Fotoquímica de compuestos heteroaromáticos tricíclicos en medios biomiméticos

[EN] The use of biomimetic media such as serum albumins (SAs), a1-acid glycloproteins (AAGs), cyclodextrins (CDs) and sodium dodecyl sulfate (SDS) micelles is combined with photophysical techniques (fluorescence (F) and laser flash photolysis (LFP)) to investigate the issue of photosensitization by drugs or drug derivatives containing a triciclyc heteroaromatic chromophore, namely carbazole (CBZ) or phenothiazine (FTZ). The photobehavior of the methyl ester of carprofen (CPFMe) in the presence of bovine serum albumin (BSA) has been addressed first. The decay of triplet excited state of CPFMe in the presence of BSA at 1:2 (at 430 nm), needed two monoexponential terms and thus two triplet lifetimes for a satisfactory fitting, which revealed binding to two different sites within the protein. The shorter value of triplet lifetime was ascribed to CPFMe present in site I, based on the quenching of 3CPFMe* by the tryptophan (Trp) residue located in this pocket, through an electron transfer mechanism. No significant differences were found between (S)- and (R)-CPFMe. Within bovine a1-acid glycloprotein (BAAG), only one binding site was observed for both CPFMe enantiomers. Stereodifferentiation was observed in the triplet lifetimes in CPFMe@BAAG complexes, with the shorter lifetimes values for the (S)-enantiomer. CPFMe photobinding to BAAG was detected by following the changes in fluorescence of CPFMe@BAAG mixtures before and after irradiation. The process was more efficient for (S)-CPFMe. The proposed mechanism involves a reductive photodehalogenation from the triplet of (S)-CPFMe, giving rise to covalent adduct (S)-CBZMe-BAAG. As a way to circumvent the unfavorable thermodynamics of homolytic C-Cl cleavage through the triplet excited state, it has been proposed that the actual operating mechanism involves formation of triplet excimers. To deepen into the mechanism of CPFMe dehalogenation, two diastereomeric dyads based on CPF (CPF-CPF and CPF-CBZ) have been synthesized and irradiated. The self-quenching of the triplet excited states in both dyads is much faster than in CPFMe, and is related to formation of charge transfer species, clearly disfavored in non-polar solvents (THF). The trends observed in the triplet lifetimes, as well as the solvent effects on photoreactivity, are in full agreement with the mechanistic picture of a photoreductive dechlorination as operating mechanism in CPF-based systems. The photochemistry of the neuroleptic drug cyamemazine (CMZ) has been investigated in biomimetic media. The encapsulation process has been followed by fluorescence, as a hypsochromic shift of the band, concomitant with enhanced quantum yields and lifetimes. LFP revealed important changes associated with encapsulation, specifically more selective generation of the triplet excited state, which turns to be longer-lived. Light-induced oxidation of CMZ afforded the corresponding radical cation, which was trapped by oxygen to afford the N,S-dioxide. The reaction has been monitored by fluorescence, by the progressive appearance of a emission band at shorter wavelengths. The process resulted to be disfavored in the employed microenvironments (lipophilics). The slowest photooxidation rate corresponds to CMZ in SDS micelles and in AAGs, where the drug is located in a more hydrophobic domain, hardly accessible from the aqueous medium, and photoionization is nearly negligible. LFP of the anti-psychotic chlorpromazine (CPZ) and two phase I metabolites, CPZ-MD and CPZ-DD (mono and didemethylated) in the presence of increasing amounts of HSA, has allowed monitoring binding to the protein, from the enhancement of the Amax value of 3CPZ* (at 470 nm). The binding degree was higher for the parent drug, in agreement with its more hydrophobic character. The use of warfarin as site I displacement probe indicated that the three compounds only bind to this site. Marginal photobinding to HSA was observed in all cases, monitored by subtle changes in the fluorescence.