Synthesis, characterization, and anticancer activity of chitosan functionalized isatin based thiosemicarbazones, and their copper(II) complexes.

The one-pot synthetic method of condensation of isatin and 5-chloroisatin on to amino group at C2 position of the pyranose ring chitosan in chitosan thiosemicarbazide was employed to get these chitosan thiosemicarbazones (TSCs). The partial incorporation of thiosemicarbazone moiety in chitosan was shown by FT-IR and 13C NMR spectroscopic studies, powder X ray diffraction, and CHNS microanalysis. The NOS tridentate coordination behavior of TSCs with copper(II) chloride to give the square planar complexes was established by FT-IR spectroscopic data, magnetic susceptibility measurement, and EPR spectral analysis. The thermal stability of these biomaterial chitosan derivatives till the commencement of chain disruption at 200C was shown by thermal studies. As revealed by colorimetric MTT assays, the in vitro anticancer activity enhancement accorded with the functionalization of chitosan as isatin based chitosan TSCs, and NOS tridentate coordination of TSCs plus a monodentate coordination of chloride ion with copper(II) ion. Only a marginal activity difference of these compounds was observed against the tumorigenic MDCK and MCF-7 cancer cell lines, irrespective of unit molecular weight (Mw) and degree of deacetylation (DDA) of ring chitosan. The 5-chloroisatin chitosan TSCs showed better activity than isatin chitosan TSCs against both the cell lines.

Pyridine-Based NNS Tridentate Chitosan Thiosemicarbazones and Their Copper(II) Complexes: Synthesis, Characterization, and Anticancer Activity.

Chitosan-functionalized pyridine-based thiosemicarbazones and their copper(II) complexes have been found to own a substantial antiproliferative activity against the tumorigenic Madin Darby canine kidney (MDCK) and MCF-7 cancer cell lines. In the current study, chitosan oligosaccharide (CS) (87% DDA, Mw < 3000 Da) and crab shell chitosan (CCS) (67% DDA, M w 350 kDa) were functionalized as chitosan pyridine-2-thiosemicarbazones and chitosan 2-acetyl pyridine-2-thiosemicarbazones, and their copper(II) complexes were synthesized. The formation of chitosan thiosemicarbazones and their NNS tridentate behavior to give the square planar copper(II) chitosan thiosemicarbazone complexes were established by spectroscopic studies, powder X-ray diffraction, elemental analysis, and magnetic moment measurements. The thermal study showed a marked stability of these derivatives before the outset of chitosan backbone degradation at 200 °C. The colorimetric MTT assay revealed a higher activity of CS thiosemicarbazones, viz., CSTSC series (IC50 375-381 µg mL-1 in the MDCK cell line and 281-355 µg mL-1 in the MCF-7 cell line) than that of high-molecular-weight CCS thiosemicarbazones, viz., CCSTSC series (IC50 335-400 µg mL-1 in the MDCK cell line and 365-400 µg mL-1 in the MCF-7 cell line), showing an enhanced activity with a decrease in Mw and an increase in DDA of constituent chitosan, a higher activity of both of these series of thiosemicarbazones than that of their native chitosan, viz., CS (IC50 370 µg mL-1 in the MCF-7 cell line and >400 µg mL-1 in the MDCK cell line) and CCS (IC50 > 400 µg mL-1 in both cell lines), and a higher activity of the Cu-CSTSC complexes (IC50 322-342 µg mL-1 in the MDCK cell line and 278-352 µg mL-1 in the MCF-7 cell line) and Cu-CCSTSC complexes (IC50 274-400 µg mL-1 in the MDCK cell line and 231-352 µg mL-1 in the MCF-7 cell line) than that of their respective ligands.

Supramolecular Cylinders Target Bulge Structures in the 5' UTR of the RNA Genome of SARS-CoV-2 and Inhibit Viral Replication*.

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5' UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents.

