Spectroscopic characterization of titanocene complexes with thionucleobases

www.elsevier.nl/locate/ica Inorganica Chimica Acta 298 (2000) 178 – 186

Spectroscopic characterization of titanocene complexes with thionucleobases Enrique Mele´ndez *, Marı´a Marrero, Carmen Rivera, Eliud Herna´ndez, Alex Segal Uni6ersity of Puerto Rico, Department of Chemistry, PO Box 9019, Mayagu¨ez, PR 00681, Puerto Rico Received 8 June 1999; accepted 1 September 1999

Abstract The reaction of Cp2TiCl2 with thionucleobases affords [Cp2Ti(L)]Cl2 complexes, where L = 2-thiouracil, 6-thiopurine, 6thioguanine, 2-thiocytosine, 6-thiopurineribose or 6-thioguanineribose. The ionic character of the chlorides has been determined by conductivity measurements and the coordination patterns of the thionucleobases have been determined by IR and proton NMR spectroscopies. The kinetics of ligand hydrolysis have been pursued to assess the stability of TiL and TiCp bonds in DMSO and DMSO–water solutions. © 2000 Elsevier Science S.A. All rights reserved. Keywords: Antitumor agent; Titanocene complexes; Thionucleobase complexes

1. Introduction In 1979, Ko¨pf-Maier and Ko¨pf published the first metallocene with antitumor activity, titanocene dichloride (Cp2TiCl2) [1]. Subsequently, other metallocene complexes with the general formula Cp2MX2 (M= V, Nb, Mo; X=halides and pseudo halides), Cp2FeX and main group analogues (C5R5)M (M =Sn, Ge; R= CH3) have been tested for antitumor activity [2–4]. These complexes showed antineoplastic activity against a wide variety of tumor cells and in particular Cp2TiCl2 was demonstrated to be more effective than platinum complexes [2,5–8]. Although the mechanism of action of these metallocenes is not clear, nucleic acids have been proposed as the target areas in the cell, most likely suppressing the synthesis of RNA or DNA [5,9–13]. Our interest in the coordination chemistry of titanocene dichloride is based on the fact that among all the metallocenes tested, Cp2TiCl2 (I) was demonstrated to be the most active antitumor agent, showing its greatest activity against colon, lung and breast cancers [2,3,5–8]. Therefore, our research program is aimed at the understanding of the interaction between titanocene dichloride and modified RNA and DNA bases, in

particular those bases where the oxygen atom is replaced by sulfur. Thionucleobases are modified RNA/ DNA bases with well recognized antitumor activity [14]. From the coordination point of view, their interaction with Cp2TiCl2 would model the interaction between RNA/DNA bases and titanocene dichloride to some extent, since these bases have similar binding interactions to their oxygen counterpart [14–16]. On the other hand, the formation of a stable complex between Cp2TiCl2 and the thionucleobase could render enhanced antitumor activity, since they contain two antineoplastic agents within the same compound [17– 20]. In fact, one example of synergism between Cp2TiCl2 and 5-fluorouracil has been reported in the literature [5]. Therefore, based on these arguments, we have spectroscopically characterized a series of titanocene complexes containing 2-thiouracil, 2-thiocytosine, 6-thiopurine, 6-thioguanine, 6-thioguanineribose and 6-thiopurineribose. Preliminary kinetic studies have been pursued to assess the stability of TiL and TiCp bonds in DMSO and DMSO–water solutions.

* Corresponding author. Tel.: + 1-787-832 4040; fax: + 1-787-265 3849. E-mail address: e – [email protected] (E. Mele´ndez) 0020-1693/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved. PII: S 0 0 2 0 - 1 6 9 3 ( 9 9 ) 0 0 4 4 1 - 7

