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Design, synthesis, antimicrobial and antioxidant activity of 3-formyl chromone hydrazone and their metal (II) complexes
Accepted Manuscript Research paper Design, synthesis, antimicrobial and antioxidant activity of 3-formyl chromone hydrazone and their metal (II) complexes Jessica Elizabeth Philip, Shanty Angamaly Antony, Sneha Jose Eeettinilkunnathil, M.R. Prathapachandra Kurup, Mohanan Puzhavoorparambil Velayudhan PII: DOI: Reference:
S0020-1693(17)30649-7 http://dx.doi.org/10.1016/j.ica.2017.09.006 ICA 17862
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
13 June 2017 1 September 2017 2 September 2017
Please cite this article as: J.E. Philip, S.A. Antony, S.J. Eeettinilkunnathil, M.R.P. Kurup, M.P. Velayudhan, Design, synthesis, antimicrobial and antioxidant activity of 3-formyl chromone hydrazone and their metal (II) complexes, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.09.006
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Design, synthesis, antimicrobial and antioxidant activity of 3-formyl chromone hydrazone and their metal (II) complexes Jessica Elizabeth Philip, Shanty Angamaly Antony, Sneha Jose Eeettinilkunnathil, M. R. Prathapachandra Kurup and Mohanan Puzhavoorparambil Velayudhan Department of Applied Chemistry, Cochin University of Science and Technology, Kochi-22 E-mail: [email protected], Corresponding author Mohanan P. V., Ph.D. Department of Applied Chemistry, Cochin University of Science and Technology, Cochin-682022, Kerala, India. Tel: +91 9447184874 Email: [email protected],
Abstract: In search of a new antimicrobial and antioxidant with improved potency, we designed and synthesized a pair of chromone hydrazones and their transition metal complexes. The characterization and elucidation of the structure of the prepared compounds were performed by elemental analysis, IR, electronic, EPR and thermo gravimetric analyzes, as well as conductivity and magnetic susceptibility measurements. The EPR spin Hamiltonian parameters of copper complexes were calculated. The spectroscopic data showed that the ligands act as a monobasic tridentate. The coordination sites with the metal (II) ion are pyrone oxygen, azomethine nitrogen and hydrazonic oxygen. Hydrazones and their metal complexes have shown antimicrobial activity against Gram-positive bacteria (S. aureus and B. subtilis); Gram-negative bacteria (P. aeruginosa and E. coli) and fungus (C. albicans) and also the synthesized ligands were tested for their antioxidant activity in vitro by DPPH scanning and the ABTS radical method. The substitution requirements for favorable antioxidant activity were investigated. Compounds containing a phenolic hydroxyl group at the imine (azomethine) position as well as an additional electron donor group exhibited lower IC50 values and the result shows that all hydrazones have a significant antioxidant activity compared to the standard antioxidants Trolox.
1
Keywords: 3-formyl chromone, tridentate ligands, EPR spectra, antioxidant activity, antimicrobial activity. 1. Introduction The current escalation of knowledge of free radicals and reactive oxygen species (ROS) in biology produces a medical revolt that promises a fresh era of health and disease management [1]. It is ironic that oxygen, a crucial element for life, in certain situations has deleterious effects on humans [2-5]. They consist of a superoxide anion radical (O2-•), a peroxy radical (RO2•), an alkoxy radical (RO•), a hydrogen peroxide (H2O2) and singlet oxygen (oxygen molecule in the electronically excited state) [6]. The hazardous effects of ROS include oxidative damage to biological molecules such as lipids, proteins, nucleic acids and sugars and also affect the balance between ROS production, cellular defense mechanisms are transferred to more oxidative conditions (the state is called oxidative stress). It has been established that oxidative stress is implicated in a wide range of diseases, such as inflammation, neurodegenerative disorders (Alzheimer's disease, Parkinson's disease), atherosclerosis, diabetes and cancer [7-13]. An antioxidant is a stable molecule appropriate to give an electron to a free radical rampaging and neutralize it, thereby dropping its capability to damage [14]. These antioxidants sustain or inhibit cell damage primarily through their free radical scavenging property [15]. These low molecular weight antioxidants can safely interact with free radicals and terminate the chain reaction before the crucial molecules are damaged. Some of these antioxidants, including glutathione, ubiquinol and uric acid, occur during normal metabolism in the body [16-17].
2
The Chromones are oxygen-containing heterocyclic compounds having a benzoannelated γ-pyrone ring (4H-chromen-4-one, 4H-1-benzopyran-4-one) as the parent compound, which is a common and integral constituent of a variety of medicinal agents [6,18-19]. These compounds show the variety of pharmacological activities and the change in their structure offer a high degree of diversity that has been found useful for the search for novel therapeutic agents [6,20-21]. Several natural and synthetic benzopyrone derivatives have been reported to exert remarkably antimicrobial, anti-tubercular and antifungal activity [22-24]. The development of new defensive and potentially beneficial agents based on chromone is an important research point in medicinal chemistry, with the core of chromone being a key element in the pharmacophores of many biologically active molecules with various medicinal applications [25]. Taking into account the biological importance and remarkable structural behavior of these compounds, in this paper we wish to report some of our findings in the preparation of a pair of hydrazone that were obtained by the condensation of the respective 3-formyl chromone with different aromatic hydrazides. The synthesized compounds were subjected to in-vitro antioxidant screening against DPPH and ABTS radical and their antimicrobial activities were screened against selected bacteria and fungi. 2. Experimental 2.1.
Materials
Reagents used for the synthesis are 3-formyl chromone, 4-hydroxy benzhydrazide, benzhydrazide, (Sigma Aldrich). All other reagents were of analytical grade and the Organic solvents (ethanol, absolute ethanol, methanol, diethylether, dimethylformamide (DMF) and dimethylsulfoxide (DMSO)) employed were either 99% purity or purified by known laboratory procedures[26]. 2.2. Physical measurements 3
Elemental analyses were performed on a Vario EL III elemental analyzer. Melting point of the ligands and their complexes were measured. IR spectra (KBr cm-1) were recorded on a JASCO FT- IR spectrometer in the range of 4000-400 cm-1 .1H/13C NMR spectra were recorded on a Bruker Advance III 400MHz FT-NMR spectrometer using CHCl3/DMSO-d 6 as solvent and TMS as an internal standard (chemical shifts in δ ppm). Electronic spectra were recorded at room temperature on a Thermo Scientific™ Evolution 201 UV-Visible spectrophotometer. Thermo gravimetric Analysis (TG/DTG) were carried out from room temperature up to 800 oC at a heating rate of 10 oC/min on Perkin Elmer Diamond TG/DTA analyzer. The molar conductivities of the complexes in DMF solutions (10-3 M) at room temperature were measured using a Systronic model 303 direct reading conductivity meter. Mass spectra of the hydrazones were recorded by direct injection on WATERS 3100 Mass Detector using Electron Spray Ionization (ESI) technique designed for routine HPLC-MS analyses. Magnetic susceptibility measurements of the complexes were carried out on a Vibrating Sample Magnetometer using Hg [Co(SCN)4] as a calibrant. The EPR spectra of the complexes in the solid state at 298 K and in DMF at 77 K were recorded on a JEOL, JapanModel: JES-FA200 ESR Spectrometer using TCNE as the standard, with 8.75-9.65 GHz microwave frequency. 2.3. Synthesis of chromone hydrazones The synthesis of chromone hydrazones are shown in scheme 1. Aromatic hydrazides (1.00 mmol), 3-formylchromone (1.00 mmol), and a few drops of acetic acid were dissolved in 25 mL of ethanol, and the mixture was stirred and refluxed for 6 h. The precipitate was collected, washed with ethanol, and dried in vacuo. All compounds were prepared similarly shown in scheme 1 and characterized as below.
4
2.3.1. N'-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide - Yield 78% as yellow powder; m.p. 171-173oC; IR (KBr, cm−1): 3206 (N-H), 1680 (C=O, pyrone), 1655 (C=O, hydrazonic), 1571 (C=N), 1238(C–O), 1035 (N–N); 1H NMR (DMSO-d6, δ, ppm): 11.72 (s, 1H, OH); 8.78 (s, 1H, CH=N); 8.59 (s, 1H, oxo ring); 7.92- 6.84 ( m, chromone ring and phenyl ring). 13C NMR (DMSO-d6, δ, ppm): 177.5(C=O), 163.77 (-N-N=CH-), 162.70, 157.20, and 152.80 (non protonated carbon), ESIMS [M+1]: 293.1. Anal. Calcd for C17H12N2O3 (292.08): C, 68.45; H, 4.28; N, 9.39; Found: C, 68.40; H, 4.19; N, 9.36%. 2.3.2. 4-hydroxy-N'-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide -Yield 76% as yellow powder; m.p. 169–170 oC; IR (KBr, cm−1): 3400-3100 (broad O-H & N-H), 1680 (C=O, pyrone), 1651 (C=O, hydrazonic), 1580 (C=N), 1232 (C–O), 1028 (N–N); 1H NMR (CHCl3, δ, ppm): 11.73 (s, 1H, OH); 8.60 (s, 1H, CH=N); 8.8 (s, 1H, oxo ring);8.15- 6.87 ( m, chromone ring and phenyl ring); 13C NMR (CHCl3, δ, ppm): 176.00 (C=O),163.71 (-NN=CH-),162.72, 160.83, 157.21and 152.83 (non protonated carbon), ESIMS [M+1]: 309.2. Anal. Calcd for C17H12N2O4 (308.18): C, 66.25; H, 3.92; N, 9.09; Found: C, 66.30; H, 3.88; N, 9.13%.
2.4. Synthesis of complexes About 1 mmol of metal (II) acetate dissolved in 30mL methanol was added gradually to 1 mmol of the ligand, dissolved in DMF. The reaction mixture was heated under reflux for 8h. The resulting precipitates were filtered, washed with methanol and hexane then diethyl ether 5
and finally air-dried. The complexes were kept in desiccators over anhydrous calcium chloride. The yield was noted. All complexes were prepared similarly. Table 1 Analytical and physical data of the chromone hydrazones (HL1 & HL2 ) and their metal (II) complexes.
No
Reaction
Complex M.F. [F.W.]
Colour
Yield %
M.P. C
1
HL1
C17 H12N2O3 [292.08]
Yellow
78
2
HL1+ Ni(OAc)2
C19 H20N2NiO8 [463.06]
Brown
62
>250
3
HL1+ Cu(OAc)2
C19 H16N2CuO6 [431.88]
Green
65
>250
4
HL1+ Zn(OAc)2
C19 H19N2ZnO7.5 [459.04]
Yellow
67
>250
HL2
C18 H14N2O4 [322.31]
Yellow
76
C19 H20N2NiO9 [479.06]
Brown
72
>250
C19 H18N2CuO8 [465.90]
Green
70
>250
C19 H14N2ZnO6 [431.73]
Yellow
66
>250
5 6 7 8
HL2+ Ni(OAc)2 HL2+ Cu(OAc)2 HL2+ Zn(OAc)2
Elemental Analysis, % Found (calac.)
o
C
H
N
68.40 (68.45) 49.33 (49.28)
4.19 (4.28) 4.47 (4.35)
9.36 (9.39) 6.15 (6.05)
M
53.03 (52.84)
3.38 (3.73)
6.40 (6.49)
14.65 (14.71)
49.47 (49.53) 66.30 (66.23) 47.58 (47.64) 48.89 (48.98) 52.79 (52.86)
4.06 (4.16) 3.88 (3.92) 4.29 (4.21) 3.93 (3.89) 3.20 (3.27)
6.13 (6.08) 9.13 (9.09) 5.72 (5.85) 6.12 (6.01) 6.41 (6.49)
14.11 (14.20)
12.53 (12.68)
12.07 (12.25) 13.51 (13.64) 15.09 (15.15)
2.5.Antioxidant activity determination 2.5.1. DPPH radical scavenging assay The DPPH (2-2'-diphenyl-1-picrylhydrazyl) radical scavenging action of the compounds was evaluated according to the method of Blios.45. The DPPH radical is a stable free radical having λmax at 517 nm and showed characteristic deep violet colour. The loss of violet colour of the DPPH solution by the abstraction of hydrogen from the substrate serves as a marker of the scavenging of DPPH radicals. A variable concentration of the test compounds was added to a solution and the final volume was made up to 3 mL with solvent. The solution was incubated at 37 °C for 20 min. The decrease in absorbance of DPPH was measured at 517 nm. The same experiment carried out without the test compounds serve as a control. Percentage inhibition can be calculated by using the equation.
