Antibacterial Activity and DNA Binding Properties of Bivalent Metal Complexes of Cuminaldehyde Acetoylhydrazone

Introduction

Hydrazones exhibit a broad spectrum of biological activities such as antibacterial¹, antitubercular¹, antioxidant², antiviral³ and anticancer⁴ activities. Hydrazones constitute an important class of compounds for new drug development⁵. Hydrazones derived from acetichydrazide are named as acetoylhydrazones.   Compared to simple hydrazone Schiff bases, acyl and aroyl hydrazones have an extra donor site in >C=O. This presents a wide range of properties in them. The discovery that acetoylhydrazones show higher activity than benzoylhydrazones evoked considerable interest ^6,7 to investigate metal complexes of former type of ligands.

Survey of literature revealed that a very few acetoylhydrazones are used as chromogenic reagents⁸ and in the study of transition ^9-11 and lanthanide^{12, 13} metal complexes. Hydrazones derived from aldehydes contain hydrogen atom on azomethine carbon atom. It is reported¹⁴   that hydrazones having azomethine hydrogen atom shows higher activity.  Hence it is of interest to investigate hydrazones derived from aldehydes rather than ketones.

Our customary foods contain carbonyl compounds¹⁵ showing beneficial effects to human health. The chemical name of cuminaldehyde is p– Isopropybenzaldehyde (IB). It is known to present in cumin, essential oils of eucalyptus, myrrh, cassia etc.  The aromatic aldehyde, viz. para-Isopropybenzaldehyde (IB) is an important ingredient of cumin. It has medicinal properties. For example, it is known to inhibits the fibrillation of alpha-synuclein¹⁶  present in cumin. Hence it is of interest to use p– Isopropybenzaldehyde in the synthesis of new hydrazone ligand and its metal complexes.

Studies on Metal-DNA interactions are expected to predict biological activity of compounds. The compound which bind DNA strongly may find application in the drug  development.  Survey of literatures revealed that metal complexes with organic ligands showed higher biological activity than metal free organic compounds.  Hence it is considered worthwhile to investigate novel hydrazone ligand originated from p– Isopropybenzaldehyde and its  metal complexes.   We have studied^17-20 nucleic acid binding of various metal complexes in the past. to develop antimicrobial agents. To renew our interests, herein we communicate our results on  spectral analysis and biological applications of  bivalent metal complexes with p-Isopropylbenzaldehyde acetoylhydrazone (IBAH)

Experimental

Acetichydrazideand p-Isopropylbenzaldehyde were bought from Sigma Aldrich and utilized without further purification. Metal salts (ZnCl₂, NiCl₂.6H₂O and CuCl₂.2H₂O)  were of Merck AR quality. Solvents were distilled before use.

Synthesis of IBAH ligand

Round bottom flask(100 mL capacity) was charged with  20 mL of methanolic  solution of  acetic hydrazide (0.91g; 0.012 mol) and 20 mL of methanolic solution  of p-Isopropylbenzaldehyde  (5ml, 0.03 mol).   Glacial acetic acid (few drops) was added as catalyst to the contents of flask.  The reactants were heated on water bath for 2.5 hrs and cooled.  The product was collected and treated repeatedly with hot H₂O & dried. Methanol solvent was used in recrystallization of ligand. Yield:70.58%;  M.P., 130-132^oC. Molecular formula: C₁₂H₁₆N₂O. Formula weight,  204.1. Synthesis of IBAH is depicted in Scheme 1.

Scheme 1: Preparation  of p-Isopropylbenzaldehyde acetoylhydrazone (IBAH) Click here to View scheme

Preparation of metallo-hydrazones

The IBAH ligand (1.5 g; 0.007mol) was transferred to 100-mL beaker and dissolved in 20 mL ethanol.  In another 100-mL beaker, CuCl₂.2 H₂O (0.007mol) was dissolved in 15 mL of ethanol. These two solutions were mixed  in a clean R.B flask and refluxed for 2 hrs. On cooling,  a green stained  product was formed. The compound was amassed and washed with few drops 50% methanol and de-solvated in vacuum. Nickel(II) and zinc(II) complexes of  IBAH were prepared similarly. Melting points and yields of compounds are delineated in Table 1.Particulars of equipment employed in the present study, deoxyribonucleic acid (DNA) binding and antibacterial activity experiments are given in our previous articles^{19, 20}

