1 Bioorgni & iinl Chemistry 12 (24) Solution hemistry n ytotoxi properties of novel orgnogol(iii) ompouns Luigi ssori,, * Giorn Mron, Mri gostin Cinellu, Mrell Coronnello, Enrio Mini, Chir Gini n Pierluigi Orioli Deprtment of Chemistry, University of Florene, Vi ell Lstrui 3, 519, Sesto Fiorentino, Florene, Itly Deprtment of Chemistry, University of Sssri, Vi Viennz, 71 Sssri, Itly Deprtment of Phrmology, University of Florene, Vile Pierini 6, 5139 Florene, Itly Reeive 13 My 24; epte 1 Septemer 24 ville online 2 Otoer 24 strt The solution ehviour of some novel orgnogol(iii) ompouns ws investigte, n their ytotoxi properties evlute ginst few humn tumour ell lines (278/S, 278/R, MCF7, HT29 n 549). Speifilly, the following ompouns were onsiere: [(ipy m H)(2,6xyliineH)][PF 6 ] (Xyl) n [(ipy m H)(ptoluiineH)][PF 6 ] (Tol) (in whih ipy m = 6(1,1imethylenzyl)2,2 ipyriine), [(py m H)(O) 2 ] (PyO) (in whih py m = 2(1,1imethylenzyl)pyriine) n [(pz Ph H) 3 ]K (Pz) (in whih pz Ph = 1phenylpyrzole). The solution hemistry of these ompouns, uner physiologillike onitions, ws investigte through UV vis sorption n 1 H MR spetrosopies. Signifint ytotoxi effets in vitro were oserve in selete ses. Ó 24 Elsevier Lt. ll rights reserve. 1. Introution Keywors: Orgnogol(III) omplexes; UV vis sorption spetrosopy; 1 H MR spetrosopy; Cytotoxiity; Gol; Cner. * Corresponing uthor. Tel.: ; fx: ; emil: We hve reently shown tht vrious gol(iii) omplexes exhiit promising ytotoxi effets in vitro n re goo nites for further evlution s ntitumour gents. 1 itionl reports on the ntitumour properties of selete gol(iii) omplexes re the result of the reserh tivities of other groups. 2 6 otly, gol(iii) ompouns seem to exert their growth inhiitory effets through mehnisms tht re istint from those of the lssil ntiner pltinum(ii) omplexes. 7 mong the reently teste ompouns, the orgnogol(iii) omplex, [(ipy m H)(OH)][PF 6 ], hrterise y n ppreile stility within physiologil onitions, hs prove to e very tive ginst the 278ell line; 8 t vrine with the pltinum(ii) omplexes, its iologil tivity seems to e unrelte to iret interferene with D funtion n metolism. 8 This prompte us to onsier the solution ehviour n the iologil properties of some itionl gol(iii) ompouns hrterise y the presene of iret ron gol(iii) on; we elieve tht this ltter feture is very importnt for the stilistion of the oxition stte +3 of the gol entre. For this purpose, we took vntge of vrious reent stuies on the synthesis, hrteristion n retivity of gol(iii) ylometllte erivtives In prtiulr, we report here on the solution hemistry n the iologil properties of four orgnogol(iii) ompouns: [(ipy m H)(2,6xyliineH)][PF 6 ] (Xyl) n [(ipy m H)(ptoluiineH)][PF 6 ] (Tol) (in whih ipy m = 6(1,1imethylenzyl) 2,2 ipyriine), 12 [((py m H)(O) 2 ] (PyO) (in whih py m = 2(1,1imethylenzyl)pyriine) 12 n [(pz Ph H) 3 ]K (Pz) (in whih pz Ph =1 phenylpyrzole). 13 The Xyl n Tol ompouns, oth feturing the sme terentte C,, lign, inspire us y the enourging results reently ttine with the prent ompoun [(ipy m H)(OH)][PF 6 ]. Speifilly, we wnte to estlish whether the introution of ulkier lign in the fourth oorintion position might hve effets on the solution ehviour n the phrmologil profile of this orgnogol(iii) ompoun. t vrine, PyO, hving ylometllte C, lign, n Pz, with monoentte C lign, represent exmples of ompouns in whih the gol(iii) entre is less protete thn in the /$ see front mtter Ó 24 Elsevier Lt. ll rights reserve. oi:1.116/j.m
