Solvent-free synthesis of novel vanillidene derivatives of Meldrum's acid: biological evaluation, DNA and BSA binding study

Abstract

A series of novel O-alkyl vanillidene derivatives containing Meldrum's acid scaffold under solvent-free conditions were synthesized. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration (MIC) using the broth microdilution. The most active compounds were 5-(4′-hydroxy-2′-iodo-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione (3a), 5-(4′-acetoxy-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione (3f), and 5-(4′-bromopropoxy-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione (3h) with the MIC values ranging from 0.039 to 10 mg mL⁻¹. Antioxidant activity was evaluated by DPPH free radical scavenging activity. 3h showed the largest scavenging activity with an IC₅₀ value of 55.61 μg mL⁻¹ (0.14 mmol L⁻¹). The interaction of 3a and 3h with DNA and bovine serum albumin (BSA) were investigated by the fluorescence spectroscopic method. The results achieved in competitive experiments with ethidium bromide (EB) indicated that 3a and 3h have an affinity to displace EB from the EB–DNA complex through intercalation. Fluorescence spectroscopy data show that the fluorescence quenching of BSA is a result of the formation of the 3a- and 3h-BSA complex species, and indicate that 3a-BSA is more stable, suggesting that 3h-BSA is less suitable for drug–cell interactions.

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Introduction

Meldrum's acid (2,2-dimethyl-1,3-dioxane-4,6-dione; isopropylidene-malonate) was prepared by A. N. Meldrum in 1908, in a reaction between acetone, malonic acid and acetic anhydride.¹ This acid is distinguished from other 1,3-dicarbonyl compounds in two main ways: first, it is extremely acidic, and second, it is highly electrophilic.² In light of this, reactions with Meldrum's acid provide easy and rapid access to various libraries of organic compounds with diverse substitution patterns, which allows for the formation of several new carbon–carbon and carbon–heteroatom bonds.³

One of the most important carbon–carbon bond formations in organic synthesis is the Knoevenagel condensation, which has been widely used in synthesis of alkenes of biological significance.^4–8 The Knoevenagel condensation of Meldrum's acid and aldehydes has been used for preparation of arylidene analogues of Meldrum's acid. These compounds exhibit different biopotentials, such as antimalarial and antioxidant activities.⁹ Also, arylidene analogues of Meldrum's acid have been reported as the key precursors in cycloaddition reactions,¹⁰ for synthesis of mono alkyl Meldrum's acid derivatives,¹¹ epoxides,¹² deuterium labelled carboxylic acids,¹³ oxopyridines,¹⁴ pyridines,¹⁵ lactones,¹⁶ β-aryl aldehydes,¹⁷ and for the synthesis of heterocyclic compounds of biological importance such as cardiotonic¹⁸ and HIV integrase inhibitory activities.¹⁹

Bering in mind the mentioned facts, there is a reasonable tendency to synthesize a novel Meldrum's acid derivatives.

Various catalysts have been used for synthesis of arylidene analogues of Meldrum's acid, such as L-tyrozine,²⁰ SbCl₃,²¹ gel-entrapped KOH,²² ionic liquids,²³ K₃PO₄,²⁴ pyrrolidinium acetate,²⁵ morpholine/acetic acid,²⁶ (R)-methyl 3-phenyl-2-(3-(pyridin-2-yl)ureido)propanoate,²⁷ Zr(O₃POK)₂ ²⁸ and NAP (3-aminopropylated silica gel).²⁹ However, the usage of these catalysts is characterized with different undesirable effects, such as complicated procedures for catalysts preparation, difficulty to isolate, the application of harmful solvents and special apparatus³⁰ (e.g., microwave irradiation), etc.

Development of green chemical methods is one of the very important purposes of organic synthesis. Reactions in the absence of solvents (solvent-free) present very powerful and green methodology for the construction of structurally different molecules in organic synthesis. These reactions are faster, usually requiring just a few minutes.

Keeping in mind these facts and environmental requirements, we investigated these reactions using three different methods (one of them in the presence of solvent and other two under solvent-free conditions) for the synthesis of 5-(arylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-diones (Scheme 1).

Scheme 1 General scheme for synthesis of arilydene Meldrum's acid derivatives 3a–j.

