The condensation reaction of the starting material N,N'-(naphthalene-1,8-diyl)bis(2-aminobenzamide) and monobenzoylacetone in presence of different transition metal salts [Fe(II),Co(II), Ni (II), Cu(II), or Pd (II)] in 1:1:1 molar ratio gives the macrocyclic complexes i.e. naphthyl-3,4:10,11-dibenzo,7-methylene,8-methyl,6- phenyl-1,5,9,13-tetraazacyclohexadecane-5,8-diene-2,12-dione metal(II) complexes. All complexes are air stable, and insoluble in most common organic solvents. However, they are soluble in DMF or DMSO. The thermal analysis, spectral, magnetic, and elemental data (Tables 1-5) are agreed with their proposed structure. From elemental analyses, all complexes are formed in 1M:1L molar ratio. X-ray crystal determination is not possible for any of complexes due to the fact that crystals of these complexes could not be grown. The molar conductivities, recorded for 10^{- 3} M solution of the metal complexes in DMF at room temperature are given in Table 1.

The molar conductance values of complexes (1-4,6-8) indicate their non-electrolytic nature [11Chandra, S.; Gupta, L.K. EPR, IR and electronic spectral studies on Mn(II), Co(II), Ni(II) and Cu(II) complexes with a new 22-membered azamacrocyclic [N4] ligand. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2004, 60(8-9), 1751-1761.
[http://dx.doi.org/10.1016/j.saa.2003.07.011] [PMID: 15248947] , 23Geary, W.G. The use of conductivity measurements in organic solvents for the characterization of coordination compounds. Coord. Chem. Rev., 1971, 7, 81-122.
[http://dx.doi.org/10.1016/S0010-8545(00)80009-0] ]. However, complexes (5,9-11) show molar conductivity value in the range 62– 66 Ω ^-1 cm² mol^-1 in DMF 10^-3 M confirming their 1:1 electrolytic nature [23Geary, W.G. The use of conductivity measurements in organic solvents for the characterization of coordination compounds. Coord. Chem. Rev., 1971, 7, 81-122.
[http://dx.doi.org/10.1016/S0010-8545(00)80009-0] , 24Kong, D.; Xie, Y. Synthesis, structural characterization of tetraazamacrocyclic ligand, five-coordinated zinc(II) complexes. Inorg. Chim. Acta, 2002, 338, 142-148.
[http://dx.doi.org/10.1016/S0020-1693(02)01025-3] ].

Table 1
Analytical data and physical properties of the macrocyclic metal complexes

The main bands and their assignments are listed in Table 2. In the IR spectra of all complexes, the presence of a single broad band in the region 3256-3176 cm^-1 may be assigned to υ(N-H) stretching vibration. The IR spectra of all complexes show the absence of carbonyl and amino groups, which showed no bands corresponding to these groups. The appearance of new strong intensity bands at 1609–1572, 1535-1504 and 1296-1277 cm^-1 is assigned to amide-I ν(C=O), amide-II ν(C-N)+ ν(N-H)] and amide-III ν(N-H) stretching vibrations, indicating the formation of macrocyclic framework. This is in consistence with the presence of a new medium band in the region 450- 410 cm^{- 1}, assignable to ν (M-N) vibrational modes [13EL-Boraey. H.A.; Emam, S.M.; Tolan, D.A.; El-Nahas, A.M.; Structural studies and anticancer activity of a novel (N6O4) macrocyclic ligand and its Cu(II) complexes. Spectrochim. Acta A, 2011, 78, 360-370.
[http://dx.doi.org/10.1016/j.saa.2010.10.021] , 25Gupta, L.K.; Chandra, S. Spectroscopic characterization and EPR spectral studies on transition metal complexes with a novel tetradentate, 12-membered macrocyclic ligand. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2006, 65(3-4), 792-796.
[http://dx.doi.org/10.1016/j.saa.2005.12.042] [PMID: 16529988] ]. It is worth to mention that, for most complexes, the region of ν (C=N) of azomethine overlapped with a strong broad peak of amide-I ν (C=O). The observed value of absorption band due to amide-I ν(C=O), azomethine ν(C=N) is lower than that that usually occur for these groups which supports the coordination of these groups to the metal atom [26Keypour, H.; Arzhangi, P.; Rahpeyma, N.; Rezaeivala, M.; Elerman, Y.; Büyükgüng, O.; Valencia, L.R.; Khavasi, H. Synthesis and characterization of Ni(II), Cu(II) and Zn(II) complexes with new macrocyclic Schiff base ligands containing piperazine moiety. J.Mol. Struct., 2010, 977, 6-11.
[http://dx.doi.org/10.1016/j.molstruc.2010.04.036] , 27Abou-Hussein, A.A.A.; Linert, W. Synthesis, spectroscopic and biological activities studies of acyclic and macrocyclic mono and binuclear metal complexes containing a hard-soft Schiff base. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2012, 95, 596-609.
[http://dx.doi.org/10.1016/j.saa.2012.04.057] [PMID: 22580137] ]. The small intensity bands present in the region 3061–2913 cm^-1 may be assigned to the ν(C–H) stretching vibrations of the aromatic and aliphatic group.

