Novel Foscan®-derived ring-fused chlorins for photodynamic therapy of cancer.

Photodynamic therapy (PDT) is an established anticancer treatment that combines the use of a photosensitiser (PS) and a light source of a specific wavelength for the generation of reactive oxygen species (ROS) that are toxic to the tumour cells. Foscan® (mTHPC) is a clinically-approved chlorin used for the PDT treatment of advanced head and neck, prostate and pancreatic cancers but is characterized by being photochemically unstable and associated with prolonged skin photosensitivity. Herein, we report the synthesis of new 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins, having the meso-tetra(3-hydroxyphenyl)macrocycle core of mTHPC, by exploring the [8π + 2π] cycloaddition of a meso-tetra(3-hydroxyphenyl)porphyrin derivative with diazafulvenium methides. These chlorins have photochemical properties similar to Foscan® but are much more photostable. Among the novel compounds, two chlorins with a hydroxymethyl group and its azide derivative present in the 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused system, are promising photodynamic agents with activity in the 100 nM range against triple-negative breast cancer cells and, in the case of azidomethyl chlorin, a safer phototherapeutic index compared to Foscan®.

Differential Tunneling-Driven and Vibrationally-Induced Reactivity in Isomeric Benzazirines.

Quantum mechanical tunneling of heavy-atoms and vibrational excitation chemistry are unconventional and scarcely explored types of reactivity. Once fully understood, they might bring new avenues to conduct chemical transformations, providing access to a new world of molecules or ways of exquisite reaction control. In this context, we present here the discovery of two isomeric benzazirines exhibiting differential tunneling-driven and vibrationally-induced reactivity, which constitute exceptional results for probing into the nature of these phenomena. The isomeric 6-fluoro- and 2-fluoro-4-hydroxy-2H-benzazirines (3-a and 3'-s) were generated in cryogenic krypton matrices by visible-light irradiation of the corresponding triplet nitrene 3 2-a, which was produced by UV-light irradiation of its azide precursor. The 3'-s was found to be stable under matrix dark conditions, whereas 3-a spontaneously rearranges (τ1/2 ∼64 h at 10 and 20 K) by heavy-atom tunneling to 3 2-a. Near-IR-light irradiation at the first OH stretching overtone frequencies (remote vibrational antenna) of the benzazirines induces the 3'-s ring-expansion reaction to a seven-member cyclic ketenimine, but the 3-a undergoes 2H-azirine ring-opening reaction to triplet nitrene 3 2-a. Computations demonstrate that 3-a and 3'-s have distinct reaction energy profiles, which explain the different experimental results. The spectroscopic direct measurement of the tunneling of 3-a to 3 2-a constitutes a unique example of an observation of a species reacting only by nitrogen tunneling. Moreover, the vibrationally-induced sole activation of the most favorable bond-breaking/bond-forming pathway available for 3-a and 3'-s provides pioneer results regarding the selective nature of such processes.

Photochromism of a Spiropyran in Low-Temperature Matrices: Unprecedented Bidirectional Switching between a Merocyanine and an Allene Intermediate.

Photochromism of spiropyrans has attracted much attention due to its potential in many light-controlled system applications. However, several fundamental aspects regarding the structure, energetics, and mechanistic details of the transformations of spiropyrans are still not well understood. Here, we report the study of the photochromism of a 6-hydroxy-spiropyran (HBPS) under conditions of matrix isolation, where monomers of the compound are frozen in a solidified noble gas (krypton, at 15 K). The structure of the matrix-isolated HBPS was first elucidated by infrared (IR) spectroscopy supported by density functional theory computations. Then, the photochromism of HBPS, from the colorless spiropyran to the colored merocyanine, was induced by ultraviolet (UV) irradiation at 310 nm. The analysis of the IR spectrum of the photoproduced species revealed the exclusive formation of the most stable merocyanine MC-TTC stereoisomer. Subsequent visible-light (550 nm) irradiation of MC-TTC generated a new colorless allenic isomeric species ALN, where the UV irradiation (310 nm) of ALN was found to convert this species back to MC-TTC. This constitutes an unprecedented bidirectional transformation between a colored merocyanine and a colorless allene species. The newly observed photoswitching reaction (or photochromism) occurs along an intramolecular hydrogen bond existing in both merocyanine and allenic species, thus suggesting that it might be generally feasible in the chemistry of spiropyrans. On the other hand, the usual assumption that, as a general rule, merocyanines photochemically revert to spiropyrans is not supported in this work.