[ES] Se ha planteado el uso de medios biomiméticos como albúminas séricas (ASs), a1-glicoproteinas ácidas (AGAs), ciclodextrinas (CDs) y micelas (SDS) combinado con técnicas fotofísicas (fluorescencia (F) y fotólisis de destello láser (LFP)) para investigar la fotosensibilización por fármacos que contienen un cromóforo carbazol (CBZ) o fenotiazina (FTZ). Se ha estudiado el fotocomportamiento del éster metílico del carprofeno (CPFMe) en presencia de albúmina sérica bovina (ASB). Las cinéticas de desaparición de los estados excitados triplete de CPFMe en presencia de ASB 1:2 (a 430 nm), reveló dos sitios de unión diferentes a la proteína. El tiempo menor se asoció al CPFMe presente en el sitio I, basado en la desactivación del 3CPFMe* por triptófano (Trp) alojado en ese sitio de la proteína a través de un mecanismo de transferencia electrónica (Te). Sin diferencias significantes entre (S)- y (R)-CPFMe. En a1-glicoproteina ácida bovina (AGAB), se observó un único sitio de unión para ambos enantiómeros de CPFMe con estereodiferenciación en los tiempos de triplete de los complejos CPFMe@AGAB, donde el más corto fue para el enantiómero (S). Se detectó fotounión de CPFMe a AGAB por los consiguientes cambios en los espectros de fluorescencia de las mezclas CPFMe@AGAB antes y después de irradiar. El proceso fue más eficiente para (S)-CPFMe. El mecanismo propuesto relaciona la fotodeshalogenación reductiva desde el estado excitado triplete de (S)-CPFMe, dando lugar al aducto covalente (S)-CBZMe-AGAB. Para evitar la termodinámica desfavorable de la ruptura homolítica C-Cl a través del estado excitado triplete, se ha propuesto que en el mecanismo real de reacción esté involucrada la formación de excímeros triplete. Para profundizar en el mecanismo de deshalogenación del CPFMe, se han sintetizado e irradiado dos diadas diastereoméricas basadas en CPF (CPF-CPF y CPF-CBZ). El "self-quenching" de los estados excitados triplete en ambas diadas es mucho más rápido que en CPFMe, y es relacionado con la formación de especies con separación de carga, claramente desfavorecido en disolventes apolares como tetrahidrofurano (THF). Los tiempos de vida de triplete, así como los efectos del disolvente en la fotorreactividad están de acuerdo con la teoría de una deshalogenación fotoreductiva como mecanismo en sistemas basados en CPF. Se ha investigado la fotoquímica del fármaco neuroléptico ciamemazina (CMZ) en medios biomiméticos. El proceso de encapsulación ha sido seguido por fluorescencia, como un desplazamiento hipsocrómico de la banda, así como un aumento de los rendimientos cuánticos y de los tiempos de vida. La FDL reveló importantes cambios asociados a la encapsulación, una generación del estado excitado triplete más selectiva, los cuales viven más tiempo. La fotooxidación de CMZ ocurre a través de fotoionización; el catión radical resultante es atrapado por oxígeno para formar su fotoproducto N,S-dióxido. La reacción ha sido monitorizada por fluorescencia, por la aparición progresiva de la banda de emisión a longitudes de onda más cortas. Éste proceso resultó ser desfavorecido en los microambentes utilizados (lipófilos). La velocidad de fotooxidación más lenta corresponde a CMZ en micelas SDS y en AGAs, donde el fármaco se encuentra en un dominio más hidrofóbico, difícilmente accesible desde el medio acuoso, donde la fotoionización es casi despreciable. La FDL del antipsicótico clorpromazina (CPZ) y dos de sus metabolitos de fase I CPZ-MD y CPZ-DD (mono y didesmetilado) en presencia de cantidades crecientes de albúmina sérica humana (ASH) ha permitido monitorizar la unión a proteína, por el aumento del valor de Amax del 3CPZ* (a 470 nm). El grado de unión fue mayor para el fármaco de origen, de acuerdo con su carácter más hidrofóbico. El uso de warfarina como sonda de desplazamiento del sitio I indicó que los tres compuestos sólo se unen a este sitio[CA] S'ha plantejat l'ús de mitjans