Journal of Molecular Structure 1079 (2015) 337–346

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Journal of Molecular Structure

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Synthesis, characterization and biological studies of homo andhetero-binuclear 13-membered pentaaza bis (macrocyclic) complexes

http://dx.doi.org/10.1016/j.molstruc.2014.08.0360022-2860/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author.E-mail address: takhan213@gmail.com (T.A. Khan).

Hina Zafar a, Abdul Kareem a, Asif Sherwani b, Owais Mohammad b, Tahir Ali Khan a,⇑a Division of Inorganic Chemistry, Department of Chemistry, Aligarh Muslim University, Aligarh 202002, Indiab Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India

h i g h l i g h t s

� A new series of homo and heterobinuclear 13-membered pentaaza bis(macrocyclic) complexes.� 1H, 13C and 119Sn NMR of the

complexes.� XRD analyses.� Thermal analyses (TGA/DTA).� In vitro anticancer activity against

different human cancer cells lines:MCF7, Hep3B and HeLa.

g r a p h i c a l a b s t r a c t

Characterization and anticancer activities of the homo and hetero-binuclear 13-membered pentaaza bis(macrocyclic) complexes. Where: M0 = Co(II), Ni(II), Cu(II) or Sn(II) and X = Cl and NO3.

a r t i c l e i n f o

Article history:Received 17 April 2014Received in revised form 19 August 2014Accepted 19 August 2014Available online 9 September 2014

Keywords:Bis (macrocyclic) complexesSpectral studiesThermal analysesAnticancer activity

a b s t r a c t

A new series of homo and hetero binuclear 13-membered pentaaza bis (macrocyclic) complexes,[MM0LX4], [M = Cu(II), M0 = Cu(II), Co(II), Ni(II) and Sn(II); L = ligand and X = Cl or NO3] have been synthe-sized by the template reaction of dichloro/dinitrato diphenyl sulphone 1,3,6,9,12-tetra hydro pentaaza-cyclo pantane copper (II) complexes with formaldehyde, triethylenetetraamine, and respective metalsalts in 1:2:1:1 molar ratio. The complexes have been characterized by elemental analyses, molar con-ductance measurements, ESI-mass, 1H, 13C and 119Sn NMR, IR, electronic and EPR spectral studies. Theresults of elemental analyses, ESI-mass and conductivity measurements confirmed the stoichiometryof the complexes while the characteristic absorption bands and resonance peaks in IR and NMR spectraconfirmed the formation of macrocyclic frameworks of the complexes. These studies showed octahedralgeometry around the metal ion. The thermal stability of copper complexes was also studied by TGA andDTA analyses. Some complexes of this series were also studied for their in vitro anticancer activity againstcancer cells lines: Hep3B, MCF7, and HeLa. The recorded IC50 values for the tested complexes show mod-erate to good cytotoxicity against these cancer cell lines.

� 2014 Elsevier B.V. All rights reserved.

Introduction

Metallamacrocyclic complexes have become an area ofparticular interest due to their appealing structures which can

