Eco-sustainable synthesis and biological evaluation of 2-phenyl 1,3-benzodioxole derivatives as anticancer, DNA binding and antibacterial agents

2.1 Chemistry

The chemicals, reagents and solvents employed for synthesis were procured from Hi-media Laboratories (Mumbai, India) and SD fine-chem limited (Mumbai, India). The progress of the reaction and purity were monitored by using TLC Silica gel 60 F₂₅₄ aluminium sheets (Merck F₂₅₄, Darmstadt, Germany) developed in mobile phase containing ethyl acetate and petroleum ether (1:1). The melting point of the synthesized compounds was determined by DRK Digital melting point apparatus. IR spectra were recorded on Shimadzu IR-Affinity spectrometer using KBr pellets. The ¹H spectra of the compounds synthesized were acquired in deuterated DMSO on a Bruker ARX 400 MHz (Bruker AG, Fallanden, Switzerland) instrument. Tetramethylsilane was used as the internal standard and all chemical shift values were expressed in parts per million (δ, ppm). The mass spectra were obtained from 6120 Quadrupole LC/MS mass spectrometer using electron spray ionization method. (Agilent Technologies, California, USA).

Compounds 1(a) to 14(a) were synthesized according to the literature (Dutta Gupta et al., 2012).

2.1.1

2.1.1 General Procedure for the synthesis of 1(b) to 14(b)

Pyrogallol (1 mol equivalent) and benzoic acid derivatives (1.05 mole equivalent) were heated in the microwave (Biotage Initiator 2.5 at 350 W, 100 °C for 30–120 s) in the presence of polyphosphoric acid (0.1 mol equivalent, Tables 1 and 2). TLC was observed to monitor the completion of the reaction by using ethyl acetate: petroleum ether in the ratio of 1:1 as the mobile phase. The reaction mass was neutralized with 10% NaOH solution and then filtered. The crude product was recrystallized using 70% alcohol (Fig. 3).

Table 1 Details of 2-phenyl benzodioxole derivatives synthesized.

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S. No.	R1	R2	R3	R4	Reactant II	Reaction time (s)	Physical description	Melting point °C	Rf value	% yield
1a	H	H	H	H	Benzoic acid	30	White crystals	50–51	0.703	80.2
2a	Cl	H	H	H	4-Chloro benzoic acid	45	Light grey powder	102–105	0.393	75.8
3a	Me	H	H	H	4-Methyl benzoic acid	60	Brownish powder	57–58	0.721	78.3
4a	Me	OMe	H	H	3-methoxy-4-methyl benzoic acid	45	Greyish powder	99–101.	0.750	82.5
5a	H	H	OH	H	2-hydroxy benzoic acid	90	White crystals	115–118	0.786	60.6
6a	NH2	H	H	H	4-amino benzoic acid	120	Brownish crystals	120–125	0.667	65.5
7a	H	NH2	H	H	3-amino benzoic acid	120	Creamish powder	130–134	0.634	68.9
8a	H	H	NH2	H	2-amino benzoic acid	105	Light brown crystals	188–190	0.626	62.8
9a	H	H	N-CH3	H	N-methyl-2-amino benzoic acid	120	Light brown powder	174–175	0.661	68.4
10a	OH	OCH3	H	H	Vanillic acid	45	White crystals	54–59	0.421	60.6
11a	Cl	H	Cl	H	2,4–dichloro benzoic acid	90	White powder	171–174	0.722	78.1.
12a	OH	OH	H	OH	Gallic acid	120	Greyish crystals	178–180	0.328	61.3
13a	H	H	OAc	H	Aspirin	105	White crystals	78–80	0.698	81.3
14a	H	H	COOH	H	Phthalic acid	60	Greyish crystals	102–105	0.746	70.5

Table 2 Details of 2-phenyl benzodioxole-4-ol derivatives synthesized.

