Ricardo G.
Teixeira
a,
Dimas C.
Belisario
b,
Xavier
Fontrodona
c,
Isabel
Romero
c,
Ana Isabel
Tomaz
a,
M. Helena
Garcia
a,
Chiara
Riganti
b and
Andreia
Valente
*a
aCentro de Química Estrutural and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal. E-mail: amvalente@ fc.ul.pt
bDepartment of Oncology, University of Torino, Torino, Italy
cDepartament de Química and Serveis Tècnics de Recerca, Universitat de Girona, C/M. Aurèlia Campmany, 69, E-17003 Girona, Spain
First published on 12th February 2021
Platinum-based therapies continue to be the main regimen used to treat non-small cell lung cancers (NSCLC), where multidrug resistance plays a key role in treatment failure and strategies to overcome this limitation are urgently sought for. In view to contribute to the development of this field, two sets of new organometallic Ru(II) compounds with general formula [Ru(η5-C5H4R′)(bipyR)(PPh3)][CF3SO3], where R′ = CHO or CH2OH and bipyR = 2,2′-bipyridine (1, 5), 4,4′-dimethyl-2,2′-bipyridine (2, 6), 4,4′-di(hydroxymethyl)-2,2′-bipyridine (3, 7) and 4,4′-dibiotin ester-2,2′-bipyridine (4), were synthesized and fully characterized. All compounds were tested against four types of NSCLC cell lines (A549, NCI-H228, Calu-3 and NCI-H1975), and four of them (1, 2, 4 and 6) presented a strong activity against cisplatin-resistant NSCLC cells. They were also able to increase cisplatin cytotoxicity up to 1390-fold (when administrated at nontoxic doses) by inhibiting MRP1 and P-gp transporters. Given the role of MRP1 in cisplatin resistance, in particular in lung cancer where cisplatin is the first-line treatment, the finding that these compounds are inducers of collateral sensitivity is of particular relevance. As far as we are aware, these are the first ruthenium-based compounds with such a mechanism of action, taking advantage of an “Achilles’ heel” and acting as MDR-selective compounds.
During the last years several research groups have developed new metallodrugs/metallodrugs formulations aiming at overcoming several forms of multidrug resistance.3–12 However, in what concerns lung cancer, promising examples are still scarce. One of the strategies used has been the co-delivery of valproic acid (VPA), a histone deacetylase inhibitor, with a metallodrug.13,14 For example, Mao and co-workers have developed VPA-functionalized cyclometalated iridium(III) complexes through a hydrolysable ester bond.13 The results have shown a significant increased activity for the new conjugates (vs. VPA alone or a mixture of complexes + VPA) validating the strategy adopted. Also, the conjugates were able to overcome cisplatin resistance in human lung carcinoma cells (A549R). In another work, Du, Meng and co-workers, used L-cysteine decorated Zr-based metal–organic frameworks (MOFs) to co-deliver cisplatin and VPA.14 Immunoblot and immunofluorescence analyses showed that the new MOFs were able to downregulate the expression of vascular endothelial growth factor (VEGF) and improved the sensitivity to cisplatin of resistant A549/CDDP. The in vivo experiments confirmed that chemotherapy using these MOFs combined with microwave thermal therapy significantly improved the therapeutic effect of cisplatin resistant lung cancer. In a different approach, a family of CoII and CoIII tris(bipyridine) compounds has shown an important cytotoxicity against a panel of cancer cell lines that included taxol-resistant, cisplatin-resistant and p53-deficient cancer cells.15 Overall, there was no direct correlation between the oxidation state of the complexes and their cytotoxicity, yet the methyl substituent at the bipyridine (vs. non-substituted or methoxy) seemed to impart a favorable response in cisplatin-resistant (A2780, SGC-7901, OV2008 and C13 vs. resistant phenotype), in taxol-resistant (MCF-7, HCT-8, and A549 vs. resistant phenotype) and in p53-deficient apoptosis-resistant (HCT116 p53+/+vs. HCT116 p53−/−) cancer cells. It was further elucidated that compound [Co(4,4′-dimethyl-2,2′-bipyridine)3]3+ was able to inhibit P-gp, which is associated to taxol-resistance, and suppressed ∼50% of tumor growth in a lung cancer xenograft model. Indeed, one of the most important mechanisms of cell resistance is the overexpression of drug transporters, such as P-gp.2 In this frame, there are a few reports on metal complexes that are able to inhibit ABC pumps. For example, Choudhuri and coworkers developed a series of [M(N-(2-hydroxy acetophenone)glycinate)(H2O)n] complexes, where the M is FeIII (n = 3),16 NiII (n = 1),17 CuII (n = 3)18 or ZnII (n = 1).19 In particular, the CuII complex was shown to directly interact with P-gp showing potential to reverse P-gp mediated drug resistance.18 Interestingly, it did not compete for the substrate binding or to verapamil-, vinblastine- and progesterone-binding sites.
