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European Journal of Medicinal Chemistry 64 (2013) 179e189

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis, characterization and in vitro pharmacological evaluationof new water soluble Ni(II) complexes of 4N-substitutedthiosemicarbazones of 2-oxo-1,2-dihydroquinoline-3-carbaldehyde

Eswaran Ramachandran a, Duraisamy Senthil Raja a, Nattamai S.P. Bhuvanesh b,Karuppannan Natarajan a,*

aDepartment of Chemistry, Bharathiar University, Coimbatore 641046, Tamil Nadu, IndiabDepartment of Chemistry, Texas A&M University, College Station, TX 77842, USA

a r t i c l e i n f o

Article history:Received 13 September 2012Received in revised form25 March 2013Accepted 27 March 2013Available online 6 April 2013

Keywords:Nickel(II) complexesDNA bindingProtein bindingAntioxidantCytotoxicity

* Corresponding author. Tel.: þ91 422 2428319; faxE-mail address: k_natraj6@yahoo.com (K. Nataraja

0223-5234/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2013.03.059

a b s t r a c t

Four new Ni(II) complexes of general formula [Ni(H2-Qtsc-R)2](NO3)2 (H2-Qtsc-R ¼ 4N-substituted thi-osemicarbazones of 2-oxo-1,2-dihydroquinoline-3-carbaldehyde, where R ¼ H (1), Me (2), Et (3), or Ph(4)) have been synthesized and characterized. The geometry of the complexes was confirmed by singlecrystal X-ray crystallography for one of the complexes (3). The binding affinity of the complexes withDNA and protein have been studied which indicate that they could interact with calf thymus DNA andbovine serum albumin protein. Investigations of antioxidative properties showed that all the complexeshave strong radical scavenging properties. Cytotoxic studies showed that the complexes exhibitedeffective cytotoxic activity against a panel of human cancer cells without affecting the normal cells much.

� 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction

Cancer has over taken the heart disease as the world’s top killer,the casualties estimated to bemore than double by 2030 [1]. Cisplatinwhich is used as the most effective anticancer drug in the treatmentof a variety of tumors [2] has its own limitation due to resistance andthe significant side effects such as nausea and kidney and liver failuretypical of heavy metal toxicity [3]. Hence, there exists a challenge toreplace this drug with more-efficient, less toxic, and target-specificnoncovalent DNA binding anticancer drugs. Nickel(II) complexesare good alternative source for metal-organic antitumor drugs sincenickel has shown expanding biological interest [4]. Moreover, anumberof nickel complexeshavebeen reported to act as antiepileptic[5], anticonvulsant [6] agents or vitamins [7], antibacterial [8,9],antifungal [9,10], antimicrobial [11] and anticancer [12e14] agents.

On the other hand, quinoline and thiosemicarbazone derivativesare biochemically important molecules that have attracted interestdue to their biological and pharmacological properties such as anti-tumor, antibacterial, antiviral, and antimalarial activities [15e21] and

: þ91 422 2422387.n).

son SAS. All rights reserved.

hence,wehave synthesized complexesof various thiosemicarbazones[22e25] and heterocycles of thiosemicarbazones [26e31] withdiverse transition metal ions many of which showed remarkablebiological activity. Since the metal complexes derived from2-oxoquinoline thiosemicarbazone and its derivatives did exhibitexcellent DNA/protein binding and antitumor activities indicating anenhanced antitumor activity due to the presence of nitrogen hetero-cycles present in the thiosemicarbazones [26e31], aneffortwasmadeto investigate the effect ofN-substituted 2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-thiosemicarbazones on their complex formationand biological activity of complexes thus formed. Herein, we reportthe synthesis, characterization, structure of somewater soluble Ni(II)complexes and their biological properties such as the binding prop-erties with calf thymus DNA (CT-DNA), competitive binding studieswith ethidium bromide (EB), antioxidant and cytotoxic activities.

2. Results and discussion

2.1. Synthesis and characterization

A series of mononuclear nickel(II) complexes of 2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-substituted thiosemicarbazones {[Ni(H2-Qtsc-H)2](NO3)2 (1), [Ni(H2-Qtsc-Me)2](NO3)2 (2),

Table 1Experimental data for crystallographic analyses.

Complex 3

CCDC deposit number 898171Empirical formula C28.49H36.98N10NiO10S2Formula weight 802.37Temperature 110(2) KWavelength 0.71073 �ACrystal system MonoclinicSpace group C2/cUnit cell dimensionsa (�A) 25.268(6)b (�A) 9.0352(19)c (�A) 15.407(3)a (�) 90b (�) 98.272(5)g (�) 90Volume (�A3) 3480.8(13)Z 4Density (calculated) 1.531 Mg/m3

Absorption coefficient 0.748 mm�1

F(000) 1672Crystal size 0.40 0.35 0.30 mm3

Theta range for data collection 1.63e27.50� .Index ranges �32 � h � 32, �11 � k � 11, �0 � l � 19Reflections collected 19,452Independent reflections 3983 [R(int) ¼ 0.0364]Completeness to theta ¼ 27.50� 99.6%Absorption correction Semi-empirical from equivalentsMax. and min. transmission 0.8068 and 0.7541Refinement method Full-matrix least-squares on F2

Data/restraints/parameters 3983/17/260Goodness-of-fit on F2 0.981Final R indices [I > 2sigma(I)] R1 ¼ 0.0422, wR2 ¼ 0.1115R indices (all data) R1 ¼ 0.0512, wR2 ¼ 0.1215Largest diff. peak and hole 0.946 and �0.593 e�A�3

