Structural modifications toward improved lead-203/lead-212 peptide-based image-guided alpha-particle radiopharmaceutical therapies for neuroendocrine tumors.

PURPOSE: The lead-203 (203Pb)/lead-212 (212Pb) elementally identical radionuclide pair has gained significant interest in the field of image-guided targeted alpha-particle therapy for cancer. Emerging evidence suggests that 212Pb-labeled peptide-based radiopharmaceuticals targeting somatostatin receptor subtype 2 (SSTR2) may provide improved effectiveness compared to beta-particle-based therapies for neuroendocrine tumors (NETs). This study aims to improve the performance of SSTR2-targeted radionuclide imaging and therapy through structural modifications to Tyr3-octreotide (TOC)-based radiopharmaceuticals. METHODS: New SSTR2-targeted peptides were designed and synthesized with the goal of optimizing the incorporation of Pb isotopes through the use of a modified cyclization technique; the introduction of a Pb-specific chelator (PSC); and the insertion of polyethylene glycol (PEG) linkers. The binding affinity of the peptides and the cellular uptake of 203Pb-labeled peptides were evaluated using pancreatic AR42J (SSTR2+) tumor cells and the biodistribution and imaging of the 203Pb-labeled peptides were assessed in an AR42J tumor xenograft mouse model. A lead peptide was identified (i.e., PSC-PEG2-TOC), which was then further evaluated for efficacy in 212Pb therapy studies. RESULTS: The lead radiopeptide drug conjugate (RPDC) - [203Pb]Pb-PSC-PEG2-TOC - significantly improved the tumor-targeting properties, including receptor binding and tumor accumulation and retention as compared to [203Pb]Pb-DOTA0-Tyr3-octreotide (DOTATOC). Additionally, the modified RPDC exhibited faster renal clearance than the DOTATOC counterpart. These advantageous characteristics of [212Pb]Pb-PSC-PEG2-TOC resulted in a dose-dependent therapeutic effect with minimal signs of toxicity in the AR42J xenograft model. Fractionated administrations of 3.7 MBq [212Pb]Pb-PSC-PEG2-TOC over three doses further improved anti-tumor effectiveness, resulting in 80% survival (70% complete response) over 120 days in the mouse model. CONCLUSION: Structural modifications to chelator and linker compositions improved tumor targeting and pharmacokinetics (PK) of 203/212Pb peptide-based radiopharmaceuticals for NET theranostics. These findings suggest that PSC-PEG2-TOC is a promising candidate for Pb-based targeted radionuclide therapy for NETs and other types of cancers that express SSTR2.

C-Sulfonylation of 4-Alkylpyridines: Formal Picolyl C-H Activation via Alkylidene Dihydropyridine Intermediates.

4-Picoline derivatives are converted to the corresponding aryl picolyl sulfones upon treatment with aryl sulfonyl chlorides and Et3N in the presence of catalytic DMAP. The reaction proceeds smoothly for a variety of alkyl and aryl picolines using a range of aryl sulfonyl chlorides. The reaction is believed to involve N-sulfonyl 4-alkylidene dihydropyridine intermediates and results in formal sulfonylation of unactivated picolyl C-H bonds.

Palladium-catalyzed allylation of 2- and 4-alkylpyridines via N-allyl alkylidene dihydropyridine intermediates.

A method to introduce allyl or cinnamyl groups to the picolyl positions of 2- or 4-alkylpyridines is described. Substituted N-allyl pyridinium salts are first treated with base (KOtBu) followed by catalytic [(η3-allyl)PdCl]2 and PPh3 to result in formal Pd-catalyzed transfer of N-allyl groups to the pyridine periphery. The reaction is believed to proceed through initial formation of nucleophilic alkylidene dihydropyridine intermediates that react with (π-allyl)Pd(II) electrophiles, thereby regenerating N-allyl pyridinium cations. Catalytic turnover and liberation of pyridine products is then achieved by oxidative addition of Pd(0) to these activated allyl groups.

Imidazolidine Hydride Donors in Palladium-Catalyzed Alkyne Hydroarylation.

