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Biological Evaluation of Newly Synthesized Schiff Bases: Crystal Structure Complemented by DFT Calculation.

Byline: Muhammad Zaheer, Zareen Akhter, Asghari Gul, Misbah Tauseef, Bushra Mirza, Michael Bolte, Muhammad Qaiser Fatmi and Moiz Uddin Ahmed

Summary: A series of Schiff bases (N1-N5) derived from aniline and different substituted aromatic aldehydes, was synthesized and characterized by elemental analysis and various spectroscopic methods (IR and NMR spectroscopy). The crystal structures of compounds N3 and N4 were determined using single crystal X-ray analysis, and their 3D geometries were optimized at hybrid B3LYP level of theory using Triple-Zeta split valence shell plus diffused and polarization function basis sets 6-311++G(d, p) employing tight convergence criteria. The calculated structural parameters as well as the FTIR vibrational frequencies strongly complemented the experimental ones. Based on this knowledge the structure of N5 was predicted using the same theory and basis sets.

The preliminary biological evaluation of the precursors and synthesized Schiff bases revealed their negligible potential to inhibit microbial growth but were found significantly cytotoxic at higher concentrations. Moreover, the compounds showed concentration dependent protection against DNA damage. Antioxidant assays showed variable behavior of precursors and derived Schiff bases.

Keywords: Schiff bases, Crystal structure, Biological applications, DFT calculations

Introduction

Schiff bases are largely studied for their interesting properties for instance, synthetic ease and flexibility, their ability to reversibly bind oxygen, [1- 9] redox system in biological systems and oxidation of DNA. [10] These compounds in general, and those derived from p-substituted aniline, are known to possess antibacterial, [11] anti-tumor, [12] antiviral, [13] anti-HIV [14] and anti-fungal activities. [15, 16] Additionally, they find their use in radiopharmaceuticals, [17] as pesticides and plant growth regulators. [18] Based on aforementioned properties, Schiff base compounds are being studied extensively for their biological activities and potential applications in the field of medicine.

We report, herein, synthesis, spectroscopic and structural characterization (experimental and calculated) of some Schiff bases. Their biological studies were also carried out by standard antibacterial, anti-fungal, cytotoxic, antioxidant and DNA protection assays in comparison to their aldehydic precursors.

Experimental

Materials and Physical Measurements

3-nitrobenzaldehyde, 2,4- dichlorobenzaldehyde, 4-bromobenzaldehyde, 3,4- dimethoxybenzaldehyde and 4-fluorbenzaldehyde were obtained from Fluka Switzerland. Solvents like ethyl alcohol, ethyl acetate, diethyl ether, toluene and hexane were obtained from Merck Germany and were freshly dried using standard methods. The elemental analysis was performed using CHNS-932 LECO instrument. Melting temperature was determined on a Mel-Temp. (Mitamura Riken Kogyo.) apparatus using open capillary tubes and is uncorrected. The solid state Fourier transform infrared spectrum was recorded on Bio-Rad Excalibur FTIR, Model 3000 MX using KBr pellets.

1H and 13C-NMR spectra were obtained on a Bruker 300MHz NMR Spectrophotometer in CDCl3 using tetramethyl silane as internal reference. The data for the crystal structure analysis were collected on a STOE IPDS-II diffractometer with monochromatic Mo-Ka radiation at 173. The structure was solved by direct methods and refined with full-matrix least- squares techniques on F2. [19]

General procedure for the synthesis of Schiff bases (N1-N5)

In a 250 mL pre-backed two-necked flask supplied with magnetic stirrer, 4 mL (43 mmol) of aniline in 20 mL of dried toluene was mixed with an equimolar amount of aromatic aldehyde in 20 mL of toluene. The reaction was stirred under reflux using Dean and Stark apparatus and its progress was monitored using TLC. Solvent was removed under vacuum and solid product was recrystallized in pentane/ethylacetate mixture.