E. Ramachandran et al. / European Journal of Medicinal Chemistry 64 (2013) 179e189180

[Ni(H2-Qtsc-Et)2](NO3)2 (3), [Ni(H2-Qtsc-Ph)2](NO3)2 (4)} has beensynthesized by the direct reactions of the Ni(NO3)$6H2O withappropriate ligands. We obtained the crystalline powder for all thecomplexes which is not suitable for X-ray studies. Hence, all thecomplexes were recrystallized with ethanol and methanol solventsmixture and we got suitable single crystals for the complex 3 only.All the new air stable nickel(II) complexes were characterized byelemental analysis and various spectroscopic techniques. In addi-tion, the molecular structure of the complex 3 was determined bysingle crystal X-ray diffraction studies. The cationic nickel com-plexes are soluble inwater and in common organic solvents such asmethanol, ethanol, DMF, DMSO. The IR peak shifts of the n(C]O),n(C]N) and n(C]S) frequencies of the complexes (given inExperimental section) with respect to the free ligand indicated thecoordination of the ligand to the nickel ion in the complex [24].From the above results, it has been observed that the thio-semicarbazones behaved as ONS neutral tridentate ligand in all thecomplexes and the complexes were neutralized by two nitratemolecules. In electronic spectra the complexes the bands exhibitedin the regions of 379e400 nmwhich are attributed to the ligand-to-metal charge-transfer (LMCT) transitions.

2.2. Crystal structure of the complex, [Ni(H2-Qtsc-Et)2](NO3)2 (3)

Molecular structure of complex 3 together with the atom la-beling scheme is given in Fig. 1. The crystallographic data and theselected bond lengths and bond angles are listed in Tables 1 and 2respectively. The complex crystallized in monoclinic lattice withspace group C2/c. Ni(II) center adopted a distorted octahedral ge-ometry comprising of two equivalent neutral tridentate ligandscoordinated in a meridional fashion using cis quinoline nitrogen,trans azomethine nitrogen and cis thione sulfur atoms positionedexactly perpendicular to each other. Two distorted methanol andethanol molecules along with two nitrate counter ions are found inthe crystal lattice. The dihedral angle between the mean planes ofthe five-membered chelate ring (comprising S2, C11, N2, N1 and Niatoms) and the six-member (comprising N1, O1, C1, C2, C3 and Niatoms) one is 2.12� which clearly indicate that the planarity of the

Fig. 1. ORTEP view of the complex 3. The nitrate ion, methan

coordinated ligand is appreciable. The nitrate anions are connectedwith the complex cations through an intricate web of hydrogenbonds. The nitrogen atoms N2 and N4, hydrogen bonded to thenitrate group oxygen atoms O32 and O31respectively. The packingdiagram of the complex 3 with hydrogen bonding networks isshown in Fig. 2.

ol and ethanol molecules have been omitted for clarity.

Table 2Selected bond lengths (�A) and bond angles (�) in Ni(II) complex.

Complex 3

Ni(1)eO(1)#1 2.0490(17) O(1)#1eNi(1)eO(1) 88.68(10)Ni(1)eO(1) 2.0490(17) O(1)#1eNi(1)eN(1)#1 89.00(7)Ni(1)eN(1)#1 2.0531(18) O(1)eNi(1)eN(1)#1 88.81(7)Ni(1)eN(1) 2.0531(18) O(1)#1eNi(1)eN(1) 88.81(7)Ni(1)eS(2) 2.3850(7) O(1)eNi(1)eN(1) 89.00(7)Ni(1)eS(2)#1 2.3851(8) N(1)#1eNi(1)eN(1) 176.94(11)S(2)eC(11) 1.695(2) O(1)#1eNi(1)eS(2) 89.02(5)C(1)eN(1) 1.293(3) O(1)eNi(1)eS(2) 172.41(5)C(1)eC(2) 1.449(3) N(1)#1eNi(1)eS(2) 98.37(6)C(2)eC(6) 1.370(3) N(1)eNi(1)eS(2) 83.73(5)C(2)eC(3) 1.459(3) O(1)#1eNi(1)eS(2)#1 172.41(5)C(3)eO(1) 1.253(3) O(1)eNi(1)eS(2)#1 89.02(5)C(3)eN(3) 1.358(3) N(1)#1eNi(1)eS(2)#1 83.73(5)C(4)eN(3) 1.380(3) N(1)eNi(1)eS(2)#1 98.37(6)C(4)eC(7) 1.402(3) S(2)eNi(1)eS(2)#1 94.15(4)

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Based on the analytical, spectroscopic characterization (FT-IR,UVevisible) and single-crystal X-ray diffraction studies for thecomplex 3, an octahedral structure has been proposed for all thenew Ni(II) complexes (Scheme 1).

2.3. DNA binding studies

DNA binding is the primary pharmacological target for evalu-ating the antitumor property of any compound, and hence, theunderstanding of the interaction between DNA and complexes tobe investigated is very important [32]. Hence, the type and thestrength of the binding of the nickel(II) complexes to CT-DNAwerestudied with different physicochemical methods.

