Cell-penetrating peptide-conjugated copper complexes for redox-mediated anticancer therapy.

Metal-based chemotherapeutics like cisplatin are widely employed in cancer treatment. In the last years, the design of redox-active (transition) metal complexes, such as of copper (Cu), has attracted high interest as alternatives to overcome platinum-induced side-effects. However, several challenges are still faced, including optimal aqueous solubility and efficient intracellular delivery, and strategies like the use of cell-penetrating peptides have been encouraging. In this context, we previously designed a Cu(II) scaffold that exhibited significant reactive oxygen species (ROS)-mediated cytotoxicity. Herein, we build upon the promising Cu(II) redox-active metallic core and aim to potentiate its anticancer activity by rationally tailoring it with solubility- and uptake-enhancing functionalizations that do not alter the ROS-generating Cu(II) center. To this end, sulfonate, arginine and arginine-rich cell-penetrating peptide (CPP) derivatives have been prepared and characterized, and all the resulting complexes preserved the parent Cu(II) coordination core, thereby maintaining its reported redox capabilities. Comparative in vitro assays in several cancer cell lines reveal that while specific solubility-targeting derivatizations (i.e., sulfonate or arginine) did not translate into an improved cytotoxicity, increased intracellular copper delivery via CPP-conjugation promoted an enhanced anticancer activity, already detectable at short treatment times. Additionally, immunofluorescence assays show that the Cu(II) peptide-conjugate distributed throughout the cytosol without lysosomal colocalization, suggesting potential avoidance of endosomal entrapment. Overall, the systematic exploration of the tailored modifications enables us to provide further understanding on structure-activity relationships of redox-active metal-based (Cu(II)) cytotoxic complexes, which contributes to rationalize and improve the design of more efficient redox-mediated metal-based anticancer therapy.

Synthesis and In Vitro Studies of Photoactivatable Semisquaraine-type Pt(II) Complexes.

The synthesis, full characterization, photochemical properties, and cytotoxic activity toward cisplatin-resistant cancer cell lines of new semisquaraine-type Pt(II) complexes are presented. The synthesis of eight semisquaraine-type ligands has been carried out by means of an innovative, straightforward methodology. A thorough structural NMR and X-ray diffraction analysis of the new ligands and complexes has been done. Density functional theory calculations have allowed to assign the trans configuration of the platinum center. Through the structural modification of the ligands, it has been possible to synthesize some complexes, which have turned out to be photoactive at wavelengths that allow their activation in cell cultures and, importantly, two of them show remarkable solubility in biological media. Photodegradation processes have been studied in depth, including the structural identification of photoproducts, thus justifying the changes observed after irradiation. From biological assessment, complexes C7 and C8 have been demonstrated to behave as promising photoactivatable compounds in the assayed cancer cell lines. Upon photoactivation, both complexes are capable of inducing a higher cytotoxic effect on the tested cells compared with nonphotoactivated compounds. Among the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells, and C8 showed a PI > 40 in A2780 cells, being also effective over cisplatin-resistant A2780cis cells (PI = 7 and PI = 4, respectively). The mechanism of action of these complexes has been studied, revealing that these photoactivated platinum complexes would actually present a combined mode of action, a therapeutically potential advantage.

Functionalized azobenzene platinum(II) complexes as putative anticancer compounds.

The synthesis and characterization of four platinum(II) complexes using azobenzenes conveniently functionalized as ligands has been carried out. The characteristic photochemical behavior of the complexes due to the presence of azobenzene-type ligands and the role of the ligands in the activation of the complexes has been studied. Their promising cytotoxicity observed in HeLa cells prompted us to study the mechanism of action of these complexes as cytostatic agents. The interaction of the compounds with DNA, studied by circular dichroism, revealed a differential activity of the Pt(II) complexes upon irradiation. The intercalation abilities of the complexes as well as their reactivity with common proteins present in the blood stream allows to confirm some of the compounds obtained as good anticancer candidates.

Copper(II) N,N,O-Chelating Complexes as Potential Anticancer Agents.