BODIPY-linked cis-dichlorido zinc(ii) conjugates: the strategic design of organelle-specific next-generation theranostic photosensitizers.

Dipicolylamine (dpa) based cis-dichlorido zinc(ii) complexes [Zn(L1-3)Cl2] (1-3), where L2 and L3 are non-iodo and di-iodo BODIPY-appended dpa in 2 and 3, and L1 is dpa in control complex 1, were prepared and characterized and their photocytotoxicity was studied. Complexes 2 and 3 were developed as potential substitutes for zinc(ii)-porphyrins/phthalocyanines that are photodynamic therapeutic agents with moderate activity owing to their inherent hydrophobicity and aggregation-induced deactivation mechanism. In our approach, we strategically designed hybrid inorganic-organic zinc-BODIPY conjugates as theranostic photosensitizers. The structurally characterized diamagnetic Zn(ii) cis-dichlorido complexes mimic cisplatin and serve as new-generation photosensitizers with enhanced aqueous solubility and mito-DNA targeting propensity while imparting significant physiological stability to the heavy atom tethered BODIPY ligand, L3. The BODIPY complexes showed a visible band near 500 nm (ε∼ 34 000-44 000 dm3 mol-1 cm-1) and an emission band at 507 nm for 2 in 1% DMSO-Dulbecco's phosphate buffered saline. The labile chlorido ligands (ΛM∼ 200 S m2 mol-1 in 9 : 1 H2O-DMSO) generated positively charged complexes inside the cellular medium enabling them to cross the mitochondrial membrane for this organelle-selective localization and singlet oxygen-mediated apoptotic photocytotoxicity at nanomolar concentrations for 3 in HeLa and MCF-7 cells in light (400-700 nm), while being less active in the dark.

Supramolecular Cylinders Target Bulge Structures in the 5' UTR of the RNA Genome of SARS-CoV-2 and Inhibit Viral Replication.

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5' UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents.

Rotaxanating Metallo-supramolecular Nano-cylinder Helicates to Switch DNA Junction Binding.

A class of rotaxane is created, not by encapsulating a conventional linear thread, but rather by wrapping a large cucurbit[10]uril macrocycle about a three-dimensional, cylindrical, nanosized, self-assembled supramolecular helicate as the axle. The resulting pseudo-rotaxane is readily converted into a proper interlocked rotaxane by adding branch points to the helicate strands that form the surface of the cylinder (like branches and roots on a tree trunk). The supramolecular cylinder that forms the axle is itself a member of a unique and remarkable class of helicate metallo-drugs that bind Y-shaped DNA junction structures and induce cell death. While pseudo-rotaxanation does not modify the DNA-binding properties, proper, mechanically-interlocked rotaxanation transforms the DNA-binding and biological activity of the cylinder. The ability of the cylinder to de-thread from the rotaxane (and thus to bind DNA junction structures) is controlled by the extent of branching: fully-branched cylinders are locked inside the cucurbit[10]uril macrocycle, while cylinders with incomplete branch points can de-thread from the rotaxane in response to competitor guests. The number of branch points can thus afford kinetic control over the drug de-threading and release.

Recent advances in uranyl binding in proteins thanks to biomimetic peptides.

Uranium is an element belonging to the actinide series. It is ubiquitous in rock, soil, and water. Uranium is found in the ecosystem due to mining and milling industrial activities and processing to nuclear fuel, but also to the extensive use of phosphate fertilizers. Understanding uranium binding in vivo is critical, first to deepen our knowledge of molecular events leading to chemical toxicity, but also to provide new mechanistic information useful for the development of efficient decorporation treatments to be applied in case of intoxication. The most stable form in physiological conditions is the uranyl cation (UO22+), in which uranium oxidation state is +VI. This short review presents uranyl coordination properties and chelation, and what is currently known about uranium binding to proteins. Although several target proteins have been identified, the UO22+ binding sites have barely been identified. Biomimetic approaches using model peptides are good options to shed light on high affinity uranyl binding sites in proteins. A strategy based on constrained cyclodecapeptides allowed recently to propose a tetraphosphate binding site for uranyl that provides an affinity similar to the one measured with the phosphoprotein osteopontin.