% ℎ
=
− × 100 6
A graph was plotted with concentration (µM) against % inhibition. The concentration at which there is 50% fall in absorbance of DPPH solution was determined from the graph (IC50). The IC50 value in µM was calculated from the above obtained concentration value. All experiments were carried out in triplicate. The 6-hydroxyl-2, 5, 7, 8-tetramethylchroman-2carboxylic acid (Trolox) was used as antioxidant reference. 2.5.2. (ABTS˙+) radical cation assay It is based on the ability of antiradical molecules to quench the ABTS radical. It is a blue green coloured chromophore shows characteristic absorption at 734 nm. By the addition of antioxidants to the radical cation reduces it to ABTS and loss its characteristic colour (decolourization). In this method, an antioxidant was added to a pre-formed ABTS radical solution, and after 20 min period, the remaining ABTS˙+ is quantified spectrophotometrically at 734 nm. The ABTS˙+ was produced by reacting 7 mM ABTS in H2O with 2.45 mM potassium persulfate (K2S2O8), stored in the dark at room temperature for 24 hrs before use (the radical was stable for more than two days under these conditions). The ABTS˙+ solution was further diluted in methanol until the initial absorbance value of 0.80 ± 0.02 at 734 nm at 30oC. Then, in a 3 mL solution of ABTS˙+, was added a methanolic solution of the compound under study so that their final concentrations vary between 0 and 15 µM. In each case, a 2080% decrease in the initial absorbance of the reaction solution was achieved. The absorbance was recorded at 0, 1, 4 and 6 min. The scavenging capacity of test compounds was calculated using the following equation: ˙" #$ $ %% &%' =
− × 100
Where Acontrol is absorbance of a control lacking any radical scavenger and Asample is absorbance of the remaining ABTS˙⁺ in the presence of scavenger. Graphs of antioxidant concentration versus percent absorbance reduction were then plotted. The concentration of
7
Trolox giving the same percentage reduction of absorbance at 734 nm as the 1 mM antioxidant solution was calculated from the three point graphs. The results were thus expressed as Trolox equivalent antioxidant capacity (TEAC) values. For each hydrazone and each concentration, measurements were performed in triplicate. Absorbance values were corrected for radical decay using blank solutions. 2.6. Antimicrobial activity The synthesized chromone hydrazones and metal complexes were tested for their in vitro antibacterial and antifungal activity against the sensitive organisms Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6635) as Gram positive bacteria, Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) as Gram negative bacteria and Candida albicans (ATCC 10231) as fungus strain using the disc diffusion method [Kirby-Bauer Test ][27](for the qualitative determination) and the serial dilutions in liquid broth method for determination of MIC values. The antibiotic chloramphenicol was used as reference in the case of bacteria, and nystatin in the case of fungi. The chromone hydrazones and metal complexes were dissolved in DMSO. The test was carried on medium potato dextrose agars (PDA) which have a mixture of 200 g potatoes, 6 g dextrose and 15 g agar [28] .Uniform dimension filter paper discs were impregnated with equal volume (10 µL) from the exact concentration of dissolved compounds and carefully located on the inoculated agar surface. After incubation for 36 h at 27 oC in the case of bacteria and for 48 h at 24 oC in the case of fungi, inhibition of the organisms was measured and used to calculate mean of inhibition zones and all experiments were carried out in parallel sets of triplicate. Data were analyzed by the analysis of variance (ANOVA) using the Origin pro 8.5 statistical software and the mean differences were separated using Tukey's studentized test at the 1% level of probability. Time kill kinetic studies were conducted for the compounds against one Gram positive, one Gram negative bacteria and against fungus. 8
In the experiment, an overnight culture of the isolates was used up. 1mL of 106 CFU mL-1 of each culture was inoculated in sterilized nutrient broth media containing 25 mg mL-1 of the compounds. The experiment was conducted for 13 h in a shaker at 30°C. Likewise, control was prepared for each microorganism without having the test compound. The CFU count was taken at regular 1 h interval. For that, 1mL of each culture was spread on nutrient agar plates from 0 h to 13 h and each plate was incubated for 24 h at 30°C. The CFU (colony forming unit) was calculated and plotted in graph. 2.6.1. Minimum Inhibitory Concentration (MIC) All the compounds showed substantial inhibition of both bacteria and fungi, were tested further for the Minimum Inhibitory Concentration (MIC). Standard pathogenic bacterial strains S. aureus, B. subtilis, P. aeruginosa, E. coli and C. albicans as fungus. The resazurin reduction method was used for finding out the MIC in 96 well microplates [29]. All the compounds were dissolved in DMSO having a final stock concentration of 10 mg mL-1. Fifty microlitres of this stock solution was serially diluted to eight times and 50 μL of each serially diluted compound was added to microplate wells. All the microbial cultures were grown in nutrient agar to reach 0.5 McFarland concentrations and 50 mL of this culture was added to each well. Microplate contents were mixed well and incubated at room temperature for 12 hours. After 12 h of incubation, 10 μL of mixture from each well was spread on the agar plate and checked for the Colony Forming Units (CFU). Further 30 μL of 0.1% resazurin solution was added to each well and incubated up to another 24 hours. Microplate well contents were observed in the change in colour from blue to pink. Those wells that have microbes growing will change the blue resazurin into pink color. The well, which remains blue after 24 hours of incubation indicates there are no microorganisms survived in the well, the minimum concentration where no microbial growth found are considered as MIC value.
9
3. Result and Discussion 3.1.Characterization of ligands and complexes The structure of the synthesized chromone hydrazones and their metal complexes were elucidated by elemental analysis, electronic, FTIR, 1H/13C NMR and ESI-MS spectra. The results of elemental analysis (C, H, N) with molecular formula (Table 1) are good agreement with those calculated for the suggested formula and the melting points (Table 1) are sharp signifying the purity of the prepared chromone hydrazones. The metal (II) complexes were isolated pure in very good yields and they are of various colours. The ligands and the complexes are very much soluble in DMF and DMSO, moderately soluble in methanol and acetonitrile and practically insoluble in water, carbon tetrachloride, chloroform and benzene. They are very air stable solids at room temperature without decomposition for a long time. Moreover, the structure of the ligands (HL1 & HL2 ) was deduced from mass spectral data which showed the [M+1] peak at 293.1 & 309.2 a.m.u. respectively, confirming its formula weight (F.W. 292.08 and 308.18 respectively; Figs. C: 1-2 (supplementary data)) it supported the suggested structure of the ligands. 3.1.1. FTIR spectra The characteristic IR bands of the chromone hydrazones (Table 3) give important information about the various functional groups present in it. Strong bands due to the ν (N-H) and ν (C=O) modes at 3220 and 1641 cm-1 are observed in the spectrum of HL1 which suggests that the hydrazone exists in the amido form in the solid state [30]. A band at 1678 cm-1 is due to the presence of (C=O, pyrone). A prominent band at 1587 cm-1 due to azomethine ν (C=N) formation of ligand HL1. It further confirms the ν (C–O), ν (N–N) bands at 1238, 1035 cm-1 respectively. Similarly in the case of ligand HL2 a strong broad band around 3394 cm-1 is due to ν (N-H & O-H). A strong band at 1650 cm-1 observed in the spectrum which suggests that the HL2 exists in the amido form in solid state. Band at 1675 cm-1 is due to the presence of 10
(C=O, pyrone). A prominent band at 1605 cm-1 due to azomethine ν (C=N) formation of ligand HL2. It further confirms the ν (C–O), ν (N–N) bands at 1235, 1048 cm-1 respectively. The comparison of the complexes with respective chromone hydrazone shown that all complexes had broad band in the range of 3690-3025 cm-1 assigned to ν (OH) of the coordinated water molecules linked with the complexes [31] and also the OH of the hydrazine moiety which are confirmed by elemental, AAS, and thermal analyses. All the metal (II) complexes displayed distinct sharp band in 1571-1531 cm-1 region corresponds to the characteristic -C=N- group stretching vibration indicating the coordination of azomethine nitrogen to metal (II) centre as observed in the literature [32]. This coordination of azomethine nitrogen is also supported by band at 1029-1013 cm-1 corresponding to ν (N-N) stretching vibration. On comparison with literature reports, ν(N-N) vibrations was found to be in higher frequencies in the complexes due to the increase in double bond character, offsetting the loss of electron density via donation to the metal[33]. The bands at 1580 cm-1 assigned to ν(C=N), in the chromone hydrazones were shifted to lower wave number region in all complexes. The band at 1655 cm-1 assigned to ν(C=O) hydrazonic in the free ligand was shifted to (1647-1598) cm-1, [34] which indicate that the azomethine nitrogen and hydrazonic (C=O) are in chelation. In complexes 1 & 4 the chelating bidentate acetate (CH3COO-) group was present due to the bands around 1424-1414 and 1337-1325cm-1. These two bands are due to νas (COO-) and νs (COO-), respectively. The separation of the two bands, ∆ ν = (νas- νs) = 88cm-1, is comparable to the values cited for the bidentate character of the acetate group ∆ ν = 75-90 cm-1[35]. On the other hand, complexes 2, 3, 5 & 6 showed new bands characteristic for νas (COO-) and νs (COO-) of acetate ion in the ranges 1549-1531 and 1413-1389cm-1. The higher difference between the two bands indicates the monodentate nature of the acetate group, in which one ‘O’ atom of acetate was coordinated to the metal center, while the second was hydrogen bonded to, a geminal H2O
11
[36]. The appearance of the non - ligand bands at 557-510 and 473-403 cm-1 were assigned to ν (M-O) and ν (M-N), respectively. Table 3 Characteristic IR spectral data of the ligand (HL1 & HL2) and its complexes. IR spectra (cm)-1 N o
Compounds
1
2
3
4
ν(OH, NH)
ν( C=O) pyrone
ν( C=O) hydrazonic
ν( C=N)
ν( C-O)
ν( N-N)
ν( M-O)
ν( M-N)
Mono/ Bidantate acetate
HL1
-, 3220
1678
1641
1587
1238
1035
-
-
-
1645
1617
1545
1220
1029
510
436
[Ni(L1)(OAc)(H2O)]
3666-2919
.2H2O
(broad)
[Cu(L1)(OAc)(H2 O)]
3445
1656
1605
1531
1228
1019
526
403
3618-3088
1641
1597
1560
1224
1027
533
460
1675
1650
1580
1232
1028
-
-
1639
1617
1571
1219
1015
520
449
3416-3025
1634
1598
1555
1229
1021
512
423
3630-2997
1654
1593
1550
1196
1013
557
473
[Zn(L1 )(OAc)] .2.5H2O
6
7
8
3394
HL2
(broad)
[Ni(L2)(OAc)(H2O)]
3690-3043
.2H2O
(broad)
[Cu(L2)(OAc) (H2O)].H2O
9
[Zn(L2 )(OAc)]
3.1.2.