2 64 L. ssori et l. / Bioorg.. Chem. 12 (24) former ses. Remrkly, PyO presents two essile oorintion positions oupie y two ette groups wheres Pz ers three exhngele hlories tht might unergo file hyrolysis within n queous environment. Overll, we hve oserve tht the orgnogol(iii) moieties of ll these ompouns, exept Pz, re generlly stle uner physiologil onitions, n exhiit signifint ytotoxi properties towrs restrite pnel of humn tumour ell lines. Bse on these oservtions, the present ompouns my e of interest for further evlution s ntiner gents Min struturl spets 2. Results The following ompouns hve een onsiere in the present stuy: Xyl, Tol, PyO n Pz (see Sheme 1). The struture in the soli stte hs een etermine only for Xyl; 12 tht of the nlogous ompoun Tol n e eue from spetrosopi t n other physiohemil mesurements. Eqully, the orgnometlli frgments of Pz n PyO re onfiently ssume to mth those of the relte omplexes [(pz Ph H)(3,5 2 py) 2 ] 13 n [ (py m H) 2 ], 14 respetively, whose rystl strutures hve een sertine y Xry iffrtion nlysis. otly, ll these ompouns re hrterise y the presene of iret ron gol on. Xyl n Tol, otine y proton exhnge of [(ipy m H)(OH)][PF 6 ] with 2,6xyliine n ptoluiine, respetively, show signifint enhnement of the hyrophoi properties ompre to the prent omplex. 12 In PyO the ylometllte enzylpyriine provies nitrogen n ron onor to the squre plnr gol(iii) entre; the remining oorintion sites of the squre plne eing oupie y two ette ligns. In Pz the gol(iii) entre is oorinte to ron tom of the 1phenylpyrzole lign n to three hlories. In ll ses, the presene of the ron gol H + PF 6 C(O)O C(O)O [(ipymh)(ptoluiineh)][pf 6 ] [(pymh)(o) 2 ] H [(ipymh)(2,6xyliineh)][pf 6 ] + PF 6 [(pzphh) 3 ]K Sheme 1. Shemti rwing of the four gol(iii) omplexes. K + on is elieve to onfer suffiient reox stility to the gol(iii) entre Solution hemistry The stility in solution of the vrious orgnogol(iii) ompouns ws ssye y simple spetrophotometri mesurements. surements were rrie out within ifferent solvents (DMSO, ihloromethne, wter n the referene physiologil uffer ontining 5mM soium phosphte 1 mm, ph = 7.4). Some signifint spetrophotometri results re shown in Figure 1. Xyl n Tol re stle in orgni solvents suh s ihloromethne or DMSO. Uner these onitions oth ompouns exhiit n intense n in the visile (entre roun 42nm) tht is tenttively ssigne s to gol(iii) hrge trnsfer. The hyrolysis of oth ompouns, either in wter or in physiologillike uffer, is very fst n results into nerly omplete ispperne of the 42nm n; notly, hyrolysis in wter is ompnie y signifint ph erese. However, the UV trnsitions hrteristi of the ipy m sffol (entre t 32nm) 8 re not perture upon hyrolysis. The (ipy m H)(OH)] + speies is most likely forme in oth ses y proton exhnge of the miogol omplexes Xyl n Tol with wter; onomitnt relese of the romti mine tkes ple. Inee, these ompouns n e quntittively otine y the retion of [(ipy m H)(OH)][PF 6 ] with n exess of the orresponing mines, only proviing tht wter, proue y the retion, is remove. 12 Solutions of PyO, either in wter or in orgni solvents, re olourless. When issolve in wter or in the referene uffer, PyO exhiits some hrteristi ns in the ner UV, roun 26nm tht re stle with time. Upon onsiering tht the ette moieties generlly ehve s goo leving groups in queous solutions, we my expet their hyrolysis n replement y hyroxie. pprently, the resulting speies is stle in solution for severl hours. When issolve within orgni solvents, Pz imprts hrteristi yellow olour to the solution. In wter, or in the referene uffer, fst fing of the olour is oserve srie to rpi hyrolysis of gol(iii) oorinte hlories s in the se of the 4 nion. 15 The proess is ompnie y rsti erese in ph. However, this proess my e reverse, lmost ompletely, y ition of exess hlorie, for exmple, Li. otly, the UV spetrum of Pz, issolve in the uffer, shows slow, progressive erese of the min n entre t 25nm (Fig. 1, IV). The proess rehes ompletion within out 12 h t 25 C. We interpret this ehviour in terms of progressive moifition of the hyrolyse speies. Overll, from the ove reporte spetrophotometri experiments, it emerges tht the first three ompouns, following quik hyrolysis of their respetive lile ligns, proue orgnogol(iii)ontining speies tht