The obtained products were subjected to biological evaluation and florescence measurements. Fluorescence spectroscopy presents a very effective experimental method that can be used to investigate the interactions between small molecules and biopolymers (e.g. DNA or carrier proteins). DNA is a major target for drugs and some harmful chemicals which can significantly influence the genetic information expression and result in some diseases related to the cell proliferation and differentiation.^31,32 Thus, the studies on the binding nature of these small molecules to DNA are important and fundamental issues in life science. Generally, small molecules interact with DNA via three kinds of noncovalent modes, such as: (a) intercalating between stacked base pairs, (b) noncovalent groove binding, or (c) electrostatic binding to the negatively charged nucleic acid sugar-phosphate skeleton.³³ A detailed investigation on the interaction of small molecules with DNA is helpful to design highly efficient drugs^34,35 or to well understand the toxic mechanisms of harmful chemicals, such as environmental pollutants, pesticides, etc.^36,37 Serum albumins are the most abundant carrier proteins in the circulatory system of a wide variety of organisms. Being the biomacromolecules contributing to the osmotic blood pressure,³⁸ they can play a dominant role in drug disposition and efficacy.³⁹ The drugs and bioactive small molecules bind reversibly to albumin and other serum components, which then function as carriers. Consequently, it is important to study the interactions of potential drugs with these proteins.

Results and discussion

Synthesis of vanillidene derivatives

In this paper, a simple, green, efficient, and convenient method for the synthesis of 5-(arylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-diones 3a–j from Meldrum's acid (1) and some vanillic aldehydes 2a–j under different catalytic conditions is presented (methods A, B and C, Scheme 1).

For this purpose, the reaction of Meldrum's acid (1) and 5-iodovanillin (2a) where the product 3a is formed was selected as a model reaction. The first task was to optimize the reaction conditions. It is worth pointing out that, in our previous investigations,^40,41 (PhNH₃)₂CuCl₄ proved to be an excellent choice for the condensation reactions under solvent and solvent-free conditions. Thus, we first examined the catalytic properties of this copper catalyst. The model reaction was performed in the presence of 10 mol% (PhNH₃)₂CuCl₄ as a catalyst at room temperature, in ethanol (method A), and under solvent-free conditions (method B). After appropriate work-up, the best results were achieved with method B. Namely, after 3, 8, 12 and 24 h, the yields of 3a were 45, 60, 68 and 71%, respectively. The method A gave lower yield of the product 3a (56%) after 24 h. In order to improve the yield in the model reaction we decided to examine the catalytic effect of the p-toluenesulfonic acid (PTSA) under solvent-free conditions (method C). The presence of PTSA (5 mol%) drastically reduced reaction time (7 min) and gave much better yields (95% of 3a) than methods A and B.

Since the method C provided the best yields in the model reaction, we decided to apply it to a series of reactions of Meldrum's acid with various O-vanillic aldehydes (2a–j). After simple work-up (see Experimental section) the newly-synthesized products 3a–j were obtained. All the products were characterized by their mp, IR, ¹H-, ¹³C-NMR, ESI-MS spectra and elemental analyses. The PTSA-promoted reactions were not accompanied by a side reaction (Michael addition). The isolated yields and structures of the products 3a–j are presented in Fig. 1. Generally, very good to excellent yields were achieved in all cases, but, the best yield (97%) was achieved in the synthesis of 3f from 1 and aldehyde 2f.

Fig. 1 Isolated yields for the products 3a–j. The reaction times are also given.

Biological evaluation

The antimicrobial activities of the investigated compounds (3a–j) against the tested microorganisms are shown in Tables 1 and 2. The tested compounds demonstrated relatively strong antimicrobial activity. The minimal inhibitory concentration (MIC) for different compounds relative to the tested microorganisms ranged from 0.039 to 2.5 mg mL⁻¹. The investigated components inhibited the growth of all the tested strains of bacteria. The strongest antibacterial activity was found in 3a, 3f and 3h compounds, which in extremely low concentrations (Table 1) inhibited all the species of bacteria. The lowest measured MIC value was 0.039 mg mL⁻¹ for compound 3h related to the B. Subtilis species.

Table 1 Antibacterial activity of compounds 3a–j; values of MIC given as mg mL⁻¹. The values are means of three replicates. In all cases the standard deviation is ± 0.002

Test compounds	Staphylococcus aureus	Bacillus subtilis	Basillus cereus	Escherichia coli	Pseudomonas aeruginosa
3a	0.312	0.156	0.156	0.625	0.156
3b	0.625	0.156	0.312	1.250	0.312
3c	1.250	0.312	0.625	1.250	0.625
3d	0.625	0.156	0.312	1.250	0.625
3e	1.250	0.312	0.312	1.250	0.625
3f	0.312	0.156	0.156	0.312	0.156
3g	0.625	0.156	0.312	0.625	0.312
3h	0.312	0.039	0.078	0.312	0.156
3i	1.25	0.156	0.312	1.250	0.312
3j	1.250	0.156	0.312	1.250	0.312
Streptomycin	0.031	0.016	0.016	0.062	0.062

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Table 2 Antifungal activity of compounds 3a–j (MIC as a mg mL⁻¹). The values are means of three replicates. In all cases the standard deviation is ± 0.001

