The Role of Hidden Conformers in Determination of Conformational Preferences of Mefenamic Acid by NOESY Spectroscopy

Abstract

Mefenamic acid has been used as a non-steroidal anti-inflammatory drug for a long time. However, its practical use is quite limited due to a number of side effects on the intestinal organs. Conformational polymorphism provides mefenamic acid with unique properties regarding possible modifications obtained during the micronization process, which can improve pharmacokinetics and minimize side effects. Micronization can be performed by decompression of supercritical fluids; methods such as rapid expansion of the supercritical solution have proven their efficiency. However, this group of methods is poorly applicable for compounds with low solubility, and the modification of the method using a pharmaceutically suitable co-solvent may be useful. In our case, addition of only 2 mol% dimethyl sulfoxide increased the solubility remarkably. Information on the conformational state may be critically important for carrying out micronization. In this work, structural analysis and estimate of conformational preferences of mefenamic acid in dimethyl sulfoxide-d₆ (at 25 °C and 0.1 MPa) and in a mixed solvent supercritical carbon dioxide + dimethyl sulfoxide-d₆ (45 °C, 9 MPa) were performed based on nuclear Overhauser effect spectroscopy. Results show changes in the conformation fractions depending on the medium used. The importance of allowing for hidden conformers in estimating the conformational state was demonstrated in the analysis. Obtained results may be useful for improving micronization parameters.

Keywords: fenamates; high-pressure NMR; spatial structure; supercritical fluid.

Figure 1

Scheme of the installation of high pressure equipment for creating and maintaining high pressure NMR experiments for the supercritical CO₂. 1—gas cylinde; 2,5,8—pressure transmitters; 3,6—high-pressure valves; 4—hand press; 7—hand valves.

Figure 2

Structural formula of MFA. Atom numbering is used below for labeling signals and cross-peaks in NMR spectra and for denoting dihedral angles. Chemical bonds forming the dihedral angle τ₁[C₂-N-C₃-C₇] are shown in red; τ₂[O=C₁-C₁₃-C₆], in blue.

Figure 3

Structure of the MFA conformers present in polymorphic forms I (black) [49], II (red) [28], and III (green) [46].

Figure 4

Structures of the MFA conformers and dihedral angles τ₁[C₂-N-C₃-C₇] (red) and τ₂[O=C₁-C₁₃-C₆] (blue).

Figure 5

¹H-¹H NOESY NMR spectrum of MFA in DMSO-d₆ (a). Observed cross-peaks correspond to hydrogen atoms separated by a distance less than 5 Å. Atom numbering is shown in the right panel of the figure (b).

Figure 6

Integral intensity as a function of the mixing time for the conformation-dependent (red and green lines) and reference (blue) distances, obtained by analysis of NOESY spectra measured for MFA in DMSO-d₆ (a) and scCO₂ + DMSO-d₆ at 45°, 9 MPa (b).

Figure 7

Distribution (a) and population (b) of conformers of MFA in DMSO-d₆ calculated from the observed conformation-dependent distance OH-H6.

Figure 8

Structure of MFA conformers AA (a) and BB (b) and dihedral angles τ₁[C₂-N-C₃-C₇] (red), τ₂[O=C₁-C₁₃-C₆] (blue), and τ₃[O=C₁-O-H] (green arrow).

Figure 9

Distribution of conformers of MFA in DMSO-d₆ calculated from the observed conformation-dependent distance OH-H6 (a) without and (b) allowing for the existence of additional conformers AA and BB. Figure 9a is a duplicate of Figure 7a for comparison.

Figure 10

Distribution of conformers of MFA in scCO₂ + DMSO-d₆ at 9 MPa calculated from the observed conformation-dependent distance OH-H6 (a) without and (b) allowing for the existence of additional conformers AA and BB.

Figure 11

Relative conformer group fractions of MFA in (a–c) DMSO-d₆ and (d–f) scCO₂ + DMSO at supercritical parameters of state, calculated from experimentally found distances OH-H6 and H9/10-H11/12.