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Metal complexes as anticancer agents
Inorganic Chemistry Communications 3 Ž2000. 453–457 www.elsevier.nlrlocaterinoche
Metal complexes as anticancer agents 2. Synthesis, spectroscopy, magnetism, electrochemistry, X-ray crystal structure and antimelanomal activity of the copper žII/ complex of 5-amino-1-tolylimidazole-4-carboxylate in B16F10 mouse melanoma cells Martin Collins a , David Ewing a , Grahame Mackenzie a,1, Ekkehard Sinn a,2 , Uday Sandbhor b, Shreelekha Padhye b, Subhash Padhye b,) a b
Department of Chemistry, UniÕersity of Hull, Hull HU6 7RX, UK Department of Chemistry, UniÕersity of Pune, Pune-411 007, India Received 1 February 2000
Abstract The copper complex wCuŽATICAR. 2 ŽH 2 O.x P 2H 2 O ŽATICARs 5-amino-1-tolylimidazole-4-carboxylate. has been prepared and characterized by its crystal structure determination. The ligand geometry around the copperŽII. center is best described as predominantly square pyramidal Ž2r3. with a trigonal bipyramidal component Ž1r3.. The ATICAR ligands act as bidentates to form the distorted square pyramid base of N2 O 2 donor atoms and a coordinated water molecule at the apex is held with a Cu–O bond that is unusually short Ž2.148 ˚ . for square pyramidal copperŽII.. Compound exhibits a dose-dependent antiproliferative effect on the growth of the B16F10 melanoma A cell line while its lower IC 50 value establish advantage by copper complexation. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Imidazole; Copper complex; Square pyramidal geometry; X-ray structure; Anticancer activity
1. Introduction Metal complexes that can bind to specific nucleobases in DNA or that can inhibit enzymes controlling nucleic acid biosynthesis are of great interest in the development of antitumor or antimycobacterial agents. For example, Kimura and co-workers w1–5x have recently shown that the ZnŽII.-cyclen complex Žcyclen is 1,4,7,10-tetraazacyclododacane. is a highly selective inhibitor of the thymidine and uridine biosynthesis and of the gene expression arising
) Corresponding author. Tel.: q91-20-565-6061; fax: q91-20-5651728. E-mail address: [email protected] ŽS. Padhye.. 1 Corresponding author. Tel.: q44-1482-465-479; fax: q44-1482-466410. 2 Corresponding author. Tel.: q44-1482-466-353; fax: q44-1482-466410.
out of them as imidazole derivatives are close structural analogs of many of the purine nucleobases. Mimics of nucleobases are, therefore, looked upon as most promising compounds for the development of useful antibacterial and anticancer agents w6x. We have described the effects of several such close structural analogs of the aminoimidazole ribonucleotides earlier as the competitive inhibitors of de novo biosynthesis of purine nuclotides w7x where the inhibition was thought to occur via the formation of a copper-substrate complex w8x. We have, therefore, undertaken synthesis of copper complexes of 1-substituted imidazole carboxylate analogs with a view to better understanding their structural properties and subsequent biological activities. In the present communication, we describe the preparation and structural characterization of the copperŽII. complex of 5-amino-1tolylimidazole-4-carboxylate and its antiproliferative activity against B16F10 mouse melanoma cells which clearly shows the advantage gained on metal complexation.
1387-7003r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 1 3 8 7 - 7 0 0 3 Ž 0 0 . 0 0 1 0 8 - 8
454
M. Collins et al.r Inorganic Chemistry Communications 3 (2000) 453–457
2. Experimental
Table 1 Selected crystallographic data for wCu ŽATICAR. 2 ŽH 2 O.x.2H 2 O
2.1. Materials
Empirical formula, formula wt. Crystal color, habit, dimensions Žmm. No. of refl. in unit cell calc. Ž2 u .
