Development of Cytotoxic GW7604-Zeise's Salt Conjugates as Multitarget Compounds with Selectivity for Estrogen Receptor- Positive Tumor Cells.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a carrier was esterified with alkenols of various lengths and coordinated through the ethylene moiety to PtCl3, similar to Zeise's salt (K[PtCl3(C2H4)]). The resulting GW7604-Alk-PtCl3 complexes (Alk = Prop, But, Pent, Hex) degraded in aqueous solution only by exchange of the chlorido ligands. For example, GW7604-Pent-PtCl3 coordinated the amino acid alanine in the cell culture medium, bound the isolated nucleotide 5'-GMP, and interacted with the DNA (empty plasmid pSport1). It accumulated in estrogen receptor (ER)-positive MCF-7 cells primarily via cytosolic vesicles, while it was only marginally taken up in ER-negative SKBr3 cells. Accordingly, GW7604-Pent-PtCl3 and related complexes were inactive in SKBr3 cells. GW7604-Pent-PtCl3 showed high affinity to ERα and ERß without mediating agonistic or ER downregulating properties. GW7604-Alk ligands also increased the cyclooxygenase (COX)-2 inhibitory potency of the complexes. In contrast to Zeise's salt, the GW7604-Alk-PtCl3 complexes inhibited COX-1 and COX-2 to the same extent.

New strategies towards advanced CT contrast agents. Development of neutral and monoanionic sulfur-bridged W(V) dimeric complexes.

Multinuclear tungsten complexes are intriguing candidates for new contrast media that can provide substantial improvements in CT imaging diagnostics. Herein, we present a ligand strategy, based on amino acids, and mono- and disubstituted EDTA derivatives, that enables the development of stable complexes with high tungsten content and reasonably low osmolality. Accordingly, a series of neutral and monoanionic di-µ-sulfido W(V) dimers have been synthesized via a convenient procedure utilizing microwave heating in combination with ion-pair HPLC reaction monitoring. The compounds were characterized in detail by various techniques, including ESI-HRMS, NMR spectroscopy, HPLC, elemental analysis, and X-ray crystallography. The aqueous stability of the complexes under physiologically relevant conditions, and during heat sterilization was also examined as an initial assessment of their potential applicability as radiocontrast agents. Monoanionic complexes featuring monosubstituted EDTA derivatives have demonstrated high stability, while producing a lower number of ions in solution (resulting in lower osmolality) in comparison to their bis-anionic EDTA counterparts. Nevertheless, they exhibited insufficient water solubility for application as intravascular contrast agents. However, our study showed that aqueous solubility of this type of complexes can be tuned by small modifications in the ligand structure.

Multifunctional Pt(iv) prodrug candidates featuring the carboplatin core and deferoxamine.

The synergistic combination of the anticancer drug carboplatin and the iron chelator deferoxamine (DFO) served as a foundation for the development of novel multifunctional prodrugs. Hence, five platinum(iv) complexes, featuring the equatorial coordination sphere of carboplatin, and one or two DFO units incorporated at axial positions, were synthesized and characterized using ESI-HRMS, multinuclear (1H, 13C, 15N, 195Pt) NMR spectroscopy and elemental analysis. Analytical studies demonstrated that the chelating properties of the DFO moiety were not compromised after coupling to the platinum(iv) core. The cytotoxic activity of the compounds was evaluated in monolayer (2D) and spheroid (3D) cancer cell models, derived from ovarian teratocarcinoma (CH1/PA-1), colon carcinoma (SW480) and non-small cell lung cancer (A549). The platinum(iv)-DFO prodrugs demonstrated moderate in vitro cytotoxicity (a consequence of their slow activation kinetics) but with less pronounced differences between intrinsically chemoresistant and chemosensitive cell lines as well as between 2D and 3D models than the clinically used platinum(ii) drug carboplatin.

Screening-based approach to discover effective platinum-based chemotherapies for cancers with poor prognosis.

