13C NMR (DMSO-d, δ ppm): 195.51(coumarin C=O), 159.24 (C–OH), 155.33, 147.49, 134.41, 130.24, 128.67, 124.99, 124.52, 118.26. 116.69; LCMS: m/z 312 [M+1]. Anal. Calcd for C15H9N3O5: C, 57.88; H, 2.91; N, 13.50, Found: C, 57.75; H, 2.85; N, 13.43.

3-[(5-Ethoxy-1,3-benzothiazol-2-yl)diazenyl]-4-hydroxy-2H-chromen-2-one (F22)

Reddish coloured solid, 75% yield, m.p. 224–226 °C. FT-IR (KBr, ʋmax, cm−1):3449 (OH), 3055(Ar-CH), 2925 (C–CH3), 1663 (C=O), 1491 (N=N), 1447 (C–N). 1H NMR (DMSO-d δ ppm): 12.50 (s, 1H, OH), 7.84–7.08 (m, 6H, Ar–H), 6.97 (t, J=5.6 Hz, 1H, Ar–H), 4.14–4.00 (m, 2H, CH2), 1.39–1.35 (m, 3H, CH3). 13C NMR (DMSO-d, δ ppm): 166.19 (coumarin C=O), 163.66 (C–OH), 136.43, 135.73, 129.66, 129.57, 126.80, 122.72, 122.34, 119.59.118.42, 116.30, 116.07, 111.41, 111.35, (CH2) 43.05, 25.48 (CH3); LCMS: m/z 368 [M+1]. Anal. Calcd for C18H13N3O4S: C, 58.85; H, 3.57; N, 11.44, Found: C, 58.74; H, 3.47; N, 11.33.

4-Hydroxy-3-[(E)-1,3-thiazol-2-yldiazenyl]-2H-chromen-2-one (F23)

Orange coloured solid, 77% yield, m.p. 194–196 °C.FT-IR (KBr, ʋmax, cm−1):3442 (OH), 2925 (Ar-CH), 1728 (C=O), 1512 (N=N), 1488 (C–N). 1H NMR (DMSO-d δ ppm): 11.37 (s, 1H, OH), 8.36–8.10 (m, 1H, Ar–H), 7.93 (d, J=7.6 Hz, 1H, Ar–H), 7.23 (t, J=8.4 Hz, 1H, Ar–H), 7.19 (m, 1H, Ar–H), 13C NMR (DMSO-d, δ ppm): 167.95 (coumarin C=O), 155.19 (C–OH), 138.73, 135.60, 132.18, 128.36, 127.03, 126.49, 126.36, 122.58. 121.00, 118.15; LCMS: m/z 274 [M+1]. Anal. Calcd for C12H7N3O3S: C, 52.74; H, 2.58; N, 15.38, Found: C, 52.67; H, 2.46; N, 15.54.

N-(4,6-dimethylpyrimidin-2-yl)-4-[(E)-(4-hydroxy-2-oxo-2H-chromen 3yl) diazenyl] benzenesulfonamide (F24)

Light yellow coloured solid, 81% yield, m.p. 215–217 °C. FT-IR (KBr, ʋmax, cm−1):3420 (OH), 3073 (NH), 2928 (Ar-CH), 1724 (C=O), 1515 (N=N), 1444 (C–N). 1H NMR (DMSO-d, δ ppm): δ 12.53 (s, 1H, OH), 7.84 (q, J=6.4 Hz, 2H, Ar–H), 7.66 (q, J=6.8 Hz, 3H, Ar–H), 7.38 (q, J=6 Hz, 4H, Ar–H),5.60 (s, 1H, NH), 2.51 (t, J=3 Hz, 6H, 2CH3).13C NMR (DMSO-d, δ ppm): 173.16 (coumarin C=O), 162.26 (C–OH), 157.13, 156.63, 144.21, 141.66, 139.84, 135.69, 129.82, 129.80, 129.68. 129.07, 127.04, 122.99, 120.97, 118.50, 117.66, 115.92, 39.72 (C–CH3); LCMS: m/z 452 [M+1]. Mol. Formula: Anal. Calcd for C21H17N5O5S: C, 55.87; H, 3.80; N, 15.51, Found: C, 52.73; H, 3.72; N, 15.43.