338 H. Zafar et al. / Journal of Molecular Structure 1079 (2015) 337–346

act as highly specific hosts used for selective recognition of neutralmolecules, cations and anions [1,2]. Bis (macrocyclic) complexes oftransition metals have attracted much attention as promisingobjects in coordination and supramolecular chemistry [3]. There-fore, efficient novel metal-based drugs with numerous advantagesover organic drugs have been developed [4]. A number of palla-dium complexes with aromatic nitrogen and nitrogen–nitrogencontaining ligands have shown very promising antitumor charac-teristics [5]. Medicinal inorganic chemistry is an area of growinginterest owing to the fact that inorganic pharmaceuticals play akey role in clinical therapy and diagnostics as MRI contrast agents[6,7]. In particular, metal-directed condensation reactionsinvolving coordinated amines and formaldehyde are useful forthe preparation of various types of saturated macrocyclic and bis(macrocyclic) complexes containing N–CH2–N linkages [8,9]. Bismacrocyclic compounds containing two binding subunits linkedby an aromatic spacer, i.e. phenylene bridge, pyridinyl, and p-xylyl,etc., owing to their diverse applications, viz., artificial nucleases,antiviral agents, and HIV antagonists, and they hold promise fora variety of clinical applications [10,11]. Significant progress hasbeen made in the synthesis of cyclam-based binucleating ligandsand structural and physical studies of the corresponding homoand hetero binuclear complexes [12–14]. In such compounds eachring may coordinate a metal ion and the two metal centers maydisplay independent or cooperative behavior depending upon thelength of the bridge joining the two subunits [15]. We haverecently reported the synthesis and characterization of variousmononuclear and binuclear cyclam, dioxo cyclam and other relatedpolyazamacrocyclic systems [16–19]. The copper complexes havegained a good reputation in the area of cancer chemotherapy[20,21]. The role of copper is significant as it can promote nucleicacid cleavage and therefore is used as metallodrug to cause DNAdamage [21,22]. The copper-based heteronuclear complexes dis-played significant nuclease properties [21–23]. Tin or organotincomplexes have displayed remarkable antiproliferative properties,owing to this, they have a crucial role to play in cancer chemother-apy [24–26]. In view of biological significance of copper and tincomplexes we synthesized and studied a new series of mono andbinuclear (homo and hetero binuclear) 13-membered pentaazamacrocyclic complexes of transition metals (Co, Ni and Cu) andtin, which were derived from 4,40-diaminodiphenyl sulphone andtriethylenetetraamine in presence of formaldehyde and respectivemetal salts.

Experimental

Materials and methods

Metal salts (all from Merck) were commercially available puresamples. The chemicals 4,40-diaminodiphenyl sulphone (Merck)and triethylenetetraamine (SRL, Sisco Research Laboratories) wereused as received. Methanol used as the solvent was of A. R. grade.Ficoll–Histopaque, Fetal calf serum, RPMI1640 and antibiotic–antimycotic solutions were procured from Sigma Aldrich, USA.Hep3B (Human Hepatocellular carcinoma), MCF7 (Human breastadenocarcinoma) and HeLa (Human cervical carcinoma) cell lineswere procured from Cell Repository–National Centre for CellScience Pune, (India).

Synthesis of the complexes

Synthesis of mononuclear [CuLX2] complexesIn this synthesis of dichloro/dinitrato diphenyl sulphone-

1,3,6,9,12 tetra hydro pentaazacyclo pantane copper (II), [CuLX2],complexes (L = ligand and X = Cl and NO3) a volume of 20 ml of a

methanolic solution of the CuX2�6H2O (10 mmol, 1.7 g) was placedin a three necked round bottom flask, then methanolic solutions oftriethylenetetraamine (10 mmol, 1.48 ml) and formaldehyde(20 mmol, 2 mL) were added dropwise with continuous stirringfollowed by a methanolic solution of 4,40-diaminodiphenyl sul-phone (10 mmol, 2.4 g) in 1:2:1 molar ratio. The resulting mixturewas stirred for about 4 h and the solid product thus obtained wasfiltered, washed several times with methanol and dried in vacuo.

Synthesis of binuclear [Cu2LX4] complexesIn this synthesis of dichlorochloro/dinitrato [1,1-diphenylesul-

phone bis (3,6,9,12-tetrahydro-1,3,6,9,12 pentaazacyclotridecane)copper (II), [Cu2LX4], complexes, a volume of 25 ml of acetonitrilesolution of [CuLX2] complex (5 mm) was placed in a round bottomflask. After this methanolic solutions of copper salt (10 mm, 1.7 g),triethylenetetraamine (10 mmol, 1.48 ml), and formaldehyde(20 mmol, 2 mL) were added dropwise. The reaction mixture wasstirred for 6 h, followed by refluxing for 4 h. A solid product thusobtained was filtered off, washed several times with methanoland dried in vacuo.

Synthesis of binuclear [CuCoLX4] complexesA similar procedure was adopted as above except that in place

of the copper salts now cobalt salts CoX2�6H2O (X = Cl or NO3) wereused.

Synthesis of binuclear [CuNiLX4] complexesA similar procedure was adopted as above except that in place

of the copper salts now nickel salts NiX2�6H2O (X = Cl or NO3) wasused.

Synthesis of binuclear [CuSnLX4] complexA similar procedure was adopted as above except that in place

of the copper salts now stannous salt SnCl2�2H2O was used.