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2. Experimental All procedures involving the handling of organometallic compounds were carried out under an atmosphere of prepurified nitrogen either in a glove box or in double-manifold vacuum lines. Solvents and solutions were added by glass syringe with stainless steel needles. Solvents were degassed (oxygen removal) using a purge of nitrogen. 1H and COSY spectra were recorded on a 300 MHz Varian Gemini spectrometer under controlled temperature. TMS was used as an internal reference. Elemental analysis was obtained from Atlantic MicroLab Inc. MS spectra were recorded on a VG–Fisions Autospec high resolution mass spectrometer using DIP mode. Peaks are only quoted if their relative intensities are at least 10% of the intensity of the strongest peak. IR spectra were taken on a Perkin–Elmer Model 2000 using KBr pellets. Titanocene dichloride, 2-thiouracil, 2-thiocytosine, 6mercaptopurine and 6-thioguanine were obtained from Aldrich and used without further purification. 6Thioguanineribose and 6-mercaptopurineribose were obtained from Sigma and used as received. The purity of the ligands and titanocene dichloride was verified by 1 H NMR spectroscopy. Methanol was reagent grade from Fisher. DMSO-d6 (from Aldrich) was distilled over CaH2 and stored under nitrogen over molecular sieves. The conductivity measurements for Ti complexes were carried out in millimolar solutions of methanol. Kinetic studies were carried out under a nitrogen atmosphere. Solvents (DMSO and water) were deoxygenated using a sparger with prepurified nitrogen. NMR samples were prepared transferring the weighted amount of Ti complex in a 5-mm NMR tube and the solvent added to form solutions of 1×10 − 2 M, under an atmosphere of nitrogen, either in the glove box or using a double-manifold vacuum line. The DMSO used was DMSO-d6 and the water was distilled and deionized. Water suppression techniques were used to suppress the water peak only in those experiments where the water signal created interference for integration or baseline distortion. Nevertheless, we found that if the peaks analyzed are 1 ppm away from the water peak, suppression was not necessary. The rate of Cp protonolysis was monitored by integration of one of the multiplets at 6.45 ppm (from the free cyclopentadiene, C5H5D) versus the bound Cp signal (6.66 ppm, TiCp). For the DMSO–water solutions, the content of water was estimated to be 1%. Solutions of 1× 10 − 3 M were used for the UV–Vis experiments. The pH reported are the average measured values of the resulting solutions. Two bands were used to monitor the thionucleobase loss, in the range 380 – 392 and 515 – 522 nm. Cp2TiCl2 has two coordination-sensitive absorption bands at 380 and 518 nm. Upon coordination to the thionucleobase, these bands shift to longer wavelength but the spectral

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shape remains unchanged. Therefore, we have used these two bands to monitor thionucleobase loss, since monitoring of this process by proton NMR is more complicated and less sensitive. Typically, the band at 380–392 nm provided more linear response, thus the observed rate constants reported are calculated using this region. For 2-thiouracil and 2-thiocytosine the wavelength used was 386 nm while for 6-thiopurine and 6-thioguanine the wavelength was 392 nm. The rate constants are averaged values of three independent measurements.

2.1. Synthesis of [Cp2Ti(L)]Cl2 In a typical reaction 0.10 g (0.4 mmol) of titanocene dichloride and the corresponding ligand (0.4 mmol) were mixed in 5 ml of methanol at room temperature under nitrogen. The reaction mixture was stirred for 8–12 h, leading to a brick-red heterogeneous mixture. The solid was isolated in a fritted funnel of fine porosity and the solid dried in vacuo at 45°C for 12 h. The yield ranged from 50–60% based on Cp2TiCl2.

2.2. Spectroscopic and analytical data 2.2.1. [Cp2Ti(6 -thioguanine)]Cl2 (1) 1 H NMR (DMSO-d6, ambient) d: 13.00 (s, 1H, NH), 12.05 (s, 1H, NH), 8.07 (s, 1H, H(8)), 6.67 (s, 10H, Cp), 6.62 (s, 2H, NH2). IR data (KBr, cm − 1): 3427(w), 3283(w), 3109(vs), 2929(w), 2845(w), 1665(s), 1618(s), 1539(m), 1483(w), 1440(m), 1376(m), 1259(m), 1231(m), 1143(w), 1107(w), 1031(m), 1016(m), 972(m), 871(w), 822(s), 779(w), 718(m), 621(m), 586(w), 565(w). MS (EI) m/z (relative intensity): 65(38), 83(16), 118(13), 120(10), 122(11), 148(58), 150(23), 169(10), 181(13), 182(10), 183(100), 184(18), 185(68), 187(13), 213(20), 248(30), 250(20). LM (cm2 ohm − 1 mol − 1)= 157. Anal. Calc.: C, 43.29; H, 3.65. Found: C, 43.24; H, 3.71%. 2.2.2. [Cp2Ti(6 -thiopurine)]Cl2 (2) 1 H NMR (DMSO-d6, ambient) d: 13.75 (d, 1H, J=1 Hz, NH), 8.18 (d, 1H, J= 2.6 Hz, H(2)), 8.38 (s, 1H, H(8)), 6.67 (s, 10H, Cp), 3.85 (bs, 1H, NH). IR data (KBr, cm − 1): 3434(m), 3327(w), 3099(m), 3049(sh), 2905(vw), 2804(m), 2678(w), 1665(w), 1602(s), 1577(sh), 1530(w), 1439(m), 1408(s), 1384(m), 1347(w), 1331(m), 1276(w), 1225(m), 1208(m), 1122(w), 1016(s), 936(m), 876(s), 821(s), 676(w), 649(m), 580(w), 509(w). MS (EI) m/z (relative intensity): 65(36), 83(17), 85(11), 118(14), 120(10), 122(11), 148(54), 149(10), 150(23), 181(12), 182(10), 183(100), 184(18), 185(70), 187(14), 213(16), 248(32), 250(23). LM (cm2 ohm − 1 mol − 1) =156. Anal. Calc.: C, 44.91; H, 3.52. Found: C, 44.66; H, 3.62%.