Drug combinations are extensively used to treat cancer and are often selected according to complementary mechanisms. Here, we describe a cell-based high-throughput screening assay for identification of synergistic combinations between broadly applied platinum-based chemotherapeutics and drugs from a library composed of 1280 chemically and pharmacologically diverse (mostly FDA approved) compounds. The assay was performed on chemoresistant cell lines derived from lung (A549) and pancreatic (PANC-1) carcinoma, where platinum-based combination regimens are currently applied though with limited success. The synergistic combinations identified during the screening were validated by synergy quantification using the combination index method and via high content fluorescent microscopy analysis. New promising synergistic combinations discovered using this approach include compounds currently not used as anticancer drugs, such as cisplatin or carboplatin with hycanthone and cisplatin with spironolactone in pancreatic carcinoma, and carboplatin and deferoxamine in non-small cell lung cancer. Strong synergy between cisplatin or carboplatin and topotecan in PANC-1 cells, compared to A549 cells, suggests that this combination, currently used in lung cancer treatment regimens, could be applied to pancreatic carcinoma as well. Several drugs used to treat diseases other than cancer, including pyrvinium pamoate, auranofin, terfenadine and haloprogin, showed strong cytotoxicity on their own and synergistic interactions with platinum drugs. This study demonstrates that non-obvious drug combinations that would not be selected based on complementary mechanisms can be identified via high-throughput screening.

The impact of whole human blood on the kinetic inertness of platinum(iv) prodrugs - an HPLC-ICP-MS study.

The potential advantage of platinum(iv) complexes as alternatives to classical platinum(ii)-based drugs relies on their kinetic stability in the body before reaching the tumor site and on their activation by reduction inside cancer cells. In this study, an analytical workflow has been developed to investigate the reductive biotransformation and kinetic inertness of platinum(iv) prodrugs comprising different ligand coordination spheres (respectively, lipophilicity and redox behavior) in whole human blood. The distribution of platinum(iv) complexes in blood pellets and plasma was determined by inductively coupled plasma-mass spectrometry (ICP-MS) after microwave digestion. An analytical approach based on reversed-phase (RP)-ICP-MS was used to monitor the parent compound and the formation of metabolites using two different extraction procedures. The ligand coordination sphere of the platinum(iv) complexes had a significant impact on their accumulation in red blood cells and on their degree of kinetic inertness in whole human blood. The most lipophilic platinum(iv) compound featuring equatorial chlorido ligands showed a pronounced penetration into blood cells and a rapid reductive biotransformation. In contrast, the more hydrophilic platinum(iv) complexes with a carboplatin- and oxaliplatin-core exerted kinetic inertness on a pharmacologically relevant time scale with notable amounts of the compound accumulated in the plasma fraction.

Impact of the equatorial coordination sphere on the rate of reduction, lipophilicity and cytotoxic activity of platinum(IV) complexes.

The impact of the equatorial coordination sphere on the reduction behavior (i.e. rate of reduction) of platinum(IV) complexes with axial carboxylato ligands was studied. Moreover, the influence of equatorial ligands on the stability, lipophilicity and cytotoxicity of platinum(IV) compounds was evaluated. For this purpose, a series of platinum(IV) complexes featuring axial carboxylato ligands (succinic acid monoesters) was synthesized; anionic carboxylato (OAc-, oxalate) and halido (Cl-, Br-, I-) ligands served as leaving groups and am(m)ine carrier ligands were provided by monodentately (isopropylamine, ammine+cyclohexaneamine) or bidentately (ethane-1,2-diamine) coordinating am(m)ines. All platinum(IV) products were fully characterized based on elemental analysis, high resolution mass spectrometry and multinuclear (1H, 13C, 15N, 195Pt) NMR spectroscopy as well as by X-ray diffraction in some cases. The rate of reduction in the presence of ascorbic acid was determined by NMR spectroscopy and the lipophilicity of the complexes was investigated by analytical reversed phase HPLC measurements. Cytotoxic properties were studied by means of a colorimetric microculture assay in three human cancer cell lines derived from cisplatin sensitive ovarian teratocarcinoma (CH1/PA-1) as well as cisplatin insensitive colon carcinoma (SW480) and non-small cell lung cancer (A549).

Oxaliplatin reacts with DMSO only in the presence of water.

Herein we show that oxaliplatin reacts rapidly with DMSO in aqueous solutions, despite being stable in pure DMSO and pure water. Furthermore, the reactivity of the clinically applied Pt(ii) drugs in water/DMSO and PBS/DMSO mixtures, and the nature of the species formed were investigated by MS, NMR and RP-HPLC techniques.

