INTERACTION OF CALCIUM-CHANNEL ANTAGONISTS WITH CALCIUM - SPECTROSCOPIC AND MODELING STUDIES ON DILTIAZEM AND ITS CA-2+ COMPLEX

Using spectral techniques, the solution conformation of diltiazem was studied in acetonitrile with special reference to the effect of Ca2+ on the drug structure. Complete assignment of the proton resonances in the H-1-NMR spectrum of the drug was made using one- and two-dimensional spectral analyses. A two-dimensional H-1-NOESY spectrum (in the phase-sensitive mode) was obtained to identify the interproton connectivities in the drug molecule. A molecular modeling program involving Monte Carlo simulation and energy minimization was employed to arrive at the structure of the drug. The program was run with and without the input of the interproton distances derived from the NOESY cross peaks. Both the protocols led to a structure of the drug which was generally similar to that reported from X-ray diffraction data on crystalline diltiazem hydrochloride (Kojic-Prodic, et al. Helv. Chim. Acta 1984, 67, 916-926). However, significant differences between the two structures were seen in the orientations of the substituent groups attached to the benzothiazepine ring. Substantial changes in the circular dichroic (CD) and H-1-NMR spectra of diltiazem were observed on addition of Ca2+ up to a mole ratio of 0.5 Ca2+ per drug. Relatively large changes were seen in H-1 resonances of the N-methyl protons and the methylene protons attached to the heterocyclic nitrogen. Analysis of the binding isotherms from CD data at 22 +/- 1-degrees-C indicated a 2:1 drug:Ca2+ ''sandwich'' complex with an estimated dissociation constant of 140 muM. One-dimensional difference NOE and two-dimensional NOESY spectra revealed interproton connectivities between two drug molecules that were compatible with the sandwich complex formation. The interproton distances derived from the volume integrals of the NOESY cross peaks were used as geometrical constraints in modeling the Ca2+-bound conformation of diltiazem. The minimum-energy conformation corresponded to the sandwich complex where Ca2+ was coordinated to three oxygens in each of the two drug molecules. Combined with our earlier data on the ability of diltiazem to translocate Ca2+ across the lipid bilayer in synthetic liposomes (Ananthanarayanan, V. S.;Taylor, L.; Pirritano, S. Biochem. Cell Biol. 1992, 70, 608-612), the structural data presented here point to a role for Ca2+ in the interaction of diltiazem with its membrane-bound receptor.