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2.2.3. [Cp2Ti(2 -thiocytosine)]Cl2 (3) 1 H NMR (DMSO-d6, ambient) d: 13.40 (bs, 1H, NH), 9.93 (s, 1H, NH2), 8.66 (s, 1H, NH2), 7.73 (d, 1H, H(6)), 6.68 (s, 10H, Cp), 6.38 (d, 1H, H(5)). IR data (KBr, cm − 1): 3340(m), 3312(m), 3145(m), 3100(m), 3051(m), 2935(m), 2856(m), 2798(m), 2751(sh), 2647(w), 2606(w), 1660(s), 1598(s), 1552(s), 1470(w), 1441(w), 1411(w), 1380(m), 1293(w), 1215(s), 1194(m), 1173(s), 1087(m), 1067(m), 1016(w), 986(m), 943(w), 872(w), 822(m), 807(m), 795(m), 721(m), 689(w), 641(m), 600(m), 543(m). MS (EI) m/z (relative intensity): 65(35), 69(12), 83(15), 85(11), 118(13), 85(10), 118(14), 120(10), 127(22), 148(42), 150(17), 181(12), 182(10), 183(100), 184(19), 185(68), 187(14), 213(11), 248(32), 250(22). LM (cm2 ohm − 1 mol − 1) = 160. Anal. Calc.: C, 44.70; H, 4.02. Found: C, 44.53; H, 4.09%. 2.2.4. [Cp2Ti(6 -thioguanineribose)]Cl2 (4) 1 H NMR (DMSO-d6, ambient) d: 12.02 (s, 1H, NH), 8.20 (s, 1H, H(8)), 6.88 (s, 2H, NH2), 6.67 (s, 10H, Cp). Ribose unit (DMSO-d6, ambient) d: 5.70 (d, 1H, J=5.7 Hz, C(1)H), 4.39 (t, 1H, J =5.2 Hz, C(2)H), 4.10 (t, 1H, J = 4.8 Hz, C(3)H), 3.88 (d, 1H, J=3.5 Hz, C(4)H), 3.63 (dd, J = 3.9, 8.2 Hz, C(5)Ha), 3.53 (dd, 1H, J= 3.9, 8.1 Hz, C(5)Hb). IR data (KBr, cm − 1): 3543(w), 3419(s), 3275(w), 3234(W), 3091(w), 2925(w), 1636(s), 1617(s), 1559(m), 1440(w), 1405(m), 1213(m), 1132(m), 1055(w), 1016(m), 968(w), 901(w), 821(m), 753(w), 634(m). MS (EI) m/z (relative intensity): 65(36), 83(19), 85(12), 118(14), 120(10), 122(12), 148(63), 149(12), 150(26), 181(12), 182(10), 183(100), 184(19), 185(69), 187(15), 213(15), 215(10), 248(33), 250(22). LM (cm2 ohm − 1 mol − 1) =172. Anal. Calc.: C, 43.81; H, 4.22. Found: C, 43.95; H, 4.26%. 2.2.5. [Cp2Ti(6 -thiopurineribose)]Cl2 (5) 1 H NMR (DMSO-d6, ambient) d: 13.82 (d, 1H, J= 1.35 Hz, NH), 8.55 (s, 1H, H(8)), 8.22 (d, 1H, J= 3.8 Hz, H(2)), 6.67 (s, 10H, Cp). Ribose unit (DMSO-d6, ambient) d: 5.89 (d, 1H, J =5.4 Hz, C(1)H), 4.47 (t, 1H, J = 5.0 Hz, C(2)H), 4.14 (t, 1H, J = 4.0 Hz, C(3)H), 3.95 (d, 1H, J =3.7 Hz, C(4)H), 3.65 (dd, 1H, J= 3.7, 8.3 Hz, C(5)Ha), 3.58 (dd, 1H, J =3.7, 8.3 Hz, C(5)Hb). IR data (KBr, cm − 1): 3413(bs), 3104(m), 3039(w), 2925(w), 2657(w), 1606(s), 1574(m), 1538(m), 1474(w), 1417(m), 1337(m), 1195(m), 1117(w), 1071(m), 1018(m), 983(m), 873(w), 821(s), 651(w). MS (EI) m/z (relative intensity): 65(40), 69(13), 83(18), 118(13), 120(10), 122(11), 148(62), 149(11), 150(25), 181(13), 183(100), 184(18), 185(69), 187(13), 213(24), 248(32), 250(20). LM (cm2 ohm − 1 mol − 1) = 170. Anal. Calc.: C, 45.05; H, 4.16. Found: C, 45.10; H, 4.13%. 2.2.6. [Cp2Ti(2 -thiouracil)]Cl2 (6) 1 H NMR (DMSO-d6, ambient) d: 12.29 (unresolved d, 1H, NH), 12.43 (s, 1H, NH), 7.39 (dd, 1H, J = 5.7,

