Ž .Inorganic Chemistry Communications 3 2000 453–457www.elsevier.nlrlocaterinoche

Metal complexes as anticancer agents 2. Synthesis, spectroscopy,magnetism, electrochemistry, X-ray crystal structure and antimelanomal

ž /activity of the copper II complex of5-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 Department of Chemistry, UniÕersity of Hull, Hull HU6 7RX, UKb Department of Chemistry, UniÕersity of Pune, Pune-411 007, India

Received 1 February 2000

Abstract

w Ž . Ž .x Ž .The copper complex Cu ATICAR H O P2H O ATICARs5-amino-1-tolylimidazole-4-carboxylate has been prepared and2 2 2Ž .characterized by its crystal structure determination. The ligand geometry around the copper II center is best described as predominantly

Ž . Ž .square pyramidal 2r3 with a trigonal bipyramidal component 1r3 . The ATICAR ligands act as bidentates to form the distorted squareŽpyramid base of N O donor atoms and a coordinated water molecule at the apex is held with a Cu–O bond that is unusually short 2.1482 2

˚ . Ž .A for square pyramidal copper II . Compound exhibits a dose-dependent antiproliferative effect on the growth of the B16F10 melanomacell line while its lower IC value establish advantage by copper complexation. q 2000 Elsevier Science S.A. All rights reserved.50

Keywords: Imidazole; Copper complex; Square pyramidal geometry; X-ray structure; Anticancer activity

1. Introduction

Metal complexes that can bind to specific nucleobasesin DNA or that can inhibit enzymes controlling nucleicacid biosynthesis are of great interest in the developmentof antitumor or antimycobacterial agents. For example,

w xKimura and co-workers 1–5 have recently shown that theŽ . ŽZn II -cyclen complex cyclen is 1,4,7,10-tetraazacyclo-

.dodacane is a highly selective inhibitor of the thymidineand uridine biosynthesis and of the gene expression arising

) Corresponding author. Tel.: q91-20-565-6061; fax: q91-20-565-1728.

Ž .E-mail address: sbpadhye@chem.unipune.ernet.in S. Padhye .1 Corresponding author. Tel.: q44-1482-465-479; fax: q44-1482-466-

410.2 Corresponding author. Tel.: q44-1482-466-353; fax: q44-1482-466-

410.

out of them as imidazole derivatives are close structuralanalogs of many of the purine nucleobases. Mimics ofnucleobases are, therefore, looked upon as most promisingcompounds for the development of useful antibacterial and

w xanticancer agents 6 .We have described the effects of several such close

structural analogs of the aminoimidazole ribonucleotidesearlier as the competitive inhibitors of de novo biosynthe-

w xsis of purine nuclotides 7 where the inhibition was thoughtto occur via the formation of a copper-substrate complexw x8 . We have, therefore, undertaken synthesis of coppercomplexes of 1-substituted imidazole carboxylate analogswith a view to better understanding their structural proper-ties and subsequent biological activities. In the presentcommunication, we describe the preparation and structural

Ž .characterization of the copper II complex of 5-amino-1-tolylimidazole-4-carboxylate and its antiproliferative activ-ity against B16F10 mouse melanoma cells which clearlyshows the advantage gained on metal complexation.

1387-7003r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved.Ž .PII: S1387-7003 00 00108-8

( )M. Collins et al.r Inorganic Chemistry Communications 3 2000 453–457454

2. Experimental

2.1. Materials

Ethyl a-amino-a-cyanoacetate was prepared accordingw xto the literature methods 9 . Triethyl orthoformate, p-

Ž .toluidine and copper nitrate Aldrich were used as re-ceived. All solvents were reagent grade and were purified

w xby standard procedures prior to use 10 .

2.1.1. Instrumental measurementsChemical micro-analyses were carried out by Butter-

Ž .worth Laboratories, Teddington UK while the details ofw xother measurements are as described earlier 11 .

