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Four new water soluble molybdenocene complexes were synthesized in aqueous solution at pH 7.0. The new species, [(η5-C5H5)2Mo(L)]Cl (L= 6-mercaptopurine, 2-amino-6-mercaptopurine, (-)-2-amino-6-mercaptopurine ribose and 6-mercaptopurine ribose), were characterized by spectroscopic methods. NMR spectroscopic data showed the presence of two coordination isomers, S(6), N(7) and S(6), N(1), in aqueous solution, being S(6), N(7) the most stable. The antiproliferative activities of the new species were investigated in HT-29 colon and MCF-7 breast cancer cell lines. The incorporation of molybdenocene (Cp2Mo2+) into the thionucleobases/thionucleosides decreases their cytotoxic activities in HT-29 colon cancer cell line. In contrast, in the MCF-7 cell line, [Cp2Mo(2-amino-6-mercaptopurine)]Cl showed a high cytotoxic activity. This is most likely a consequence of the enhanced lipophilic character on the thionucleobase combined with synergism between Cp2Mo2+ and the thionucleobase ligand.
During the last century, metals have been used to treat fungal infections, arthritis, ulcers and other conditions. However, the interest of metals in medicine was awakened by the accidental discovery of cis-platin, Pt(NH3)2Cl2, as potential anticancer agent in 1970 . In subsequent years, other metals were explored for medicinal applications ranging from cancers to diagnostic, immunoassay, biosensors, enzymes and proteins .
The first non-platinum complex tested in clinical trials as antitumor agent was cis-[(CH3CH2O)2(bzac)2Ti(IV)], bzac = 1-phenylbutane-1,3-dioanate . This complex is active against a wide variety of ascites and solid tumors [3,4]. Other cis-[X2(bzac)2Ti(IV)], X = halides, complexes have been investigated exhibiting similar biological activity as the ethoxide complex . However, the interest for non-platinum complex proceeded slowly because of the pharmacological efficacy and potent antitumor properties of cis-platin.
The discovery of metallocene-based organometallic anticancer agent, titanocene dichloride (Cp2TiCl2), in 1979 by Köpf and Köpf-Maier  stimulated much interest to investigate other non-platinum complexes with a different mechanism of anticancer activity. Other diacido metallocene complexes, Cp2MX2 (Cp = cyclopentadienyl, M = Ti, V, Nb, Mo; X = halides and pseudo-halides), have been investigated for antitumor activity [6-11]. Among these metallocene complexes, Cp2TiCl2 has been studied in details but it possesses a major drawback: hydrolyzes extensively at physiological pH . In contrast, molybdenocene dichloride is stable at physiological pH, producing Cp2Mo(H2O)(OH)+ . Thus, there is a great potential to synthesize water soluble and stable molybdenocene species and study their biological properties.
Several water soluble thiols derivatives of molybdenocene have been prepared and cell uptake and cytotoxic studies were pursued by Harding and co-workers . It was found that the molybdenocenes containing thiols as ancillary ligands are robust toward hydrolysis but yielded non-cytotoxic complexes . Strong coordination of molybdenocene by the thiolates apparently hinders the formation of vacant coordination sites on the metal center and the net result is inactivation. In the past, we reported a series of water soluble molybdenocenes containing thionucleobases . These species were synthesized under non-aqueous solution. These complexes are stable at physiological pH but under these conditions, three or more species are produced in solution with unknown chemical formula. However, it is unclear as to which species have anticancer properties. Therefore, we decided to revisit the synthesis of molybdenocene-thionucleobase/thionulceoside complexes in aqueous solution at pH of 7.0, leading to the formation of more water stable species with features different to the previously synthesized molybdenocene-thionucleobase/thionulceoside complexes under non-aqueous solution. Herein we report the synthesis, characterization and cytotoxic activity of these molybdenocenes in HT-29 colon cancer and MCF-7 breast cancer cell lines.