Results and Discussion

The ligand (IBAH) is characterized on the basis of spectroscopic studies. FT-IR spectroscopy: 3264, 3082, 2945, 1648 and 1542 are designated to secondary amine (N-H), aromatic C- H, aliphatic C-H , >C=O and azomethine (>C=N) elongating vibrations correspondingly.  ¹H-NMR spectroscopy (in deuterated chloroform):  δ 10.64 (s, 1H) 8.95 (s, 1H ) (7.29 – 7.75) (m, 4H), 2.32 (s, 3H) 1.271 (d, 6H) 2.97 (septet 1H)   are respectively assigned to  azomethine (CH=N-),  >NH , Ar-H, CH₃ of acetoyl,  -CH₃ protons of isopropyl and -CH methine protons of isopropyl group sequentially..  Mass spectrometry:  Mass plot of IBAH (Fig. 1) shows a peak at m/z = 204.1 which coincides with formula (C₁₂H₁₆N₂O) weight of IBAH. 

Figure 1: Mass spectrum of IBAH Click here to View figure

Based on spectral analysis the structure (Fig. 2) of IBAH ligand is proposed.  Mass spectral fragmentation pattern of IBAH (Fig. 3) is shown below.

Figure 2: Structure of IBAH  ligand Click here to View figure

Figure 3: Fragmentation pattern of IBAH ligand Click here to View figure

Structural characterization of metal complexes

Metal chlorides reacted with p-Isopropylbenzaldehyde acetoylhydrazone (IBAH)  in basic medium under reflux conditions to produce corresponding metal complexes. The complexes are stable, non-hygroscopic, moderately soluble in CH₃OH and C₂H₅OH but easily soluble in dimethylformamide(DMF)  and dimethyl sulfoxide(DMSO). Color, yield, ESI-MS & conductivity data of Cu(II), Ni(II) and Zn(II) compounds are stated in Table 1.  Molar conductivity outcome show that the coordination compounds are non-electrolytes²¹.

Table 1: Physicochemical and Analytical data* of complexes

Compound	Colour (Yield, %)	ESI-MS (F.W)	Molar                    Conductivity@
Cu(IBAH)2	Dark green (74.36%)	471.2 (469.5)	12.75
Ni( IBAH)2	Parrot green (79.85%)	464.8 (464.7)	9.66
Zn( IBAH)2	Yellow (75.87%)	468.3  (471.4)	15.12

*Calculated values are given in parenthesis.  ^@ Units, Ω^-1cm²mol^-1

Electronic spectroscopy

UV-Visible data of metal complexes and designation of peaks are summarized in Table 2. The electronic spectrum of Cu(II) complex shows(Fig. 4)  peak at 14,792 cm^-1  (676 nm) having shoulder peak at 16,447 cm^-1 in the  visible region. This band is assigned to ²Eg  → ²T_2g electron transfer in favour of octahedral geometry. The presence of shoulder peak at 16,447    cm^-1 (608 nm) may be due to Jahn-Teller effect which is due to geometric distortion generally noticed for copper(II) octahedral compounds where the two axial bonds can be shorter or longer than those of the equatorial bonds.

Figure 4: UV-Visible spectrum of Cu(IBAH)2 complex showing Jahn-Teller effect Click here to View figure

Table 2: UV-Visible spectral data of metal compounds with IBAH ligand

Complex	Wavelength λmax (nm)  (nm)	Frequency  (cm-1)	Transition
Cu(IBAH)2	285	35087	π→π*
	608 676	16447 14792	d→d       d→d
Ni(IBAH)2
	287	34843	π→π*
	611	16366	d→d
	989	10111	d→d

 

Peaks are observed at 16,366 and 10,111 in the electronic spectrum of nickel complex. These peaks are related to ³A_{2g →} ³T_1g(F)   and ³A_2g  → ³T_1g(P)    electron transfers respectively in   support of octahedral geometry.  A peak in higher energy region due to ³A_2g → ³T_2g transition is not observed due to the domination of  π-→π* transition.  

