Electrochemical Detection of a Novel Pt(IV) Prodrug with the Metronidazole Axial Ligand in the Hypoxic Area.

We report herein a Pt(IV) prodrug with metronidazole in axial positions Pt-Mnz. The nitroaromatic axial ligand was conjugated with a cisplatin scaffold to irreversibly reduce under hypoxic conditions, thereby retaining the Pt(IV) prodrug in the area of hypoxia. X-ray near-edge adsorption spectroscopy (XANES) on dried drug-preincubated tumor cell samples revealed a gradual release of cisplatin from the Pt-Mnz prodrug instead of rapid intracellular degradation. The ability of the prodrug to penetrate into three-dimensional (3D) spheroid cellular cultures was evaluated by a novel electrochemical assay via a platinum-coated carbon nanoelectrode, capable of single-cell measurements. Using a unique technique of electrochemical measurements in single tumor spheroids, we were able to both detect the real-time response of the axial ligand to hypoxia and establish the depth of penetration of the drug into the tumor model.

Synthesis and Preclinical Evaluation of Small-Molecule Prostate-Specific Membrane Antigen-Targeted Abiraterone Conjugate.

Prostate cancer is the second most common type of cancer among men. The main method of its treatment is androgen deprivation therapy, which has a wide range of side effects. One of the solutions to this challenge is the targeted delivery of drugs to prostate cancer cells. In this study, we performed the synthesis of a novel small-molecule PSMA-targeted conjugate based on abiraterone. Cytotoxicity, the induction of intracellular reactive oxygen species, and P450-cytochrome species inhibition were investigated for this conjugate PSMA-abiraterone. The conjugate demonstrated a preferential effect on prostate tumor cells, remaining inactive at up to 100 µM in human fibroblast cells. In addition, it revealed preferential efficacy, specifically on PSMA-expressing lines with a 65% tumor growth inhibition level on 22Rv1 (PSMA+) xenografts after 14-fold oral administration of PSMA-Abi at a single dose of 500 mg/kg (7.0 g/kg total dose) was observed. This compound showed significantly reduced acute toxicity with comparable efficacy compared to AbiAc.

Ullmann-type C-Se Cross-Coupling in the Hydantoin Family: Synthesis, Mechanistic Studies, and Tests of Biological Activity.

An attractive strategy for C-Se bond formation by Ullmann-type copper(I)-promoted cross-coupling is developed. A wide range of aryliodides reacts with various disubstituted 2-selenohydantoins under mild conditions and provides Se-arylated imidazolines in moderate to high yields. Computational mechanistic studies show the oxidative addition/intramolecular reductive elimination likely to be the lowest-energy pathway. Cytotoxic activity of all 43 reaction products has been tested in vitro against MCF7 and A549 cancer cell lines with VA13 and MCF10a control cells.

Synthesis and biological activity of 5-aryliden-2-thiohydantoin S-aryl derivatives.

Three new and complementary approaches to S-arylation of 2-thiohydantoins have been developed: copper-catalyzed cross coupling with either arylboronic acids or aryl iodides under mild conditions, or direct nucleophilic substitution in activated aryl halides. For 38 diverse compounds, reaction yields for all three methods have been determined. Selected by molecular docking, they have been tested on androgen receptor activation, and p53-Mdm2 regulation, and A549, MCF7, VA13, HEK293T, PC3, LnCAP cell lines for cytotoxicity, Two of them turned out to be promising as androgen receptor activators (likely by allosteric regulation), and another one is shown to activate the p53 cascade. It is hoped that 2-thiohydantoin S-arylidenes are worth further studies as biologically active compounds.

Polypeptide-Based Molecular Platform and Its Docetaxel/Sulfo-Cy5-Containing Conjugate for Targeted Delivery to Prostate Specific Membrane Antigen.