Ruthenium complexes are nowadays considered promising alternatives to the metal-based drugs in clinical use with some compounds still in clinical trials, namely KP1019 sodium salt NKP1339/IT-139 (sodium trans-[tetrachlorobis(indazole)ruthenate(III)])20,21 and TLD1433 (Ru(II) polypyridyl complex; ClinicalTrials.gov Identifier: NCT03053635).22 In the scope of ruthenium MDR modulators, i.e., compounds able to block the drug efflux, only a few examples are known. Among them, the compound [Ru(η6-p-cymene)Cl2(N-(Anthracen-9-yl)-imidazole)] (Fig. 1, left), developed by Juillerat-Jeanneret, Dyson and coworkers, showed good cytotoxicity against A549 lung, HT29 colon, and T47D breast carcinoma and inhibited P-gp close to the levels of the reference inhibitor verapamil.23 In this regard, we have been also engaged in the last years in the development of ruthenium(II) compounds with ATP Binding Cassette (ABC) efflux pumps inhibitory properties. Notably, during structure–activity studies on compounds from the [RuII(η5-C5H4R)(2,2′-bipyridine-4,4′-R′)(PPh3)]+ family, one compound (LCR134 for R = H, R′ = biotin ester, Fig. 1, right) has shown remarkable ability to inhibit P-gp (even better than the reference inhibitor verapamil)24 and another stood out as a possible MRP1 inhibitor (RT11 for R = CH3 and R′ = CH3) (Fig. 1, bottom).25 In addition, both compounds (LCR134 and RT11) were also cytotoxic for breast (MCF-7 and MDA-MB-231) and ovarian (A2780/A2780cisR) cancer cells, respectively, exhibiting a rare dual behavior as both cytotoxic agents and ABC pump inhibitors. It was further observed that the substituent at the bipyridine is key to provide inhibitory properties to this family of compounds. Thus, in the continuation of these studies other substituents were added at the cyclopentadienyl ring (–CHO or –CH2OH) allowing the synthesis of a new family of compounds. The results disclosed in this work will highlight the importance that specific chemical groups have on the inhibitory properties of ABC pumps and how this might contribute to improve the activity of the compounds and to sensitize NSCLC cells to cisplatin.
1H, 13C{1H} and 31P{1H} NMR characterization of the new ruthenium-based compounds fully supported the proposed formulations (see ESI†). 2D NMR techniques (COSY, HMQC and HMBC) were used to assist on the assignment of all signals. Overall, analysis of the 1H NMR spectra shows that Sigma coordination of the bipyridyl derivatives to the ruthenium-cyclopentadienyl scaffold induces a common deshielding effect of the monosubstituted η5-cyclopentadienyl protons along with a deshielding of H6 (ΔH6 up to 0.82 ppm) and a shielding of H3 (ΔH3 up to −0.41 ppm) bipyridyl protons, as it was reported for related ruthenium cationic compounds.29 Additionally, other resonances in the aromatic region (6.95 ppm < δ < 7.42 ppm) were ascribed to the aromatic ortho, meta and para protons of the triphenylphosphane co-ligand. As expected, compounds 5, 6 and 7 exhibit shielded resonances for the hydroxymethylcyclopentadienyl protons when compared to their formylcyclopentadienyl analogues, which is in good agreement with the electron donating character of the newly introduced pendant group. Detailed spectroscopic data concerning APT-13C{1H} and 31P{1H}-NMR experiments are included in the Experimental section and are in accordance with the effects discussed in this 1H NMR analysis.
The solid state FTIR of the complexes (KBr pellets) of the novel organometallic ruthenium-cyclopentadienyl compounds 1–7 shows the bands for the νC–H stretching vibration of the phosphane, cyclopentadienyl and bipyridyl ligands in the range 3100–3050 cm−1 and bands for νCC ranging from 1489 to 1390 cm−1. In addition, the presence of the triflate counter-ion was observed at 1250–1260 cm−1. Characteristic stretching vibrations of the formyl and hydroxymethyl appended groups were also found at the expected region at ∼1670 cm−1 and ∼3420 cm−1, respectively.
Optical absorption spectra of complexes 1–7 were recorded at room temperature using ∼10−4 to ∼10−6 M solutions in dichloromethane and dimethylsulfoxide. Table S1† presents molar absorptivity coefficient (ε) values and the correspondent wavelength (λmax) for the bands observed. Fig. 3A shows the spectra of compounds 1 and 5 which are representative of the behavior of formyl- or hydroxymethylcyclopentadienyl series of compounds (which exhibit very similar spectral features), whereas Fig. 3B illustrates the solvatochromic phenomenon observed for compound 3. The same trend is observed in the electronic spectra of all complexes with two very intense absorption bands at high energy values (λ = 240–330 nm) that are ascribed to electronic transitions occurring in the organometallic fragment ({[Ru(η5-C5H4R)((PPh3)]+} R = CHO or CH2OH) and the coordinated ligands. In the visible range, one or two medium-strength absorption bands assigned to metal-to-ligand charge transfer transitions (MLCT) from Ru 4d orbitals to the π* orbitals of the phosphane and bipyridyl ligands (λ = 330–550 nm), as previously reported for related compounds.25,30 The charge transfer character of these bands is corroborated by solvatochromic studies in DMSO where a clear blue-shift is observed with the increase of polarity of the solvent for compound 3 (379 nm in CH2Cl2vs. 366 nm in DMSO).