2.3.1. Electronic absorption titrationElectronic absorption spectroscopy is in general employed to

investigate the mode of interaction of metal complexes with DNA[33]. Any compound binding to DNA through intercalation usuallyresults in hypochromism with or without a small red or blue shift,due to a strong stacking interaction between the planar aromaticchromophore of the compound and the base pairs of DNA [33].Hence, the change in the electronic absorption spectra of all thenickel complexes on the addition increasing amount of CT-DNAwasstudied and Fig. 3 shows the results of absorption spectra of thenew nickel complexes in the absence and presence of CT-DNA.Upon increasing the concentration of DNA, the absorption bandsof the complexes 1 and 2 exhibited hypochromism of 30.39% and31.34% with red shift of 2 and 1 nm at 379 and 396 nm respectively,whereas the absorption bands of the complex 3 at 379 nmexhibited a hypochromism of about 32.16% with a blue shift of1 nm. However, the complex 4 at 400 nm exhibited a hypo-chromism of about 46.29% with red shift of 3 nm. These experi-mental results suggested that all the complexes bind to the DNA

Fig. 2. Packing diagram of the complex 3 with hydrogen bonding networks.

helix via intercalation, due to stacking interaction between theplanar aromatic chromophore of the complexes under investiga-tion and the base pairs of DNA. These observations were compa-rable to those reported earlier for various metallointercalators [34].Among the four Ni(II) complexes, the complex 4 showed morehypochromicity compared to the other complexes, indicating thatthe binding strength of the complex 4 is much stronger than theremaining complexes. To get further idea about the relativestrength of the binding of the new nickel(II) complexes, theirintrinsic binding constants (Kb) have been determined from thefollowing equation [35].

½DNA�=� 3a � 3f� ¼ ½DNA�=� 3b � 3f

�þ 1=Kb�

3b � 3f�

where, [DNA] is the concentration of DNA in the base pairs, 3a is theapparent absorption coefficient corresponding to Aobs/[complex], 3fis the extinction coefficient of the free complex and 3b is theextinction coefficient of the complex when fully bound to DNA.From the plot of [DNA]/( 3a � 3f) versus [DNA] (Fig. 4), intrinsicbinding constant (Kb) was calculated from the ratio of the slope andthe intercept. The Kb values have been found to be 3.53(�0.08)� 103 M�1, 5.82(�0.04) � 103 M�1, 7.30(�0.06) � 103 M�1 and3.17(�0.08) � 104 M�1 for the complexes, 1, 2, 3 and 4 respectively.From the results obtained, it has been found that complex 4 stronglybinds with CT-DNAwhen compared to that of other complexes andthe order of binding affinity is 1<2< 3< 4. Even though the resultsof the electronic spectral studies indicated that the complexes bindto DNAvia intercalation, the bindingmode has to be proved throughsome more experiments.

2.3.2. Ethidium bromide (EB) displacement studiesIn order to further confirm the binding mode and to compare

their binding affinities, ethidium bromide displacement experi-ments were carried out. EB is a planar cationic dye which is widelyused as a sensitive fluorescence probe for native DNA. Generally, EBemits intense fluorescent light in the presence of DNA due to itsstrong intercalation between the adjacent DNA base pairs [36].Hence, EB displacement technique can be used to get an indirectevidence for the DNA binding mode. The displacement technique isbased on the decrease of fluorescence resulting from thedisplacement of EB from a DNA sequence by a quencher and thequenching is due to the reduction of the number of binding sites onthe DNA that is available to the EB. Fig. 5 depicts the fluorescencequenching spectra of DNA-bound EB by the tested complexes,which illustrate that as the concentration of the complexes in-creases, the observed fluorescence intensity decreases, clearlyindicating that the EB molecules are displaced from their DNAbinding sites by the complexes under investigation [32]. Thequenching parameter has been analyzed according to the SterneVolmer equation,

F0=F ¼ Kq½Q � þ 1

where F0 is the emission intensity in the absence of complex, F isthe emission intensity in the presence of complex, Kq is thequenching constant, and [Q] is the concentration of the compound.The Kq value has been obtained as a slope from the plot of F0/F versus [Q]. From the SterneVolmer plot (Fig. 6) of F0/F versus [Q],the quenching constants (Kq) were obtained from the slope,they were 5.03(�0.01) � 103 M�1, 7.49(�0.02) � 103 M�1,1.17(�0.04) � 104 M�1 and 1.85(�0.03) � 104 M�1 for the com-plexes 1e4 respectively. Further, the apparent DNA binding con-stants (Kapp) were calculated using the following equation,

KEB½EB� ¼ Kapp½complex�

NH

O

N

HN

HN

S

RNH

O

N

HN

HN

S

R

HNO

NNH

NH

S

R

Ni.2NO3NiNO3.6H2O

MeOHReflux 1h

R = H(1), Me(2), Et (3), Ph(4)

Scheme 1. The synthetic route of the nickel(II) complexes.

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(where the complex concentration is the value at a 50% reduction inthe fluorescence intensity of EB, KEB (1.0 � 107 M�1) is the DNAbinding constant of EB, [EB] is the concentration of EB ¼ 12 mM),and they were found to be 8.76 � 105 M�1, 8.99 � 105 M�1,2.01 � 106 M�1 and 2.21 � 106 M�1 for the complexes 1e4respectively. From these results, it is seen that the complex 4 re-places the EB more than the other complexes, which is in agree-ment with the results observed from the electronic absorptionspectra. Since these changes indicate only one kind of quenchingprocess, it may be concluded that the all complexes bind to DNAviathe intercalation mode. Furthermore, the observed quenchingconstants and binding constants of the new complexes suggest thatthe interaction of all the complexes with DNA should be of inter-calation [37].