A strategy for stereoselective synthesis of molecular platform for targeted delivery of bimodal therapeutic or theranostic agents to the prostate-specific membrane antigen (PSMA) receptor was developed. The proposed platform contains a urea-based, PSMA-targeting Glu-Urea-Lys (EuK) fragment as a vector moiety and tripeptide linker with terminal amide and azide groups for subsequent addition of two different therapeutic and diagnostic agents. The optimal method for this molecular platform synthesis includes (a) solid-phase assembly of the polypeptide linker, (b) coupling of this linker with the vector fragment, (c) attachment of 3-aminopropylazide, and (d) amide and carboxylic groups deprotection. A bimodal theranostic conjugate of the proposed platform with a cytostatic drug (docetaxel) and a fluorescent label (Sulfo-Cy5) was synthesized to demonstrate its possible sequential conjugation with different functional molecules.

Thiourea Modified Doxorubicin: A Perspective pH-Sensitive Prodrug.

A novel approach to the synthesis of pH-sensitive prodrugs has been proposed: thiourea drug modification. Resulting prodrugs can release the cytotoxic agent and the biologically active 2-thiohydantoin in the acidic environment of tumor cells. The concept of acid-catalyzed cyclization of thioureas to 2-thiohydantoins has been proven using a FRET model. Dual prodrugs of model azidothymidine, cytotoxic doxorubicin, and 2-thiohydantoin albutoin were obtained, which release the corresponding drugs in the acidic environment. The resulting doxorubicin prodrug was tested on prostate cancer cells and showed that the thiourea-modified prodrug is less cytotoxic (average IC50 ranging from 0.5584 to 0.9885 µM) than doxorubicin (IC50 ranging from 0.01258 to 0.02559 µM) in neutral pH 7.6 and has similar toxicity (average IC50 ranging from 0.4970 to 0.7994 µM) to doxorubicin (IC50 ranging from 0.2303 to 0.8110 µM) under mildly acidic conditions of cancer cells. Cellular and nuclear accumulation in PC3 tumor cells of Dox prodrug is much higher than accumulation of free doxorubicin.

Synthesis and biological evaluation of Doxorubicin-containing conjugate targeting PSMA.

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), has recently emerged as a prominent biomarker of prostate cancer (PC) and as an attractive protein trap for drug targeting. At the present time, several drugs and molecular diagnostic tools conjugated with selective PSMA ligands are actively evaluated in different preclinical and clinical trials. In the current work, we discuss design, synthesis and a preliminary biological evaluation of PSMA-specific small-molecule carrier equipped by Doxorubicin (Dox). We have introduced an unstable azo-linker between Dox and the carrier hence the designed compound does release the active substance inside cancer cells thereby providing a relatively high Dox concentration in nuclei and a relevant cytotoxic effect. In contrast, we have also synthesized a similar conjugate with a stable amide linker and it did not release the drug at all. This compound was predominantly accumulated in cytoplasm and did not cause cell death. Preliminary in vivo evaluation has showed good efficiency for the degradable conjugate against PC3-PIP(PSMA+)-containing xenograft mine. Thus, we have demonstrated that the conjugate can be used as a template to design novel analogues with improved targeting, anticancer activity and lower rate of potential side effects. 3D molecular docking study has also been performed to elucidate the underlying mechanism of binding and to further optimization of the linker area for improving the target affinity.

Synthesis and biological evaluation of PSMA-targeting paclitaxel conjugates.

Prostate cancer (PC) is the second most commonly occurring cancer in men. Conventional chemotherapy has wide variety of disadvantages such as high systemic toxicity and low selectivity. Targeted drug delivery is a promising approach to decrease side effects of therapy. Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer cells while low level of expression is observed in normal cells. In this study we describe the development of Glu-urea-Lys based PSMA-targeting conjugates with paclitaxel. A series of new PSMA targeting conjugates with paclitaxel was designed and synthesized. The cytotoxicity of conjugates was evaluated against prostate (LNCaP, 22Rv1 and PC-3) and non-prostate (Hek293T, VA13, A549 and MCF-7) cell lines. The most promising conjugate 21 was examined in vivo using 22Rv1 xenograft mice model. It demonstrated good efficiency comparable with paclitaxel, while reduced toxicity. 3D molecular docking study was also performed to understand underlying mechanism of binding and further optimization of the linker substructure and conjugates structure for improving the target affinity. These conjugates may be useful for further design of novel PSMA targeting delivery systems for PC.