All complexes display a strong as well as broadband at 3429-3321 cm ^-1 due to ν(OH) vibration. The appearance of new medium intensity band in the region 541-498 cm ^-1, assigned to ν(M-O) [25Gupta, L.K.; Chandra, S. Spectroscopic characterization and EPR spectral studies on transition metal complexes with a novel tetradentate, 12-membered macrocyclic ligand. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2006, 65(3-4), 792-796.
[http://dx.doi.org/10.1016/j.saa.2005.12.042] [PMID: 16529988] ]. The additional bands observed due to anions (Table 2). Complex (3) shows IR bands at 1383 (ν₅), 1296(ν₁), 1041(ν₂) cm^-1. The value of Δ(ν₅−ν₁), stretching vibration is, separated by 87 cm^-1 suggesting unicoordinated nitrate ion [28El-Boraey, H.A. Coordination behavior of tetraaza [N₄] ligand towards Co(II), Ni(II), Cu(II), Cu(I) and Pd(II) complexes: Synthesis, spectroscopic characterization and anticancer activity. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2012, 97, 255-262.
[http://dx.doi.org/10.1016/j.saa.2012.05.077] [PMID: 22765944] , 29Swamy, S.J.; Reddy, E.R.; Raju, D.N.; Jyothi, S. Synthesis and spectral investigations of manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II) complexes of new polydentate ligands containing a 1,8-naphthyridine moiety. Molecules, 2006, 11(12), 1000-1008.
[http://dx.doi.org/10.3390/11121000] [PMID: 18007404] ], whereas complex (9) shows IR bands at 1370 and 1284 cm^-1 characterizing the ionic nitrate [13EL-Boraey. H.A.; Emam, S.M.; Tolan, D.A.; El-Nahas, A.M.; Structural studies and anticancer activity of a novel (N6O4) macrocyclic ligand and its Cu(II) complexes. Spectrochim. Acta A, 2011, 78, 360-370.
[http://dx.doi.org/10.1016/j.saa.2010.10.021] , 14El-Boraey, H.A.; Serag El-Din, A.A. Transition metal complexes of a new 15-membered [N5] penta-azamacrocyclic ligand with their spectral and anticancer studies. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2014, 132, 663-671.
[http://dx.doi.org/10.1016/j.saa.2014.05.018] [PMID: 24892547] ]. These observations are further confirmed from molar conductivities (Table 1). Complexes (4,6) show two bands at 1586-1575 and1330-1323 cm ^-1. The energy separation between ν_as and ν_s is found to be >144 cm ^-1 indicating the monodentate coordination of the acetate ion [14El-Boraey, H.A.; Serag El-Din, A.A. Transition metal complexes of a new 15-membered [N5] penta-azamacrocyclic ligand with their spectral and anticancer studies. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2014, 132, 663-671.
[http://dx.doi.org/10.1016/j.saa.2014.05.018] [PMID: 24892547] , 28El-Boraey, H.A. Coordination behavior of tetraaza [N₄] ligand towards Co(II), Ni(II), Cu(II), Cu(I) and Pd(II) complexes: Synthesis, spectroscopic characterization and anticancer activity. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2012, 97, 255-262.
[http://dx.doi.org/10.1016/j.saa.2012.05.077] [PMID: 22765944] ]. The presence of uncoordinated perchlorate anion in the complex (10) is confirmed from the bands around 1114 and 629 cm^-1 [30Sherif, O.E.; Abdel-Kader, N.S. Spectroscopic and biological activities studies of bivalent transition metal complexes of Schiff bases derived from condensation of 1,4-phenylenediamine and benzopyrone derivatives. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2014, 117, 519-526.
[http://dx.doi.org/10.1016/j.saa.2013.08.037] [PMID: 24025671] ]. On the basis of the IR spectra, it is concluded that the macrocyclic ligand behaves as a neutral quadridentate one, with the lone electron pairs of two amide nitrogen atoms and two azomethine nitrogen atoms, or as neutral bidentate one with the lone electron pairs of two azomethine nitrogen atoms only for Pd(II) complex (11).