biomimètics com albúmines sèriques (ASs), a1-glicoproteïnes àcides (AGAs), ciclodextrines (CDs) i micel·les de dodecil sulfat sòdic (SDS) combinat amb tècniques fotofísiques com a fluorescència (F) i fotòlisi de llampada làser (FLL) per a investigar la fotosensibilització per fàrmacs que contenen un cromòfor tipus carbazol (CBZ) o fenotiazina (FTZ). S'ha estudiat el comportament de l'èster metílic del carprofén (CPFMe) en presencia d'albúmina sèrica bovina (ASB). Les cinètiques de desaparició dels estats excitats triplet de CPFMe en presencia de ASB 1:2 (a 430 nm) van revelar dos llocs d'unió de CPFMe a la proteïna; el temps menor es va associar al lloc I a causa de la desactivació del 3CPFMe* per triptòfan (Trp) allotjat en eixe lloc de la proteïna a través d'un mecanisme de transferència electrònica (Te); no es van observar diferències significatives entre (S)- i (R)-CPFMe. En a1-glicoproteïna àcida bovina (AGAB), CPFMe es va encapsular en un únic lloc d'unió amb estereodiferenciació en els temps de vida de triplet dels complexos CPFMe@AGAB, amb un valor més curt per a l'enantiòmer (S). A més, es va detectar fotounió de CPFMe a AGAB pels consegüents canvis als espectres de fluorescència de les mescles CPFMe@AGAB abans i després d'irradiar. El procés va ser més eficient per a (S)-CPFMe. El mecanisme proposat relaciona la fotodeshalogenació reductiva des de l'estat excitat triplet de (S)-CPFMe i que forma el corresponent adducte (S)-CBZMe-AGAB. Per a evitar la termodinàmica desfavorida de la ruptura homolítica C-Cl a través del estat excitat triplet, s'ha proposat que al mecanisme real de reacció estigui involucrat la formació de excímers triplet. En aquest context, per aprofundir en el mecanisme de deshalogenació del CPFMe, s'ha sintetitzat e irradiat dos diades diastereomèriques (CPF-CPF i CPF-CBZ). El "self-quenching" dels estats excitats triplet de ambdues diades es molt més ràpid que en CPFMe, i es relaciona amb la formació d'espècies amb separació de càrrega, clarament desfavorit en dissolvents apolars com tetrahidrofurà (THF). Les tendències observades en els temps de vida de triplet, així com els efectes del dissolvent en la fotorreactivitat estan d'acord amb la teoria d'una deshalogenació fotoreductiva com a mecanisme en sistemes basats en CPF. S'ha investigat la fotoquímica del fàrmac neurolèptic ciamemazina (CMZ) en medis biomimètics. El procés d'encapsulació ha estat seguit per fluorescència, com un desplaçament hipsocròmic de la banda, així com un augment dels rendiments quàntics i dels temps de vida. La FLL va revelar canvis importants associats a l'encapsulació, específicament una generació de l'estat excitat triplet més selectiva i amb temps de vida més llarg. La fotooxidació de CMZ ocorre a través de fotoionització; el catió radical resultant es atrapat per oxigen per a formar el seu fotoproducte N,S-diòxid. La reacció ha estat monitoritzada per fluorescència, per l'aparició progressiva de la banda de emissió a longituds d'ona més curtes. Este procés resultà ser desfavorit als microambients utilitzats (lipofílics). La velocitat de fotooxidació més lenta correspon a CMZ en micel·les SDS i en AGAs, on el fàrmac està en un domini més hidrofòbic, difícilment accessible des del medi aquós i on la fotoionització és quasi menyspreable. La FLL de l'antipsicòtic clorpromazina (CPZ) i dos dels seus metabòlits CPZ-MD i CPZ-DD (mono i didesmetilat) en presència de quantitats creixents d'albúmina sèrica humana (ASH) ha permès monitoritzar la unió a proteïna, mitjançant l'augment del valor de Amax del 3CPZ* (a 470 nm). El grau d'unió va ser major per al fàrmac d'origen, d'acord amb el seu caràcter més hidrofòbic. L'ús de warfarina com a sonda de desplaçament del lloc I va indicar que els tres compostos només s'uneixen a eixe lloc. Es va observar una lleugera fotounió a ASHLimones Herrero, D. (2015). Fotoquímica de compuestos heteroaromáticos tricíclicos en medios biomiméticos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/59392TESI