Three novel dinuclear Cu(II) complexes based on a N,N,O-chelating salphen-like ligand scaffold and bearing varying aromatic substituents (-H, -Cl, and -Br) have been synthesized and characterized. The experimental and computational data obtained suggest that all three complexes exist in the dimeric form in the solid state and adopt the same conformation. The mass spectrometry and electron paramagnetic resonance results indicate that the dimeric structure coexists with the monomeric form in solution upon solvent (dimethyl sulfoxide and water) coordination. The three synthesized Cu(II) complexes exhibit high potentiality as ROS generators, with the Cu(II)/Cu(I) redox potential inside the biological redox window, and thus being able to biologically undergo Cu(II)/Cu(I) redox cycling. The formation of ROS is one of the most promising reported cell death mechanisms for metal complexes to offer an inherent selectivity to cancer cells. In vitro cytotoxic studies in two different cancer cell lines (HeLa and MCF7) and in a normal fibroblast cell line show promising selective cytotoxicity for cancer cells (IC50 about 25 µM in HeLa cells, which is in the range of cisplatin and improved with respect to carboplatin), hence placing this N,N,O-chelating salphen-like metallic core as a promising scaffold to be explored in the design of future tailor-made Cu(II) cytotoxic compounds.

Studying the reactivity of "old" Cu(II) complexes for "novel" anticancer purposes.

Reactive oxygen species (ROS) formation appears as one of the most promising pathways to induce cell death. The interesting Cu(II)/Cu(I) redox pair has been reported to biologically generate ROS and induce cell damage. Simple metal complexes, such as cisplatin, sometimes offer even better properties than others highly accurately synthesized, which imply considerable time and economical efforts. This work relies on the synthesis and characterisation of four existing Cu(II) complexes bearing N-donor ligands, previously used for a totally different intend, but tested now for anticancer purposes. Furthermore, a relationship between their coordinating features, i.e. their redox behaviour, with their biological activity have been inferred to further understand the medicinal role of the Cu(II)/Cu(I) redox pair. Cytotoxicity studies and interactions towards DNA have been assessed, studying both covalent and non-covalent modes of binding via mass spectrometry (MS), UV-Vis and fluorescence, evaluating the cleaving properties of the assayed compounds, as well as their capacity to generate ROS inside the cells. The role of the ligand for one of the complexes has been evaluated by a computational approach. The idea of using "old" complexes for "novel" anticancer purposes can offer promising results in the future, being a simple but interesting approach to study, as we demonstrate here for most of the complexes analysed, showing a non-expected "new" and beneficial role.

Squaramide-Based Pt(II) Complexes as Potential Oxygen-Regulated Light-Triggered Photocages.

Two new squaramide-based platinum(II) complexes C1 and C2 have been synthesized and fully characterized. Their photoresponse has been assessed and is discussed. A remarkable enhancement in the DNA binding activity has been observed for both complexes, up on irradiation. For C2, the release of Pt(II) provoked by its irradiation has been studied. The response of C2 has been found to be regulated by the presence of oxygen. In vitro cytotoxicity tests show an enhancement in the activity of complex C2 after selective irradiation under hypoxic conditions. Resulting Pt(II) species have been isolated and characterized by various analytical methods establishing this type of squaramido-based complexes as a proof of concept for new Pt(II) photocages.

Recent advances in the synthesis of azaphenalene alkaloids: first enantioselective approaches.

Azaphenalene alkaloids are biosynthesised and segregated by diverse insects of the Coccinellidae family (ladybirds) and are believed to play an important role in the defensive mechanism against their natural predators. The particular unique framework of these alkaloids, along with their potential in the field of biological pest control, has led to several research groups developing synthetic sequences to prepare these compounds. The main purpose of the present review is to provide an update of the more recent synthetic progress towards these alkaloids, including the pioneering enantioselective approaches to chiral congeners.

Iterative Synthetic Strategy for Azaphenalene Alkaloids. Total Synthesis of (-)-9a epi-Hippocasine.