Measurements

The elemental analyses for carbon, hydrogen and nitrogen wereobtained from the Central Drug Research Institute (Lucknow,India). The 1H, 13C and 119 Sn NMR recorded on a JEOL GSX 300MHz FX-1000 spectrometer using DMSO-d6 as a solvent and tetra-methylsilane (Me4Si) as an internal standard. The IR spectra wererecorded in the region 4000–400 cm�1 by using FT-IR Perkin Elmer(2400) spectrometer and in the region 700–30 cm�1 by usingFT-IR/FIR Perkin Elmer (Frontier) spectrometer. Electro sprayionization-mass spectra (ESI-MS) of the complexes were recordedon a Q-Tof micro Mass Spectrometer. Metal and chloride ions weredetermined volumetrically [27] and gravimetrically [28], respec-tively. The electronic spectra of the compounds in DMSO wererecorded on a Pye-Unicam 8800 spectrophotometer at room tem-perature. The EPR spectra were recorded on a JEOL JES RE2X EPRspectrometer. The electrical conductivities were obtained witha systronics type 302 conductivity bridge equilibrated at25 ± 0.01 �C using 10�3M solution. The TGA and DTA wereperformed on a Schimadzu Thermal Analyze under nitrogenatmosphere using alumina powder as reference. The XRD patternand different parameters were collected with a Rigaku, Mini FlexII powder diffractometer using X-ray radiation.

Cell viability assay (MTT)

The fresh Blood (10–15 mL) provided by Blood bank of Jaw-aharlal Nehru Medical College, A.M.U, Aligarh (India), was dilutedwith the same volume of phosphate buffered saline (PBS). Thediluted blood sample was carefully layered on Ficoll–Histopaque.

H. Zafar et al. / Journal of Molecular Structure 1079 (2015) 337–346 339

The mixture was centrifuged under at 900 � g for 10 min at 20–22 �C. The undisturbed lymphocyte layer was carefully transferredout. The lymphocyte was washed and pelleted down with threevolumes of PBS for twice and resuspended RPMI-1640 media with10% antibiotic and antimycotic solution v/v fetal calf serum (FCS).Cell counting was performed to determine the PBMC cell numberwith equal volume of trypan blue [29,30]. The anticancer activityin vitro was measured using the 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. The HeLa (HumanCervical Carcinoma), Hep3B (Human Hepatocellular carcinoma),MCF7 (Human Breast Adenocarcinoma) and PBMC (Blood Periphe-ral Mononuclear) cells were maintained in RPMI 1640 culturemedium supplemented with 10% heat-inactivated fetal calf serum.The cells were plated at a density of 5 � 103 cells per well in a96-well plate, and cultured for 24 h at 37 �C. The cells weresubsequently exposed to drugs. The plates were incubated for48 h, and cell proliferation was measured by adding 20 lL ofMTT dye (5 mg/mL in phosphate buffered saline) per well. The

Scheme 1. Formation and suggested structure of 13-membered bis (macrocyclic) binucl[MM0LX4] respectively, [M = Cu(II), M0 = Co(II), Ni(II), Cu(II) or Sn(II), L = ligand and X = C

plates were incubated for a further 4 h at 37 �C in a humidifiedchamber containing 5% CO2. Formazan crystals formed due toreduction of dye by viable cells in each well were dissolved in150 lL dimethyl sulfoxide, and absorbance was read at 570 nm.The absorption values were expressed as the cell percent viability,according to the control group as 100%.

Results and discussion

A new series of 13-membered bis (macrocyclic) complexes of thetype [MM0LX4] (M = Cu(II) M0 = Cu(II), Co(II) Ni(II) or Sn(II), L = ligandand X = Cl/NO3) were synthesized as shown in Scheme 1. All thecomplexes were polycrystalline, stable in atmosphere and fairly sol-uble in DMF (Dimethylformamide) and DMSO (Dimethyl sulfoxide)and slightly soluble in MeCN. The formation of macrocyclic com-plexes was deduced on the basis of results of elemental analyses,molecular ion peak in mass spectra (Table 1), characteristic bands

ear transition homo and hetro metal pentaaza macrocyclic complexes [MMLX4] andl and NO3].

Table 1Elemental analyses, m/z values, colors, yields, molar conductance and melting points of the complexes.

Complexes m/z Found(calc.)