2.2. Synthesis

2.2.1. Ethyl 5-amino-1-tolylimidazole-4-carboxylate( ) ( )EATICAR 1

Ž .A mixture of ethyl a-amino-a-cyanoacetate 5.6 g andŽ . Ž .triethyl orthoformate 7.0 g in acetonitrile 40 ml was

boiled under reflux for 45 min. On cooling in the reactionŽ .mixture, p-toluidine 5.0 g was added to produce a red

solution and was set aside overnight at room temperatureŽ .to give a crystalline precipitate of the aminoimidazole 1 .

It was recrystallised from ethanol as colorless needles.Ž .Yield: 5.7 g 58% , m.p. 1568C, Anal. Calc.: for

C H N O requires C, 63.66; H, 6.17; N, 17.13. Found:13 15 3 2Ž y1 . y1C, 63.50; H, 6.25; N, 17.2%. IR: KBr, cm : 3414 cm

Ž . y1 Ž y. y1 Ž .n C–NH , 1678 cm n COO , 1620 cm n CsN .2

Ž . ŽStructure of 1 EATICAR ethyl 5-amino-1-tolylimida-.zole-4-carboxylate

[ ( ) ( )] ( )2.2.2. Cu ATICAR H O .2H O 22 2 2Ž .Alkaline hydrolysis of 1 by the reported procedure

w x12 yielded the corresponding acid. It was further reactedŽ .with the solution of Cu NO P3H O in a metal: ligand3 2 2

ratio of 1:2 in methanol maintaining the pH of the reactionmixture at 7.0 by addition of 2 M sodium acetate. Thereaction mixture was refluxed for 2 h and then stored in a

Ž .refrigerator overnight to yield a green precipitate of 2which was recrystallised from methanol–water solventŽ .9:1 as green needles suitable for the single crystal X-ray

Ž .diffraction studies. Yield 2.1g 78% . Anal. Calc.: forCuO N C H requires C, 48.05; H, 4.77; N, 15.28; Cu,7 6 22 26

11.55. Found: C, 48.3; H, 4.81; N, 15.04; Cu, 11.45%. IRŽ . y1 y1 Ž . y1Nujol cm : 3414 cm n C–NH , 1651 cm2Ž y. y1 Ž .n COO , 1578 cm n CsN .

Table 1w Ž . Ž .xSelected crystallographic data for Cu ATICAR H O .2H O2 2 2

Empirical formula, CuO N C H , 550.07 6 22 26

formula wt.Crystal color, habit, green, fragment, 0.24=0.22=0.50

Ž .dimensions mmŽ .No. of refl. in unit 26 17.5–29.3

Ž .cell calc. 2u

˚Ž . Ž . Ž .Unit cell lengths a, b, c 12.134 5 , 12.631 7 , 7.797 3 AŽ . Ž . Ž .Unit cell angles a , b , g 93.08 4 , 92.94 4 , 93.37 7 8

3˚Ž .Cell volume 1189 2 A

Space group P1, triclinic3Z value, D 2, 1.554 grcmcalc

y1Ž .F , m MoK a 570, 9.72 cm000

Diffractometer Rigaku AFC6S˚Ž .Radiation MoK a ls0.71069 A

Temperature 228CŽ .Scan type, rate v –2u , 48rmin in v

o Ž .2u 48.1 to hs9, ks13, ls8max

No. of reflections Total 2142, Unique 2001Ž .measured R s0.074int

Ž .Structure solution Heavy atom PattersonRefinement Full-matrix least-squares

2 2Ž .Least-squares weights 4F rs 2 F0 0

p-factor 0.05Residuals: R; R , 0.066; 0.054, 1.25w

goodness-of-fit3˚Max., min. peak 0.50, y0.44 erA

in final diff. map

2.3. Crystallographic studies

w xMeasurements were made as previously described 13using a Rigaku AFC6S diffractometer with graphitemonochromated MoK a radiation on a green fragment

w Ž . Ž .xcrystal of Cu C H N O H O P 2H O, CuO -11 10 3 2 2 2 2 7

N C H having dimensions 0.24=0.22=0.50 mm6 22 26

mounted on a glass fibre. Least-squares refinement of thesetting angles of 26 reflections yielded a triclinic cell with

˚ ˚ ˚Ž . Ž . Ž .as12.134 5 A, bs12.631 7 A; cs7.797 3 A, as˚3Ž . Ž . Ž . Ž .93.08 4 8, bs92.95 4 8, gs93.37 7 8, Vs1189 2 A .