At neutral pH molybdenocene dichloride undergoes chloride hydrolysis forming a stable species, Cp2Mo(OH)(H2O)+ [13,16]. Taking advantage of the stability of molybdenocene dichloride at neutral pH, we performed the reaction at pH 7.0. The reaction of Cp2MoCl2 with the corresponding thionulceobase or thionucleoside in degassed water at pH 7.0 leads to the formation of, after column chromatography using lipophilic Sephadex, orange solids of general formula [Cp2Mo(L)]Cl, L= 6-mercaptopurine, 2-amino-6-mercaptopurine, (-)-2-amino-6-mercaptopurine ribose and 6-mercaptopurine ribose. [Cp2Mo(L)]Cl species are highly soluble in water and stable at physiological pH for several days without decomposition. Previous report on the reaction of Cp2MoCl2 with the corresponding thionulceobase or thionucleoside in methanol afforded slightly different species, [Cp2Mo(L)]Cl2 . The difference between the previous reported species and the new species is the reaction media: methanol (former) vs. water and NaOH to adjust the pH and deprotonate the thionucleobase.
The new complexes where characterized by a series of analytical and spectroscopic techniques. Attempts to obtain single crystals suitable for x-ray diffraction were not successful. However, mass-spectrometric characterization by electrospray positive ion mode in the presence of formic acid was performed on the new complexes. Since in the [Cp2Mo(L)]Cl complexes the chloride is in the outer sphere coordination (ionic), although it could be involved in hydrogen bonding with the thionucleobase, [Cp2Mo(L)]+ is the principal species in aqueous solution. The calculated molecular ion m/z peaks for [Cp2Mo(6-mercaptopurine)]+, [Cp2Mo(6-mercaptopurine ribose)]+, [Cp2Mo(2-amino-6-mercaptopurine)]+ and [Cp2Mo(2-amino-6-mercaptopurine ribose)]+ are 383, 515, 398 and 530 respectively. First, upon analysis of the experimental mass spectrometric data showed the presence of one thionucleobase or thionulceoside per Cp2Mo2+. Second, the experimental results demonstrated that all the species exhibited parent peaks corresponding to [Cp2Mo(L-H)]+ ions, see Table 1S, Supplementary Material. The theoretical mass-spectrometric isotopic distributions for [Cp2Mo(L) - H]+ ions were calculated and compared with the experimental ones. The experimental values have a pattern very similar to the predicted theoretical values, supporting the proposed formula for these new species. In addition, IR spectra corroborated the presence of functional groups such as N-H, C=N, C=C, C-N and S-C corresponding to the thionucleobase/thionucleoside ligands.
The 1H NMR spectra of [Cp2Mo(L)]Cl complexes are particularly informative regarding their structures and coordination patterns. Spectral features of 6-mercaptopurine and its complex have been used to propose the structure of [Cp2Mo(6-mercaptopurine)]Cl. An identical strategy was pursued for the remaining three complexes. The 1H NMR spectrum in dimethyl sulfoxide-d6 (DMSO-d6) of 6-mercaptopurine shows four signals: one broad singlet of two averaged N-H groups (N(1)-H/N(9)-H) at 13.58 ppm and two singlets for H(2) and H(8) at 8.21 and 8.47 ppm, respectively. Due to the reaction medium (pH = 7.0), 6-mercaptopurine undergoes deprotonation at N(1)-H site. There are several resonant structures possible but the thiolate structure shown below (II) could explain our experimental results.
Upon examination of structure II, two possible coordination isomers can exist: S(6),N(7) (forming a 5-member chelate ring) and S(6),N(1) (forming a 4-member chelate ring) thionucleobase coordination modes, see Figure 1. Of these two possible coordination isomers, we believe, based on thermodynamic considerations, that only the 5-membered chelate, S(6),N(7) thionucleobase coordination (Figure 1A) is observed in the 1H NMR spectrum of [Cp2Mo(6-mercaptopurine)]Cl in DMSO-d6. The predominance of this structure might be explained by the stability of the 5-member chelate ring over the 4-member chelate ring. However, the S(6),N(1) (4-member chelate) thionucleobase coordination (Figure 1B) cannot be completely ruled out.