Switching on H-Tunneling through Conformational Control.

H-tunneling is a ubiquitous phenomenon, relevant to fields from biochemistry to materials science, but harnessing it for mastering the manipulation of chemical structures still remains nearly illusory. Here, we demonstrate how to switch on H-tunneling by conformational control using external radiation. This is outlined with a triplet 2-hydroxyphenylnitrene generated in an N2 matrix at 10 K by UV-irradiation of an azide precursor. The anti-orientation of the nitrene's OH moiety was converted to syn by selective vibrational excitation at the 2ν(OH) frequency, thereby moving the H atom closer to the vicinal nitrene center. This triggers spontaneous H-tunneling to a singlet 6-imino-2,4-cyclohexadienone. Computations reveal that such fast H-tunneling occurs through crossing the triplet-to-singlet potential energy surfaces. Our experimental realization provides an exciting novel strategy to attain control over tunneling, opening new avenues for directing chemical transformations.

Evidence of IR-Induced Chemistry in a Neat Solid: Tautomerization of Thiotropolone by Thermal, Electronic, and Vibrational Excitations.

Thiotropolone isolated in argon and xenon matrices (as monomers) or in a neat solid (as the crystalline or amorphous state) at low temperature was found to exist only in the thione-enol form. Visible light irradiation (λ ≥ 400 nm) leads to thione-enol → thiol-keto tautomerization in matrices and under neat solid conditions at 15 K. The assignment of the IR spectra of the two thiotropolone tautomers (thione-enol and thiol-keto) was carried out with the support of B3LYP/6-311+G(2d,p) computations. The thiol-keto form generated in situ in a neat solid was found to tautomerize back to the thione-enol upon annealing up to 100 K. Gaussian-4 (G4) computations estimate that such a tautomerization process has an energy barrier of ∼25 kJ mol-1, which is consistent with the observations. Moreover, it was found that narrowband IR irradiation of the thiol-keto form in a neat solid, at the frequency of its CH stretching overtones/combination modes, also induces tautomerization to the thione-enol form. Such a result constitutes an important demonstration of vibrationally induced chemistry under neat solid conditions.

Synthesis of chiral hexacyclic steroids via [8π + 2π] cycloaddition of diazafulvenium methides.

First examples of [8π + 2π] cycloaddition of 16-dehydropregnenolone (16-DPA) acetate with diazafulvenium methides leading to chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused steroids are reported. These hexacyclic steroids were obtained exclusively or selectively with the approach of the 1,7-dipole by the less hindered α-face of 16-DPA. Quantum chemical calculations at the DFT level were carried out, using the cycloaddition of 1-methyl- and 1-benzyl-diazafulvenium methides with N-phenylmaleimide as model reactions, in order to rationalize the stereochemistry outcome. The results indicate that endo cycloadditions of the more stable dipole conformation, having the 1-substituent pointing outward, are significantly more favorable than the alternative exo cycloaddition.

Ring-Fused meso-Tetraarylchlorins as Auspicious PDT Sensitizers: Synthesis, Structural Characterization, Photophysics, and Biological Evaluation.

Novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused meso-tetraarylchlorins, with different degrees of hydrophilicity (with methyl ester, hydroxymethyl, and carboxylic acid moieties), have been synthesized and their photophysical characterization as well as in vitro photocytotoxicity assessment against human melanoma and esophageal and bladder carcinomas was carried out. An integrated analysis of the photosensitizers' performance, considering the singlet oxygen generation data, cell internalization, and intracellular localization, allowed to establish relevant structure-photoactivity relationships and the rationalization of the observed photocytotoxicity. In the diacid and monoalcohol series, chlorins derived from meso-tetraphenylporphyrin proved to be the most efficient photodynamic therapy agents, showing IC50 values of 68 and 344 nM against A375 cells, respectively. These compounds were less active against OE19 and HT1376 cells, the diacid chlorin with IC50 values still in the nano-molar range, whereas the monohydroxymethyl-chlorin showed significantly higher IC50 values. The lead di(hydroxymethyl)-substituted meso-tetraphenylchlorin confirmed its remarkable photoactivity with IC50 values below 75 nM against the studied cancer cell lines. Subcellular accumulation of this chlorin in the mitochondria, endoplasmic reticulum, and plasma membrane was demonstrated.

Isolation and Identification of Cytotoxic Compounds Present in Biomaterial Life®.

