Design, synthesis and antimicrobial evaluation of dihydropyrimidone based organic–inorganic nano-hybrids

Tilak Raja, Beant Kaur Billingb, Navneet Kaur*b and Narinder Singh*a
aDepartment of Chemistry, Indian Institute of Technology Ropar (IIT Ropar), Rupnagar, Panjab 140001, India. E-mail: nsingh@iitrpr.ac.in; Tel: +91 1881242176
bCentre for Nanoscience and Nanotechnology (UIEAST), Panjab University, Chandigarh, 160014, India. E-mail: navneetkaur@pu.ac.in; Tel: +91 9815245098

Received 11th May 2015 , Accepted 12th May 2015

First published on 18th May 2015


Abstract

Substituted dihydropyrimidone derivatives were synthesized through one pot multicomponent Zn(ClO4)2 catalysed Biginelli reactions between differently substituted aromatic aldehydes, urea and ethylacetoacetate. Purified products were characterized and further engineered into metal inorganic conjugates of silver, copper and gold. Size, morphology and stability of nanoaggregates were analysed using FTIR, EDX, TEM, XRD and TGA analysis. Antimicrobial activities of all the synthesized compounds and their metal conjugates were evaluated against various bacterial and fungal strains. Results of investigations revealed that metal nanoaggregate Au@4c has shown maximum inhibition of MIC 8 against bacteria S. aureus. Further, bacterial SEM cell imaging carried out against S. aureus before and after treatment of Au@4c advocates that formation of blebs due to the accumulation of the cytoplasm are possible cause of bacterial cell death.


Introduction

Microbial infections are a leading cause of the large number of deaths every year due to the development of multidrug resistance strains in immune compromised hosts.1–9 Many new drugs have been devised from time to time to combat the microbial infections due to resistant strains. However, pharmaceutical industry faces major challenges in the discovery and development of antimicrobial drugs due to long step organic synthesis and high cost.10 Additionally, use of the harmful chemicals and undesirable side effects are other major drawbacks.11 Therefore, development new antimicrobial agents with significantly low cost and reduced or low toxicity is highly desirable in pharmaceutical industry.12–15

Recently, Biginelli single step multicomponent reactions (MCRs) are emerged possible route in synthesis of the dihydropyrimidone derivatives, which possess wide range of the biological activities including antibacterial activities.16–29 At the same time, metal nanoaggregates of silver (Ag), copper (Cu), gold (Au), titanium (Ti), and zinc (Zn) and their conjugates are known for antimicrobial activity30 due to its different in their physical and chemical properties than bulk materials due to its large surface area and quantum sized effects, which makes them highly effective in the treatment.31–42 However, detrimental effect of some metal nanoaggregates to human cells are the major drawback, which can be controlled by using biocompatible metal nanoaggregates and controlling the size of metal nanoaggregates.43

Reported literature on antimicrobial potential of both dihydropyrimidones and metal nanoaggregates, encouraged us to coupled organic dihydropyrimidones with inorganic biocompatible metals44 such as silver (Ag), copper (Cu), gold (Au) to obtain organic–inorganic nanohybrids for evaluation of their antimicrobial activities against various bacterial and fungal strains. To best of our knowledge this is the first report on antimicrobial evaluation on organic metal nanoaggregates of the Biginelli compounds.

Experimental

All the chemicals were purchased from commercial supplier and were used without further purification. 1H NMR and 13C NMR spectra were recorded on JEOL II 400 spectrometer (400 MHz with TMS as internal standard; chemical shifts are expressed in ppm). Fourier transform infrared (FT-IR) of dried compounds were measured on Bruker Tensor 27 spectrophotometer, using a KBr pellet technique. Mass analysis was performed on Shimadzu 79037 GCMS GC-2010 plus. The CHN analysis was performed on Perkin Elmer 2400 CHN Elemental Analyser. Scanning electron microscopic (SEM) studies were conducted with drying the aqueous solutions of materials (10 μM). EDX images were taken with Jeol JSM-6610LV scanning electron microscope, which was operated at 15 keV. TEM images were recorded on Hitachi (H-7500) instrument worked at 80 kV. A 400-mesh carbon-coated copper grid was used for sample preparation. The nanoaggregates were suspended in aqueous medium and single drop of solution is placed onto the grid and draw-off the excess solvent after 1 min. TGA/DSC-I Star system Mettler Toledo was used to record TGA nanoaggregates. The crystal structure of the ZnO powders were analyzed with X-ray diffraction (XRD) with a PANalytical X'PERT PRO diffractometer operated at 45 kV and 40 mA using Ni-filtered Cu Kα radiations with a scan speed of 10° min−1 for 2θ in a range from 10 to 90. For measurement, an air-dried fine powder was taken and made a uniform layer on a zero background sample holder. The average crystalline size was estimated with Dewithe–Scherrer's equation using XRD analysis data.