A new strategy for the stereoselective synthesis of alkaloids with perhydro-9b-azaphenalene skeleton has been developed. The starting material is the substituted glutarimide derivative 1, readily available in either enantiomeric form through the palladium-catalyzed asymmetric allylic alkylation of glutarimide. The strategy relies on an iterative methodology encompassing two nucleophilic allylations and two ring closing metathesis processes. The approach has been used in the first synthesis of (-)-9a- epi-hippocasine.

Reprint of "Anticancer activity of hydroxy- and sulfonamide-azobenzene platinum(II) complexes in cisplatin-resistant ovarian cancer cells".

The syntheses of three platinum(II) complexes bearing sulfonamide-((E)-2-(4-methylphenylsulfonamido)-2',6'-difluoroazobenzene, HL1) and hydroxy-azo-2,6-difluorobenzene ((E)-2-((2,6-difluorophenyl)diazenyl)phenol, HL2) bidentate ligands is described. These complexes, [Pt(L1)(DMSO)Cl] (1), [Pt(L2)(DMSO)Cl] (2), and [Pt(L2)2] (3), were characterized by multinuclear NMR spectroscopy, mass spectrometry, and X-ray crystallography. Despite bearing azobenzene functional groups, none of the three complexes undergo photoisomerization. The anticancer activities of these complexes were evaluated in wild-type (A2780) and cisplatin-resistant (A2780CP70) ovarian cancer cells. All three complexes exhibited IC50 values below 10µM and displayed similar activity in both A2780 and A2780CP70 cell lines, indicating that they are not cross-resistant with cisplatin. The DNA-binding properties of 1-3 were investigated by circular dichroism spectroscopy and by agarose gel electrophoresis. Both studies suggest that 1 and 2 form monofunctional DNA adducts.

Anticancer activity of hydroxy- and sulfonamide-azobenzene platinum(II) complexes in cisplatin-resistant ovarian cancer cells.

The syntheses of three platinum(II) complexes bearing sulfonamide- ( (E)-2-(4-methylphenylsulfonamido)-2',6'-difluoroazobenzene, HL1) and hydroxy-azo-2,6-difluorobenzene ((E)-2-((2,6-difluorophenyl)diazenyl)phenol, HL2) bidentate ligands is described. These complexes, [Pt(L1)(DMSO)Cl] (1), [Pt(L2)(DMSO)Cl] (2), and [Pt(L2)2] (3), were characterized by multinuclear NMR spectroscopy, mass spectrometry, and X-ray crystallography. Despite bearing azobenzene functional groups, none of the three complexes undergo photoisomerization. The anticancer activities of these complexes were evaluated in wild-type (A2780) and cisplatin-resistant (A2780CP70) ovarian cancer cells. All three complexes exhibited IC50 values below 10µM and displayed similar activity in both A2780 and A2780CP70 cell lines, indicating that they are not cross-resistant with cisplatin. The DNA-binding properties of 1-3 were investigated by circular dichroism spectroscopy and by agarose gel electrophoresis. Both studies suggest that 1 and 2 form monofunctional DNA adducts.

How a diversity-oriented approach has inspired a new hypothesis for the gabosine biosynthetic pathway. A new synthesis of (+)-gabosine C.

A new synthesis of (+)-gabosine C has been accomplished as part of a general diversity-oriented approach that also delivered the previously unknown (-)-4-epi-gabosine C. The identification of the unexpected intermediate (+)-8, together with the isolation of ketones 9 and 10 in previous investigations, prompted us to formulate a new hypothesis for the biosynthesis of gabosines, based on a keto-enol equilibrium cascade pathway starting from 2-epi-5-epi-valiolone, along which the necessary precursors for all the different types of gabosines are generated.

Stereoselective synthesis and relative configuration assignment of gabosine J.

The first total synthesis of (+)-gabosine J and that of the epimer at C4 of its enantiomer have been accomplished through an enantioselective approach from a common intermediate 1. These syntheses have allowed us to establish the correct relative configuration of the natural metabolite, which was originally misassigned. This work, together with our former syntheses of other gabosines and related compounds, validates enone 1 as a general synthetic precursor for this kind of carbasugars.

Enantioselective approach to Securinega alkaloids. Total synthesis of securinine and (-)-norsecurinine.