CuO 7 N6 C 22 H 26 , 550.0
Unit cell lengths a, b, c Unit cell angles a , b , g
˚ 12.134Ž5., 12.631Ž7., 7.797Ž3. A 93.08Ž4., 92.94Ž4., 93.37Ž7.8 ˚3 1189Ž2. A
Ethyl a-amino-a-cyanoacetate was prepared according to the literature methods w9x. Triethyl orthoformate, ptoluidine and copper nitrate ŽAldrich. were used as received. All solvents were reagent grade and were purified by standard procedures prior to use w10x. 2.1.1. Instrumental measurements Chemical micro-analyses were carried out by Butterworth Laboratories, Teddington ŽUK. while the details of other measurements are as described earlier w11x. 2.2. Synthesis 2.2.1. Ethyl 5-amino-1-tolylimidazole-4-carboxylate (EATICAR) (1) A mixture of ethyl a-amino-a-cyanoacetate Ž5.6 g. and triethyl orthoformate Ž7.0 g. in acetonitrile Ž40 ml. was boiled under reflux for 45 min. On cooling in the reaction mixture, p-toluidine Ž5.0 g. was added to produce a red solution and was set aside overnight at room temperature to give a crystalline precipitate of the aminoimidazole Ž1.. It was recrystallised from ethanol as colorless needles. Yield: 5.7 g Ž58%., m.p. 1568C, Anal. Calc.: for C 13 H 15 N3 O 2 requires C, 63.66; H, 6.17; N, 17.13. Found: C, 63.50; H, 6.25; N, 17.2%. IR: ŽKBr, cmy1 .: 3414 cmy1 n ŽC–NH 2 ., 1678 cmy1 n ŽCOOy. , 1620 cmy1 n ŽC s N..
Structure of Ž1. EATICAR Žethyl 5-amino-1-tolylimidazole-4-carboxylate. 2.2.2. [Cu (ATICAR)2 (H2 O)].2H2 O (2) Alkaline hydrolysis of Ž1. by the reported procedure w12x yielded the corresponding acid. It was further reacted with the solution of Cu ŽNO 3 . 2 P 3H 2 O in a metal: ligand ratio of 1:2 in methanol maintaining the pH of the reaction mixture at 7.0 by addition of 2 M sodium acetate. The reaction mixture was refluxed for 2 h and then stored in a refrigerator overnight to yield a green precipitate of Ž2. which was recrystallised from methanol–water solvent Ž9:1. as green needles suitable for the single crystal X-ray diffraction studies. Yield 2.1g Ž78%.. Anal. Calc.: for CuO 7 N6 C 22 H 26 requires C, 48.05; H, 4.77; N, 15.28; Cu, 11.55. Found: C, 48.3; H, 4.81; N, 15.04; Cu, 11.45%. IR ŽNujol. cmy1 : 3414 cmy1 n ŽC–NH 2 ., 1651 cmy1 n ŽCOOy. , 1578 cmy1 n ŽC s N..
Cell volume Space group Z value, Dcalc F000 , m ŽMoK a . Diffractometer Radiation Temperature Scan type, rate 2 uma x No. of reflections measured Structure solution Refinement Least-squares weights p-factor Residuals: R; R w , goodness-of-fit Max., min. peak in final diff. map
green, fragment, 0.24=0.22=0.50 26 Ž17.5–29.3.
P1, triclinic 2, 1.554 grcm3 570, 9.72 cmy1 Rigaku AFC6S ˚. MoK a Ž l s 0.71069 A 228C v –2 u , 48rmin Žin v . 48.1o Žto hs9, k s13, l s8. Total 2142, Unique 2001 Ž R int s 0.074. Heavy atom ŽPatterson. Full-matrix least-squares 4 F02 r s 2Ž F02 . 0.05 0.066; 0.054, 1.25
˚3 0.50, y0.44 erA
2.3. Crystallographic studies Measurements were made as previously described w13x using a Rigaku AFC6S diffractometer with graphite monochromated MoK a radiation on a green fragment crystal of wCuŽC 11 H 10 N 3 O 2 . 2 ŽH 2 O.x P 2H 2 O, CuO 7 N6 C 22 H 26 having dimensions 0.24 = 0.22 = 0.50 mm mounted on a glass fibre. Least-squares refinement of the setting angles of 26 reflections yielded a triclinic cell with ˚ b s 12.631Ž7. A; ˚ c s 7.797Ž3. A, ˚ as a s 12.134Ž5. A, ˚ 3. 93.08Ž4.8, b s 92.95Ž4.8, g s 93.37Ž7.8, V s 1189Ž2. A Space group P1. Data were collected at 228C using v-2 u scans Ž1.30 q 0.33tan u . at 4.08rmin Žin v . with stationary background counts on both sides of each reflection. Of the 2142 reflections collected, 2001 were unique Ž R int s 0.074.; equivalent reflections were averaged. Of these, 1795 reflections had F02 ) 0.1 s Ž F02 ., where s Ž F02 . was estimated from counting statistics w13,14x. Lorentz-polarization and absorption corrections were applied.3 The intensities of three standard reflections measured after every 150 reflections showed no greater variation than those expected from Poisson statistics.