Biological studies

Antimicrobial activity

The antimicrobial activity of the coumarin based azo dyes was screened using three bacterial strains Escherichia coli, Pseudomonas aeruginosa, (gram-negative bacteria), Enterococcusfaecalis (gram-positive bacteria) and three fungal strains Candida albicans, Aspergillus flavus and Aspergillus nigerby tube dilution assay [25,26]. The Brain heart infusion (BHI) was used as a medium for growing the target microorganisms, the ciprofloxacin and fluconazole were used as a positive control and DMSO as a negative control. Further, the inhibitory effect of the compounds against microbial strains was computed in terms of minimum inhibitory concentration (MIC) along with the standard drugs.

Antitubercular activity

Mycobacterium tuberculosis is a deadly microorganism and causes respiratory diseases to the human beings. Even though, number of drugs are available to reduce the toxic effects of the pathogen, the death rates due to tuberculosis are continuously increasing. It is due to the increasing of toxicity of the existing drugs and multi drug resistance strain and some other clinical and environmental problems [27]. Therefore, in the present study we have synthesized coumarin based-azo dyes (F–F) and studied the antitubercular activity against M. tuberculosis by Microplate Alamar Blue Assay (MABA) method [28]. The obtained results were compared with the standard drugs Pyrazinamide, Ciprofloxacin and Streptomycin. The colour change in the microplate from pink to blue suggesting no bacterial growth where as pink colour stipulates the growth.

DNA cleavage

The target compounds (F–F) were studied for their cleavage study against super coiled pBR-322 DNA by electrophoresis technique [29]. The experimental technique involves the use of accurately weighed DNA sample (0.35 μg/test), this was added to the target compounds (F–F) and these mixtures were incubated at 37 °C for 30 min. Then, the 250mg of agarose was dissolved in 25 mL of TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 ltr) and was added to the reaction mixture. When the agarose gel attains ~55 °C, it was poured into the gel cassette fitted with a comb. After solidification, the gel was detached carefully and shifted into the electrophoresis chamber containing TAE buffer. The mixture containing equimolar ratio of bromophenol blue dye and 20 μL of the DNA sample was loaded into the wells along with the standard DNA marker. Later, electricity of 50 V was constantly supplied to the chamber for about 45 min. After this period, the gel was removed from the chamber and stained with ETBR solution (10 μg/ml) for 10–15 min and the bands were observed under UV transilluminator.

In silico molecular docking studies

The antimicrobial activity results of synthesized compounds inspired us to investigate the interaction of the target compounds with the biological receptors theoretically by in silico molecular docking using RpsA receptor. The molecular structures of the coumarin derivatives (F–F) were optimized by using Chem Bio Draw tool (Chem Bio Office Ultra 14.0 suite) with 2D-orientation and then they were converted into 3D-format with the minimization energy by Schrodinger Maestro. The 2XCT-Protein Data Bank (PDB) was used to get the 3D coordinates of the target receptor and the best docked conformation of the tested structures were obtained on the basis of glide energy, docking score, active hydrogen bonding sites and hydrophobic interactions [30].

Result and discussion

The purpose of this study is to explore the structural and biological properties of the novel coumarin based azo dyes. We adopted the simple and conventional diazo-coupling reaction to synthesize the azo dyes containing 4-hydroxy coumarin nucleus. The reaction pathway involved in getting the target compounds was depicted in Scheme 1.

Scheme 1

Synthetic route adopted for the preparation of coumarin based azo dyes (F–F).

IR spectral data

The FTIR spectra of the synthesized compounds (F–F) were recorded using KBr pellets in the region 4000-400 cm−1. The important IR bands exhibited by azo dyes were displayed in Table 1. Further, the absorption frequencies of the compounds obtained experimentally were compared with theoretical values calculated by DFT/B3LYP method using 6-31G(d,p) bases set at gaseous state which have been summarized in the Table 1.

Table 1

The comparative analysis of experimental and calculated vibrational frequencies for the coumarin based azo dyes (F–F).