IR spectroscopy

The coordination sites ofIBAH ligand are uncovered by examining IR spectra of metal- free ligand and complexes. Spectral data are stated in Table 3. In the spectrum of the IBAH bands  are observed due to ν_N-H, ν_C=C,ν_C=O andν_C=N stretching modes. Both  ν_N-H  (3264 cm^-1) and υ_C=O (1648 cm ^-1) bands of ligand are absent in the spectra of complexes due to enolization and subsequent  deprotonation of ligand during  complex formation (Scheme-2).

Scheme 2: Mechanism showing complex formation between IBAH ligand and bivalent metal  ions [Where,  M(II)  =   Cu(II), Ni(II), Zn(II)] Click here to View scheme

Table 3: Infrared Spectral data of IBAH ligand and its complexes

IBAH	Cu(IBAH)2	Ni(IBAH)2	Zn(IBAH)2	Assignment
3264	–	–	–	υN-H
3082	3014	3027	3024	υC-H aromatic
2945	2920	2915	2913	υC-H aliphatic
1648	–	–	–	υC=O
1648	1615	1620	1612	υC=N
1542 1465	1528	1522	1532	υC=C (aromatic)
1378	1367	1318	1312	υC=C (aromatic)
1264	1260	1242	1248
1185	1173	1178	1165	υC-O
	604	616	632	υM-O
	574 513	538 520	542 524	υM-N

 

The bands of  ν_C=O andν_C=N  areshifted to lower wave numbers indicating involvement of acetoyl >C=Oand azomethine (>C=N-) nitrogen groups in chelation.  Spectral data suggest that the IBAH behaves as uninegative bidentate ligand in complexes. Bands in low energy regions, 604- 632 and 513-524 cm^-1 are related to υ_M-O and υ_M-N vibrations respectively.

ESR spectroscopy

ESR spectrum of Cu complex in DMF at liquid nitrogen temperature (LNT) is depicted in Figure 5.  Table-4 gives ESR data of copper(II) complex in solid state & in DMF solvent. The g_║, g_┴ and α² and λ values of Cu complex suggest covalent nature of Metal- Ligand (M-L) bond and the location of unpaired electron in d_x2 _{– y}2 orbital. This finding indicates covalent nature ²² of metal- ligand (M-L) bond.   The orbital reduction parameters (K_║,  K_┴) reveal the occurrence of in-plane π- bonding in the complex. The axial symmetry parameter values of complex in solid and solution states are below 4.0. The values indicate absence of quid pro quo interaction.    

Figure 5: ESR spectrum of Cu(IBAH)2 at LNT Click here to View figure

Table 4: Spectral data† of [Cu (IBAH)₂]  complex in DMF at  LNT

g||	g^	g avg	G	A||×10-5	A ^  ×10-5	K ||	K ^	λ	α2
2.38 (2.20)	2.07 (2.12)	2.17 (2.15)	5.64 (1.62)	0.00262	0.00014	0.9970	1.091	520	0.3816

ESR data of complex at RT are given in brackets.

Based on analytical, molar conductivity, UV-Visible, FT-IR   ESR spectroscopy studies a general structure (Fig. 6) is tentatively suggested.

Figure 6: Proposed Structure for complexes in DMF medium Click here to view figure

Deoxyribonucleic acid Interactions

Interactivity of complexes with CT-DNA was examined by UV-visible spectroscopy. Spectra of copper complex are shown in Figure 7.

Figure 7: UV-Visible spectra of copper complex with increasing amount of DNA. (Top most curve is spectrum of complex without DNA) Click here to View figure

Metal derivatives of IBAH showed strong peak due to M→L charge transfer transitions. The binding constant (Kb) is calculated using standard equation.²³ DNA – binding constants of complexes are given in the Table-5. Metal complexes (except Cu(II) complex) showed considerable  hypsochromic  shift (Blue shift)  (∆ λ_max = 0.8 – 1.5 nm) with raising amounts of DNA.  High binding constants (4.3- 17.2 x 10⁶ M^-1) of present complexes are suggestive ^24-28 of intercalation mode of binding.  The binding constant (K_b) order is Cu(IBAH)₂   >Ni(IBAH)₂    >Zn(IBAH)₂.  The observed trend indicates that Cu(IBAH)₂  interacts DNA more firmly.

