Clofazimine pKa Determination by Potentiometry and Spectrophotometry: Reverse Cosolvent Dependence as an Indicator of the Presence of Dimers in Aqueous Solutions.

The weakly basic antibiotic and anti-inflammatory drug, clofazimine (CFZ), was first described in 1957. It has been used therapeutically, most notably in the treatment of leprosy. However, the compound is extremely insoluble in aqueous media, and, indeed, there is poor consensus about what its intrinsic solubility is since the reported values range from 0.04 to 11 ng/mL. To understand the speciation and solubilization of CFZ as a function of pH, it is of paramount importance to know the true aqueous pKa. However, there is also poor consensus about the value of the pKa (reported measured values range from 6.08 to 9.11). In the present study, we report the determination of the CFZ ionization constant using two independent techniques. A state-of-the-art potentiometric analysis was performed, drawing on titration data in methanol-water solutions (46-75 wt % MeOH) of CFZ, using the bias-reducing consensus of two different procedures of extrapolating the apparent psKa values to zero cosolvent to approximate the true aqueous pKa as 9.43 ± 0.12 (25 °C, I = 0.15 M reference ionic strength). In parallel, spectrophotometric UV/vis titration data were acquired (250-600 nm at different pH) in 10 mM HEPES buffer solutions containing up to 54 wt % MeOH. The alternating least squares (ALS) method was used in the analysis of the absorbance-pH spectra. Uncharacteristically, the cosolvent UV/vis data in our study showed reverse cosolvent dependence (apparent pKa values increased with increasing cosolvent) which could be explained by a dimerization of the free base. The analysis of UV/vis data obtained from 54 wt % MeOH-water solution containing 20 µM CFZ yielded the apparent pKa 9.51 ± 0.17 (I ≈ 0.005 M). To assess whether self-assembly of CFZ was energetically feasible, density functional theory (DFT) calculations were used to study the putative CFZ dimers in aqueous and methanol media. The DFT-optimized geometries and infrared spectra of CFZ dimers using water and methanol as solvents were calculated and analyzed. Based on the lack of negative frequencies in calculated infrared spectra, it was confirmed that optimized geometries correspond to the true energetic minima. Visual analysis of optimized structures indicates the presence of stacking interactions between two CFZ molecules. The protonation site (the imine nitrogen atom) was determined by 1H NMR spectroscopy.

Nortriptyline Hydrochloride Solubility-pH Profiles in a Saline Phosphate Buffer: Drug-Phosphate Complexes and Multiple pHmax Domains with a Gibbs Phase Rule "Soft" Constraints.

The solubility of a model basic drug, nortriptyline (Nor), was investigated as a function of pH in phosphate and/or a chloride-containing aqueous suspension using experimental practices recommended in the previously published "white paper" (Avdeef et al., 2016). The pH-Ramp Shake-Flask (pH-RSF) method, introduced in our earlier work (Markovic et al., 2019), was applied. An improved and more detailed experimental design of the Nor solubility measurement allowed us to exploit the full capacity of the pH-RSF method. Complex equilibria in the aqueous phase (cationic and anionic complex formation between Nor and the phosphate) and solid-phase transformations (Nor free base, 1:1 Nor hydrochloride salt, 1:1 and 1:2 Nor phosphate salts) were characterized by a detailed analysis of the solubility measurements using the computer program pDISOL-X. The solid phases were characterized by thermogravimetric analysis, differential scanning calorimetry, powder X-ray diffraction, and elemental analyses. The results of the present investigation illustrate the influence of competing counterions, such as buffering agents, complexing agents, salt coformers, tonicity adjusters, and so forth, on the aqueous solubility of drugs and interconversion of salts. Careful attention given to these factors can be helpful in the formulation of drug products.

5-Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human carbonic anhydrases IX and XII.

Inhibitory activity of a congeneric set of 23 phenyl-substituted 5-phenyl-pyrazole-3-carboxylic acids toward human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II, IX and XII was evaluated by a stopped-flow CO2 hydrase assay. These compounds exerted a clear, selective inhibition of hCA IX and XII over hCAI and II, with Ki in two to one digit micromolar concentrations (4-50 µM). Derivatives bearing bulkier substituents in para-position of the phenyl ring inhibited hCA XII at one-digit micromolar concentrations, while derivatives having alkyl substituents in both ortho- and meta-positions inhibited hCA IX with Kis ranging between 5 and 25 µM. Results of docking experiments offered a rational explanation on the selectivity of these compounds toward CA IX and XII, as well as on the substitution patterns leading to best CA IX or CA XII inhibitors. By examining the active sites of these four isoforms with GRID generated molecular-interaction fields, striking differences between hCA XII and the other three isoforms were observed. The field of hydrophobic probe (DRY) appeared significantly different in CA XII active site, comparing to other three isoforms studied. To the best of our knowledge such an observation was not reported in literature so far. Considering the selectivity of these carboxylates towards membrane-associated over cytosolic CA isoforms, the title compounds could be useful for the development of isoform-specific non-sulfonamide CA inhibitors.