2.4. Protein binding studies

2.4.1. Fluorescence quenching of protein by nickel complexesThe interaction of drugs with plasma protein is an important

step in the transport of metal ions and metal complexes with drugsthrough the blood stream. Binding of these compounds to theproteins may lead to either a loss or enhancement of the biological

Fig. 3. Electronic spectra of the complexes 1(A), 2(B), 3(C) and 4(D) in TriseHCl buffer upon aintensities decrease upon increasing DNA concentration.

properties of the original drug and provide paths for drug trans-portation. An analysis of the binding of any compound to bovineserum albumin (BSA) is commonly detected by examining thefluorescence spectra. It is well known that the fluorescence of BSAis caused by two intrinsic characteristics of the protein, namelytryptophan and tyrosine. Commonly, changes in the emissionspectra of tryptophan can be seen in response to protein confor-mational transitions, subunit associations, substrate binding, ordenaturation. Therefore, the intrinsic fluorescence of BSA can pro-vide considerable information on their structure and dynamics andit can be utilized in the study of protein folding and associationreactions. Hence, we have studied the interaction of BSA with thenew nickel(II) complexes by fluorescence measurement at roomtemperature. A solution of BSA (1 mM) was titrated with variousconcentrations of the complexes (0e25 mM). Fluorescence spectrawere recorded in the range of 290e450 nm upon excitation at280 nm. The changes observed on the fluorescence emissionspectra of solution of BSA on the addition of increasing amounts ofthe nickel complexes are given in Fig. 7. Upon the addition of thenew complexes to the BSA solution, a significant decrease of thefluorescence intensity of BSA at 348 nm of up to 65.37, 71.25, 74.97and 81.81% from the initial fluorescence intensity of BSA

ddition of CT-DNA. [Complex] ¼ 25 mM, [DNA] ¼ 0e50 mM. Arrow shows the absorption

Fig. 4. Plots of [DNA]/( 3a � 3f) versus [DNA] for the complexes 1e4 with CT-DNA.Fig. 6. SterneVolmer plots of the EB-DNA fluorescence titration for the complexes1e4.

E. Ramachandran et al. / European Journal of Medicinal Chemistry 64 (2013) 179e189 183

accompanied by a hypsochromic shift of 6, 3, 5 and 4 nm for thecomplexes 1e4 respectively have been observed. The observed blueshift is mainly due to the fact that the active site in protein is buriedin a hydrophobic environment. These results suggested a definiteinteraction of all of the complexes with the BSA protein [32,34].Further, fluorescence quenching data were analyzed with theSterneVolmer equation and Scatchard equation. The quenchingconstant (Kq) was calculated using the plot of I0/I versus [Q] (Fig. 8).If it is assumed that the binding of complexes with BSA occurs atequilibrium, the equilibrium binding constant can be analyzed ac-cording to the Scatchard equation:

log ½ðI0 � IÞ=I� ¼ log Kbin þ n log½Q �

Fig. 5. The emission spectra of DNA-EB system, lexi ¼ 515 nm, lemi ¼ 530e750 nm, in the75 mM, [EB] ¼ 12 mM. Arrow shows the emission intensity changes upon increasing compl

where Kbin is the binding constant of the compound with BSA and nis the number of binding sites. The values of ‘n’ and Kbin have beenobtained from the plot of log (I0 � I)/I versus log [Q] (Fig. 9). Thecalculated Kq and Kbin value of the complexes 1e4 are given inTable 3. The calculated values of Kq and Kbin for all of the complexessuggested that the complexes are interacting strongly with BSA.

The fluorescence quenching of BSA by our complexes can beclassified as either dynamic or static. A dynamic quenching denotesto a process in which the fluorophore and the quencher come intocontact during the transient existence of the excited state and astatic quenching refers to fluorophoreequencher complex forma-tion in the ground state. This can be easily found out by using UVevisible absorption spectroscopy. UVevisible spectra of BSA in the

presence of the complexes 1(A), 2(B), 3(C) and 4(D). [DNA] ¼ 12 mM, [Complex] ¼ 0eex concentration.

Fig. 7. The emission spectrum of BSA (1 mM; lexi ¼ 280 nm; lemi ¼ 348 nm) in the presence of increasing amounts of the complexes 1 (A), 2 (B), 3 (C) and 4 (D) (0e25 mM). Arrowshows the emission intensity changes upon increasing complex concentration.

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absence and presence of the complexes (Fig. 10) that we have car-ried out showed that the absorption intensity of BSAwas enhancedas the complexes were added, and there was a little blue shift ofabout 1e2 nm for the new complexes. From the results obtained itis concluded that only a static interaction takes place between thetested complexes and BSA [31].

2.4.2. Characteristics of synchronous fluorescence spectraThe structural changes occurred to BSA upon the addition of

new complexes, particularly in the vicinity of the fluorophorefunctional groups can be obtained from synchronous fluorescencespectral studies [38]. It is known that the fluorescence of BSA isnormally due to the presence of tyrosine, tryptophan and

Fig. 8. SterneVolmer plot of the BSA fluorescence titration for the complexes 1e4.

phenylalanine residues and hence, spectroscopic methods areusually applied to study the conformation of serum protein. Ac-cording to Miller [39], the difference between the excitationwavelength and emission wavelength (Dl ¼ lemi � lexi) indicatesthe type of chromophores. While a higher Dl value such as 60 nm isindicative of the characteristic of tryptophan residue, a lower Dlvalue such as 15 nm is characteristic of tyrosine residue [40].Figs. 11 and 12 show the synchronous fluorescence spectra of BSAwith various concentrations of test complexes recorded atDl ¼ 15 nm and Dl ¼ 60 nm respectively. In the synchronousfluorescence spectra of BSA at Dl¼ 15, addition of the complexes tothe solution of BSA resulted in a decrease of the fluorescence in-tensity of BSA at 302 nm, up to 15.13, 39.45, 52.76 and 58.84% of the

Fig. 9. Scatchard plot of the BSA fluorescence titration for the complexes 1e4.

Table 3Quenching constant (Kq), binding constant (Kbin) and number of binding sites (n) forthe interactions of complexes with BSA.