General procedure for synthesis of compounds 4a–e

All the reactions was carried out by reacting differently substituted aromatic aldehydes (15.4 mmol), ethylacetoacetate (15.4 mmol) and urea (23.1 mmol) in 15 ml of methanol by heating at reflux for 8–12 h using Zn(ClO4)2 as a catalyst. Completion of reaction was monitored by TLC. After completion of reaction, solvent was evaporated under reduced pressure and obtained residue was washed three times with methanol to obtain solid product, which was further recrystallized from acetone/water 2[thin space (1/6-em)]:[thin space (1/6-em)]1 mixture to obtain pure product.
Compound 4a. Compound 4a–d was synthesized and characterized spectroscopically in comparison with reported data.45,46
Compound 4e. A yellow solid product was obtained in 90% yield (175 mg); IR (KBr): 3349.1, 1698.1; 1H NMR (400 MHz, DMSO): δ = 11.36 (s, 1H, OH), 9.18 (s, 1H, NH), 8.01 (s, 1H, NH), 7.81 (d, 1H, ArH), 7.36 (s, 1H, ArH), 6.91 (d, 1H, ArH), 5.48 (s, 1H, CH), 3.92 (q, 2H, CH2), 2.28 (s, 2H, CH3), 1.03 (t, 3H, CH3); 13C NMR (75 MHz, DMSO): δ = 165.1, 161.5, 151.8, 149.2, 139.1, 131.0, 124.9, 123.9, 115.9, 96.7, 59.0, 49.9, 17.6, 13.8; mass (m/z) 322 (M − 1), anal. calcd for C14H15N3O6: C, 52.34; H, 4.71; N, 13.08; found: C, 52.22; H, 4.63; N, 12.99%.

Synthesis of organic nanoaggregates and their metal conjugates

Synthesis of organic nanoaggregates (ONAs). The ONAs of compounds 4a–e were synthesized by reprecipitation method.47 Briefly 1 ml of organic solution of organic compounds in tetrahydrofuran was injected with microsyringe at a constant rate to double distilled water (50 ml) under vigorous stirring. The solution was then sonicated for 20 min at constant temperature to produce ONAs of 5–6 nm size range, which provided a slight turbidity. The size of ONAs was simply controlled by varying the concentration of organic compounds in the solution.
Synthesis of organic–inorganic hybrid of silver. Organic–inorganic hybrid silver nanoaggregates48 of compounds 4a–e were prepared by taking an aqueous suspension of ONAs (5 ml) along with 0.2 mM of AgNO3. Heating this solution at constant temperature of 70 °C initiated the reduction of Ag (+1) to Ag (0) resulting in the formation of AgNPs immobilized on the surface of ONAs. The colour of final suspension was pale yellow indicating the formation of Ag@ONAs.
Synthesis of organic–inorganic hybrid of copper. Organic–inorganic hybrid of copper nanoaggregates49 of compounds 4a–e were prepared by taking an aqueous suspension of ONAs (5 ml) along with 0.2 mM of CuNO3 and continues stirring for 5 min. After 5 min, an aqueous solution of NaBH4 was added (4 μmol; 0.1 M), and red sols were immediately formed. The obtained colloidal solutions were left under stirring 5 hours to complete the reaction. The colour of final suspension was shining brown indicating the formation of Cu@ONAs.
Synthesis of organic–inorganic hybrid of gold. Organic–inorganic hybrid gold nanoaggregates Au@ONAs50 of all compounds 4a–e were prepared by taking mixture of aqueous suspension of ONAs (5 ml) along with 0.2 mM of HAuCl4 were 5 min. After 5 min, an aqueous solution of NaBH4 was added (4 μmol; 0.1 M), and red sols were immediately formed. The obtained colloidal solutions were left under stirring 10 hours to complete the reaction.