Color Yield (%) Found (calc.)% Molar Conductivity(ohm�1cm2mol�1)/m.p.(�C)

M C H N Cl O

[CuL(NO3)2] 606.40 10.32 38.90 4.50 18.55 – 20.90 13.2/330C20H30N8SCuO8 (606.1) Light green [72] (10.48) (39.59) (4.98) (18.49) (21.11)[CuLCl2] 550.00 12.40 43.00 5 .48 15.30 11.90 – 15.4/280C20H30N6 O2SCuCl2 (553.01) Green [70] (12.82) (43.30) (5.49) (15.20) (12.82)[Cu2L(NO3)4 ] 963.79 13.17 34.70 4.90 20.20 – 23.40 17.5/320C28H48N14SCu2O14 (963. 86) Dark green [70] (13.18) (34.88) (5.00) (20.34) (23.23)[Cu2LCl4 ] 852.00 16.66 45.6 6.29 16.10 18.61 – 15.5/300C28H48N10 O2SCu2Cl4 (857.62) Green [75] (16.76) (44.38) (6.38) (16.32) (18.71)[CuCoL(NO3)4] 959.29 12.62 35.01 5.05 20.30 – 23.17 21.5/320C28H48N14SCuCoO14 (959.34) Dark green [69] (12.76) (35.06) (5.04) (20.45) (23.19)[CuCoLCl4] 853.21 16.25 44.60 5.91 16.90 18.50 – 20.5/300C28H48N10 O2SCuCoCl4 (853.00) Forest green [75] (16.26) (44.68) (6.42) (16.41) (18.73)[CuNiL(NO3)4] 951.00 12.72 35.01 5.05 20.30 – 23.17 22.5/320C28H48N14SCuNiO14 (959.10) Dark green [69] (12.74) (35.06) (5.04) (20.45) (23.19)[CuNiLCl4] 852.79 16.25 44.60 5.91 16.90 18.50 – 20.5/300C28H48N10 O2SCuNiCl 4 (852.76) Dark green [73] (16.23) (44.68) (6.42) (16.41) (18.73)[CuSnLCl4] 910.00 Sky blue [60] 19.00 36.40 5.10 14.90 15.00 – 19.5/300C28H48N10 O2SCuSnCl4 (912.98) (19.93) (36.80) (5.20) (15.34) (15.55)

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in FT-IR, and resonance signals in the 1H NMR. The overall geometryof the complexes was inferred from the observed values of magneticmoments and the position of the bands in electronic spectra. Themolar conductance data of the 10�3 M solutions of the complexesmeasured in DMSO indicate the non-electrolytic nature of all thecomplexes [31]. The analytical results (Table 1) are in support of pro-posed stoichiometry.

ESI-mass spectra

Mass spectra of 13-membered mono and binuclear complexesshowed m/z peaks at 606.40, 550.00, 963.79, 852.00, 959.29,853.21, 951.00, 852.79, 910.00 that corresponded to C20H30N8

SCuO8, C20H30N6O2SCuCl2, C28H48N14SCu2O14, C28H48N10O2SCu2Cl4,C28H48N14SCuCoO14 and C28H48N10O2SCuCoCl4, C28H48N14SCuNiO14,C28H48N10O2SCuNiCl4, C28H48N10O2SCuSnCl4 moieties, respectively.The proposed molecular formulae of synthesized complexes wasconfirmed by comparing their molecular formula weights withrespective m/z values (Table 1) that are in good agreement foraforementioned complexes shown in Fig. 1.

FT-IR spectra

The preliminary information regarding the formation of pro-posed macrocyclic complexes has been obtained by comparing the

Fig. 1. Mass spectra of the complexes:

IR spectra (Table 2) of complexes with their starting materials. Astrong band appeared in IR spectra in the region 3210–3260 cm�1

of all the bimetallic complexes which may be assigned [32,33] tot(N–H) of the coordinated secondary amines moiety. The appear-ance of a doublet in the range about 3350 cm�1 in the mononuclearcopper complexes may be due to the t(N–H) of the uncoordinatedprimary amino group of the 4,4-diaminodiphenyl sulphone moiety.The IR spectra show no bands assignable to primary amino groups inthe bis (macrocyclic) bimetallic complexes. All the complexesshow a strong band in the region 1140–1180 cm�1 assignable tothe t(C–N). The weak-intensity band appearing in the region1430–1450 cm�1 is assigned to d(C–H) vibrations for secondaryamine. All of the complexes show strong bands in the regions2900–2920 which may correspond to t(C–H) vibrations. The coordi-nation of nitrato groups has been ascertained by appearance ofbands in 230–240 cm�1 regions, which may reasonably be assigned[34,35] to t(M–O) and t(M0–O) of the O–NO2 group in [MM0L(NO3)4]complexes [36,37]. In the chloro complexes the bands appearing inthe region 310–315 cm�1 and 315–320 cm�1 are assigned to thet(M–Cl) and t(M0–Cl) stretching vibrations respectively.