3 An empirical absorption correction, based on azimuthal scans of several reflections, was applied which resulted in transmission factors ranging from 0.78 to 1.22.
M. Collins et al.r Inorganic Chemistry Communications 3 (2000) 453–457
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humid 5% CO 2 atmosphere and fed on alternate days. These culture conditions were found to be optimal for the evaluation of antineoplastic compounds in B16F10 cell line w15x. 2.3.2. Cell proliferation studies Exponentially growing cells were trypsinized, counted and plated in a multi-well plate at a density of 4.5 = 10 4 cellsrwell in 2 ml of media. After 2 days of incubation, when the cells were in an exponentially growth phase, the test compounds were added. Controlwells received the same amount of the vehicle alone. After a 24 h exposure period the exponentially growing cells were counted by the hemocytometer using the Trypan blue exclusion method to quantify cell viability. The cytotoxicity of the compound was determined on the basis of treatment-induced cell viability and calculated as follows: cytotoxicity % cell viability Ž control. -% cell viability Ž treated. =100 s % cell viability
.
3. Results and discussion
Fig. 1. ORTEP representation of wCuŽATICAR. 2 ŽH 2 O.xP2H 2 O.
2.3.1. Cell culture methods The B16F10 cancer cell line was grown in Dulbecco’s Modified Eagle’s Medium ŽDMEM. supplemented with penicillin Ž100 mgrml., streptomycin Ž100 mgrml. and 5% fetal calf serum. Cultures were grown at 378C in a
The crystallographic parameters for compound Ž2. are summarized in Table 1. The structure wCuŽATICAR. 2ŽH 2 O.x P 2H 2 O consists of a hydrogen-bonded network linking the monomeric complex unit wCuŽATICAR. 2 ŽH 2 O.x and two lattice water molecules. The ligand geometry around copperŽII. center is best described as predominantly square pyramidal with a trigonal bipyramidal component Žt . of 0.32 wt s Ž b y a .r60x, with a and b being the two largest coordination angles.. For perfect square pyramidal geometry t is 0, while it is 1 in a perfect trigonal bipyramid w16x. The ATICAR ligands act as bidentates to form the distorted square pyramid base of N2 O 2 donor atoms while a coordinated water molecule is held at the apex with a Cu–O bond that is longer than the others
Table 2 ˚ . and bond angles Ž8. for wCuŽATICAR. 2 ŽH 2 O.x P 2H 2 O Selected bond distances ŽA
˚. Bond distances ŽA Cu–OŽ1w. Cu–OŽ1a. Cu–OŽ1b. Cu–NŽ3a. Bond angles Ž8. OŽ1w. –Cu–OŽ1a. OŽ1w. –Cu–OŽ1b. OŽ1w. –Cu–NŽ3a. OŽ1w. –Cu–NŽ3b. OŽ1a. –Cu–OŽ1b.
2.148Ž6. 1.991Ž4. 2.025Ž5. 1.948Ž5.
Cu–NŽ3b. OŽ1w. –OŽ1b. OŽ1w. –OŽ1a. OŽ1a. –OŽ1b.
1.946Ž7. 3.26Ž1. 3.310Ž8. 3.888Ž7.
106.1Ž2. 102.9Ž2. 96.5Ž3. 93.3Ž3. 151.0Ž2.
OŽ1a. –Cu–NŽ3b. OŽ1b. –Cu–NŽ3a. OŽ1b. –Cu–NŽ3b. NŽ3a. –Cu–NŽ3b. OŽ1a. –Cu–NŽ3a.
94.8Ž2. 94.7Ž2. 83.0Ž3. 170.2Ž2. 82.5Ž2.
456
M. Collins et al.r Inorganic Chemistry Communications 3 (2000) 453–457
Fig. 2. Dose-dependent antiproliferative response of Ž2. on B16F10 melanoma cells.