N-(3-nitrobenzylidene) aniline (N1)

4 mL (43 mmol) of freshly distilled aniline was mixed with 4.97 g (43 mmol) of 3- nitrobenzaldehyde in dry toluene. The reaction mixture was refluxed for 13 h and solid obtained after rotary evaporation was recrystallized from a mixture of ethyl acetate and n-hexane. Yield 80%; m.p. 64-65oC; Anal.cal. for C13H10N2O2: C, 69.0; H, 4.4; N, 12.4. Found C, 68.9; H, 4.4, N, 12.3; IR (KBr) : 1660 (C=N), 3000 (Ar-H), 1528, 1350 (NO2); 1H NMR (300 MHz, CDCl3) d: 8.54 (s, 1H, -CH=N), 8.48 (m, 1H, Ar-H), 8.25 (m, 1H, Ar-H), 8.15 (m, 1H, Ar-H), 7.70(m, 1H, Ar-H), 7.46 (d, 2H, J = 8.4 Hz, Ar-H), 7.33(d, 2H, J = 8.4 Hz, Ar-H), 7.02 (m, 1H, Ar-H); 13C-NMR (100 MHz, CDCl3) d: 160.1 (- CH=N), 153.0 (Ar-C-N), 148.2, (Ar-NO2), 135.5, 130.6, 129.8, 127.3, 122.3 (Ar-C).

N-(2, 4-dichlorobenzylidene) aniline (N2)

4 mL (43 mmol) of aniline was reacted with 5.76 g (43 mmol) of 2, 4-dichlorobenzaldehyde following the same procedure as discussed above. Solid product obtained was recrystallized from a mixture of ethyl acetate and n-hexane. Yield 83%; m.p. 90-91oC; Anal. Cal. For C13H9NCl2: C, 62.4; H, 3.6; N, 5.6. Found: C, 62.3; H, 3.6; N, 5.6. IR (KBr) : 1662 (C=N), 3070 (Ar-H), 1619, 1484 (Ar-H), 1048, 1097 (Ar-Cl); 1H NMR (300 MHz, CDCl3) d: 8.64 (s, 1H, -CH=N), 7.90 (d, 1H, J = 8.4 Hz, Ar-H), 7.70 (s, 1H, Ar-H), 7.50 (d, 1H, J = 8.4 Hz, Ar-H), 7.41 (d, 2H, J = 8.4 Hz, Ar-H), 7.38 (d, 2H, J = 8.4 Hz, Ar-H), 7.06 (m, 1H, Ar-H); 13C-NMR (100 MHz, CDCl3) d: 160.8 (-CH=N), 153.4 (Ar-C-N), 138.0, 135.5 (Ar-C-Cl), 130.7, 127.3, 122.3 (Ar-C).

N-(4-bromobenzylidene) aniline (N3)

For the synthesis of N3, 4 mL (43 mmol) of aniline was refluxed for 13 h with 6.09 g (43 mmol) of 4-bromobenzaldehyde in dry toluene. Solvent was rotary evaporated and solid product obtained was recrystallized from n-pentane. Yield 86%; m.p. 74oC; Anal. Cal. For C13H10NBr: C, 60.0; H, 3.8; N, 5.3 found C, 60.1; H, 3.8; N, 5.3; IR (KBr) : 1663 (C=N), 3085 (Ar-H), 1619, 1483 (Ar-H), 1069 ( Ar- Br); H NMR (300 MHz, CDCl3) d: 8.69 (s, 1H, - CH=N), 7.80 (d, 2H, J = 8.4 Hz, Ar-H), 7.68 (d, 2H, J = 8.4 Hz, Ar-H), 7.40 (d, 2H, J = 8.4 Hz, Ar-H), 7.37 (d, 2H, J = 8.4 Hz, Ar-H); 13C-NMR (100 MHz, CDCl3) d: 160.1 (-CH=N), 153.2 (Ar-C-N), 131.7, 131.3, 130.5, 127.3 (Ar-C) 125.5 (Ar-C-Br).

N-(3, 4-dimethoxybenzylidene) aniline (N4)

Compound N4 was synthesized by the reaction of 4 mL (43 mmol) of aniline with 7.13 g (43 mmol) of 3, 4-dimethoxybenzaldehyde in dry toluene. Solid product obtained after the removal of solvent was recrystallized from ethanol. Yield 79%; m.p. 73oC; Anal. Cal. For C15H15O2N: C, 74.7; H, 6.2; N, 5.8 found: C, 74.6; H, 6.2; N, 5.8; IR (KBr) : 1631 (C=N), 3041(Ar-H), 1583, 1467 (Ar-H), 2812 (C-H aliphatic); 1H NMR (300 MHz, CDCl3) d: 8.60 (s, 1H, -CH=N), 7.58 (s, 1H, Ar-H), 7.40 (d, 1H, J = 8.4 Hz, Ar-H), 7.44 (d, 2H, J = 8.4 Hz, Ar-H), 7.30 (d, 2H, J = 8.4 Hz, Ar-H), 6.90 (d, 1H, J = 8.4 Hz, Ar-H), 3.88 (s, 3H, OCH3), 3.85 (s, 3H, OCH3); 13C-NMR (100 MHz, CDCl3) d: 160.3 (-CH=N), 153.2 (Ar-C-N), 152.0 (Ar-C-O), 150.0 (Ar-C-O), 130.0, 127.5, 122.5, 115.4 (Ar-C), 56.1 (OCH3).