Ruthenium(II) Conjugates of Boron-Dipyrromethene and Biotin for Targeted Photodynamic Therapy in Red Light.

The ruthenium(II) complexes [RuCl(L1)(L3)]Cl (1), [RuCl(L1)(L4)]Cl (2), [RuCl(L2)(L4)]Cl (3), [RuCl(L1)(L5)]Cl (4), and [RuCl(L2)(L5)]Cl (5) of NNN-donor dipicolylamine (dpa) bases (L4, L5) having BODIPY (boron-dipyrromethene) moieties, NN-donor phenanthroline derivatives (L1, L2), and benzyldipicolylamine (bzdpa, L3) were prepared and characterized by spectroscopic techniques and their cellular localization/uptake and photocytotoxicity studied. Complex 1, as its PF6 salt (1a), has been structurally characterized with help of a single-crystal X-ray diffraction technique. It has a RuN5Cl core with the Cl bonded trans to the amine nitrogen atom of bzdpa. The complexes showed intense absorption spectral bands near 500 nm (ε ≈ 58000 M-1 cm-1) in 2 and 3 and 654 nm (ε ≈ 80000 M-1 cm-1) in 4 and 5 in 1/1 DMSO/DPBS (v/v). Complex 5 having biotin and PEGylated-disteryl BODIPY gave a singlet oxygen quantum yield (ΦΔ) of ∼0.65 in DMSO. Complex 5 exhibited remarkable PDT (photodynamic therapy) activity (IC50 ≈ 0.02 µM) with a photocytotoxicity index (PI) value of >5000 in red light of 600-720 nm in A549 cancer cells. The biotin-conjugated complexes showed better photocytotoxicity in comparison to nonbiotinylated analogues in A549 cells. The complexes displayed less toxicity in HPL1D normal cells in comparison to A549 cancer cells. The emissive BODIPY complexes 3 and 5 (ΦF ≈ 0.07 in DMSO) showed significant mitochondrial localization.

Crystal structure, DNA crosslinking and photo-induced cytotoxicity of oxovanadium(IV) conjugates of boron-dipyrromethene.

Cis-dichloro-oxovanadium(IV) complexes [VO(L1/L2)Cl2], where L1 is N-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4ʎ4,5ʎ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine in 1 and L2 is N-(4-(5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-5H-4ʎ4,5ʎ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine in 2) having 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene as boron-dipyrromethene (BODIPY) appended dipicolylamine bases were prepared, characterized and their photocytotoxicity studied. X-ray crystal structure of 1 showed distorted octahedral geometry with a VIVON3Cl2 core having Cl-V-Cl angle of 91.93(4)°. The complexes showed variable solution conductivity properties. They were non-electrolytes in dry DMF at 25 °C but showed 1:1 electrolytic behavior in an aqueous medium due to dissociation of one chloride ligand as evidenced from the mass spectral study. Complexes 1 and 2 showed absorption bands at 500 and 535 nm, respectively. The calf thymus DNA melting study revealed their interaction through DNA crosslinking on exposure to light which was further confirmed from the alkaline agarose gel electrophoresis using plasmid supercoiled pUC19 DNA. Complex 2 showed disruption of the mitochondrial membrane potential in the JC-1 (1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloroimidacarbocyanine iodide) assay. The complexes were photocytotoxic in visible light (400-700 nm, power: 10 J cm-2) in cervical cancer HeLa and breast cancer MCF-7 cells. Complex 2 having a photoactive diiodo­boron-dipyrromethene moiety gave a singlet oxygen quantum yield (ΦΔ) value of ~0.6. It showed singlet oxygen mediated apoptotic photodynamic therapy activity with remarkably low IC50 (half maximal inhibitory concentration) value of ~0.15 µM. The cis-disposition of chlorides gave a cis-divacant 4-coordinate intermediate structure from the density functional theory (DFT) study thus mimicking the DNA crosslinking property of cisplatin.