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S. No.	R1	R2	R3	R4	Reactant II	Reaction time (s)	Physical description	Melting point °C	Rf value	% yield
1b	H	H	H	H	Benzoic acid	30	White crystals	113–114	0.323	72.6
2b	Cl	H	H	H	4-Chloro benzoic acid	30	Pale white crystals	99–103	0.256	78.2
3b	Me	H	H	H	4-Methyl benzoic acid	90	Greyish powder	122–125	0.465	82.4
4b	Me	OMe	H	H	3-Methoxy-4-methyl benzoic acid	60	Dark brown powder	87–89	0.333	68.6
5b	H	H	OH	H	2-Hydroxy benzoic acid	60	White crystals	110–112	0.286	70.8
6b	NH2	H	H	H	4-Amino benzoic acid	90	Light brown crystals	165–168	0.376	65.1
7b	H	NH2	H	H	3-Amino benzoic acid	90	Creamish powder	154–156	0.365	60.6
8b	H	H	NH2	H	2-Amino benzoic acid	120	Brownish crystals	180–182	0.343	61.4
9b	H	H	N-CH3	H	N-Methyl-2-amino benzoic acid	120	Brown powder	164–168	0.423	68.8
10b	OH	OCH3	H	H	Vanillic acid	30	Cream coloured crystals	72-73	0.289	62.6
11b	Cl	H	Cl	H	2,4-Dichloro benzoic acid	90	White powder	184–185	0.523	80.5
12b	OH	OH	H	OH	Gallic acid	120	Cream coloured powder	180–183	0.275	60.2
13b	H	H	OAc	H	Aspirin	90	White crystals	87–90	0.462	75.5
14b	H	H	COOH	H	Phthalic acid	45	Greyish powder	112–113	0.491	76.8

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Figure 3

Scheme of synthesis.

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2.2 In vitro cytotoxicity studies

The in-vitro cytotoxicity potential of the test compounds was evaluated on A549 human lung carcinoma cells using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] based cell proliferation assay (Van Meerloo et al., 2011; Ferrari et al., 1990). The carcinoma cell lines were obtained from National Centre for Cell Science (NCCS), Pune (India) and cultivated in Dulbecco's modified Eagle's red medium (DMEM) (Sigma Life Science, USA) containing 10% foetal bovine serum (FBS). An equal number of cells were incubated at 37 °C with 5% CO₂ in a 96 well micro plates. Thereafter, the cells were treated with test compounds and standard at concentration of 100 μg/ml. Control cells were supplemented with 0.5% DMSO. After 72 h treatment, 5 μl of MTT reagent along with 45 ml of phenol red and FBS free DMEM was added to each well and plates were incubated at 37 °C with 5% CO₂ for 4 h. Subsequently, 50 ml of solubilization buffer was added to each well to solubilize the coloured formazan crystals produced by the reduction of MTT. After 24 hrs, the optical density was measured at 550 nm using a spectrophotometer in a microplate reader (Bio-Rad, USA). Cisplatin was used as standard reference compound.

2.3 DNA binding studies

The interaction of the compounds with calf thymus DNA was studied in phosphate buffer of pH 7 using UV visible spectrophotometer (Shimadzu, UV 1800) (Mansuri-Torshizi et al., 2001). The hypochromic effect observed in the absorption spectra of the molecules with increasing concentrations of DNA (0–100 μM) is shown in Fig 4. The change in absorbance was measured at 210–320 nm for synthetic compounds–DNA complex. Scatchard equation was utilized to build the binding isotherm. The half reciprocal UV plot obtained on the basis of absorbance data for synthetic compounds-DNA complex is depicted in Fig. 5. The linear plots obtained indicate the involvement of one binding process with independent binding sites on DNA. The parameters, λ_max, hypochromicity, isobestic point and binding constant were found from the absorption spectra (Mansouri-Torshizi et al., 2008). The intrinsic binding constant (K_i) for a given complex with DNA was obtained from a plot of D/Δε_app versus D according to equation, D/Δε_app = D/Δε + 1/Δε × K, where D = concentration of DNA in base molarities, Δε_app = |ε_a − ε_f| and Δε = |ε_b − ε_f|, where ε_a and ε_f are respective extinction coefficients of the complex in the presence and absence of DNA. The apparent extinction coefficient ε_a is obtained by calculating Aobs/[Acridones]. The data were fitted to the equation with a slope equal to 1/Δε and Y-intercept equal to 1/(Δε × K). Thereafter the intrinsic binding constant (K_i) was determined from the slope of Y-intercept.

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Figure 4

Effect of CT DNA on synthesized compound 3b.

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Figure 5

Half reciprocal UV plots for the binding of synthesized compound 3b to DNA.

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2.4 Antibacterial studies

The microorganisms were procured from MTCC, Mumbai, India. The in vitro antibacterial studies were carried out by disc diffusion method in nutrient agar medium (Dutta Gupta et al., 2010, Ednie et al., 2000) The screening of the compounds was performed against Escherichia coli (MTCC 40, Gram negative), Pseudomonas aeruginosa (MTCC 424, Gram negative) Bacillus subtilis (MTCC 441, Gram positive) and Staphylococcus aureus (MTCC 3160, Gram positive) at concentration of 60, 80, 100, 120 and 140 μg/ml. Cinoxacin was used as standard reference drug. The required concentrations of the compounds were prepared in sterile DMSO. The sterile growth medium was poured into the petri plate and allowed to solidify. Subsequently, the microbial suspension was swabbed on agar bed using a sterile cotton swab. This was followed by placing a sterile paper disc uniformly on the agar bed. The synthesized compounds and standard drug contained in sterile paper disc were allowed to diffuse for 10 min. Thereafter, the petri plates were incubated for 24 h at 37 °C. The zone of inhibition was measured in mm to determine the antimicrobial potency of the compounds.