1
νas 1431, νs 1328 νas 1531, νs 1323 νas 1529, νs 1397 -
νas 1451, νs 1364 νas 1511, νs 1374 νas 1536, νs 1388
HNMR spectra
Proton Nuclear Magnetic Resonance (1 H NMR) Spectroscopy is a powerful tool used for the determination of the structure of compounds. The 1H NMR spectra of the chromone hydrazones and Zn (II) (because of their diamagnetic nature) have been recorded with DMSO as solvent. 1H and 13C NMR spectral data (δ ppm) of the ligand relative to TMS (0 ppm) in DMSO-d 6, Tables 2; Figs. A: 1-2 & B: 1-2 (supplementary data), give further support of the suggested structure of the ligand. Table 2
1
H & 13C NMR Spectral data of ligands HL1 & HL2
1 HNMR δ ppm (DMSO)
Assignments
13CNMR δ ppm (DMSO)
Assignments
HL1 11.72 8.78 8.59 7.92 -6.84
(s,1H,OH) 177.5 (s,1H,CH=N-) 163.77 (s,1H,oxo ring) 162.70,157.20,152.80 (m, chromone & phenyl ring)
-C=O -N-N=CHNon protonated carbon
12
HL2 11.73 10.12 8.80
(s,1H,OH) (s,1H,OH) (s,1H,oxo ring)
8.60
(s,1H,CH=N-)
7.88-6.84
(m, chromone & phenyl ring)
176.00
C=O
163.06 162.72,160.83, 157.21,152.83
-N-N=CHNon protonated carbon
The comparison of the NMR spectra of the complexes with the spectra of ligands gave valuable information regarding the coordination mode of ligands during complexation. The 1H NMR spectrum obtained for complexes 3 and 6 was not good due to the poor solubility of the compound. In the spectra of the free hydrazones there are sharp singlets in the range of 11-12 ppm showing the existence of iminol form in solution. They also gave singlets in the range of 10-11 ppm with an area integral of one which is due to phenolic OH protons. Syntheses and characterization of Zn (II) complexes derived from ONO donor chromone hydrazones the intensity of these signals significantly decreases, which suggests that these protons are easily exchangeable and confirm the assignment. Peaks for aromatic protons were found in the region 6.8-8 ppm. Peaks are in the region 11-12 ppm corresponds to iminol protons found in the spectra of free hydrazones were absent in the spectra of complexes indicating the coordination of iminol oxygen to zinc. In the spectrum of complexes, a singlet with an integral area of three is found at 2.0-1.9 ppm and it is assigned to the methyl group present in acetate which is coordinated to zinc. Also a singlet with area integral one is appeared at 8.27 & 8.7 ppm may indicate the presence of -CH- proton. All other peaks observed in the spectra of free hydrazones are slightly shifted. The 1 H NMR spectra of complexes 3 and 6 are shown in the Figs. A: 3-4 (supplementary data). 3.1.3. Solution studies UV -Vis spectra and Molar conductivity measurements
13
The electronic spectral data of the ligand in DMF (Table 3) showed two bands λmax (DMF) nm (ε in 103 dm3 mol-1cm-1): 237 (13.7), 341 (8.6) for HL1 and 241(14.1), 347 (7.9) for HL2. The higher energy band may be assigned to π-π* transitions of the azomethine linkage and the aromatic rings. The medium energy band may be assigned to n-π* transitions of the C=O and C=N groups. The metal complexes in DMF solutions present absorption maxima attributable to the hydrazone ligand together with the absorptions, around 419-697 nm, due to ligand to metal charge transfer and d-d transitions of the metals in the complexes [37]. The bands in the range 340 and 241 nm in the electronic spectra of hydrazones due n→π* and to π→π* transitions suffered marginal shifts (350 and 252 nm) upon complexation. This may be due to the weakening of the C=O bond and the extension of conjugation upon complexation. The shift occurs also due to the coordination of the metal (II) ion are pyrone oxygen, azomethine nitrogen and hydrazonic oxygen. The electronic spectral data of the metal (II) complexes are given in Table 4. The molar conductance values of the synthesized Ni (II), Cu (II) and Zn (II) complexes in DMF (10-3) were measured at room temperature and the results were shown in Table 4. The values showed that all complexes have non-electrolytic nature [38]. Table 4 Electronic spectra, magnetic moments and molar conductivity data of the ligands (HL1&HL2) and its metal complexes.
No
Compounds
1
HL1
2
[Ni(L1 )(OAc)(H2O)].2H2O
3
[Cu(L1)(OAc)(H2 O)]
4
[Zn(L1)(OAc)].2.5H2O
6
HL2
Electronic spectral bandsa (nm) λmax a (nm)/(εmax L cm-1 mol-1 ) 237 (1.37) - π→π* 361 (8.6) - n→π* 260 (4.57) - π→π* 316 (4.42) - π→π* 366 (4.79) - n→π* 429 (2.45) - CT 589 – dd 254 (4.46) - π→π* 310(1.88) - π→π* 407(5.91) - n→π* 428 (6.03) - CT 642 (1.14) – dd 272 (4.77) - π→π* 358 (2.85) - n→π* 410 (2.52) - CT 241 (1.41) - π→π* 359 (7.9) - n→π*
µ eff. b B.M.
conductancea (Ω -1 cm2 mol-1)
-
-
3.45
12.5
1.65
9.4
D
8.6
-
-
14
7
[Ni(L2 )(OAc)(H2O)].2H2O
8
[Cu(L2)(OAc)(H2 O)].H2 O
9
[Zn(L2)(OAc)]
257 (3.83) - π→π* 316 (5.19) - π→π* 353 (6.12) - n→π* 611 - dd 257 (5.20) - π→π* 377 (5.70) - π→π* 437(5.31) - n→π* 462 (2.85) - CT 629 (1.24) - dd 260 (4.26) - π→π* 349 (3.30) - n→π* 430(1.12) - CT
3.32
15.4
1.72
13.7
D
9.5
a
Solutions in DMF (10-3 M), values of εmax are in parentheses and multiplied by 104 (L mol-1 cm-1). μ eff. is the magnetic moment of the complex. D - diamagnetic b
3.1.4. TG/DTG The thermal (thermo gravimetric) analysis was mainly used to confirmation of the associated water or solvent molecule to be in the coordination sphere or in the outer sphere of the complex [39] and information on their properties, nature of intermediate and final products of thermal decomposition can be obtained. From TGA curves, the weight loss was calculated for the different steps and compared with theoretically calculated weight for the suggested formulae based on the results obtained from elemental analyses as well as molar conductance and AAS measurements. TGA indicated the formation of metal oxide as the end product from which the metal content could be calculated and compared with that obtained from analytical determination. Thermo grams of metal complexes indicate decompositions around 100 oC is due to hydrated water (except complex 2 & 6) and also (complex 1, 2, 4 & 5) around 200 °C is due to coordinated water molecules. The results of thermal analysis (Table - 5, Figs. E: 1-6 (supplementary data)) of these complexes are in agreement with elemental analyses. Table 5 TG/DTG of metal (II) complexes
Complexes
Temperature range(oC)
Mass loss (%) Obsd. (Cald.)
Decomposition product
[Ni(L1)(OAc)(H2 O)].2H2 O
80-105 297-354
7.62 (7.77) 17.47 (18.03)
Loss of 2H2 O (hyd) Loss of H2 O (coordinated) & acetate
15
[Cu(L1 )(OAc)(H2O)]
[Zn(L1 )(OAc)].2.5H2 O
[Ni(L2)(OAc)(H2 O)].2H2 O
[Cu(L2 )(OAc)(H2O)].H2O
[Zn(L2 )(OAc)]
229 258-306 307-369 50-120 450-477 50-130 173-248 277-368 60-114 200-248 290-429 150-280 419-484
4.55 (4.16) 13.93 (14.25) 22.35(22.07) 9.96 (9.80) 14.44(14.89) 7.16 (7.51) 4.33 (4.06) 22.14(22.05) 4.05 (3.86) 4.26 (4.01) 32.33 (33.08) 43.66 (43.01) 6.58 (6.97)
Loss of H2 O (coordinated) Acetate C6 H6 Loss of 2.5 H2 O (hyd) C2 H4O2 Loss of 2H2 O (hyd) Loss of H2 O (coordinated) C6 H6O Loss of H2O (hyd) Loss of H2 O (coordinated) C7 H9 NO2 C10 H8N2O2 CH4
3.1.5. Magnetic measurements Due to the Jahn–Teller distortion of Cu II-ion (d9) it lower the symmetry, complete interpretations of the spectra and magnetic properties are somewhat difficult [40]. The magnetic moment values of the copper complexes are 1.68, 1.72 B.M., which is consistent with the presence of one unpaired electron. In nickel complexes the magnetic moment values are 3.45 and 3.32 B.M; indicate that complexes follows octahedral geometry. 3.1.6. EPR studies The EPR spectra of the copper (II) complexes in DMF at 77 K and in a polycrystalline state at 298 K were recorded in the X-band, using 100-kHz modulation frequency and 9.5 GHz microwave frequency. All the EPR spectra are simulated using Easy Spin 5.0.20 package[41] and the experimental (red) and simulated (blue) best fits are included. EPR parameters of the copper (II) complexes are presented in Table 6 and Figs. 1- 4. Copper (II) complex (2 & 5) displayed Figs. 1- 4 axial spectra in the polycrystalline state at 298 K with gǁ and g⊥ values. The variation in the gǁ and g⊥ values indicates that in the solid state, the geometry of the compounds is affected by the nature of coordinating ligands. Fig 1: EPR spectrum of Complex 2 at 298 K; Fig 2: EPR spectrum of Complex 2 in DMF at 77K; Fig 3: EPR spectrum of Complex 5 at 298 K; Fig 4: EPR spectrum of Complex 5 in DMF at 77K
16
1 2
3
4
For complex (2 & 5), displayed axial features; since it is magnetically concentrated, hyperfine splitting was not clear in both parallel and perpendicular regions. The geometric parameter G, represents the exchange interaction between copper canters in the polycrystalline compound and is calculated for each complexes using the equation, G = (gǁ 2.0023)/(g⊥ - 2.0023) for axial spectra [42]. If G > 4.4, exchange interaction is negligible and if it less than 4.4, considerable exchange interaction is indicated in the solid complex. The calculated G values are greater than 4.4 for complexes which indicate that there are no copper-copper exchange interaction is present in the polycrystalline state of the complexes. As gǁ > g⊥ > 2.0023, a square pyramidal geometry consistent with dx2-y2 ground state can be assigned to the copper (II) complexes (2 & 5), thereby ruling out the possibility of a trigonal bipyramidal structure (where gǁ < g⊥). At 77 K in DMF solution, complexes 2 & 5 (Figs.1-4) exhibits axial spectrum. The EPR parameters gǁ and g⊥ incorporated with the energies of d-d transition were used to evaluate the bonding parameters. The following simplified expression were used to calculate the bonding parameters K2ǁ = (gǁ - 2.0023) Ed-d/8λ0 K2⊥ = (g⊥ - 2.0023) Ed -d/2λ0 17
Where Kǁ and K⊥ are orbital reduction factors and λ0 represents the one electron spin orbit coupling constant which equals -828 cm-1. Hathaway [42] has pointed out that for pure sigma bonding Kǁ ≈ K⊥ ≈ 0.77, for in plane π-bonding Kǁ < K⊥ and for out-of-plane π-bonding, K⊥< Kǁ. For complexes (2 & 5), it is observed Kǁ < K⊥ indicates significant in-plane π-bonding is present in the complex. The gǁ values also provide information regarding the nature of metal-ligand bond [43]. The gǁ value is normally 2.3 or larger for ionic and less than 2.3 for covalent metalligand bonds. The gǁ values obtained for complexes (2) and (5) indicate a significant degree of covalency in the metal-ligand bonds [44]. Table 6 Spin Hamiltonian and bonding parameters of copper (II) chromone hydrazones Polycrystalline state DMF solution (77 K) (298 K)
Complex
gǁ
g⊥
gav
G
gǁ
g⊥
Kǁ
K⊥
[Cu(L1 )(OAc)(H2O)] (2)
2.190
2.066
2.1073
2.9466
2.190
2.066
0.6609
0.7700
[Cu(L2 )(OAc)(H2O)].H2O(2)
2.19
2.059
2.1027
3.3104
2.20
2.057
0.6829
0.7185
Fig 5: Proposed structure of complex 1, 2, 3, 4, 5 & 6
O O
H2O
O Ni
O O
N O
.2 H2O N
O
N
Cu O
N
.H2O
O O
18
O
O
O Zn O
N
N
O
. 2.5 H 2O H2O
O
Ni O
N O
O
OH
O
O
O
H2O
O
N
Cu O O
. 2 H2O
N
.H2O
O
N
N
Zn O
O OH
N
O O
OH
3.2. Antioxidant activity The antioxidant activity of chromones and their derivatives has attracted increasing interests and been extensively investigated, mainly in- vitro system. It has been reported that over production of free radicals may induce some oxidative damages to bio molecules such as carbohydrates, proteins, lipids and DNA, thus accelerating aging, cancer, cardiovascular diseases, and inflammation and so on. 3.2.1. Scavenging radical activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical In this method the decrease in absorbance (stable free radical DPPH) at 517 nm, in presence of chromone hydrazones were tested. All tested chromone hydrazones shows UV absorbance between 230 and 350 nm ranges. These values do not interfere with the kinetic measurement of DPPH absorbance at 517 nm. The absorbance of DPPH radical decrease in presence of antioxidants is due to hydrogen transfer from the antioxidant, thus forming the DPPH-H stable compound. Methanol solutions of DPPH radical at different concentrations of
19
hydrazones were checked over a 20 min period. The chromone hydrazones were studied to investigate their antiradical ability by comparing their scavenging effects with standard Trolox. We find that the inhibitory effect of the compounds tested on DPPH radical is concentration dependent and the suppression ratio increases with increasing sample concentrations in the range tested. The IC50 values of the chromone hydrazones were determined (HL1 & HL2 = 3.71 & 1.54 mg/mL respectively) and standard Trolox is 6.25mg/mL. Compounds having a hydroxyl group at para position show the lowest IC50 values. A lower IC50 value means higher DPPH free radical scavenging activity. Hydrogen atom abstraction from phenolic ring under the examined conditions is quite easier for compounds HL2 as shown in Scheme 2. As a consequence the reaction of DPPH radicals may create nitrogen radical on the antioxidant considered. This is in good agreement with Dolenc et al. [24] concerning the auto oxidation process of hydrazones. Studying structurally different hydrazones, we proposed for those compounds having at least one hydrogen atom on nitrogen, the formation of nitrogen cantered radical (hydrazonyl radical) which is stabilized through conjugation forming delocalised carbon cantered radicals. The in vitro antioxidant activities of synthesized chromone hydrazones may be due to the relative ability for the N-H hydrogen atom abstraction from the compound and also substitutions on aromatic or hetero aromatic hydrazides.