13C NMR spectra

The 13C NMR spectra of the bis (macrocyclic) complexesrecorded in DMSO-d6 at room temperature gave 13C NMR signals

(a) [CuLCl2] and (b) [CuNiL(NO3)4].

Table 2IR spectral data of the complexes (cm�1).

Complexes t(N–H) t(C–H) t(C-N) d(C–H) t(M–N)/t(M0–N) t(M–Cl)/t(M0–Cl) t(M-O)/t(M0–O)

[CuL(NO3)2] 3225 2900 1140 1440 420 – 235[CuLCl2] 3220 2910 1150 1450 410 310 –[Cu2L(NO3)4] 3240 2900 1160 1450 440/450 – 235/240[Cu2LCl4] 3240 2920 1180 1440 450/450 312/320 –[CuCoL(NO3)4] 3220 2920 1160 1430 445/445 – 230/235[CuCoLCl4] 3210 2910 1170 1450 450/440 310/315 –[CuNiL(NO3)4] 3255 2910 1170 1435 450/445 – 240/235[CuNiLCl4] 3225 2920 1160 1430 445/440 310/315 –[CuSnLCl4] 3260 2920 1180 1450 450/440 315/320 –

Fig. 2. 13C NMR spectra of bis (macrocyclic) complexes: (a) [CuCoLCl4] and (b) [CuNiL(NO3)4].

H. Zafar et al. / Journal of Molecular Structure 1079 (2015) 337–346 341

characteristic of carbon atoms of the macrocyclic skeleton at theirappropriate positions corresponding to the proposed structure.However, the positions of resonance signals were found to beslightly downfield shifted in complexes [38,39] (Fig. 2).

1H NMR spectra

The 1H NMR spectra of bis (macrocyclic) complexes recorded inDMSO-d6 show multiplets in the region 3.32–3.97 ppm which

Fig. 3. 1H NMR spectra of bis (macrocyclic) complexes: (a) [CuCoLCl4] and (b) [CuNiL(NO3)4].

342 H. Zafar et al. / Journal of Molecular Structure 1079 (2015) 337–346

correspond [40] to the secondary amino (C–NH–C, 8H) and meth-ylene (N–CH2–N, 8H) protons, respectively. Another multipletobserved for the complex in the 2.37–2.87 ppm region may bedue to the methylene (N–CH2–C, 24H) protons of the triethylene-tetramine moiety. Another multiplet observed for the complexesin the region 7.39–8.50 ppm corresponds to the sulphone aromaticring protons [41] and the relative intensity for the biphenyl pro-tons is expected to show 1:l ratio (Fig. 3).

119Sn NMR spectra

The 119Sn NMR spectroscopy has been found to be useful tech-nique for structure elucidation and the nature of coordination oftin atom in complexes. The 119Sn NMR spectrum of the complex[CuSnLCl4] displayed a signal at d �170.05 ppm (Fig. 4) which isin support of octahedral geometry around tin atom [42].

EPR spectra

The EPR spectra of the copper complexes were recorded at roomtemperature. The hyperfine lines could not be resolved which indi-cate the strong dipolar and exchange interaction between copper(II) ions in the unit cell [43,44]. A single broad signal with two gvalues, consistent with an axial symmetry (gII > g\) in the moleculewas exhibited. This indicates [44] essentially a dx2 � y2 groundstate for the copper (II) ion. The reported complexes gave gII andg\ values in the regions 2.15–2.28 and 2.05–2.11, respectively,which are tabulated in (Table 3). The g values are related by theexpression G = (gII � 2)/(g\ � 2), which measures the exchangeinteraction between copper centres in the polycrystalline solids.The calculated G values for these complexes appeared in the range2.18–3.00 which suggest the existence of considerable exchangeinteraction. It should be noted [45] that for an ionic environment

Fig. 4. 119Sn spectra of the [CuSnLCl4] complex.

