Synthesis and Preclinical Evaluation of Urea-Based Prostate-Specific Membrane Antigen-Targeted Conjugates Labeled with 177Lu.

177Lu-labeled small-molecule prostate-specific membrane antigen (PSMA) targeted tracers are therapeutic agents for metastatic castration-resistant prostate cancer. Optimizing molecular design holds the potential to further enhance the pharmacokinetic properties of PSMA-targeted agents while preserving their potent therapeutic effects. In this study, six novel N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-l-lysine (DCL) urea-based PSMA ligand 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid conjugates were synthesized. These conjugates feature polypeptide linkers containing the Phe-Phe peptide sequence and an aromatic fragment at the ε-NH-Lys group of the DCL fragment. The synthesis yielded products with satisfactory yields ranging from 60% to 72%, paving the way for their preclinical evaluation. The labeling of the new variants of urea-based PSMA inhibitors provided a radiochemical yield of over 95%. The 177Lu-labeled conjugates demonstrated specific and moderate affinity binding to PSMA-expressing human cancer cells PC3-pip in vitro and specific accumulation in PSMA-expressing xenografts in vivo. Based on the results, both the lipophilicity and the type of substituent in the linker significantly influence the binding properties of the PSMA inhibitor and its biodistribution profile. Specifically, the studied variants containing a bromine substituent or a hydroxyl group introduced into the aromatic fragment of the phenylalanyl residue in DCL exhibit higher affinities to PSMA compared to variants with only a chlorine-substituted aromatic fragment or variants without any substituents. The [177Lu]Lu-13C with the bromine substituent was characterized by the highest activity accumulation in blood, salivary glands, muscle, bone, and gastrointestinal tract and had inasmuch as an unfavorable pharmacokinetic profile. The negative charge of the carboxyl group in the phenyl moiety of the [177Lu]Lu-13A variant has demonstrated a positive effect on reducing the retention of activity in the liver and the kidneys (the ratio of tumor to kidneys was 1.3-fold). Low accumulation in normal tissues in vivo indicates that this novel PSMA-targeting inhibitor is a promising radioligand.

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.

Biotinylated Pt(IV) prodrugs with elevated lipophilicity and cytotoxicity.

A design of Pt(IV) prodrugs with tumor cell targeting moieties leading to increased selectivity is of interest. Herein, we designed a novel Pt(IV) prodrugs with COX-inhibitor naproxen, long-chain hydrophobic stearic acid moiety and biotin as axial ligands. We have established that for Pt(IV) prodrugs with biotin and naproxen or stearate in axial position, the lipophilicity rather than biotin receptors expression is the main factor of cytotoxicity. We also monitored the reduction speed of Pt(IV) prodrug 3 with naproxen and biotin in axial positions in A549 cells using XANES and demonstrated that the prodrug gradually releases cisplatin within 20 hours of incubation.

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.

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.

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.

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.

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.

Synthesis and Biological Evaluation of S-, O- and Se-Containing Dispirooxindoles.

A series of novel S-, O- and Se-containing dispirooxindole derivatives has been synthesized using 1,3-dipolar cycloaddition reaction of azomethine ylide generated from isatines and sarcosine at the double C=C bond of 5-indolidene-2-chalcogen-imidazolones (chalcogen was oxygen, sulfur or selenium). The cytotoxicity of these dispiro derivatives was evaluated in vitro using different tumor cell lines. Several molecules have demonstrated a considerable cytotoxicity against the panel and showed good selectivity towards colorectal carcinoma HCT116 p53+/+ over HCT116 p53-/- cells. In particular, good results have been obtained for LNCaP prostate cell line. The performed in silico study has revealed MDM2/p53 interaction as one of the possible targets for the synthesized molecules. However, in contrast to selectivity revealed during the cell-based evaluation and the results obtained in computational study, no significant p53 activation using a reporter construction in p53wt A549 cell line was observed in a relevant concentration range.

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.

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.

PSMA-targeted low-molecular double conjugates for diagnostics and therapy.

This review presents data on dual conjugates of therapeutic and diagnostic action for targeted delivery to prostate cancer cells. The works of the last ten years on this topic were analyzed. The mail attention focuses on low-molecular-weight conjugates directed to the prostate-specific membrane antigen (PSMA); the comparison of high and low molecular weight PSMA-targeted conjugates was made. The considered conjugates were divided in the review into two main classes: diagnostic bimodal conjugates (which are containing two fragments for different types of diagnostics), theranostic conjugates (containing both therapeutic and diagnostic agents); also bimodal high molecular weight therapeutic conjugates containing two therapeutic agents are briefly discussed. The data of in vitro and in vivo studies for PSMA-targeted double conjugates available by the beginning of 2021 have been analyzed.

Alternative mechanism of action of the DNP PtIV prodrug: intracellular cisplatin release and the mitochondria-mediated apoptotic pathway.

In a recent research paper Dr. Suxing Jin et al. reported two multispecific PtIV complexes DNP and NP with non-steroidal anti-inflammatory drug naproxen (NPX) as the axial ligand(s). Herein, we clarify the mechanism of action of DNP, its therapeutic target and intracellular redox-status.

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.

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.

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.

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.

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 of 1,3-diaryl-spiro[azetidine-2,3'-indoline]-2',4-diones via the Staudinger reaction: cis- or trans-diastereoselectivity with different addition modes.

A new synthetic approach for realizing biologically relevant bis-aryl spiro[azetidine-2,3'-indoline]-2',4-diones was developed based on Staudinger ketene-imine cycloaddition through the one-pot reaction of substituted acetic acids and Schiff bases in the presence of oxalyl chloride and an organic base. A series of [azetidine-2,3'-indoline]-2',4-diones were synthesized using this method. For comparison, the same compounds were obtained using a known technique, where ketene is generated from pre-synthesized acyl chloride. It was shown that the use of oxalyl chloride for ketene generation in the one-pot reaction at room temperature allows for the reversal of the diastereoselectivity of spiro-lactam formation, unlike previously described procedures.

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.