Compounds	Assignments	FT-IR absorption frequencies (cm−1)
		Experimental	Theoretical
F21	ʋOH	3421	3686
	ʋAr-CH	3072	3185
	ʋC=O	1660	1672
	ʋN=N	1461	1458
	ʋC-N	1421	1409
F22	ʋOH	3449	3569
	ʋAr-CH	3055	3047
	ʋC=O	1663	1821
	ʋN=N	1491	1515
	ʋC-N	1447	1496
F23	ʋOH	3442	3642
	ʋAr-CH	2925	3154
	ʋC=O	1728	1814
	ʋN=N	1512	1531
	ʋC-N	1488	1473
F24	ʋOH	3420	3598
	ʋNH	3073	3229
	ʋAr-CH	2928	3052
	ʋC=O	1724	1672
	ʋN=N	1515	1502
	ʋC-N	1444	1458

A medium intensity broad peak observed in the region between 3449-3420 cm−1 is due to the presence of O–H group attached to the coumarin ring and the corresponding theoretical value was appeared between 3686-3569 cm−1 which were in well agreement with the literature. Another absorption band for the presence of N–H functionality of the sulfamethazine moiety in compound F was appeared at 3073 cm−1 and its corresponding theoretical N–H stretching value was found to be at 3229 cm−1. The absorption band appeared in the region 3100-2925 cm−1 was assigned to the aromatic C–H stretching vibrations for all the compounds and its respective theoretical value was observed at 3185-3052 cm−1. The carbonyl (C=O)and –N=N- functionalities exhibited medium intensity bands in the region 1728-1660and 1515- 1461 cm−1 respectively and their corresponding theoretical values obtained in the region 1821–1672 and 1531-1458cm−1 respectively. Another low intensity absorption band was appeared in the region 1488-1421 cm−1 due to the C–N stretching vibrations in all the dyes and their corresponding theoretical values lies in the region of 1496–1409 cm−1. Thus, from the above discussion it is clear that the experimentally recorded IR spectra were almost matches with the computational IR spectral data [31,32].

Electronic absorption spectra

The effect of solvent polarity and the electronic substitution was studied for the title compounds (F–F) in four different solvents such as DMSO, DMF, THF and DCM at a concentration of 10−6 M at ambient temperature and the typical absorption spectra were displayed in Figure 1. The absorption maxima (λmax) and its corresponding logarithmic molar extinction coefficient for all the compounds in studied solvents were obtained from the plot and summarized in the Table 2. The electronic spectra of the synthesized compounds showed broad peaks in the region 461–436, 389–382, 389–362 and 365–369 nm in DMSO, DMF, THF and DCM solvents due to π→π∗or n→π∗ transitions respectively [33]. From the close observation of the spectral data (Table 2), it is clearly evident that as the polarity of the solvent increases, the absorption maxima shift towards longer wavelength so that the bathochromic shift observed in all the compounds. This may be due to the effective interaction between the solvent molecules and the lone pair of electrons present on the diazo component. The presence of electron releasing substituents on the aromatic ring bearing the azo group also contributes to the bathochromic shift. From this study, it concludes that solvent polarity and electronic substitution played very important role in the shift of λmax for all the studied azo dyes [34].

Figure 1

The electronoic spectra of the compounds (F–F) in various solvents recorded at room temperature at 10−6 M concentration.

Table 2

The electronic spectral data of the compounds (F–F) obtained indifferent solvents.

Compounds	λmax(nm)				Logε
	DMSO	DMF	THF	DCM	DMSO	DMF	THF	DCM
F21	436	382	362	369	5.84	5.57	6.05	5.87
F22	461	386	389	366	5.64	5.88	5.81	6.12
F23	443	389	371	367	5.98	5.99	5.93	6.09
F24	441	388	376	365	5.53	5.99	5.80	5.93