Mitochondria-localizing BODIPY-copper(ii) conjugates for cellular imaging and photo-activated cytotoxicity forming singlet oxygen.

Copper(ii) acetylacetonates of N,N,N-donor dipicolylamine (dpa) ligands, viz. [Cu(L1)(acac)]ClO4 (1), [Cu(L2)(acac)]ClO4 (2) and [Cu(L3)(acac)]ClO4 (3), where L1 is benzyldipicolylamine (bzdpa), L2 and L3 are non-iodinated and diiodinated BODIPY (borondipyrromethene) ligands and Hacac is acetylacetone, were synthesized and characterized and their photocytotoxicity was studied. The BODIPY complex 2, structurally characterized by X-ray crystallography, has copper(ii) in a distorted square-pyramidal geometry (degree of trigonality, τ5 = 0.28). The one-electron paramagnetic and redox active copper(ii) complexes displayed 1 : 1 electrolytic behaviour in polar organic solvents. The BODIPY complexes 2 and 3 showed respective visible bands at 498 and 539 nm in 5% DMSO-phosphate buffered saline (PBS). Complex 2 displayed an emission band at 511 nm in 5% DMSO-PBS (λex = 465 nm) with a fluorescence quantum yield (ΦF) value of 0.15. Cellular imaging using this complex showed significant mitochondrial localization in HeLa and MCF-7 cancer cells. Complex 3 with a diiodo-BODIPY moiety was non-emissive (ΦF = 0.01) but acted as an efficient photosensitizer with a singlet oxygen quantum yield value of 0.59 (ΦΔ). Complex 3 showed a remarkable PDT effect with apoptotic cell death due to singlet oxygen giving IC50 values within 0.04-0.06 µM in HeLa and MCF-7 cells using visible light (400-700 nm), while being less toxic in the dark.

Glucose-Appended Platinum(II)-BODIPY Conjugates for Targeted Photodynamic Therapy in Red Light.

Platinum(II) complexes [Pt(L1)(R-BODIPY)]Cl (1) and [Pt(L2)(R-BODIPY)]Cl (2), where R-BODIPY is 8-(4-ethynylphenyl)-distyryl-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3, L1 is 4'-phenyl-2,2':6',2″-terpyridine, and L2 is (2,2':6',2″-terpyridin-4'-oxy)ethyl-ß-d-glucopyranoside, were synthesized and characterized, and their photocytotoxicity was studied. The phenylacetylide complex [Pt(L1)(C≡CPh)]Cl (3) was prepared and used as a control. Complexes 1 and 2 showed near-IR absorption bands at 713 nm (ε = 3.47 × 104 M-1 cm-1) and 715 nm (3.2 × 104 M-1 cm-1) in 10% dimethyl sulfoxide (DMSO)-Dulbecco's Modified Eagle's Medium (DMEM) (pH 7.2). The BODIPY complexes are emissive in 10% DMSO-DMEM at pH 7.2 with λem (λex, Φf) = 822 nm (710 nm, 0.022) for complex 1 and λem (λex, Φf) = 825 nm (710 nm, 0.026) for complex 2. They generated singlet oxygen (1O2) in red light as evidenced from 1,3-diphenylisobenzofuran (DPBF) titration experiments. The singlet oxygen quantum yield (ΦΔ) values for 1 and 2 were ∼0.6 signifying their photosensitizing ability. They were remarkably photodynamic therapy (PDT) active in red light showing significant red light-induced cytotoxicity in cervical HeLa, lung cancer A549, and breast cancer MCF-7 cells (IC50: 2.3-24.7 µM in light) with negligible dark toxicity (IC50 > 100 µM). A significant enhancement in cellular uptake was observed for 2 having glucose-appended terpyridine ligand compared to 1. The confocal microscopy showed significant mitochondrial localization of the complexes as evidenced from the JC-1 assay. The complexes released the photoactive R-BODIPY ligand upon red light-irradiation as evidenced from the mass and 1H NMR spectral studies. Complex 2 is remarkable in satisfying the essential requirements of targeted PDT in red light.