Test compounds	Mucor mucedo	Trichoderma viride	Cladosporium cladosporioides	Fusarium oxysporum	Alternaria alternata	Aspergillus flavus	Aspergillus niger	Candida albicans	Penicillium expansum	Penicillium italicum
3a	0.625	0.156	0.312	0.312	0.625	2.5	2.5	0.312	0.625	0.312
3b	1.25	0.625	0.625	0.625	1.25	2.5	2.5	0.625	1.25	1.25
3c	1.25	0.625	1.25	0.625	1.25	2.5	2.5	0.625	2.5	1.25
3d	0.625	1.25	0.625	1.25	1.25	2.5	2.5	1.25	1.25	0.625
3e	1.25	0.312	0.312	0.625	1.25	2.5	2.5	0.312	1.25	0.625
3f	0.312	0.312	0.312	0.312	0.625	0.625	1.25	0.312	0.625	0.312
3g	0.625	0.625	0.625	0.312	0.625	1.25	1.25	0.312	1.25	0.312
3h	0.312	0.156	0.156	0.156	0.156	1.25	0.625	0.156	0.312	0.312
3i	1.25	0.312	0.312	0.625	1.25	2.5	2.5	0.312	1.25	0.625
3j	1.25	0.312	0.156	0.312	0.625	2.5	2.5	0.312	0.625	0.312
Ketoconazole	0.156	0.078	0.039	0.078	0.078	0.078	0.078	0.039	0.078	0.156

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The investigated compounds also inhibited the growth of all the tested fungi (Table 2) but in slightly higher concentrations (MIC values were from 0.156 to 2.5 mg mL⁻¹). The compounds 3f and 3h showed the strongest antifungal activity with MIC values ranging from 1.25 to 1.56 mg mL⁻¹. The most sensitive, among the bacteria, was B. subtilis, and the highest resistance was shown in E. coli and S. aureus. Among the fungi, the most sensitive appeared to be C. albicans, F. oxysporum, T. viride and C. cladosporioides.

The antimicrobial activity was compared to the standard antibiotics, streptomycin (for bacteria) and ketoconazole (for fungi). The results showed that standard antibiotics exhibited slightly stronger activity than tested samples, as shown in Tables 1 and 2. In a negative control, DMSO had no inhibitory effect on the tested organisms.

In these experiments, the compounds examined at the same concentrations showed a slightly stronger antibacterial than antifungal activity. These results were not unexpected due to the fact that numerous tests proved that bacteria are more sensitive to the antibiotic compared to fungi.⁴² The reason for different sensitivities between fungi and bacteria can be found in different permeability of the cell wall. The cell wall of the Gram-positive bacteria consists of peptidoglycans (murein) and teichoic acids, while the cell wall of Gram-negative bacteria consists of lipopolysaccharides and lipopoliproteins,⁴³ whereas, the cell wall of fungi consists of polysaccharides such as chitin and glucan.⁴⁴

DPPH free radical scavenging assay

DPPH radical scavenging activity of the investigated compounds (3a–j) was compared to that ascorbic acid. The results showed that standard antioxidant had stronger activity than tested samples. The IC₅₀ values (μg mL⁻¹) of the studied compounds are shown in Table 3. These values range from 55.61–1026.21 μg mL⁻¹. There was a statistically significant difference between tested samples and blank (P < 0.05). Among the tested compounds, 3h showed largest DPPH radical scavenging activity (IC₅₀ = 55.61 μg mL⁻¹). The 3a and 3i compounds also demonstrated moderate DPPH radical scavenging activity. The IC₅₀ values for these compounds were 161.61 and 162.93 μg mL⁻¹, respectively. The weakest antioxidant activity was found in the case of 3c.

Table 3 DPPH free radical scavenging activity of tested compounds. The values are means of three replicates ± standard deviation

Tested compounds	IC50 (μg mL^−1)
3a	161.61 ± 3
3b	965.22 ± 6
3c	1026.21 ± 7
3d	866.12 ± 5
3e	571.59 ± 4
3f	532.01 ± 4
3g	600.77 ± 4
3h	55.61 ± 1
3i	162.93 ± 3
3j	467.25 ± 4
Ascorbic acid	6.42 ± 1

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Fluorescence measurements

Florescence quenching on EB–DNA. An intermolecular deactivation of EB–DNA (complex between ethidium bromide and DNA) was investigated in the presence of 3a and 3h. We used the molecular fluorophore EB because, when bound to DNA, it shows intense fluorescence light due to its strong intercalation between the adjacent DNA base pairs.⁴⁵ The molecules of 3a and 3h were selected because they are planar and rigid, so that one can expect that they can bind to DNA. In addition, these products exhibit most pronounced biological activities (Tables 1 and 2).

