Histidine-Specific Peptide Modification via Visible-Light-Promoted C-H Alkylation.

Histidine (His) carries a unique heteroaromatic imidazole side chain and plays irreplaceable functional roles in peptides and proteins. Existing strategies for site-selective histidine modification predominantly rely on the N-substitution reactions of the moderately nucleophilic imidazole group, which inherently suffers from the interferences from lysine and cysteine residues. Chemoselective modification of histidine remains one of the most difficult challenges in peptide chemistry. Herein, we report peptide modification via radical-mediated chemoselective C-H alkylation of histidine using C4-alkyl-1,4-dihydropyridine (DHP) reagents under visible-light-promoted conditions. The method exploits the electrophilic reactivity of the imidazole ring via a Minisci-type reaction pathway. This method exhibits an exceptionally broad scope for both peptides and DHP alkylation reagents. Its utility has been demonstrated in a series of important peptide drugs, complex natural products, and a small protein. Distinct from N-substitution reactions, the unsubstituted nitrogen groups of the modified imidazole ring are conserved in the C-H alkylated products.

Prediction of photosensitivity of 1,4-dihydropyridine antihypertensives by quantitative structure-property relationship.

A quantitative structure-property relationships (QSPR) model, correlating the light sensitivity against theoretical molecular descriptors, was developed for a set of 1,4-dihydropyridine calcium channel antagonist drugs. These compounds are characterized by a high tendency to degradation when exposed to light, furnishing in the most of cases a related oxidation product from aromatization of the dihydropyridinic ring. Photodegradation was forced by exposing the drugs to a Xenon lamp, in accordance with the ICH international rules, and degradation kinetics was monitored by spectrophotometry. The photodegradation rates combined with a series of descriptors related to the chemical structures were computed by Partial Least Squares (PLS) multivariate analysis. An accurate selection of the variables, fitting at the best the PLS model, was performed. Two descriptors related to the substituent information on both the dihydropyridinic and benzenic rings and four molecular descriptors, were selected. The QSPR model was fully cross validated and then optimized with an external set of novel 1,4-dihydropyridine drugs, obtaining very satisfactory statistical results. The good agreement between predicted and measured photodegradation rate (R(2)=0.8727) demonstrated the high accuracy of the QSPR model in predicting the photosensitivity of the drugs belonging to this class. The model was finally proposed as an effective tool to design new congeneric molecules characterized by high photostability.

Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: classification criteria and application to drug candidates.

We have previously demonstrated that the phototoxic potential of chemicals could be partly predicted by the determination of reactive oxygen species (ROS) from photo-irradiated compounds. In this study, ROS assay strategy was applied to 39 marketed drugs and 210 drug candidates in order to establish provisional classification criteria for risk assessment of drug-induced phototoxicity. The photosensitizing properties of 39 model compounds consisting of phototoxic and non-phototoxic chemicals, as well as ca. 210 drug candidates including 11 chemical series were evaluated using ROS assay and the 3T3 neutral red uptake phototoxicity test (NRU PT). With respect to marketed drugs, most phototoxic drugs tended to cause type I and/or II photochemical reactions, resulting in generation of singlet oxygen and superoxide. There seemed to be a clear difference between phototoxic drugs and non-phototoxic compounds in their abilities to induce photochemical reactions. A plot analysis of ROS data on the marked drugs provided classification criteria to discriminate the photosensitizers from non-phototoxic substances. Of all drug candidates tested, 35.2% compounds were identified as phototoxic or likely phototoxic on the basis of the 3T3 NRU PT, and all ROS data for these phototoxic compounds were found to be over the threshold value. Furthermore, 46.3% of non-phototoxic drug candidates were found to be in the subthreshold region. These results verify the usefulness of the ROS assay for understanding the phototoxicity risk of pharmaceutical substances, and the ROS assay can be used for screening purposes in the drug discovery stage.

Microwave-assisted combinatorial synthesis of hexa-substituted 1,4-dihydropyridines scaffolds using one-pot two-step multicomponent reaction followed by a S-alkylation.

A sequential one-pot two-step protocol for microwave-assisted synthesis of Hantzsch-type hexa-substituted 1,4-dihydropyridines has been developed. The three-component reactions of beta-aroylthioamides with aldehydes and acetonitrile derivatives produce the intermediates in situ followed by a S-alkylation to afford hexa-substituted 1,4-dihydropyridines. The reaction presumably proceeds via a Knoevenagel condensation-Michael addition-cyclocondensation-rearrangement-S N2 reaction sequence. Target compounds were obtained in high yields and simply purified by recrystallization. The novel method is complementary to the classical Hantzsch synthesis in that it is well-suited to the preparation of hexa-substituted 1,4-dihydropyridines.