Table 3Electronic spectral bands with their assignments and EPR data of the complexes.

Complexes Electronicspectra (cm�1)

Assignments EPR parameters

gII g\ G

[CuL(NO3)2] 16,000 2B1g ?2Eg – – –

19,100 2B1g ?2B2g

[CuLCl2] 18,600 2B1g ?2Eg 2.28 2.10 2.80

19,400 2B1g ?2B2g

[Cu2L(NO3)4] 18,800 2B1g ?2Eg 2.28 2.11 2.50

20,600 2B1g ?2B2g

[Cu2LCl4] 18,600 2B1g ?2Eg – – –

22,200 2B1g ?2B2g

[CuCoL(NO3)4] 18,800 4T1g (F) ? 4T1g 2.24 2.11 2.1820,600 4T1g (F) ? 4A2g

[CuCoLCl4] 17,600 4T1g (F) ? 4T1g – – –21,600 4T1g (F) ? 4A2g

[CuNiL(NO3)4] 18,600 3A2g ?3T1g 2.15 2.05 3.00

19,900 3A2g ?3T1g

[CuNiLCl4] 19,800 3A2g ?3T1g – – –

20,600 3A2g ?3T1g

[CuSnLCl4] 16,600 2B1g ?2Eg – – –

17,800 2B1g ? 2A1g

H. Zafar et al. / Journal of Molecular Structure 1079 (2015) 337–346 343

gII > 2.30. The fact that gII < 2.30 indicates (Table 3) that the com-plexes exhibit considerable covalent character.

Electronic spectra

The electronic spectra of mononuclear and binuclear coppercomplexes show two spin allowed transitions in the 16,000–18,800 cm�1 and 19,100–22,200 cm�1 range assignable to the2B1g ?

2Eg and 2B1g ?2B2g transitions respectively, corresponding

to an octahedral geometry around the metal ion [46]. The cop-per–cobalt bis (macrocyclic) complexes show two bands appearingin the 17,600–18,800 and 20,600–21,600 cm�1 regions assignableto the 4T1g(F) ? 4T1g(P) and 4T1g(F) ? 4A2g(F) transitions, respec-tively, consistent with an octahedral geometry [47] around metalions. The copper–nickel bis (macrocyclic) complexes show twobands in their electronic spectra centred in the 18,600–19,800and 19,900–20,600 cm�1 regions which may reasonably beassigned to the 3A2g ?

3T1g and 3A2g ?3T1g transitions, respec-

tively, indicating an octahedral geometry around the metals ions[48]. The copper-tin complex show bands at 16,600 cm�1 and17,800 cm�1 assignable to the 2B1g ?

2Eg and 2B1g ?2A1g

transitions, respectively which is consistent with an octahedralgeometry around Cu(II) ion [49](Table 3).

X-ray diffraction analysis

The X-ray powder diffractions (XRD) were used to determinethe type of structure ordering of the 13-membered pentaaza bis(macrocyclic) complexes. The XRD pattern of the mono and binu-clear copper complexes, [CuLCl2], [CuCuLCl4] and [CuSnLCl4]recorded from polycrystalline sample exhibited some sharp peaksin the spectrum (Fig. 5) correlated to the crystalline behavior ofmono and binuclear complexes. Other parameters were calculatedfrom Fig. 5 which are tabulated in Table 4.

Thermal Analyses (TGA/DTA)

The thermal stability of the complexes was investigated usingTGA Fig. 6. The thermo gravimetric analyses (TGA) were carriedout at a heating rate of 20 �C/min in a nitrogen atmosphere overa temperature range of 20–800 �C. The mono and binuclear coppercomplexes have a different decomposition process. The relativehigh thermal stability observed in all thermally investigatedcomplexes may correspond to the absence of any solvent/watermolecules in/out of the coordination sphere. This is proposed refer-ring to the higher thermal stability observed for all investigatedcomplexes in which the first decomposition step is started at arelatively higher temperature. The thermo grams of TGA of monoand binuclear complexes exhibit decomposition between 335and 420 �C, which may be due to the removal of the coordinatedchloride ion. The second decomposition stage followed the previ-ous one ended at 800 �C, may be attributed to the removal of theorganic part of the compounds into their corresponding oxides.Further horizontal constant curve may be due to the presence ofmetal oxides residue in the remaining part. The differentialthermal analysis (DTA) curves (Fig. 6) of the mono and binuclearcopper complexes show endothermic peak in the temperaturerange 180–200 �C assigned to loss of coordinated chloride ion.The sharp decomposition corresponding to the loss of organicmoiety in complexes can be seen in the DTA curves that containedone sharp exothermic peak falling in the range of 350–415 �C andindicate the formation of metal oxides [50,51].