The crystal structures of complexes 1–3 and 5–7 have been solved by X-ray diffraction analysis. Fig. 4 displays the ORTEP diagrams of their molecular structures whereas the main crystallographic data and selected bond distances and angles can be found in the ESI† section (Tables S2–S3†). All compounds crystallize in the monoclinic system, space group C12/c1 (1), P121/c1 (3, 7) or P121/n (5, 6) with exception of compound 2 which crystallizes in the triclinic system, space group P. In all cases the corresponding unit cells of the complexes display two enantiomers in the racemic crystal.
All complexes adopt the classical pseudo-octahedral three-legged piano stool geometry and, hence, the cyclopentadienyl arene rings exhibit the usual π-bonded η5 coordination mode, whereas the bipyridyl ligands are coordinated in a bidentate fashion. The sixth coordination site is occupied by the triphenylphosphane ligands. The distances between Ru and the centroids of the cyclopentadienyl moiety in all the complexes, are within a narrow interval (1.828–1.841 Å), similar to other cyclopentadienyl ruthenium complexes described in the literature.29
The Ru–P bond distances in the complexes are all in the same range (2.3086(11)–2.3351(3) Å), similar to the Ru–Nbipy bond distances, which are in the same order for all the compounds (2.0685(11)–2.1112(3) Å). The Ru–C distances are in general higher than Ru–Nbipy, probably due to the σ-donor character of the cyclopentadienyl rings. Moreover, the distances between Ru and the monosubstituted carbon on the η5-cyclopentadienyl ring in complexes 1–3 (Ru–C21 = 2.1901(13) 1; Ru–C2 = 2.212(3) 2; Ru–C21 = 2.1820(17) 3 Å) are in general slightly shorter than in complexes 5–7 (Ru–C21 = 2.230(2) 5; Ru–C2 = 2.213(3) 6; Ru–C21 = 2.235(3) 7 Å). This behavior could be due to the presence of the –CH2OH substituent onto the arene ring in the latter, which presents an electron-withdrawing character a lower than that of the –CHO substituent. In the case of complex 1 (see Fig. S29†) an intramolecular hydrogen bond is formed between one pyridyl H atom and the O atom from the substituent –CHO (H-bond distances are H29–O27 = 2.580 Å), since the O atom of –CHO is orientated toward the bipyridine ligand.
The angles N–Ru–N and C–Ru–C in the complexes, show the geometrical restrictions imposed by the bipyridyl and cyclopentadienyl ligands, and the values found are similar to other compounds described in the literature.25,29
It is worth mentioning the packing structure of the compounds, where additional intermolecular hydrogen bonds can be observed between oxygen atoms of the triflate anions and the hydrogen atoms of the triphenylphosphane (1) or the bipyridine ligands (2, 3); in addition, intermolecular hydrogen bonds are displayed between the oxygen atom of the –CH2OH substituent and the hydrogen atoms of the bipyridine ligand of a neighboring molecule (5, 6, 7). In the case of 7 additional intermolecular hydrogen bonds are observed between O atom of the triflate anions and H atoms of the bipyridine ligand (Fig. S30†).
Analyzing the different IC50 obtained for cisplatin (Table 1), we observed that the higher the level of MRP1 in the cells analyzed (A549 and NCI-H228), the greater their resistance to cisplatin (i.e., a higher IC50 value is obtained), suggesting that MRP1 plays a key role in the efflux of cisplatin in our cellular models.
1 | 2 | 3 | 4 | 5 | 6 | 7 | Cisplatin | |
---|---|---|---|---|---|---|---|---|
Viability of cells measured after 72 h incubation with increasing concentrations (0–100 μM) of each compound, measured with a spectrophotometric assay. Data are means ± SD (n = 4). | ||||||||
A549 | 10.8 ± 1.3 | 12.4 ± 3.6 | >100 | 15.4 ± 2.6 | >100 | 12.5 ± 2.1 | >100 | >100 |
NCI-H228 | 4.3 ± 0.7 | 3.8 ± 1.4 | >100 | 16.5 ± 1.3 | >100 | 7.8 ± 1.2 | >100 | >100 |
Calu-3 | 24.7 ± 4.1 | 4.9 ± 1.6 | >100 | 28.9 ± 0.8 | >100 | 5.9 ± 1.2 | >100 | 63.4 ± 8.7 |
NCI-H1975 | 91.8 ± 10.4 | >100 | >100 | >100 | >100 | >100 | >100 | 3.8 ± 1.1 |
Based on the IC50 values obtained, the resistance to cisplatin varies following the rank order: A549 > NCI-H228 > Calu-3 > NCI-H1975 cells.
The cytotoxicity of cisplatin, commonly evaluated with the MTT assay, often results in lower IC50 values in NSCLC cells32 than those measured in the present work with the WST-1 assay. The different methods as well as the incubation time of the dye in the MTT assay (ranging from 1 to 4 h in different experimental works) can explain such discrepancies.