The fluorescence emission titrations were recorded in the range of 550–740 nm. After the addition of 3a and 3h into the EB–DNA solution a significant decrease in the intensity of the florescence emission band (at 607 and 613 nm, respectively) was observed. In addition, the maximum wavelength of the EB–DNA was red-shifted (by about 5 nm). The observed quenching of EB–DNA indicates a competition between the added compounds and EB in binding to DNA. Namely, these molecules displace EB from the EB–DNA complex, and interact with DNA by intercalation.

The fluorescence quenching of 3a and 3h was described by means of the Stern–Volmer eqn (1),⁴⁶ implying that the dependence of I₀/I on [Q] was examined (Fig. 2).

I₀/I = 1 + k_qτ₀[Q] = 1 + K_sv[Q] (1)

Fig. 2 Top: emission spectra of EB bound to DNA in the absence (red lines) and presence of compounds 3a and 3h. The black lines denote solutions: buffer + quencher. [EB] = 50 μM; [DNA] = 50 μM; [3a] and [3h] = 0–50 μM; pH = 7.4; λ_ex = 520 nm. The arrows show the emission intensity changes with increasing the concentrations of the quenchers. Bottom: plots of I₀/I versus [Q].

In eqn (1) I₀ and I are the emission intensities in the absence and presence of the quenchers, [Q] is the total concentration of the quenchers, k_q is the bimolecular quenching rate constant, and τ₀ is the average lifetime of DNA in the absence of a quencher (10⁻⁸ s). K_sv is the Stern–Volmer quenching constant whose values were obtained from the slopes of the plots of I₀/I versus [Q]. Fig. 1 clearly shows that fluorescence intensity of EB–DNA decreases continually with the increasing concentrations of the quenchers.

Quenching parameters are presented in Table 4. As can be seen, 3a and 3h showed high K_sv values of (1.6 ± 0.1) × 10⁴ and (2.4 ± 0.2) × 10⁴ M⁻¹, respectively. This finding indicates their large affinity and efficiency to substitute EB from the EB–DNA complex, and bind strongly with DNA.

Table 4 The bimolecular quenching rate constant (k_q), Stern–Volmer constant (K_sv), and correlation coefficient (R) for the quenchers 3a and 3h

Compound	kq [M^−1 s^−1]	Ksv [M^−1]	R
3a	(1.6 ± 0.1) × 10^12	(1.6 ± 0.1) × 10^4	0.989
3h	(2.4 ± 0.2) × 10^12	(2.4 ± 0.2) × 10^4	0.992

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Protein binding experiments

Bearing in mind that the efficiency of drugs strongly depends on their protein binding ability, we decided to investigate the affinity of compounds 3a and 3h to bind to bovine serum albumin (BSA). This task was realized by means of the fluorescence emission titration of BSA with 3a and 3h in the range of 300–475 nm. It is apparent from Fig. 3 that the fluorescence intensity of BSA continually decreased with the increasing concentrations of 3a and 3h.

Fig. 3 Top: emission spectra of BSA in the absence (red lines) and presence of compounds 3a and 3h. The black lines denote solutions: buffer + quencher. [BSA] = 1.6 μM; [3a] and [3h] = 0.0, 8.0, 9.6, 11.2, 12.8, 14.4, 16.0, 17.6, 19.2, 20.8 and 22.4; pH = 7.4; λ_ex = 285 nm.

The fluorescence quenching data were analysed by using the eqn (2):⁴⁷

log(I₀ − I/I) = log K_a + n log[Q] (2)

where I₀ and I are the emission intensities in the absence and presence of the quencher, K_a is the binding constant for 3a or 3h-protein interaction, n is the number of binding sites per BSA molecule, and [Q] is the concentration of the quencher. The plots of log[(I₀ − I)/I] versus log[Q] were depicted (Fig. 3). The values of K_a and n were obtained from the intercept and slope of the obtained straight lines. The values of binding parameters for 3a- and 3h-BSA complexes are given in Table 5.

Table 5 Binding parameters (K_a and n) and the correlation coefficient (R) for interaction of 3a and 3h with BSA

Compounds	Ka (M)	n	R
3a	(2.8 ± 0.2) × 10^6	3.30	0.988
3h	(2.3 ± 0.1) × 10^4	2.16	0.994

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Based on the values of n (Table 5) we can conclude that 3a and 3h bind to BSA in the molar ratios of 3 : 1 and 2 : 1, respectively. The larger K_a value ((2.8 ± 0.2) × 10⁶) for 3a-BSA than for 3h-BSA complex ((2.3 ± 0.1) × 10⁴) indicates that this complex is more stable, which means that 3h-BSA complex is less suitable for drug–cell interactions.