Scheme 2: Proposed reaction of DPPH with p-hydroxybenzhydrazide derivative
20
3.3.2. Scavenging radical activity on 2, 2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS˙+) radical cation The antioxidant activity of chromone hydrazones were evaluated by using the Trolox equivalent antioxidant capacity values (TEAC). In this method we measure the relative ability of antioxidant compound to scavenge the radical cation [2, 2′-azinobis (3ethylbenzthiazoline-6-sulfonic acid) (ABTS˙+)] and compared with a standard amount of synthetic antioxidant Trolox [6-hydroxy- 2, 5, 7, 8-tetramethylchroman-2-carboxylic acid], it’s a vitamin E analogue. The TEAC value means the concentration of standard Trolox with the same antioxidant capacity as 1 mM concentration of the antioxidant compounds here chromone hydrazones under investigation. This method based on the generation and detection of coloured long-lived radical cation ABTS is chemically oxidized by potassium persulphate to give the relatively stable (over 24 h) ABTS radical. The concentration of ABTS˙+ is measured at 734 nm [25]. Thus the antioxidant-induced reduction of the ABTS˙+ concentration is directly related to the antioxidant capacity of the compound being tested. ABTS radicals are involved in an electron transfer process. All screening compounds exhibited at various levels radical cation scavenging activity. There were no differences between the TEAC values for the chromone hydrazones at 1 min, 4 min and 6 min (data not shown). As seen in Fig. 6, compounds HL2 were the most effective ABTS˙+ radical scavengers depending on concentration range (0-15 μM). ABTS˙+ absorbance decreases significantly with all chromone hydrazone concentrations, the effect being less pronounced for hydrazone HL1 at concentrations above 12 µM. The scavenging effects on ABTS˙+ decreased in the order Trolox>HL1>HL2. Table 7 summarised the results obtained in terms of Trolox equivalents capacity. All chromone hydrazones possess TEAC values equivalent or inferior values. This might be due to a better
21
transfer of radical and better resonance stabilisation of the radical species for these compounds (Table 7). Fig.6 ABTS radical scavenging activity of chromone hydrazones compared with standard Trolox. Values are average of 3 independent measurements of each compound.
HL1 HL2
1.0 0.9 0.8
Absorbance
0.7 0.6 0.5 0.4 0.3 0.2 0.1
0
2
4
6
8
10
12
14
16
Conc mg/mL
Table 7: Antioxidant activity of chromone hydrazones by ABTS radical. Values are average of 3 independent measurements of each compound.
Compounds
TEAC
3a
3.23
3b
1.48
Trolox
3.09
3.3.Antimicrobial activity The antibacterial activity of all compounds against Mycobacterium were plotted in a bar diagram and represented in Fig. 7. Fig. 7 represents the zone of inhibition for bacteria and fungi at three different concentrations (1, 0.5 and 0.25 mg mL-1) with a standard deviation of the triplicate values (Table 8). Antifungal zone of inhibition showed by the synthetic compounds represented in Fig. 7. Figure 8 and table 9 represent the MIC of the compounds synthesized against the microorganisms. Chloramphenicol was used as a reference in the
22
case of bacteria and nystatin in the case of fungi were tested at a concentration of 10 mg mL-1. Most of the compounds showed good microbial inhibition for all tested microorganisms such as S. aureus and B. subtilis, two Gram negative bacteria P. aeruginosa and E. coli and the pathogenic fungus C. albicans in the disc diffusion method. Complexes 2 and 5 showed a good zone against S. aureus, in the case of B. subtilis complex 2, 3, 5 and 6; for P. aeruginosa complex 2, 5 and 6; for E. coli the complex 5 showed good inhibition zone diameter. All the other compounds with an inhibition zone of up to 35 mm. But in the case of C. albicans complex 5 shows good inhibition zone diameter. The ratio of structural activity of the synthesized compounds was explained based on the microorganisms tested. Among all compounds, complex 5 has shown that the best inhibition against all tested microorganisms may be due to the copper (II) complex. The presence of the p-hydroxyl group has increased the activity of complex 5 against all microorganisms tested. Complex 2 has shown a significant inhibition against Gram-positive bacteria, which is due to the presence of copper (II). The compound HL2 showed greater inhibition of bacteria and less for fungi indicating that the compound may be a better antibacterial than antifungal activity. This indicates that the synthesized compounds are biologically active as antimicrobials. B. subtilis was inhibited to the maximum extent by most compounds. Fig 7: Comparison of zone diameters of compounds
23
Table 8: Inhibitory activity of the compounds expressed as zone of inhibition (mm)a Mean of zone diameter, nearest wholea (mm) Gram-positive bacteria
Gram-negative bacteria
Yeasts
Pseudomonas aeruginosa
Candida albicans
Organism
Staphylococcus aureus (ATCC 25923)
Bacillus subtilis (ATCC 6635)
(ATCC 27853)
Escherichia coli (ATCC 25922)
(ATCC 10231)
HL1
22.4± ±0.2
26.4± ±0.3
21.2 ± 0.2
22.1± ±0.3
21.2± ±0.2
(1)
27.3± ±0.4
30.5± ±0.1
27.6± ±0.1
23.5± ±0.1
22.5± ±0.1
(2)
31.7± ±0.2
34.9± ±0.2
31.0± ±0.3
28.8± ±0.2
28.7± ±0.2
(3)
29.5± ±0.1
33.6± ±0.2
28.2± ±0.2
24.3± ±0.2
24.8± ±0.1
HL2
23.5± ±0.2
25.3± ±0.3
22.4± ±0.2
22.3± ±0.1
20.8± ±0.3
(4)
28.2± ±0.4
31.5± ±0.4
29.3± ±0.1
24.3± ±0.2
25.6± ±0.2
(5)
31.2± ±0.1
36.2± ±0.1
35.7± ±0.4
30.4± ±0.2
30.7± ±0.2
(6)
29.4± ±0.3
32.6± ±0.3
31.3± ±0.1
26.8± ±0.2
26.3± ±0.4
Control
00
00
00
00
00
Chloramphenicol
34.0± ±0.1
35.1±0.2
35.8± ±0.1
32.1± ±0.3
-
Nystatin
-
-
-
-
31.5± ±0.1
a
The zone diameters have been calculated in mm by digital vernier caliper. Each value represents a mean ± standard deviation (SD) of three replications. Values followed by the same letter(s) in each column are not statistically different according to Tukey's test (P < 0.01).
Fig 8: The Minimum Inhibitory Concentration (MIC) for the compounds
24
Table 9: The Minimum Inhibitory Concentration (MIC) for the compounds Minimum Inhibitory Concentration (MIC) in mg mL-1 Gram-positive bacteria
Gram-negative bacteria
Fungi
Staphylococcus aureus
Bacillus subtilis
Pseudomonas aeruginosa
Escherichia coli
Candida albicans
HL1
125.00
62.50
125
250
125
(1)
31.25
15.60
31.25
62.50
62.50
(2)
15.60
15.60
15.6
31.25
31.25
Organism
(3)
31.25
31.25
31.25
62.50
62.50
HL2
62.50
62.50
62.50
125
62.50
(4)
31.25
31.25
31.25
62.50
31.25
(5)
15.60
15.60
15.6
15.6
15.6
(6)
31.25
15.60
15.6
31.25
31.25
Chloramphenicol
12.00
12.00
12.00
12.00
-
Nystatin
-
-
-
-
15.00
–: not detected inhibition; control; dimethylsulfoxide.
After screening for the antimicrobial properties of compounds, the minimum inhibitory concentration (MIC) of compounds were tested and compared with the marketed drugs as shown in Table 9. The MIC of complex 5 showed that, they are most active against bacteria S. aureus, B. subtilis, P. aeruginosa and E. coli and fungi C. albicans with value of 15.6 mg mL-1. The complex 2 were shown moderate MIC value of 31.25 mg mL-1 against and E. coli and fungi C.albicans. It was revealed that compound showed a prominent 25
bacterial zone of inhibition and found to vary statistically insignificantly up to 1% level with chloramphenicol of the same concentration. Similarly, compounds show a prominent fungal zone of inhibition and comparable to the marketed antifungal drug nystatin. Further, to understand the time for actual inhibition of test microorganisms with the application of the compounds, time-kill kinetic study was performed. Time-kill kinetic study exhibits basic pharmacodynamic information on the relationship between the synthesized compound and the growth of microorganisms. This test thereby contributes to a better understanding of current and future application of the compound against the diseases caused by the respective bacteria or fungi. Time kill kinetics study for complex 5 against one Gram positive, one Gram negative bacteria and on one fungus is shown in Fig. 9. As shown in Fig. 9, the untreated controls in each case represented the normal growth curve of P. aeruginosa, S. aureus and C. albicans, where lag period remained for 1 h. After that, the exponential growth or the log phase occurred followed by a stationary phase. Whereas, in case of complex (5) for both the microorganisms, a very short exponential growth phase was observed in compare to the untreated control. The growth inhibition of Pseudomonas sp. and Staphylococcus sp. was observed at 3-4 h of incubation period in case of complex (5). At 4th hour of incubation the bacterial CFU enters in the declining phase i.e. death phase. When compound complex (5) was applied to the Candida sp. a negligible growth phase was seen to occur. Consequently, the growth inhibition was found at 2 nd hour of incubation. After an hour of growth inhibition, the cells entered in the death phase. Thus this observation revealed that the complexes show promising bactericidal and fungicidal activities respectively. Fig 9 : Time kill kinetics study for complex 5 against one Gram positive (a. Staphylococcus sp.), one Gram negative (b. Pseudomonas sp. ) bacteria and one fungus(c. Candida sp).
26
a
b
c
3.4. Conclusion The condensation reaction of 3-formyl chromone with benzhydrazide and 4hydoxybenzhydrazide, afforded the tridentate chromone hydrazones, HL1 and HL2, ligands. The reactions of the ligands with nickel (II), copper (II) and zinc (II) salts gave neutral mononuclear complexes. The spectroscopic data showed that the ligand acts as monobasic tridentate ligand through the γ - pyrone oxygen, azomethine nitrogen and hydrazonic oxygen. The ligand and complexes showed antimicrobial activity. From the time-kill kinetic study revealed that the complexes show promising bactericidal and fungicidal activities. In the case of ligands shows better antioxidant activity than standard Trolox. Acknowledgements One of the authors (Jessica Elizabeth Philip) is grateful to the University Grand Commission (UGC), Government of India for financial assistance in the form of Senior Research Fellowship and also authors are thankful to the Department of Applied Chemistry, Cochin University of Science and Technology, Cochin-22, India; Central Institute of Fisheries Technology, Indian Council of Agricultural Research, Kochi-29,India; SAIF, IIT Bombay, India and SAIF CUSAT, India for providing necessary facilities to carry out this work. REFERENCES 1. B. Halliwell, How to characterize an antioxidant: An update, in: C. RiceEvans, B. Halliwell, G.G. Lunt (Eds.) Free Radicals and Oxidative Stress: Environment, Drugs and Food Additives, 1995, 73-101.