Aldehyde-derived imidazolidines participate as hydride donors in intramolecular reductive Heck-type reactions. N,N'-Diphenylimidazolidines prepared from ortho-alkynyl benzaldehydes underwent regio- and stereoselective palladium-catalyzed hydroarylation followed by formal 1,5-hydride transfer and reductive elimination to afford substituted alkenes and imidazolium moieties, the latter conveniently converted in situ to ring-opened benzanilides to simplify product isolation. Internal alkynes were converted to trisubstituted alkenes via a syn hydroarylation process, while a terminal alkyne was converted to a cis alkene via a formal trans hydroarylation reaction. Benzanilide products could be converted to carboxylic acid derivatives under basic conditions, resulting in the net conversion of alkynyl aldehydes to alkenyl carboxylic acids. A styrene derivative with an attached N,N'-dimethylbenzimidazoline hydride donor was also found to undergo an analogous hydroarylation/benzimidazoline oxidation to give a diarylethane product.

A Mild and Highly Diastereoselective Preparation of N-Alkenyl-2-Pyridones via 2-Halopyridinium Salts and Aldehydes.

An experimentally simple one-pot preparation of N-alkenyl-2-pyridones is reported. The reaction features mild conditions using readily available 2-halopyridinium salts and aldehydes. N-Alkenyl-2-pyridone formation proceeds with high diastereoselectivity, and a wide range of aldehyde reaction partners is tolerated. Pyridone products are also amenable to further manipulation, including conversion to N-alkyl pyridones and polycyclic ring systems.

Expanding the Toolbox for Label-Free Enzyme Assays: A Dinuclear Platinum(II) Complex/DNA Ensemble with Switchable Near-IR Emission.

Switchable luminescent bioprobes whose emission can be turned on as a function of specific enzymatic activity are emerging as important tools in chemical biology. We report a promising platform for the development of label-free and continuous enzymatic assays in high-throughput mode based on the reversible solvent-induced self-assembly of a neutral dinuclear Pt(II) complex. To demonstrate the utility of this strategy, the switchable luminescence of a dinuclear Pt(II) complex was utilized in developing an experimentally simple, fast (10 min), low cost, and label-free turn-on luminescence assay for the endonuclease enzyme DNAse I. The complex displays a near-IR (NIR) aggregation-induced emission at 785 nm in aqueous solution that is completely quenched upon binding to G-quadruplex DNA from the human c-myc oncogene. Luminescence is restored upon DNA degradation elicited by exposure to DNAse I. Correlation between near-IR luminescence intensity and DNAse I concentration in human serum samples allows for fast and label-free detection of DNAse I down to 0.002 U/mL. The Pt(II) complex/DNA assembly is also effective for identification of DNAse I inhibitors, and assays can be performed in multiwell plates compatible with high-throughput screening. The combination of sensitivity, speed, convenience, and cost render this method superior to all other reported luminescence-based DNAse I assays. The versatile response of the Pt(II) complex to DNA structures promises broad potential applications in developing real-time and label-free assays for other nucleases as well as enzymes that regulate DNA topology.

Rhenium Complexes of Bis(benzothiazole)-Based Tetraarylethylenes as Selective Luminescent Probes for Amyloid Fibrils.

Probes for monitoring aggregation of amyloid beta (Aß) peptides are crucial to advance understanding of the molecular pathogenesis of Alzheimer's Disease (AD). Here, we report luminescent tricarbonyl rhenium complexes of tetraarylethylene (TAE) ligands featuring bis(benzothiazole) chelating groups in combination with (oligo)thiophene units that have been designed for monitoring amyloid fibrillation. Variation in the number of thiophenes influenced the photophysical properties of these complexes, as well as their binding affinities toward Aß42 fibrils. All complexes displayed submicromolar Kd's for binding Aß42 aggregates accompanied by up to 34-fold enhanced luminescence and red-shifted emission wavelengths. The high binding affinities and desirable photophysical properties of these complexes render them potential alternatives to established fluorescent Aß probes such as thioflavin T. Additionally, the general and modular design approach implemented in this study should facilitate development of second-generation TAE-based diagnostic tools for studying protein aggregation in AD and other neurological diseases.

Platinum(II) Complexes with Sterically Expansive Tetraarylethylene Ligands as Probes for Mismatched DNA.