N-(4-fluorobenzylidene) aniline (N5)

4 mL (43 mmol) of aniline was refluxed with 4.63 mL (43 mmol) of 4-fluorobenzaldehyde in dry toluene for 14 h to get compound N5. Final solid product was recrystallized from a mixture of Ethanol and ethyl acetate. Yield 85%; m.p. 44oC; Anal. Cal. For C13H10NF: C, 78.4; H, 5.0; N, 7.0 found: C, 78.4; H, 5.0; N, 7.0; IR (KBr) : 1629 (C=N), 3075, 3001(Ar-H), 1139, 1211, 1237 (Ar-F); 1H NMR (300 MHz, CDCl3) d: 8.65 (s, 1H, -CH=N), 8.01 (d, 2H, J = 8.4 Hz, Ar-H), 7.40 (d, 2H, J = 8.4 Hz, Ar-H), 7.32 (d, 2H, J = 8.4 Hz, Ar-H), 7.30 (d, 2H, J = 8.4 Hz, Ar-H); C-NMR (100 MHz, CDCl3) d: 165.5 (Ar-C- F), 160.6 (-CH=N), 153.4 (Ar-C-N), 130.7, 130.0, 130.5, 127.3, 122.5 (Ar-C).

Biological Activities

Antibacterial Assay

Agar well diffusion method [20] was used for the determination of inhibition zone. Single colony from each bacterial culture plate was transferred to nutrient broth (pH 7) and incubated at 37oC for 24 h. A volume of 0.75 mL of broth culture containing ca.106 colony forming units per mL of test strain was added to the 75 mL of nutrient agar medium, mixed well, and then poured into a 14 cm sterile agar plate. Reaction was performed in triplicate. Wells were prepared by using 8 mm sterilized metallic borer, sealed with medium and filled with 100 uL of 1 mg/mL solution of each compound. Roxythromycine (1 mg/mL) and Cefixime (1 mg/mL) were used as standard drugs while DMSO was used as negative control. Plates were incubated at 37oC aerobically and zone of inhibition was measured after 24 h.

Antifungal Assay

The agar tube dilution method [21] was used for the antifungal activity of test compounds with some modification as reported earlier.[22] Screw caped test tubes containing sabouraud dextrose agar medium (SDA) were autoclaved at 121oC for 20 minutes. The tubes were allowed to cool to 50oC and non-solidified SDA was loaded with 66.6 uL of compound from stock solution (12 mg/mL in DMSO). The tubes were then allowed to solidify in slanting position at room temperature. The tubes were prepared in triplicate for each fungus species. The tubes containing solidified media and test compound were inoculated with 4 mm diameter piece of inoculums from a seven days old fungal culture.

Media supplemented with DMSO and reference antifungal drug were used as negative and positive control respectively. The tubes were incubated at 28degC for 7 days. Fungal growth was determined by measuring linear growth (mm) and growth inhibition was calculated with reference to negative control.

Cytotoxicity Assay

The test compounds were subjected to Brine Shrimp Lethality Assay [23] for cytotoxic activity. Artificial sea water was prepared by dissolving 20 g commercial sea salt (sigma) in 0.5 L distilled water and aerated for two h with continuous stirring. Brine shrimps (Artemia salina) eggs (sera, Heidelberg Germany) were hatched in a narrow rectangular dish filled with artificial sea water at 37oC. After 48 hrs incubation, larvae were collected and 10 larvae were transferred per vial containing 50 uL of test compound (at concentration of 10000 ppm, 1000 ppm and 100 ppm) and then volume was made up to 5 mL. In final reaction mixture concentration became 1000 ppm, 100 ppm and 10 ppm. Negative control was run with 50 uL of DMSO instead of test sample. Vials were placed uncovered at room temperature and illuminated for 24 h. After 24 h of incubation survivors were counted. Results were analyzed by statistical computer program Finney.