In vitro phototoxicity of dihydropyridine derivatives: a photochemical and photobiological study.

Some photosensitizing drugs can cause phototoxic skin responses even after systemic administration; therefore, avoidance of undesired side-effects is a key consideration in drug discovery and development. As a prediction tool for phototoxic risk, we previously proposed the monitoring of reactive oxygen species (ROS) generated from compounds irradiated with UVA/B, which can be effective for understanding photochemical/photobiological properties. In this investigation, we evaluated the photosensitizing properties of a novel dihydropyridine derivative, with bradykinin B(2) receptor antagonist activity (compound A) using our ROS assay and several analytical/biochemical techniques. Exposure of compound A, and several dihydropyridine-type calcium channel antagonists to simulated sunlight resulted in the significant production of singlet oxygen, superoxide, or both, which indicates their photosensitive/phototoxic potential. This is consistent with the observation that compound A under UVA/B light exposure caused significant photodegradation and even peroxidation of fatty acid, which could lead to phototoxic dermatitis. Interestingly, the addition of radical scavengers, especially GSH, MPG and BHA, could attenuate the lipid peroxidation, suggesting the involvement of ROS generation in the phototoxic pathways of compound A. In the 3T3 neutral red uptake phototoxicity test, compound A also showed a phototoxic effect on 3T3 mouse fibroblast cells. These findings also support the usefulness of the ROS assay for the risk assessment studies on the drug-induced phototoxicity even at the early stages of pharmaceutical development.

Ultrasound-assisted photochemical oxidation of unsymmetrically substituted 1,4-dihydropyridines.

The combination of ultrasound and photochemistry has been used for the oxidation of unsymmetrical 1,4-dihydropyridines to the pyridine derivatives. An ultrasonic probe of 24 kHz frequency and a Hg-lamp of 100 W have been used for this study. The effect of parameters such as ultrasonic intensity, the presence of oxygen and argon atmospheres and also the separate usage of one of these irradiation sources have been studied. Whereas sonication of these compounds alone did not result in the oxidation of them, the use of ultrasound increases the rate of photo-oxidation. The presence of oxygen decreases or increases the rate of reaction, depending on the type of excited state of 1,4-dihydropyridines involved in the reaction.

The effect of ionizing radiation of 1,4-dihydropyridine derivatives in the solid state.

Electron paramagnetic resonance (EPR) spectroscopy was used to investigate the gamma-radiation damage in the crystalline powder form of nine calcium channel blockers from the 1,4-dihydropyridine derivatives, which are in clinical use for treatment of arteria hypertension and ischemic heart disease. EPR studies have been carried out, showing the influence of irradiation and storage parameters on the nature and concentration of the free radicals trapped. EPR spectra of isardipine and felodipine showed single EPR line. EPR spectra of nifedipine, nisoldipine, nitrendipine, nimodipine, nicardipine and nilvadipine reveal a broad anisotropic signal of hyperfine interaction. No EPR signal was observed from amlodipine.

Photophysics and photochemical studies of 1,4-dihydropyridine derivatives.

The absorption and fluorescence properties of nifedipine (NPDHP), felodipine (CPDHP) and a series of structurally related 1,4-dihydropyridines were studied in aqueous solution and organic solvents of different properties. The absorption and fluorescence spectra were found to depend on the chemical nature of the substituents at the position 4 of the 1,4-dihydropyridine ring (DHP) and on solvent properties. In aqueous solution, the fluorescence spectra of 4-phenyl substituted compounds are blue-shifted with respect to the alkyl substituted compounds. The more fluorescent compound is CPDHP. Nifedipine is not fluorescent. All compounds, with the exception of CPDHP, present monoexponential fluorescence decay with very short lifetime (0.2-0.4 ns). CPDHP showed a biexponential emission decay with a long-lived component of 1.7 ns; this behavior is explained in terms of different conformers because of the hindered rotation of the phenyl group by the ortho-substitution. Analysis of the solvent effect on the maximum of the absorption spectrum by using the linear solvent-energy relation solvato-chromic equation indicates the redshifts are influenced by the polarizability, hydrogen bonding ability and the hydrogen bond acceptance of the solvent. Whereas, the fluorescence characteristics (spectra, quantum yields and lifetimes) are sensitive to the polarizabilty and hydrogen bond ability of the solvents. Photo-decomposition of nifedipine is dependent on the solvent properties. Faster decomposition rates were obtained in nonprotic solvents. The 4-carboxylic derivative goes to decarboxylation. Under similar conditions, the other DHP compounds did not show appreciable photodecomposition.

Photostabilization of 1,4-dihydropyridine antihypertensives by incorporation into beta-cyclodextrin and liposomes.