Repositioning approved drugs for the treatment of problematic cancers using a screening approach.

Advances in treatment strategies together with an earlier diagnosis have considerably increased the average survival of cancer patients over the last four decades. Nevertheless, despite the growing number of new antineoplastic agents introduced each year, there is still no adequate therapy for problematic malignancies such as pancreatic, lung and stomach cancers. Consequently, it is important to ensure that existing drugs used to treat other types of cancers, and potentially other diseases, are not overlooked when searching for new chemotherapy regimens for these problematic cancer types. We describe a screening approach that identifies chemotherapeutics for the treatment of lung and pancreatic cancers, based on drugs already approved for other applications. Initially, the 1280 chemically and pharmacologically diverse compounds from the Prestwick Chemical Library® (PCL) were screened against A549 (lung cancer) and PANC-1 (pancreatic carcinoma) cells using the PrestoBlue fluorescent-based cell viability assay. More than 100 compounds from the PCL were identified as hits in one or both cell lines (80 of them, being drugs used to treat diseases other than cancer). Selected PCL hits were further evaluated in a dose-response manner. Promising candidates for repositioning emanating from this study include antiparasitics, cardiac glycosides, as well as the anticancer drugs vorinostat and topotecan.

The role of the equatorial ligands for the redox behavior, mode of cellular accumulation and cytotoxicity of platinum(IV) prodrugs.

The current study aims to elucidate the possible reasons for the significantly different pharmacological behavior of platinum(IV) complexes with cisplatin-, carboplatin- or nedaplatin-like cores and how this difference can be related to their main physicochemical properties. Chlorido-containing complexes are reduced fast (within hours) by ascorbate and are able to unwind plasmid DNA in the presence of ascorbate, while their tri- and tetracarboxylato analogs are generally inert under the same conditions. Comparison of the lipophilicity, cellular accumulation and cytotoxicity of the investigated platinum compounds revealed the necessity to define new structure-property/activity relationships (SPRs and SARs). The higher activity and improved accumulation of platinum(IV) complexes bearing Cl(-) in equatorial position cannot only be attributed to passive diffusion facilitated by their lipophilicity. Therefore, further platinum accumulation experiments under conditions where active/facilitated transport mechanisms are suppressed were performed. Under hypothermic conditions (4°C), accumulation of dichloridoplatinum(IV) complexes is reduced down to 10% of the amount determined at 37°C. These findings suggest the involvement of active and/or facilitated transport in cellular uptake of platinum(IV) complexes with a cisplatin-like core. Studies with ATP depletion mediated by oligomycin and low glucose partially confirmed these observations, but their feasibility was severely limited in the adherent cell culture setting.

Prediction of logP for Pt(II) and Pt(IV) complexes: Comparison of statistical and quantum-chemistry based approaches.

The octanol/water partition coefficient, logP, is one of the most important physico-chemical parameters for the development of new metal-based anticancer drugs with improved pharmacokinetic properties. This study addresses an issue with the absence of publicly available models to predict logP of Pt(IV) complexes. Following data collection and subsequent development of models based on 187 complexes from literature, we validate new and previously published models on a new set of 11 Pt(II) and 35 Pt(IV) complexes, which were kept blind during the model development step. The error of the consensus model, 0.65 for Pt(IV) and 0.37 for Pt(II) complexes, indicates its good accuracy of predictions. The lower accuracy for Pt(IV) complexes was attributed to experimental difficulties with logP measurements for some poorly-soluble compounds. This model was developed using general-purpose descriptors such as extended functional groups, molecular fragments and E-state indices. Surprisingly, models based on quantum-chemistry calculations provided lower prediction accuracy. We also found that all the developed models strongly overestimate logP values for the three complexes measured in the presence of DMSO. Considering that DMSO is frequently used as a solvent to store chemicals, its effect should not be overlooked when logP measurements by means of the shake flask method are performed. The final models are freely available at http://ochem.eu/article/76903.

Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups - A rational approach toward exploiting the platinum(IV) prodrug strategy.