Table 2
Important IR spectral bands (cm^-1)^a of the macrocyclic complexes.

The electronic spectra of the macrocyclic complexes were measured in DMF solution. The absorption bands and the magnetic moment values at room temperature (μ_eff B.M.) are given in Table 3.

2.3.5. Palladium(II) Complex

The electronic absorption spectrum of Pd(II) complex (11) exhibits band at 500 nm, which may be assigned to ¹A_lg→^lA_2g transitions [39Geeta, B.; Shravankumar, K.; Reddy, P.M.; Ravikrishna, E.; Sarangapani, M.; Reddy, K.K.; Ravinder, V. Binuclear cobalt(II), nickel(II), Copper(II) and Palladium(II) complexes of a new Schiff-base as ligand: Synthesis, structural characterization, and antibacterial activity. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2010, 77(4), 911-915.
[http://dx.doi.org/10.1016/j.saa.2010.08.004] [PMID: 20801709] , 40Chandra, S.; Raizada, S.; Rani, S. Structural and spectral studies of palladium(II) and platinum(II) complexes derived from N,N,N,N-tetradentate macrocyclic ligands. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2008, 71(2), 720-724.
[http://dx.doi.org/10.1016/j.saa.2007.12.051] [PMID: 18343190] ], indicating square-planar structure around the Pd(II) ion. The coordination of the chloride to the square-planar bivalent palladium centre is confirmed by its non-electrolytic nature in DMF solution. Pd(II) complex is diamagnetic confirming the square planar geometry .

Table 3
Electronic spectral data (nm) and magnetic moment (B.M.) of the macrocyclic metal complexes.