Complexes Kq (M�1) Kbin (M�1) N

Complex 1 7.44(�0.08) � 104 3.36(�0.03) � 105 1.07Complex 2 1.03(�0.09) � 105 5.62(�0.04) � 105 1.17Complex 3 1.19(�0.16) � 105 1.51(�0.04) � 106 1.24Complex 4 1.35(�0.86) � 105 5.05(�0.09) � 106 1.36

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initial fluorescence intensity of BSA for the nickel(II) complexes 1e4respectively. However, in the case of synchronous fluorescencespectra of BSA at Dl ¼ 60, addition of the complexes to the solutionof BSA resulted in a significant decrease of the fluorescence in-tensity of BSA at 343 nm, up to 63.99, 70.26, 72.88 and 80.63% of theinitial fluorescence intensity of BSA accompanied by a small blueshift of 1 nm for the complexes 1e4 respectively. Hence, the syn-chronous fluorescence spectral studies clearly suggested that thefluorescence intensities of both the tryptophan and tyrosine weredecreased but the emission wavelength of the tryptophan residuesis blue shifted with increasing concentration of complexes. And yet,therewas no change in the emissionwavelength of tyrosine. So, it isclear that the interaction of complexes with BSA affects theconformation of tryptophan micro-region revealing that the hy-drophobicity around tryptophan residues is strengthened [26]. Thehydrophobicity observed in fluorescence and synchronous mea-surements confirmed the effective binding of all the complexeswith the BSA. The observed strong interaction of these new nick-el(II) octahedral complexes with BSA suggests that the complexesmay be fit for anticancer studies.

2.5. Evaluation of antioxidant properties of the complexes

Since the experiments conducted so far revealed that the newnickel(II) complexes exhibit good DNA and protein binding affinity,it is considered worthwhile to study the radical scavenging prop-erties of these complexes. The antioxidative properties of 2-oxo-1,2-dihydroquinoline-3-carbaldehyde Schiff bases and their metalcomplexes have attracted a lot of interests recently and have beenextensively investigated, mainly in the in vitro systems [41,42]. Theradical scavenging activities of our complexes along with stan-dards, butylated hydroxyanisole (BHA) and butylated hydrox-ytoluene (BHT) in cell free system have been examined withreference to hydroxyl radicals (OH�), DPPH radicals (DPPH�), nitricoxide (NO), superoxide anion radicals (O2

��) and their corresponding

Fig. 10. UV absorption spectra of BSA (10 mM) in the presence of the complexes 1 (A), 2(B), 3 (C) and 4 (D) (0 and 5 mM).

IC50 (determination of 50% activity) values have been tabulated inTable 4. It is to be noted that no significant radical scavenging ac-tivities were observed in all the experiments carried out withNi(NO3)2 under the same experimental conditions. The IC50 values(Table 4) indicated that all the four water soluble complexesshowed excellent antioxidant activity over the standard antioxi-dants in all the experiments except DPPH� scavenging activity. TheOH� scavenging power of the tested complexes was found to bebetter than that for the other radicals studied. However, the overallobserved antioxidant activity indicated that the terminal N-sub-stitution in the ligands does not have any appreciable influencemuch on the antioxidative properties of the resulting complexes.This may be due the structural homology (octahedral geometry) ofall the complexes.

2.6. In vitro cytotoxic activity evaluation of the compounds

The results from the previous biological studies namely, DNAbinding, BSA binding and antioxidative studies for the new watersoluble Ni(II) complexes, 1, 2, 3 and 4 encouraged us to test theircytotoxicity against a panel of human cancer cell lines, humancervical cancer cells (HeLa), human skin cancer cells (A431), humanliver carcinoma cells (Hep G2) and normal NIH 3T3 cells (mouseembryonic fibroblasts cells) with cisplatin as the positive control.Complexes were dissolved in DMSO and blank samples containingsame volume of DMSO are taken as controls to identify the activityof solvent in this cytotoxicity experiment. The results wereanalyzed bymeans of cell inhibition expressed as (50% activity) IC50values and are shown in Table 5. It is to be noted that the ligandsand the Ni(II)nitrate salt did not show any significant activity evenup to 500 mMof concentration on all the cells, which confirmed thatthe chelation of the ligand with the Ni(II) ion is the only responsiblefactor for the observed cytotoxic properties of the new complexes.The results of in vitro cytotoxic activity studies have indicated thatthe IC50 value of the complexes against NIH 3T3 mouse embryonicfibroblasts (normal cells) is found to be above 350 mM, whichconfirmed that the complexes are very specific on cancer cells. In allthe cases, the activity of the four complexes has been found to besignificantly lower than the well-known anticancer drug cisplatin.In general, Table 5 indicated that the complexes have more cyto-toxic specificity on human cervical cancer cells over the other twocancer cells. The enhanced cytotoxic properties of Ni(II) complexesover the ligands may be attributed to the extended planar structureinduced by the p / p* conjugation resulting from the chelating ofthe Ni(II) with ligand and the cationic nature of the complexes [31].

3. Conclusion

A new series of four cationic nickel(II) complexes of 2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-substituted thiosemicarbazone complexes have been synthesized and characterized. Themolecular structure of one of the complexes has been establishedby single crystal X-ray diffraction studies. The DNA interaction of allthe complexes has been evaluated by various photo physicalmethods which revealed that all the complexes can bind to DNAviaintercalation in the order of 1<2< 3< 4. Further, the experimentalresults show that the complex 4 can bind to DNA more stronglythan the other three complexes. It may be noted that the bindingaffinity of the nickel complexes with BSA showed a significantresult and the synchronous spectral studies of the complexesshowed that the complexes bound with BSA in both tyrosineand tryptophan residues. From the above experimental resultswe observed that the DNA interaction and protein bindinghas been enriched while increasing the size of the substitution atterminal nitrogen of the thiosemicarbazone ligand. The

Fig. 11. Synchronous spectra of BSA (1 mM) in the presence of increasing amounts of the complexes 1(A), 2(B), 3(C) and 4(D) (0e25 mM) in the wavelength difference of Dl ¼ 15 nm.Arrow shows the emission intensity changes upon the increasing concentration of complex.