˚ . for square pyramidal but still unusually short Ž2.148 A copperŽII. w17–19x. The plane formed by the N2 O 2 donor set is slightly distorted along O–M–O axis as indicated by the unequal O–M–O Ž151.08. and N–M–N Ž170.28. angles and is further reflected in non-equivalent metal– ˚ . distances. The central copper oxygen Ž1.991 and 2.025 A atom is slightly lifted out of the plane formed by the four in-plane ligand atoms as indicated by the sum of angles subtended by the donor atoms with the central metal atom. The unit atom labeling scheme is shown in Fig. 1 while intramolecular bond distances and bond angles are included in Table 2. In the IR spectrum of the copper complex the broad band at 3250–3460 cmy1 can be assigned to the coordinated water molecule w20x. The band at 3414 cmy1 which remains unaffected even after complexation is assigned to NH 2 stretching frequency. The vibrational frequencies due to carboxylate Ž1672 cmy1 . and C s N of imidazole Ž1620 cmy1 . linkages are shifted to 1651 cmy1 and 1578 cmy1 respectively upon complexation indicating the involvement of these groups in coordination. The room temperature magnetic moment of the copper complex using the Faraday method is found to be 1.96 BM which is typical of the copper complexes with square pyramidal geometries w21x. The value suggests no intermolecular interactions and mixing of orbitals in the present case. The electronic spectrum of the copper complex in methanol shows a broad absorption around 15152 cmy1 which can be assigned to the d z 2 d x 2yy 2 transition w22x. A similar band is observed in the case of square pyramidal copper complex wCuŽ2-pic.ŽNO 3 . 2 x Žwhere pic s picolinic acid.. Other d–d transitions are not resolved in the present compound. The absorption peaks in the range of 40000– 27027 cmy1 are due to ligand transitions w23x. The cyclic voltammogram of the copper complex in DMSO solvent shows three reduction peaks at q0.14V corresponding to the CuŽII.rCuŽI. redox couple w24x, a quasi-reversible peak at y0.14V and an irreversible reduction peak at y1.0V corresponding to the ligand. The positive potential observed for the copper redox-couple in
™
the present compound makes it easier to reduce intracellularly. Treatment of the melanoma cultures with the copper compound of Ž1. leads to dose-dependent survival rates ŽFig. 2.. The IC 50 value calculated for Ž2. from the doseresponse curve for B16F10 is 15 mM, which is lower than the one found for the ligand alone Ž50 mM. or the starting material copper nitrate Ž) 100 mM. respectively. It clearly establishes the advantage of copper complexation. The facile intracellular reduction of the copper Žpromoted by its positive redox potential. and its subsequent interaction with the cellular thiols has been postulated as the possible mechanism of anticancer activity of the such copper conjugates w25x.
4. Supplementary material Atomic coordinates, details of bond lengths and angles and thermal parameters are available from the author E.S. on request.
Acknowledgements U.S. and S.P. would like to thank the British Council for the visitorships under HE link program. Thanks are also due to Dr. Gopal Kundu of NCCS, Pune, for his keen interest and encouragement.
References w1x M. Shionoya, E. Kimura, M. Shiro, J. Am. Chem. Soc. 115 Ž1993. 6730. w2x M. Shionoya, T. Ikeda, E. Kimura, M. Shiro, J. Am. Chem. Soc. 116 Ž1994. 3848. w3x E. Kimura, T. Koike, Comments Inorg. Chem. 11 Ž1991. 285. w4x E. Kimura, Tetrahedron 48 Ž1992. 6175. w5x E. Kimura, Prog. Inorg. Chem. 41 Ž1994. 443. w6x R.I. Chistoperson, S.D. Lyons, Med. Res. Rev. 10 Ž1990. 505. w7x G. MacKenzie, A. Scott Frame, R.H. Wightman, Tetrahedron 27 Ž1996. 9219. w8x N.J. Kusak, G. Shaw, G.J. Litchfield, J. Chem. Soc. ŽC. Ž1971. 1501. w9x G. Shaw, R.N. Warrener, D.N. Butler, R.K. Ralph, J. Chem. Soc. Ž1959. 1648. w10x D.D. Perrin, W.L.F. Armarego, Purification of Laboratory Chemicals, Pergamon Press, New York, 1988. w11x A. Murugkar, S. Padhye, S. Guha-Roy, U. Wagh, Inorg. Chem. Commun. 2 Ž1999. 545. w12x M. Fraunks, C.P. Green, G. Shaw, G.J. Litchfield, J. Chem. Soc. ŽC., Ž1966. 2270. w13x J.R. Backhouse, H.M. Lowe, E. Sinn, S. Suzuki, S. Woodward, J. Chem. Soc., Dalton Trans. Ž1950. 1489. w14x P.W.P. Corfield, R.J. Doedens, J.A. Ibers, Inorg. Chem. 6 Ž1967. 197. w15x S.A. Burchill, D.C. Banett, A. Holmes, A.J. Thody, Pathobiology 59 Ž1991. 335. w16x A.W. Addison, T.N. Rao, J. Reedijk, J. van Rijn, G.C. Vershoor, J. Chem. Soc., Dalton Trans . Ž1984. 1349.