DPPH Free Radical Scavenging Assay

Radical scavenging activity of test compounds was measured spectrophotometrically by using modified protocol as reported earlier.[24] Antioxidant activity of compound (Z2-Z5) was tested at three concentrations 100 ppm, 50 ppm and 25 ppm. While three compounds (Z1, N1-N5) were tested at two additional concentrations 10 ppm and 5 ppm in order to get data for IC25 calculation. A volume of 100 uL of each test compound was added to 2 mL of 0.1 mM DPPH solution in ethanol and 0.1 mL of 0.1mM tris-HCl buffer. Reaction was performed in triplicate. Vials were capped and reaction mixture was incubated for 30 minutes at room temperature in dark. Absorbance of reaction mixture was measured at 517 nm. In negative control 100 uL of DMSO was used. Blank was prepared by mixing distilled water, ethanol and DMSO in ratio of 10:1:9 respectively. The percentage scavenging of DPPH free radical for each concentration of compound was calculated with reference to absorbance of negative control.

DNA Damage Protection Assay

Determination of antioxidant (protective) or pro-oxidant (damaging) activity of test compounds was conducted according to the reported protocol. [25] The reaction was conducted in PCR micro-tubes in a total volume of 15 uL. A volume of 3 uL of pBR322 plasmid DNA solution (containing 0.5 ug DNA) was transferred to each micro tube followed by 5 uL of stock solution of test compound at three different concentrations 3000 ppm, 300 ppm and 30 ppm to make final concentrations of reaction mixtures 1000 ppm, 100 ppm and 10 ppm. Then, 3 uL of 2 mM FeSO4 and 4 uL of 30% H2O2 were added successively. Four different controls were used in this assay including positive control (X) which contained 3 uL of phosphate buffer instead of test sample. Negative control (P) was plasmid DNA and phosphate buffer only, third control was plasmid DNA with hydrogen peroxide treatment (H) and fourth control was plasmid DNA with Fe (II) treatment (F). Reaction mixtures were incubated at 37degC in dark for one hour.

Each reaction mixture was run in 1X TBE buffer at 60 volts for 1 hour in horizontal electrophoresis apparatus. For each run, a 1kb ladder and four controls P, F, H and X were run simultaneously. The gels were photographed under UV light.

Computational Details

The input 3D structures of N3 and N4 were taken from the crystallography followed by the optimization at B3LYP/6-311++G(d,p) using Gaussian03 (G03) software [26]. The Br atom in the crystal structure of N3 was substituted by F to create the N5 structure. The geometry optimization of compounds N3-N5 was performed in the gaseous phase using tight convergence criteria. The energy minimum for all three compounds was confirmed by frequency calculations on the optimized geometries giving the imaginary frequency value at zero. The N1 and N2 structures were also built from the crystal structures of N3 followed by geometry optimization and frequency calculations using the same parameters. All vibrational frequency values have been scaled by a factor of 0.965. The Gabedit [27] and Avogadro [28] programs were used to build the 3D structures and Gaussian input files of three compounds. All calculations were carried out on Core i5 desktop machine with windows operating system.

The 3D coordinates for optimized N3-N5 structures, and their corresponding electronic energies have been given in the supplementary material.

Results and Discussion

Synthesis and Spectral Characterization

Schiff bases (N1-N5) were synthesized by the condensation reaction of substituted aromatic aldehydes and aniline using toluene as solvent. The reactions were carried out under reflux and water was azeotropically removed using Dean and Stark apparatus. Progress of the reaction was monitored by TLC and products were recrystallized from pentane/ethylacetate mixture Scheme-1. The synthesized compounds were characterized by melting points, CHN, FT-IR, 1H and 13C-NMR spectral studies. Elemental analysis data of all products is in good agreement with the calculated values. IR spectra of these compounds show all characteristic peaks. Absorption bands in the range of 1640-1690 cm-1 may be assigned to the stretching vibration of C=N bond while those in the range 3000-3150 cm-1 may be due to aromatic C-H stretch. Absence of any peak at 3500-3300 cm-1 confirms the formation of Schiff bases.

