Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 µM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

Advantages of Induced Circular Dichroism Spectroscopy for Qualitative and Quantitative Analysis of Solution-Phase Cyclodextrin Host-Guest Complexes.

The presence of a chiral or chirally perturbed chromophore in the molecule under investigation is a fundamental requirement for the appearance of a circular dichroism (CD) spectrum. For native and for most of the substituted cyclodextrins, this condition is not applicable, because although chiral, cyclodextrins lack a chromophore group and therefore have no characteristic CD spectra over 220 nm. The reason this method can be used is that if the guest molecule has a chromophore group and this is in the right proximity to the cyclodextrin, it becomes chirally perturbed. As a result, the complex will now provide a CD signal, and this phenomenon is called induced circular dichroism (ICD). The appearance of the ICD spectrum is clear evidence of the formation of the complex, and the spectral sign and intensity is a good predictor of the structure of the complex. By varying the concentration of cyclodextrin, the ICD signal changes, resulting in a saturation curve, and from these data, the stability constant can be calculated for a 1:1 complex. This article compares ICD and NMR spectroscopic and molecular modeling results of cyclodextrin complexes of four model compounds: nimesulide, fenbufen, fenoprofen, and bifonazole. The results obtained by the different methods show good agreement, and the structures estimated from the ICD spectra are supported by NMR data and molecular modeling.

Effect of Substitution Degree and Homogeneity on Cyclodextrin-Ligand Complex Stability: Comparison of Fenbufen and Fenoprofen Using CD and NMR Spectroscopy.

The stability of host-guest complexes of two NSAID drugs with similar physicochemical properties, fenbufen and fenoprofen, was investigated by comparing induced circular dichroism and 1H nuclear magnetic resonance methods using eight cyclodextrins of different degrees of substitution and isomeric purity as guest compounds. These cyclodextrins include native ß-cyclodextrin (BCyD), 2,6-dimethyl-ß-cyclodextrin 50 (DIMEB50), 80 (DIMEB80) and 95% (DIMEB95) isomerically pure versions, low-methylated CRYSMEB, randomly methylated ß-cyclodextrin (RAMEB) and 4.5 and 6.3 average substitution grade hydroxypropyl-ß-cyclodextrin (HPBCyD). The stability constants obtained by the two methods show good agreement in most cases. For fenbufen complexes, there is a clear trend that the stability constant increases with the degree of substitution while isomer purity has a smaller effect on the magnitude of stability constants. A significant difference was found in the case of DIMEB50 when compared to DIMEB80/DIMEB95, while the latter two are similar. In the fenbufen-fenoprofen comparison, fenbufen, with its linear axis, gives a more stable complex, while fenoprofen shows lower constants and poorly defined trends.

Cyclodextrin encapsulation enabling the anticancer repositioning of disulfiram: Preparation, analytical and in vitro biological characterization of the inclusion complexes.

Drug repositioning is a high-priority and feasible strategy in the field of oncology research, where the unmet medical needs are continuously unbalanced. Disulfiram is a potential non-chemotherapeutic, adjuvant anticancer agent. However, the clinical translation is limited by the drug's poor bioavailability. Therefore, the molecular encapsulation of disulfiram with cyclodextrins is evaluated to enhance the solubility and stability of the drug. The present work describes for the first time the complexation of disulfiram with randomly methylated-ß-cyclodextrin. A parallel analytical andin vitrobiological comparison of disulfiram inclusion complexes with hydroxypropyl-ß-cyclodextrin, randomly methylated-ß-cyclodextrin and sulfobutylether-ß-cyclodextrin is conducted. A significant drug solubility enhancement by about 1000-folds and fast dissolution in 1 min is demonstrated. Thein vitrodissolution-permeation studies and proliferation assays demonstrate the solubility-dependent efficacy of the drug. Throughout the different cancer cell lines' characteristics and disulfiram unspecific antitumoral activity, the inhibitory efficacy of the cyclodextrin encapsulated drug on melanoma (IC50 about 100 nM) and on glioblastoma (IC50 about 7000 nM) cell lines differ by a magnitude. This pre-formulation screening experiment serves as a proof of concept of using cyclodextrin encapsulation as a platform tool for further drug delivery development in repositioning areas.