In the 1H NMR spectrum of [Cp2Mo(6-mercaptopurine)]Cl in DMSO-d6, the 6-mercaptopurine ligand bound to Cp2Mo2+ experiences downfield shifts for N-H proton H(9) (13.90 ppm vs.13.58 ppm for free ligand) and H(8) proton (8.51 ppm vs. 8.47 ppm for free ligand). The Cp protons also experience a downfield shift at 5.70 ppm when compared to the Cp signal for Cp2MoCl2, at 5.53 ppm. The opposite behavior is observed for H(2). H(2) shows an upfield shift for H(2) at 8.01 ppm vs. 8.21 ppm for free ligand. In contrast to DMSO-d6, in D2O the 1H NMR spectrum of [Cp2Mo(6-mercaptopurine)]Cl is different. Two coordination isomers are present in aqueous medium: S(6),N(7) and S(6),N(1) thionucleobase coordination modes in a 7:1 ratio. Three signals (singlets) are expected for each isomer for a total six peaks. The signals at 5.60 (Cp), 7.83 (H(8) and 8.30 H(2) ppm are attributed to the S(6),N(1) (4-member chelate ring) coordination isomer, whereas the singlets at 5.73 (Cp), 7.78 (H(2), and 8.21 (H(8) ppm correspond to the S(6),N(7) (5-member chelate ring) coordination isomer. The attribution of H(2) resonance peak at 8.30 ppm (down shifted) in the 4-member chelate ring is due to the deshielding effect induced by coordination of N(1) to Mo(IV) center. This is compared to the more shielded resonance peak at 7.78 ppm for H(2) of the isomer with the 5-member chelate ring. It should be mentioned that all four complexes exhibited two coordination isomers in aqueous solution but only [Cp2Mo((-)-2-amino-6-mercaptopurine ribose)]Cl showed two coordination isomers in DMSO-d6 solution. In all cases, S(6),N(7) thionucleobase/thionucleoside coordination mode forming a 5-member chelate ring should be the preferred isomer.
The cytotoxic studies of [Cp2Mo(L)]Cl complexes in colon cancer HT-29 and breast cancer MCF-7 cell lines were performed after exposing the cell to the compounds for 72 hours, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell viability assay [17,18]. We have previously reported the cytotoxic activity of Cp2MoCl2 at 72 hours on HT-29 and MCF-7 cell lines . The IC50 value for HT-29 is 2.6(±0.3) mM. In contrast, in MCF-7, Cp2MoCl2 has a proliferative effect instead of antiproliferative. Control experiments were run in 100% growth medium, demonstrating that the medium does not have any cytotoxic effect on these cells.
The objective of this study is to investigate the role of the ancillary ligands on molybdenocene complex and the resulting anticancer properties on colon and breast cancer cell lines. In particular, many thionucleobases have anticancer properties . Thus, it is of fundamental importance to determine what effects have these bases on molybdenocene antiproliferative properties. Table 1 summarizes the results of the in vitro cytotoxicity experiments on HT-29 colon cancer and MCF-7 breast cancer cell lines as determined by MTT assay.
From the analysis of Table 1, it can be observed that all the [Cp2Mo(L)]Cl complexes are more cytotoxic on HT-29 colon cancer cell line than Cp2MoCl2. Therefore thionucleobases and thionucleosides as ancillary ligands apparently enhance the cytotoxic properties on molybdenocene. But, particularly noteworthy is that these thionulceobases and thionucleosides have cytotoxic activities in several cancer cell lines including HT-29 colon cancer cell line in the low micromolar range (IC50 < 100 μM), see Table 1. Therefore, the apparent enhancement in cytotoxic activity of molybdenocenes with thionucleobases and thionucleosides ligands in HT-29 colon cancer cell line proceeds from the thionucleobase/thionucleoside activity in this cell line and not from molybdenocene or any possible synergism between both moieties. In fact, the incorporation of molybdenocene into the thionucleobases/thionucleosides decreases their cytotoxic activities.
The cytotoxic study of [Cp2Mo(L)]Cl complexes on the MCF-7 breast cancer cell line is more pronounced and different from the HT-29 colon cancer cell line. Cp2MoCl2 possesses proliferative activity on MCF-7 as reported by Meléndez and coworkers . In addition, 6-mercaptopurine and 2-amino-6-mercaptopurine have low cytotoxic activities in breast cancer cell line with IC50 100 μM (Table 1). [Cp2Mo(6-mercaptopurine)]Cl has an IC50 of 203 μM on MCF-7 which suggests that the complex cytotoxicity proceeds apparently, mainly from the ancillary ligand, 6-mercaptopurine. However, the role of molybdenocene cannot be overlooked. The substantial improvement in the cytotoxicity of [Cp2Mo(6-mercaptopurine)]Cl could proceed from two sources: the increase in the lipophilic character of the drug by incorporating a Cp2Mo2+ organometallic group or by possible synergism from between molybdenocene and the thionulceobase moieties, as explained below.