Table 1: Experimental and calculated IR values in cm-1. The calculated IR vibration frequencies have been scaled with a factor of 0.965 as suggested in a published paper. [29]

Codes###FTIR cm-1 Experimental###FTIR cm-1 Calculated (multiplied by scaling factor of 0.965)

###1660 (C=N),###1632 (C=N),

###N1###3000 (Ar-H),###3076 (Ar-H),

###1528, 1350 (NO2)###1526, 1322 (NO2),

###1662 (C=N),###1618 (C=N),

###3070 (Ar-H),###3075 (Ar-H),

###N2

###1619, 1484 (Ar-H),###1558, 1442 (Ar-H),

###1048, 1097 (Ar-Cl)###1024, 1072 (Ar-Cl),

###1663 (C=N),###1628 (C=N),

###3085 (Ar-H),###3075 (Ar-H),

###N3

###1619, 1483 (Ar-H),###1564, 1465 (Ar-H),

###1069 (Ar-Br)###1041 (Ar-Br),

###1631 (C=N),###1621 (C=N),

###3041 (Ar-H),###3072 (Ar-H),

###N4

###1583, 1467 (Ar-H),###1554, 1489 (Ar-H),

###2812 (C-H aliphatic)###2901 (C-H aliphatic),

###1629 (C=N),###1630 (C=N),

###N5###3075, 3001 (Ar-H),###3074, 3050 (Ar-H),

###1139, 1211, 1237 (Ar-F)###1196 (Ar-F),

The IR frequencies have been also calculated using hybrid B3LYP with DFT- optimized 6-311++G(d, p) basis sets; the frequencies have been scaled as suggested in a published paper. (Table-1). 1H and 13C-NMR spectra of the synthesized Schiff bases were recorded in CDCl3 relative to TMS as reference. In 1H NMR spectra of all compounds azomethine proton (CH=N) is the most deshielded proton and gives a singlet at 8.5. 8.9 ppm while aromatic protons show their signals in the range 7.0-8.0 ppm. 13C-NMR spectra show the signal of azomethine carbon as the most deshielded which appears at 155-166 ppm. Aromatic carbon atoms show their signal at 122, 128, 134 and 153 ppm respectively.

Single Crystal X-Ray Analysis of Schiff Bases

Crystal structures of compounds N1 and N2 have already been reported [26, 27] and those of N3 and N4 are presented here. Attempts to crystallize compound N5 for single crystal X-ray analysis proved unsuccessful; therefore, the structure has been predicted using DFT method. The optimized structures of N3 and N4 showed very small root mean square deviation (RMSD) values, i.e. 0.24 and 0.27 A, respectively, when compared with the corresponding crystal structure. As expected, the optimized 3D structure of N5 also exhibited negligible RMSD value, 0.28 A and 0.14 A, when compared with the N3 crystal structure and the optimized N3 structure, respectively. The structures of N3 and N4 (Fig 1 and 2) do not show any unusual features. Geometric parameters are in the usual ranges.

Table-2: Crystal data and structure refinement for N3 and N4.

###Identification code###N4###N3

###Empirical formula###C15 H15 N O2###C13 H10 Br N

###Formula weight###241.28###260.13

###Temperature###173(2) K###173(2) K

###Wavelength###0.71073 A###0.71073 A

###Crystal system###Orthorhombic###Orthorhombic

###Space group###Pbca###Pbcn

###Unit cell dimensions###a = 15.9829(16) A, a = 90deg###a = 29.2445(12) A, a = 90deg

###b = 6.1574(4) A,###b = 90deg###b = 5.9215(2) A,###b = 90deg

###c = 25.6344(18) A, Y = 90deg###c = 25.5126(8) A,###Y = 90deg

###Volume###3###3

###2522.8(3) A###4418.1(3) A

###Z###8###16

###Density (calculated)###3###3

###1.271 Mg/m###1.564 Mg/m

###Absorption coefficient###-1###-1

###0.084 mm###3.685 mm

###F(000)###1024###2080

###Crystal size###3###3

###0.49 x 0.47 x 0.23 mm###0.24 x 0.11 x 0.11 mm

###Theta range for data collection###3.00 to 26.78deg.###1.39 to 25.03deg.

###Index ranges###-12<=h<=20, -7<=k<=7, -32<=l<=26###-34<=h<=34, -6<=k<=7, -30<=l2sigma(I)]###R1 = 0.0420, wR2 = 0.1048###R1 = 0.0404, wR2 = 0.0938

###R indices (all data)###R1 = 0.0563, wR2 = 0.1098###R1 = 0.0583, wR2 = 0.1012

###Extinction coefficient###0.027(2)###0.0024(2)

###Largest diff. peak and hole###-###-3

###0.219 and -0.192 e.A###0.615 and -0.455 e.A

Table-3: Selected Bond lengths [A] and angles [deg] for N3, N4 and N5. The calculations have been performed at B3LYP/6-311++G(d,p) level of theory and basis sets.