Space group P1. Data were collected at 228C using v-2u

Ž . Ž .scans 1.30q0.33tanu at 4.08rmin in v with station-ary background counts on both sides of each reflection. Of

Žthe 2142 reflections collected, 2001 were unique R sint.0.074 ; equivalent reflections were averaged. Of these,

2 Ž 2 . Ž 2 .1795 reflections had F )0.1s F , where s F was0 0 0w xestimated from counting statistics 13,14 . Lorentz-polar-

ization and absorption corrections were applied.3 The in-tensities of three standard reflections measured after every150 reflections showed no greater variation than thoseexpected from Poisson statistics.

3 An empirical absorption correction, based on azimuthal scans ofseveral reflections, was applied which resulted in transmission factorsranging from 0.78 to 1.22.

( )M. Collins et al.r Inorganic Chemistry Communications 3 2000 453–457 455

w Ž . Ž .xFig. 1. ORTEP representation of Cu ATICAR H O P2H O.2 2 2

2.3.1. Cell culture methodsThe B16F10 cancer cell line was grown in Dulbecco’s

Ž .Modified Eagle’s Medium DMEM supplemented withŽ . Ž .penicillin 100 mgrml , streptomycin 100 mgrml and

5% fetal calf serum. Cultures were grown at 378C in a

humid 5% CO atmosphere and fed on alternate days.2

These culture conditions were found to be optimal for theevaluation of antineoplastic compounds in B16F10 cell

w xline 15 .

2.3.2. Cell proliferation studiesExponentially growing cells were trypsinized, counted

and plated in a multi-well plate at a density of 4.5=104

cellsrwell in 2 ml of media. After 2 days of incubation,when the cells were in an exponentially growth phase, thetest compounds were added. Controlwells received thesame amount of the vehicle alone. After a 24 h exposureperiod the exponentially growing cells were counted by thehemocytometer using the Trypan blue exclusion method toquantify cell viability. The cytotoxicity of the compoundwas determined on the basis of treatment-induced cellviability and calculated as follows:

cytotoxicity

Ž . Ž .% cell viability control -% cell viability treated =100s .

% cell viability

3. Results and discussion

Ž .The crystallographic parameters for compound 2 arew Ž .summarized in Table 1. The structure Cu ATICAR -2

Ž .xH O P2H O consists of a hydrogen-bonded network2 2w Ž . Ž .xlinking the monomeric complex unit Cu ATICAR H O2 2

and two lattice water molecules. The ligand geometryŽ .around copper II center is best described as predomi-

nantly square pyramidal with a trigonal bipyramidal com-Ž . w Ž . xponent t of 0.32 ts bya r60 , with a and b being

.the two largest coordination angles . For perfect squarepyramidal geometry t is 0, while it is 1 in a perfect

w xtrigonal bipyramid 16 . The ATICAR ligands act as biden-tates to form the distorted square pyramid base of N O2 2

donor atoms while a coordinated water molecule is held atthe apex with a Cu–O bond that is longer than the others

Table 2˚Ž . Ž . w Ž . Ž .xSelected bond distances A and bond angles 8 for Cu ATICAR H O P2H O2 2 2

˚Ž .Bond distances AŽ . Ž . Ž . Ž .Cu–O 1w 2.148 6 Cu–N 3b 1.946 7Ž . Ž . Ž . Ž . Ž .Cu–O 1a 1.991 4 O 1w –O 1b 3.26 1Ž . Ž . Ž . Ž . Ž .Cu–O 1b 2.025 5 O 1w –O 1a 3.310 8Ž . Ž . Ž . Ž . Ž .Cu–N 3a 1.948 5 O 1a –O 1b 3.888 7