Antimicrobial activities

Antibacterial and antifungal activities of synthesized compounds 4a–e and their organic–inorganic nanohybrids were evaluated using disc-diffusion test for pre-screening of antibacterial and antifungal potential of agents and broth micro-dilution method to determine the minimum inhibitory concentration.

Antibacterial activity – materials and methods

The following bacterial strains were used: the gram negative strains used were Escherichia coli (MTCC-119, facultative anaerobic), Pseudomonas aeruginosa (MTCC-741, aerobic) and Shigella flexneri (MTCC-1457, facultative anaerobic). Gram positive strain used is Staphylococcus aureus (MTCC-740, facultative anaerobic) from Microbial Type Culture Collection, IMTECH, Chandigarh, India. Bacteria were cultivated at 37 °C in Nutrient Agar. For MIC determination Nutrient Broth was used. All compounds and standards were dissolved in dimethylsulfoxide (1/10) and applied in different concentrations. Dimethylsulfoxide was used as a negative control and antibiotics (ciprofloxacin) as positive controls.

Antibacterial activity

The disk-diffusion assay was applied to determine the growth inhibition of bacteria by compounds to be tested. Further, 4 h grown bacterial cultures in Nutrient Broth (100 ml) were spread onto mueller hinton agar (MHA) and compounds 4a–e were applied to 8 mm disks (Whatman paper no. 1). After 24 h of incubation at 37 °C, the diameter of growth inhibition zones was measured.
Minimum inhibitory concentration (MIC). It is the concentration mg ml−1 required to inhibit bacterial cell proliferation by 50% after exposure of cells to test compounds. Inhibitory potential of the compounds was determined in terms of MIC (mg ml−1) using turbidimetry method.
MIC determination. The broth dilution test was performed in test tubes. In twofold serial dilutions of the compounds, a standardized suspension (Sarthak turbidity standard) of test bacteria (100 ml) was added to obtain a final concentration of 5 × 105 CFU ml−1. A growth control tube and sterility control tube were used in each test. After overnight incubation at 37 °C, the MIC was determined by measuring optical density at 600 nm as the lowest concentration that inhibits growth, evidenced by the absence of turbidity.

Antifungal activity – materials and methods

The following fungal strain was used: A. niger (MTCC-281), G. candidum (MTCC-3993), C. albicans (MTCC-227) and C. tropicalis (MTCC-230). Fungus was cultivated at 25 °C on Soyabean Casein Digest Medium (Tryptose Soya Broth) and MIC was determined. The compounds and standard were dissolved in dimethylsulfoxide (DMSO, 1/10) and applied in different concentrations. Dimethylsulfoxide was used as a negative control and antifungal (fluconazole) as positive controls.

Antifungal activity

The disk-diffusion assay was applied to determine the growth inhibition of fungi by compounds to be tested. Overnight fungal cultures (100 ml) were spread onto Soyabean Casein Digest Medium (Tryptose Soya Broth). The compounds were applied to 8 mm disks (Whatman paper no. 1). After 48 h of incubation at 25 °C, the diameter of growth inhibition zones was measured.
MIC determination. The broth dilution test was performed in test tubes. The conidial suspension, which gave the final concentration of 1 × 105 CFU ml−1, was prepared. A growth control tube and sterility control tube were used in each test. After 24–72 hours of incubation at 25 °C, the MIC was determined visually as the lowest concentration that inhibits growth, evidenced by the absence of turbidity.

Result and discussions

Chemistry

All the reactions were carried using equimolar concentration of substituted benzaldehyde (1), ethylacetoacetate (2) and urea (3) in refluxing methanol using Zn(ClO4)2 as a catalyst to obtain product (4) in high yield. Progress of reaction was monitored with TLC and after completion of reaction solvent was evaporated under reduced pressure. Solid residue obtained was filtered, washed three times with methanol to get solid product. Finally, pure products were recrystallized from acetone/water 2[thin space (1/6-em)]:[thin space (1/6-em)]1 mixture of solvent (Scheme 1, Table 1). All the products 4a–e were characterized using 1H and 13C NMR, IR, mass (Fig. S1–S4) and elemental analysis, and in comparison with reported literature data.
image file: c5ra08765a-s1.tif
Scheme 1 Synthesis of dihydropyrimidone derivatives.
Table 1 Reaction yield (%) of the products 4
Entry Aldehydes Product Reaction time (h) Yield (%)
1 1a 4a 5 80
2 1b 4b 4 85
3 1c 4c 4 90
4 1d 4d 6 86
5 1e 4e 8 85