7.43 Hz, H(6)), 6.65 (s, 10H, Cp), 5.80 (d, 1H). IR data (KBr, cm − 1): 3100(w), 3087(s), 3048(m), 2926(m), 2655(vw), 1704(s), 1684(s), 1628(m), 1564(s), 1440(m), 1422(m), 1394(w), 1368(w), 1239(s), 1211(s), 1176(m), 1157(m), 1072(w), 1016(w), 1003(w), 983(w), 926(w), 913(w), 893(m), 874(m), 821(s), 759(m), 736(w), 709(m), 648(w), 547(s), 526(m). MS (EI) m/z (relative intensity): 65(33), 69(13), 83(16), 85(10), 118(18), 120(11), 122(10), 128(38), 148(45), 150(18), 181(11), 182(10), 183(100), 184(19), 185(68), 187(14), 213(14), 248(33), 250(23). LM (cm2 ohm − 1 mol − 1)= 165. Anal. Calc.: C, 44.59; H, 3.74. Found: C, 44.49; H, 3.76%.

3. Results and discussion The reaction of titanocene dichloride with one equivalent of the corresponding thionucleobase in methanol affords a weak but stable Lewis acid–base adduct between the Cp2Ti2 + fragment and the thionucleobase, in a 1:1 stoichiometric ratio, Eq. (1). MeOH

Cp2TiCl2 + L “ [Cp2Ti(L)]Cl2

(1)

L= 6-thiopurine, 6-thioguanine, 2-thiocytosine, 6-thiopurineribose, 6-thioguanineribose, 2-thiouracil. The ionic character of the compounds was determined by conductivity measurements in methanol. Millimolar solutions of the compounds showed conductivities in the range 155–172 mS, indicating the presence of an electrolyte in the ratio of 1:2 (cation:anion) [21]. This suggests that the two chlorides are mainly ionic. In contrast to previously reported titanocene complexes containing nucleosides, the isolated species did not contain any methanol molecule coordinated to titanium [21]. Addition of a second equivalent of the thionucleobases did not consistently afford the disubstituted complex, [Cp2T(L)2]Cl2, suggesting a possible equilibrium between the mono- and disubstituted complex, Eq. (2). [Cp2Ti(L)]Cl2 ? [Cp2Ti(L)2]Cl2

(2)

The chemical composition of the complexes has been verified by elemental analysis and their structures have been determined by IR and NMR spectroscopies. The complexes are soluble in DMSO therefore, parts of their characterization have been performed in this solvent. In addition, to determine the stability of these complexes in DMSO and DMSO–water solutions, kinetic studies of their ligand hydrolysis under pseudo-first order conditions were investigated.