In a first experimental set, we evaluated the cell viability of the cell lines in the presence of increasing concentrations of the new ruthenium compounds (Table 1).
According to the IC50 values obtained for the Ru compounds it can be noticed that four compounds – 1, 2, 4 and 6 – were more active. For the panel of compounds tested herein, the presence of the substituent –CH2OH seems to be detrimental for the activity of the overall compound, whether placed in the cyclopentadienyl ring (in 5 and 7) or in the bipyridine ligand (in 3). The formyl substituent in the cyclopentadienyl ring seems advantageous, with compounds 1, 2 and 4 being active. The methyl substituent in the bipyridine co-ligand (complexes 2 and 6) is, in this panel of complexes, highly beneficial for the activity, rendering active complexes. Indeed, compounds 1, 2, 4 and 6, were stronger killers of A549 and NCI-H228 cells, i.e. the wo cell lines showing the highest resistance to cisplatin among those analyzed, suggesting that they could be inducers of collateral sensitivity of drug resistant cells.
This is not the first time that metal-based compounds are reported to exert a selective cytotoxicity against drug resistant cells: for instance, cobalt-based complexes15 and ferrocene-based compounds33 overcome resistance to taxol-resistant tumors. Ruthenium-based compounds have been described as potent anti-cancer drugs,34–37 but their potential as collateral sensitivity inducers and/or MDR-reversing agents remains poorly known.
To verify if the compounds can improve the sensitivity to cisplatin, in particular in the cell lines with the highest degree of resistance to this drug, we incubated cells with increasing concentrations of cisplatin together with 1, 2, 4 and 6 at 1 μM, a concentration equivalent to IC25 of the Ru compounds for all cell lines. Working at these poorly toxic concentrations, it is indeed easier to evaluate the effect of the compounds as enhancers of cisplatin toxicity, not as cytotoxic drugs. The ratio IC50(cisplatin)/IC50(cisplatin + Ru compound), i.e. the Resistance factor (Rf) reported in Table 2, was considered an index of sensitization toward cisplatin38 and indicates how many folds the compound enhances the efficacy of cisplatin.
1 | 2 | 4 | 6 | |
---|---|---|---|---|
Viability of cells measured after 72 h incubation with increasing concentrations (0–100 μM) of cisplatin, co-incubated with 1 μM of each compound, measured with a spectrophotometric assay. Values are data from Rf = IC50(cisplatin)/IC50(cisplatin + Ru compound) (n = 4). | ||||
A549 | 71.4 | 555.6 | 1250 | 243.9 |
NCI-H228 | 333.3 | 1389.9 | 588.2 | 344.8 |
Calu-3 | 26.3 | 126 | 33.2 | 78.7 |
NCI-H1975 | 2.9 | 0.6 | 1.6 | 0.7 |
All the compounds strongly sensitized A549 and NCI-H228 cell lines, showing a moderate sensitization effect in the mild cisplatin-resistant Calu-3 cells, and losing their effect on the cisplatin-sensitive NCI-H1975 cells.
Of note, A549 and NCI-H228 cells were the cell lines with the highest expression of MRP1, whereas in Calu-3 the most abundant transporter was P-gp. Thus, we decided to investigate if the Ru compounds can directly modulate the activity of these transporters by measuring the ATPase activity of cell extracts after incubation with the compounds (Fig. 5). This assay indicates if the compounds, once entered within the cells, bind the ABC transporters and interfere with their catalytic activity that is based on the hydrolysis of two ATPs associated to the efflux of the drug. Therein, a reduction in the ATPase activity of MRP1 or P-gp may suggest that the compounds inhibit the efflux activity of these transporters, e.g. by acting as inhibitors, substrates or negative allosteric modulators, as previously reported.39
MRP1 and P-gp activities differed between the cell lines, in agreement with the expression levels of the protein, as observed for the controls. Interestingly, all Ru compounds inhibited MRP1 ATPase activity (Fig. 5A). They also inhibited P-gp activity in Calu-3 cells, that expresses P-gp to a greater extent (Fig. 5B). We could not detect any significant decrease of P-gp ATPase activity in A549 and NCI-H228 cells, most likely as a consequence of the lower amount of P-gp expressed. From the four complexes tested, compounds 4 and 6 seem to be the most efficient in inhibiting P-gp activity in Calu-3 cell extracts.