A series of novel symmetrically and unsymmetrically coordinated platinum(IV) complexes with monodentate carboxylato ligands was synthesized. The compounds exhibit a general coordination sphere of [Pt(en)(OCOR)2(OCOR')(OCOR″)], where the carboxylato ligands are represented by acetato and succinic acid monoester ligands. Dicarboxylatoplatinum(II) complexes were synthesized and oxidized symmetrically or unsymmetrically to obtain platinum(IV) complexes, which were subsequently carboxylated with noncyclic anhydrides. The compounds were investigated in detail by elemental analysis, mass spectrometry, infrared and multinuclear ((1)H, (13)C, (15)N, (195)Pt) NMR spectroscopy as well as by X-ray diffraction in some cases. The reduction behavior was followed by NMR spectroscopy, while stability and lipophilicity were examined by analytical reversed phase HPLC measurements. Cytotoxic properties were studied in three human cancer cell lines derived from cisplatin sensitive ovarian teratocarcinoma (CH1/PA-1), cisplatin insensitive colon carcinoma (SW480) and non-small cell lung cancer (A549). Thereby, the most lipophilic (yet water soluble) platinum(IV) complexes showed promising IC50 values in the low micromolar and even nanomolar range, demonstrating the significant advantage of using equatorially coordinated monodentate carboxylato ligands.

Tumor microenvironment in focus: LA-ICP-MS bioimaging of a preclinical tumor model upon treatment with platinum(IV)-based anticancer agents.

The selection of drug candidates for entering clinical development relies on in vivo testing in (solid) tumor animal models. However, the heterogeneity of tumor tissue (e.g. in terms of drug uptake or tissue composition) is rarely considered when testing novel drug candidates. Therefore, we used the murine colon cancer CT-26 tumor model to study the spatially-resolved drug distribution in tumor tissue upon repetitive treatment of animals over two weeks with three investigational platinum(IV)-based anticancer agents, oxaliplatin or satraplatin. A quantitative laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging method revealed a heterogeneous platinum distribution, which correlated well with the histologic features of the tumor and surrounding tissue at the microscopic level. In most of the cases, higher amounts of intratumoral platinum were found in the surrounding tissue than in the malignant parts of the sample. This indicates that determination of average platinum amounts (e.g. by microwave-assisted digestion of the sample followed by analysis with ICP-MS) might overestimate the drug uptake in tumor tissue causing misleading conclusions. In addition, we studied the platinum distribution in the kidneys of treated animals to probe if accumulation in the cortex and medulla predict potential nephrotoxicity. A 10-fold increase of platinum in the cortex of the kidney over the medulla was observed for oxaliplatin and satraplatin. Although these findings are similar to those in the platinum distribution of the nephrotoxic anticancer drug cisplatin, treatment with the compounds of our study did not show signs of nephrotoxicity in clinical use or clinical trials (oxaliplatin, satraplatin) and did not result in the alteration of renal structures. Thus, predicting the side effects based on bioimaging data by LA-ICP-MS should be considered with caution. To the best of our knowledge, this is the first LA-ICP-MS study on spatially-resolved platinum accumulation in tissues after repetitive platinum-based anticancer drug treatment of mice bearing a preclinical tumor model.

Comparative in vitro and in vivo pharmacological investigation of platinum(IV) complexes as novel anticancer drug candidates for oral application.

Platinum(IV) complexes are promising candidates as prodrugs for oral application in anticancer chemotherapy. However, only a few Pt(IV) compounds entered (pre)clinical trials, e.g. satraplatin, while most of the others were only tested in vitro. Aim of the study was investigation of the in vivo pharmacological behavior as well as the anticancer activity of two novel platinum(IV) complexes vs. satraplatin. The drugs were selected due to significantly different in vitro cytotoxicity while sharing some physicochemical properties (e.g. lipophilicity). Initial experiments indicated that the highly in vitro cytotoxic compound 1 ((OC-6-33)-dichloridobis((4-ethoxy)-4-oxobutanoato)-bis(ethylamine)platinum(IV)) was also characterized by high drug absorption and tissue platinum levels after oral application. Interestingly, analysis of serum samples using SEC-ICP-MS revealed that the administered drugs have completely been metabolized and/or bound to proteins in serum within 2 h after treatment. With regard to the activity in vivo, the outcomes were rather unexpected: although potent anticancer effect of 1 was observed in cell culture, the effects in vivo were rather minor. Nevertheless, 1 was superior to 2 ((OC-6-33)-diammine(cyclobutane-1,1-dicarboxylato)-bis((4-cyclopentylamino)-4-oxobutanoato)platinum(IV)) after i.p. administration, which was, at least to some extent, in accordance to the cell culture experiments. After oral gavage, both compounds exhibited comparable activity. This is remarkable considering the distinctly lower activity of 2 in cell culture as well as the low platinum levels detected both in serum and tissues after oral application. Consequently, our data indicate that the prediction of in vivo anticancer activity by cell culture experiments is not trivial, especially for orally applied drugs.