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31
Graphical abstract
32
Highlights • • • •
A tridentate chromone hydrazone ligand was synthesized. Nickel, Copper, Zinc (II) complexes were synthesized and characterized by spectral and analytical methods. The spin Hamiltonian parameters of copper complexes were calculated and discussed. The ligand and metal (II) complexes showed antioxidant and antimicrobial activity.
33
S0020-1693(17)30649-7 http://dx.doi.org/10.1016/j.ica.2017.09.006 ICA 17862
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
13 June 2017 1 September 2017 2 September 2017
Please cite this article as: J.E. Philip, S.A. Antony, S.J. Eeettinilkunnathil, M.R.P. Kurup, M.P. Velayudhan, Design, synthesis, antimicrobial and antioxidant activity of 3-formyl chromone hydrazone and their metal (II) complexes, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.09.006
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Design, synthesis, antimicrobial and antioxidant activity of 3-formyl chromone hydrazone and their metal (II) complexes Jessica Elizabeth Philip, Shanty Angamaly Antony, Sneha Jose Eeettinilkunnathil, M. R. Prathapachandra Kurup and Mohanan Puzhavoorparambil Velayudhan Department of Applied Chemistry, Cochin University of Science and Technology, Kochi-22 E-mail: [email protected], Corresponding author Mohanan P. V., Ph.D. Department of Applied Chemistry, Cochin University of Science and Technology, Cochin-682022, Kerala, India. Tel: +91 9447184874 Email: [email protected],
Abstract: In search of a new antimicrobial and antioxidant with improved potency, we designed and synthesized a pair of chromone hydrazones and their transition metal complexes. The characterization and elucidation of the structure of the prepared compounds were performed by elemental analysis, IR, electronic, EPR and thermo gravimetric analyzes, as well as conductivity and magnetic susceptibility measurements. The EPR spin Hamiltonian parameters of copper complexes were calculated. The spectroscopic data showed that the ligands act as a monobasic tridentate. The coordination sites with the metal (II) ion are pyrone oxygen, azomethine nitrogen and hydrazonic oxygen. Hydrazones and their metal complexes have shown antimicrobial activity against Gram-positive bacteria (S. aureus and B. subtilis); Gram-negative bacteria (P. aeruginosa and E. coli) and fungus (C. albicans) and also the synthesized ligands were tested for their antioxidant activity in vitro by DPPH scanning and the ABTS radical method. The substitution requirements for favorable antioxidant activity were investigated. Compounds containing a phenolic hydroxyl group at the imine (azomethine) position as well as an additional electron donor group exhibited lower IC50 values and the result shows that all hydrazones have a significant antioxidant activity compared to the standard antioxidants Trolox.
1
Keywords: 3-formyl chromone, tridentate ligands, EPR spectra, antioxidant activity, antimicrobial activity. 1. Introduction The current escalation of knowledge of free radicals and reactive oxygen species (ROS) in biology produces a medical revolt that promises a fresh era of health and disease management [1]. It is ironic that oxygen, a crucial element for life, in certain situations has deleterious effects on humans [2-5]. They consist of a superoxide anion radical (O2-•), a peroxy radical (RO2•), an alkoxy radical (RO•), a hydrogen peroxide (H2O2) and singlet oxygen (oxygen molecule in the electronically excited state) [6]. The hazardous effects of ROS include oxidative damage to biological molecules such as lipids, proteins, nucleic acids and sugars and also affect the balance between ROS production, cellular defense mechanisms are transferred to more oxidative conditions (the state is called oxidative stress). It has been established that oxidative stress is implicated in a wide range of diseases, such as inflammation, neurodegenerative disorders (Alzheimer's disease, Parkinson's disease), atherosclerosis, diabetes and cancer [7-13]. An antioxidant is a stable molecule appropriate to give an electron to a free radical rampaging and neutralize it, thereby dropping its capability to damage [14]. These antioxidants sustain or inhibit cell damage primarily through their free radical scavenging property [15]. These low molecular weight antioxidants can safely interact with free radicals and terminate the chain reaction before the crucial molecules are damaged. Some of these antioxidants, including glutathione, ubiquinol and uric acid, occur during normal metabolism in the body [16-17].
2
The Chromones are oxygen-containing heterocyclic compounds having a benzoannelated γ-pyrone ring (4H-chromen-4-one, 4H-1-benzopyran-4-one) as the parent compound, which is a common and integral constituent of a variety of medicinal agents [6,18-19]. These compounds show the variety of pharmacological activities and the change in their structure offer a high degree of diversity that has been found useful for the search for novel therapeutic agents [6,20-21]. Several natural and synthetic benzopyrone derivatives have been reported to exert remarkably antimicrobial, anti-tubercular and antifungal activity [22-24]. The development of new defensive and potentially beneficial agents based on chromone is an important research point in medicinal chemistry, with the core of chromone being a key element in the pharmacophores of many biologically active molecules with various medicinal applications [25]. Taking into account the biological importance and remarkable structural behavior of these compounds, in this paper we wish to report some of our findings in the preparation of a pair of hydrazone that were obtained by the condensation of the respective 3-formyl chromone with different aromatic hydrazides. The synthesized compounds were subjected to in-vitro antioxidant screening against DPPH and ABTS radical and their antimicrobial activities were screened against selected bacteria and fungi. 2. Experimental 2.1.
Materials
Reagents used for the synthesis are 3-formyl chromone, 4-hydroxy benzhydrazide, benzhydrazide, (Sigma Aldrich). All other reagents were of analytical grade and the Organic solvents (ethanol, absolute ethanol, methanol, diethylether, dimethylformamide (DMF) and dimethylsulfoxide (DMSO)) employed were either 99% purity or purified by known laboratory procedures[26]. 2.2. Physical measurements 3
Elemental analyses were performed on a Vario EL III elemental analyzer. Melting point of the ligands and their complexes were measured. IR spectra (KBr cm-1) were recorded on a JASCO FT- IR spectrometer in the range of 4000-400 cm-1 .1H/13C NMR spectra were recorded on a Bruker Advance III 400MHz FT-NMR spectrometer using CHCl3/DMSO-d 6 as solvent and TMS as an internal standard (chemical shifts in δ ppm). Electronic spectra were recorded at room temperature on a Thermo Scientific™ Evolution 201 UV-Visible spectrophotometer. Thermo gravimetric Analysis (TG/DTG) were carried out from room temperature up to 800 oC at a heating rate of 10 oC/min on Perkin Elmer Diamond TG/DTA analyzer. The molar conductivities of the complexes in DMF solutions (10-3 M) at room temperature were measured using a Systronic model 303 direct reading conductivity meter. Mass spectra of the hydrazones were recorded by direct injection on WATERS 3100 Mass Detector using Electron Spray Ionization (ESI) technique designed for routine HPLC-MS analyses. Magnetic susceptibility measurements of the complexes were carried out on a Vibrating Sample Magnetometer using Hg [Co(SCN)4] as a calibrant. The EPR spectra of the complexes in the solid state at 298 K and in DMF at 77 K were recorded on a JEOL, JapanModel: JES-FA200 ESR Spectrometer using TCNE as the standard, with 8.75-9.65 GHz microwave frequency. 2.3. Synthesis of chromone hydrazones The synthesis of chromone hydrazones are shown in scheme 1. Aromatic hydrazides (1.00 mmol), 3-formylchromone (1.00 mmol), and a few drops of acetic acid were dissolved in 25 mL of ethanol, and the mixture was stirred and refluxed for 6 h. The precipitate was collected, washed with ethanol, and dried in vacuo. All compounds were prepared similarly shown in scheme 1 and characterized as below.
4
2.3.1. N'-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide - Yield 78% as yellow powder; m.p. 171-173oC; IR (KBr, cm−1): 3206 (N-H), 1680 (C=O, pyrone), 1655 (C=O, hydrazonic), 1571 (C=N), 1238(C–O), 1035 (N–N); 1H NMR (DMSO-d6, δ, ppm): 11.72 (s, 1H, OH); 8.78 (s, 1H, CH=N); 8.59 (s, 1H, oxo ring); 7.92- 6.84 ( m, chromone ring and phenyl ring). 13C NMR (DMSO-d6, δ, ppm): 177.5(C=O), 163.77 (-N-N=CH-), 162.70, 157.20, and 152.80 (non protonated carbon), ESIMS [M+1]: 293.1. Anal. Calcd for C17H12N2O3 (292.08): C, 68.45; H, 4.28; N, 9.39; Found: C, 68.40; H, 4.19; N, 9.36%. 2.3.2. 4-hydroxy-N'-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide -Yield 76% as yellow powder; m.p. 169–170 oC; IR (KBr, cm−1): 3400-3100 (broad O-H & N-H), 1680 (C=O, pyrone), 1651 (C=O, hydrazonic), 1580 (C=N), 1232 (C–O), 1028 (N–N); 1H NMR (CHCl3, δ, ppm): 11.73 (s, 1H, OH); 8.60 (s, 1H, CH=N); 8.8 (s, 1H, oxo ring);8.15- 6.87 ( m, chromone ring and phenyl ring); 13C NMR (CHCl3, δ, ppm): 176.00 (C=O),163.71 (-NN=CH-),162.72, 160.83, 157.21and 152.83 (non protonated carbon), ESIMS [M+1]: 309.2. Anal. Calcd for C17H12N2O4 (308.18): C, 66.25; H, 3.92; N, 9.09; Found: C, 66.30; H, 3.88; N, 9.13%.
2.4. Synthesis of complexes About 1 mmol of metal (II) acetate dissolved in 30mL methanol was added gradually to 1 mmol of the ligand, dissolved in DMF. The reaction mixture was heated under reflux for 8h. The resulting precipitates were filtered, washed with methanol and hexane then diethyl ether 5
and finally air-dried. The complexes were kept in desiccators over anhydrous calcium chloride. The yield was noted. All complexes were prepared similarly. Table 1 Analytical and physical data of the chromone hydrazones (HL1 & HL2 ) and their metal (II) complexes.
No
Reaction
Complex M.F. [F.W.]
Colour
Yield %
M.P. C
1
HL1
C17 H12N2O3 [292.08]
Yellow
78
2
HL1+ Ni(OAc)2
C19 H20N2NiO8 [463.06]
Brown
62
>250
3
HL1+ Cu(OAc)2
C19 H16N2CuO6 [431.88]
Green
65
>250
4
HL1+ Zn(OAc)2
C19 H19N2ZnO7.5 [459.04]
Yellow
67
>250
HL2
C18 H14N2O4 [322.31]
Yellow
76
C19 H20N2NiO9 [479.06]
Brown
72
>250
C19 H18N2CuO8 [465.90]
Green
70
>250
C19 H14N2ZnO6 [431.73]
Yellow
66
>250
5 6 7 8
HL2+ Ni(OAc)2 HL2+ Cu(OAc)2 HL2+ Zn(OAc)2
Elemental Analysis, % Found (calac.)
o
C
H
N
68.40 (68.45) 49.33 (49.28)
4.19 (4.28) 4.47 (4.35)
9.36 (9.39) 6.15 (6.05)
M
53.03 (52.84)
3.38 (3.73)
6.40 (6.49)
14.65 (14.71)
49.47 (49.53) 66.30 (66.23) 47.58 (47.64) 48.89 (48.98) 52.79 (52.86)
4.06 (4.16) 3.88 (3.92) 4.29 (4.21) 3.93 (3.89) 3.20 (3.27)
6.13 (6.08) 9.13 (9.09) 5.72 (5.85) 6.12 (6.01) 6.41 (6.49)
14.11 (14.20)
12.53 (12.68)
12.07 (12.25) 13.51 (13.64) 15.09 (15.15)
2.5.Antioxidant activity determination 2.5.1. DPPH radical scavenging assay The DPPH (2-2'-diphenyl-1-picrylhydrazyl) radical scavenging action of the compounds was evaluated according to the method of Blios.45. The DPPH radical is a stable free radical having λmax at 517 nm and showed characteristic deep violet colour. The loss of violet colour of the DPPH solution by the abstraction of hydrogen from the substrate serves as a marker of the scavenging of DPPH radicals. A variable concentration of the test compounds was added to a solution and the final volume was made up to 3 mL with solvent. The solution was incubated at 37 °C for 20 min. The decrease in absorbance of DPPH was measured at 517 nm. The same experiment carried out without the test compounds serve as a control. Percentage inhibition can be calculated by using the equation.