Investigation into novel thiophene- and furan-based 4-amino-7-chloroquinolines afforded antimalarials that cure mice.

We herein report the design and synthesis of a novel series of thiophene- and furan-based aminoquinoline derivatives which were found to be potent antimalarials and inhibitors of ß-hematin polymerization. Tested compounds were 3-71 times more potent in vitro than CQ against chloroquine-resistant (CQR) W2 strain with benzonitrile 30 being as active as mefloquine (MFQ), and almost all synthesized aminoquinolines (22/27) were more potent than MFQ against multidrug-resistant (MDR) strain C235. In vivo experiments revealed that compound 28 showed clearance with recrudescence at 40 mg/kg/day, while 5/5 mice survived in Thompson test at 160 mg/kg/day.

A novel method of molecular imprinting applied to the template cholesterol.

A novel method is successfully tested for non-covalent imprinting. Conditions are used which practically exclude the formation of prepolymerization complexes. The template is cholesterol, and no so-called functional monomer is used. The polymers contain only an acrylic diester crosslinker. The porogen isopropanol prevents even hydrogen bonding between the template and the monomer in the prepolymerization solution. Despite of these apparently very disadvantageous conditions, appreciable imprinting factors for cholesterol and imprinted selectivity against some other steroids are observed, similar to other cholesterol MIPs with proven analytical usefulness.

Solubility-pH profile of desipramine hydrochloride in saline phosphate buffer: Enhanced solubility due to drug-buffer aggregates.

Although solubility-pH data for desipramine hydrochloride (DsHCl) have been reported previously, the aim of the present study was to critically examine the aqueous solubility-pH behavior of DsHCl in buffer-free and buffered solutions, in the presence of physiologically-relevant chloride concentration, using experimental practices recommended in the recently-published "white paper" (Avdeef et al., 2016). The computer program pDISOL-X was used to design the structured experiments (pH-RSF method), to process the data, and to refine the equilibrium constants. Low-to-high and high-to-low pH assays (using HCl, H3PO4, or NaOH to adjust pH) were performed on phosphate-buffered (0.12­0.15 M) saturated solutions of DsHCl in the pH 1.3-11.6 range. After equilibration (stirring 6 h, followed by 18 h stir-free sedimentation), filtration or centrifugation was used for phase separation. Concentration was measured using HPLC with UV/VIS detection. The 2:1 drug-phosphate solubility product (Ksp2:1 = [DsH+]2[HPO42-]) was determined from data in the pH 4-9 region. The free base of desipramine was prepared and used to determine the Ksp1:1 ([DsH+][H2PO4-]) in chloride-free acidified suspension. In addition, phosphate-free titrations were conducted to determine the intrinsic solubility, S0, and the 1:1 drug-chloride solubility product, KspDsHCl = [DsH+][Cl-]. Under the assay conditions, only the phosphate-free solutions showed some supersaturation near pHmax 8.0. In phosphate-containing solutions, pHmax was indicated at higher pH (8.8-9.6). Oils mixed with solids were observed to form in alkaline solutions (pH > 11). Notably, soluble drug-phosphate complexes appeared to form below pH 3.9 and above pHmax in saturated phosphate­containing saline solutions. This was indicated by the systematic pH shift to higher values in the log S-pH curve in alkaline solution than expected from the Henderson-Hasselbalch equation. For pH < 3.9, saturated phosphate-containing saline solutions exhibited elevated solubility, with drug-hydrochloride as the sole precipitate. Salt solubility products, intrinsic solubility, and complexation constants, which rationalized the data, were determined. Elemental, thermogravimetric (TGA), differential scanning calorimetric (DSC), and powder X-ray diffraction (PXRD) analyses were used to characterize the precipitates isolated from suspensions at different pH.

Human serum albumin binding of certain antimalarials.

