Conformational —exibility within the chelate rings of [Pt(en)(CBDCA-O,Oº) ] , an analogue of the antitumour drug carboplatin : X-ray crystallographic and solid-state NMR studies Zijian Guo,a Abraha Habtemariam,a Peter J. Sadler,*a Rex Palmerb and Brian S. Potterb a Department of Chemistry, University of Edinburgh, Kingœs Buildings, W est Mains Road, Edinburgh, UK EH9 3JJ b Department of Crystallography, Birkbeck College, University of L ondon, Malet Street, L ondon, UK W C1E 7HX The X-ray crystal structure of [Pt(en)(CBDCA-O,O@)] 1, an analogue of the anticancer drug carboplatin, shows that platinum has an approximate square-planar coordination.The crystals are orthorhombic with space group Pnma. The Pt-CBDCA chelate ring adopts a —attened-boat conformation, similar to that found for carboplatin, and the Pt-ethylenediamine chelate ring exhibits both d and k conformations with equal populations.Ethylenediamine chelate ring inversion was observed by 13C CP/MAS NMR spectroscopy. The exchange rate and the activation free energy (*Gt) at 317 K were determined to be ca. 415 s~1 and 62 kJ mol~1, respectively. The CBDCA ligand appears to have a direct eÜect on the dynamics of the en ring.Conformational —exibility of the CBDCA ring is also discussed. Such dynamic processes within chelated platinum complexes could play a role in the biological recognition of anticancer complexes. Several side eÜects of cisplatin, such as nephro- and neurotoxicity, have led to the search for the second generation of platinum drugs that circumvent these problems. Such eÜorts have led to the discovery of carboplatin.1 The replacement of the chloride ligand by chelated cyclobutane dicarboxylate maintains the high antitumour activity but greatly lowers the toxicity.There are a large number of clinical trials in progress on analogues that contain chelated diamine ligands and/or chelated dicarboxylates.2 These may recognize biological targets such as DNA by a diÜerent mechanism from cisplatin.In particular, chelated complexes may not hydrolyse before reacting with DNA bases,3 and initial second-sphere interactions may therefore direct the complex to selective regions of DNA. Outer-sphere recognition probably also plays an important role in dictating the target site of cisplatin.4 It is notable that Natile and coworkers have elegantly illustrated that stereospeci–c substitution in diamino chelate rings can have a major in—uence on nucleotide recognition at the trans positions.5 Conformational —exibility within the coordination sphere of platinum complexes is therefore likely to be an important feature in drug design.In this work we have studied dynamic processes within the bis-chelated complex [Pt(en)(CBDCA)] 1 by X-ray crystallography and solid-state NMR spectroscopy, and compared its behaviour with that of carboplatin,6,7 and some related PtII and PdII complexes. Experimental Cyclobutane-1,1@-dicarboxylic acid was pur- (H2CBDCA) chased from Sigma, and from K2PtCl4, LiOH ÆH2O AgNO3 Johnson Matthey, ethylenediamine (en) and other chemicals from Aldrich.and [Pt(en)Cl2], [Pt(en)I2], [Pd(en)2][PdCl4] (carboplatin) were prepared according [Pt(CBDCA)(NH3)2] to literature methods.8,9 [Pt(en)(CBDCA)] 1 was prepared as follows. A suspension of (127.8 mg, 0.39 mmol) and 2 mol equiv of [Pt(en)Cl2] in (2 mL) was stirred for 24 h. The AgCl was AgNO3 H2O –ltered oÜ. To the –ltrate, (56.5 mg, 0.39 mmol) H2CBDCA * Fax ](44) 131 650 6452.E-mail: P.J.Sadler=ed.ac.uk and 2 mol equiv of were added (the pH of the LiOH ÆH2O solution was ca. 6.4). Crystals of 1 were obtained by slow evaporation of the above solution and used for the X-ray diffraction and solid-state NMR studies. Elemental analysis of 1: Calcd (%) for C 24.19 ; H 3.55 ; N 7.05. C8H14N2O4Pt: Found: C 24.28 ; H 3.44 ; N 7.07. X-Ray crystallography The X-ray crystallographic analysis showed that crystals of complex 1 were orthorhombic with space group Pnma.Crystal data and re–nement data are listed in Table 1. Intensity data were measured at room temperature on a Nonius CAD4 diÜractometer in the x/2h scanning