Cytotoxic potential of macrocyclic complexes

The cytotoxic potential of the inhouse synthesized macrocycliccomplexes against Hep3B, HeLa and MCF7 cell lines was assessedby determining the number of viable cells surviving after incuba-tion with the macrocyclic complexes for the stipulated time periodusing the MTT method as given in the experimental section. The

Fig. 5. XRD pattern for macrocyclic complexes: (a) [CuLCl2], (b) [CuCuLCl4] and (c)[CuSnLCl4].

Fig. 6. TGA/DTA curve for 13-membered macrocyclic complexes: (a) [CuLCl2] and(b)[Cu2LCl4].

344 H. Zafar et al. / Journal of Molecular Structure 1079 (2015) 337–346

cytotoxicity assay suggests a variable cytotoxicity of inhouse syn-thesized complexes for Hep3B, HeLa as well as MCF7 cell lineswhich can be attributed to the intrinsic anticancer property ofthese macrocyclic complexes. Curves of dose-dependent effectsof all the complexes on cell viability of different human cancer celllines (Hep3B, MCF7 and HeLa) and normal cells (PBMC) are dis-played in Fig. 7. Experiments revealed that there was substantialincrease in cytotoxicity in the cell lines with increasing exposureto drug concentration. The absorption values were expressed asthe cell viability in percent, according to the control group as100%. Assays were performed in triplicate on three independentexperiments. The concentration required for 50% inhibition of cellviability (IC50) was calculated using the software ‘‘Prism 3.0’’. Foreach of the tested complexes IC50 was calculated and the results

Table 4The XRD parameters of [CuLCl2], [Cu2LCl4] and [CuSnLCl4] complexes.

Identification Complex [CuLCl2] [CuCu

Empirical formula C20H30N6 O2SCuCl2 C28H4

Formula weight (553.1) (g mol�1) (852.Wave length 1.670598 Å 1.670Crystal system (Powder) Hexagonal HexaLattice parameter a = 4.4568 Å, b = 5.9168 Å, c = 5.2089 Å a = 5.Lattice parameter Alpha = 90�, Beta = 90�, Gama = 120� Alpha2h min–max 5–65.00 00.00Lattice type p p

are summarized in Table 5. It is apparent from the IC50 values thatall the tested complexes show moderate to good cytotoxicityagainst different human cancer cell lines. The complex [CuLCl2],led to remarkable potency with IC50 values of 15.7 ± 1.3 lM againstMCF7 and 18.56 ± 1.5 lM against HeLa cell lines. The another com-plex [Cu2LCl4] is more effective against Hep3B and MCF7 cell lineswith IC50 values of 13.4 ± 0.9 lM and 19.4 ± 1.9 lM, respectively.The complex [CuCoLCl4] is effective against Hep3B cells lines withIC50 values of 18.4 ± 2.6 lM. It is remarkable that among all thesecomplexes the bimetallic complex of tin with copper, [CuSnLCl4],exhibits comparatively high cytotoxicity against all cancerous celllines with IC50 values of 11.9 ± 2.8 lM against Hep3B,12.53 ± 1.9 lM against MCF7 and 17.2 ± 1.7 lM against HeLa celllines, respectively. Further, IC50 values for normal cells, (PBMC),are comparatively much higher than the cancerous cell linesindicating that normal cells are almost unaffected by these testedcompounds. It is reported that tin and organotin complexes exhibit

LCl4] [CuSnLCl4]

8N10 O2SCu2Cl4 C28H48N10 O2SCuSnCl4]0) (g mol�1) (910.0) (g mol�1)598 Å 1.6705 Ågonal Hexagonal4568 Å, b = 6.9168 Å, c = 5.4089 Å a = 5.4568 Å, b = 6.9168 Å, c = 5.4089 Å

= 90�, Beta = 90�, Gama = 120� Alpha = 90�, Beta = 90�, Gama = 120�–60.00 00.00–75.00

p

Fig. 7. Dose-dependent effects of [CuL(NO3)2], [CuLCl2], [Cu2LCl4], [CuCoLCl4] and [CuSnLCl4] complexes on different human cancer cell lines i.e., Hep3B, HeLa and MCF7 andnormal cells (PBMC).