Ž .Bond angles 8

Ž . Ž . Ž . Ž . Ž . Ž .O 1w –Cu–O 1a 106.1 2 O 1a –Cu–N 3b 94.8 2Ž . Ž . Ž . Ž . Ž . Ž .O 1w –Cu–O 1b 102.9 2 O 1b –Cu–N 3a 94.7 2Ž . Ž . Ž . Ž . Ž . Ž .O 1w –Cu–N 3a 96.5 3 O 1b –Cu–N 3b 83.0 3Ž . Ž . Ž . Ž . Ž . Ž .O 1w –Cu–N 3b 93.3 3 N 3a –Cu–N 3b 170.2 2Ž . Ž . Ž . Ž . Ž . Ž .O 1a –Cu–O 1b 151.0 2 O 1a –Cu–N 3a 82.5 2

( )M. Collins et al.r Inorganic Chemistry Communications 3 2000 453–457456

Ž .Fig. 2. Dose-dependent antiproliferative response of 2 on B16F10melanoma cells.

˚Ž .but still unusually short 2.148 A for square pyramidalŽ . w xcopper II 17–19 . The plane formed by the N O donor2 2

set is slightly distorted along O–M–O axis as indicated byŽ . Ž .the unequal O–M–O 151.08 and N–M–N 170.28 an-

gles and is further reflected in non-equivalent metal–˚Ž .oxygen 1.991 and 2.025 A distances. The central copper

atom is slightly lifted out of the plane formed by the fourin-plane ligand atoms as indicated by the sum of anglessubtended by the donor atoms with the central metal atom.The unit atom labeling scheme is shown in Fig. 1 whileintramolecular bond distances and bond angles are in-cluded in Table 2.

In the IR spectrum of the copper complex the broadband at 3250–3460 cmy1 can be assigned to the coordi-

w x y1nated water molecule 20 . The band at 3414 cm whichremains unaffected even after complexation is assigned toNH stretching frequency. The vibrational frequencies due2

Ž y1 . Žto carboxylate 1672 cm and CsN of imidazole 1620y1 . y1 y1cm linkages are shifted to 1651 cm and 1578 cm

respectively upon complexation indicating the involvementof these groups in coordination.

The room temperature magnetic moment of the coppercomplex using the Faraday method is found to be 1.96 BMwhich is typical of the copper complexes with square

w xpyramidal geometries 21 . The value suggests no inter-molecular interactions and mixing of orbitals in the presentcase.

The electronic spectrum of the copper complex inmethanol shows a broad absorption around 15152 cmy1

w x2 2 2which can be assigned to the d ™d transition 22 .z x yy

A similar band is observed in the case of square pyramidalw Ž .Ž . x Žcopper complex Cu 2-pic NO where picspicolinic3 2

.acid . Other d–d transitions are not resolved in the presentcompound. The absorption peaks in the range of 40000–

y1 w x27027 cm are due to ligand transitions 23 .The cyclic voltammogram of the copper complex in

DMSO solvent shows three reduction peaks at q0.14VŽ . Ž . w xcorresponding to the Cu II rCu I redox couple 24 , a

quasi-reversible peak at y0.14V and an irreversible reduc-tion peak at y1.0V corresponding to the ligand. Thepositive potential observed for the copper redox-couple in

the present compound makes it easier to reduce intra-cellularly.

Treatment of the melanoma cultures with the copperŽ .compound of 1 leads to dose-dependent survival rates

Ž . Ž .Fig. 2 . The IC value calculated for 2 from the dose-50

response curve for B16F10 is 15 mM, which is lower thanŽ .the one found for the ligand alone 50 mM or the starting

Ž .material copper nitrate )100 mM respectively. It clearlyestablishes the advantage of copper complexation. The

Žfacile intracellular reduction of the copper promoted by its.positive redox potential and its subsequent interaction

with the cellular thiols has been postulated as the possiblemechanism of anticancer activity of the such copper conju-

w xgates 25 .