Deficiencies in DNA mismatch repair (MMR) machinery result in greater incidence of DNA base pair mismatches in many types of cancer cells relative to normal cells. Consequently, luminescent probes capable of signaling the presence of mismatched DNA hold promise as potential cancer diagnostic and therapeutic tools. In this study, a series of cyclometalated platinum(II) complexes with sterically expansive tetraarylethylene ligands were synthesized and examined for selective detection of mismatched DNA. Increased steric bulk of the tetraarylethylene ligands in these complexes was observed to correlate with greater preferential luminescence enhancement in the presence of hairpin DNA oligonucleotides containing a mismatched site compared to well-matched oligonucleotides, with the most effective complex displaying ∼14-fold higher emission upon binding CC mismatched oligonucleotides compared to well-matched oligonucleotides. The results indicate binding to mismatched sites in DNA oligonucleotides occurs through metalloinsertion, and the luminescence response increases as a function of thermodynamic destabilization of the mismatch. Luminescence quenching experiments with Cu(phen)22+ and NaI further indicate mismatch binding from the minor groove, consistent with metalloinsertion. Binding to CC mismatched oligonucleotides was also investigated by isothermal titration calorimetry and UV-melting studies. These results demonstrate the efficacy of tetraarylethylene-based platinum(II) complexes for detection of mismatched DNA and establish a new molecular platform for development of organometallic DNA binding agents.

Synthesis and Evaluation of Tetraarylethylene-based Mono-, Bis-, and Tris(pyridinium) Derivatives for Image-Guided Mitochondria-Specific Targeting and Cytotoxicity of Metastatic Melanoma Cells.

Metastatic melanoma is the most aggressive and lethal form of skin cancer. Emerging evidence suggests that differences in melanoma metabolism relative to nonmalignant cells represent potential targets for improved therapy for melanoma. Specifically, melanoma cells exhibit increased mitochondrial electron transport chain (ETC) activity and concomitant hyperpolarized mitochondrial membrane potential relative to nonmalignant cells. We have synthesized several new fluorescent lipophilic vinylpyridinium cations built from tetraarylethylene scaffolds that target mitochondria via attraction to the hyperpolarized mitochondrial membrane potential. Mitochondria-specific accumulation in melanoma cells relative to normal human fibroblasts was demonstrated using confocal fluorescence microscopy and resulted in the disruption of oxidative metabolism leading to melanoma specific cell death in vitro. Thus, the pyridinium tetraarylethylene platform represents a promising new mitochondrial-targeted delivery vehicle with potential imaging and therapeutic properties.

Alkylidene Dihydropyridines As Synthetic Intermediates: Model Studies toward the Synthesis of the Bis(piperidine) Alkaloid Xestoproxamine C.

Results of model studies demonstrating a stereoselective synthetic route to tricyclic analogues of the bis(piperidine) alkaloid xestoproxamine C are presented. Dearomatization of a tricyclic pyridine derivative to afford an alkylidene dihydropyridine (anhydrobase) intermediate followed by catalytic heterogeneous hydrogenation was used to install the correct relative stereochemistry about the bis(piperidine) ring system. Other key features of these model studies include development of an efficient ring-closing metathesis procedure to prepare macrocyclic derivatives of 3,4-disusbstituted pyridines, intramolecular cyclizations of alkylidene dihydropyridines to establish pyridine-substituted pyrrolidines and piperidines, successful homologation of pyridine-4-carboxaldehydes using formaldehyde dimethyl thioacetal monoxide (FAMSO), and application of B-alkyl Suzuki coupling to assemble substituted pyridines.

Brønsted acid catalyzed intramolecular benzylic cyclizations of alkylpyridines.

Aldehyde and ketone electrophiles incorporated into the side chains of 2- and 4-alkylpyridines participate in intramolecular aldol-like condensations with pyridine benzylic carbons in the presence of Brønsted acid catalysts. Pyridines featuring ß-ketoamide side chains undergo cyclization in the presence of 10 mol% TfOH to afford pyridyl-substituted hydroxy lactams in good yield. These products were found to be resistant to further dehydration under a variety of conditions, however treatment with thionyl chloride elicited an unusual dehydration/oxidation reaction sequence. In contrast, acid-catalyzed cyclization of pyridines tethered to aliphatic aldehydes with amine linkers gives pyridyl-substituted dehydro-piperidine products. Similarly, intramolecular condensation of salicylaldehyde- and salicylketone-substituted pyridines affords pyridyl-substituted benzofurans.

Detection and disaggregation of amyloid fibrils by luminescent amphiphilic platinum(II) complexes.