% ℎ
=
− × 100 6
A graph was plotted with concentration (µM) against % inhibition. The concentration at which there is 50% fall in absorbance of DPPH solution was determined from the graph (IC50). The IC50 value in µM was calculated from the above obtained concentration value. All experiments were carried out in triplicate. The 6-hydroxyl-2, 5, 7, 8-tetramethylchroman-2carboxylic acid (Trolox) was used as antioxidant reference. 2.5.2. (ABTS˙+) radical cation assay It is based on the ability of antiradical molecules to quench the ABTS radical. It is a blue green coloured chromophore shows characteristic absorption at 734 nm. By the addition of antioxidants to the radical cation reduces it to ABTS and loss its characteristic colour (decolourization). In this method, an antioxidant was added to a pre-formed ABTS radical solution, and after 20 min period, the remaining ABTS˙+ is quantified spectrophotometrically at 734 nm. The ABTS˙+ was produced by reacting 7 mM ABTS in H2O with 2.45 mM potassium persulfate (K2S2O8), stored in the dark at room temperature for 24 hrs before use (the radical was stable for more than two days under these conditions). The ABTS˙+ solution was further diluted in methanol until the initial absorbance value of 0.80 ± 0.02 at 734 nm at 30oC. Then, in a 3 mL solution of ABTS˙+, was added a methanolic solution of the compound under study so that their final concentrations vary between 0 and 15 µM. In each case, a 2080% decrease in the initial absorbance of the reaction solution was achieved. The absorbance was recorded at 0, 1, 4 and 6 min. The scavenging capacity of test compounds was calculated using the following equation: ˙" #$ $ %% &%' =
− × 100
Where Acontrol is absorbance of a control lacking any radical scavenger and Asample is absorbance of the remaining ABTS˙⁺ in the presence of scavenger. Graphs of antioxidant concentration versus percent absorbance reduction were then plotted. The concentration of
7
Trolox giving the same percentage reduction of absorbance at 734 nm as the 1 mM antioxidant solution was calculated from the three point graphs. The results were thus expressed as Trolox equivalent antioxidant capacity (TEAC) values. For each hydrazone and each concentration, measurements were performed in triplicate. Absorbance values were corrected for radical decay using blank solutions. 2.6. Antimicrobial activity The synthesized chromone hydrazones and metal complexes were tested for their in vitro antibacterial and antifungal activity against the sensitive organisms Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6635) as Gram positive bacteria, Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) as Gram negative bacteria and Candida albicans (ATCC 10231) as fungus strain using the disc diffusion method [Kirby-Bauer Test ][27](for the qualitative determination) and the serial dilutions in liquid broth method for determination of MIC values. The antibiotic chloramphenicol was used as reference in the case of bacteria, and nystatin in the case of fungi. The chromone hydrazones and metal complexes were dissolved in DMSO. The test was carried on medium potato dextrose agars (PDA) which have a mixture of 200 g potatoes, 6 g dextrose and 15 g agar [28] .Uniform dimension filter paper discs were impregnated with equal volume (10 µL) from the exact concentration of dissolved compounds and carefully located on the inoculated agar surface. After incubation for 36 h at 27 oC in the case of bacteria and for 48 h at 24 oC in the case of fungi, inhibition of the organisms was measured and used to calculate mean of inhibition zones and all experiments were carried out in parallel sets of triplicate. Data were analyzed by the analysis of variance (ANOVA) using the Origin pro 8.5 statistical software and the mean differences were separated using Tukey's studentized test at the 1% level of probability. Time kill kinetic studies were conducted for the compounds against one Gram positive, one Gram negative bacteria and against fungus. 8
In the experiment, an overnight culture of the isolates was used up. 1mL of 106 CFU mL-1 of each culture was inoculated in sterilized nutrient broth media containing 25 mg mL-1 of the compounds. The experiment was conducted for 13 h in a shaker at 30°C. Likewise, control was prepared for each microorganism without having the test compound. The CFU count was taken at regular 1 h interval. For that, 1mL of each culture was spread on nutrient agar plates from 0 h to 13 h and each plate was incubated for 24 h at 30°C. The CFU (colony forming unit) was calculated and plotted in graph. 2.6.1. Minimum Inhibitory Concentration (MIC) All the compounds showed substantial inhibition of both bacteria and fungi, were tested further for the Minimum Inhibitory Concentration (MIC). Standard pathogenic bacterial strains S. aureus, B. subtilis, P. aeruginosa, E. coli and C. albicans as fungus. The resazurin reduction method was used for finding out the MIC in 96 well microplates [29]. All the compounds were dissolved in DMSO having a final stock concentration of 10 mg mL-1. Fifty microlitres of this stock solution was serially diluted to eight times and 50 μL of each serially diluted compound was added to microplate wells. All the microbial cultures were grown in nutrient agar to reach 0.5 McFarland concentrations and 50 mL of this culture was added to each well. Microplate contents were mixed well and incubated at room temperature for 12 hours. After 12 h of incubation, 10 μL of mixture from each well was spread on the agar plate and checked for the Colony Forming Units (CFU). Further 30 μL of 0.1% resazurin solution was added to each well and incubated up to another 24 hours. Microplate well contents were observed in the change in colour from blue to pink. Those wells that have microbes growing will change the blue resazurin into pink color. The well, which remains blue after 24 hours of incubation indicates there are no microorganisms survived in the well, the minimum concentration where no microbial growth found are considered as MIC value.
9
3. Result and Discussion 3.1.Characterization of ligands and complexes The structure of the synthesized chromone hydrazones and their metal complexes were elucidated by elemental analysis, electronic, FTIR, 1H/13C NMR and ESI-MS spectra. The results of elemental analysis (C, H, N) with molecular formula (Table 1) are good agreement with those calculated for the suggested formula and the melting points (Table 1) are sharp signifying the purity of the prepared chromone hydrazones. The metal (II) complexes were isolated pure in very good yields and they are of various colours. The ligands and the complexes are very much soluble in DMF and DMSO, moderately soluble in methanol and acetonitrile and practically insoluble in water, carbon tetrachloride, chloroform and benzene. They are very air stable solids at room temperature without decomposition for a long time. Moreover, the structure of the ligands (HL1 & HL2 ) was deduced from mass spectral data which showed the [M+1] peak at 293.1 & 309.2 a.m.u. respectively, confirming its formula weight (F.W. 292.08 and 308.18 respectively; Figs. C: 1-2 (supplementary data)) it supported the suggested structure of the ligands. 3.1.1. FTIR spectra The characteristic IR bands of the chromone hydrazones (Table 3) give important information about the various functional groups present in it. Strong bands due to the ν (N-H) and ν (C=O) modes at 3220 and 1641 cm-1 are observed in the spectrum of HL1 which suggests that the hydrazone exists in the amido form in the solid state [30]. A band at 1678 cm-1 is due to the presence of (C=O, pyrone). A prominent band at 1587 cm-1 due to azomethine ν (C=N) formation of ligand HL1. It further confirms the ν (C–O), ν (N–N) bands at 1238, 1035 cm-1 respectively. Similarly in the case of ligand HL2 a strong broad band around 3394 cm-1 is due to ν (N-H & O-H). A strong band at 1650 cm-1 observed in the spectrum which suggests that the HL2 exists in the amido form in solid state. Band at 1675 cm-1 is due to the presence of 10
(C=O, pyrone). A prominent band at 1605 cm-1 due to azomethine ν (C=N) formation of ligand HL2. It further confirms the ν (C–O), ν (N–N) bands at 1235, 1048 cm-1 respectively. The comparison of the complexes with respective chromone hydrazone shown that all complexes had broad band in the range of 3690-3025 cm-1 assigned to ν (OH) of the coordinated water molecules linked with the complexes [31] and also the OH of the hydrazine moiety which are confirmed by elemental, AAS, and thermal analyses. All the metal (II) complexes displayed distinct sharp band in 1571-1531 cm-1 region corresponds to the characteristic -C=N- group stretching vibration indicating the coordination of azomethine nitrogen to metal (II) centre as observed in the literature [32]. This coordination of azomethine nitrogen is also supported by band at 1029-1013 cm-1 corresponding to ν (N-N) stretching vibration. On comparison with literature reports, ν(N-N) vibrations was found to be in higher frequencies in the complexes due to the increase in double bond character, offsetting the loss of electron density via donation to the metal[33]. The bands at 1580 cm-1 assigned to ν(C=N), in the chromone hydrazones were shifted to lower wave number region in all complexes. The band at 1655 cm-1 assigned to ν(C=O) hydrazonic in the free ligand was shifted to (1647-1598) cm-1, [34] which indicate that the azomethine nitrogen and hydrazonic (C=O) are in chelation. In complexes 1 & 4 the chelating bidentate acetate (CH3COO-) group was present due to the bands around 1424-1414 and 1337-1325cm-1. These two bands are due to νas (COO-) and νs (COO-), respectively. The separation of the two bands, ∆ ν = (νas- νs) = 88cm-1, is comparable to the values cited for the bidentate character of the acetate group ∆ ν = 75-90 cm-1[35]. On the other hand, complexes 2, 3, 5 & 6 showed new bands characteristic for νas (COO-) and νs (COO-) of acetate ion in the ranges 1549-1531 and 1413-1389cm-1. The higher difference between the two bands indicates the monodentate nature of the acetate group, in which one ‘O’ atom of acetate was coordinated to the metal center, while the second was hydrogen bonded to, a geminal H2O
11
[36]. The appearance of the non - ligand bands at 557-510 and 473-403 cm-1 were assigned to ν (M-O) and ν (M-N), respectively. Table 3 Characteristic IR spectral data of the ligand (HL1 & HL2) and its complexes. IR spectra (cm)-1 N o
Compounds
1
2
3
4
ν(OH, NH)
ν( C=O) pyrone
ν( C=O) hydrazonic
ν( C=N)
ν( C-O)
ν( N-N)
ν( M-O)
ν( M-N)
Mono/ Bidantate acetate
HL1
-, 3220
1678
1641
1587
1238
1035
-
-
-
1645
1617
1545
1220
1029
510
436
[Ni(L1)(OAc)(H2O)]
3666-2919
.2H2O
(broad)
[Cu(L1)(OAc)(H2 O)]
3445
1656
1605
1531
1228
1019
526
403
3618-3088
1641
1597
1560
1224
1027
533
460
1675
1650
1580
1232
1028
-
-
1639
1617
1571
1219
1015
520
449
3416-3025
1634
1598
1555
1229
1021
512
423
3630-2997
1654
1593
1550
1196
1013
557
473
[Zn(L1 )(OAc)] .2.5H2O
6
7
8
3394
HL2
(broad)
[Ni(L2)(OAc)(H2O)]
3690-3043
.2H2O
(broad)
[Cu(L2)(OAc) (H2O)].H2O
9
[Zn(L2 )(OAc)]
3.1.2.