Inclusion compounds of eleven dihydropyridine drugs were formed and investigated for protection against photo-induced drug degradation. Formulations of cyclodextrins and liposomes were prepared and their photoprotective ability for the encapsulated drug was monitored. Drug photodegradation was spectrophotometrically followed during exposure of the formulations to light of a Xenon lamp. ICH guidelines for photostability testing were applied. A comparison with common pharmaceutical formulations revealed optimal protection for both formulations. The use of the liposome and cyclodextrin inclusion complexes resulted in a mean drug recovery of 77 and more then 90% respectively, after a light exposure until to 30 minutes with an intensity of 21 kJ x min(-1) m(-2). Lercanidipine and Manidipine only did not show a satisfactory increase of photostabilization in the studied supramolecular complexes, due to their low inclusion in both the systems.

Fractional wavelet analysis for the simultaneous quantitative analysis of lacidipine and its photodegradation product by continuous wavelet transform and multilinear regression calibration.

Fractional wavelet transform (FWT) was applied to the original absorption spectra of lacidipine (LAC) and its photodegradation product (LACD), and the resulting FWT spectra were processed by continuous wavelet transform (CWT) and multilinear regression calibration (MLRC) for the simultaneous quantitative analysis of both products in their binary mixtures. These methods do not require any chemical separation step and chemical complex reaction to obtain a detectable signal for the degradation product. By using the Mexican hat function, 2 calibration functions for LAC and LACD were obtained by measuring the CWT transformed signals at 416.1 nm for LAC and 414.6 nm for LACD, after FWT processing of the original absorption spectra. The calibration graphs were linear in the concentration range of 5.08-40.64 microg/mL for LAC and 0.51-8.16 microg/mL for LACD. The limit of detection and the limit of quantitation were found to be 0.289 and 0.956 microg/mL for LAC and 0.036 and 0.118 microg/mL for LACD, respectively. For comparison, the MLRC algorithm was applied to the linear regression functions for the individual drug and its photoproduct. In this approach, a set of linear regression functions was obtained from the relationship between concentrations and FWT signals in the wavelength range 411.0-412.4 nm. Both methods were applied to the quantitative evaluation of LAC and LACD in laboratory and pharmaceutical samples, and produced very satisfactory results.

Photoprotection of 1,4-dihydropyridine derivatives by dyes.

The possibility of increasing the photochemical stability of nisoldipine by using indigotine and azorubine as photoprotectors has been studied. The course of the photodegradation was monitored by means of UV-vis spectrophotometry and HPLC. Quantitative assessments of the nisoldipine photodegradation included evaluation of the quantum yields and kinetic parameters. In order to establish the light intensity absorbed by a system, Reinecke salt was used as a chemical actinometer. The values of the quantum yields (phi) of photodegradation decreased with increasing dye concentration and were 0.24-0.15 and 0.27-0.25 for indigotine and azorubine, respectively. Furthermore, our attention was focused on determination the role of the selected dyes during the photodegradation of nisoldipine and the calculations were made to eliminate an inner filter effect. The values obtained were used to construct a Stern-Volmer plot and calculate the Stern-Volmer constants (KSV). On the basis of the KSV and the values of the quenching constant (kq) the exited state lifetime (tauo) of nisoldipine in the presence of indigotine and azorubine were estimated. The calculated values of tauo for NS were 6.66x10(-6)s--in the presence of indigotine and 2.76x10(-6)s--in the presence of azorubine, indicating that the photodegradation of nisoldipine occurs from the lowest triplet excited state.

Solid state photochemistry of 1,4-dihydropyridines.

Photochemical behavior of some 1,4-dihydropyridines has been investigated in the solid state. Whereas upon irradiation of 1,4-dihydropyridines in solution phase, their photo-oxidation and formation of pyridine derivatives have been observed, irradiation of these compounds in the solid state decreases their light sensitivity. In many cases photo-oxidation has been observed only in very low yields.

Multivariate least squares regression applied to the spectrophotometric analysis of manidipine and its main photoproduct.

The simultaneous quantitative assay of 1,4-dihydropyridine calcium antagonist manidipine and its main photodegradation by-product has been defined by using a multivariate calibration on UV spectra based on a classical least squares regression. Optimization of the procedure was achieved by means of a calibration model using suitable wavelength ranges singled out from a fractionation scheme thereat defined. Recovery values of 99 and 96% for the drug and the by-product, respectively, were found either in appropriately prepared mixtures and commercial formulations. Quantification limit for the photoproduct concentration was estimated as 1.0% for the reference samples.

Photo- and radiostability of some 1,4-dihydropyridine derivatives in the solid state.