Cyclometallated Pt(II) complexes possessing hydrophobic 2-phenylpyridine (ppy) ligands and hydrophilic acetonylacetone (acac) ligands have been investigated for their ability to detect amyloid fibrils via luminescence response. Using hen egg-white lysozyme (HEWL) as a model amyloid protein, Pt(II) complexes featuring benzanilide-substituted ppy ligands and ethylene glycol-functionalized acac ligands demonstrated enhanced luminescence in the presence of HEWL fibrils, whereas Pt(II) complexes lacking complementary hydrophobic/hydrophilic ligand sets displayed little to no emission enhancement. An amphiphilic Pt(II) complex incorporating a bis(ethylene glycol)-derivatized acac ligand was additionally found to trigger restructuring of HEWL fibrils into smaller spherical aggregates. Amphiphilic Pt(II) complexes were generally non-toxic to SH-SY5Y neuroblastoma cells, and several complexes also exhibited enhanced luminescence in the presence of Aß42 fibrils associated with Alzheimer's disease. This study demonstrates that easily prepared and robust (ppy)PtII(acac) complexes show promising reactivity toward amyloid fibrils and represent attractive molecular scaffolds for design of small-molecule probes targeting amyloid assemblies.

Preclinical Evaluation of a Lead Specific Chelator (PSC) Conjugated to Radiopeptides for 203Pb and 212Pb-Based Theranostics.

203Pb and 212Pb have emerged as promising theranostic isotopes for image-guided α-particle radionuclide therapy for cancers. Here, we report a cyclen-based Pb specific chelator (PSC) that is conjugated to tyr3-octreotide via a PEG2 linker (PSC-PEG-T) targeting somatostatin receptor subtype 2 (SSTR2). PSC-PEG-T could be labeled efficiently to purified 212Pb at 25 °C and also to 212Bi at 80 °C. Efficient radiolabeling of mixed 212Pb and 212Bi in PSC-PEG-T was also observed at 80 °C. Post radiolabeling, stable Pb(II) and Bi(III) radiometal complexes in saline were observed after incubating [203Pb]Pb-PSC-PEG-T for 72 h and [212Bi]Bi-PSC-PEG-T for 5 h. Stable [212Pb]Pb-PSC-PEG-T and progeny [212Bi]Bi-PSC-PEG-T were identified after storage in saline for 24 h. In serum, stable radiometal/radiopeptide were observed after incubating [203Pb]Pb-PSC-PEG-T for 55 h and [212Pb]Pb-PSC-PEG-T for 24 h. In vivo biodistribution of [212Pb]Pb-PSC-PEG-T in tumor-free CD-1 Elite mice and athymic mice bearing AR42J xenografts revealed rapid tumor accumulation, excellent tumor retention and fast renal clearance of both 212Pb and 212Bi, with no in vivo redistribution of progeny 212Bi. Single-photon emission computed tomography (SPECT) imaging of [203Pb]Pb-PSC-PEG-T and [212Pb]Pb-PSC-PEG-T in mice also demonstrated comparable accumulation in AR42J xenografts and renal clearance, confirming the theranostic potential of the elementally identical 203Pb/212Pb radionuclide pair.

Intramolecular cyclization manifolds of 4-alkylpyridines bearing ambiphilic side chains: construction of spirodihydropyridines or benzylic cyclization via anhydrobase intermediates.

4-Alkylpyridines possessing nucleophilic ß-dicarbonyl side chains have been converted to spirodihydropyridines upon treatment with ethyl chloroformate and sub-stoichiometric amounts of Ti(O(i)Pr)(4). Alternatively, inclusion of mild base in the reaction medium was found to facilitate generation of anhydrobase intermediates. Subsequent aldol-like condensations with electrophilic side chain moieties followed by hydrolysis delivered benzylically cyclized pyridines in good yield. In situ hydrogenation of cyclized anhydrobase intermediates afforded 4-substituted piperidines.

Semiconducting organic assemblies prepared from tetraphenylethylene tetracarboxylic acid and bis(pyridine)s via charge-assisted hydrogen bonding.

Principles of crystal engineering have been applied toward the construction of supramolecular assemblies between an acid-functionalized tetraphenylethylene derivative and three different bis(pyridine)s [4,4'-bis(pyridyl)ethylene, 4,4'-bis(pyridyl)ethane, and 4,4'-bipyridine]. Each assembly was structurally characterized, and charge transfer interactions within each sample were visually apparent. Quantum chemical calculations were used to determine crystal band structure and band gap magnitude, and electrical properties of the materials were measured using conducting probe atomic force microscopy (CP-AFM). The crystals displayed charge-carrier capability, and the magnitude of semiconductivity varied systematically as a function of conjugation in the bis(pyridine) component. Crystals incorporating 4,4'-bis(pyridyl)ethylene and 4,4'-bipyridine displayed conductivities comparable to those of established organic semiconductors (µ(eff) = 0.38 and 1.7 × 10(-2) cm(2)/V·s, respectively).