1
νas 1431, νs 1328 νas 1531, νs 1323 νas 1529, νs 1397 -
νas 1451, νs 1364 νas 1511, νs 1374 νas 1536, νs 1388
HNMR spectra
Proton Nuclear Magnetic Resonance (1 H NMR) Spectroscopy is a powerful tool used for the determination of the structure of compounds. The 1H NMR spectra of the chromone hydrazones and Zn (II) (because of their diamagnetic nature) have been recorded with DMSO as solvent. 1H and 13C NMR spectral data (δ ppm) of the ligand relative to TMS (0 ppm) in DMSO-d 6, Tables 2; Figs. A: 1-2 & B: 1-2 (supplementary data), give further support of the suggested structure of the ligand. Table 2
1
H & 13C NMR Spectral data of ligands HL1 & HL2
1 HNMR δ ppm (DMSO)
Assignments
13CNMR δ ppm (DMSO)
Assignments
HL1 11.72 8.78 8.59 7.92 -6.84
(s,1H,OH) 177.5 (s,1H,CH=N-) 163.77 (s,1H,oxo ring) 162.70,157.20,152.80 (m, chromone & phenyl ring)
-C=O -N-N=CHNon protonated carbon
12
HL2 11.73 10.12 8.80
(s,1H,OH) (s,1H,OH) (s,1H,oxo ring)
8.60
(s,1H,CH=N-)
7.88-6.84
(m, chromone & phenyl ring)
176.00
C=O
163.06 162.72,160.83, 157.21,152.83
-N-N=CHNon protonated carbon
The comparison of the NMR spectra of the complexes with the spectra of ligands gave valuable information regarding the coordination mode of ligands during complexation. The 1H NMR spectrum obtained for complexes 3 and 6 was not good due to the poor solubility of the compound. In the spectra of the free hydrazones there are sharp singlets in the range of 11-12 ppm showing the existence of iminol form in solution. They also gave singlets in the range of 10-11 ppm with an area integral of one which is due to phenolic OH protons. Syntheses and characterization of Zn (II) complexes derived from ONO donor chromone hydrazones the intensity of these signals significantly decreases, which suggests that these protons are easily exchangeable and confirm the assignment. Peaks for aromatic protons were found in the region 6.8-8 ppm. Peaks are in the region 11-12 ppm corresponds to iminol protons found in the spectra of free hydrazones were absent in the spectra of complexes indicating the coordination of iminol oxygen to zinc. In the spectrum of complexes, a singlet with an integral area of three is found at 2.0-1.9 ppm and it is assigned to the methyl group present in acetate which is coordinated to zinc. Also a singlet with area integral one is appeared at 8.27 & 8.7 ppm may indicate the presence of -CH- proton. All other peaks observed in the spectra of free hydrazones are slightly shifted. The 1 H NMR spectra of complexes 3 and 6 are shown in the Figs. A: 3-4 (supplementary data). 3.1.3. Solution studies UV -Vis spectra and Molar conductivity measurements
13
The electronic spectral data of the ligand in DMF (Table 3) showed two bands λmax (DMF) nm (ε in 103 dm3 mol-1cm-1): 237 (13.7), 341 (8.6) for HL1 and 241(14.1), 347 (7.9) for HL2. The higher energy band may be assigned to π-π* transitions of the azomethine linkage and the aromatic rings. The medium energy band may be assigned to n-π* transitions of the C=O and C=N groups. The metal complexes in DMF solutions present absorption maxima attributable to the hydrazone ligand together with the absorptions, around 419-697 nm, due to ligand to metal charge transfer and d-d transitions of the metals in the complexes [37]. The bands in the range 340 and 241 nm in the electronic spectra of hydrazones due n→π* and to π→π* transitions suffered marginal shifts (350 and 252 nm) upon complexation. This may be due to the weakening of the C=O bond and the extension of conjugation upon complexation. The shift occurs also due to the coordination of the metal (II) ion are pyrone oxygen, azomethine nitrogen and hydrazonic oxygen. The electronic spectral data of the metal (II) complexes are given in Table 4. The molar conductance values of the synthesized Ni (II), Cu (II) and Zn (II) complexes in DMF (10-3) were measured at room temperature and the results were shown in Table 4. The values showed that all complexes have non-electrolytic nature [38]. Table 4 Electronic spectra, magnetic moments and molar conductivity data of the ligands (HL1&HL2) and its metal complexes.
No
Compounds
1
HL1
2
[Ni(L1 )(OAc)(H2O)].2H2O
3
[Cu(L1)(OAc)(H2 O)]
4
[Zn(L1)(OAc)].2.5H2O
6
HL2
Electronic spectral bandsa (nm) λmax a (nm)/(εmax L cm-1 mol-1 ) 237 (1.37) - π→π* 361 (8.6) - n→π* 260 (4.57) - π→π* 316 (4.42) - π→π* 366 (4.79) - n→π* 429 (2.45) - CT 589 – dd 254 (4.46) - π→π* 310(1.88) - π→π* 407(5.91) - n→π* 428 (6.03) - CT 642 (1.14) – dd 272 (4.77) - π→π* 358 (2.85) - n→π* 410 (2.52) - CT 241 (1.41) - π→π* 359 (7.9) - n→π*
µ eff. b B.M.
conductancea (Ω -1 cm2 mol-1)
-
-
3.45
12.5
1.65
9.4
D
8.6
-
-
14
7
[Ni(L2 )(OAc)(H2O)].2H2O
8
[Cu(L2)(OAc)(H2 O)].H2 O
9
[Zn(L2)(OAc)]
257 (3.83) - π→π* 316 (5.19) - π→π* 353 (6.12) - n→π* 611 - dd 257 (5.20) - π→π* 377 (5.70) - π→π* 437(5.31) - n→π* 462 (2.85) - CT 629 (1.24) - dd 260 (4.26) - π→π* 349 (3.30) - n→π* 430(1.12) - CT
3.32
15.4
1.72
13.7
D
9.5
a
Solutions in DMF (10-3 M), values of εmax are in parentheses and multiplied by 104 (L mol-1 cm-1). μ eff. is the magnetic moment of the complex. D - diamagnetic b
3.1.4. TG/DTG The thermal (thermo gravimetric) analysis was mainly used to confirmation of the associated water or solvent molecule to be in the coordination sphere or in the outer sphere of the complex [39] and information on their properties, nature of intermediate and final products of thermal decomposition can be obtained. From TGA curves, the weight loss was calculated for the different steps and compared with theoretically calculated weight for the suggested formulae based on the results obtained from elemental analyses as well as molar conductance and AAS measurements. TGA indicated the formation of metal oxide as the end product from which the metal content could be calculated and compared with that obtained from analytical determination. Thermo grams of metal complexes indicate decompositions around 100 oC is due to hydrated water (except complex 2 & 6) and also (complex 1, 2, 4 & 5) around 200 °C is due to coordinated water molecules. The results of thermal analysis (Table - 5, Figs. E: 1-6 (supplementary data)) of these complexes are in agreement with elemental analyses. Table 5 TG/DTG of metal (II) complexes
Complexes
Temperature range(oC)
Mass loss (%) Obsd. (Cald.)
Decomposition product
[Ni(L1)(OAc)(H2 O)].2H2 O
80-105 297-354
7.62 (7.77) 17.47 (18.03)
Loss of 2H2 O (hyd) Loss of H2 O (coordinated) & acetate
15
[Cu(L1 )(OAc)(H2O)]
[Zn(L1 )(OAc)].2.5H2 O
[Ni(L2)(OAc)(H2 O)].2H2 O
[Cu(L2 )(OAc)(H2O)].H2O
[Zn(L2 )(OAc)]
229 258-306 307-369 50-120 450-477 50-130 173-248 277-368 60-114 200-248 290-429 150-280 419-484
4.55 (4.16) 13.93 (14.25) 22.35(22.07) 9.96 (9.80) 14.44(14.89) 7.16 (7.51) 4.33 (4.06) 22.14(22.05) 4.05 (3.86) 4.26 (4.01) 32.33 (33.08) 43.66 (43.01) 6.58 (6.97)
Loss of H2 O (coordinated) Acetate C6 H6 Loss of 2.5 H2 O (hyd) C2 H4O2 Loss of 2H2 O (hyd) Loss of H2 O (coordinated) C6 H6O Loss of H2O (hyd) Loss of H2 O (coordinated) C7 H9 NO2 C10 H8N2O2 CH4
3.1.5. Magnetic measurements Due to the Jahn–Teller distortion of Cu II-ion (d9) it lower the symmetry, complete interpretations of the spectra and magnetic properties are somewhat difficult [40]. The magnetic moment values of the copper complexes are 1.68, 1.72 B.M., which is consistent with the presence of one unpaired electron. In nickel complexes the magnetic moment values are 3.45 and 3.32 B.M; indicate that complexes follows octahedral geometry. 3.1.6. EPR studies The EPR spectra of the copper (II) complexes in DMF at 77 K and in a polycrystalline state at 298 K were recorded in the X-band, using 100-kHz modulation frequency and 9.5 GHz microwave frequency. All the EPR spectra are simulated using Easy Spin 5.0.20 package[41] and the experimental (red) and simulated (blue) best fits are included. EPR parameters of the copper (II) complexes are presented in Table 6 and Figs. 1- 4. Copper (II) complex (2 & 5) displayed Figs. 1- 4 axial spectra in the polycrystalline state at 298 K with gǁ and g⊥ values. The variation in the gǁ and g⊥ values indicates that in the solid state, the geometry of the compounds is affected by the nature of coordinating ligands. Fig 1: EPR spectrum of Complex 2 at 298 K; Fig 2: EPR spectrum of Complex 2 in DMF at 77K; Fig 3: EPR spectrum of Complex 5 at 298 K; Fig 4: EPR spectrum of Complex 5 in DMF at 77K
16
1 2
3
4
For complex (2 & 5), displayed axial features; since it is magnetically concentrated, hyperfine splitting was not clear in both parallel and perpendicular regions. The geometric parameter G, represents the exchange interaction between copper canters in the polycrystalline compound and is calculated for each complexes using the equation, G = (gǁ 2.0023)/(g⊥ - 2.0023) for axial spectra [42]. If G > 4.4, exchange interaction is negligible and if it less than 4.4, considerable exchange interaction is indicated in the solid complex. The calculated G values are greater than 4.4 for complexes which indicate that there are no copper-copper exchange interaction is present in the polycrystalline state of the complexes. As gǁ > g⊥ > 2.0023, a square pyramidal geometry consistent with dx2-y2 ground state can be assigned to the copper (II) complexes (2 & 5), thereby ruling out the possibility of a trigonal bipyramidal structure (where gǁ < g⊥). At 77 K in DMF solution, complexes 2 & 5 (Figs.1-4) exhibits axial spectrum. The EPR parameters gǁ and g⊥ incorporated with the energies of d-d transition were used to evaluate the bonding parameters. The following simplified expression were used to calculate the bonding parameters K2ǁ = (gǁ - 2.0023) Ed-d/8λ0 K2⊥ = (g⊥ - 2.0023) Ed -d/2λ0 17
Where Kǁ and K⊥ are orbital reduction factors and λ0 represents the one electron spin orbit coupling constant which equals -828 cm-1. Hathaway [42] has pointed out that for pure sigma bonding Kǁ ≈ K⊥ ≈ 0.77, for in plane π-bonding Kǁ < K⊥ and for out-of-plane π-bonding, K⊥< Kǁ. For complexes (2 & 5), it is observed Kǁ < K⊥ indicates significant in-plane π-bonding is present in the complex. The gǁ values also provide information regarding the nature of metal-ligand bond [43]. The gǁ value is normally 2.3 or larger for ionic and less than 2.3 for covalent metalligand bonds. The gǁ values obtained for complexes (2) and (5) indicate a significant degree of covalency in the metal-ligand bonds [44]. Table 6 Spin Hamiltonian and bonding parameters of copper (II) chromone hydrazones Polycrystalline state DMF solution (77 K) (298 K)
Complex
gǁ
g⊥
gav
G
gǁ
g⊥
Kǁ
K⊥
[Cu(L1 )(OAc)(H2O)] (2)
2.190
2.066
2.1073
2.9466
2.190
2.066
0.6609
0.7700
[Cu(L2 )(OAc)(H2O)].H2O(2)
2.19
2.059
2.1027
3.3104
2.20
2.057
0.6829
0.7185
Fig 5: Proposed structure of complex 1, 2, 3, 4, 5 & 6
O O
H2O
O Ni
O O
N O
.2 H2O N
O
N
Cu O
N
.H2O
O O
18
O
O
O Zn O
N
N
O
. 2.5 H 2O H2O
O
Ni O
N O
O
OH
O
O
O
H2O
O
N
Cu O O
. 2 H2O
N
.H2O
O
N
N
Zn O
O OH
N
O O
OH
3.2. Antioxidant activity The antioxidant activity of chromones and their derivatives has attracted increasing interests and been extensively investigated, mainly in- vitro system. It has been reported that over production of free radicals may induce some oxidative damages to bio molecules such as carbohydrates, proteins, lipids and DNA, thus accelerating aging, cancer, cardiovascular diseases, and inflammation and so on. 3.2.1. Scavenging radical activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical In this method the decrease in absorbance (stable free radical DPPH) at 517 nm, in presence of chromone hydrazones were tested. All tested chromone hydrazones shows UV absorbance between 230 and 350 nm ranges. These values do not interfere with the kinetic measurement of DPPH absorbance at 517 nm. The absorbance of DPPH radical decrease in presence of antioxidants is due to hydrogen transfer from the antioxidant, thus forming the DPPH-H stable compound. Methanol solutions of DPPH radical at different concentrations of
19
hydrazones were checked over a 20 min period. The chromone hydrazones were studied to investigate their antiradical ability by comparing their scavenging effects with standard Trolox. We find that the inhibitory effect of the compounds tested on DPPH radical is concentration dependent and the suppression ratio increases with increasing sample concentrations in the range tested. The IC50 values of the chromone hydrazones were determined (HL1 & HL2 = 3.71 & 1.54 mg/mL respectively) and standard Trolox is 6.25mg/mL. Compounds having a hydroxyl group at para position show the lowest IC50 values. A lower IC50 value means higher DPPH free radical scavenging activity. Hydrogen atom abstraction from phenolic ring under the examined conditions is quite easier for compounds HL2 as shown in Scheme 2. As a consequence the reaction of DPPH radicals may create nitrogen radical on the antioxidant considered. This is in good agreement with Dolenc et al. [24] concerning the auto oxidation process of hydrazones. Studying structurally different hydrazones, we proposed for those compounds having at least one hydrogen atom on nitrogen, the formation of nitrogen cantered radical (hydrazonyl radical) which is stabilized through conjugation forming delocalised carbon cantered radicals. The in vitro antioxidant activities of synthesized chromone hydrazones may be due to the relative ability for the N-H hydrogen atom abstraction from the compound and also substitutions on aromatic or hetero aromatic hydrazides.