The effect of UV (254 nm) and beta-irradiation at doses 10-100 kGy on physical and chemical properties of four 1,4-dihydropyridyne derivatives in the solid state (nifedipine, nitrendipine, felodipine and nimodipine) has been studied. It has been established that the irradiation causes the appearance of free radicals (EPR) and decomposition products (TLC, GC), a decrease in the melting point (DSC) and the content (GLC, IR) and an increase in microbiological purity. The changes appear faster under the effect of UV than by beta-irradiation and their intensity and character depend on the derivative and dose. The most sensitive to UV irradiation is nifedipine (about 300 times more sensitive than the most resistant nimodipine), while the most sensitive to beta-irradiation is felodipine (about twice as sensitive as the most resistant nitrendipine). The results of our study indicate that 1,4-dihydropyridyne derivatives in solid state can be safely subjected to sterilization by beta-irradiation at doses < 20 kGy.

The effect of ionizing radiation on some derivatives of 1,4-dihydropyridine in the solid state.

The effect of gamma and beta radiation in doses between 10 and 100 kGy on physico-chemical properties of four derivatives of 1,4-dihydropyridine (nifedipine, nitrendipine, felodipine and nimodipine) in the solid state was analysed. A number of qualitative and quantitative methods such as UV, IR, TLC, GLC, DSC, EPR as well as organoleptic and gravimetric analysis were used to determine and analyse any changes resulting from irradiation. In order to determine the effectiveness of sterilization with ionizing radiation of doses from 10 to 25 kGy, various microbiological tests were used. It was established that only doses 10-20 kGy of both kinds of radiation ensure total sterilization without any degradation of physico-chemical properties of the compounds studied. For the doses 50-100 kGy a decrease in the content of the compounds, appearance of the products of their decomposition and changes in the melting point and IR spectra appeared. Felodipine (with chlorophenyl substituent) was found to be much more sensitive to ionising radiation than nifedipine, nitrendipine and nimodipine (all with nitrophenyl substituent).

Photodegradation studies on lacidipine in solution: basic experiments with a cis-trans reversible photoequilibrium under UV-A radiation exposure.

The photostability of Lacidipine, a dihydropyridine drug used in the treatment of mild and moderate hypertension, was studied in solutions exposed to UV-A radiations. The effects of the solvent (ethanol, acetone, dichloromethane), drug concentration and radiation wavelength on the drug photostability were evaluated. Lacidipine and its photoproducts were separated by a selective liquid chromatographic (HPLC) method, under normal phase conditions (CN-column), using n-hexane:ethanol 97:3 (v/v) as mobile phase, at a flow rate of 2.0 ml/min. The main photodegradation products were isolated and characterised and a photodegradation pathway was proposed for Lacidipine in solution. The cis-isomer and a photocyclic isomer proved to be the main photodegradation products.

Photodecomposition of a new 1,4-dihydropyridine: furnidipine.

The photodecomposition of new 1,4-dihydropyridine, furnidipine (CRE-319); [2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-tetrahydrofurfuryl 5-methyl diester) was studied by voltammetric, UV-vis spectrophotometric, and HPLC technique with three different light conditions (artificial daylight, UV light, and room daylight). The artificial daylight photodecomposition of furnidipine follows 0.5-order kinetics as assessed by the above-mentioned techniques. Furthermore, the daylight photoderivative was isolated and identified by NMR and IR as 2,6-dimethyl-4-(2-nitros-ophenyl)pyridine-3,5-dicarboxylic acid 3-tetrahydrofurfuryl 5-methyl diester. Quantitative kinetic data for the UV photodecomposition of furnidipine cannot be obtained due to both the high rate of degradation (< 1 min) and intermediate reactions. However, polarographic, spectroscopic, and chromatographic evidence permits us to identify this photoproduct as 2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 3-tetrahydrofurfuryl 5-methyl diester. Polarography was the most useful technique to assess the photodegradation of this drug from the qualitative point of view, and quantitative kinetic data were similar to those obtained by HPLC and UV-vis spectroscopy.

New 4-alkyl-1,4-dihydropyridines: evaluation of photostability and phototoxic potential.

The photostability and phototoxic potential of two new 4-alkyl-1,4-dihydropyridines (PCA-4230 and PCA-4248) were investigated. When these 4-alkyl-1,4-dihydropyridines were irradiated with a multilamp photoreactor (band centred at 350 nm), both exhibited a slow photodegradation showing first-order kinetics. The photodegradation rate constants were 0.37 h-1 for PCA-4248 and 0.39 h-1 for PCA-4230 in oxygenated conditions. The photodecomposition was slower for both drugs in the absence of oxygen. In order to evaluate the phototoxicity induced by these drugs, red blood cells and Hep-2 (human laringo carcinoma cell line) were irradiated using a minisolarium, which emits UVA radiation (350-390 nm). The results showed that PCA-4248 and PCA-4230 did not exhibit a phototoxic effect in the two models tested.