mode, Table 1 Crystal data and structure re–nement for [Pt(en)(CBDCAO, O@)] Empirical formula C8H14N2O4Pt Formula weight 397.30 Temperature 291^2 K Wavelength 1.54178 ” Crystal system Orthorhombic Space group Pnma Unit cell dimensions a\8.660 (3) ” a\90° b\9.614 (3) ” b\90° c\12.713 (5) ” c\90° Volume 1058.4 (6) ”3 Z 4 Density (calculated) 2.493 g cm~3 Absorption coef–cient 24.825 mm~1 F(000) 744 Crystal size 0.2]0.3]0.2 mm h range for data collection 5.77° to 71.87° Index ranges 0OhO10, 0Ok\11, [15OlO15 Re—ections collected 2661 Independent re—ections 1103 [R(int)\0.1194] Re–nement method Full-matrix least-squares on F2 Data/restraints/parameters 1054/4/78 Goodness-of-–t on F2 1.130 Final R indices [I[2r(I)] R1\0.0614, W r\0.1691 R indices (all data) R1\0.0771, W r2\0.2225 Extinction coef–cient 0.002 (1) Largest diÜ.peak and hole 4.451 and [4.808 e ”~3 New J. Chem., 1998, Pages 11»14 11employing graphite monochromated CuKa radiation.Two asymmetric units of data were recorded to 72° h. The crystals were weakly diÜracting and suÜered from statistical disorder, factors which aÜected the quality of the X-ray data measurements. The structure was solved using the heavy atom method, employing the program SHELXS-86.10 In addition to the usual Lorentz and polarization corrections, absorption corrections were calculated using the DIFABS method.11 The absorption corrections ranged from 0.678 to 1.832 with an average value of 0.964.The structure was re–ned by fullmatrix least-squares on oF2 o with SHELXL-93.12 Anisotropic displacement parameters were used for the non-H atoms and isotropic ones for the H atoms in the geometrically –xed, riding mode. Re–nement of the structure in the related space group was tried and proved to be unsatisfactory.Pn21a, Solid-state NMR 13C solid-state NMR spectra were recorded on a Bruker MSL-300 NMR spectrometer at 75 MHz, using cross polarization, proton decoupling and magic-angle spinning (CP/ MAS). The sample was inserted into a 7 mm diameter sample rotor. Spinning speeds of 4.5 to 5.0 kHz were employed, and a 1 ms contact time was used.Acquisition times were 30»40 ms and the recycle time between scans was 3 to 8 s. Typically the 90° pulse length for 1H was 4.5 ms and usually 2000 to 8000 scans were acquired. The chemical shifts are externally referenced to liquid TMS (0.0 ppm). Solution NMR Solution 13CM1HN NMR spectra were acquired on a JEOL GSX-270 NMR spectrometer at 67.5 MHz, using 16 K data points, relaxation delay of 2 s, and 4000 to 8000 transients. Tubes of 5 and 10 mm diameter were used.The chemical shifts are referenced internally to dioxane (67.3 ppm). Results and Discussion Crystal structure of [Pt(en)(CBDCA)] 1 The X-ray crystal structure of 1 is shown in Fig. 1(a) and (b). The atomic coordinates are listed in Table 2 and selected bond distances and angles of the molecule are shown in Table Fig. 1 View of [Pt(en)(CBDCA-O,O@)] 1, showing the numbering scheme with the H atoms as spheres of arbitrary diameter. (a) A view perpendicular to the least-squares plane; (b) view perpendicular to the CBDCA plane Table 2 Atomic coordinates (]104) for [Pt(en)(CBDCA-O,O@)] x y z Pt 1149(1) 2500 435(1) O3 1943(12) 4002(9) [531(5) O5 3316(13) 4710(13) [1854(8) N1 168(9) 3908(9) 1411(5) C2A* [1047(32) 3124(34) 2050(24) C2B* [291(25) 1806(19) 2365(15) C4 3051(13) 3810(13) [1188(7) C6 4027(14) 2500 [1089(10) C7 5463(15) 2500 [1814(11) C8 6487(25) 2500 [825(14) C9 5068(21) 2500 [104(14) * Occupancy 0.5. 