NMR spectral data

The structural confirmations of the synthesized compounds (F–F) were accomplished by recording their 1H and 13C NMR spectra in DMSO-d In 1H NMR spectra, the hydroxyl proton attached to the coumarin ring present in all the compounds was appeared in the region 16.22–11.37 ppm as singlet. The aromatic protons were resonated as multiplet in the region 8.36–6.79 ppm. A broad peak appeared at δ 5.6 in compound F was due to the presence of NH proton. The –CH2 protons attached to the benzothiazole ring in the compound F was appeared as doublet at δ 3.92–3.81 and the methyl protons of compounds F and F were appeared as singlet in the region 2.50–1.32 ppm. These 1H NMR spectral results are in close agreement with the proposed structure of the compounds. The 13C NMR spectrum of the compound F shows signals at δ 195.51, 159.24, 155.33, 147.49,134.41,130.24,128.67,124.99,124.52,118.26 and 116.69 ppm corresponding to the carbon atoms C11, C5, C7, C6, C2, C1, C3, C4, C10, C8 and C9 respectively. The compound F displayed the 13C NMR signals at δ 166.19, 163.66, 136.41, 135.73, 129.66, 129.57, 126.80, 122.72, 122.34, 119.57, 118.42, 116.30, 116.07, 111.41, 43.05 and 30.98 ppm related to the carbon atoms C16, C5, C1, C2, C3, C4, C6, C7, C8, C9, C10, C11, C12, C13, C14 and C15 respectively. The 13C NMR spectrum of compound F showed the signals at δ 167.95, 155.19, 138.73, 135.60, 132.16, 128.36, 127.03, 126.49, 126.36, 122.58, 121.00 and 118.15 ppm related to the carbon atoms of C12, C7, C5, C1, C2, C3, C4, C6, C7, C8, C9, C10 and C11. The 13C NMR spectrum of the synthesized compound F exhibits the signals at δ 173.16, 162.26, 144.21, 141.66, 139.84, 135.69, 129.82, 129.80, 129.68, 129.07, 127.04, 122.99, 120.97, 118.50, 117.66, 39.72 and 36.38 ppm resultant to the carbon atoms of C17, C5, C1, C2, C3, C4, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15 and C16 respectively.

The FT-IR, 1H NMR, 13C NMR and Mass spectra of the compounds (F–F) have been given in the supplementary data file (S1–S20).

Geometrical optimization

In order to understand the molecular properties of the synthesized compounds (F–F), we carried out their structural optimization and the energies of the Highly Occupied Molecular Orbital (HOMO) and Least Unoccupied Molecular Orbital (LUMO) were evaluated from the optimized structures. Further, the energies of HOMO and LUMO were utilized to calculate some of the parameters like electronegativity (χ), chemical potential (α), hardness (η), electrophilicity index (ω), electron affinity (I) and ionization potential (A) to understand the reactivity of the studied molecules. The global parameters have been calculated by using the following equations.

The calculated HOMO-LUMO energies and global parameters of the prepared azo molecules (F–F) are displayed in Table 3.

Table 3

The quantum chemical parameters evaluated for the azo dyes (F–F) by DFT method at B3YLP/6-31G(d,p).

Electronic parameters	F21	F22	F23	F24
EHOMO (eV)	-5.52	-4.47	-4.38	-5.96
ELUMO (eV)	-2.51	-1.53	-1.47	-2.70
EHOMO-ELUMO (eV)	3.00	2.97	2.90	3.25
Electronegativity (χ)	4.01	3.00	2.93	1.62
Chemical potential(μ)	-4.01	-3.00	-2.93	-1.62
Hardness(η)	1.50	1.474	1.45	1.62
Electrophilicity index (ω)	5.37	2.035	2.01	1.00
Ionization potential (A)	5.52	4.47	4.38	5.96
Electron affinity (I)	2.51	1.53	1.47	2.70
Dipole moment (D)	1.72	3.02	1.14	0.84

The tabulated data reveals that, low energy gap, chemical hardness and electrophilicity index values are responsible for the good biological activity of the azo dyes (F21–F24) [35].

The optimized molecular structures and HOMO-LUMO energy level diagrams of the azo dyes (F–F) were shown in Figures 2, 3, 4, and 5 and Table 4. The reactivity of molecules often decided by using density functional theory (DFT) and that is based on the energy differences between the HOMO and LUMO. From the literature review, it was observed that if the difference between the HOMO and LUMO is small, the energy required to excite an electron to higher energy state is less and therefore the molecules become more reactive chemically and biologically [36,37]. If the gap is large, then the promotion of electron becomes difficult and requires lot of energy, so that the molecules become more stable towards any reaction. Thus, from the above discussion it is inferred that the theoretical modeling is most useful in the interpretation of chemical reactivity, kinetic stability, polarizability and biological properties of the molecules [38,39,40].

Figure 2

The optimized molecular structure of the compound F.

Figure 3

The optimized molecular structure of the compound F.

Figure 4

The optimized molecular structure of the compound F.