Cp2MoCl2 has proliferative activity on hormone dependent MCF-7 breast cancer cells must likely because Mo(IV) could be coordinated by the nearby amino acids in the estrogen receptor ligand binding domain behaving as an agonist . While this idea is speculative, previous molecular modeling studies performed by Jaouen and collaborators with Ti4+ support the idea that the amino acids in helix 12 (histidine 547) and helix 4 (cysteine 381, glutamic acid 380) can coordinate Ti4+, closing the gap between the helices 4 and 12, activating the receptor thus, the metal behaves like an agonist . Likewise Cd2+ coordinates into the ER ligand binding domain to cysteines 381 and 447, glutamic acid 523, histidine 524 and aspartic acid 538 demonstrating estrogenic effects . While we have not performed any molecular modeling studies with molybdenocene, based on the Lewis acid character of Mo(IV) which is softer that Ti4+ and harder than Cd2+, we can envision that Cp2Mo2+ could be coordinated by the amino acids of the ER ligand binding domain, in particular to cysteines, exhibiting estrogenic effects. But, once molybdenocene has 6-mercaptopurine as ligand, there are no vacant coordination sites available on Mo(IV) center thus, the amino acids on the ER ligand binding domain cannot coordinate Mo(IV) and cannot recognize it as an agonist. Then, Cp2Mo2+ organometallic group can express its intrinsic antiproliferative properties and in synergism with 6-mercaptopurine ligand as well as increased lipophilicity leads to highly activity anticancer drug.
Similar to [Cp2Mo(6-mercaptopurine)]Cl, but even more prominent is the cytotoxic activity of [Cp2Mo(2-amino-6-mercaptopurine)]Cl on MCF-7 cell line (IC50 of 16 μM, Table 1). Neither 2-amino-6-mercaptopurine nor Cp2MoCl2 have such antiproliferative activity. It is very likely that this enhancement comes from the incorporation of a lipophilic organometallic group, “Cp2Mo” (Cp2Mo2+) into the 2-amino-6-mercaptopurine, increasing the lipophilicity of the thionucleobase as well as its aqueous solubility and increasing permeability across the lipophilic cell membrane but synergism between the two moieties cannot be ruled out [23-25], analogous to the [Cp2Mo(6-mercaptopurine)]Cl complex.
Finally, 6-thioguanine and 6-mercaptopurine are antimetabolites that inhibit the novo purine synthesis [26,27]. While the IC50 values of [Cp2Mo(2-amino-6-mercaptopurine)]Cl are almost identical, which may suggest that it is only the 2-amino-6-mercaptopurine that is expressing its genotoxic effect, the fact that the free ligand has different IC50 values in HT-29 and MCF-7 cell lines supports the above hypothesis that Cp2Mo2+ is contributing with the resulting cytotoxic activity of the complex.
We have synthesized a series of molybdenocene-thionucleobase and –thionucleoside complexes in aqueous solution at pH 7.0. This simple synthetic route provided highly soluble and stable complexes in water. Two coordination isomers (S(6), N(7) and S(6), N(1)) were observed by NMR spectroscospy, forming 5-member and 4-member chelate rings. We hypothesize that S(6), N(7) coordination is the major isomer in aqueous solution.
The cytotoxic properties of these new species were investigated on HT-29 colon cancer and MCF-7 breast cancer cell lines. The replacement of chlorides for thionucleobases and thionucleosides as ancillary ligands enhances the molybdenocene solubility in aqueous environment as well as cytotoxic activities on HT-29 colon cancer cells when compared to Cp2MoCl2. But if we consider that the thionulceobases/thionucleosides ligands have IC50 values in the range of 7 to 32 μM, then the incorporation (by coordination) of the organometallic group, Cp2Mo2+, renders detrimental to the cytotoxic activity of the ligands.
The cytotoxic studies on MCF-7 breast cancer are more remarkable. Cp2MoCl2 has proliferative effect on MCF-7 whereas 6-mercaptopurine and 2-amino-6-mercaptopurine ligands have low cytotoxic activities on MCF-7 (IC50 > 100 μM) [19,20]. Surprisingly, [Cp2Mo(2-amino-6-mercaptopurine)]Cl showed to be a highly active species on MCF-7 cell line with an IC50 of 16 μM. In general, the increase in the 6-mercaptopurine and 2-amino-6-mercaptopurine biological activities could be the result of an increase in aqueous solubility and lipophilicity induced by Cp2Mo2+ allowing the thionulceobases to cross the cell membrane and reach the target place inside the cell but potential synergism between the metallic species and the ligand cannot be ruled out [21-23]. Finally, we have synthesized a metallic species, [Cp2Mo(2-amino-6-mercaptopurine)]Cl, which is highly active in two cancer cell lines, HT-29 colon and MCF-7 breast cancers.