The powder ESR spectra were carried out for the 16-membered N₄ macrocyclic copper(II) complexes (7, 9) at room temperature on the X-band frequency 9. 719 GHz. The EPR parameters are tabulated in Table 4. The complexes give axial spectra (Fig. 1). Analysis of spectra gives g_// = 2.16 and g_┴ = 2. 039 - 2. 033. The trend g_// >g_┴ > 2.0023 suggests d_x²_-y² ground state for the Cu(II) ion, indicating that the unpaired electron is in the d_x²_-y² orbital of the Cu(II) ion [14El-Boraey, H.A.; Serag El-Din, A.A. Transition metal complexes of a new 15-membered [N5] penta-azamacrocyclic ligand with their spectral and anticancer studies. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2014, 132, 663-671.
[http://dx.doi.org/10.1016/j.saa.2014.05.018] [PMID: 24892547] , 15EL-Boraey. H.A.; EL-Gammal, O.A.; New 15-membered tetraaza (N4) macrocyclic ligand and its transition metal complexes: spectral, magnetic, thermal and anticancer activity. Spectrochim. Acta A, 2015, 138, 533-562.
[http://dx.doi.org/10.1016/j.saa.2014.11.015] , 35Shebl, M.; Khalil, S.M.E.; Al-Gohani, F.S. Preparation, spectral characterization and antimicrobial activity of binary and ternary Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Ce(III) and UO2(VI) complexes of a thiocarbohydrazone ligand. J. Mol. Struct., 2010, 980, 78-87.
[http://dx.doi.org/10.1016/j.molstruc.2010.06.040] ]. The greater value of g_// compared to g_┴ indicates a tetragonal distortion around the Cu(II) ions [41Kavitha, N.; Anantha Lakshmi, P.V. Synthesis, characterization and thermogravimetric analysis of Co(II), Ni(II), Cu(II) and Zn(II) complexes supported by ONNO tetradentate Schiff base ligand derived from hydrazino benzoxazine. J. Saudi Chem. Soc., 2015, 21, S457-66.
[http://dx.doi.org/10.1016/j.jscs.2015.01.003] , 42Gurumoorthy, P.; Ravichandran, J.; Karthikeyan, N.; Palani, P.; Kalilur Rahiman, A. Template synthesis of polyaza macrocyclic copper(II) and nickel(II) complexes: Spectral characterization and antimicrobial studies. Bull. Korean Chem. Soc., 2012, 33(7), 2279-2286.
[http://dx.doi.org/10.5012/bkcs.2012.33.7.2279] ]. Moreover, the observed g_// values ˂ 2.3 revealing an appreciable covalent nature for Cu-L bond [43Kivelson, D.; Neiman, R. ESR studies on the bonding in copper complexes. J. Chem. Phys., 1961, 35, 149-155.
[http://dx.doi.org/10.1063/1.1731880] ]. The exchange interaction between the copper centers in polycrystalline sample is explained by Hathaway expression G=g_//-2.0023/g_┴-2.0023 [44Hathaway, B.J.; Billing, D.E. The electronic properties and stereochemistry of mononuclear complexes of the copper(II) ion. Coord. Chem. Rev., 1970, 5, 143-207.
[http://dx.doi.org/10.1016/S0010-8545(00)80135-6] ]. The calculated G value for complexes is higher than 4 indicating no exchange interaction between copper centers.

Fig. (1) EPR spectra for Cu(II) complexes (7,9).

Table 4
: EPR spectral data of Cu(II) complexes.

The thermal properties of the 16-membered N₄ macrocyclic metal(II) complexes were carried out by thermogravimetric analysis (TGA/DTG) in N₂ atmosphere from 25 to 800 °C, at a rate of 10 °C min^-1. The obtained data are represented in Table 5.

2.5.4. Copper(II) Complexes

TG thermograms of copper(II) complexes (7-9) show a mass loss from room temperature up to 97 °C, associated with one endothermic DTG peak, that is attributed to the loss of solvent molecules (Table 5). The TG/ DTG curves also show that complexes (7,8) decomposed at 300,230 °C, respectively. The thermogram of complex (9) displayed two degradation stages. The first stage at 97-297 °C is corresponding to the loss of (NO₃+OH) (weight loss; Calc./Found%; 11.47/11.8%). The next stage at 297-408 °C is attributed to the completion of the decomposition process. The final decomposition products can be CuO for complexes (7-8) and Cu for complex (9).

Table 5
Thermal data of the macrocyclic metal complexes.

The kinetic and thermodynamic parameters of the decomposition stages of the complexes (1, 4-7) were calculated from TGA thermogram using the Coats–Redfern equation [45Coats, A.W.; Redfern, J.P. Kinetic parameters from thermogravimetric data. Nature, 1964, 201, 68-122.
[http://dx.doi.org/10.1038/201068a0] ]. The Horowitz and Metzger [46Horowitz, H.H.; Metzger, G. A New analysis of thermogravimetric traces. J. Anal. Chem., 1963, 35, 1464-1468.
[http://dx.doi.org/10.1021/ac60203a013] ] equation: was used for the evaluation of the value of the reaction order, and given by: where is the weight fraction of the substance present at DTG peak temperature T_s, is the remaining weight at T_s, and are the initial and final weights of the substance, respectively. The estimated values of for the thermal decomposition of the desolvated complexes were found in the range of 0.28-0.35 (Table 6 and Fig. 2), indicating that the degradation suggests first order reaction [14El-Boraey, H.A.; Serag El-Din, A.A. Transition metal complexes of a new 15-membered [N5] penta-azamacrocyclic ligand with their spectral and anticancer studies. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2014, 132, 663-671.
[http://dx.doi.org/10.1016/j.saa.2014.05.018] [PMID: 24892547] , 15EL-Boraey. H.A.; EL-Gammal, O.A.; New 15-membered tetraaza (N4) macrocyclic ligand and its transition metal complexes: spectral, magnetic, thermal and anticancer activity. Spectrochim. Acta A, 2015, 138, 533-562.
[http://dx.doi.org/10.1016/j.saa.2014.11.015] ]. Thermodynamic parameters such as the activation energy E*, Arrhenius constant A, the activation entropy S*, the activation enthalpy H* and the free energy of activation G* were calculated using Coats–Redfern equation for n = 1:

(1)

Fig. (2) TG/DTA curves of complexes (2,4, 5,9).

Where: x is the fraction decomposed, R: is the gas constant and θ is the heating rate. Since (1-2RT/E*) ≈ 1, a plot of the left-hand side of Eq. (1) against 1/T gives a straight line from its slope and intercept, E* and A were determined. The entropy of activation S*, enthalpy of activation H* and Gibbs free energy G* were calculated by applying the following equations:,and .

The calculated kinetic and thermodynamic values are listed in Table 6. From the results, the values reveal high stability of the complexes. Since the positive sign of indicates that decomposition stages are endothermic processes. The reaction for which is positive and is negative reveals that the free energy of the final residue is higher than that of the initial compound, and all decomposition steps are non-spontaneous processes. On the other hand, the negative values of indicate more ordered activated complex than the corresponding reactants and that the reaction is slow [47Sivakumar, P.; Parthiban, K.S.; Sivakumar, P.; Vinoba, M.; Renganathan, S. Optimization of extraction process and kinetics of sterculia foetida Seed oil and its process augmentation for biodiesel production. Ind. Eng. Chem. Res., 2012, 52, 8992-8998.
[http://dx.doi.org/10.1021/ie300882t] ]. The correlation coefficients of the Arrhenius plots of the thermal decomposition steps were found to lie in the range 0.97 to 0. 95 showing the best fit with a linear function.

Table 6
Kinetic and thermodynamic parameters for macrocyclic metal complexes (1,4-7)

From the interpretation of elemental analyses, molar conductivity, magnetic susceptibilities, spectral and thermal studies, suggested structures for the complexes (1–11) are given in Fig. (3).

Fig. (3) Suggested structure of the macrocyclic complexes (1-11).

The anticancer activity of the16-membered N₄ macrocyclic metal complexes (1,2,5,7,9) was detected in vitro against human breast cancer cell (MCF-7) and human hepatocarcinoma cell (Hep-G2) by SRB assay. The results of the cytotoxic activity in vitro expressed as IC₅₀ are given in Table 7 and plotted in (Fig. 4). The tested complexes show IC₅₀ value in the range of 3.2-1.6 and 3.7-1.7 μg/mL towards human breast cancer cell lines (MCF-7) and human hepatocarcinoma cells (Hep- G2), respectively. According to Sheir [48Shier, W.T. Mammalian cell culture on $5 a day: A laboratory manual of low cost methods., 1991, ], these complexes are considered to be very active towards the two cell lines. The results indicate that Ni(II) complex (5) showed highest cytotoxic activity on both human breast cancer cell line and hepatocarcinoma cell (IC₅₀ value = 1.6 and 1.7 μg/mL, respectively). The enhanced activity of the investigated complexes agrees well with and better than the documented activity of similar metal complexes as antitumor agents [13EL-Boraey. H.A.; Emam, S.M.; Tolan, D.A.; El-Nahas, A.M.; Structural studies and anticancer activity of a novel (N6O4) macrocyclic ligand and its Cu(II) complexes. Spectrochim. Acta A, 2011, 78, 360-370.
[http://dx.doi.org/10.1016/j.saa.2010.10.021] -15EL-Boraey. H.A.; EL-Gammal, O.A.; New 15-membered tetraaza (N4) macrocyclic ligand and its transition metal complexes: spectral, magnetic, thermal and anticancer activity. Spectrochim. Acta A, 2015, 138, 533-562.
[http://dx.doi.org/10.1016/j.saa.2014.11.015] , 30Sherif, O.E.; Abdel-Kader, N.S. Spectroscopic and biological activities studies of bivalent transition metal complexes of Schiff bases derived from condensation of 1,4-phenylenediamine and benzopyrone derivatives. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2014, 117, 519-526.
[http://dx.doi.org/10.1016/j.saa.2013.08.037] [PMID: 24025671] , 49Osowole, A.A.; Akpan, E.J. Synthesis, spectroscopic characterisation, In-vitro anticancer and antimicrobial activities of some metal(II) complexes of 3-{4, 6-dimethoxy pyrimidinyl) iminomethylnaphthalen-2-ol. Eur. J. Appl. Sci., 2012, 4(1), 14-20.]. The macrocycle, as well as the type of metal ions, may be the reason for their different anticancer activity. These compounds seem to be promising as an anticancer agent because of their high cytotoxic activity. The active sequence of the complexes follows the trend:

Ni(II) > Co(II) = Cu(II) NO₃ > Fe(II) > Cu(II) Cl₂ for MCF-7 cells;

NiII) > Cu(II) NO₃ > Co(III) > Fe(II) > Cu(II)Cl₂ for Hep-G2 cells

Table 7
Lethal concentration (IC₅₀) of the macrocyclic metal complexes on MCF-7 and Hep-G2 cell lines.

Fig. (4) Lethal concentration (IC50) of the macrocyclic metal complexes on MCF-7 and Hep-G2 cell lines.

The metal salts (E.Merck) were commercially available as pure samples. All the chemicals used in this study were of AnalaR grade and procured from Sigma, Aldrich and Fluka. All solvents were used as received. All reactions were carried out under normal atmospheric conditions.

Microanalyses (C,H and N) of these complexes were carried out at Cairo University, Giza, Egypt using CHNS- 932 (LECO) Vario Elemental Analyzer. Metal contents were estimated using standard methods [50West, T.S. Complexometry with EDTA and related reagents., (3rd ed. ), 1969, ]. The Fourier Transform Infrared (FT-IR) measurements in the range of 4000– 400 cm^-1 were recorded in KBr discs using Nenexeus-Nicolidite-640-MSA FT-IR. Thermo-Electronics Co. Electronic spectra were determined in Nujol mull using 4802 UV/vis double beam spectrophotometer. The ¹H NMR spectrum was recorded in DMSO- d₆ using Varian Gemini 200 NMR spectrophotometer at 300 MHz. The EPR spectra were measured using a Varian E-109C model X-band spectrometer. The molar conductance was measured at 30 ^oC in DMSO solution (10^{- 3} M) using a CON 6000 conductivity meter. Magnetic measurements (Gouy balance) were carried out at room temperature. The effective magnetic moments were calculated using the relation μ_eff=2.828 (χ_mT)^1/2 B.M., where χ_m is the molar susceptibility corrected for diamagnetism of all atoms in the compounds. The complexes were studied by thermogravimetry (TG /DTG) in a static nitrogen atmosphere with a sample heating rate of 10 ^oC/min, using a Shimadzu DTA/TG-50 Thermal Analyzer.

The cytotoxicity of the compounds was tested at the National Cancer Institute, Cairo University Egypt by SRB assay using the method of Skehan et al. [51Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.; Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst., 1990, 82(13), 1107-1112.
[http://dx.doi.org/10.1093/jnci/82.13.1107] [PMID: 2359136] ]. Cells were plated in 96- multiwell plate (104 cells/ well) for 24 h before treatment with the compounds to allow attachment of a cell to the wall of the plate. Different concentrations of the compound under test (0, 2.5, 5, 10, 20 μg /mL) added to the cell monolayer triplicate wells were prepared for each individual dose. Monolayer cells were incubated with the compounds for 48 h at 37 ^oC and an atmosphere of 5% CO₂. After 48 h, cells were fixed, washed and stained with Sulfo-Rhoda- mine-B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to obtain the survival curve of each tumor cell line after the specified compound.