3 L. ssori et l. / Bioorg.. Chem. 12 (24) (I) (II) (III) (IV) Figure 1. UV vis spetr of Xyl (I), Tol (II), PyO (III) n Pz (IV). Tres represent the ompouns issolve in DMSO t 5mM onentrtion. Then the DMSO mother solutions of the four ompouns were ilute 1:4 either in pure DMSO (tres ) or in the referene phosphte uffer (tres ) or in wter (tres ). re stle for severl hours uner physiologillike onitions. t vrine, the hyrolysis prout of Pz mnifests lower stility n unergoes further trnsformtions. The ehviour in solution of Xyl n Tol ws further nlyse y 1 H MR spetrosopy. The spetr were reore either in euterte DMSO or in euterte queous solutions. otly, the 1 H MR spetr of Xyl n Tol, in DMSO, exhiit the resonnes hrteristi of the ylometllte ipyriine lign, plus resonnes ssigne to the xyliine or toluiine moieties oorinte to the gol(iii) entre. Upon hyrolysis within the referene euterte uffer, the 1 H MR spetr exhiit some hrteristi moifitions tht re srie to relese of either xyliine or toluiine from gol(iii) oorintion; t the sme time loss of the hrteristi olour is etete. In the finl 1 H MR spetr signls hrteristi of free xyliine or free toluiine re esily reognise (t 6.87, 6.6n 2.6 ppm for xyliine; t 6.97, 6.65 n 2.13ppm for toluiine) in greement with our hypothesis. Thus, the 1 H MR results onfirm the piture oming out from the spetrophotometri mesurements. Xyl n Tol issolve in the uffer rpily hyrolyse their mie ligns. However, the moleulr sffol of these ompouns, hrterise y the presene of iret rontogol on, is pretty stle n oes not show ny sign of reution of the gol(iii) entre Cytotoxi effets Owing to the ppreile stility of the vrious orgnogol(iii)ontining frgments uner physiologillike onitions, ll the ove ompouns oul e evlute in vitro s potentil ytotoxi gents. These orgnogol(iii) ompouns were teste ginst the following humn tumour ell lines: 278/S, 278/R (ovrin ner), MCF7 (rest ner), HT29 (olon ner) n 549 (lung ner). Their growth inhiitory properties were nlyse through the lssil sulforhomineb metho. 12 Results re shown in Tle 1. For omprison purposes, results otine with [(ipy m H)(OH)][PF 6 ] the preursor of Xyl n Tol; oxlipltin n ispltin (oth urrently use s ntiner rugs in the linis) re lso reporte. otly, PyO n Xyl prove to e signifintly tive ginst the two ovrin rinom ell lines, with IC 5 vlues of 2.9±.34 lm n 2.5±.43 lm, respetively, on the 278/S ell line. Crossresistne is reltively moest s it emerges from inspetion of the t otine on the 278/S n 278/R lines. Lower ut still importnt ytotoxi effets were proue y Tol (6.15 ± 1.4 lm) n [(ipy m H)(OH)][PF 6 ] (8.2± 2.lM) on the sme 278/S line. Moest effets were oserve on the MCF7 ell line, exept for Xyl (5.2±.4lM) (5.3±.87 lm); negligile effets were generlly mesure on the HT29 n 549 lines. t vrine with the other orgnogol(iii) ompouns, Pz turne out to e virtully evoi of ytotoxiity towrs ll teste ell lines, proly in reltion to its lower hemil stility profile. It is worthwhile noting tht the three ompouns [ (ipy m H)(OH)][PF 6 ], [(ipy m H)(2,6xyliine H)][PF 6 ] n [(ipy m H)(ptoluiineH)][PF 6 ]