###N3###N4###N5

###Atoms###Experimental###Calculated###Atoms###Experimental###Calculated###Atoms###Calculated

###Br(1)-C(14)###1.900(4)###1.915###N(1)-C(1)###1.272(17)###1.278###F(1)-C(14)###1.353

###N(1)-C(1)###1.279(4)###1.276###N(1)-C(21)###1.422(17)###1.405###N(1)-C(1)###1.276

###N(1)-C(21)###1.422(4)###1.406###O(1)-C(13)###1.366(15)###1.358###N(1)-C(21)###1.406

C(1)-N(1)-C(21)###119.2(3)###120.4###O(1)-C(17)###1.432(15)###1.422###C(1)-N(1)-C

###(21)###120.3

N(1)-C(1)-C(11)###122.2(3)###122.7###O(2)-C(14)###1.363(14)###1.354###N(1)-C(1)-C###122.8

###(11)

N(1)-C(1)-H(1)###118.9###121.7###O(2)-C(18)###1.430(16)###1.422###N(1)-C(1)-H(1)###121.6

C(26)-C(21)-N(1)###116.5(3)###117.8###C(1)-N(1)-C(21)###117.5(11)###120.1###C(26)-C(21)-N(1)###117.9

C(22)-C(21)-N(1)###124.3(3)###123.1###C(13)-O(1)-C(17)###117.1(10)###118.1###C(22)-C(21)-N(1)###123.1

###C(14)-O(2)-C(18)###117.3(10)###118.5

###N(1)-C(1)-C(11)###123.5(12)###123.3

###N(1)-C(1)-H(1)###118.2###121.4

###C(26)-C(21)-N(1)###122.2(12)###123.0

###C(22)-C(21)-N(1)###118.7(12)###118.1

Biological Studies of Schiff bases (N1-N5) and their Precursors (Z1-Z5, AN) Antibacterial Assay The antibacterial activity was tested against six bacterial strains; three Gram-positive i.e. (B. subtilis (ATCC 6633), S. aureus (ATCC 6538), M. luteus (ATCC 10240) and three Gram-negative strains i.e. E. coli (ATCC 15224), E. aerogenes (ATCC 13048) and S. setubal (ATCC 19196). The agar well diffusion method was used in this assay and the experiment was performed in triplicate. Only compounds Z1, Z4 (aldehyde precursors) and N1 (Schiff base) showed some activity but the diameter of the zone of inhibition was below 7 mm (non- significant level of activity). However, precursor Z1 was active against all the bacterial strains tested except E. coli with inhibition zone ranging from 2.3 mm for M. luteus to 4.5 mm for B. subtilis (Table-4) but its Schiff base derivative (N1) did not show any appreciable activity against the tested bacterial strains.

Antifungal Assay

All test compounds were subjected to antifungal activity against five fungal strains; A. fumigatus (66), A. flavus (0064), F. solanni (0291), Mucor species (0300) and A. niger (0198). The results (Table 5) show effective interaction of compounds with the tested fungal species. Mucour species were found the most susceptible to the test compounds with seven out of ten compounds having more than 40% activity. All other compounds, except Z2, have growth promoting effect on A. niger, as indicated by negative values of percentage of inhibition.

Aldehydic precursor Z 2 (2-4 dichloro benzaldehyde) was found most effective against all strains with 100% growth inhibition of A. flavus and F. solanni. This activity might be due to the lipophillic character of polar C-Cl bond which favours its permeation through lipoid layer of microbial membrane. It can be concluded from table 4 that the antifungal potential of Schiff bases is relatively lower than their corresponding aldehyde precursors except Schiff base N1 which exhibits enhanced activity against A. flavus and Mucor strains than its aldehydic precursor Z1.

Cytotoxicity Assay

Cytotoxicty of the compounds in solution (1000 ppm, 100 ppm and 10 ppm) was tested using Brine Shrimp lethality assay. LD50 values were calculated from the data (Table-6). Most of the compounds were found significantly cytotoxic at 1000 ppm and 100 ppm. Highest cytotoxicity was exhibited by Z3 with least LD50 value i.e. 4.25 ug/mL. Schiff bases N4 and N5 were found considerably cytotoxic (LD50 values of 9.27 ug/mL and 9.47ug/mL) than their precursors Z4 and Z5 (with LD50 of 106.44 ug/mL and 89.52 ug/mL respectively).

Table-4: Antibacterial activities of the test compounds.