All starting materials were obtained from Aldrich and used without further purification. The purity of molybdenocene dichloride and ligands were checked by IR and/or by 1H NMR spectroscopy to determine possible decomposition. Water was doubly distilled, deionized and thoroughly saturated with dried nitrogen. All solvents for NMR measurements were 99.9% D purity grade. The 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (yellow tetrazolium MTT) was obtained from Sigma and used as purchased.
The complexes were dissolved in a mixture of water/methanol (1:1) containing 0.2% of formic acid prior to mass spectral (MS) analysis. Solutions of 1ppm, for both complexes were used. A Bruker Daltonics Esquire 6000 instrument was used to record the mass spectral data. The electrospray positive ion was used as ionization mode during the MS experiment. The experimental intensities were reported relative to the parent peak on each mass spectrum. In order to determine the theoretical isotopic distribution patter of the [M]+ m/z peaks, the Molecular Weight Calculator Software available on http://jjorg.chem.unc.edu/personal/monroe was used. Since the infrared characterizations were performed using a reflectance IR, pure complexes do not require any sample preparation before the analysis. The IR spectra were obtained using a reflectance IR-FT spectrophotometer Scimitar Series Digilab FIS 1000 instrument, equipped with a Digilab software resolution 4. The 1H spectra were recorded on a 500 MHz Avance Bruker spectrometers under controlled temperature. 3-(trimethylsilyl) propanesulfonic acid (DSS) or solvent peak were used as internal reference. Elemental analysis was performed by Atlantic Microlab.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays  were performed into two different cell lines, HT-29 and MCF-7, both obtained from American Type Culture Collection (ATCC HTB-38 and ATCC HTB-22). The colon cancer cell line, HT-29, was grown under 95% Air / 5% CO2 (USP grade) atmosphere at 37 °C. The growth medium used was McCoy's 5A (ATCC) complete medium adjusted by supplier to contain 1.5 mM L-glutamine and 2.2g/L sodium bicarbonate. In addition, this medium was supplemented with 10% (v/v) fetal bovine serum (ATCC) and with 1% (v/v) antibiotic-antimycotic (Sigma).
The breast cancer cell line, MCF-7, was grown and maintained, as well as HT-29, at 37°C and 95% Air/5% CO2 (USP grade). This cell line was grown in Dubelcco's Modification of Eagle's Media (DMEM) from Cellgro, which is supplemented by supplier with L-glutamine, 4.5g/L glucose and sodium pyruvate. This complete media was supplemented with 10% (v/v) fetal bovine serum (ATCC), and with 1% (v/v) antibiotic-antimycotic (Sigma).
A 100 μL suspension with an initial population of 10,000 to 15,000 (for HT-29 cell line) cells per well were seeded in a 96 well plates (VWR) and after 24 hrs of incubation, a dose of the metal complex was added. Complexes' concentrations were from 0.01 to 0.000001 M (ten data points evenly distributed, one concentration per column of eight wells) dissolved in 100% medium. Experiments were performed in quadruplicate plates. The plates were leave at 37°C and 95% air/5% CO2 for 72 hours. Two to four hours before the completion of the 72 hours of incubation, a solution of MTT (1.0 mg/mL) was added and incubated. When time was completed and the purple formazan insoluble product was observed, the cell media was removed and plates were washed with cold Phosphate Buffer Solution (PBS). The PBS was prepared with sodium chloride, potassium chloride, sodium phosphate and potassium phosphate (all from Sigma-Aldrich) dissolved in double distilled, deionized and autoclaved water. The PBS solution was autoclaved and filtered through cellulose-acetate 0.2 μm filters. At this point 200 μL per well of a detergent solution, 10% (v/v) Triton X-100 (Sigma) in 2-propanol (Fisher), was added and left at 37 °C in order to dissolve the formazan product.
The absorbances of the resulted colored solutions were measured at 570nm in a Micro Plate Reader with background subtraction at 630 nm. The instrument used was the 340 ATTC Microplate Reader from SLT Lab Instruments equipped with a temperature control unit and interfaced with a computer with WinSeLecT software. For the MCF-7 cell line, an initial population of cells per well greater than that for the HT-29 was required because MCF-7 cells have a doubling time (ATCC) greater than HT-29. The IC50, a metal complex concentration necessary to inhibit cell proliferation by 50%, was calculated by fitting the data to a four-parameter logistic plot using the SigmaPlot software from SPSS Company.