4 642 L. ssori et l. / Bioorg.. Chem. 12 (24) Tle 1. IC 5 (lm) vlues of orgnogol(iii) ompouns in humn tumour ells, sensitive or resistnt to ispltin Compouns IC 5 (lm) 278/S 278/R MCF7 HT Pz 3.2 ± () 33.7 ± 2.15 >5 >5 [(ipy m H)(OH)][PF 6 ] 8.2± ± 1.42 (1.6) 35.3± >5 PyO 2.9 ± ±.1 (2.2) 17.7 ± Xyl 2.5 ± ±.3 (2.3) 5.2 ± Tol 6.15 ± ±.94 (2.3) 18.1 ± Cispltin.33 ± ±.87 (19.3) 5.3 ± ±.23 Oxlipltin.4 ±.12.2 ±.2 (.5).13 ±.1.4 ±.16 Dt were ollete fter 72h exposure to the rugs. IC 5 is efine s the onentrtion of rug require to inhiit ell growth y 5% ompre to ontrol; it is expresse s men ± SE of t lest three etermintions or men of two etermintions; vlues in prentheses ( ) enote the rtios of IC 5 of ispltinresistnt ell line n IC 5 of prentl sensitive ell line. exhiit signifint vritions in their ytotoxi properties ginst the teste ell lines, in spite of the ft tht ll of them most likely trnsform into the sme speies when issolve in the referene uffer. This oservtion implies tht the presene of the 2,6 2 C 6 H 3 H or 4C 6 H 5 H lign ffets somehow the iologil tivities of the ommon orgnogol(iii) entre. 3. Disussion Owing to the enourging in vitro ntitumour properties reently oserve for (ipy m H)(OH)][PF 6 ], new orgnogol(iii) omplexes were prepre, hrterise n teste s potentil ytotoxi gents. The solution ehviour n the stility within physiologillike environment of these novel orgnogol(iii) ompouns ws investigte. In ll ses, the gol(iii) entre oes not unergo reution, eing equtely stilise y the iret rontogol on. However, importnt hyrolysis proesses tke ple. Speifilly, in the se of Xyl n Tol, hyrolysis n relese of either xyliine or toluiine re lerly oserve resulting in the formtion of the prent hyroxoomplex. Both PyO n Pz unergo to some extent hyrolysis of the nioni oligns. fterwrs, further egrtion is oserve in the se of the pyrzolto erivtive, whih ers monoentte C lign. The ytotoxi properties of ll these ompouns were teste ginst the 278/S, 278/R, MCF7, HT29 n 549 humn tumour ell lines. otly, the investigte orgnogol(iii) ompouns, with the exeption of Pz, prove signifintly tive ginst the former tumour ell lines, with ytotoxiity vlues flling in the low miromolr rnge. Some ifferenes were oserve in the tivity ptterns of Xyl n Tol ompre to the referene [(ipy m H)(OH)][PF 6 ] ompoun. 7 Moerte effets were oserve on the MCF7 n HT29 lines, wheres negligile effets were mesure on the 549 line. Overll, similr trens of iologil tivity were ientifie mong the three tive ompouns tht re suggestive of similr mehnism of tion. We elieve tht the speifiity of their iologil effets my e srie to the presene of gol(iii) entre, hrterise y n eptle stility profile uner physiologillike onitions. In the light of these oservtions, the gol(iii) ontining moleulr frgment is very likely to e responsile for the oserve ytotoxi effets, lthough the tul mehnism is not yet unerstoo Chemils 4. Experimentl Common hemil regents were purhse from SIG M Chemil Co. (Milno, Itly) n Phrmi. Fetl lf serum (FCS), ntiiotis, n RPMI164meium were otine from Gio Life Tehnologies Itli (Milno, Itly). Synthesis of [(ipy m H)(2,6xyliineH)][PF 6 ] (Xyl), 12 [(ipy m H)(ptoluiineH)][PF 6 ] ( Tol), 12 [(py m H)(CH 3 COO) 2 ] (PyO) 12 n [(pz Ph H) 3 ]K (Pz). 13 The title ompoun ws prepre oring to Refs. 12 n Eletroni spetr The sorption spetr in the UV vis region were reore on Perkin Elmer Lm 2Bio spetrophotometer operting t room temperture. The eletroni spetr were reore t room temperture, ing smll mounts of freshly prepre, onentrte solutions of the iniviul omplexes in DMSO to, respetively, DMSO, wter or to the referene uffer (5mM phosphte, ph 7.4, 1 mm ); the onentrtions of eh gol(iii) ompoun ws The hyrolysis experiments were rrie out monitoring the eletroni spetr of M gol(iii) omplexes solutions in DMSO over 24 h H MR stuies Solution 1 H MR spetr of the gol(iii) omplexes were reore on Vrin Gemini 2 spetrometer operting t 3MHz. The M solutions were prepre in 6 DMSO n in 5mM PO 3 4, 4mM, ph 7.4 euterte uffer. The spetr were reore immeitely fter issolution.