Table 5The IC50 values (lmol L�1) of the complexes with reference drugs.

Complexes Hep3B MCF7 HeLa PBMC

[CuL(NO3)2] 36.9 ± 1.4 29.7 ± 2.5 39.1 ± 1.9 41.7 ± 2.5[CuLCl2] 23.6 ± 2.9 15.7 ± 1.3 18.56 ± 1.5 51.2 ± 1.1[Cu2LCl4] 13.4 ± 0.9 19.4 ± 1.9 21.2 ± 2.9 49.1 ± 2.9[CuCoLCl4] 18.4 ± 2.6 21.2 ± 1.4 20.2 ± 2.6 46.5 ± 1.2[CuSnLCl4] 11.9 ± 2.8 12.53 ± 1.9 17.2 ± 1.7 48.2 ± 1.8(Doxo)a 2.3 ± 0.6 2.9 ± 2.5 3.1 ± 0.7 7.1 ± 1.55-FUa 4.6 ± 1.2 4.8 ± 1.7 5.2 ± 0.8 11.7 ± 1.9

a Doxorubicin (Dox) and 5-Fluorouracil are drugs of reference.

H. Zafar et al. / Journal of Molecular Structure 1079 (2015) 337–346 345

significant in vitro antiproliferative activity which, in some cases, iseven higher than the corresponding activity of cisplatin and someother drugs used for clinical treatment in cancer chemotherapy

[20,21]. Although the mechanism of this antiproliferative activityis not well established, it has been suggested that organotin (IV)compounds show antiproliferative effects through binding to thiolgroups of the proteins hence differing from the behavior of severalcytotoxic complexes of other metals, like platinum which effec-tively interact with DNA [25,26].

Concluding remarks

The physicochemical studies of the new series of 13-memberedpentaaza homo and hetero binuclear macrocyclic complexes,[MM0LX4], [M = Cu(II), M0 = Cu(II), Co(II), Ni(II) or Sn(II); L = ligandand X = Cl/NO3) indicate octahedral geometry around metal ions.In view of biological significance of copper complexes first we syn-thesized mononuclear complexes of copper. Then from these

346 H. Zafar et al. / Journal of Molecular Structure 1079 (2015) 337–346

mononuclear we synthesized binuclear Cu–Cu, Cu–Co, and Cu–Sncomplexes. Then we studied their anticancer activities with theview to compare anticancer activity of copper with other metalse.g. Co and Sn. The results of cytotoxic study by MTT assay revealedthat these mono and binuclear complexes showed a broad spec-trum of anticancer activity against the three tested human cancercell lines (Hep3B, MCF7 and HeLa). The recorded IC50 values formononuclear and binuclear copper complexes show an enhance-ment in anticancer activity in case of binuclear copper complexin comparison to mononuclear complexes. Further these resultsshow that although there is not much difference in anticanceractivity between Cu–Cu and Cu–Co bimetallic complexes but thereappears much difference in anticancer activity of Cu–Cu and Cu–Snbimetallic complexes. This indicates that anticancer activity of thebimetallic complex enhanced after replacing Cu by Sn. These stud-ies presented here provided a new structural type for the develop-ment of novel anticancer agents. Ultimately, it is comprehensiblethat further development of macrocyclic complexes of differentmetals can serve as new templates for antitumor chemotherapyand could probably be lead to more active complexes in the areaof cancer chemotherapy.

Acknowledgements

The authors thank to the Chairman, Department of Chemistry,AMU, Aligarh (India) for providing necessary research facilitiesand also Department of Applied Physics, AMU, Aligarh (India) forXRD analysis. The co-authors Hina Zafar and Asif Sherwani(Research Scholars) also thank UGC and DBT (DBT-JRF/2011-12/160) for financial assistance, respectively.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.molstruc.2014.08.036.

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