Experimental

All solvents (CHCl₃ and EtOAc), substrate (Meldrum's acid and vanillin) and catalyst (PTSA), calf-thymus DNA (CT-DNA), BSA (bovine serum albumin) and ethidium bromide (EB) were purchased from Sigma. Phosphate buffered saline (PBS) tablets were purchased from Fisher BioReagents. The complex salt, (PhNH₃)₂CuCl₄, was synthesized by procedure described earlier.⁴⁸ The used O-alkyl vanillic aldehydes were synthesized according to the previously described methodology.⁴⁹ A freshly solution of CT-DNA and EB in doubly distilled water was prepared in 15 mM Tris–HCl/100 mM NaCl buffer at pH = 7.4. The DNA solution gave a ratio of UV absorbance at 260 nm and 280 nm (A₂₆₀/A₂₈₀) of ca. 1.8–1.9, indicating that the DNA was sufficiently free of proteins. CT-DNA concentration was measured by the UV absorbance at 260 nm (ε = 6600 M⁻¹ cm⁻¹).⁵⁰ BSA solution in doubly distilled water in 10 mM PBS buffer at pH = 7.4, was prepared.

Melting-points (mp) were determined on a Mel-Temp apparatus and are uncorrected. Thin-layer chromatography (TLC) was carried out on 0.25 mm Sigma-Aldrich coated silica gel plates (60F-254) using eluent CHCl₃ : EtOAc (4 : 1) mixture as a mobile phase and UV light for visualization. The IR spectra were recorded by a Perkin-Elmer Spectrum One FT-IR spectrometer on KBr pellet. The NMR spectra of compounds 3a–j were performed in CDCl₃ or DMSO-d₆, with TMS as internal standard on a Varian Gemini 200 MHz NMR spectrometer (¹H at 200 and ¹³C at 50 MHz). Abbreviations for the NMR signal that were used are: s = singlet, d = doublet, t = triplet, m = multiplet, and dd = doublet of doublet. All NMR (¹H and ¹³C) spectral data given in ESI (see Fig. S1–S20†). Mass spectrometry was performed by Waters Micromass Quattro II triple quadrupole mass spectrometer and MassLynx software for control and data processing. Electrospray ionization in the positive mode was used. The electro spray capillary was set at 3.0 kV and the cone at 20 V. The ion source temperature was set at 120 °C and the flow rates for nitrogen bath and spray were 500 L h⁻¹ and 50 L h⁻¹, respectively. The collision energy was 20 eV. Microanalyses of C and H were obtained on CarloErba EA1108. The RF-1501 PC spectrofluorometer (Shimadzu, Japan) for fluorescence measurements were used.

The following strains of bacteria were used as test organisms in this study: Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), Bacillus cereus (ATCC 10987), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). All of the bacteria used were obtained from the American Type Culture Collection (ATCC). The bacterial cultures were maintained on Müller–Hinton agar substrates (Torlak, Belgrade). The strains of fungi used as test organisms were: Aspergillus flavus (ATCC 9170), Aspergillus niger (ATCC 16888), Candida albicans (ATCC 10259), Penicillium expansum (ATCC 20466), Penicillium italicum (ATCC 10454), Mucor mucedo (ATCC 20094), Trichoderma viride (ATCC 13233), Cladosporium cladosporioides (ATCC 11680), Fusarium oxysporum (ATCC 62506) and Alternaria alternata (ATCC 36376). All of the fungi were from the American Type Culture Collection (ATCC). The fungal cultures were maintained on potato dextrose (PD) agar, except for Candida albicans that was maintained on Sabouraud dextrose (SD) agar (Torlak, Belgrade). All of the cultures were stored at 4 °C and subcultured every 15 days.

Bacterial inoculi were obtained from bacterial cultures incubated for 24 h at 37 °C on Müller–Hinton agar substrates and brought up by dilution according to the 0.5 McFarland standard to approximately 108 CFU mL⁻¹. Suspensions of fungal spores were prepared from freshly mature (3- to 7-day-old) cultures that grew at 30 °C on a PD agar substrate. The spores were rinsed with sterile distilled water, used to determine turbidity spectrophotometrically at 530 nm, and were then further diluted to approximately 106 CFU mL⁻¹ according to the procedure recommended by NCCLS.⁵¹

Minimal inhibitory concentration (MIC)

The minimal inhibitory concentration (MIC) was determined by the broth microdilution method using 96-well micro-titer plates.⁵² A series of dilutions with concentrations ranging from 20 to 0.0195 mg mL⁻¹ of the tested compounds was used in the experiment against every microorganism tested. The starting solutions of tested compounds were obtained by measuring off a certain quantity of the compounds and dissolving it in 5% DMSO. Two-fold dilutions of the compounds were prepared in a Müller–Hinton broth for bacterial cultures and a SD broth for fungal cultures. The MIC was determined with resazurin. Resazurin is an oxidation–reduction indicator used for the evaluation of microbial growth. It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. The boundary dilution without any changing in color of resazurin was defined as the MIC for the tested microorganism at a given concentration. As a positive control of growth inhibition, streptomycin was used in the case of bacteria and ketoconazole in the case of fungi. A 5% DMSO solution was used as a negative control for the influence of the solvents.