5 L. ssori et l. / Bioorg.. Chem. 12 (24) Cell ulture n ytotoxiity ssy Cytotoxiity stuies were rrie out on the 278/S, 278/R, MCF7, HT29 n 549 humn tumour ell lines. The ispltinresistnt 278/R ell line ws proue y repete 1h weekly exposure to 5lM of the sensitive prentl ell line. 8 Cell lines were ulture in RPMI164meium (GIBCO Life tehnologies Prout tlog, RPMI 164meium, Ct. no 11817) supplemente with 1% FCS n ntiiotis (streptomyin 1lg/mL n peniillin 1U/mL) t 37 C in5% CO 2 tmosphere n suulture twie weekly. Experiments were onute on exponentilly growing ells. Drugs were issolve in DMSO. Inhiition of ell growth ws etermine fter 72 h rug exposure through the SulforhomineB (SRB) ssy 16 for the ovrin rinom ell lines. Referenes n notes 1. Mron, G.; ssori, L.; Orioli, P. Expert Rev. ntiner Ther. 22, 2, Prish, R. V.; Howe, B. P.; Wright, J. P.; Mk, J.; Prithr, R. G.; Bukley, R. G.; Elsome,. M.; Friker, S. P. Inorg. Chem. 1996, 35, Bukley, R. G.; Elsome,. M.; Friker, S. P.; Henerson, G. R.; Theol, B. R. C.; Prish, R. V.; Howe, B. P.; Kelln, L. R. J.. Chem. 1996, 39, Css, J. S.; Cstño, M. V.; Cifuentes, M. C.; Grí Monteguo, J. C.; Sánhez,.; Soro, J.; rm, U.; J. Inorg. Biohem. 24, 98, Che, C. M.; Sun, R. W.; Yu, W. Y.; Ko, C. B.; Zhu,.; Sun, H. Chem. Commun. (Cm.) 23, 21, Tiekink, E. R. T. Crit. Rev. Onol. Hemtol. 22, 42, ssori, L.; Mron, G. In tl Ions in Biologil Systems; Sigel,., Sigel, H., Es.; Mrel Dekker: etherlns, 24; Vol. 42, pp Mron, G.; Crotti, S.; Colonnello, M.; ssori, L.; Mini, E.; Orioli, P.; Mzzei, T.; Cinellu, M..; Minghetti, G. J.. Chem. 22, 45, Fn, D.; Yng, C.T.; Rnfor, J. D.; Lee, P. F.; Vittl, J. J. J. Chem. So., Dlton Trns. 23, Fn, D.; Yng, C.T.; Rnfor, J. D.; Vittl, J. J.; Lee, P. F. J. Chem. So., Dlton Trns. 23, Goss, C. H..; Henerson, W.; Wilkins,. L.; Evns, C. J. Orgnomet. Chem. 23, 679, Cinellu, M..; Minghetti, G.; Pinn, M. V.; Stooro, S.; Zu,.; Mnssero, M. Eur. J. Inorg. Chem. 23, 12, Minghetti, G.; Cinellu, M..; Pinn, M. V.; Stooro, S.; Zu,.; Mnssero, M. J. Orgnomet. Chem. 1998, 568, Cinellu, M..; Zu,.; Stooro, S.; Minghetti, G.; Mnssero, M.; Snsoni, S. J. Chem. So., Dlton Trns. 1995, Puephtt, R. J. In The Chemistry of Gol; rk, R. J. H., E.; Elsevier: msterm, Shehn, P.; Storeng, R.; Suiero, D.; Monks,.; Mmhon, J.; Visti, D.; Wrren, J. T.; Bokesh, H.; Kenney, S.; Boy, M. R. J. tl. Cner Inst. 199, 82, 117.