3.1. NMR and IR spectroscopies of [Cp2Ti(L)]Cl2 complexes Caution in the analysis of the IR and NMR spectra should be taken since they are recorded in different

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media and phases, NMR in a DMSO solution and IR in a KBr pellet. This sometimes points to a situation where perhaps in the solid state there is only one predominant isomer while in solution more than one may exist. It is important to notice that in DMSO the complexes loose the thionucleobases slowly, forming the solvated titanocene cation species. Nevertheless, DMSO is the only solvent where the complexes are completely soluble. The complexes are partially soluble in methanol and insoluble in pure water. Included in the supplementary material are tables containing IR and proton NMR data for ligands and their complexes.

3.1.1. [Cp2Ti(6 -thioguanine)]Cl2 The 1H NMR and IR data for the ligand (6-thioguanine) and its complex have been examined to propose the most likely structure of the complex. Similar arguments will be extended to the other thionucleobases

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(vide infra). The proton NMR of 6-thioguanine, Fig. 1, shows the presence of two tautomers in a ratio of 1.2:0.8, both having two NH resonances around 11.8 to 13 ppm. In addition, two H(8) and NH2 peaks are observed for each corresponding tautomer. This indicates that in DMSO solutions, two isomers exist in their thione tautomeric form, most probably N1,7(H) and N1,9(H) and not in the thiol form [22]. Upon coordination, only one tautomer is recognized depicting two NH resonances at 12.05 (singlet) and 13.00 (broad singlet) ppm. The H(8) is located at 8.07 ppm, somewhat upfield when compared to one tautomer and downfield when compared to the other. The IR spectrum exhibits some minor perturbations in the CS vibration. The band at 1231 cm − 1, attributed to CS stretching, decreased in intensity and the band at 1202 cm − 1 vanished. The decrease in intensity of the CS band has been accounted for as the substitution on

Fig. 1. 1H NMR of 6-thioguanine tautomers (upper trace) and [Cp2Ti(6-thioguanine)]Cl2 (bottom trace). * indicates impurity.

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sulfur by either a methyl group or metal coordination [23,24]. Furthermore, around ca. 1600 cm − 1, where the CN and CC appear, the band at 1637 cm − 1 vanishes and the one at 1618 cm − 1 decreases in intensity. Based on this information, we believe the thioguanine is likely to be engaged in a N(7) coordination to the Ti center, probably assisted by a weak S···Ti interaction.

To further elucidate the coordination features of 6-thioguanine, we studied the N(9) ribose substituted ligand, 6-thioguanineribose. The ribose substitution ruled out the coordination of N(9) to titanium. Assignments of the ligand and [Cp2Ti(6-thioguanineribose)]Cl2 complex 1H NMR resonances were assisted by COSY spectra (supplementary material). Surprisingly, the proton NMR pattern of [Cp2Ti(6-thioguanineribose)]Cl2 is virtually identical to the 6-thioguanine complex. The resonance previously observed at 13.00 ppm (N(9)H) in the thioguanine complex does not appear since it has been substituted by the ribose unit. Additional resonances are observed due to the ribose unit. Interestingly the OH resonances of the ribose, which are observed in the free ligand, collapse in a broad peak at 5 ppm and show no coupling to the CH or CH2 groups. This behavior suggests that the OH groups are possibly undergoing chemical exchange or being perturbed due to hydrogen bonding to the chlorides. Hydrogen bonding to chloride has been identified in [Cp2Ti(L)2]Cl2 (L = amino acids) complexes causing distortion of the NH3 protons [25–27]. On the other hand, coordination of the OH groups to Ti may not only shift the OH resonances downfield, but would also change the H(8) and NH2 chemical shifts. Nevertheless, other dimeric or oligomeric structures may be possible and cannot be ruled out.

3.1.2. [Cp2Ti(6 -thiopurine)]Cl2 Likewise, the spectral features of 6-thiopurine and its complex have been utilized to propose the structure of the Ti-(6-thiopurine) complex. The proton NMR spectrum of 6-thiopurine shows only one resonance (a singlet) for the NH proton (13.79 ppm) and two singlets for H(2) and H(8) (8.23 and 8.43 ppm) with a broad peak centered about 4.1 ppm, which suggests an exchange between the imino proton and traces of water in DMSO. Based on this information together with the IR data, thiopurine is predominantly in the N1,9(H)

tautomeric form. Upon coordination, the 6-thiopurine experiences some minor but significant upfield shifts in all their proton signals. More importantly, the NH resonance becomes an unresolved doublet along with H(2). This indicates that N(1)H is coupled to H(2) perhaps due to a more rigid conformation of the thiopurine when attached to the metal center. The exchangeable NH is also shifted upfield. While a downfield shift in the proton resonances have been taken as an indication of coordination of RNA/DNA bases to metal centers [28,29], in metallocene systems the opposite behavior has also been observed when the protons fall below the Cp plane and has been attributed to the magnetic anisotropy induced by the Cp rings [30]. The IR spectrum shows stretching bands attributed to two different NH groups. The bands corresponding to CN and CC bonds are displaced to lower wavenumbers. These two pieces of information lead to a structure where 6-thiopurine is most likely attached to N(7), S coordination sites (III) analogous to the 6thioguanine complex.