3.1 Chemistry

A series of 2 phenyl substituted 1,4-benzodioxole derivatives were synthesized (Fig. 3) by green chemistry approach and characterized as per literature (Dutta Gupta et al., 2012). In a similar environment friendly fashion, one-pot synthesis of a series of 2 phenyl substituted 1,4-benzdioxole-4-ol derivatives were carried out by using polyphosphoric acid as solvent/catalyst under microwave irradiation (Fig. 3). The physical properties of the synthesized compounds are summarized in Tables 1 and 2. The reaction is believed to proceed by the attack of the hydroxyl group of benzoic acid by ‘H’ of polyphosphoric acid to form the protonated benzoic acid which is attacked by catechol/pyrogallol to form a tetrahedral intermediate that further cyclizes to form the 1,3-benzodioxole ring. The formation of the synthesized compounds was confirmed by IR, ¹H NMR and mass spectral analysis. In the IR spectra of compounds (1b–14b), the stretching bands due to one OH group were detected in the range of 3498–3317 cm⁻¹. The compounds 6b–9b showed a characteristic NH stretching band between 3325 and 3381 cm⁻¹. In case of compound 2b and 11b, corresponding C–Cl stretching was observed at 732 cm⁻¹ and 761 cm⁻¹, respectively. In ¹H NMR spectra of the compounds (1b–14b), a single peak corresponding to the CH proton of the dioxole ring system was observed between 7.0 and 7.5. The physical properties and percentage yield of the synthesized compounds along with their IR, ¹H NMR and mass spectral data are given below.

3.2 In vitro cytotoxicity studies

All the compounds exhibited considerable amount of anticancer activity against A 549 human lung carcinoma cells in terms of percentage cell proliferation inhibition (% CPI) at a concentration of 100 μgm/ml. Compound 3a (% CPI = 78.80) and 9b (% CPI = 52.90) were found to be more potent than the standard (% CPI = 49.50). It was revealed that mostly compounds belonging to benzodioxole-4-ol category (2b, 6b–14b) showed better anticancer activity than the compounds without a hydroxy group at fourth position of the benzodioxole ring (2a, 6a–14a). However this phenomenon was reversed for compounds with unsubstituted phenyl ring (1a, 1b), methyl (3a, 3b) or hydroxy (4a, 5a, 4b, 5b) substituted phenyl ring. Subsequently it was observed that among 2-phenyl benzodioxole derivatives, mono substitution at para position of the phenyl ring with a methyl group (3a) was the most potent. In case of 2-phenyl benzodioxole-4-ol derivatives, mono substitution with a methyl amino group at second position of the phenyl ring (9b) was found to be the most active compound. The percentage cell proliferation inhibition of the synthesized compounds is depicted in Table 3.

3.3 DNA binding studies

The ability of the synthesized compounds to bind with DNA as determined by binding constant value was found to be moderate to mild. This can be attributed to a different mechanism of action via which the compounds exhibit their cytotoxic effects. The 2-phenyl benzodioxole-4-ol derivatives (1b–14b) were found to be more effective than the 2-phenyl benzodioxole class of compounds (1a–14a). Compound with a hydroxyl group at second position of the phenyl ring (5a and 5b) was found to be the most active in their respective series. The binding constant (K_i), λ_max, % hypochromicity and isobestic point is highlighted in Table 4.

3.4 Antibacterial studies

Antibacterial studies on the compounds bearing 2-phenyl benzodioxole ring system (1a–14a) revealed that compound 3a is more potent than the standard against all the strains of bacteria (Table 5). Compound 4a exhibited better activity than the standard in terms of inhibiting S. aureus. It was further disclosed that the activity of the compounds either remained same or decreased when the phenyl ring is substituted with any group other than methyl at para position. Substitution at other position of the phenyl ring did not result in any significant increase in potency. The activity data of compounds belonging to 2-phenyl benzodioxole-4-ol category (1b–14b) showed that all have considerable antibacterial activity but none are potent than the reference standard. Subsequently, a closer look at the activity profile of this series of compounds demonstrated that there is no marked improvement in antibacterial property of the compounds with a substitution in the phenyl ring at any position as compared to unsubstituted phenyl ring (1b). However, in this series also, compound 3b (methyl group at para position of the phenyl ring, i.e. analogue of 3a) was the exception which exhibited enhanced activity among all the derivatives of the series. The result of antibacterial studies of the synthesized compounds against various strains of bacteria is depicted in Table 5.