Synthesized compounds 4a–e were tailored to their organic nanoaggregates and their biocompatible metal conjugates of silver, copper and gold.44 Formation of the metal organic framework leads to the change in physical and chemical properties of the organic compounds (Fig. 1).50 Formation of the nanoaggregates were observed metal nanoaggregates were observed with visible colour change and shift in the IR spectral peaks (Fig. S4–S7).


image file: c5ra08765a-f1.tif
Fig. 1 Formation of metal organic nanohybrids.

Size of metal conjugated nanostructures (silver, copper, gold) on ONAs of compound 4a, were dissolved in CHCl3 to separate organic matter from metal particles. Size and morphology of compound 4a on Ag@ONAs was investigated using TEM analysis. Stability of nanoaggregates was determined using TGA analysis (Fig. S8–S10). TEM images determine the formation of the uniform sized nanoparticles of 100 nm and elemental mapping advocates the presence of silver, nitrogen, carbon and sulphur in the sample (Fig. 2A and B). XRD analysis of the sample indicates the nanoparticle size range 134 nm (Fig. S8). TGA analysis of the 4a capped nanoaggregates validates the stability (Fig. S9) with variable temperature range.


image file: c5ra08765a-f2.tif
Fig. 2 (A) EDS spectra of silver capped compound 4a nanoaggregates. (B) TEM image of compound 4a nanoaggregates.

TEM results revealed the formation of needle shape Cu@ONAs of compound 4a with size 20 nm (Fig. 3A and B). Additionally, EDS profile confirmed the presence of copper, carbon and oxygen in the sample. XRD analysis of the sample indicates the nanoparticle size range 49 nm (Fig. S10) TGA of the sample confirmed the variable temperature stability range (Fig. S11).


image file: c5ra08765a-f3.tif
Fig. 3 (A) EDS spectra of copper capped 4a nanoaggregates, (B) TEM image of copper capped 4a nanoaggregates.

TEM analysis of Au@ONAs of compound 4a indicate the formation nanoaggregates with size range <100 nm (Fig. 4A and B) and XRD nanoparticle size 80 nm (Fig. S12) with high TGA stability range (Fig. S13). EDS analysis confirmed the presence of gold, carbon and oxygen in the sample.


image file: c5ra08765a-f4.tif
Fig. 4 (A) EDS spectra of gold capped compound 4a nanoaggregates. (B) TEM image of compound 4a nanoaggregates.

In vitro antifungal51,52 and antibacterial activities53 of all the synthesized compounds (4a–e) and their metal conjugates i.e., Ag@ONAs, Cu@ONAs and Au@ONAs were carried out against various fungal and bacterial strains.

Initially, in vitro antibacterial studies of all dihydropyrimidones 4a–e including their silver, copper and gold nanoaggregates were carried out against Escherichia coli (MTCC-119, facultative anaerobic), Shigella flexneri (MTCC-1457, facultative anaerobic) and Pseudomonas aeruginosa (MTCC-741, aerobic) and Staphylococcus aureus (MTCC-740, facultative anaerobic) strains by disk-diffusion assay.

The result of the disk diffusion method was utilized to determine the activity of compounds and their metal nanoaggregates against gram negative and gram positive bacteria. Results of the investigations revealed that some of the compounds including metal nanoaggregates are active against different strains of bacteria. Further, MIC values were determined using turbidimetry method (Table 2) to authenticate the results. Ciprofloxacin was used as positive controls. It was observed that some of the compounds have shown good antibacterial activities; when capped over the inorganic nanoaggregates. In the case of aerobic gram positive bacteria Staphylococcus aureus compound 4c showed inhibition of MIC 20 ± 3, however, their metal nanoaggregates of Ag@ONAs and Cu@ONAs showed inhibition of MIC 18 ± 1 and Au@ONAs showed promising MIC 8 ± 0.5. Similarly, organic compound 4a showed inhibition (MIC 15 ± 1) and their Ag@ONAs (MIC 12 ± 0.5) and Au@ONAs (MIC 10 ± 0.5). Compound 4b showed MIC 15 ± 1 and its Ag@ONAs and Au@ONAs (MIC 10 ± 0.5) and Cu@ONAs of MIC 9 ± 1. P. aeruginosa bacteria the maximum growth inhibition observed (MIC 18 ± 1) for 4c, and its Ag@ONAs (MIC 12 ± 1) and Au@ONAs (MIC 10 ± 0.5). In the case of bacteria S. flexneri no significant inhibition was observed however, some of the compounds and their nanoaggregates showed mild inhibition i.e., compound 4abc MIC 71 ± 3, >100, 73 ± 2 and their Au@ONAs MIC 60 ± 2, 72 ± 3 and 52 ± 2, respectively. Compounds 4a had shown maximum inhibition of MIC 72 ± 2 and their Ag@ONAs (MIC 18 ± 2), Cu@ONAs MIC 14 ± 1 and Au@ONAs MIC 10 ± 0.5 against anaerobic gram negative bacteria E. coli.