Photochemotherapy of Infrared Active BODIPY-Appended Iron(III) Catecholates for in Vivo Tumor Growth Inhibition.

Iron(III) catecholates of BODIPY (boron-dipyrromethene)-conjugated dipicolylamine ligands, viz. [Fe(L1)(cat)Cl] (1) and [Fe(L2)(cat)Cl] (2) (H2cat = catechol), with a ligand-to-metal charge transfer band at ∼800 nm were studied for their in vivo activity in dark and infrared light in luciferase-expressing human breast adenocarcinoma (BT474luc) cells. Complex 2 displayed in vitro photocytotoxicity in BT474luc cells (IC50: 6 µM) in infrared light of 600-720 nm with moderate dark toxicity (IC50: 18 µM) as evidenced from the MTT and Annexin-V FITC/PI staining assays. The mitochondria-localizing complexes showed apoptotic cell death involving reactive oxygen species whose generation was evidenced from 2,7-dichlorofluorescein diacetate assay. In vivo studies showed tumor growth inhibition in mice with an optimized complex 2 dose of 5 mg per kg body weight on exposure to infrared light of 685 nm (dose of 20 mW/cm2). The in vivo results exemplify complex 2 as a unique iron-based infrared-active photochemotherapeutic agent.

Curcumin "Drug" Stabilized in Oxidovanadium(IV)-BODIPY Conjugates for Mitochondria-Targeted Photocytotoxicity.

Ternary oxidovanadium(IV) complexes of curcumin (Hcur), dipicolylamine (dpa) base, and its derivatives having pendant noniodinated and di-iodinated boron-dipyrromethene (BODIPY) moiety (L1 and L2, respectively), namely, [VO(dpa)(cur)]ClO4 (1), [VO(L1)(cur)]ClO4 (2), and [VO(L2)(cur)]ClO4 (3) and their chloride salts (1a-3a) were prepared, characterized, and studied for anticancer activity. The chloride salts were used for biological studies due to their aqueous solubility. Complex 1 was structurally characterized by single-crystal X-ray crystallography. The complex has a VO2+ moiety bound to dpa ligand showing N,N,N-coordination in a facial mode, and curcumin is bound in its mono-anionic enolic form. The V-O(cur) distances are 1.950(18) and 1.977(16) Å, while the V-N bond lengths are 2.090(2), 2.130(2), and 2.290(2) Å. The bond trans to V═O is long due to trans effect. The complexes are stable in a solution phase over a long period of time of 48 h without showing any apparent degradation of the curcumin ligand. The diiodo-BODIPY ligand (L2) or Hcur alone showed limited solution stability in dark. The emissive BODIPY (L1) containing complex 2a showed preferential mitochondrial localization in MCF-7 cells in cellular imaging experiments. The cytotoxicity of the complexes was studied by MTT assay. The BODIPY complex 3a showed excellent photodynamic therapy effect in visible light (400-700 nm) giving IC50 values of 2-6 µM in HeLa and MCF-7 cancer cells, while being less toxic in dark (∼100 µM). The cell death was apoptotic in nature involving reactive oxygen species (ROS). Mechanistic data from pUC19 DNA photocleavage studies revealed photogenerated ROS as primarily 1O2 from the BODIPY moiety and ·OH radicals from the curcumin ligand.

Monofunctional BODIPY-Appended Imidazoplatin for Cellular Imaging and Mitochondria-Targeted Photocytotoxicity.