Figure 5

The optimized molecular structure of the compound F.

Table 4

The structure of the HOMO and LUMO of the compounds (F–F) generated from B3LYP method using 6-31G(d,p) basis set.

Compounds	Structure of HOMO	Structure of LUMO
F21	Image 1	Image 2
F22	Image 3	Image 4
F23	Image 5	Image 6
F24	Image 7	Image 8

Biological evaluation

A multidrug resistance of bacterial strains remains to be a great challenge due to their biochemical and morphological modifications and essential for the development of novel drugs. Therefore, the heterocyclic molecules having azo chromospheres received significant importance due to their wide spectrum of biological applications [41]. Thus, we have studied in vitro antibacterial activities of the synthesized azo dyes by tube dilution method and the results of the study have been summarized in Table 5. The results indicated that all the studied compounds showed efficient antibacterial activity against tested microbial strains. In particular, among the tested pathogens, the compounds exhibited effective inhibition against E. coli and C. albicans, whereas moderate activity exhibited against rest of the organisms.

Table 5

Antimicrobial activity results of the azo dyes (F–F) in terms of MIC (mg/mL).

Compounds (mg/mL)	100	50	25	12.5	6.25	3.12	1.6	0.8	0.4	0.2
E. coli
F21	S	S	S	S	S	S	S	S	S	R
F22	S	S	S	S	S	S	S	S	R	R
F23	S	S	S	S	S	S	S	S	R	R
F24	S	S	S	S	S	S	S	S	S	R
P. aeruginosa
F21	S	R	R	R	R	R	R	R	R	R
F22	S	S	S	R	R	R	R	R	R	R
F23	S	S	R	R	R	R	R	R	R	R
F24	S	S	R	R	R	R	R	R	R	R
E. faecalis
F21	S	S	S	R	R	R	R	R	R	R
F22	S	S	R	R	R	R	R	R	R	R
F23	S	S	S	R	R	R	R	R	R	R
F24	S	S	S	S	R	R	R	R	R	R
Ciprofloxacin	S	S	S	S	S	S	S	S	S	S
C. albicans
F21	S	S	S	S	S	S	R	R	R	R
F22	S	S	S	S	S	S	R	R	R	R
F23	S	S	S	R	R	R	R	R	R	R
F24	S	S	S	S	S	R	R	R	R	R
A. flavus
F21	S	S	S	S	S	S	S	S	R	R
F22	S	S	R	R	R	R	R	R	R	R
F23	S	S	S	S	S	S	S	S	R	R
F24	S	S	S	S	S	S	S	S	R	R
A. niger
F21	S	S	S	R	R	R	R	R	R	R
F22	S	S	R	R	R	R	R	R	R	R
F23	S	S	R	R	R	R	R	R	R	R
F24	S	R	R	R	R	R	R	R	R	R
Fluconazole	S	S	S	S	S	S	S	S	S	R

Here, S: Sensitive, R: Resistance.

The highest antibacterial activity of the synthesized compounds against E. coli and C. albicans pathogens may be due to the presence of electron donating atoms, delocalization of pi electrons, solubility and dipole moment. It increases the lipophilic environment around the microorganisms and allows the dye molecules to penetrate into the cell and finally an arrest the functions of the cell thereby suppresses the activity.

The antitubercular activity of the compounds (F–F) was carried out against M. tuberculosis by MABA method and the result of the study was interpreted in terms of MIC depicted in Table 6 and Figure 6. From the results of the study it is inferred that, the synthesized compound can be able to show effective inhibitory action with MIC value equal to 1.6 μg/mL and it is almost equal to the MIC of the standard drugs. Therefore, from our study it is concluded that, the synthesized compounds could be able to show appreciable antitubercular activity against M. tuberculosis and they may be used as better agents in the development of antitubercular drugs in future.

Table 6

The results of the antitubercular activity against M. tuberculosis (MIC, mg/mL) of the compounds (F–F).

Sample	100 μg/mL	50μg/mL	25μg/mL	12.5 μg/mL	6.25 μg/mL	3.12 μg/mL	1.6μg/mL	0.8μg/mL
F1	S	S	S	S	S	S	S	R
F2	S	S	S	S	S	S	S	R
F3	S	S	S	S	S	S	S	R
F4	S	S	S	S	S	S	R	R

Figure 6

Antitubercular activity results of the compounds (F–F).