E. Ramachandran et al. / European Journal of Medicinal Chemistry 64 (2013) 179e189186

antioxidation results indicated that all the complexes have almostcomparable values which may be due the structural homology(octahedral geometry) of all the complexes. In addition, all thecomplexes showed significant cytotoxic activity against HeLa, A431

Fig. 12. Synchronous spectra of BSA (1 mM) in the presence of increasing amounts of the comArrow shows the emission intensity changes upon the increasing concentration of complex

and Hep G2 cancer cell lines without affecting the normal NIH 3T3cells much. Moreover, the above observations are very muchcomparable with our earlier results of Ni(II) thiosemicarbazonecomplexes [31].

plexes 1(A), 2(B), 3(C) and 4(D) (0e25 mM) in the wavelength difference of Dl ¼ 60 nm..

Table 4IC50 values (in mM) calculated from various radical scavenging assays of the com-plexes (1, 2, 3 and 4) and standards (BHA and BHT).

Compound OH� NO DPPH� O2��

1 17.3 � 0.6 12.3 � 0.9 22.3 � 1.7 27.2 � 2.32 16.8 � 1.9 10.8 � 1.3 28.9 � 2.4 26.1 � 1.53 17.2 � 1.1 15.4 � 0.7 20.3 � 0.3 18.3 � 1.44 15.2 � 2.1 9.97 � 0.96 25.2 � 3.1 22.5 � 2.7BHA 312 � 10 613 � 10 9.19 � 0.52 288 � 9BHT 287 � 4 715 � 9 9.37 � 0.71 292 � 6

E. Ramachandran et al. / European Journal of Medicinal Chemistry 64 (2013) 179e189 187

4. Experimental section

4.1. Materials and instrumentation

All starting materials used in all the experiments were ofanalytical or chemically pure grade. The ligands were preparedaccording to the literature procedure [26]. Solvents were purifiedand dried according to standard procedures [43]. The reagents werepurchased commercially and used without further purificationunless otherwise noted. CT-DNA, BSA, Agarose and EB were ob-tained from SigmaeAldrich and used as received. Elemental ana-lyses (C, H, N, S) were performed on Vario EL III Elemental analyzerinstrument. IR spectra (4000e400 cm�1) for KBr disks wererecorded on a Nicolet Avatar Model FT-IR spectrophotometer.Melting points were determined with a Lab India instrument.Electronic absorption spectra were recorded using Jasco V-630spectrophotometer. Emission spectra were measured with Jasco FP6600 spectrofluorometer.

4.2. Preparation of the complexes

4.2.1. [Ni(H2L1)2](NO3)2 (1)A warm methanolic solution (20 mL) containing 2-oxo-1,2-

dihydroquinoline-3-carbaldehydethiosemicarbazone (H2L1) (123mg, 0.5 mmol) was added to a methanolic solution (20 mL) ofNi(NO3)2$6H2O (145 mg, 0.5 mmol). The resulting green color so-lution was refluxed for an hour. Green colored crystalline powderwas obtained on slow evaporation. They were filtered off, washedwith cold methanol, and dried under vacuum. Yield: 83%. MP: 271e272 �C, Anal. calcd. for C22H20N10NiO8S2 (%): C, 39.13; H, 2.99; N,20.74; S, 9.54. Found (%): C, 39.25; H, 3.01; N, 20.81; S, 9.49. IR (KBr,cm�1): 3291(ms) n(NH); 1625(s) n(C]O); 1596(s) n(C]N); 821(m)n(C]S). UVevisible (solvent 5% DMSO in buffer, nm): 379 (LMCT).

4.2.2. [Ni(H2L2)2](NO3)2 (2)Complex 2was prepared using the same procedure as described

for 1 with 2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-meth-ylthiosemicarbazone (130 mg, 0.5 mmol) and Ni(NO3)2$6H2O(145 mg, 0.5 mmol). Brown colored crystalline powder was ob-tained. Yield: 81%. MP: 283e285 �C, Anal. calcd. forC24H24N10NiO8S2 (%): C, 40.96; H, 3.44; N, 19.92; S, 9.12. Found (%):C, 40.87; H, 3.49; N, 19.97; S, 9.08. IR (KBr, cm�1): 3321(ms) n(NH);

Table 5The cytotoxic activity of the compounds.

Compound IC50 values (mM)

HeLa Hep G2 A431 NIH 3T3

1 17.7 � 1.5 32.1 � 1.5 25.2 � 1.2 374 � 112 18.4 � 2.1 30.5 � 1.2 28.6 � 1.9 365 � 103 16.5 � 1.6 26.2 � 0.7 26.4 � 1.7 390 � 84 15.2 � 1.3 29.3 � 1.9 31.6 � 3.1 355 � 8Cisplatin 12.3 � 0.7 9.87 � 0.41 11.4 � 1.3 265 � 9

1610(s) n(C]O); 1589(s) n(C]N); 823(m) n(C]S). UVevisible(solvent 5% DMSO in buffer, nm): 396 (LMCT).