These results provided evidence that the selected ruthenium compounds increased the sensitivity to cisplatin in the resistant cell lines by directly impairing the catalytic activity of the main drug efflux transporters expressed the panel of lung cancer cell lines chosen. We are now investigating if this inhibition is exerted on other ABC transporters and by which molecular mechanisms. Considering our previous results on anti-cancer ruthenium cyclopentadienyl compounds, it seems that a trend is gaining some consistency: concerning their cytotoxicity, hydroxylated [Ru(η5-C5H4R)(PPh3)(bipy)]+ (R = H, CH3, CHO or CH2OH) based compounds are less active or inactive in any of the cell lines tested (lung, breast or ovarian). This could possibly be related to their behavior as substrates of ABC pumps as we have previously observed for [Ru(η5-C5H5)(4,4′-di(hydroxymethyl)-2,2′-bipyridine)(PPh3)]+ (pmc79) and [Ru(η5-C5H4CH3)(4,4′-di(hydroxymethyl)-2,2′-bipyridine)(PPh3)]+ (RT12) compounds that were possibly P-gp substrates.25,28RT12 was also a poor substrate for MRP1, MRP2 and BCRP. On the contrary, [Ru(η5-C5H5)(4,4′-dibiotin ester-2,2′-bipyridine)(PPh3)]+ (LCR134) and [Ru(η5-C5H4CH3)(4,4′-dimethyl-2,2′-bipyridine)(PPh3)]+ (RT11) were found to be P-gp and MRP1/MRP2 inhibitors, respectively.25,28 We will now focus on understanding how these structural changes influence the ability of the compounds to act as inhibitors at the molecular level.
These four complexes were further tested for their ability to sensitize cisplatin-resistant cells. Complexes 1, 2, 4 and 6 increased cisplatin cytotoxicity up to 1390-fold by inhibiting MRP1 and P-gp transporters. This is of particular interest since, besides exerting an important role in cisplatin resistance,40 MRP1 has been proposed as a potential mediator of collateral sensitivity.41–43 Few compounds targeting MRP1 are inducers of collateral sensitivity44 but the research in this field is in great expansion. In lung cancer, where cisplatin is the first-line treatment and MRP1 is one of the main responsible for resistance to this drug,45 finding MRP1-targeting agents, inducing collateral sensitivity is absolutely preeminent. In this context, complexes 1, 2, 4 and 6 emerge as valuable prospective agents for lung cancer chemotherapy, specially compounds 4 and 6, both quite active as cytotoxic agents and able to inhibit both MRP1 and P-gp, suggesting an important ability to overcome drug resistance mechanisms.
1H NMR [(CD3)2CO, Me4Si, δ/ppm]: 9.35 (broad, 2H, H6), 9.24 (s, 1H, (η5-C5H4CO), 8.20 (d, 2H, 3JHH = 8.0,
3), 7.97 (t, 2H, 3JHH = 7.6,
4), 7.46 (m, 5H,
pPPh3 +
5), 7.36 (m, 6H,
mPPh3), 7.12 (t, 6H, 3JHH = 8.4,
oPPh3), 5.80 (broad, 2H,
β-η5-C5H4CHO), 4.93 (broad, 2H,
γ-η5-C5H4CHO).
APT-13C{1H} NMR [(CD3)2CO, δ/ppm]: 189.1 (η5-C5H4HO), 156.8 (
6), 138.1 (
4), 133.9 (d, 2JCP = 11,
HoPPh3), 131.8 (d, 1JCP = 43.8,
qPPh3), 131.4 (d, 4JCP = 1.9,
HpPPh3), 129.5 (d, 3JCP = 9.8,
HmPPh3), 126.7 (
5), 124.4 (
3), 100.8 (
α-η5-C5H4CHO), 85.2 (
β-η5-C5H4CHO), 79.1 (
γ-η5-C5H4CHO).
31P{1H} NMR [(CD3)2CO, δ/ppm]: 48.81 (s, PPh3).
FTIR [KBr, cm−1]: 3105–3055 (νC–H aromatic rings), 1667 (νCO, η5-C5H4CHO), 1433 (νC
C aromatic rings), 1262 (νCF3SO3 counter ion).
UV-vis [DMSO, λmax/nm (ε × 103/M−1 cm−1)]: 289 (24.49), 362 (6.46), 415 (Sh).
UV-vis [CH2Cl2, λmax/nm (ε × 103/M−1 cm−1)]: 289 (21.71), 357 (Sh), 409 (Sh), 457 (Sh).
Elemental analysis calc. for C35H28F3N2O4PRuS (761.71): C, 55.2, H, 3.7; N, 3.7; S, 4.2. Found: C, 54.9; H, 3.5; N, 3.6; S, 4.3.
ESI-MS: [1-CF3SO3]+ calc. for [C34H28N2OPRu]+: 613.10 found: 613.05.
1H NMR [(CD3)2CO, Me4Si, δ/ppm]: 9.25 (s, 1H, (η5-C5H4CO)), 9.16 (broad, 2H,
6), 8.04 (s, 2H,
3), 7.47 (m, 3H,
pPPh3), 7.36 (m, 6H,
mPPh3), 7.30 (br s, 2H,
5), 7.12 (t, 6H, 3JHH = 8.4,
oPPh3), 5.75 (broad, 2H,
β-(η5-C5H4CHO), 4.85 (broad, 2H,
γ-(η5-C5H4CHO), 2.48 (s, 6H, C
3).