Table 5: UV-Visible Spectral Data Complexes with and without DNA

Complex	λ max, nm  (n)		Δ λ	H%	Kb [M-1] X106
	Free	Bound
Cu(IBAH) 2	285.0	285.5	0.5	19.32	17.2
Ni(IBAH)2	286.0	284.5	-1.5	11.26	13.4
Zn(IBAH)2	242.2	241.4	-0.8	16.42	4.3

 

Antibacterial activity studies

The compounds under investigation are tested for antibacterial activity as outlined before⁵ by taking Gram-positive (Micrococcus luteus, Bacillus subtilis ) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa)  bacteria using agar well diffusion method against ciproflaxin as positive  control. About 24 hrs old bacterial cultures are used to prepare bacterial lawns. Agar wells were prepared and injected with sample solutions prepared using DMF. The agar plates are incubated at 35^oC for 24 hrs and inhibition zones are measured and expressed (Table 6) in millimeter units.

Table 6: Zone inhibition (in mm) data of IBAH ligand and its metal complexes

Compound	Amount Taken (μg/μL)	E.coli	P.aeruginosa	M.luteus	B.substilis
Ciprofloxacin	5	11.68±0.05	10.24±0.01	9.78±0.02	10.36±0.04
IBAH	100	1.57±0.19	1.56±0.11	1.49±0.37	1.03±0.43
	200	1.82±0.35	2.48±0.01	2.73±0.25	2.87±0.04
	300	2.64±0.44	2.65±0.28	2.68±0.08	2.29±0.22
Cu( IBAH) 2	100	6.45±0.25	3.42±0.39	4.09±0.32	3.93±0.16
	200	7.05±0.36	5.46±0.05	5.92±0.42	5.84±0.38
	300	6.31±0.08	6.29±0.03	6.38±0.14	5.68±0.24
Ni( IBAH)2	100	2.14±0.06	1.75±0.06	1.92±0.42	2.81±0.21
	200	3.95±0.18	2.22±0.03	2.33±0.50	2.70±0.13
	300	4.09±0.22	4.86±0.36	3.57±0.27	4.58±0.11
Zn( IBAH)2	100	2.02±0.17	3.08±0.23	2.73±0.04	1.89±0.19
	200	4.94±0.01	4.33±0.18	4.52±0.41	2.51±0.28
	300	5.73±0.23	6.14±0.31	3.43±0.29	4.73±0.35

Results in Table 6 indicate that the present coordination compounds show higher activity than metal-free IBAH ligand.  Data are depicted in the form of bar graph (Fig. 8) which indicates that Cu(IBAH)₂ complex shows more activity possibly due to its strong  binding to DNA.

Figure 8: Graphical representation of inhibition zones (in mm) of metal complexes Click here to View figure

Metal complexes displayed higher antibacterial activity^28,29 than the metal free ligand. Increased activity of coordination compounds may be explained by using Tweedy’s chelation theory³⁰ and Overtone’s concept³¹ . The ligand forms organic coat around the metal ion in the formation of complex. As a result, the complex becomes lipophilic. According to latter concept, the cell membrane allows the passage of complexes, as they are lipid-soluble. Hence, lipid solubility of compound is principal requirement for showing this action. On complex formation, the polarity of metal ion is significantly reduced due to delocalization of π-electrons. On entering into the cell, the complex releases metal ion , which inhibits enzymatic activity of microorganisms more effectively³².

Conclusion

A new organic ligand viz. p– Isopropylbenzaldehyde acetoylhydrazone (IBAH) and it’s metal complexes are synthesized and characterized for the first time. A general structures for the complexes are proposed based on analytical and electronic spectral data. High DNA binding constants suggest that the complexes bind DNA via intercalation. Complexes showed higher antibacterial activity.  Among all compounds, the copper complex binds DNA more firmly and inhibits bacteria effectively. The Cu(IBAH)₂ complex  not only binds DNA strongly but also inhibits bacteria  more effectively. The observation revealed a direct relationship between DNA binding constant and antibacterial activity. Thus, antibacterial activities of metal complexes are concomitant with their DNA binding constants. 

Acknowledgement

K. H. Reddy is grateful to UGC, New Delhi for the award of BSR Faculty Fellowship.

Conflicts of interest

Regarding the publication of this article, the authors declare that there is no conflict of interests.

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