All MTT protocol was performed in a dark room. Experiments were designed in order to contain blanks well (controls), which contained only cell with the medium and test wells, which were cells treated with the metal compound at different concentrations.
In a three neck round bottom flask of 50 mL, 0.050 g (0.17 mmol) of Cp2MoCl2 and one equivalent (0.17 mmol) of the ligand were loaded and dissolved with 20mL of deionized and degassed water. The pH was adjusted to 7.0 with 1.0 M NaOH and the reaction was carried out at room temperature under an atmosphere of nitrogen. The reaction times for [Cp2Mo(6-mercaptopurine)]Cl and [Cp2Mo(6-mercaptopurine ribose)]Cl were 18, 24 hours, respectively, and 48 hours for [Cp2Mo(2-amino-6-mercaptopurine)]Cl and [Cp2Mo(2-amino-6-mercaptopurine ribose)]Cl. The resulting orange solution was filtered through celite and the solvent removed under vacuum. The solid was dissolved in methanol and filtered by gravity. The filtrate was purified by column chromatography using lipophilic Sephadex(20-100 μm, from Aldrich) as stationary phase and methanol as mobile phase. The complexes were obtained in 60-70% yield. The complexes are hygroscopic. The proton assigment is based on the following figure:
[Cp2Mo(6-mercaptopurine)]Cl: 1H NMR (dmso-d6) δ ppm: 13.90 (bs, 1H, N(9)-H), 8.51 (s, 1H, H(8)), 8.01 (s, 1H, H(2), 5.70 (s, 10H, Cp). Major isomer, 5-member chelate, 1H NMR (D2O) δ ppm: 8.21 (s, 1H, H(8)), 7.78 (s, 1H, H(2)), 5.73 (s, 10H, Cp). Minor isomer, 4-member chelate, 1H NMR (D2O) δ (ppm): 8.30 (s, 1H, H(2)), 7.83(s, 1H, H(8)), 5.60 (s, 10h, Cp). IR (cm-1): 3315(υN-H), 3083(υC-H), 3075(υCp), 1614(υC=N), 1570(υC=C), 1421(υ(C=C, Cp), 1330(υC-N), 1224 (υS=C) 884, 839 (δC-H, Cp), 522 (υMo-N). ESI-MS (positive mode), m/z (relative intensity): [Cp2Mo(6-mercaptopurine) - H]+ 382(7), 381(42), 380(18), 379(100), 378(52), 377(71), 376(59), 375(34), 374(9), 373(49). Anal. calcd. for C15H13N4SClMo·H2O, [Cp2Mo(6-mercaptopurine)]Cl·H2O: C, 41.82; H, 3.51. Found: C, 41.82; H, 3.62.
[Cp2Mo(6-mercaptopurine ribose)]Cl: 1H NMR (dmso-d6) δ ppm: 8.75(s, 1H, H(8)), 8.10(s, 1H, H(2)), 5.95(s, 10H, Cp) 5.92(d, 1H, H(C′1), 5.55(s, 1H, OH(C′2)), 5.33(s, 1H, OH(C′3)), 5.10(s, 1H, OH(C′(5)), 4.45(t, 1H, H(C′2)), 4.15(t, 1H, H(C′3)), 3.96(m, 1H, H(C′4)), 3.70-3.45(m, 2H, H(C′5)). Major isomer, 5-member chelate, 1H NMR (D2O) δ ppm: 8.41(s, 1H, H(8)), 7.90(s, 1H, H(2)), 5.99 (d, 1H, H(C′1)), 5.75(s, 10H, Cp). Minor isomer, 4-member chelate, 1H NMR (D2O) δ (ppm): 8.57(s, 1H, H(2)), 8.43(s, 1H, H(8)), 5.97(d, 1H, H(C′1)), 5.63(s, 10H, Cp). IR (cm-1): 3386(υN-H), 3075(υCp), 2933(υC-H), 1623(υC=N), 1582(υC=C), 1418(υ(C=C, Cp), 1340(υC-N), 1203 (υS=C) 883, 840 (δC-H, Cp), 490 (υMo-N). ESI-MS (positive mode), m/z (relative intensity): [Cp2Mo(6-mercaptopurine ribose) - H]+ 514(11), 513(40), 512(24), 511(100), 510(55), 509(78), 508(68), 507(39), 506(12), 505(50). Anal. calcd. for C20H21O4N4SClMo·3H2O, [Cp2Mo(6-mercaptopurine ribose)]Cl·3H2O: C, 40.11; H, 4,54. Found: C, 40.43; H, 4.18.