Antioxidant activity

Scavenging DPPH radicals. The free radical scavenging activity of samples was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH). The method used is similar to the method previously used by some authors^53,54 but was modified in details. Two milliliters of methanolic solution of DPPH radical in the concentration of 0.05 mg mL⁻¹ and 1 mL of test samples (1000, 500, 250, 125 and 62.5 mg mL⁻¹) were placed in cuvettes. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm in a Jenway spectrophotometer (Bibby Scientific Limited, Stone, UK). Ascorbic acid was used as positive control.

The DPPH radical concentration was calculated using the following eqn (3):

DPPH scavenging effect (%) = [(A₀ − A₁)/A₀] × 100 (3)

where: A₀ is the absorbance of the negative control and A₁ is the absorbance of the reaction mixture or the standard.

The inhibition concentration at 50% inhibition (IC₅₀) was the parameter used to compare the radical scavenging activity.

Synthetic procedure

Meldrum's acid (1.58 g, 11 mmol) and vanillic aldehyde (10 mmol) are homogenized in a mortar, and finally 5 mol% of PTSA (0.086 g, 0.5 mmol) was added at room temperature. Solid mixture very fast changed the colour, from white to light green. The reaction was monitored by TLC (eluent CHCl₃ : EtOAc, v/v 4 : 1). The solid was washed with small portions of cold ethanol and then dried at room temperature to afford the desired product with good purity grade (without recrystallization).

3a. 5-(4′-Hydroxy-2′-iodo-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 95%; mp = 227–228 °C; IR (KBr): 3283, 1739, 1703, 1566, 1523, 1496, 1418, 1398, 1292 cm⁻¹; ¹H NMR (200 MHz, DMSO-d₆): δ = 1.73 (s, 6H), 3.86 (s, 3H), 8.01 (d, 1H, J = 1.8 Hz), 8.22 (s, 1H), 8.35 (d, 1H, J = 1.8 Hz), 10.65 (br. s, 1H); ¹³C NMR (50 MHz, DMSO-d₆): δ = 27.1, 56.4, 84.5, 104.3, 111.7, 117.8, 125.1, 139.1, 146.3, 152.4, 155.8, 160.4, 163.3; ESI-MS: m/z (%) = 427 [M⁺ + Na], 404 [M⁺], 302, 277; anal. calcd C₁₄H₁₃O₆I (%): C 41.61, H 3.24; found: C 41.49, H 3.31.

3b. 5-(3′-Methoxy-4′-propoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 85%; mp = 147 °C; IR (KBr): 1749, 1713, 1578, 1560, 1523, 1392, 1274 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 1.07 (t, 3H, J = 7.6 Hz), 1.79 (s, 6H), 1.87–1.97 (m, 2H), 3.94 (s, 3H), 4.10 (t, 2H, J = 6.8 Hz), 6.94 (d, 1H, J = 8.6 Hz), 7.64 (dd, J = 8.6, 2.0 Hz, 1H), 8.29 (d, 1H, J = 2.2 Hz), 8.36 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 10.3, 22, 27.4, 56.2, 70.6, 104, 110.3, 111.3, 116.1, 124.6, 132.6, 148.8, 154.7, 158.2, 160.6, 164.1; ESI-MS: m/z (%) = 320 (15.6%) [M]⁺, 277 (54.6%), 218 (100%), 130 (89.8%); anal. calcd C₁₇H₂₀O₆ (%): C 63.74, H 6.29; found: C 63.77, H 6.30.

3c. 5-(4′-Isopropoxy-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 90%; mp = 151–152 °C; IR (KBr): 1745, 1712, 1548, 1521, 1428, 1397, 1273 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 1.45 (d, 6H, J = 6.2 Hz), 1.79 (s, 6H), 3.93 (s, 3H), 4.68–4.80 (m, 1H), 6.94 (d, 1H, J = 8.6 Hz), 7.64 (dd, J = 8.6, 2.2 Hz, 1H), 8.29 (d, 1H, J = 2.0 Hz), 8.36 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 21.7, 27.4, 55.9, 71.6, 104, 110.3, 112.7, 116.1, 124.6, 132.3, 149.4, 153.4, 158.2, 160.6, 164.1; ESI-MS: m/z (%) = 320 (15.7%) [M]⁺, 277 (26.3%), 218 (100%), 150 (43.5%); anal. calcd C₁₇H₂₀O₆ (%): C 63.74, H 6.29; found: C 63.80, H 6.36.