Interactions between eight in-house synthesized aminoquinolines, along with well-known chloroquine, and human serum albumin (HSA) have been studied by fluorescence spectroscopy. The synthesized aminoquinolines, despite being structurally diverse, were found to be very potent antimalarials. Fluorescence measurements indicate that three compounds having additional thiophene or benzothiophene substructure bind more strongly to HSA than other studied compounds. Competitive binding experiments indicate that these three compounds bind significantly stronger to warfarin compared to diazepam binding site. Fluorescence quenching at three temperatures (20, 25, and 37°C) was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. The enthalpy and entropy changes upon sulphur-containing compound-HSA interactions were calculated using Van't Hoff equation. Positive values of enthalpy and entropy changes indicate that non-specific, hydrophobic interactions are the main contributors to HSA-compound interaction. Molecular docking and calculated lipophilicity descriptors indicate the same, pointing out that the increased lipophilicity of sulphur-containing compounds might be a reason for their better binding to HSA. Obtained results might contribute to design of novel derivatives with improved pharmacokinetic properties and drug efficacy.

Design, synthesis and biological evaluation of novel aryldiketo acids with enhanced antibacterial activity against multidrug resistant bacterial strains.

Antimicrobial resistance (AMR) is a major health problem worldwide, because of ability of bacteria, fungi and viruses to evade known therapeutic agents used in treatment of infections. Aryldiketo acids (ADK) have shown antimicrobial activity against several resistant strains including Gram-positive Staphylococcus aureus bacteria. Our previous studies revealed that ADK analogues having bulky alkyl group in ortho position on a phenyl ring have up to ten times better activity than norfloxacin against the same strains. Rational modifications of analogues by introduction of hydrophobic substituents on the aromatic ring has led to more than tenfold increase in antibacterial activity against multidrug resistant Gram positive strains. To elucidate a potential mechanism of action for this potentially novel class of antimicrobials, several bacterial enzymes were identified as putative targets according to literature data and pharmacophoric similarity searches for potent ADK analogues. Among the seven bacterial targets chosen, the strongest favorable binding interactions were observed between most active analogue and S. aureus dehydrosqualene synthase and DNA gyrase. Furthermore, the docking results in combination with literature data suggest that these novel molecules could also target several other bacterial enzymes, including prenyl-transferases and methionine aminopeptidase. These results and our statistically significant 3D QSAR model could be used to guide the further design of more potent derivatives as well as in virtual screening for novel antibacterial agents.

New Steroidal 4-Aminoquinolines Antagonize Botulinum Neurotoxin Serotype A in Mouse Embryonic Stem Cell Derived Motor Neurons in Postintoxication Model.

The synthesis and inhibitory potencies against botulinum neurotoxin serotype A light chain (BoNT/A LC) using in vitro HPLC based enzymatic assay for various steroidal, benzothiophene, thiophene, and adamantane 4-aminoquinoline derivatives are described. In addition, the compounds were evaluated for the activity against BoNT/A holotoxin in mouse embryonic stem cell derived motor neurons. Steroidal derivative 16 showed remarkable protection (up to 89% of uncleaved SNAP-25) even when administered 30 min postintoxication. This appears to be the first example of LC inhibitors antagonizing BoNT intoxication in mouse embryonic stem cell derived motor neurons (mES-MNs) in a postexposure model. Oral administration of 16 was well tolerated in the mouse up to 600 mg/kg, q.d. Although adequate unbound drug levels were not achieved at this dose, the favorable in vitro ADMET results strongly support further work in this series.

Synthesis and characterisation of bismacrocyclic DO3A-amide derivatives - an approach towards metal-responsive PARACEST agents.

Three new bismacrocyclic Ln(3+) chelates consisting of triamide derivatives of cyclen with glycine, methyl and tert-butyl substituents (, respectively) linked to an acyclic EGTA-derived calcium chelator were synthesised as potential MRI contrast agents (EGTA - ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Eu(3+) and Yb(3+) complexes of were investigated as chemical exchange saturation transfer (CEST) agents. Moderate to minor CEST effects were observed for , and complexes in the absence of Ca(2+), with negligible changes upon addition of this metal ion. Luminescence steady-state emission and lifetime experiments did not reveal any changes in the coordination environment of the complexes, while the number of inner-sphere water molecules remained constant in the absence and presence of Ca(2+). The protonation constants of and and stability constants of their complexes with Ca(2+), Mg(2+) and Zn(2+) were determined by means of potentiometric titrations. The results show that the charge of the complex dramatically affects the protonation constants of the EGTA-binding unit. The stability constants of the complexes formed with Ca(2+), Mg(2+) and Zn(2+) are several orders of magnitude lower than those of EGTA. These findings indicate that the nature of Ln(3+) chelates and their charge are the main reasons for the observed results and weaker response of these EGTA-derived triamide derivatives compared to their tricarboxylate analogues.