Improved synthesis and biological evaluation of chelator-modified α-MSH analogs prepared by copper-free click chemistry.

Radionuclide chelators (DOTA, NOTA) functionalized with a monofluorocyclooctyne group were prepared. These materials reacted rapidly and in high yield with a fully deprotected azide-modified peptide via Cu-free click chemistry under mild reaction conditions (aqueous solution, room temperature). The resulting bioconjugates bind with high affinity and specificity to their cell-surface receptor targets in vitro and appear stable to degradation in mouse serum over 3h of incubation at 37°C.

N-alkyl triphenylvinylpyridinium conjugated dihydroartemisinin perturbs mitochondrial functions resulting in enhanced cancer versus normal cell toxicity.

Dihydroartemisinin (DHA) is an FDA-approved antimalarial drug that has been repurposed for cancer therapy because of its preferential antiproliferative effects on cancer versus normal cells. Mitochondria represent an attractive target for cancer therapy based on their regulatory role in proliferation and cell death. This study investigates whether DHA conjugated to innately fluorescent N-alkyl triphenylvinylpyridinium (TPVP) perturbs mitochondrial functions resulting in a differential toxicity of cancer versus normal cells. TPVP-DHA treatments resulted in a dose-dependent toxicity of human melanoma and pancreatic cancer cells, whereas normal human fibroblasts were resistant to this treatment. TPVP-DHA treatments resulted in a G1-delay of the cancer cell cycle, which was also associated with a significant inhibition of the mTOR-metabolic and ERK1/2-proliferative signaling pathways. TPVP-DHA treatments perturbed mitochondrial functions, which correlated with increases in mitochondrial fission. In summary, TPVP mediated mitochondrial targeting of DHA enhanced cancer cell toxicity by perturbing mitochondrial functions and morphology.

A DOTA-peptide conjugate by copper-free click chemistry.

Attachment of DOTA to a novel monofluoro-cyclooctyne facilitates bioconjugation to an azide-modified peptide via Cu-free click chemistry. The resulting conjugate was radiolabeled with (111)In to afford a potential targeted molecular imaging agent with high specific activity and an excellent radiochemical purity.

Triphenylphosphonium derivatives disrupt metabolism and inhibit melanoma growth in vivo when delivered via a thermosensitive hydrogel.

Despite dramatic improvements in outcomes arising from the introduction of targeted therapies and immunotherapies, metastatic melanoma is a highly resistant form of cancer with 5 year survival rates of <35%. Drug resistance is frequently reported to be associated with changes in oxidative metabolism that lead to malignancy that is non-responsive to current treatments. The current report demonstrates that triphenylphosphonium(TPP)-based lipophilic cations can be utilized to induce cytotoxicity in pre-clinical models of malignant melanoma by disrupting mitochondrial metabolism. In vitro experiments demonstrated that TPP-derivatives modified with aliphatic side chains accumulated in melanoma cell mitochondria; disrupted mitochondrial metabolism; led to increases in steady-state levels of reactive oxygen species; decreased total glutathione; increased the fraction of glutathione disulfide; and caused cell killing by a thiol-dependent process that could be rescued by N-acetylcysteine. Furthermore, TPP-derivative-induced melanoma toxicity was enhanced by glutathione depletion (using buthionine sulfoximine) as well as inhibition of thioredoxin reductase (using auranofin). In addition, there was a structure-activity relationship between the aliphatic side-chain length of TPP-derivatives (5-16 carbons), where longer carbon chains increased melanoma cell metabolic disruption and cell killing. In vivo bio-distribution experiments showed that intratumoral administration of a C14-TPP-derivative (12-carbon aliphatic chain), using a slow-release thermosensitive hydrogel as a delivery vehicle, localized the drug at the melanoma tumor site. There, it was observed to persist and decrease the growth rate of melanoma tumors. These results demonstrate that TPP-derivatives selectively induce thiol-dependent metabolic oxidative stress and cell killing in malignant melanoma and support the hypothesis that a hydrogel-based TPP-derivative delivery system could represent a therapeutic drug-delivery strategy for melanoma.

Hexagonal crystalline inclusion complexes of 4-iodophenoxy trimesoate.

Bifurcated Ipi and IO[double bond, length as m-dash]C halogen bonding interactions assist in formation of unique iodo-arene trimers leading to nanoscale channels in inclusion complexes of trimesic acid iodophenolate.