Investigation of metabolite-protein interactions by transient absorption spectroscopy and in silico methods

[EN] Transient absorption spectroscopy in combination with in silico methods has been employed to study the interactions between human serum albumin (HSA) and the anti-psychotic agent chlorpromazine (CPZ) as well as its two demethylated metabolites (MCPZ and DCPZ). Thus, solutions containing CPZ, MCPZ or DCPZ and HSA (molar ligand:protein ratios between 1:0 and 1:3) were submitted to laser flash photolysis and the Delta A(max) value at lambda = 470 nm, corresponding to the triplet excited state, was monitored. In all cases, the protein-bound ligand exhibited higher Delta Amax values measured after the laser pulse and were also considerably longer-lived than the non-complexed forms. This is in agreement with an enhanced hydrophilicity of the metabolites, due to the replacement of methyl groups with H that led to a lower extent of protein binding. For the three compounds, laser flash photolysis displacement experiments using warfarin or ibuprofen indicated Sudlow site I as the main binding site. Docking and molecular dynamics simulation studies revealed that the binding mode of the two demethylated ligands with HSA would be remarkable different from CPZ, specially for DCPZ, which appears to come from the different ability of their terminal ammonium groups to stablish hydrogen bonding interactions with the negatively charged residues within the protein pocket (Glu153, Glu292) as well as to allocate the methyl groups in an apolar environment. DCPZ would be rotated 180 degrees in relation to CPZ locating the aromatic ring away from the Sudlow site I of HSA. (C) 2019 Elsevier B.V. All rights reserved.Financial support from Ministerio de Economia, Industria y Competitividad (CTQ2016-78875-P, SAF2016-75638-R, BES-2011-043706), Generalitat Valenciana (Prometeo 2017/075), Xunta de Galicia [Centro Singular de Investigacion de Galicia accreditation 2016-2019 (ED431G/09, ED431B 2018/04) and post-doctoral fellowship to E. L.] and European Union (European Regional Development Fund-ERDF) is gratefully acknowledged. I. A. holds a "Miguel Servet" contract (CP1116/00052) funded by the Carlos III Health Institute. We are grateful to the Centro de Supercomputacion de Galicia (CESGA) for computational facilities.Limones Herrero, D.; Palumbo, F.; Vendrell Criado, V.; Andreu Ros, MI.; Lence, E.; González-Bello, C.; Miranda Alonso, MÁ.... (2020). Investigation of metabolite-protein interactions by transient absorption spectroscopy and in silico methods. 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Enhanced photo(geno)toxicity of demethylated chlorpromazine metabolites

Chlorpromazine (CPZ) is an anti-psychotic drug widely used to treat disorders such as schizophrenia or manic-depression. Unfortunately, CPZ exhibits undesirable side effects such as phototoxic and photoallergic reactions in humans. In general, the influence of drug metabolism on this type of reactions has not been previously considered in photosafety testing. Thus, the present work aims to investigate the possible photo(geno)toxic potential of drug metabolites, using CPZ as an established reference compound. In this case, the metabolites selected for the study are demethylchlorpromazine (DMCPZ), didemethylchlorpromazine (DDMCPZ) and chlorpromazine sulfoxide (CPZSO). The demethylated CPZ metabolites DMCPZ and DDMCPZ maintain identical chromophore to the parent drug. In this work, it has been found that the nature of the aminoalkyl side chain modulates the hydrophobicity and the photochemical properties (for instance, the excited state lifetimes), but it does not change the photoreactivity pattern, which is characterized by reductive photodehalogenation, triggered by homolytic carbon-chlorine bond cleavage with formation of highly reactive aryl radical intermediates. Accordingly, these metabolites are phototoxic to cells, as revealed by the 3T3 NRU assay; their photo-irritation factors are even higher than that of CPZ. The same trend is observed in photogenotoxicity studies, both with isolated and with cellular DNA, where DMCPZ and DDMCPZ are more active than CPZ itself. In summary, side-chain demethylation of CPZ, as a consequence of Phase I biotransformation, does not result a photodetoxification. Instead, it leads to metabolites that exhibit in an even enhanced photo(geno)toxicity.This work was supported by the MINECO (Grant: CTQ2013-47872), by Carlos III Institute of Health (Grants: RD12/0013/0009 and CP11/00154) and by the Generalitat Valenciana (Prometeo II/2013/005).Palumbo, F.; García Lainez, G.; Limones Herrero, D.; Coloma, MD.; Escobar, J.; Jiménez Molero, MC.; Miranda Alonso, MÁ.... (2016). Enhanced photo(geno)toxicity of demethylated chlorpromazine metabolites. Toxicology and Applied Pharmacology. 313:231-237. https://doi.org/10.1016/j.taap.2016.10.024S23123731