4.2.3. [Ni(H2L3)2] (NO3)2 (3)Complex 3was prepared using the same procedure as described

for 1 with 2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-ethyl-thiosemicarbazone (137 mg, 0.5 mmol) and Ni(NO3)2$6H2O(145 mg, 0.5 mmol). Dark brown colored crystalline powder wasobtained. Yield: 86%. MP: 297e299 �C, Anal. calcd. forC26H28N10NiO8S2 (%): C, 42.70; H, 3.86; N, 19.15; S, 8.77. Found (%):C, 42.68; H, 4.81; N, 19.23; S, 8.73. IR (KBr, cm�1): 3152(ms) n(NH);1642(s) n(C]O); 1553(s) n(C]N); 835(m) n(C]S). UVevisible(solvent 5% DMSO in buffer, nm): 379 (LMCT). The crystallinepowder sample was recrystallized with ethanol and methanolsolvent mixture and the suitable crystals were obtained.

4.2.4. [Ni(H2L4)2] (NO3)2 (4)Complex 4was prepared using the same procedure as described

for 1 with 2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-phe-nylthiosemicarbazone (161 mg, 0.5 mmol) and Ni(NO3)2$6H2O(145 mg, 0.5 mmol). Brown colored crystalline powder was ob-tained. Yield: 89%. MP: 302e304 �C, Anal. calcd. forC34H28N10NiO8S2 (%): C, 49.35; H, 3.41; N, 16.93; S, 7.75. Found (%):C, 49.42; H, 3.47; N, 16.89; S, 7.71. IR (KBr, cm�1): 3135(ms) n(NH);1644(s) n(C]O); 1534(s) n(C]N); 864(m) n(C]S). UVevisible(solvent 5% DMSO in buffer, nm): 400 (LMCT).

4.3. Single-crystal X-ray diffraction studies

A BRUKER SMART 1000 X-ray (three-circle) diffractometer wasemployed for crystal screening, unit cell determination, and datacollection. Integrated intensity information for each reflection wasobtained by reduction of the data frames with APEX2 [44]. The X-ray radiation employed was generated from a Mo sealed X-ray tube(Ka ¼ 0.70173�A with a potential of 50 kV and a current of 40 mA)fitted with a graphite monochromator in the parallel mode(175 mm collimator with 0.5 mm pinholes). The integrationmethod employed a three dimensional profiling algorithm and alldata were corrected for Lorentz and polarization factors, as well asfor crystal decay effects with the program SADABS [45]. A solutionwas obtained readily using SHELXTL (SHELXS) [46]. All non-hydrogen atoms were refined with anisotropic thermal parame-ters. While the hydrogen atoms bound to carbon were placed inidealized positions, hydrogen bound to nitrogen were located fromFourier difference map andwere set riding on the parent atomwithidealized distances. Disordered ethanol and methanol were foundsolvated and were modeled. The hydrogen atoms attached to thecorresponding O atoms were placed only to satisfy the formula andhas no physical meaning. The structures were refined (weightedleast squares refinement on F2) to convergence [46].

4.4. DNA binding studies

All of the experiments involving the binding of compounds withCT-DNA were carried out in double distilled water withtris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodiumchloride (50 mM) and adjusted to pH 7.2 with hydrochloric acid. Asolution of CT-DNA in the buffer gave a ratio of UV absorbance ofabout 1.9 at 260 and 280 nm, indicating that the DNA was suffi-ciently free of protein. The DNA concentration per nucleotide wasdetermined by absorption spectroscopy using the molar extinctioncoefficient value of 6600M�1 cm�1 at 260 nm. The complexes weredissolved in a mixed solvent of 5% DMSO and 95% TriseHCl bufferfor all of the experiments. Absorption titration experiments wereperformed with a fixed concentration of the compounds (25 mM)

E. Ramachandran et al. / European Journal of Medicinal Chemistry 64 (2013) 179e189188

while gradually increasing the concentration of DNA (0e50 mM).While measuring the absorption spectra, an equal amount of DNAwas added to both the test solution and the reference solution toeliminate the absorbance of DNA itself. The same experimentalprocedure was followed for emission studies also. Further supportfor the complexes binding to DNA via intercalation is given throughemission quenching experiments. DNA was pretreated withethidium bromide for 30min. Then the test solutions were added tothis mixture of EB-DNA, and the change in the fluorescence in-tensity was measured. The excitation and the emission wavelengthwere 515 nm and 604e605 nm, respectively.

4.5. Protein binding studies

The excitationwavelength of BSA at 280 nm and the emission at348 nm were monitored for the protein binding studies. The exci-tation and emission slit widths and scan rates were maintainedconstant for all the experiments. Samples were carefully degassedusing pure nitrogen gas for 15 min. Quartz cells (4 � 1 �1 cm) withhigh vacuum Teflon stopcocks were used for degassing. Stock solu-tion of BSAwas prepared in 50mM phosphate buffer (pH¼ 7.2) andstored in the dark at 4 �C for further use. Concentrated stock solutionof the complexes prepared as mentioned for the DNA binding ex-periments except that the phosphate buffer was used instead ofTriseHCl buffer for all the experiments. Titrations were manuallydone by using micropipette for the addition of the complexes. Forsynchronous fluorescence spectra also, the same concentration ofBSA and the complexeswere used and the spectraweremeasured attwo different Dl (difference between the excitation and emissionwavelengths of BSA) values such as 15 and 60 nm.

4.6. Antioxidant assays

The hydroxyl radical scavenging activity of the compounds hasbeen investigated using the Nash method [47]. In vitro hydroxylradicals were generated with the Fe3þ/ascorbic acid system. Thedetection of hydroxyl radicals was carried out by measuring theamount of formaldehyde formed from the oxidation reaction withDMSO. The formaldehyde produced was detected spectrophoto-metrically at 412 nm. A mixture of 1.0 mL of an ironeEDTA solution(0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 mL of EDTAsolution (0.018%), and 1.0 mL of DMSO (0.85% DMSO (V/V) in 0.1 Mphosphate buffer, pH 7.4) was sequentially added in the test tubescontaining test solutions. The reaction was initiated by adding0.5 mL of ascorbic acid (0.22%) and incubated at 80e90 �C for15 min in a water bath. After incubation, the reaction was termi-nated by the addition of 1.0 mL of ice-cold trichloroacetic acid(17.5% W/V). Subsequently, 3.0 mL of Nash reagent was added toeach tube and left at room temperature for 15 min. The intensity ofthe color formed was measured spectrophotometrically at 412 nmagainst a reagent blank.