3d. 5-(4′-Butoxy-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 88%; mp = 140 °C; IR (KBr): 1748, 1713, 1577, 1559, 1523, 1392, 1278 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 0.99 (t, 3H, J = 7.4 Hz), 1.43–1.61 (m, 2H), 1.79 (s, 6H), 1.79–1.95 (m, 2H), 3.94 (s, 3H), 4.14 (t, 2H, J = 6.8 Hz), 6.94 (d, 1H, J = 8.4 Hz), 7.64 (dd, 1H, J = 8.6, 2.0 Hz), 8.29 (d, 1H, J = 2.0 Hz), 8.36 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 13.7, 19.1, 27.4, 30.8, 56, 68.9, 104.1, 110.3, 111.4, 116.1, 124.8, 132.6, 148.9, 154.6, 158.3, 160.6, 164.2; ESI-MS: m/z (%) = 334 (8.4%) [M]⁺; 277 (25.4%), 232 (26.1%), 151 (100%); anal. calcd C₁₈H₂₂O₆ (%): C 64.66, H 6.63; found: C 64.57, H 6.60.

3e. (E)-5-(4′-(But-2′-enyloxy)-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 96%; mp = 155–157 °C; IR (KBr): 1742, 1705, 1546, 1519, 1395, 1286, 1268 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 1.75–1.79 (m, 9H), 3.94 (s, 3H), 4.65 (d, 1H, J = 6.0 Hz), 4.79 (d, H, J = 5.0 Hz), 5.77–5.97 (m, 2H), 6.95 (d, 1H, J = 8.6 Hz), 7.63 (dd, 1H, J = 8.6, 2.0 Hz), 8.29 (d, 1H, J = 2.0 Hz), 8.36 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 17.8, 27.4, 55.9, 69.7, 104, 110.4, 111.8, 115.9, 124.8, 124.9, 131.9, 132.4, 148.9, 154, 158.2, 164.1; ESI-MS: m/z (%) = 332 (45.4%) [M]⁺, 277 (69.1%), 230 (45%), 175 (100%). Anal. calcd C₁₈H₂₀O₆ (%): C 65.05, H 6.07; found: C 64.98, H 6.02.

3f. 5-(4′-Acetoxy-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 97%; mp = 170 °C; IR (KBr): 1764, 1732, 1615, 1582, 1513, 1396, 1373, 1282 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 1.80 (s, 6H), 2.35 (s, 3H), 3.91 (s, 3H), 7.15 (d, 1H, J = 8.2 Hz), 7.57 (dd, 1H, J = 8.2, 2.0 Hz), 8.22 (s, 1H), 8.36 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 20.6, 27.5, 56, 104.5, 114.2, 116.9, 122.9, 129.1, 130.3, 144.4, 144.6, 151.1, 157.1, 159.9, 163.3, 168.2; ESI-MS: m/z (%) = 320 (10.9%) [M]⁺, 277 (52%), 175 (32.5%), 107 (100%); anal. calcd C₁₆H₁₆O₇ (%): C 60.00, H 5.04; found: C 59.95, H 5.01.

3g. Ethyl-2-(4-((2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methyl)-2-methoxyphenoxy)acetate; yield 95%; mp = 104–105 °C; IR (KBr): 1774, 1748, 1728, 1703, 1577, 1556, 1508, 1384, 1274 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 1.29 (t, 3H, J = 7.0 Hz), 1.79 (s, 6H), 3.96 (s, 3H), 4.28 (q, 2H, J = 7.2 Hz), 4.80 (s, 2H), 6.82 (d, 1H, J = 8.6 Hz), 7.61 (dd, 1H, J = 8.6, 2.2 Hz), 8.29 (d, 1H, J = 2.0 Hz), 8.35 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 14.1, 27.4, 56, 61.6, 65.8, 104.2, 111.5, 112.2, 116.5, 126, 131.5, 148.9, 152.6, 157.8, 160.4, 163.9, 167.8; ESI-MS: m/z (%) = 364 (5.2%) [M]⁺, 291 (2%), 262 (30.4%), 79 (100%); anal. calcd C₁₈H₂₀O₈ (%): C 59.34, H 5.53; found: C 59.30, H 5.46.

3h. 5-(4′-Bromopropoxy-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 91%; mp = 135 °C; IR (KBr): 1748, 1711, 1580, 1563, 1522, 1390, 1273 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 1.80 (s, 6H), 2.36–2.47 (m, 2H), 3.64 (t, 2H, J = 6.4 Hz), 3.94 (s, 3H), 4.28 (t, 2H, J = 6.0 Hz), 6.98 (d, 1H, J = 8.4 Hz), 7.64 (dd, 1H, J = 8.4, 1.8 Hz), 8.29 (d, 1H, J = 2.2 Hz), 8.36 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 27.5, 29.5, 31.9, 56, 66.5, 104.1, 111.1, 111.8, 116.2, 125.3, 132.3, 149, 153.9, 158.1, 160.6, 164.1; ESI-MS: m/z (%) = 399 (18.3%) [M]⁺, 319 (29.7%), 297 (100%), 276 (40.1%); anal. calcd C₁₇H₁₉BrO₆ (%): C 51.14, H 4.80; found: C 51.18, H 4.84.