M. Collins et al.r Inorganic Chemistry Communications 3 (2000) 453–457 w17x W.D. Harrison, B.J. Hathaway, Acta Crystallogr. Sect. B 35 Ž1979. 2910, and references therein. w18x J. Ellis, G.M. Mockler, E. Sinn, Inorg. Chem. 20 Ž1981. 1206. w19x A.W. Addison, T.N. Rao and E. Sinn, Inorg. Chem . 23 Ž1984. 1957 and references therein. w20x K. Nakamoto, Infrared Spectra of Inorganic and Coordination Compounds, John Wiley, New York, 1970.
457
w21x B. Morrison, Acta Crystallogr. Sect. B 25 Ž1969. 19. w22x A.F. Cameron, R.H. Nuttall, D.W. Taylor, Chem. Commun. Ž1970. 865. w23x D. Sutton, Electronic Spectra of Transition Metal Complexes, McGraw-Hill, London, 1968. w24x G.S. Patterson, R.H. Holm, Bioinorg. Chem. 4 Ž1975. 1975. w25x C.J. Reed, K.T. Douglas, Biochemistry J. 275 Ž1991. 601.
Metal complexes as anticancer agents 2. Synthesis, spectroscopy, magnetism, electrochemistry, X-ray crystal structure and antimelanomal activity of the copper žII/ complex of 5-amino-1-tolylimidazole-4-carboxylate in B16F10 mouse melanoma cells Martin Collins a , David Ewing a , Grahame Mackenzie a,1, Ekkehard Sinn a,2 , Uday Sandbhor b, Shreelekha Padhye b, Subhash Padhye b,) a b
Department of Chemistry, UniÕersity of Hull, Hull HU6 7RX, UK Department of Chemistry, UniÕersity of Pune, Pune-411 007, India Received 1 February 2000
Abstract The copper complex wCuŽATICAR. 2 ŽH 2 O.x P 2H 2 O ŽATICARs 5-amino-1-tolylimidazole-4-carboxylate. has been prepared and characterized by its crystal structure determination. The ligand geometry around the copperŽII. center is best described as predominantly square pyramidal Ž2r3. with a trigonal bipyramidal component Ž1r3.. The ATICAR ligands act as bidentates to form the distorted square pyramid base of N2 O 2 donor atoms and a coordinated water molecule at the apex is held with a Cu–O bond that is unusually short Ž2.148 ˚ . for square pyramidal copperŽII.. Compound exhibits a dose-dependent antiproliferative effect on the growth of the B16F10 melanoma A cell line while its lower IC 50 value establish advantage by copper complexation. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Imidazole; Copper complex; Square pyramidal geometry; X-ray structure; Anticancer activity
1. Introduction Metal complexes that can bind to specific nucleobases in DNA or that can inhibit enzymes controlling nucleic acid biosynthesis are of great interest in the development of antitumor or antimycobacterial agents. For example, Kimura and co-workers w1–5x have recently shown that the ZnŽII.-cyclen complex Žcyclen is 1,4,7,10-tetraazacyclododacane. is a highly selective inhibitor of the thymidine and uridine biosynthesis and of the gene expression arising
) Corresponding author. Tel.: q91-20-565-6061; fax: q91-20-5651728. E-mail address: [email protected] ŽS. Padhye.. 1 Corresponding author. Tel.: q44-1482-465-479; fax: q44-1482-466410. 2 Corresponding author. Tel.: q44-1482-466-353; fax: q44-1482-466410.
out of them as imidazole derivatives are close structural analogs of many of the purine nucleobases. Mimics of nucleobases are, therefore, looked upon as most promising compounds for the development of useful antibacterial and anticancer agents w6x. We have described the effects of several such close structural analogs of the aminoimidazole ribonucleotides earlier as the competitive inhibitors of de novo biosynthesis of purine nuclotides w7x where the inhibition was thought to occur via the formation of a copper-substrate complex w8x. We have, therefore, undertaken synthesis of copper complexes of 1-substituted imidazole carboxylate analogs with a view to better understanding their structural properties and subsequent biological activities. In the present communication, we describe the preparation and structural characterization of the copperŽII. complex of 5-amino-1tolylimidazole-4-carboxylate and its antiproliferative activity against B16F10 mouse melanoma cells which clearly shows the advantage gained on metal complexation.