Scheme 2: Proposed reaction of DPPH with p-hydroxybenzhydrazide derivative
20
3.3.2. Scavenging radical activity on 2, 2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS˙+) radical cation The antioxidant activity of chromone hydrazones were evaluated by using the Trolox equivalent antioxidant capacity values (TEAC). In this method we measure the relative ability of antioxidant compound to scavenge the radical cation [2, 2′-azinobis (3ethylbenzthiazoline-6-sulfonic acid) (ABTS˙+)] and compared with a standard amount of synthetic antioxidant Trolox [6-hydroxy- 2, 5, 7, 8-tetramethylchroman-2-carboxylic acid], it’s a vitamin E analogue. The TEAC value means the concentration of standard Trolox with the same antioxidant capacity as 1 mM concentration of the antioxidant compounds here chromone hydrazones under investigation. This method based on the generation and detection of coloured long-lived radical cation ABTS is chemically oxidized by potassium persulphate to give the relatively stable (over 24 h) ABTS radical. The concentration of ABTS˙+ is measured at 734 nm [25]. Thus the antioxidant-induced reduction of the ABTS˙+ concentration is directly related to the antioxidant capacity of the compound being tested. ABTS radicals are involved in an electron transfer process. All screening compounds exhibited at various levels radical cation scavenging activity. There were no differences between the TEAC values for the chromone hydrazones at 1 min, 4 min and 6 min (data not shown). As seen in Fig. 6, compounds HL2 were the most effective ABTS˙+ radical scavengers depending on concentration range (0-15 μM). ABTS˙+ absorbance decreases significantly with all chromone hydrazone concentrations, the effect being less pronounced for hydrazone HL1 at concentrations above 12 µM. The scavenging effects on ABTS˙+ decreased in the order Trolox>HL1>HL2. Table 7 summarised the results obtained in terms of Trolox equivalents capacity. All chromone hydrazones possess TEAC values equivalent or inferior values. This might be due to a better
21
transfer of radical and better resonance stabilisation of the radical species for these compounds (Table 7). Fig.6 ABTS radical scavenging activity of chromone hydrazones compared with standard Trolox. Values are average of 3 independent measurements of each compound.
HL1 HL2
1.0 0.9 0.8
Absorbance
0.7 0.6 0.5 0.4 0.3 0.2 0.1
0
2
4
6
8
10
12
14
16
Conc mg/mL
Table 7: Antioxidant activity of chromone hydrazones by ABTS radical. Values are average of 3 independent measurements of each compound.
Compounds
TEAC
3a
3.23
3b
1.48
Trolox
3.09
3.3.Antimicrobial activity The antibacterial activity of all compounds against Mycobacterium were plotted in a bar diagram and represented in Fig. 7. Fig. 7 represents the zone of inhibition for bacteria and fungi at three different concentrations (1, 0.5 and 0.25 mg mL-1) with a standard deviation of the triplicate values (Table 8). Antifungal zone of inhibition showed by the synthetic compounds represented in Fig. 7. Figure 8 and table 9 represent the MIC of the compounds synthesized against the microorganisms. Chloramphenicol was used as a reference in the
22
case of bacteria and nystatin in the case of fungi were tested at a concentration of 10 mg mL-1. Most of the compounds showed good microbial inhibition for all tested microorganisms such as S. aureus and B. subtilis, two Gram negative bacteria P. aeruginosa and E. coli and the pathogenic fungus C. albicans in the disc diffusion method. Complexes 2 and 5 showed a good zone against S. aureus, in the case of B. subtilis complex 2, 3, 5 and 6; for P. aeruginosa complex 2, 5 and 6; for E. coli the complex 5 showed good inhibition zone diameter. All the other compounds with an inhibition zone of up to 35 mm. But in the case of C. albicans complex 5 shows good inhibition zone diameter. The ratio of structural activity of the synthesized compounds was explained based on the microorganisms tested. Among all compounds, complex 5 has shown that the best inhibition against all tested microorganisms may be due to the copper (II) complex. The presence of the p-hydroxyl group has increased the activity of complex 5 against all microorganisms tested. Complex 2 has shown a significant inhibition against Gram-positive bacteria, which is due to the presence of copper (II). The compound HL2 showed greater inhibition of bacteria and less for fungi indicating that the compound may be a better antibacterial than antifungal activity. This indicates that the synthesized compounds are biologically active as antimicrobials. B. subtilis was inhibited to the maximum extent by most compounds. Fig 7: Comparison of zone diameters of compounds
23
Table 8: Inhibitory activity of the compounds expressed as zone of inhibition (mm)a Mean of zone diameter, nearest wholea (mm) Gram-positive bacteria
Gram-negative bacteria
Yeasts
Pseudomonas aeruginosa
Candida albicans
Organism
Staphylococcus aureus (ATCC 25923)
Bacillus subtilis (ATCC 6635)
(ATCC 27853)
Escherichia coli (ATCC 25922)
(ATCC 10231)
HL1
22.4± ±0.2
26.4± ±0.3
21.2 ± 0.2
22.1± ±0.3
21.2± ±0.2
(1)
27.3± ±0.4
30.5± ±0.1
27.6± ±0.1
23.5± ±0.1
22.5± ±0.1
(2)
31.7± ±0.2
34.9± ±0.2
31.0± ±0.3
28.8± ±0.2
28.7± ±0.2
(3)
29.5± ±0.1
33.6± ±0.2
28.2± ±0.2
24.3± ±0.2
24.8± ±0.1
HL2
23.5± ±0.2
25.3± ±0.3
22.4± ±0.2
22.3± ±0.1
20.8± ±0.3
(4)
28.2± ±0.4
31.5± ±0.4
29.3± ±0.1
24.3± ±0.2
25.6± ±0.2
(5)
31.2± ±0.1
36.2± ±0.1
35.7± ±0.4
30.4± ±0.2
30.7± ±0.2
(6)
29.4± ±0.3
32.6± ±0.3
31.3± ±0.1
26.8± ±0.2
26.3± ±0.4
Control
00
00
00
00
00
Chloramphenicol
34.0± ±0.1
35.1±0.2
35.8± ±0.1
32.1± ±0.3
-
Nystatin
-
-
-
-
31.5± ±0.1
a
The zone diameters have been calculated in mm by digital vernier caliper. Each value represents a mean ± standard deviation (SD) of three replications. Values followed by the same letter(s) in each column are not statistically different according to Tukey's test (P < 0.01).
Fig 8: The Minimum Inhibitory Concentration (MIC) for the compounds
24
Table 9: The Minimum Inhibitory Concentration (MIC) for the compounds Minimum Inhibitory Concentration (MIC) in mg mL-1 Gram-positive bacteria
Gram-negative bacteria
Fungi
Staphylococcus aureus
Bacillus subtilis
Pseudomonas aeruginosa
Escherichia coli
Candida albicans
HL1
125.00
62.50
125
250
125
(1)
31.25
15.60
31.25
62.50
62.50
(2)
15.60
15.60
15.6
31.25
31.25
Organism
(3)
31.25
31.25
31.25
62.50
62.50
HL2
62.50
62.50
62.50
125
62.50
(4)
31.25
31.25
31.25
62.50
31.25
(5)
15.60
15.60
15.6
15.6
15.6
(6)
31.25
15.60
15.6
31.25
31.25
Chloramphenicol
12.00
12.00
12.00
12.00
-
Nystatin
-
-
-
-
15.00
–: not detected inhibition; control; dimethylsulfoxide.
After screening for the antimicrobial properties of compounds, the minimum inhibitory concentration (MIC) of compounds were tested and compared with the marketed drugs as shown in Table 9. The MIC of complex 5 showed that, they are most active against bacteria S. aureus, B. subtilis, P. aeruginosa and E. coli and fungi C. albicans with value of 15.6 mg mL-1. The complex 2 were shown moderate MIC value of 31.25 mg mL-1 against and E. coli and fungi C.albicans. It was revealed that compound showed a prominent 25
bacterial zone of inhibition and found to vary statistically insignificantly up to 1% level with chloramphenicol of the same concentration. Similarly, compounds show a prominent fungal zone of inhibition and comparable to the marketed antifungal drug nystatin. Further, to understand the time for actual inhibition of test microorganisms with the application of the compounds, time-kill kinetic study was performed. Time-kill kinetic study exhibits basic pharmacodynamic information on the relationship between the synthesized compound and the growth of microorganisms. This test thereby contributes to a better understanding of current and future application of the compound against the diseases caused by the respective bacteria or fungi. Time kill kinetics study for complex 5 against one Gram positive, one Gram negative bacteria and on one fungus is shown in Fig. 9. As shown in Fig. 9, the untreated controls in each case represented the normal growth curve of P. aeruginosa, S. aureus and C. albicans, where lag period remained for 1 h. After that, the exponential growth or the log phase occurred followed by a stationary phase. Whereas, in case of complex (5) for both the microorganisms, a very short exponential growth phase was observed in compare to the untreated control. The growth inhibition of Pseudomonas sp. and Staphylococcus sp. was observed at 3-4 h of incubation period in case of complex (5). At 4th hour of incubation the bacterial CFU enters in the declining phase i.e. death phase. When compound complex (5) was applied to the Candida sp. a negligible growth phase was seen to occur. Consequently, the growth inhibition was found at 2 nd hour of incubation. After an hour of growth inhibition, the cells entered in the death phase. Thus this observation revealed that the complexes show promising bactericidal and fungicidal activities respectively. Fig 9 : Time kill kinetics study for complex 5 against one Gram positive (a. Staphylococcus sp.), one Gram negative (b. Pseudomonas sp. ) bacteria and one fungus(c. Candida sp).
26
a
b
c
3.4. Conclusion The condensation reaction of 3-formyl chromone with benzhydrazide and 4hydoxybenzhydrazide, afforded the tridentate chromone hydrazones, HL1 and HL2, ligands. The reactions of the ligands with nickel (II), copper (II) and zinc (II) salts gave neutral mononuclear complexes. The spectroscopic data showed that the ligand acts as monobasic tridentate ligand through the γ - pyrone oxygen, azomethine nitrogen and hydrazonic oxygen. The ligand and complexes showed antimicrobial activity. From the time-kill kinetic study revealed that the complexes show promising bactericidal and fungicidal activities. In the case of ligands shows better antioxidant activity than standard Trolox. Acknowledgements One of the authors (Jessica Elizabeth Philip) is grateful to the University Grand Commission (UGC), Government of India for financial assistance in the form of Senior Research Fellowship and also authors are thankful to the Department of Applied Chemistry, Cochin University of Science and Technology, Cochin-22, India; Central Institute of Fisheries Technology, Indian Council of Agricultural Research, Kochi-29,India; SAIF, IIT Bombay, India and SAIF CUSAT, India for providing necessary facilities to carry out this work. REFERENCES 1. B. Halliwell, How to characterize an antioxidant: An update, in: C. RiceEvans, B. Halliwell, G.G. Lunt (Eds.) Free Radicals and Oxidative Stress: Environment, Drugs and Food Additives, 1995, 73-101.
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31
Graphical abstract
32
Highlights • • • •
A tridentate chromone hydrazone ligand was synthesized. Nickel, Copper, Zinc (II) complexes were synthesized and characterized by spectral and analytical methods. The spin Hamiltonian parameters of copper complexes were calculated and discussed. The ligand and metal (II) complexes showed antioxidant and antimicrobial activity.
33