Photo(geno)toxicity changes associated with hydroxylation of the aromatic chromophores during diclofenac metabolism

[EN] Diclofenac (DCF) can cause adverse reactions such as gastrointestinal, renal and cardiovascular disorders; therefore, topical administration may be an attractive alternative to the management of local pain in order to avoid these side effects. However, previous studies have shown that DCF, in combination with sunlight, displays capability to induce photosensitivity disorders. In humans, DCF is biotransformed into hydroxylated metabolites at positions 4¿ and 5 (4¿OH-DCF and 5OH-DCF), and this chemical change produces non negligible alterations of the drug chromophore, resulting in a significant modification of its light-absorbing properties. In the present work, 5OH-DCF exhibited higher photo(geno)toxic potential than the parent drug, as shown by several in vitro assays (3T3 NRU phototoxicity, DNA ssb gel electrophoresis and COMET), whereas 4¿OH-DCF did not display significant photo(geno)toxicity. This could be associated, at least partially with their more efficient UV-light absorption by 5OH-DCF metabolite and with a higher photoreactivity. Interestingly, most of the cellular DNA damage photosensitized by DCF and 5OH-DCF was repaired by the cells after several hours, although this effect was not complete in the case of 5OH-DCF.This work was supported by the Carlos III Institute of Health (Grants: RD16/0006/0030, PI16/01877), by the MINECO (Grants: CTQ2013-47872, CTQ2016-78875), and by the Generalitat Valenciana (Prometeo 2017/075).García -Laínez, G.; Ana M Marínez-Reig; Limones Herrero, D.; Jiménez Molero, MC.; Miranda Alonso, MÁ.; Andreu Ros, MI. (2018). Photo(geno)toxicity changes associated with hydroxylation of the aromatic chromophores during diclofenac metabolism. Toxicology and Applied Pharmacology. 341:51-55. https://doi.org/10.1016/j.taap.2018.01.005S515534

Bypassing the energy barrier of homolytic photodehalogenation in chloroaromatics through self-quenching

Reductive photodehalogenation of chloroaromatics is assumed to proceed from the triplet excited state, although its energy is often insufficient to promote a clean homolytic C¿Cl cleavage. A clear-cut experimental proof is provided that correlates self-quenching of the directly observed triplet excited states of chlorocarbazole-based dyads 1 and 2 with photoreactivity via intramolecular charge transfer.Financial support from the Spanish Government (CTQ2010-14882, BES-2011-043706, JCI-2010-06204) and from the Generalitat Valenciana (Prometeo 2008/090 and GV/2012/041) is gratefully acknowledged.Limones Herrero, D.; Pérez Ruiz, R.; Jiménez Molero, MC.; Miranda Alonso, MÁ. (2013). Bypassing the energy barrier of homolytic photodehalogenation in chloroaromatics through self-quenching. Organic Letters. 15(6):1314-1317. doi:10.1021/ol400251sS1314131715

Bypassing the Energy Barrier of Homolytic Photodehalogenation in Chloroaromatics through Self-Quenching

Reductive photodehalogenation of chloroaromatics is assumed to proceed from the triplet excited state, although its energy is often insufficient to promote a clean homolytic C–Cl cleavage. A clear-cut experimental proof is provided that correlates self-quenching of the directly observed triplet excited states of chlorocarbazole-based dyads <b>1</b> and <b>2</b> with photoreactivity via intramolecular charge transfer