4. Supplementary material

Atomic coordinates, details of bond lengths and anglesand thermal parameters are available from the author E.S.on request.

Acknowledgements

U.S. and S.P. would like to thank the British Councilfor the visitorships under HE link program. Thanks arealso due to Dr. Gopal Kundu of NCCS, Pune, for his keeninterest and encouragement.

References

w x Ž .1 M. Shionoya, E. Kimura, M. Shiro, J. Am. Chem. Soc. 115 19936730.

w x2 M. Shionoya, T. Ikeda, E. Kimura, M. Shiro, J. Am. Chem. Soc. 116Ž .1994 3848.

w x Ž .3 E. Kimura, T. Koike, Comments Inorg. Chem. 11 1991 285.w x Ž .4 E. Kimura, Tetrahedron 48 1992 6175.w x Ž .5 E. Kimura, Prog. Inorg. Chem. 41 1994 443.w x Ž .6 R.I. Chistoperson, S.D. Lyons, Med. Res. Rev. 10 1990 505.w x7 G. MacKenzie, A. Scott Frame, R.H. Wightman, Tetrahedron 27

Ž .1996 9219.w x Ž . Ž .8 N.J. Kusak, G. Shaw, G.J. Litchfield, J. Chem. Soc. C 1971

1501.w x9 G. Shaw, R.N. Warrener, D.N. Butler, R.K. Ralph, J. Chem. Soc.

Ž .1959 1648.w x10 D.D. Perrin, W.L.F. Armarego, Purification of Laboratory Chemi-

cals, Pergamon Press, New York, 1988.w x11 A. Murugkar, S. Padhye, S. Guha-Roy, U. Wagh, Inorg. Chem.

Ž .Commun. 2 1999 545.w x12 M. Fraunks, C.P. Green, G. Shaw, G.J. Litchfield, J. Chem. Soc.

Ž . Ž .C , 1966 2270.w x13 J.R. Backhouse, H.M. Lowe, E. Sinn, S. Suzuki, S. Woodward, J.

Ž .Chem. Soc., Dalton Trans. 1950 1489.w x Ž .14 P.W.P. Corfield, R.J. Doedens, J.A. Ibers, Inorg. Chem. 6 1967

197.w x15 S.A. Burchill, D.C. Banett, A. Holmes, A.J. Thody, Pathobiology 59

Ž .1991 335.w x16 A.W. Addison, T.N. Rao, J. Reedijk, J. van Rijn, G.C. Vershoor, J.

Ž .Chem. Soc., Dalton Trans . 1984 1349.

( )M. Collins et al.r Inorganic Chemistry Communications 3 2000 453–457 457

w x Ž .17 W.D. Harrison, B.J. Hathaway, Acta Crystallogr. Sect. B 35 19792910, and references therein.

w x Ž .18 J. Ellis, G.M. Mockler, E. Sinn, Inorg. Chem. 20 1981 1206.w x Ž .19 A.W. Addison, T.N. Rao and E. Sinn, Inorg. Chem . 23 1984 1957

and references therein.w x20 K. Nakamoto, Infrared Spectra of Inorganic and Coordination Com-

pounds, John Wiley, New York, 1970.

w x Ž .21 B. Morrison, Acta Crystallogr. Sect. B 25 1969 19.w x Ž .22 A.F. Cameron, R.H. Nuttall, D.W. Taylor, Chem. Commun. 1970

865.w x23 D. Sutton, Electronic Spectra of Transition Metal Complexes, Mc-

Graw-Hill, London, 1968.w x Ž .24 G.S. Patterson, R.H. Holm, Bioinorg. Chem. 4 1975 1975.w x Ž .25 C.J. Reed, K.T. Douglas, Biochemistry J. 275 1991 601.