3i. 5-(4′-Bromobutoxy-3′-methoxybenzylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 91%; mp = 118–121 °C; IR (KBr): 1748, 1713, 1580, 1563, 1398, 1272 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 1.80 (s, 6H), 2.06–2.09 (m, 2H), 3.51 (t, 2H, J = 6.2 Hz), 3.94 (s, 3H), 4.17 (t, 2H, J = 5.8 Hz), 6.93 (d, 1H, J = 8.6 Hz), 7.63 (dd, 1H, J = 8.4, 2.0 Hz), 8.28 (d, 1H, J = 2.2 Hz), 8.35 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 27.4, 29.2, 33.1, 55.9, 68.1, 104.1, 110.6, 111.5, 116.1, 125, 132.4, 148.9, 154.1, 158.1, 160.5, 164.1; ESI-MS: m/z (%) = 413 (35.4%) [M]⁺, 313 (86.2%), 311 (100%), 277 (34.1%); anal. calcd C₁₈H₂₁BrO₆ (%): C 52.31, H 5.12; found: C 52.28, H 5.15.

3j. 5-(4′-Bromopentyloxy-3′-methoxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione; yield 91%; mp = 103–105 °C; IR (KBr): 1748, 1713, 1578, 1561, 1523, 1391, 1273 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ = 1.65–1.76 (m, 2H), 1.79 (s, 6H), 1.83–2.03 (m, 4H), 3.45 (t, 2H, J = 6.8 Hz), 3.94 (s, 3H), 4.15 (t, 2H, J = 6.6 Hz), 6.93 (d, 1H, J = 8.6 Hz), 7.64 (dd, 1H, J = 8.6, 2.0 Hz), 8.28 (d, 1H, J = 2.0 Hz), 8.35 (s, 1H); ¹³C NMR (50 MHz, CDCl₃): δ = 24.6, 27.4, 28, 32.3, 33.3, 55.9, 68.8, 104.1, 110.5, 111.5, 116.1, 124.9, 132.4, 148.9, 154.2, 158.1, 160.6, 164.1; ESI-MS: m/z (%) = 427 (37.8%) [M]⁺, 325 (80%), 261 (100%), 166 (48.2%); anal. calcd C₁₉H₂₃BrO₆ (%): C 53.41, H 5.43; found: C 53.20, H 5.38.

Conclusions

In summary, a facile and efficient synthetic route to novel 5-(arylidenyl)-2,2-dimethyl-1,3-dioxane-4,6-diones (3a–j) has been developed via solvent-free condensation reactions of vanillic aldehydes and Meldrum's acid in the presence of PTSA as a catalyst at ambient temperature. Simple work-up procedure, inexpensive catalyst, shorter reaction times along with excellent product yields are the significant features of this environmentally friendly method. Biological evaluations for the newly synthesized compounds 3a–j were investigated via treatment of series of bacteria and fungi strains. The strongest antibacterial activity was found in 3a, 3f and 3h compounds, which in extremely low concentrations inhibited all the species of bacteria. The lowest measured MIC value was 0.039 mg mL⁻¹ for 3h related to the Bacilus subtilis species. Based on the MIC value, the compounds 3f and 3h showed the strongest antifungal activity, and the most sensitive among the fungi were Fusarium oxysporum, Trichoderma viride and Cladosporium cladosporioides.

DPPH free scavenging assay showed that 3h interacts well with DPPH radical and exhibits high antioxidant activity. This factor indicates that 3h can be used as an antioxidant.

DNA interaction with selected compounds (3a and 3h) in the presence of ethidium bromide (EB) showed that both quenchers partially replace EB from the EB–DNA complex, and quench fluorescence intensity. The measured quenching constants for 3a and 3h of (1.6 ± 0.1) × 10⁴ and (2.4 ± 0.2) × 10⁴ M⁻¹, respectively, indicate their large affinity and efficiency to substitute EB from EB–DNA complexes as classical intercalators, and bind strongly with DNA. In interaction with BSA the measured K_a value ((2.8 ± 0.2) × 10⁶) for 3a-BSA complex indicates that it is more stable and more suitable for drug–cell interaction than 3h-BSA complex (K_a = (2.3 ± 0.1) × 10⁴).

Acknowledgements

The authors are grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia for financial support (Grants 172011 and 172034).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Characterization of compounds 3a–j, copies of ¹H and ¹³C NMR spectrums. See DOI: 10.1039/c6ra0771k

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