###Zone of Inhibition (mm)

###Compounds

###B. subtilis###S. setubal###M. luteus###E. coli###E. aerogenus###S. aureus

###Z1###4.5+-2.36###4+-2.79###2.3+-1.66###-###4.4+- 3.11###4.2+- 2.96

###Z2###-###-###-###-###-###-

###Z3###-###-###-###-###-###-

###Z4###1.1+-0.57###-###1.5+-1.53###-###1.2+- 0.69###2.15+- 1.64

###Z5###-###-###-###-###-###-

###AN###-###-###-###-###-###-

###N1###1.6+-1.19###2+-1.17###-###2.1+-1.21###2.066+- 1.19###-

###N3###-###-###-###-###-###-

###N4###-###-###-###-###-###-

###N5###-###-###-###-###-###-

###Roxithromycine###6.5+-1.02###5.5+-.06###6.2+-.049###6+-.022###6.9+-2.96###18.5

###Cefixime###20+-.05###28+-.055###15+-.84###26+-.75###25.8+-2.12###14.5+-.707

###(-)ve control###-###-###-###-###-###-

Table-5: Antifungal activities of the test compounds.

###A. fumigatus###A. flavus###F. solanii###Mucour Species.###A. niger

###Compounds

###Percentage of Growth Inhibition +- STDEV

###Z1###43.89+-19.51###17.99+-5.29###-0.9+-4.76###30.35+-19.31###-17.026+-7.67

###Z2###90.24+-2.43###100+-0###100+-0###89.28+-8.18###68.08+-3.68

###Z3###-34.15+-12.19###1.99+-5.29###24.32+-6.24###85.71+-3.57###-14.89+-3.68

###Z4###-11.22+-9.74###-4+-4###8.1+-0###68.21+-5.18###-19.15+-2.12

###Z5###-14.63+-17.07###-12+-4###-6.3+-1.8###46.42+-6.18###-27.66+-0

###AN###-24.39+-7.31###-4.+-5.29###-4.50+-3.6###21.42+-12.87###-2.13+-0

###N1###34.14+-11.17###51.99+-5.02###6.3+-7.85###39.64+-20.23###-0.00426+-5.62

###N3###-26.83+-4.87###3.99+-9.16###8.1+-8.25###49.28+-20.29###-14.89+-1.26

###N4###-14.63+-8.79###-8+-3.46###-0.9+-3.6###62.85+-11.41###-14.89+-1.26

###N5###-4.88+-13.58###3.99+-3.46###-0.9+-1.8###35.71+-16.36###-23.4+-4.25

###Terbinafine###100+-0.0###100+-0.0###100+-0.0###100+-0.0###100+-0.0

###(-)ve Control###-###-###-###-###-

Table-6: Cytotoxicty data of the test compounds.

###No of Shrimps Used at###No. of Shrimps Killed###LD50 value (ppm) or

###Compounds

###Each Dose Level.###1000ppm###100ppm###10ppm###(ug/ml)

###Z1###30###30###30###2

###Z2###30###30###30###8###17.7164

###Z3###30###30###30###20###4.2534

###Z4###30###30###13###1###106.44

###Z5###30###30###10###3###89.52

###AN###30###30###29###27

###N1###30###30###28###11###15.26

###N3###30###30###30###27

###N4###30###30###30###15###9.27

###N5###30###30###28###15###9.47

Table-7: Percentage scavenging and IC25 data of the test compounds.

Sample Code###Percentage Scavenging###IC25 ug/ml###Remarks

###100ppm###50ppm###25ppm###10ppm###5ppm

###Z1###-2.3926###-3.5889###-0.9244###-3.2###-2.7###-###Pro-oxidant

###Z2###31.5850###26.4568###21.1538###47.87###Antioxidant

###Z3###4.8643###3.2770###18.1771###>100###Antioxidant

###Z4###-0.9787###1.08754###-2.7732###-###Pro-oxidant

###Z5###-5.0363###-4.7248###-3.0114###-###Pro-oxidant

###AN###31.6614###24.7126###19.5924###56.28###Antioxidant

###N1###15.4554###11.9186###9.06###7.41###1.16###>100###Antioxidant

###N3###7.5691###3.7845###-4.5123###-2.03###1.16###>100###Antioxidant

###N4###19.5251###16.0852###13.1298###6.39###6.1###>100###Antioxidant

###N5###22.8682###11.4825###15.2131###6.68###5.08###>100###Antioxidant

DPPH Free Radical Scavenging Assay

DPPH is a stable free radical and antioxidants react with it by donating electron or hydrogen thus neutralizing it to diphenyl picrylhydrazine. [30] Reduction of DPPH radical by an antioxidant can be determined by the decrease in absorbance at 517 nm spectrophotometrically. The test compounds were subjected to DPPH assay to determine their antioxidant or prooxidant behavior.