[Cp2Mo(2-amino-6-mercaptopurine)]Cl: 1H NMR (dmso-d6) δ ppm: 12.70(s, 1H, N(9)-H), 7.97(s, 1H, H(8)), 6.15(s, 2H, NH2), 5.80(s, 10H, Cp). Major isomer, 5-member chelate, 1H NMR (D2O) δ ppm: 7.84(s, 1H, H(8), 5.75(s, 10H, Cp). Minor isomer, 4-member chelate, 1H NMR (D2O) δ (ppm): 7.65(s, 1H, H(8)), 5.55(s, 10H, Cp). IR (cm-1): 3289(υN-H), 3081(υCp), 2912(υC-H), 1642(υC=N), 1599(υC=C), 1414(υ(C=C, Cp), 1385(υC-N), 1257 (υS=C) 883, 808 (δC-H, Cp), 457 (υMo-N)). ESI-MS (positive mode), m/z (relative intensity): [Cp2Mo(2-amino-6-MP) - H]+ 397(7), 396(40), 395(19), 394(100), 393(54), 392(81), 391(68), 390(37), 389(11), 388(56). Anal. calcd. for C15H14N5SClMo·2H2O, [Cp2Mo((-)-2-amino-6-mercaptopurine)]Cl·2H2O: C, 38.83; H, 3.91. Found: C, 38.89; H, 3.67.
[Cp2Mo(2-amino-6mercaptopurine ribose)]Cl: Major isomer, 1H NMR (dmso-d6) δ ppm: 8.25(s, 1H, H(8)), 6,64(s, 2H, NH2), 5.89(s, 10H, Cp), 5.79(d, 1H, H(C′1), 5.71(d, 1H, OH(C′2)), )), 5.57(s, 1H, OH(C′3)), 5.47(s, 1H, OH(C′(5)), 5.07(t, 1H, H(C′2)), 4.41(t, 1H, H(C′3)), 3.95(m, 1H, H(C′4)), 3.51(m, 2H, H(C′5)). Minor isomer, 1H NMR (dmso-d6) δ ppm: 8.44(s, 1H, H(8)), 6.57(s, 2H, NH2), 5.78(s, 10H, Cp), 5.77(d, 1H, H(C′1)), 5.67(d, 1H, OH(C′2)), 5.55(s, 1H, OH(C′3)), 5.52(s, 1H, OH(C′(5)), 4.81(t, 1H, H(C′2)), 4.51(t, 1H, H(C′3), 3.90(m, 1H, H(C′4)), 3.62(m, 2H, H(C′5)). Major isomer, 5-member chelate, 1H NMR (D2O) δ ppm: 8.02(S, 1H, H(8)), 5.77(s, 10H, Cp). Minor isomer, 4-member chelate, 1H NMR (D2O) δ (ppm): 8.14(s, 1H, H(8)) 5.63(s, 10H, Cp). IR (cm-1): 3339(υN-H), 3090(υCp), 2924(υC-H), 1619(υC=N), 1580(υC=C), 1418(υ(C=C,Cp), 1340(υC-N), 1205 (υS=C) 893, 840 (δC-H, Cp), 530(υMo-N). ESI-MS (positive mode), m/z (relative intensity): [Cp2Mo(2-amino-6-MPR) - H]+ 529(10), 528(40), 527(23), 526(100), 525(55), 524(79), 523(69), 522(41), 521(12). Anal. calcd. for C20H22N5O4SClMo·2H2O, [Cp2Mo((-)-2-amino-6-mercaptopurine ribose)]Cl·2H2O: C, 40.3; H, 4.3. Found: C, 40.2; H, 4.1.
E.M. acknowledges the NIH-MBRS SCORE Programs at both the University of Puerto Rico Mayagüez and the Ponce School of Medicine (PSM)for financial support via NIH-MBRS-SCORE Program grant #S06 GM008103-37 and #S06 GM008239-23 and the PSM-Moffitt Cancer Center Partnership 1U56CA126379-01 grant. In addition, EM thanks NSF-MRI Program for providing funds for the purchase of the 500 MHz NMR instrument.
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