A novel class of bis- and tris-chelate diam(m)inebis(dicarboxylato)platinum(IV) complexes as potential anticancer prodrugs.

A novel class of platinum(IV) complexes of the type [Pt(Am)(R(COO)2)2], where Am is a chelating diamine or two monodentate am(m)ine ligands and R(COO)2 is a chelating dicarboxylato moiety, was synthesized. For this purpose, the reaction between the corresponding tetrahydroxidoplatinum(IV) precursors and various dicarboxylic acids, such as oxalic, malonic, 3-methylmalonic, and cyclobutanedicarboxylic acid, was utilized. All new compounds were characterized in detail, using 1D and 2D NMR techniques, ESI-MS, FTIR spectroscopy, elemental analysis, TGA, and X-ray diffraction. Their in vitro cytotoxicity was determined in a panel of human tumor cell lines (CH1, SW480 and A549) by means of the MTT colorimetric assay. Furthermore, the lipophilicity and redox properties of the novel complexes were evaluated in order to better understand their pharmacological behavior. The most promising drug candidate, 4b (Pt(DACH)(mal)2), demonstrated low in vivo toxicity but profound anticancer activity against both the L1210 leukemia and CT-26 colon carcinoma models.

Theoretical investigations and density functional theory based quantitative structure-activity relationships model for novel cytotoxic platinum(IV) complexes.

Octahedral platinum(IV) complexes are promising candidates in the fight against cancer. In order to rationalize the further development of this class of compounds, detailed studies on their mechanisms of action, toxicity, and resistance must be provided and structure-activity relationships must be drawn. Herein, we report on theoretical and QSAR investigations of a series of 53 novel bis-, tris-, and tetrakis(carboxylato)platinum(IV) complexes, synthesized and tested for cytotoxicity in our laboratories. The hybrid DFT functional wb97x was used for optimization of the structure geometry and calculation of the descriptors. Reliable and robust QSAR models with good explanatory and predictive properties were obtained for both the cisplatin sensitive cell line CH1 and the intrinsically cisplatin resistant cell line SW480, with a set of four descriptors.

Novel tetracarboxylatoplatinum(IV) complexes as carboplatin prodrugs.

It is widely accepted that platinum(IV) complexes act as prodrugs and have to be activated by reduction to the respective platinum(II) analogs. Recently it could be shown that introduction of lipophilic carboxylato ligands in the axial position leads to diaminedichloridoplatinum(IV) compounds with exceptionally high cytotoxicity. With the aim of improving the antiproliferative properties of carboplatin, a series of twenty-one novel Pt(IV) complexes, featuring the equatorial ligand sphere of carboplatin as well as lipophilic axial carboxylato ligands, was synthesized. In depth characterization is based on elemental analysis, ESI-MS, ATR-IR, and multinuclear ((1)H, (13)C, (15)N, and (195)Pt) NMR spectroscopy. Their cytotoxic activity in four cell lines (CH1, SK-OV-3, SW480, and A549), lipophilicity, electrochemistry and additionally the rate of reduction in the presence of ascorbic acid were investigated. In contrast to analogous diaminedicarboxylatodichloridoplatinum(IV) compounds, the cytotoxicity of novel diaminetetracarboxylato counterparts could not substantially be increased by simply enhancing their lipophilic character. It seems that not only the reduction potential, but also the rate of reduction has a tremendous influence on the cytotoxic properties. This has to be taken into account for the development of novel anticancer platinum(IV) agents.