All the compounds were tested at three concentrations 1000 ppm, 100 ppm and 10 ppm and tests were performed in triplicate. Data in Table-7 indicates mean percentage scavenging at all concentrations and IC25 values. Precursors Z1, Z4 and Z5 had pro-oxidant behavior as indicated by negative values of percentage scavenging.

Their absorbance values were found >negative control. In terms of color change these compounds enhanced the violet color of DPPH in reaction mixture by contributing free radicals. Two compounds Z4 and N3 behaved differently at different concentrations. While other compounds (Z2, Z3, AN, N1, N4 and N5) showed antioxidant behaviour at all concentrations. In our study the compound Z1 with a -nitro group at position 3 in benzene ring has pro-oxidant behavior. But, another study [31] reported contradictory behavior of -nitro at the same position on benzyl ring. On the other hand net substitution effect resulted in poor inhibition of lipid pro-oxidation observed when -nitro group was substituted at position 2. So, these results confirm that -nitro group is biologically significant but its position plays pivotal role in determining behavior of the compound.

DNA Damage Protection Assay

Active oxygen species such as superoxide anion radical (O-3), hydrogen peroxide (H2O2), and hydroxyl radical (-OH) can damage almost all cell components, including DNA, membranes, and proteins. Reduction of H2O2 by reduced transition metals results in the formation of -OH and related oxidants via the Fenton reaction. Organic compounds were subjected to H2O2 induced DNA damage assay as a test of DNA damage protection activity of these test compounds.

All the compounds were tested at concentrations of 1000 ppm, 100 ppm and 10 ppm. Effect was then compared with four controls P (untreated plasmid), F (plasmid treated with FeSO4), H (plasmid treated with H2O2) and X (plasmid treated with both FeSO4 and H2O2). Untreated, FeSO4 and H2O2 treated plasmids appeared predominantly as supercolied DNA as indicated by thickness of bands while control X indicates totally damaged DNA. Commonly, pBR322 plasmid DNA exists as supercoiled form in water solution. When nuclease or chemicals attack the pBR322, it could cut supercoiled form to be nicked form (or open circular form, relaxed circular form). Further attacks could cut the nicked DNA to be linear form. The three different forms of DNA show different migration speed in gel electrophoresis; therefore, it can be characterized using ethidium bromide stain.

Organic Schiff bases and their precursors showed protecting behaviour against DNA damage (Fig. 3-6). Furtnermore, the protecting effect was found concentration dependent. At highest concentration DNA was found in undamaged supercoiled form. All the Schiff bases and their precursors protected DNA at all three tested concentrations. Aniline (AN) was unable to protect DNA at lowest test concentration. Effect is visible as streak and less distinct bands of coiled and supercoiled plasmid. [31-33].

Conclusion

Schiff bases (N1-N5) comprising various substitutes were synthesized and characterized successfully. The data for single crystal X-ray analysis showed that the geometric parameters lie within the usual range for N3 and N4. The calculated bond distances, angles and vibrational IR frequencies showed excellent agreement with the experimental data. The N5 structure predicted theoretically by DFT is very similar to N3. Schiff bases and their precursors did not show any significant antibacterial activity. Anifungal potential of Schiff bases was found lower than their aldehydic precursors. All compounds exhibited significant cytotoxic activity and protecting behavior against DNA damage. Antioxidant assays showed variable behaviour of precursors and derived Schiff bases.

Supplementary Material

Supplementary data of the crystal structure and refinement, like full bond lengths, bond angles dihedral angles, and anisotropic displacement parameters, have been deposited with Cambridge crystallographic data centre, CCDC No. 776069 (N3), 776068 (N4). These data can be obtained, free of charge, using the link: www.ccdc.cam.ac.uk or from the CCDC, 12 Union Road Cambridge CB2 1EZ, UK, Fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk.

Acknowledgements

The authors are grateful to the Department of Chemistry and Dep ar tment o f B iochemistr y, Quaid-I-Azam University, Islamabad, Pakistan for providing laboratory and analytical facility. We also like to thank Prof. Dr. Rumana Qureshi for providing us the computational software facility.

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Author:Zaheer, Muhammad; Akhterm, Zareen; Gul, Asghari; Tauseef, Misbah; Mirza, Bushra; Bolte, Michael; Fat
Publication:Journal of the Chemical Society of Pakistan
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Date:Aug 31, 2016
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