APT-13C{1H} NMR [(CD3)2CO, δ/ppm]: 189.0 (η5-C5H4CO), 156.5 (
2), 156.0 (
6), 150.5 (
4), 134.0 (d, 2JCP = 11,
HoPPh3), 132.2 (d, 1JCP = 43,
qPPh3), 131.3 (d, 4JCP = 2,
HpPPh3), 129.5 (d, 3JCP = 9,
HmPPh3), 127.7 (
5), 125.1 (
3), 103.0 (
α-η5-C5H4CHO), 84.8 (
β-η5-C5H4CHO), 78.6 (
γ-η5-C5H4CHO), 20.8 (
H3).
31P{1H} NMR [(CD3)2CO, δ/ppm]: 48.94 ppm (s, Ph3).
FTIR [KBr, cm−1]: 3071–3050 cm−1 (νC–H aromatic rings), 2922 cm−1 (νC–H alkanes), 1670 (νCO, η5-C5H4CHO), 1440 (νCC aromatic rings), 1258 cm−1 (νCF3SO3 counterion).
UV-vis [DMSO, λmax/nm (ε × 103/M−1 cm−1)]: 289 (26.27), 371 (7.21), 412 (Sh).
UV-vis [CH2Cl2, λmax/nm (ε × 103/M−1 cm−1)]: 290 (26.10), 366 (6.91), 412 (Sh).
Elemental analysis calc. for C37H32F3N2O4PRuS (789.76): C, 56.3, H, 4.1; N, 3.6; S, 4.1. Found: C, 54.8; H, 3.9; N, 3.4; S, 4.0.
ESI-MS: [2-CF3SO3]+ calc. for [C36H32N2OPRu]+: 641.13 found: 641.02.
1H NMR [(CD3)2CO, Me4Si, δ/ppm]: 9.26 (m, 3H, (η5-C5H4CHO) + 6), 8.09 (s, 2H,
3), 7.43 (m, 5H,
pPPh3 +
5), 7.35 (m, 6H,
mPPh3), 7.12 (t, 6H, 3JHH = 8,
oPPh3), 5.78 (broad, 2H,
β-η5-C5H4CHO), 4.87–4.80 (m, 8H,
γ-η5-C5H4CHO + C
2OH + CH2O
).
APT-13C{1H} NMR [(CD3)2CO, δ/ppm]: 189.0 (η5-C5H4HO), 156.5 (
2), 156.2 (
6), 154.9 (
4), 134.0 (d, 2JCP = 11,
HoPPh3), 132.1 (d, 1JCP = 44,
qPPh3), 131.3 (d, 4JCP = 2,
HpPPh3), 129.5 (d, 3JCP = 10,
HmPPh3), 123.9 (
5), 121.3 (
3), 103.0 (
α-η5-C5H4CHO), 89.4 (
β-η5-C5H4CHO), 78.7 (
γ-η5-C5H4CHO), 62.4 (
H2OH).
31P NMR [(CD3)2CO, δ/ppm]: 48.94 (s, Ph3).
FTIR [KBr, cm−1]: 3464 (νO–H), 3084–2916 (νC–H aromatic rings); 2884–2857 (νC–H alkanes), 1678 (νCO, η5-C5H4CHO), 1440 (νCC aromatic rings), 1246 cm−1 (νCF3SO3 counter ion), 1221 cm−1 (νC–O).
UV-vis [DMSO, λmax/nm (ε × 103/M−1 cm−1)]: 290 (27.15), 366 (7.27), 416 (Sh).
UV-vis [CH2Cl2, λmax/nm (ε × 103/M−1 cm−1)]: 277 (Sh), 289 (28.43), 379 (7.43), 451 (Sh).
Elemental analysis calc. for C37H32F3N2O6PRuS (821.76): C, 54.1; H, 3.9; N, 3.4; S, 3.9. Found: C, 54.1; H, 3.9; N, 3.4; S, 3.9.
ESI-MS: [3-CF3SO3]+ calc. for [C36H32N2O3PRu]+: 673.12 found: 673.02.
Yield: 88% (135 mg).
1H NMR [(CD3)2CO, Me4Si, δ/ppm]: 9.35 (broad, 2H, 6), 9.26 (s, 1H, η5-C5H4C
O), 8.15 (s, 2H,
3), 7.48 (m, 3H, 3JHH = 7.6,
4), 7.46 (m, 5H,
pPPh3 +
5), 7.36 (m, 6H,
mPPh3), 7.12 (t, 6H, 3JHH = 8,
oPPh3), 6.11 (broad s, 2H, NH) 5.90 (d, 2H, NH), 5.83 (broad, 2H,
β-η5-C5H4CHO), 5.28 (m, 4H, bipy-C
2Biotin), 4.93 (broad, 2H,
γ-η5-C5H4CHO), 4.50 (SCH2-C
Biotin), 4.32 (m, 2H, C
Biotin), 3.22 (m, 2H, S-C
Biotin), 2.94 (m, 2H, SC
2Biotin), 2.67 (m, 2H, H), 2.52 + 1.75–1.49 (3× m, 16H, C
2C
2C
2C
2Biotin).