Novel compstatin family peptides inhibit complement activation by drusen-like deposits in human retinal pigmented epithelial cell cultures.

We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment.

NMR assignments and the acid-base characterization of the pomegranate ellagitannin punicalagin in the acidic pH-range.

In exploring the capability of nuclear magnetic resonance (NMR) spectroscopy for pomegranate juice analysis, the eight aromatic singlet resonances of α- and ß-punicalagin were clearly identified in the (1)H NMR spectra of juice samples. The four downfield resonances were found to be sensitive to small pH changes around pH 3.50 where the NMR spectra of the juice samples were recorded. To understand this unusual behavior, the (1)H and (13)C resonance assignments of the punicalagin anomers were determined in aqueous solution and pH titrations with UV and (1)H NMR detection carried out to characterize the acid-base properties of punicalagin over the pH range 2-8. Simultaneous fitting of all of the pH-sensitive (1)H NMR signals produced similar but significantly different pKa values for the first two deprotonation equilibria of the gallagic acid moiety of the punicalagin α- (pKa1 = 4.57 ± 0.02, pKa2 = 5.63 ± 0.03) and ß- (pKa1 = 4.36 ± 0.01, pKa2 = 5.47 ± 0.02) anomers. Equivalent pKa values, (α : 6.64 ± 0.01, ß : 6.63± 0.01) were measured for the third deprotonation step involving the ellagic acid group, in good agreement with a prior literature report. The punicalagin anomer equilibrium readjusts in parallel with the proton dissociation steps as the pH is raised such that ß-punicalagin becomes the most abundant anomer at neutral pH. The unusual upfield shifts observed for the glucose H3 and H5 resonances with increasing pH along with the shift in the α/ß anomer equilibrium are likely the consequence of a conformational rearrangement.

Population, basicity and partition of short-lived conformers. Characterization of baclofen and pregabalin, the biaxial, doubly rotating drug molecules.

Populations, protonation constants and octanol-water partition coefficients were determined and assigned specifically to fast interconverting individual conformers, exemplified in baclofen and pregabalin, the GABA-related drug molecules of biaxial, double rotations. Rotamer statuses along both axes in water and octanol were elucidated from 1H NMR vicinal coupling constants. Conformer abundances were obtained by the appropriate combination of the rotamer populations in the two adjacent moieties in the molecule. The bulky aromatic group in baclofen versus the aliphatic side chain of pregabalin explains why baclofen exists mainly in trans-trans conformeric form, throughout the pH range, unlike pregabalin that has no any highly dominant form. Characteristically enough, for pregabalin, the lipophilicity of the conformers is primarily influenced by the conformation state. Conformers in gauche state are of higher lipophilicity. The conformers of the two compounds were ranked by their membrane-influx and -outflow propensities.

Physico-chemical profiling of semisynthetic opioids.

Species-specific acid-base and partition equilibrium constants were experimentally determined for the therapeutically important semisynthetic opioid receptor agonist hydromorphone, dihydromorphine, and mixed agonist-antagonist nalorphine and nalbuphine. The acid-base microequilibria were characterized by combining pH-potentiometry and deductive methods using synthesized auxiliary compounds. Independent of the pH, there are approximately 4.8 times as many zwitterionic microspecies than non-charged ones in nalbuphine solutions, while for nalorphine it is the non-charged form that predominates by the same ratio. The non-charged microspecies is the dominant one also in the case of hydromorphone, although its concentration exceeds only 1.3 times that of its zwitterionic protonation isomer. The pH-independent partition coefficients of the individual microspecies were determined by a combination of experimentally measured, pH-dependent, conditional distribution constants and a custom-tailored evaluation method, using highly similar auxiliary compounds. The pH-independent contribution of the zwitterionic microspecies to the distribution constant is 1380, 1070, 3160 and 72,440 times smaller than that of the inherently more lipophilic non-charged one for hydromorphone, dihydromorphine, nalbuphine and nalorphine, respectively.