The coordination pattern of the thiopurine was corroborated by using the N(9)-ribose substituted purine, 6-thiopurineribose. The 1H NMR spectrum of [Cp2Ti(6-thiopurineribose)]Cl2 shows an almost identical proton chemical shift pattern (in the purine framework) to the thiopurine complex. Again, N(1)H is coupled to H(2) as identified by a COSY experiment. In a similar manner as the 6-thioguanineribose complex, a major perturbation for the OH groups is observed at 5 ppm as compared to the free ligand where a broad peak now appears. Furthermore, all the coupling of these OH groups to the CH and CH2 groups, vanished analogous to the 6-thioguanineribose complex, perhaps due to hydrogen bonding, as previously explained for the 6-thioguanineribose complex, or due to chemical exchange.

3.1.3. [Cp2Ti(2 -thiocytosine)]Cl2 The analysis of the proton NMR (Fig. 2) of this complex clearly indicates that the NH2 is engaged in bonding and probably assisted by N(3) making a fourmembered chelate ring. For instance the NH2 signals have been shifted downfield from 7.70 and 7.54 ppm to 9.93 and 8.66 ppm, respectively. This marked difference in the NH2 signals strongly suggest that the nitrogen

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Fig. 2. 1H NMR of 2-thiocytosine (upper trace) and [Cp2Ti(2-thiocytosine)]Cl2 (bottom trace).

lone pair is coordinated to the Ti center, although, Zhang et al. have suggested hydrogen bonding between coordinated H2O and NH2 groups [31,32]. However, the low content of water (if any) in a dried DMSO-d6 makes this binding interaction less likely, but on the present information it cannot be ruled out altogether. The H(5) and H(6) signals were shifted downfield by more than 0.3 ppm. The NH signal was also shifted downfield. Based on this data, the most probable structure involves mainly NH2 and N(3) coordination (IV). A four-membered ring has been observed in Cp2Mo(L)PF6, (L=1-methylcytosyl, 9-methyladenyl) solid-state structures [33]. Although Ti(IV) has a smaller radius than Mo(IV), Ti(IV) is a stronger Lewis acid than

molybdenum, this acidic character being perhaps the driving force for such a strained coordination mode. Nevertheless, other structures cannot be ruled out.

3.1.4. [Cp2Ti(2 -thiouracil)]Cl2 The interaction of 2-thiouracil with the Ti center appears to be very weak. Thus, coordination of Ti to

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2-thiouracil creates some minor perturbations in the spectra, but significant enough to account for some sort of binding interaction. Analysis of the proton NMR spectrum of 2-thiouracil shows two doublets corresponding to hydrogen atoms attached to C(5) and C(6), 5.87 (H(5)) and 7.41 (H(6)) ppm. In addition, two singlets are observed for N(1)H and N(3)H protons at 12.28 and 12.44 ppm, respectively. On the other hand, the [Cp2Ti(2-thiouracil)]Cl2 proton NMR spectrum shows two NH signals, but one of them, at 12.29 ppm corresponding to the N(1)H proton, becomes an unresolved doublet. Furthermore, H(6) is split into a doublet of doublets. This is due to additional splitting of the H(6) to N(1)H proton. Both H(5) and H(6) show upfield shifts. The IR spectrum of the complex exhibits a band attributed to the n(CO) of 2-thiouracil at 1707 cm − 1 shifted to 1703 cm − 1. Other perturbations involve the disappearance of the band at 1604 cm − 1 assigned to the CC stretching vibration and a decrease in the intensity of the CS vibration at 1211 cm − 1. These minor but evident perturbations have been observed in the past for thiouracil when the ligand is engaged in weak binding interactions [23,24]. Therefore, the spectral data suggest that thiouracil is most likely engaged through both oxygen (O(4)) and sulfur (S(2)) coordination most probably bridging two Cp2Ti2 + units.