Table 2 In vitro antibacterial activities of compounds
Entry Compounds MIC (μg ml−1)
S. aureus MTCC-740 P. aeruginosa MTCC-741 S. flexneri MTCC-1457 E. coli MTCC-119
a Ag@4, Cu@4 and Au@4 are Ag@ONAs, Cu@ONAs and Au@ONAs nanoaggregates, respectively.
1 4a 15 ± 1 30 ± 0.5 71 ± 3 72 ± 2
2 4b 15 ± 1 >100 >100 >100
3 4c 20 ± 3 18 ± 1 73 ± 2 75 ± 2
4 4d 80 ± 4 82 ± 2 >100 90 ± 4
5 4e 90 ± 2 91 ± 2 >100 85 ± 4
6 Ag@4aa 12 ± 0.5 40 ± 3 >100 18 ± 2
7 Ag@4b 10 ± 0.5 >100 >100 >100
8 Ag@4c 18 ± 1 12 ± 1 >100 22 ± 1
9 Ag@4d 70 ± 2 70 ± 2 >100 70 ± 1
10 Ag@4e 72 ± 2 75 ± 2 >100 80 ± 2
11 Cu@4aa >100 >100 >100 14 ± 1
12 Cu@4b 9 ± 1 >100 >100 >100
13 Cu@4c 18 ± 2 90 ± 2 >100 40 ± 1
14 Cu@4d 72 ± 3 >100 >100 >100
15 Cu@4e 85 ± 2 80 ± 2 >100 >100
16 Au@4aa 10 ± 0.5 20 ± 1 60 ± 2 10 ± 0.5
17 Au@4b 10 ± 0.5 >100 72 ± 3 >100
18 Au@4c 8 ± 0.5 10 ± 0.5 52 ± 2 72 ± 1
19 Au@4d 75 ± 2 60 ± 2 >100 80 ± 2
20 Au@4e 82 ± 1 75 ± 2 >100 80 ± 1
21 Ciprofloxacin 1.0 0.7 1.6 1.2


In order to determine the mechanism of the cell death in the case of S. aureus, cell imaging of bacteria S. aureus was conducted by keeping samples were overnight fixed with glutaraldehyde. Cell suspensions were centrifuged and washed three times with deionised water that had previously been filtered through a 0.22 nm micropore filter. After resuspension in deionised water, one drop (0.025 ml) of each suspension was added to 1 ml of absolute ethyl alcohol, which was applied to a standard metal support plate and allowed to dry in air. All the images are recorded at different interval of time after and were examined in a scanning mode, with Jeol JSM-6610LV scanning electron microscope which operated at 15 keV (Fig. 5).


image file: c5ra08765a-f5.tif
Fig. 5 SEM Images of the S. aureus at different interval of time: (A) before addition of Au@4c, (B) immediate after treatment of compound with Au@4c, (C) after 12 h incubaction after treatment, (D) 18 h incubaction after treatment, (E) 24 h incubaction after treatment, (F) close cell image of the bacteria after 24 h incubaction.

Results of investigations revealed that bacteria showed regular spherical shape before addition of the Au@4c. Bacterial cell images were taken at different interval of incubation time after addition of compound Au@4c (Fig. 5B–D). Further, after 18 h of the treatment morphological changes were observed in the bacterial cells as shown in Fig. 4D through the formation of blebs54 on the surface of the bacterial cells which might be due to the accumulation of the cytoplasm. After 24 h incubation cells starts rupturing, which lead to the bacterial cell death (Fig. 5E and F).