3. As expected, the platinum atom is square-planar and coordinated to two bidentate ligands.The crystal contains the monomeric molecule, which lies across the crystallographic mirror perpendicular to the b axis. Platinum and the en ring atoms C6, C7, C8, C9 lie exactly in the mirror plane. The en ring is thus constrained to be exactly planar. Atoms O3, C4, O5 and N1 and their mirror-related counterparts complete the molecule, which thus exhibits mirror symmetry. Atom (Cs) C2 is disordered in two sites, C2A and C2B, with a distance of 0.77 apart, giving rise to two conformers, related by re—ec- ” tion across the mirror plane.The crystallographic space group is thus satis–ed statistically by averaging the two conformers. The N1wC2A and C2AwC2B bond distances for the disordered en ring were constrained to the values shown in Table 3. The Pt-CBDCA chelate ring also possesses exact symmetry Cs in a —attened boat conformation.There are no unusual features in the bond lengths and angles of the molecule. The PtwN bond distance of 2.023(7) is very similar to ” values reported for other Pt-ethylenediamine compounds such as dichloroethylenediamineplatinum(II) (2.034 ”),13 chloro(ethylenediamine)M([)-2,3,5,6-tetrahydro-6-phenylimidazo[ 2,1-b]thiazoleNplatinum(II) chloride (2.024 chloro- ”),14 uracil-ethylenediamineplatinum(II) chloride (2.03 trans- ”),15 S,S-[N,N@-bis(2-hydroxyethyl)ethylenediamine(oxalato-O,O@)- platinum(II)] (2.025 ”). The PtwO distance [2.017(8) is also consistent with ”] those found in Pt-bicarboxylate complexes such as carbo- Table 3 Selected bond length and angles (°) for [Pt(en)(CBDCA- (”) O,O@)] C6wC7 1.548(9) C4wC6 1.522(14) C7wC8 1.538(10) C4wO5 1.232(14) C8wC9 1.533(10) PtwN1 2.023(7) C6wC9 1.543(9) N1wC2A 1.48(3) PtwO3 2.017(8) C2AwC2B 1.54(3) O3wC4 1.285(13) C9wC6wC7 90.8(12) C4@wC6wC4 111.7(11) C8wC7wC6 88.7(13) O5wC4wO3 119.0(11) C9wC8wC7 91.5(13) O5wC4wC6 122.3(10) C8wC9wC6 89.0(13) O3@wPtwN1 174.7(4) C4@wC6wC7 113.4(7) O3wPtwN1 92.2(3) C4wC6wC7 113.4(7) C4@wC6wC9 113.0(7) N1wPtwN1@ 84.0(5) C4wC6wC7 113.0(7) C2AwN1wPt 106.6(12) C2BwC2AwN1 105(2) O3@wPtwO3 91.5(5) N1@wC2BwC2A 107(2) C4wO3wPt 123.2(8) C2B@wN1wPt 108.2(9) O3wC4wC6 118.7(9) Ring (PtwO3wC4wC6wC4@wO3@) and ring (PtwN1wC2Aw C2BwN1@) are completed by the operation of m perpendicular to b. 12 New J. Chem., 1998, Pages 11»14platin [(2.029 and (2.025 [Pt(NH3)2(CBDCA-O,O@)] ”)6 ”)7] trans-S,S-[N,N@-bis(2-hydroxyethyl)ethylenediamine(oxalato- O,O@)platinum(II)] (2.039 cis-1R,2R-cyclohexanediamine- ”),16 N,N@-oxalatoplatinum(II) (2.01, 2.04 Pt(malonato-O,O@)- ”),17 cis-1R, 2R- cyclohexanediamine -N,N@ - malonatoplatinum(II) (2.02 potassium anti-bis(2-methylmalonate)platinum(II) ”),17 dihydrate (2.01, 2.00 and potassium dichloro- ”),18 (oxalato)platinate(II) hydrate (2.04, 2.03 ”).18 The O3wPtwO3@ angle [91.5(5)°] is typical for a sixmembered chelate ring in a PtII-bicarboxylate complex.6,7,17,18 The six-membered Pt-CBDCA ring adopts a —attened boat conformation which is similar to that observed in carboplatin crystals.6,7 The N1wPtwN1@ [84.0(5)°] angle has also a normal value for a –ve-membered ethylenediamine chelate ring.The Pt-en chelate ring exhibits both d and l twisted sites C2A and C2B in the alternative conformations (d and k), and both conformations were equally populated in the crystal, with N(1)wCwCwN(1@) torsion angles of 58.7(8)° (d conformer) and [58.7(8)° (k conformer), similar to those reported for other Pt-ethylenediamine complexes.16 CCDC reference number 440/001.NMR studies of [Pt(en)(CBDCA)] 1 The 13CM1HN NMR solution spectrum of 1 at 298 K contains –ve signals that can be assigned to the ring carbons C8 (16.0 ppm), C7 and C9 (31.7 ppm), C6 (56.9 ppm), the carboxyl carbons C4 and C4@ (182.3 ppm) of CBDCA, and the methylene carbons C2A and C2B (48.7 ppm) of ethylenediamine.Similar 13C chemical shifts for the CBDCA ligand have been reported for carboplatin.6 Despite the inequivalence of the cyclobutane ring carbons in the solid-state structure of 1, only one resonance was seen for C7 and C9, showing that rapid ring inversion occurs as for carboplatin.6 Similarly, the d and k conformations of the Pt-ethylenediamine ring are in rapid exchange, only one set of signals being observed for the methylene carbons.The solid-state 13C CP/MAS NMR spectrum of complex 1 at 297 K is shown in Fig. 2(a). Now two broad signals are observed at 49.6 and 50.3 ppm, which are assignable to the en carbons. On increasing the temperature, the two signals CH2 became closer, began to merge at 317 K, and gave rise to a single sharp signal at 390 K [Fig. 2(b), peak c]. Based on the assumption that the two carbon signals are well separated at 