3.2. Kinetic studies The stability of these complexes in DMSO and DMSO–water is of fundamental importance since it provides insight into which species reaches the target area in the cell. In aqueous solution, the rate of chloride hydrolysis and Cp protonolysis in titanocene dichloride have been studied by Toney and Marks [34]. Toney and Marks reported rapid hydrolysis of the two chlorides in which the Cp remains bound to Ti at least for a period of 24 h, as long as the pH is kept below 5 [34]. At physiological pH, extensive Cp protonolysis occurs leading to the formation of cyclopentadiene (and its dimer) and an uncharacterized hydrolysis product.

Below pH 5, Cp2Ti(H2O)22 + is the proposed principal species, although several other intermediate species may exist as products of incomplete chloride hydrolysis. More recently, Mokdsi and Harding published two studies on the stability of Cp2TiX2 complexes (X=Cl, O2CCH2NH3Cl and O2CCCl3) in water, DMSO and DMSO–water [35,36]. Again, at low pH, in aqueous solution, the existing species is Cp2Ti(H2O)22 + and the presence of DMSO promotes Cp protonolysis. In particular, for the complexes containing O2CCH2NH3Cl and O2CCCl3 ligands, the Cp protonolysis in DMSO is promoted and more extensive than in the chloride complex [36]. Our stability studies are somewhat different from those reported by Harding and co-workers and could be due to the presence of the thionucleobases as ligands. We have noticed that these complexes are stable in dried degassed DMSO with respect to the TiCp bond but, DMSO slowly replaces the thionucleobase forming possible solvated species, such as Cp2Ti-h1L(DMSO)2 + or Cp2Ti(DMSO)22 + . In fact these complexes in dried, degassed DMSO-d6 show only very minor Cp protonolysis, B 4% as evidenced by 1H NMR spectroscopy (supplementary material), but the presence of Ti–DMSO interaction was evident from the appearance of a multiplet at 2.9 ppm, which has been attributed to coordinated DMSO [36]. However, UV– Vis spectroscopy proved to be a better technique for monitoring the thionucleobase loss (dissociation). Two bands were used to monitor the progress of the reaction about 380–392 and 518–522 nm. When these regions between Cp2TiCl2 and [Cp2Ti(L)]Cl2 complexes were compared, it was observed that the bands move to longer wavelengths for [Cp2Ti(L)]Cl2 compared to Cp2TiCl2 and maintain similar spectral shape. Therefore, these regions in the UV–Vis spectrum are excellent places to monitor thionucleobase loss. Table 1 presents the kinetic data under pseudo-first order conditions in dried DMSO, DMSO–H2O (1%) and DMSO–H2O (1%), 0.1 M NaCl for reactions monitored at 380–392 nm region. The pH reported are average measured values for the resulting solution.

Table 1 Initial rate of thionucleobase loss at 37°C (UV–Vis) [Cp2Ti(L)]Cl2

kobs (DMSO–H2O, pH 4.2–4.9) (min−1)

kobs (DMSO) (min−1)

kobs, (DMSO/0.1 M NaCl, pH 4.2–4.9) (min−1)

L =2-thiouracil L =6-thiopurine L =6-thioguanine L =2-thiocytosine

1.13(0.06)×10−2 9.8(0.5)×10−3 1.19(0.04)×10−2 3.52(0.07)×10−3

4.9(0.3)×10−2 1.6(0.2)×10−2 2.0(0.2)×10−2 8.8(0.3)×10−4

3.13(0.04)×10−3 5.7(0.2)×10−3 1.47(0.09)×10−2 1.0(0.6)×10−3

E. Mele´ndez et al. / Inorganica Chimica Acta 298 (2000) 178–186 Table 2 Initial rate of cyclopentadienyl loss at 37°C [Cp2Ti(L)]Cl2

kobs (DMSO–H2O, pH 4.2–4.9) (min−1)

kobs (DMSO–0.1 M NaCl, pH 4.2–4.9) (min−1)