1387-7003r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 1 3 8 7 - 7 0 0 3 Ž 0 0 . 0 0 1 0 8 - 8
454
M. Collins et al.r Inorganic Chemistry Communications 3 (2000) 453–457
2. Experimental
Table 1 Selected crystallographic data for wCu ŽATICAR. 2 ŽH 2 O.x.2H 2 O
2.1. Materials
Empirical formula, formula wt. Crystal color, habit, dimensions Žmm. No. of refl. in unit cell calc. Ž2 u .
CuO 7 N6 C 22 H 26 , 550.0
Unit cell lengths a, b, c Unit cell angles a , b , g
˚ 12.134Ž5., 12.631Ž7., 7.797Ž3. A 93.08Ž4., 92.94Ž4., 93.37Ž7.8 ˚3 1189Ž2. A
Ethyl a-amino-a-cyanoacetate was prepared according to the literature methods w9x. Triethyl orthoformate, ptoluidine and copper nitrate ŽAldrich. were used as received. All solvents were reagent grade and were purified by standard procedures prior to use w10x. 2.1.1. Instrumental measurements Chemical micro-analyses were carried out by Butterworth Laboratories, Teddington ŽUK. while the details of other measurements are as described earlier w11x. 2.2. Synthesis 2.2.1. Ethyl 5-amino-1-tolylimidazole-4-carboxylate (EATICAR) (1) A mixture of ethyl a-amino-a-cyanoacetate Ž5.6 g. and triethyl orthoformate Ž7.0 g. in acetonitrile Ž40 ml. was boiled under reflux for 45 min. On cooling in the reaction mixture, p-toluidine Ž5.0 g. was added to produce a red solution and was set aside overnight at room temperature to give a crystalline precipitate of the aminoimidazole Ž1.. It was recrystallised from ethanol as colorless needles. Yield: 5.7 g Ž58%., m.p. 1568C, Anal. Calc.: for C 13 H 15 N3 O 2 requires C, 63.66; H, 6.17; N, 17.13. Found: C, 63.50; H, 6.25; N, 17.2%. IR: ŽKBr, cmy1 .: 3414 cmy1 n ŽC–NH 2 ., 1678 cmy1 n ŽCOOy. , 1620 cmy1 n ŽC s N..
Structure of Ž1. EATICAR Žethyl 5-amino-1-tolylimidazole-4-carboxylate. 2.2.2. [Cu (ATICAR)2 (H2 O)].2H2 O (2) Alkaline hydrolysis of Ž1. by the reported procedure w12x yielded the corresponding acid. It was further reacted with the solution of Cu ŽNO 3 . 2 P 3H 2 O in a metal: ligand ratio of 1:2 in methanol maintaining the pH of the reaction mixture at 7.0 by addition of 2 M sodium acetate. The reaction mixture was refluxed for 2 h and then stored in a refrigerator overnight to yield a green precipitate of Ž2. which was recrystallised from methanol–water solvent Ž9:1. as green needles suitable for the single crystal X-ray diffraction studies. Yield 2.1g Ž78%.. Anal. Calc.: for CuO 7 N6 C 22 H 26 requires C, 48.05; H, 4.77; N, 15.28; Cu, 11.55. Found: C, 48.3; H, 4.81; N, 15.04; Cu, 11.45%. IR ŽNujol. cmy1 : 3414 cmy1 n ŽC–NH 2 ., 1651 cmy1 n ŽCOOy. , 1578 cmy1 n ŽC s N..
Cell volume Space group Z value, Dcalc F000 , m ŽMoK a . Diffractometer Radiation Temperature Scan type, rate 2 uma x No. of reflections measured Structure solution Refinement Least-squares weights p-factor Residuals: R; R w , goodness-of-fit Max., min. peak in final diff. map
green, fragment, 0.24=0.22=0.50 26 Ž17.5–29.3.