Characterization of ester hydrolysis in terms of microscopic rate constants.

Hydroxide-catalyzed ester hydrolysis for molecules of coexisting species is quantitated in terms of microscopic rate constants, a new, species-specific physicochemical parameter. Relationships between the overall and component reactions, as well as the macroscopic and microscopic rate constants are deduced. Experimental techniques, evaluation methods, and feasibility are discussed. Species-specific, pH-independent rate constants of four coexisting, differently hydrolyzing microspecies are determined for the first time. Protonation of an alpha-amino and beta-imidazolyl site in amino acid esters has been found to accelerate the hydroxide-catalyzed hydrolysis by factors of 120 and 7.5, respectively, whereas they jointly exert a nearly 3000-fold acceleration. A total of 20 microscopic protonation equilibrium constants, as component parameters in the rate equations, have also been determined. The species-specific rate constants have been found to correlate with the site- and species-specific basicity of the leaving group and the NMR chemical shift of an adjacent proton. Individual contributions of the various microforms to the overall hydrolysis rate are depicted in microscopic reaction fraction diagrams.

Site- and species-specific hydrolysis rates of heroin.

The hydroxide-catalyzed non-enzymatic, simultaneous and consecutive hydrolyses of diacetylmorphine (DAM, heroin) are quantified in terms of 10 site- and species-specific rate constants in connection with also 10 site- and species-specific acid-base equilibrium constants, comprising all the 12 coexisting species in solution. This characterization involves the major and minor decomposition pathways via 6-acetylmorphine and 3-acetylmorphine, respectively, and morphine, the final product. Hydrolysis has been found to be 18-120 times faster at site 3 than at site 6, depending on the status of the amino group and the rest of the molecule. Nitrogen protonation accelerates the hydrolysis 5-6 times at site 3 and slightly less at site 6. Hydrolysis rate constants are interpreted in terms of intramolecular inductive effects and the concomitant local electron densities. Hydrolysis fraction, a new physico-chemical parameter is introduced and determined to quantify the contribution of the individual microspecies to the overall hydrolysis. Hydrolysis fractions are depicted as a function of pH.

Determination of conformer-specific partition coefficients in octanol/water systems.

The first conformer-specific experimental partition coefficients are presented for octanol/water, the most widespread solvent system to predict lipophilicity of drugs. Rotamer populations in octanol and water were elucidated from 1H NMR vicinal coupling constants and were combined with classical partition coefficients to obtain the conformer-specific ones. Feasibility of the determination of conformer-specific partition coefficients is exemplified on amphetamine and clenbuterol, two flexible drug molecules. Partition capacities of the amphetamine rotamers have been proven to be essentially equal. The conformers of clenbuterol, however, have been found to be greatly different in partition properties, which could be interpreted in terms of intramolecular interactions between the vicinal polar sites and the solvent-accessibility of the groups. The conformers could be put into order of their membrane-influx and -outflow propensities. Deviations between experimental and calculated log P values could also be interpreted in view of the species-specific partition coefficients.

The interaction of enoxaparin and fondaparinux with calcium.

The main sites of calcium binding were determined for the low molecular weight heparin drug enoxaparin and the synthetic pentasaccharide Arixtra (fondaparinux). [(1)H,(13)C] HSQC pH titrations were carried out to characterize the acid-base properties of these samples both in the presence and absence of calcium. The differences in the titration curves were used to determine the structural components of enoxaparin and fondaparinux responsible for Ca(2+) binding. In enoxaparin both unsubstituted and 2-O-sulfated iduronic acid residues are important in calcium binding and the presence of the 2-O-sulfo group does not seem to influence the Ca(2+) binding capability of the iduronate ring. In fondaparinux changes in chemical shifts upon Ca(2+) binding were smaller than observed for enoxaparin, and were observed for both the glucuronic acid and 2-O-sulfated iduronic acid residues. In enoxaparin significant perturbations of the chemical shift of the N-sulfoglucosamine anomeric carbon in residues connected to 2-O-sulfated iduronic acid were detected on Ca(2+) binding, however it was not possible to determine whether these changes reflect direct involvement in calcium complexation or result from through space interactions or conformational changes.