Antifungal evaluations were also carried out against various fungal strains A. niger (MTCC-281), G. candidum (MTCC-3993), C. albicans (MTCC-227) and C. tropicalis (MTCC-230) using turbidimetry method and determined in terms of MIC (μg ml−1) (Table 3). Few strains have shown marginal activity against fungal strain. In the case of Geotrichum candidum, compound 4a with MIC 40 ± 1 μg ml−1 and its Ag@ONAs (MIC 30 ± 2) and Au@ONAs (MIC 25 ± 2) showed maximum inhibition.

Table 3 In vitro antifungal activities of compounds
Entry Compds MIC (μg ml−1)
A. niger (MTCC-281) G. candidum (MTCC-3993) C. albicans (MTCC-227) C. tropicalis (MTCC-230)
a Ag@4, Cu@4 and Au@4 are Ag@ONAs, Cu@ONAs and Au@ONAs of 4a–e, respectively.
1 4a >100 40 ± 1 99 ± 1 >100
2 4b >100 60 ± 3 >100 >100
3 4c >100 >100 99 ± 2 >100
4 4d >100 >100 >100 >100
5 4e >100 >100 >100 >100
6 Ag@4aa >100 30 ± 2 60 ± 2 >100
7 Ag@4b >100 55 ± 4 >100 >100
8 Ag@4c >100 >100 95 ± 1 >100
9 Ag@4d >100 >100 >100 >100
10 Ag@4e >100 >100 >100 >100
11 Cu@4aa >100 >100 99 ± 2 >100
12 Cu@4b >100 45 ± 2 94 ± 3 >100
13 Cu@4c >100 97 ± 1 >100 >100
14 Cu@4d >100 >100 >100 >100
15 Cu@4e >100 >100 >100 >100
16 Au@4aa >100 25 ± 2 40 ± 3 >100
17 Au@4b >100 40 ± 4 >100 >100
18 Au@4c >100 96 ± 1 >100 >100
19 Au@4d >100 >100 >100 >100
20 Au@4e >100 >100 >100 >100
21 Fluconazole 42 12 24 20


To determine the cytotoxicity of organic–inorganic nanohybrids of Ag@4b, Cu@4b and Au@4c was carried out using MTT assay with HeLa cells cultured in nutrient mixture. As per the standard protocol54 the cells were incubated at 37 °C with 5% CO2 environment for 24 h. Further, on addition of Ag@4b, Cu@4b and Au@4c, only marginal cell death (96–98% cell survival) at concentration (20 μM). However, in order to investigate the potential cytotoxic effects of metal nanoaggregates at higher concentrations, the dose dependent studies were performed. The results indicate that all given organic–inorganic nanohybrids are not much toxic to HeLa cells, if all given organic–inorganic nanohybrids concentrations remains under 200 μM, that is with 84–86% cell survival over 24 h for 200 μM concentration of Ag@4b, Cu@4b and Au@4c.

Conclusion

Biginelli compounds 4a–e were synthesized and processed to organic nanoaggregates (ONAs) for further conversion to their metal conjugates of silver, copper and gold. All the compounds and their metal conjugates were evaluated for antimicrobial activities against different bacterial and fungal strains. Results of the investigations revealed that antimicrobial activity of some of compounds increased upon conversion to their organic–inorganic nanohybrids i.e., organic compound 4c showed MIC 20 ± 3 and their Au@4c showed MIC 08 ± 0.5 against S. aureus bacteria. Further, mechanism of the cell death preliminarily was determined using time dependent SEM analysis. The results of investigations revealed that possible cause of the cell death in the case of S. aureus is due to accumulation of the cytoplasm on the outer membrane of the bacteria leading to bleb formation. These organic–inorganic nanoaggregates can be used as ‘lead’ compounds for further antimicrobial drug development using organic–inorganic nanohybrids.

Acknowledgements

Dr Tilak Raj is thankful to UGC, New Delhi, India for Post Doctoral fellowship Grant no. PDFSS-SC-PUN-34.

Notes and references

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Footnote

Electronic supplementary information (ESI) available: 1H, 13C and mass spectra of compounds and cytotoxicty results. See DOI: 10.1039/c5ra08765a

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