Synthesis, isomerization and biological activity of novel 2-selenohydantoin derivatives.

A set of novel selenohydantoins were synthesized via a convenient and versatile approach involving the reaction of isoselenocyanates with various amines. We also revealed an unexpected Z→E isomerization of pyridin-2-yl-substituted selenohydantoins in the presence of Cu(2+) cations. The detailed mechanism of this transformation was suggested on the basis of quantum-chemical calculations, and the key role of Cu(2+) was elucidated. The obtained compounds were subsequently evaluated against a panel of different cancer cell lines. As a result, several molecules were identified as promising micromolar hits with good selectivity index. Instead of analogous thiohydantoins, which have been synthesized previously, selenohydantoins demonstrated a relatively high antioxidant activity comparable (or greater) to the reference molecule, Ebselen, a clinically approved drug candidate. The most active compounds have been selected for further biological trials.

Design, synthesis and biological evaluation of novel potent MDM2/p53 small-molecule inhibitors.

Regioselective synthesis, biological evaluation and 3D-molecular modeling for a series of novel diastereomeric 2-thioxo-5H-dispiro[imidazolidine-4,3-pyrrolidine-2,3-indole]-2,5(1H)-diones are described. The studied compounds have been tentatively identified as potent small molecule MDM2/p53 PPI inhibitors and can therefore be reasonably regarded as promising anticancer therapeutics.

Photoinduced Reduction of Novel Dual-Action Riboplatin Pt(IV) Prodrug.

Controlled photoreduction of Pt(IV) prodrugs is a challenging task due to the possibility of targeted light-controlled activation of anticancer agents without affecting healthy tissues. Also, a conjugation of photosensitizers and clinically used platinum drugs into one Pt(IV) prodrug allows combining photodynamic therapy and chemotherapy approaches into one molecule. Herein, we designed the cisplatin-based Pt(IV) prodrug Riboplatin with tetraacetylriboflavin in the axial position. A novel Pt(IV) prodrug is able to act both as a photodynamic therapy (PDT) agent through the conversion of ground-state 3O2 to excited-state 1O2 and as an agent of photoactivated chemotherapy (PACT) through releasing of cisplatin under gentle blue light irradiation, without the requirement of a reducing agent. The light-induced behavior of Riboplatin was investigated using an electrochemical sensor in MCF-7 tumor spheroids. Photocontrolled cisplatin release and ROS generation were detected electrochemically in real time. This appears to be the first confirmation of simultaneous photoactivated release of anticancer drug cisplatin and ROS from a dual-action Pt(IV) prodrug observed from the inside of living tumor spheroids.

Copper coordination compounds with (5Z,5Z')-2,2'-(alkane-α,ω-diyldiselenyl)-bis-5-(2-pyridylmethylene)-3,5-dihydro-4H-imidazol-4-ones. Comparison with sulfur analogue.