Direct pulp capping consists of a procedure in which a material is directly placed over the exposed pulp to maintain dental vitality. Although still widely used in clinical practice, previous in vitro studies found that the biomaterial Life® presented high cytotoxicity, leading to cell death. This study aimed to identify the Life® constituents responsible for its cytotoxic effects on odontoblast-like cells (MDPC-23). Aqueous medium conditioned with Life® was subjected to liquid-liquid extraction with ethyl acetate. After solvent removal, cells were treated with residues isolated from the organic and aqueous fractions. MTT and Trypan blue assays were carried out to evaluate the metabolic activity and cell death. The organic phase residue promoted a significant decrease in metabolic activity and increased cell death. On the contrary, no cytotoxic effects were observed with the mixture from the aqueous fraction. Spectroscopic and spectrometric methods allowed the identification of the toxic compounds. A mixture of the regioisomers ortho, para, and meta of N-ethyl-toluenesulfonamide was identified as the agent responsible for the toxicity of biomaterial Life® in MDPC-23 cells. These findings contribute to improving biomaterial research and development.

Inducing molecular reactions by selective vibrational excitation of a remote antenna with near-infrared light.

We demonstrate here that selective vibrational excitation of a moiety, remotely attached in relation to the molecular reaction site, might offer a generalized strategy for inducing bond-breaking/bond-forming reactions with exquisite precision. As a proof-of-principle, the electrocyclic ring-expansion of a benzazirine to a ketenimine was induced, in a cryogenic matrix, by near-IR light tuned at the overtone stretching frequency of its OH remote antenna. This accomplishment paves the way for harnessing IR vibrational excitation as a tool to guide a variety of molecular structure manipulations in an exceptional highly-selective manner.

Novel fluorinated ring-fused chlorins as promising PDT agents against melanoma and esophagus cancer.

Investigation of novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins, derived from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, as PDT agents against melanoma and esophagus cancer is disclosed. Diol and diester fluorinated ring-fused chlorins, including derivatives with 2-(2-hydroxyethoxy)ethanamino groups at the phenyl rings, were obtained via a two-step methodology, combining SNAr and [8π + 2π] cycloaddition reactions. The short-chain PEG groups at the para-position of the phenyl rings together with the diol moiety at the fused pyrazole ring promote a red-shift of the Soret band, a decrease of the fluorescence quantum yield and an increase of the singlet oxygen formation quantum yield, improving the photophysical characteristics required to act as a photosensitizer. Introduction of these hydrophilic groups also improves the incorporation of the sensitizers by the cells reaching cellular uptake values of nearly 50% of the initial dose. The rational design led to a photosensitizer with impressive IC50 values, 13 and 27 nM against human melanoma and esophageal carcinoma cell lines, respectively.

Bond-Breaking/Bond-Forming Reactions by Vibrational Excitation: Infrared-Induced Bidirectional Tautomerization of Matrix-Isolated Thiotropolone.

Infrared vibrational excitation is a promising approach for gaining exceptional control of chemical reactions, in ways that cannot be attained via thermal or electronic excitation. Here, we report an unprecedented example of a bond-breaking/bond-forming reaction by vibrational excitation under matrix isolation conditions. Thiotropolone monomers were isolated in cryogenic argon matrices and characterized by infrared spectroscopy and vibrational computations (harmonic and anharmonic). Narrowband near-infrared irradiations tuned at frequencies of first CH stretching overtone (5940 cm-1) or combination modes (5980 cm-1) of the OH tautomer, the sole form of the compound that exists in the as-deposited matrices, led to its conversion into the SH tautomer. The tautomerization in the reverse direction was achieved by vibrational excitation of the SH tautomer with irradiation at 5947 or 5994 cm-1, corresponding to the frequencies of its CH stretching combination and overtone modes. This pioneer demonstration of bidirectional hydroxyl ↔ thiol tautomerization controlled by vibrational excitation creates prospects for new advances in vibrationally induced chemistry.

Platinum(II) ring-fused chlorins as efficient theranostic agents: Dyes for tumor-imaging and photodynamic therapy of cancer.

