Determination of enantiomeric purity of a novel COX-2 anti-inflammatory drug by capillary electrophoresis using single and dual cyclodextrin systems

E-6087 is the most advanced compound among the cyclooxygenase-2 (COX-2) inhibitor drugs developed in our company. Its activity is mainly associated with the S(-)-enantiomer (E-6232), whereas the R(-)-enantiomer (E-6231) becomes an impurity whose content should be determined. Five main impurities and degradation products of E-6232 have been found (E-6144, E-6024, E-6072, E-6397 and E-6132), and some of them co-elute with the distomer when using a chiral high-performance liquid chromatography (HPLC) method. Consequently, we have optimized the separation of all the impurities from the two enantiomers of E-6087 by capillary electrophoresis (CE), in order to use the method for the enantiomeric purity determination of E-6232. The effect of the methanol (MeOH) content in the background electrolyte (BGE), the sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD) and heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) concentration, and the capillary temperature have been studied. Separation of all compounds could be achieved in different systems, either in a single CD-system (with SBE-beta-CD) or in a dual CD-system (with DM-beta-CD as a neutral CD). By using the dual CD system a limit of detection (LOD) and a limit of quantitation (LOQ) of 0.03% and 0.1% of distomer, respectively, were achieved*.     

Unravelling the intrinsic features of NO binding to iron(II)- and iron(III)-hemes

Electrospray ionization of appropriate precursors is used to deliver [Fe (III)-heme] (+) and [Fe (II)-hemeH] (+) ions as naked species in the gas phase where their ion chemistry has been examined by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. In the naked, four-coordinate [Fe (II)-hemeH] (+) and [Fe (III)-heme] (+) ions, the intrinsic reactivity of iron(II)- and iron(III)-hemes is revealed free from any influence due to axial ligand, counterion, or solvent effects. Ligand (L) addition and ligand transfer equilibria with a series of selected neutrals are attained when [Fe (II)-hemeH] (+), corresponding to protonated Fe (II)-heme, is allowed to react in the FT-ICR cell. A Heme Cation Basicity (HCB) ladder for the various ligands toward [Fe (II)-hemeH] (+), corresponding to -Delta G degrees for the process [Fe (II)-hemeH] (+) + L --> [Fe (II)-hemeH(L)] (+) and named HCB (II), can thus be established. The so-obtained HCB (II) values are compared with the corresponding HCB (III) values for [Fe (III)-heme] (+). In spite of pronounced differences displayed by various ligands, NO shows a quite similar HCB of about 67 kJ mol (-1) at 300 K toward both ions, estimated to correspond to a binding energy of 124 kJ mol (-1). Density Functional Theory (DFT) computations confirm the experimental results, yielding very similar values of NO binding energies to [Fe (II)-hemeH] (+) and [Fe (III)-heme] (+), equal to 140 and 144 kJ mol (-1), respectively. The kinetic study of the NO association reaction supports the equilibrium HCB data and reveals that the two species share very close rate constant values both for the forward and for the reverse reaction. These gas phase results diverge markedly from the kinetics and thermodynamic behavior of NO binding to iron(II)- and iron(III)-heme proteins and model complexes in solution. The requisite of either a very labile or a vacant coordination site on iron for a facile addition of NO to occur, suggested to explain the bias for typically five-coordinate iron(II) species in solution, is fully supported by the present work.     

The conformational free energy landscape of beta-D-glucopyranose. Implications for substrate preactivation in beta-glucoside hydrolases

Using ab initio metadynamics we have computed the conformational free energy landscape of beta-D-glucopyranose as a function of the puckering coordinates. We show that the correspondence between the free energy and the Stoddard's pseudorotational itinerary for the system is rather poor. The number of free energy minima (9) is smaller than the number of ideal structures (13). Moreover, only six minima correspond to a canonical conformation. The structural features, the electronic properties, and the relative stability of the predicted conformers permit the rationalization of the occurrence of distorted sugar conformations in all the available X-ray structures of beta-glucoside hydrolase Michaelis complexes. We show that these enzymes recognize the most stable distorted conformers of the isolated substrate and at the same time the ones better prepared for catalysis in terms of bond elongation/shrinking and charge distribution. This suggests that the factors governing the distortions present in these complexes are largely dictated by the intrinsic properties of a single glucose unit.     