Monofunctional platinum(II) complexes of formulation cis-[Pt(NH3)2(L)Cl](NO3), where L is an imidazole base conjugated to 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) with emissive (L1 in 1) and nonemissive (L2 in 2) moieties were prepared and characterized, and their singlet oxygen-mediated photoinduced cytotoxicity was studied. The 1-methylimidazole (1-MeIm) complex 3 was prepared as a control and for structural characterization by X-ray crystallography. Complexes 1 and 2 showed strong visible absorption bands at 500 nm (ε = 2.7 × 104 M-1 cm-1) and 540 nm (1.4 × 104 M-1 cm-1). Complex 1 is emissive with a band at 510 nm (ΦF = 0.09) in 1% dimethyl sulfoxide/Dulbecco's Modified Eagle's Medium (pH 7.2). Singlet oxygen generation upon photoirradiation with visible light (400-700 nm) was evidenced from 1,3-diphenylisobenzofuran titration experiments showing significant photosensitizing ability of the BODIPY complexes. Both 1 and 2 were remarkably photocytotoxic in visible light (400-700 nm, 10 J cm-2) in skin keratinocyte HaCaT and breast cancer MCF-7 cells giving IC50 values in nanomolar concentration. The complexes were, however, essentially nontoxic to the cells in the dark (IC50 > 80 µM). Complex 2 having a diiodo-BODIPY unit is nonemissive but an efficient photosensitizer with high singlet oxygen generation ability in visible light (400-700 nm). Confocal microscopy using the emissive complex 1 showed significant mitochondrial localization of the complex. Cell death via apoptotic pathway was observed from the Annexin-V-FITC/PI assay. The formation of Pt-DNA adducts was evidenced from the binding experiments of the complexes 1 and 2 with 9-ethylguanine as a model nucleobase from 1H NMR and mass spectral studies.

Photorelease and Cellular Delivery of Mitocurcumin from Its Cytotoxic Cobalt(III) Complex in Visible Light.

Ternary cobalt(III) complexes of curcumin (Hcur) and mitocurcumin [Hmitocur, a dicationic bis(triphenylphosphonium) derivative of curcumin] having a tetradentate phenolate-based ligand (H2L), namely, [Co(cur)(L)] (1) and [Co(mitocur)(L)]Cl2 (2), were prepared and structurally characterized, and their photoinduced cytotoxicity was studied. The diamagnetic cobalt(III) complexes show an irreversible Co(III)-Co(II) redox response and a quasireversible curcuminoid-based reduction near -1.45 and -1.74 V SCE, respectively, in DMF/0.1 M [(n)Bu4N](ClO4). The complexes exhibit a curcumin/mitocurcumin-based absorption band near 420 nm. Complex 1 was structurally characterized by X-ray crystallography. The structure contains the metal in a CoN2O4 distorted octahedral coordination arrangement with curcumin binding to the metal in its enolic form. Binding to cobalt(III) increases the hydrolytic stability of curcumin. Complex 2, having a dicationic curcuminoid, shows significant cellular uptake and photoinduced cytotoxicity compared to its curcumin analogue 1. The dicationic cobalt(III) complex 2 has significantly better cellular uptake and bioactivity than the neutral species 1. Complex 2 with mitochondrial localization releases the mitocurcumin dye upon exposure to visible light (400-700 nm) in human breast cancer MCF-7 cells through photoreduction of cobalt(III) to cobalt(II). Complex 2 displays a remarkable photodynamic therapy (PDT) effect, giving an IC50 value of ∼3.9 µM in visible light (400-700 nm) in MCF-7 cells while being much less toxic in the dark (>50 µM). The released mitocurcumin acts as a phototoxin, generating intracellular reactive oxygen species (ROSs). The overall process leads to light-controlled delivery of a curcuminoid (mitocur) into the tumor cells while the dye alone suffers from hydrolytic instability and poor bioavailability.