Triprotic site-specific acid-base equilibria and related properties of fluoroquinolone antibacterials.

The complete macro- and microequilibrium analyses of six fluoroquinolone drugs - ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, ofloxacin and moxifloxacin - are presented. Previous controversial literature data are straightened up, the protonation centers are unambiguously identified, and the protonation macro- and microconstant values are reported. The macroconstants were determined by (1)H NMR-pH titrations while the microconstants were determined by a multi-modal spectroscopic-deductive methodology, in which methyl ester derivatives were synthesized and their NMR-pH titration data contributed to the evaluation of all the microconstants. The full (1)H, (13)C and (15)N NMR assignments, NMR-pH profiles, macro- and microprotonation schemes and species-specific diagrams are included. Our studies show that the fluoroquinolones have three protonation centers: the carboxylate group, the N-1' and N-4' piperazine nitrogens and concentration of the uncharged microspecies is way below the values published earlier. The results could be well interpreted in terms of structural properties. The protonation macro- and microconstant values allow the pre-planned method development in techniques such as capillary zone electrophoresis and also, the interpretation of fluoroquinolone mechanism of biological action, including the pharmacokinetic properties, and antibacterial activities that are all heavily influenced by the states of protonation.

Determination of microscopic acid-base parameters from NMR-pH titrations.

The theory and practice of proton microspeciation based on NMR-pH titrations are surveyed. Principles of bi-, tri-, tetra-, and n-protic microequilibrium systems are discussed. Evaluation methods are exemplified by case studies on bi- and tetraprotic biomolecules. Selection criteria and properties of 'reporter' NMR nuclei are described. Literature data on complete microspeciations of small ligands and site-specific basicity characterizations of peptides and proteins are critically reviewed.

Determination of rotamer populations and related parameters from NMR coupling constants: a critical review.

Methods to determine rotamer populations from NMR homo- and heteronuclear vicinal coupling constants are reviewed. Theory and practice related to the elucidation of various gauche and trans limiting coupling constants as key parameters in the characterization of discrete rotational isomers are discussed. Properties and capacities of Karplus-type equations are assessed. Continuous models of rotational isomerism are compared to discrete ones. Principles of the highly specific physicochemical parameters rotamer-specific basicity and rotamer-specific partition coefficient are also described.

Interaction of indomethacin and ciprofloxacin in the cornea following phototherapeutic keratectomy.

BACKGROUND: We report a case in which stromal deposits were produced due to drug interaction in a 61-year-old patient following phototherapeutic keratectomy (PTK). METHODS: In our case, the patient herself added the use of indomethacin eye drops to the prescribed topical antibiotic treatment and eye gel (5x ciprofloxacin plus 3x indomethacin, eye gel overnight) following PTK. At 5 days the central stroma was not reepithelialised; instead, whitish, round deposits were found in the stroma. Mixing indomethacin and ciprofloxacin eye drops 1:1, the pH of the mixture changed to 5.6, and a yellow precipitate was formed; this was analysed by (1)H-NMR spectroscopy. To investigate the solubility of the two drugs at the pH of the mixture, the pH of each solution was independently set to 5.6 by addition of hydrochloric acid or sodium hydroxide. RESULTS: In the precipitate both indomethacin and ciprofloxacin were detected. After setting the pH to 5.6, a yellow precipitate was observed in the indomethacin solution; however, the ciprofloxacin solution remained clear. CONCLUSIONS: Interaction may occur if ciprofloxacin and indomethacin are used together. It is better to avoid the use of the two drugs at the same time, particularly in the case of a large epithelial defect when stromal deposition of the drugs may be produced.