APT-13C{1H} NMR [(CD3)2CO, δ/ppm]: 189.3 (η5-C5H4HO), 173.5 (
O, bipy-Biotin ester), 164.1 (CO, bipy-Biotin, urea), 157.1 (C1), 156.6 (C2), 148.7 (C4), 133.9 (2JCP = 11, Cortho-PPh3), 131.9 (1JCP = 40, Cq, PPh3), 131.5 (4JCP = 1, Cpara-PPh3), 129.6 (3JCP = 10, Cmeta-PPh3), 123.9 (d, 4JCP = 6,
5), 122.6 (d, 4JCP = 8,
3), 107.5 (
α-η5-C5H4CHO), 85.1 (2 signals, s, Cβ-η5C5H4CHO), 79.1 (2 signals, s, Cγ-η5-C5H4CHO), 64.0 (2 signals, s, bipy-
H2Biotin), 62.6 (2 signals, s,
HBiotin), 60.9 (2 signals, s, SCH2-
HBiotin), 56.7 (2 signals, s, S
HBiotin), 41.1 (2 signals, s, S
H2Biotin), 34.3 + 29.2* + 25.6 (sets of 2 signals,
H2
H2
H2
H2Biotin).
31P NMR [(CD3)2CO, δ/ppm]: 48.70 (s, Ph3).
FTIR [KBr, cm−1]: 3073 (νC–H aromatic rings), 2928–2859 (νC–H alkanes), 1736–1620 (νCO η5-C5H4CHO, urea, ester), 1433 (νC
C aromatic rings), 1260 cm−1 (νCF3SO3 counter ion).
UV-vis [DMSO, λmax/nm (ε × 103/M−1 cm−1)]: 293 (47.69), 358 (11.94), 414 (Sh) 468 (Sh).
UV-vis [CH2Cl2, λmax/nm (ε × 103/M−1 cm−1)]: 291 (12.59), 345 (Sh), 416 (2.39), 479 (Sh).
Elemental analysis calc. for C57H60F3N6O10PRuS3 (1274.35): C, 53.7; H, 4.8; N, 6.6; S, 7.6. Found: C, 52.8; H, 5.2; N, 6.7; S, 7.0.
ESI-MS: [4-CF3SO3]+ calc. for [C56H60N6O7PRuS2]+: 1125.27 found: 1125.09.
1H NMR [(CD3)2CO, Me4Si, δ/ppm]: 9.49 (broad, 2H, 6), 8.17 (d, 2H, 3JHH = 8,
3), 7.88 (t, 2H, 3JHH = 8,
4), 7.41–7.32 (m, 11H,
pPPh3 +
mPPh3 +
5), 7.11 (t, 6H, 3JHH = 8,
oPPh3), 4. 94 (broad, 2H,
β-η5-C5H4CH2OH), 4.62 (broad, 2H,
γ-η5-C5H4CH2OH), 4.11 (s, 3H, η5-C5H4C
2OH + η5-C5H4CH2O
).
APT-13C{1H} NMR [(CD3)2CO, δ/ppm]: 157.0 (d, JCP = 2, 6), 156.7 (
2), 136.9 (
4), 133.8 (d, 2JCP = 11,
HoPPh3), 132.6 (d, 1JCP = 41,
qPPh3), 130.9 (d, 4JCP = 2,
HpPPh3), 129.3 (d, 3JCP = 10,
HmPPh3), 126.1 (
5), 124.1 (
3), 104.9 (
α-η5-C5H4CH2OH), 76.7 + 76.5 (
β-η5-C5H4CH2OH +
γ-η5-C5H4CH2OH), 57.7 (η5-C5H4
H2OH).
31P NMR [(CD3)2CO, δ/ppm]: 51.25 (s, Ph3).
FTIR [KBr, cm−1]: 3412 (νO−H), 3075–3055 (νC–H aromatic rings), 2962–2853 (νC–H alkanes), 1435 (νCC aromatic rings), 1256 (νCF3SO3 counter ion).
UV-vis [DMSO, λmax/nm (ε × 103/M−1 cm−1)]: 292 (16.59), 344 (Sh), 411 (2.93), 483 (Sh).
UV-vis [CH2Cl2, λmax/nm (ε × 103/M−1 cm−1)]: 291 (24.89), 348 (Sh), 423 (4.38), 486 (Sh).
Elemental analysis calc. for C35H30F3N2O4PRuS (763.73): C, 55.0; H, 4.0; N, 3.7; S, 4.2. Found: C, 53.6; H, 3.9; N, 3.5; S, 4.2.
ESI-MS: [5-CF3SO3]+ calc. for [C34H30N2OPRu]+: 615.11 found: 615.02.
1H NMR [(CD3)2CO, Me4Si, δ/ppm]: 9.29 (broad, 2H, 6), 8.01 (s, 2H,
3), 7.41 (m, 3H,
pPPh3), 7.32 (m, 6H,
mPPh3), 7.20 (broad, 2H,
5), 7.12 (t, 6H, 3JHH = 8,
oPPh3), 4.88 (broad, 2H,
β-η5-C5H4CH2OH), 4.56 (broad, 2H,
γ-η5-C5H4CH2OH), 4.10 (s, 3H, η5-C5H4C
2OH + η5-C5H4CH2O
), 2.45 (s, 6H, C
3).