Toeplitz operators on weighted Hardy and Besov spaces

We obtain estimates of the norm of Toeplitz operators on weighted Hardy and Besov spaces. As an application we give characterizations of some spaces of pointwise multipliers.     

Littlewood-Paley Formulas and Carleson Measures for Weighted Fock Spaces Induced by A ∞ $A_{\infty }$ -Type Weights

Potential Analysis - We obtain Littlewood-Paley formulas for Fock spaces ${\mathcal {F}}_{\beta ,\omega }^{q}$ induced by weights $\omega \in {A}_{\infty }^{restricted} = \cup _{1 \le p <...     

Trace Inequalities of Sobolev Type in the Upper Triangle Case

We give a new non-capacitary characterization of positive Borelmeasures µ on Rⁿ such that the potential space I*L^p isimbedded in L^q(dµ) for $1qp+, that is, the trace inequality holds, for Riesz potentials I = (- )². A weak-type trace inequality is also characterized. A non-isotropic version on the unit sphere Sⁿ is studied,as well as the holomorphic case for Hardy–Sobolev spaces in the ball. 1991 MathematicsSubject Classification: primary 31C15, 42B20; secondary 32A35.     

Photo-oxygenation of Styrenic Estrogens: Structural Analysis of 8,9-Didehydro-, 6,7-Didehydro-, and 9,11-Didehydroestrone Derivatives and Their Reactivity towards Singlet Oxygen

A series of didehydro-3-O-methyl-estrones having a styrenic framework, with the ring-A-conjugated double bond in all three possible positions (8,9-didehydro- ( 6 ),9,11-didehydro- ( 1b ), 6,7-didehydro- ( 9 ), and the 12,18-di-nor-8,9-didehydroestrone analog 11 ), were compared for their reactivity towards singlet oxygen. Under dye-sensitized photo-oxygenation conditions, both, products derived from ene-type reactions with the isolation of a stable hydroperoxide and a fragmentation product, were obtained from 6 (see Scheme 3), while only fragmentation took place for 1b (Scheme 1), Kinetic studies indicated that 6 is more reactive towards ¹O₂ than 1b (β = 9.2·10^?3 mol·1^?1 vs 3.3·10^?2 mol·1^?1, resp.). The observed reactivity, apparently, does not match with ene-type reaction and [2 + 2]cycloaddition being in competition, since the most activated substrate 6 preferentially yields ene-type products and their derivatives. Conformational analysis on the structure of 6 and 1b , both calculated by molecular-mechanic techniques (MMPMI) and determined by X-ray diffraction, show that the allylic H-atoms satisfy the orthogonality rule for ene-type reactions. The product distribution is best rationalized by applying Fukui's rule which takes into account a combination of electronic and geometric factors. Substrates 9 and 11 yielded photo-products arising from ene-type reaction with no stable primary products isolated (Scheme 4). Geometric considerations based on the calculated structures by molecular mechanics are consistent with the observed results.     

On Integral Equations Related to Weighted Toeplitz Operators

For weighted Toeplitz operators T^N_j{{\mathcal T}^N_\varphi} defined on spaces of holomorphic functions in the unit ball, we derive regularity properties of the solutions f to the equation T^N_j(f)=h{{\mathcal T}^N_\varphi(f)=h} in terms of the regularity of the symbol φ and the data h. As an application, we deduce that if f\not o 0{f\not\equiv0} is a function in the Hardy space H ¹ such that its argument [`(f)]/f{\bar f/f} is in a Lipschitz space on the unit sphere \mathbb S{{\mathbb S}}, then f is also in the same Lipschitz space, extending a result of Dyakonov to several complex variables.     

On Weighted Toeplitz, Big Hankel Operators and Carleson Measures

We compute the norm of pointwise multiplication operators, Toeplitz and Big Hankel operators with antiholomorphic symbols, defined on Besov spaces. These norms will be given in terms of Carleson measures for Besov spaces related to the symbol.