P1, triclinic 2, 1.554 grcm3 570, 9.72 cmy1 Rigaku AFC6S ˚. MoK a Ž l s 0.71069 A 228C v –2 u , 48rmin Žin v . 48.1o Žto hs9, k s13, l s8. Total 2142, Unique 2001 Ž R int s 0.074. Heavy atom ŽPatterson. Full-matrix least-squares 4 F02 r s 2Ž F02 . 0.05 0.066; 0.054, 1.25
˚3 0.50, y0.44 erA
2.3. Crystallographic studies Measurements were made as previously described w13x using a Rigaku AFC6S diffractometer with graphite monochromated MoK a radiation on a green fragment crystal of wCuŽC 11 H 10 N 3 O 2 . 2 ŽH 2 O.x P 2H 2 O, CuO 7 N6 C 22 H 26 having dimensions 0.24 = 0.22 = 0.50 mm mounted on a glass fibre. Least-squares refinement of the setting angles of 26 reflections yielded a triclinic cell with ˚ b s 12.631Ž7. A; ˚ c s 7.797Ž3. A, ˚ as a s 12.134Ž5. A, ˚ 3. 93.08Ž4.8, b s 92.95Ž4.8, g s 93.37Ž7.8, V s 1189Ž2. A Space group P1. Data were collected at 228C using v-2 u scans Ž1.30 q 0.33tan u . at 4.08rmin Žin v . with stationary background counts on both sides of each reflection. Of the 2142 reflections collected, 2001 were unique Ž R int s 0.074.; equivalent reflections were averaged. Of these, 1795 reflections had F02 ) 0.1 s Ž F02 ., where s Ž F02 . was estimated from counting statistics w13,14x. Lorentz-polarization and absorption corrections were applied.3 The intensities of three standard reflections measured after every 150 reflections showed no greater variation than those expected from Poisson statistics.
3 An empirical absorption correction, based on azimuthal scans of several reflections, was applied which resulted in transmission factors ranging from 0.78 to 1.22.
M. Collins et al.r Inorganic Chemistry Communications 3 (2000) 453–457
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humid 5% CO 2 atmosphere and fed on alternate days. These culture conditions were found to be optimal for the evaluation of antineoplastic compounds in B16F10 cell line w15x. 2.3.2. Cell proliferation studies Exponentially growing cells were trypsinized, counted and plated in a multi-well plate at a density of 4.5 = 10 4 cellsrwell in 2 ml of media. After 2 days of incubation, when the cells were in an exponentially growth phase, the test compounds were added. Controlwells received the same amount of the vehicle alone. After a 24 h exposure period the exponentially growing cells were counted by the hemocytometer using the Trypan blue exclusion method to quantify cell viability. The cytotoxicity of the compound was determined on the basis of treatment-induced cell viability and calculated as follows: cytotoxicity % cell viability Ž control. -% cell viability Ž treated. =100 s % cell viability
.
3. Results and discussion
Fig. 1. ORTEP representation of wCuŽATICAR. 2 ŽH 2 O.xP2H 2 O.
2.3.1. Cell culture methods The B16F10 cancer cell line was grown in Dulbecco’s Modified Eagle’s Medium ŽDMEM. supplemented with penicillin Ž100 mgrml., streptomycin Ž100 mgrml. and 5% fetal calf serum. Cultures were grown at 378C in a
The crystallographic parameters for compound Ž2. are summarized in Table 1. The structure wCuŽATICAR. 2ŽH 2 O.x P 2H 2 O consists of a hydrogen-bonded network linking the monomeric complex unit wCuŽATICAR. 2 ŽH 2 O.x and two lattice water molecules. The ligand geometry around copperŽII. center is best described as predominantly square pyramidal with a trigonal bipyramidal component Žt . of 0.32 wt s Ž b y a .r60x, with a and b being the two largest coordination angles.. For perfect square pyramidal geometry t is 0, while it is 1 in a perfect trigonal bipyramid w16x. The ATICAR ligands act as bidentates to form the distorted square pyramid base of N2 O 2 donor atoms while a coordinated water molecule is held at the apex with a Cu–O bond that is longer than the others
Table 2 ˚ . and bond angles Ž8. for wCuŽATICAR. 2 ŽH 2 O.x P 2H 2 O Selected bond distances ŽA
˚. Bond distances ŽA Cu–OŽ1w. Cu–OŽ1a. Cu–OŽ1b. Cu–NŽ3a. Bond angles Ž8. OŽ1w. –Cu–OŽ1a. OŽ1w. –Cu–OŽ1b. OŽ1w. –Cu–NŽ3a. OŽ1w. –Cu–NŽ3b. OŽ1a. –Cu–OŽ1b.