297 K Hz), and two-site exchange with equal (*t1@2\186 populations,19 the exchange rate constant was determined to be 415 s~1.At the coalescence temperature of ca. 317 K, the activation energy *Gt was calculated to be 62 kJ mol~1. The energy barriers for this conformational change in an unsubstituted –ve-membered chelate ring, with a metal» nitrogen bond length of 2.0 have been estimated by strain- ”, energy minimization calculations to be 20 kJ mol~1 in solution.20 In the solid state, the energy barrier would be expected to be much higher.Because there are very limited solid-state NMR data available for ethylenediamine complexes, we also studied the 13C NMR spectra of two other platinum(II) and palladium(II) ethylenediamine complexes at 297 K. Only one signal was observed for the two ring carbons of both (50.1 [Pt(en)I2] ppm) and (46.5 ppm).We have recently [Pd(en)2][PdCl4] reported solid-state NMR data for the outer-sphere macrochelate complexes [Pt(en)(5@-GMP) (49.3 ppm) and 2] [Pd(en)(5@-GMP) (48.9 ppm), where again the ethyl- 2] enediamine ligand also gave rise to only one signal.21 Therefore it appears that the slower exchange rate in complex 1 is due to the presence of the CBDCA chelate ring.The other –ve major peaks observed in the solid-state 13C NMR spectrum of 1 [Fig 2(a)] can be assigned to C8 (16.8 ppm), C6 (56.0 ppm), C7 (27.5 ppm), C9 (35.9 ppm) and C4 Fig. 2 The 13C CP/MAS spectra of [Pt(en)(CBDCA-O,O@)] 1 at (a) 297 K and (b) 390 K. Peaks h and j are assigned to the groups of CH2 ethylenediamine and coalesce to give peak c ; other assignments are : a, C8; f and g, C7 and C9; d, C6; e, C4 and C4@.The * denotes minor peaks which may due to a diÜerent conformation of the CBDCA ring (see text) and C4@ (181.3 ppm) of the CBDCA ligand. A series of minor signals were also observed [denoted by * in Fig. 2(a)]. These signals decreased in intensity or disappeared at 390 K [Fig. 2(b)]. From their chemical shifts and temperature dependences, they can tentatively be assigned to a minor alternative conformation of the CBDCA ligand. Further support for the presence of conformational —exibility within the CBDCA ligand of 1 comes from the observation that the cyclobutane ring was not perfectly located in the X-ray diÜraction electron density map. Similarly, in crystalline carboplatin the CBDCA ring also exhibits substantial motion at room temperature, which has been related to the dynamic puckering between two conformations.7 The potential barrier was estimated to be below 6 kJ mol~1.We recorded the solid-state 13C CP/MAS NMR spectrum of crystalline carboplatin at 298 K, but only –ve signals were observed at 16.4, 28.1, 35.3, 56.5 and 182.0 ppm, which can be assigned to the –ve magnetically non-equivalent types of carbon atoms in the CBDCA ligand (four cyclobutane resonances, one peak for the two carboxylates).The shifts are very similar to those of 1 [Fig. 2(a)]. Since no minor signals were observed in the spectrum, it can be concluded that the energy barrier for the dynamic puckering is lower than for 1. Taken together, it seems clear that two chelate rings in bis(chelated)-PtII complexes exhibit interactive conformational dynamics.Such eÜects may be important to molecular recognition processes involving platinum drugs and may be relevant to the design of anticancer drugs. Acknowledgements thank the Association for International Cancer Research, We BBSRC for their support for this work, Dr.P. J. Barrie (University College London) for recording the solid-state NMR spectra and for helpful discussions, and University of London Intercollegiate Research Service for the provision of NMR facilities. We thank the EC support of COST group D1/0002/92. New J. Chem., 1998, Pages 11»14 13References 1 Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy 2, ed.H. M. Pinedo and J. H. Schornagel, Plenum, New York, 1996. 2 J. Reedijk, J. Chem. Soc., Chem. Commun., 1996, 801. 3 U. Frey, J. D. Ranford and P. J. Sadler, Inorg. Chem., 1993, 32, 1333. 4 S. K. C. Elmroth and S. J. 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