The discovery of Pt-chlorin-type theranostic agents is described. Luminescent Pt(II) 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins, with different degrees of hydrophilicity, have been synthesized and their in vitro photocytotoxicity against human melanoma, oesophageal and bladder carcinomas was studied. A di(hydroxymethyl)-substituted chlorin was identified as a privileged molecule to explore imaging-guided photodynamic therapy. In addition to the high activity as PDT agent and absence of cytotoxicity per se, this molecule showed the ideal photophysical and photochemical properties. In vivo studies using a A375 melanoma mouse model, proved the extraordinary properties of this chlorin as a luminescent probe and the ability to impair tumor growth, making image guided treatment and follow up a possibility.

A Review on (Hydro)Porphyrin-Loaded Polymer Micelles: Interesting and Valuable Platforms for Enhanced Cancer Nanotheranostics.

Porphyrins are known therapeutic agents for photodynamic therapy of cancer and also imaging agents for NIR fluorescence imaging, MRI, or PET. A combination of interesting features makes tetrapyrrolic macrocycles suitable for use as theranostic agents whose full potential can be achieved using nanocarriers. This review provides an overview on nanotheranostic agents based on polymeric micelles and porphyrins developed so far.

Ring-Fused Diphenylchlorins as Potent Photosensitizers for Photodynamic Therapy Applications: In Vitro Tumor Cell Biology and in Vivo Chick Embryo Chorioallantoic Membrane Studies.

Ring-fused diphenylchlorins as potent low-dose photosensitizers for photodynamic therapy of bladder carcinoma and esophageal adenocarcinoma are described. All studied molecules were very active against HT1376 urinary bladder carcinoma and OE19 esophageal adenocarcinoma cell lines, showing IC50 values below 50 nM. The in vivo evaluation of the more promising photosensitizer, using an OE19 tumor/chick embryo chorioallantoic membrane model, showed a tumor weight regression of 33% with a single photodynamic therapy treatment with the photosensitizer dose as low as 37 ng/embryo.

Advances on photodynamic therapy of melanoma through novel ring-fused 5,15-diphenylchlorins.

The synthesis, photophysical behaviour and photosensitization ability of novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused 5,15-diphenylchlorins against melanoma cells are described. All studied chlorins were found to be extremely active against melanoma cell lines A375 showing IC50 values below 20 nM. Furthermore, a dihydroxymethyl diphenylchlorin was identified as an excellent candidate to allow modulating of different types of cell death, apoptosis vs. necrosis, by varying its concentration. This can be explored as a tool to improve the effectiveness of PDT since inflammatory response resulting from necrotic cell death after PDT can activate the antitumor immune response with implications also regarding the vascular damage. This feature combined with very low cytotoxicity against human melanoma cells in the absence of light activation and against human fibroblast HFF-1 cells makes this chlorin a candidate of choice as a photosensitizer for PDT. A comprehensive photophysical investigation including the determination of quantum yields for fluorescence, singlet oxygen sensitization and internal conversion, lifetimes and rate constants of all the excited state deactivation processes has been undertaken.

Platinum(II) Ring-Fused Chlorins as Near-Infrared Emitting Oxygen Sensors and Photodynamic Agents.

Novel near-infrared luminescent compounds based on platinum(II) 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins are described. These compounds have high photostability and display light emission, in particular simultaneous fluorescence and phosphorescence emission in solution at room temperature, in the biologically relevant 700-850 nm red and near-infrared (NIR) spectral region, making them excellent materials for biological imaging. The simultaneous presence of fluorescence and phosphorescence emission at room temperature, with the phosphorescence strongly quenched by oxygen whereas fluorescence remains unaffected, allows these compounds to be used as ratiometric oxygen sensors in chemical and biological media. Both steady-state (fluorescence vs phosphorescence intensities) and dynamic (dependence of phosphorescence lifetimes upon oxygen concentration) luminescence approaches can be used. Photocytotoxicity studies against human melanocytic melanoma cells (A375) indicate that these compounds display potential as photosensitizers in photodynamic therapy.

Novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine fused chlorins as very active photodynamic agents for melanoma cells.

The development of new stable 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins with high absorption properties at 650 nm, and their impressive photosensitizer ability against melanotic and amelanotic cancer cells is described. Comparison between a diester-substituted chlorin with the corresponding dihydroxymethyl derivative demonstrated that the increased hydrophilicity of the latter is crucial to ensure nanomolar activity against melanoma cells. The new photosensitizer leads to death of human melanoma cells being both apoptosis and necrosis in equal parts involved in the treatment response. The dihydroxymethyl-chlorin was particularly active against human melanocytic melanoma A375 cells, which can be viewed as a solution to overcome the resistance of melanotic melanoma to photodynamic therapy.