APT-13C{1H} NMR [(CD3)2CO, δ/ppm]: 156.4 (2), 156.1 (d, JCP = 2,
6), 149.1 (
4), 133.9 (d, 2JCP = 11,
HoPPh3), 133.0 (d, 1JCP = 41,
qPPh3), 130.8 (d, 4JCP = 2,
HpPPh3), 129.6 (d, 3JCP = 9,
HmPPh3), 127.1 (
5), 124.6 (
3), 104.5 (d, 2JCP = 6,
αη5-C5H4CH2OH), 76.2 (
β-η5-C5H4CH2OH), 76.1 (d, 2JCP = 2,
γ-η5-C5H4CH2OH), 57.8 (
H2OH), 20.8 (
H3).
31P NMR [(CD3)2CO, δ/ppm]: 51.28 [s, PPh3].
FTIR [KBr, cm−1]: 3415 (νO−H), 3071–3050 cm−1 (νC–H aromatic rings), 2922 cm−1 (νC–H alkanes), 1440 (νCC aromatic rings), 1258 cm−1 (νCF3SO3 counterion).
UV-vis [DMSO, λmax/nm (ε × 103/M−1 cm−1)]: 291 (4.11), 351 (Sh), 418 (6.86), 472 (Sh).
UV-vis [CH2Cl2, λmax/nm (ε × 103/M−1 cm−1)]: 287 (29.20), 345 (Sh), 417 (5.28), 470 (Sh).
Elemental analysis calc. for C37H34F3N2O4PRuS (791.78): C, 56.1; H, 4.3; N, 3.5; S, 4.1. Found: C, 55.4; H, 4.1; N, 3.3; S, 4.0.
ESI-MS: [6-CF3SO3]+ calc. for [C36H34N2OPRu]+: 643.15 found: 643.00.
1H NMR [(CD3)2CO, Me4Si, δ/ppm]: 9.39 (broad, 2H, 6), 8.07 (s, 2H,
3), 7.41 (m, 3H,
pPPh3), 7.32 (m, 8H,
mPPh3 +
5), 7.12 (t, 6H, 3JHH = 8,
oPPh3), 4.91 (broad, 2H,
β-η5-C5H4CH2OH), 4.79 (s, 6H, C
2OH + CH2O
), 4.60 (broad, 2H,
γ-η5-C5H4CH2OH), 4.11 (m, 3H, η5-C5H4C
2OH + η5-C5H4CH2O
).
APT-13C{1H} NMR [(CD3)2CO, δ/ppm]: 156.4 (2), 156.3 (d, 3JCP = 2,
6), 153.7 (
4), 133.8 (d, 2JCP = 11,
HoPPh3), 132.8 (d, 1JCP = 40,
qPPh3), 130.8 (d, 4JCP = 2,
HpPPh3), 129.3 (d, 3JCP = 10,
HmPPh3), 123.4 (
5), 120.9 (
3), 104.5 (
α-η5-C5H4CH2OH), 76.3 + 76.2 (
β-η5-C5H4CH2OH +
γ-η5-C5H4CH2OH), 62.5 (
H2OH), 57.8 (η5-C5H4
H2OH).
31P NMR [(CD3)2CO, δ/ppm]: 51.27 (s, Ph3).
FTIR [KBr, cm−1]: 3486–3470 (νO–H), 2922–2864 (νC–H alkanes), 1440 (νCC aromatic rings), 1261 (νCF3SO3 counter ion), 1234 (νC–O).
UV-vis [DMSO, λmax/nm (ε × 103/M−1 cm−1)]: 292 (5.67), 348 (Sh), 416 (1.02), 478 (Sh).
UV-vis [CH2Cl2, λmax/nm (ε × 103/M−1 cm−1)]: 291 (4.11), 351 (Sh), 418 (6.86), 472 (Sh).
Elemental analysis calc. for C37H34F3N2O6PRuS (823.78): C, 53.9; H, 4.1; N, 3.4; S, 3.9. Found: C, 52.3; H, 4.0; N, 3.1; S, 3.0.
ESI-MS: [7-CF3SO3]+ calc. for [C36H34N2O3PRu]+: 675.14 found: 675.01.
The crystallographic data as well as details of the structure solution and refinement procedures are reported in ESI.† CCDC 2042407–2042412† contain the supplementary crystallographic data for this paper.
Footnote |
† Electronic supplementary information (ESI) available: Schemes forsynthesis of compounds 1–7; characterization of compounds 1–7 (ESI-MS and NMR spectra and UV-vis data); crystallographic data and structural refinement details for X-ray data for 1, 2, 3, 4, 5, and 6; stability curves in DMSO/DMEM; P-gp and MRP1 expression in non-small cell lung cancer cell lines (immunoblotting). CCDC 2042407–2042412. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0qi01344g |
This journal is © the Partner Organisations 2021 |