A series of new organic ligands (5Z,5Z')-2,2'-(alkane-α,ω-diyldiselenyl)-bis-5-(2-pyridylmethylene)-3,5-dihydro-4H-imidazol-4-ones (L) consisting of two 5-(2-pyridylmethylene)-3,5-dihydro-4H-imidazol-4-one units linked with polymethylene chains of various lengths (n = 2-10, where n is the number of CH2 units) have been synthesized. The reactions of these ligands with CuCl2·2H2O and CuClO4·6H2O gave Cu2+ or Cu1+ containing mono- and binuclear complexes with Cu2LCl x (x = 2-4) or CuL(ClO4) y (y = 1, 2) composition. It was shown that the agents reducing Cu2+ to Cu1+ in the course of complex formation can be both a ligand and an organic solvent in which the reaction is carried out. This fundamentally distinguishes the selenium-containing ligands L from their previously described sulfur analogs, which by themselves are not capable of reducing Cu2+ during complexation under the same conditions. A higher cytotoxicity and reasonable selectivity to cancer cell lines for synthesized complexes of selenium-containing ligands was shown; unlike sulfur analogs, ligands L themselves demonstrate a high cytotoxicity, comparable in some cases to the toxicity of copper-containing complexes.

Pt(IV) Prodrugs with Non-Steroidal Anti-inflammatory Drugs in the Axial Position.

We report herein the design, synthesis, and biological investigation of a series of novel Pt(IV) prodrugs with non-steroidal anti-inflammatory drugs naproxen, diclofenac, and flurbiprofen, as well as these with stearic acid in the axial position. Six Pt(IV) prodrugs 5-10 were designed, which showed superior antiproliferative activity compared to cisplatin as well as an ability to overcome tumor cell line resistance to cisplatin. By tuning the drug lipophilicity via variation of the axial ligands, the most potent Pt(IV) prodrug 7 was obtained, with an enhanced cellular accumulation of up to 153-fold that of cisplatin and nanomolar cytotoxicity both in 2D and 3D cell cultures. Pt2+ species were detected at different depths of MCF-7 spheroids after incubation with Pt(IV) prodrugs using a Pt-coated carbon nanoelectrode. Cisplatin accumulation in vivo in the murine mammary EMT6 tumor tissue of BALB/c mice after Pt(IV) prodrug injection was proved electrochemically as well. The drug tolerance study on BALB/c mice showed good tolerance of 7 in doses up to 8 mg/kg.

PSMA-targeted small-molecule docetaxel conjugate: Synthesis and preclinical evaluation.

Prostate cancer is one of the most commonly diagnosed men's cancers and remains one of the leading causes of cancer death. The development of approaches to the treatment of this oncological disease is an ongoing process. In this work, we have carried out the selection of ligands for the creation of conjugates based on the drug docetaxel and synthesized a series of three docetaxel conjugates. In vitro cytotoxicity of these molecules was evaluated using the MTT assay. Based on the assay results, we selected the conjugate which showed cytotoxic potential close to unmodified docetaxel. At the same time, the molar solubility of the resulting compound increased up to 20 times in comparison with the drug itself. In vivo evaluation on 22Rv1 (PSMA+) xenograft model demonstrated a good potency of the synthesized conjugate to inhibit tumor growth: the inhibition turned out to be more than 80% at a dose of 30 mg/kg. Pharmacokinetic parameters of conjugate distribution were analyzed. Also, it was found that PSMA-targeted docetaxel conjugate is less toxic than docetaxel itself, the decrease of molar acute toxicity in comparison with free docetaxel was up to 20%. Obtained conjugate PSMA-DOC is a good candidate for further expanded preclinical trials because of high antitumor activity, fewer side toxic effects and better solubility.

cis-Diastereoselective Synthesis of Spirooxindolo-ß-Lactams by Staudinger Cycloaddition with TsCl as Activating Co-reagent.

A convenient and versatile one-pot method for synthesis of 1,3-bis-aryl spirooxindolo-ß-lactams from isatin Schiff bases and substituted phenylacetic acids using ketene-imine cycloaddition reaction with TsCl for a ketene generation has been developed. The reaction procedure does not require absolute solvents and unstable starting reagents. The studied reactions lead to cis-diastereoselective ß-lactam formation for all tested phenylacetic acids except 4-MeOC6H4CH2COOH. An increase of trans-diastereomers yields with increasing temperature and solvent polarity was demonstrated.