DNA cleavage activity

The examination on the cleaving affinity of compounds to DNA is very attractive since it can provide to understand the toxicity mechanism of them and to develop new artificial nuclease. Thus, the cleavage ability of the newly synthesized compounds (F–F) was studied by agarose gel electrophoresis assay against super coiled pBR322 plasmid DNA. The Figure 7 provided below is the gel picture showing cleavage properties of the tested compounds against pBR322 DNA. From the results of the study indicated that, the prepared azo compounds F and F cleaved Form II DNA and has not cleaved Form I DNA, whereas rest of the compounds (F andF) did not showed much cleavage activity.

Figure 7

The picture showing cleavage of the supercoiled pBR322 DNA by the azo dyes (F21–F24), here M: marker DNA, C: control DNA, F21–F24: coumarin based azo dyes.

Molecular docking studies

Most of the drug design for the treatment of various diseases requires theoretical modelling of the structure of the drug. Nowadays, rather than going to synthesize the suitable compounds for the treatment, researchers chose to design the structure of the drugs by using theoretical modelling. One such method is the molecular docking, in which the interaction of the drugs with the appropriate proteins were checked. The in silico molecular docking is one of the most advanced technique to understand the bonding interaction, toxicity and multi drug resistance strain of the new drugs. One such attempt was made in the present work to check the mode of interaction of the synthesized compounds (F–F) with target receptor RpsA and the docking results of the studied compounds were depicted in the Table 7.

Table 7

The results of the in silico molecular docking studies of the azo dyes (F–F) against RpsA enzyme.

Compounds	Affinity (kcal/mol)	H-bonds	H-bond length (Å)	H-bond with	Hydrophobic interactions
F21	-5.0	3	3.03	4NNI:Arg357::F21:O2	Tyr280, Lys303, Phe310, Glu318, Arg355, Arg356
			3.07	4NNI:Arg357::F21:O2
F22	-5.1	1	2.80	4NNI:Arg357::F22:O4	Lys303, Phe310, Glu318, Arg355
F23	-5.4	2	2.92	4NNI:Tyr::F23:O2	Lys303, Phe310, Glu318, Arg355
			3.19	4NNI:Arg356::F23:N3
F24	-5.9	1	2.85	4NNI:Arg357::F24:O3	Lys303, Phe310, Glu318, Arg355
Pyrazinamide	-5.3	2	3.01	4NNI:Arg357::PYZ:O	Phe307, Leu320, His322
			3.06	4NNI:Arg357::PYZ:O

The 2D and 3D representations the interaction of compounds F–F interacted with the target receptor RpsA have been shown in Figure 8 and Figure 9 respectively.

Figure 8

2D representation of the interaction of compounds F, F, F, F and standard drugs with pyrazinamide against RpsA receptor.

Figure 9

3D representation of the interaction of compounds F, F, F, F and standard drugs with pyrazinamide against RpsA receptor.

From the obtained results, it is noticed that the studied compounds were significantly interacted with the amino acids of the target protein RpsA with appreciable binding energy ranging from -5.0 to -5.9 kcal/mol and it is almost equal to the binding energy of the standard drug pyrazinamide. Therefore, our synthesized compounds were potentially efficient in interacting with the target receptor and can be utilised in the designing of potential drugs in the treatment of various diseases.

Conclusion

The present work aimed to synthesize novel heterocyclic coumarin based azo molecules by simple diazo-coupling reaction with good yield. The newly synthesized compounds were thoroughly characterized by elemental, FT-IR, UV-Visible, NMR and mass spectrometric studies. The structures of synthesized compounds were also studied by DFT method at B3YLP/6-31G(d,p). Further, these coumarin based azo dyes were screened for their antimicrobial, antitubercular, DNA cleavage and in silico molecular docking studies. The results of all the activities indicated that, the bioactive coumarin nucleus in the compounds could be able to show appreciable pharmacological properties against tested organisms. Thus, the newly synthesized azo compounds can be useful in the drug designing.

Declarations

Author contribution statement

Nagaraja O: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Yadav D. Bodke: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Itte Pushpavathi, Ravi Kumar S: Contributed reagents, materials, analysis tools or data.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing interest statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.