L = 2-thiouracil L =6-thiopurine L=6-thioguanine L=2-thiocytosine

1.1(0.2)×10−3 2.1(0.2)×10−3 2.9(0.5)×10−3 3.8(0.2)×10−4

1.6(0.1)×10−3 2.8(0.6)×10−4 5.4(0.5)×10−4 1.1(0.1)×10−3

There are several points that deserve to be mentioned about Table 1. First, for the DMSO – water solutions, the measured pH is B 5, a pH where the TiCp bond is stable, at least more stable than TiL (where L = Cl, O2CCH2NH3Cl and O2CCCl3) [34 – 36] and in our case, more stable than Ti – thionucleobase (Table 2). Second, except from 2-thiocytosine, the Ti – thionucleobase interaction is less stable in DMSO than in DMSO – water. The opposite behavior is observed for the TiCp bond. Furthermore, addition of NaCl to the solvent medium does not dramatically change the rate of thionucleobase loss, implying that the chloride does not have a major effect on this decomposition pattern. The 2-thiocytosine complex was shown to be the most stable in all solvent media tested compared to others. Table 2 presents the results of Cp protonolysis. In general terms, Cp loss is about one order of magnitude slower than thionucleobase loss when the reaction medium is DMSO– water. The presence of NaCl in the solution decreases the rate of the Cp loss for the 6-thioguanine and 6-thiopurine complexes and increases the rate for 2-thiocytosine. For 2-thiouracil, NaCl has no notable effect on the reaction rate. In addition, in DMSO – NaCl medium, Cp and thionucleobase loss becomes a competitive (most probably parallel) processes for the 2-thiocytosine and 2-thiouracil complexes, while for the purine-containing ligands, the Cp protonolysis is a process that occurs at a slower rate than the thionucleobase loss. In all the experiments performed in DMSO or DMSO–water solutions we did not observe the formation of a precipitate as reported in Cp2TiX2 (X= O2CCl3 and O2CCH2NH3Cl) [36]. Attempts to isolate the Cp protonolysis product using Marks procedure [34] failed, leading to insoluble material in the water or DMSO. In any event, the material isolated, as judged by its solubility, may not correspond to the species formed in the kinetic studies. However, what is clear at this point is that the thionucleobase is lost in DMSO or DMSO–water to form either Cp2Ti(DMSO)22 + or Cp2Ti(H2O)22 + [34– 36]. Evidence of Ti – DMSO interaction can be supported by the appearance of a multiplet at 2.90 ppm as reported by Harding [35]. While

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[Cp2Ti(X)O(X)TiCp2] has been proposed to be the possible product of Cp2TiX2 (X=O2CCl3 and O2CCH2NH3Cl) hydrolysis [35,36], we have not detected the presence of this complex by 1H NMR spectroscopy, suggesting that the concentration of this complex, if formed, is very small. On the other hand, the nature of the Cp protonolysis product is uncertain. Toney and Marks suggested ‘TiCp1.66O4.5(OH)4.4’ as a product for Cp protonolysis at pH B 7 [34]. At the present time, we cannot rule out the formation of this species but this awaits further investigations.

4. Concluding remarks This contribution presents the possible coordination patterns of modified DNA/RNA bases to Cp2TiCl2. 6-Thiopurine and 6-thioguanine bases appear to coordinate through N(7), assisted by S(6). Such coordination has been identified in the solid state for palladium complexes [37], but in our case the binding interaction appears to be weak since the change in the proton chemical shift between the free and the coordinated ligand is not dramatic. Particularly interesting is the fact that molecular recognition between one tautomer of 6-thioguanine and Cp2Ti2 + can be identified. On the other hand, involvement of the NH2 group in 2-thiocytosine base can be easily envisioned based on proton NMR spectroscopy. Why can the NH2 group of 2-thiocytosine be involved in binding while the NH2 of the 6-thioguanine can not, is not clear at the present time, but again it could be part of the molecular recognition. For the 2-thiouracil, the perturbation created by Cp2Ti2 + is minimum, suggesting that its binding interaction is very weak, at least in DMSO. Finally, these complexes are labile in DMSO with respect to the thionucleobase but stable with respect to the TiCp bond as long as water is excluded. This behavior is the opposite to that of the titanocene complexes containing amino acid [36]. In fact, we have observed [Cp2Ti(L-cysteine)2]Cl2 and [Cp2Ti(D-penicilamine)2]Cl2 complexes to be soluble and stable in water and the presence of DMSO promotes extensive hydrolysis and formation of cloudy solutions [38]. Apparently, DMSO replaces the amino acid easily forming very unstable species. At this point, the chemical nature of the hydrolysis products as well as the stability of these complexes in other DMSO–water mixtures are still under investigation.

Acknowledgements We are grateful to the NIH-MBRS Program and the Department of Chemistry for support of this research.

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