The assay of nitric oxide scavenging activity was based on themethod [48] where sodium nitroprusside in aqueous solution atphysiological pH spontaneously generates nitric oxide, which in-teractswithoxygen toproducenitrite ions thatcanbeestimatedusingthe Greiss reagent. Scavengers of nitric oxide compete with oxygen,leading to reduced production of nitrite ions. For the experiment,sodium nitroprusside (10 mM) in phosphate buffered saline wasmixed with a fixed concentration of the compound and incubated atroom temperature for 150min. After the incubation period, 0.5mL oftheGriess reagent containing1% sulfanilamide, 2%H3PO4, and0.1%N-(1-naphthyl) ethylenediamine dihydrochloride was added. Theabsorbance of the chromophore formed was measured at 546 nm.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavengingactivity of the compounds was measured according to the method

of Blois [49]. The DPPH radical is a stable free radical having a lmaxat 517 nm. A fixed concentration of the experimental compoundwas added to a solution of DPPH in methanol (125 mM, 2 mL), andthe final volume was made up to 4 mL with double distilled water.The solution was incubated at 37 �C for 30 min in the dark. Thedecrease in absorbance of DPPH was measured at 517 nm.

The superoxide anion radical scavenging assay was based on thecapacity of the compounds to inhibit formazan formation byscavenging the superoxide radicals generated in a riboflavin-light-nitroblue tetrazolium system [50]. Each 3 mL reaction mixturecontained 50 mM sodium phosphate buffer (pH 7.6), 20 mg ofriboflavin, 12 mM EDTA, 0.1 mg of nitroblue tetrazolium, and 1 mLof test solution (20e100 mg/mL). The reaction was started by illu-minating the reaction mixture with different concentrations of thecompounds for 90 s. Immediately after illumination, the absor-bance was measured at 590 nm. The entire reaction assembly wasenclosed in a box lined with aluminum foil. Identical tubes with thereaction mixture kept in the dark served as blanks.

For the above four assays, all of the tests were run in triplicate,and various concentrations of the compounds were used to fix aconcentration at which the compounds showed in and around 50%of activity. In addition, the percentage of activity was calculatedusing the formula, % of suppression ratio ¼ [(A0 � AC)/A0] � 100. A0

and AC are the absorbance in the absence and presence of the testedcompounds, respectively. The 50% activity (IC50) can be calculatedusing the percentage of activity.

4.7. In vitro anticancer activity evaluation by MTT assays

Cytotoxicity studies of the nickel complexes along with cisplatinwere carried out on human cervical cancer cells (HeLa), humanliver carcinoma cells, (Hep G2) human skin cancer cells (A431) andNIH 3T3 normal cells (mouse embryonic fibroblasts), which wereobtained from National Centre for Cell Science, Pune, India. Cellviability was carried out using the MTT assay method [51]. TheHeLa, Hep G2, A431, and cells were grown in Eagles minimumessential medium containing 10% fetal bovine serum (FBS), whileNIH 3T3 fibroblasts were grown in Dulbeccos modified Eaglesmedium (DMEM) containing 10% FBS. For the screening experi-ment, the cells were seeded into 96-well plates in 100 mL of therespective medium containing 10% FBS, at a plating density of10 000 cells/well, and incubated at 37 �C, under conditions of 5%CO2, 95% air, and 100% relative humidity for 24 h prior to theaddition of complexes. The complexes were dissolved in DMSO anddiluted in the respective medium containing 1% FBS. After 24 h, themedium was replaced with the respective medium with 1% FBScontaining the compounds at various concentrations and incu-bated at 37 �C under conditions of 5% CO2, 95% air, and 100%relative humidity for 48 h. Triplication was maintained, and themedium not containing the compounds served as the control. After48 h, 10 mL of MTT (5 mg/mL) in phosphate buffered saline (PBS)was added to each well and incubated at 37 �C for 4 h. The mediumwith MTT was then flicked off, and the formed formazan crystalswere dissolved in 100 mL of DMSO. The absorbance was thenmeasured at 570 nm using a microplate reader. The percentage ofcell inhibition was determined using the following formula, and agraph was plotted with the percentage of cell inhibition versusconcentration. From this, the IC50 value was calculated: %inhibition ¼ [mean OD of untreated cells (control)/mean OD oftreated cells (control)] � 100.

Acknowledgments

Council of Scientific and Industrial Research (CSIR), New Delhi,India [Grant No. 21(0745)/09/EMR-II] for the financial assistance

E. Ramachandran et al. / European Journal of Medicinal Chemistry 64 (2013) 179e189 189

received and for the award of SRF to E. Ramachandran is gratefullyacknowledged.

Appendix A. Supplementary material

Crystallographic data for the structure reported in this paperhave been deposited with the Cambridge Crystallographic DataCentre (CCDC) as supplementary publication number CCDC-898171for the nickel complex 3. Copies of the data can be obtained free ofcharge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK;Tel.: þ44-1223-336408; Fax: þ44-1223-336003; e-mail: deposit@ccdc.cam.ac.uk; Web site http://www.ccdc.cam.ac.uk).

Appendix B. Supplementary data

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.ejmech.2013.03.059.

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