2.148Ž6. 1.991Ž4. 2.025Ž5. 1.948Ž5.
Cu–NŽ3b. OŽ1w. –OŽ1b. OŽ1w. –OŽ1a. OŽ1a. –OŽ1b.
1.946Ž7. 3.26Ž1. 3.310Ž8. 3.888Ž7.
106.1Ž2. 102.9Ž2. 96.5Ž3. 93.3Ž3. 151.0Ž2.
OŽ1a. –Cu–NŽ3b. OŽ1b. –Cu–NŽ3a. OŽ1b. –Cu–NŽ3b. NŽ3a. –Cu–NŽ3b. OŽ1a. –Cu–NŽ3a.
94.8Ž2. 94.7Ž2. 83.0Ž3. 170.2Ž2. 82.5Ž2.
456
M. Collins et al.r Inorganic Chemistry Communications 3 (2000) 453–457
Fig. 2. Dose-dependent antiproliferative response of Ž2. on B16F10 melanoma cells.
˚ . for square pyramidal but still unusually short Ž2.148 A copperŽII. w17–19x. The plane formed by the N2 O 2 donor set is slightly distorted along O–M–O axis as indicated by the unequal O–M–O Ž151.08. and N–M–N Ž170.28. angles and is further reflected in non-equivalent metal– ˚ . distances. The central copper oxygen Ž1.991 and 2.025 A atom is slightly lifted out of the plane formed by the four in-plane ligand atoms as indicated by the sum of angles subtended by the donor atoms with the central metal atom. The unit atom labeling scheme is shown in Fig. 1 while intramolecular bond distances and bond angles are included in Table 2. In the IR spectrum of the copper complex the broad band at 3250–3460 cmy1 can be assigned to the coordinated water molecule w20x. The band at 3414 cmy1 which remains unaffected even after complexation is assigned to NH 2 stretching frequency. The vibrational frequencies due to carboxylate Ž1672 cmy1 . and C s N of imidazole Ž1620 cmy1 . linkages are shifted to 1651 cmy1 and 1578 cmy1 respectively upon complexation indicating the involvement of these groups in coordination. The room temperature magnetic moment of the copper complex using the Faraday method is found to be 1.96 BM which is typical of the copper complexes with square pyramidal geometries w21x. The value suggests no intermolecular interactions and mixing of orbitals in the present case. The electronic spectrum of the copper complex in methanol shows a broad absorption around 15152 cmy1 which can be assigned to the d z 2 d x 2yy 2 transition w22x. A similar band is observed in the case of square pyramidal copper complex wCuŽ2-pic.ŽNO 3 . 2 x Žwhere pic s picolinic acid.. Other d–d transitions are not resolved in the present compound. The absorption peaks in the range of 40000– 27027 cmy1 are due to ligand transitions w23x. The cyclic voltammogram of the copper complex in DMSO solvent shows three reduction peaks at q0.14V corresponding to the CuŽII.rCuŽI. redox couple w24x, a quasi-reversible peak at y0.14V and an irreversible reduction peak at y1.0V corresponding to the ligand. The positive potential observed for the copper redox-couple in
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the present compound makes it easier to reduce intracellularly. Treatment of the melanoma cultures with the copper compound of Ž1. leads to dose-dependent survival rates ŽFig. 2.. The IC 50 value calculated for Ž2. from the doseresponse curve for B16F10 is 15 mM, which is lower than the one found for the ligand alone Ž50 mM. or the starting material copper nitrate Ž) 100 mM. respectively. It clearly establishes the advantage of copper complexation. The facile intracellular reduction of the copper Žpromoted by its positive redox potential. and its subsequent interaction with the cellular thiols has been postulated as the possible mechanism of anticancer activity of the such copper conjugates w25x.
4. Supplementary material Atomic coordinates, details of bond lengths and angles and thermal parameters are available from the author E.S. on request.
Acknowledgements U.S. and S.P. would like to thank the British Council for the visitorships under HE link program. Thanks are also due to Dr. Gopal Kundu of NCCS, Pune, for his keen interest and encouragement.
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