The most representative securinega alkaloids have been synthesized through a new strategy involving the palladium-catalyzed enantioselective allylation of a cyclic imide, a vinylogous Mannich reaction, and a ring-closing metathesis process, as the key steps. The diastereoselectivity of the vinylogous Mannich reaction was in partial agreement with DFT theoretical calculations performed in a model system. The synthesis of (-)-norsecurine has been accomplished in nine steps from succinimide and 14% overall yield and that of securinine in 10 steps from glutarimide and 20% overall yield. Both syntheses compare favorably with those previously described. The three key transformations have been performed in a synthetically useful scale (more than 500 mg). Moreover, since the enantioselectivity was originated by a chiral phosphine ligand, the antipode of which is readily available, the same route is expected to give access to (+)-norsecurinine and virosecurinine.

Diastereoselective synthesis of allosecurinine and viroallosecurinine from menisdaurilide.

A new and versatile synthetic route to Securinega alkaloids is reported. The first synthesis of allosecurinine has been accomplished in seven steps and 40% yield, starting from (+)-menisdaurilide, using a vinylogous Mannich reaction as the key transformation. Similarly, viroallosecurinine has been synthesized from (-)-menisdaurilide.

An efficient protocol for the enantioselective preparation of a key polyfunctionalized cyclohexane. New access to (R)- and (S)-4-Hydroxy-2-cyclohexenone and (R)- and (S)-trans-cyclohex-2-ene-1,4-diol.

Starting from very accessible raw materials such as p-methoxyphenol, ethylene glycol, and thiophenol, a protocol has been developed to prepare multigram quantities of the polyfunctionalized cyclohexane (+/-)- 7. A highly efficient resolution of (+/-)- 7 has been achieved through enantioselective acetylation catalyzed by Candida antarctica lipase B. Straightforward and enantioselective syntheses of 4-hydroxy-2-cyclohexenone, 1, trans-cyclohex-2-ene-1,4-diol, 2, and their O-protected derivatives 18 and 19 have been readily accomplished from 7.

Enantioselective synthesis and absolute configuration assignment of gabosine O. Synthesis of (+)- and (-)-gabosine N and (+)- and (-)-epigabosines N and O.

[reaction: see text] A rational approach to the synthesis of gabosines and other related carba-sugars starting from a masked p-benzoquinone has been designed. The enantioselective acetylation of the hydroxyketal 2 provides a practical entry to either enantiomer of the target products. The strategy has been applied to the synthesis of (+)- and (-)-gabosines N and O and (+)- and (-)-epigabosines N and O. The absolute configuration of natural gabosine O has been established.

An effective enantioselective approach to the securinega alkaloids: total synthesis of (-)-norsecurinine.

[reaction: see text] A highly versatile approach to the enantioselective synthesis of securinega alkaloids is presented. Crucial steps are a palladium-catalyzed enantioselective imide alkylation, a vinylogous Mannich reaction, and a ring-closing metathesis process. Through this strategy, the synthesis of (-)-norsecurinine has been accomplished in nine steps and 11% overall yield.

Optically active aromatic and heteroaromatic alpha-amino acids by a one-pot catalytic enantioselective addition of aromatic and heteroaromatic C-H bonds to alpha-imino esters.

The development of a practical one-pot catalytic enantioselective procedure for the synthesis of non-natural aromatic and heteroaromatic alpha-amino acids is reported. Starting from readily available starting materials and application of a chiral BINAP-Cu(I) catalyst, the optically active products are formed with readily removable N-protecting groups. The scope of the reaction is demonstrated by the addition of substituted furans, thiophenes, pyrroles, and aromatic compounds to N-alkoxycarbonyl-alpha-imino esters in good yields and with enantioselectivities up to 96% ee for the furans, 93% ee for the thiophenes, and 98% for the aromatic compounds. The protecting groups are readily removed, and various transformations of the aromatic and heteroaromatic alpha-amino acids are demonstrated. The coordination of the N-alkoxycarbonyl alpha-imino ester to the chiral BINAP-Cu(I) complex and the enantioselectivity of the catalyst is discussed on the basis of the DFT calculations and X-ray crystallographic data.