Synthesis, Characterization, and Preclinical Evaluation of a Small-Molecule Prostate-Specific Membrane Antigen-Targeted Monomethyl Auristatin E Conjugate.

Prostate cancer is the second most common type of cancer among men. Its main method of treatment is chemotherapy, which has a wide range of side effects. One of the solutions to this challenge is targeted delivery to prostate cancer cells. Here we synthesized a novel small-molecule PSMA-targeted conjugate based on the monomethyl auristatin E. Its structure and conformational properties were investigated by NMR spectroscopy. Cytotoxicity, intracellular reactive oxygen species induction, and stability under liver microsomes and P450-cytochrome species were investigated for this conjugate. The conjugate demonstrated 77-85% tumor growth inhibition levels on 22Rv1 (PSMA (+)) xenografts, compared with a 37% inhibition level on PC-3 (PSMA (-)) xenografts, in a single dose of 0.3 mg/kg and a sufficiently high therapeutic index of 21. Acute, chronic, and subchronic toxicities and pharmacokinetics have shown that the synthesized conjugate is a promising potential agent for the chemotherapy of prostate cancer.

Synthesis and Biological Evaluation of PSMA Ligands with Aromatic Residues and Fluorescent Conjugates Based on Them.

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is a suitable target for specific delivery of antitumor drugs and diagnostic agents due to its overexpression in prostate cancer cells. In the current work, we describe the design, synthesis, and biological evaluation of novel low-molecular PSMA ligands and conjugates with fluorescent dyes FAM-5, SulfoCy5, and SulfoCy7. In vitro evaluation of synthesized PSMA ligands on the activity of PSMA shows that the addition of aromatic amino acids into a linker structure leads to a significant increase in inhibition. The conjugates of the most potent ligand with FAM-5 as well as SulfoCy5 demonstrated high affinities to PSMA-expressing tumor cells in vitro. In vivo biodistribution in 22Rv1 xenografts in Balb/c nude mice of PSMA-SulfoCy5 and PSMA-SulfoCy7 conjugates with a novel PSMA ligand demonstrated good visualization of PSMA-expressing tumors. Also, the conjugate PSMA-SulfoCy7 demonstrated the absence of any explicit toxicity up to 87.9 mg/kg.

Novel Copper-Containing Cytotoxic Agents Based on 2-Thioxoimidazolones.

A series of 73 ligands and 73 of their Cu+2 and Cu+1 copper complexes with different geometries, oxidation states of the metal, and redox activities were synthesized and characterized. The aim of the study was to establish the structure-activity relationship within a series of analogues with different substituents at the N(3) position, which govern the redox potentials of the Cu+2/Cu+1 redox couples, ROS generation ability, and intracellular accumulation. Possible cytotoxicity mechanisms, such as DNA damage, DNA intercalation, telomerase inhibition, and apoptosis induction, have been investigated. ROS formation in MCF-7 cells and three-dimensional (3D) spheroids was proven using the Pt-nanoelectrode. Drug accumulation and ROS formation at 40-60 µm spheroid depths were found to be the key factors for the drug efficacy in the 3D tumor model, governed by the Cu+2/Cu+1 redox potential. A nontoxic in vivo single-dose evaluation for two binuclear mixed-valence Cu+1/Cu+2 redox-active coordination compounds, 72k and 61k, was conducted.

Copper-Promoted C-Se Cross-Coupling of 2-Selenohydantoins with Arylboronic Acids in an Open Flask.

The modification of Chan-Lam-Evans cross-coupling reaction for the selective Se-arylation of 2-selenohydantoins under base-free mild conditions via aryl boronic acids is described herein. This approach was used to synthesize novel 5-arylidene-3-substituted-2-(arylselanyl)-imidazoline-4-ones with high yields. The anticancer activity of the final compounds was evaluated in vitro against different cancer cells, and thus, the possibility of 5-arylidene-3-substituted-2-(arylselanyl)-imidazoline-4-ones successful application as cytotoxic agents was demonstrated.