Fast 19F Magic Angle Spinning NMR Crystallography for Structural Characterization of Fluorine-Containing Pharmaceutical Compounds

Abstract

Fluorine-containing compounds comprise 20 to 30 percent of all commercial drugs, and the proportion of fluorinated pharmaceuticals is rapidly growing. While magic angle spinning (MAS) NMR spectroscopy is a popular technique for analysis of solid pharmaceutical compounds, fluorine has been underutilized as a structural probe so far. Here, we report a fast (40-60 kHz) MAS ¹⁹F NMR approach for structural characterization of fluorine-containing crystalline pharmaceutical compounds at natural abundance, using the antimalarial fluorine-containing drug mefloquine as an example. We demonstrate the utility of 2D ¹⁹F-¹³C and ¹⁹F-¹⁹F dipolar-coupling-based correlation experiments for ¹⁹F and ¹³C resonance frequency assignment, which permit identification of crystallographically inequivalent sites. The efficiency of ¹⁹F-¹³C cross-polarization and the effect of ¹H and ¹⁹F decoupling on spectral resolution and sensitivity were evaluated in a broad range of experimental conditions. We further demonstrate a protocol for measuring accurate interfluorine distances based on 1D DANTE-RFDR experiments combined with multispin numerical simulations.

Figure 1.

a) Chemical and 3D structure of mefloquine. b) Arrangement of mefloquine molecules in the crystal. The three types of inequivalent molecules are colored in purple, orange and cyan. The fluorine atoms are shown as spheres. The 8CF₃ and 2CF₃ groups are colored in light purple and light cyan, respectively.

Figure 2.

¹⁹F MAS spectra of mefloquine acquired at MAS frequencies of 60 kHz (top two traces), 40 kHz (middle two traces), and 10 kHz (bottom two traces), with or without SW_f-TPPM ¹H decoupling at the RF field strength, as indicated next to each spectrum. The blue traces are expansions around the isotropic peaks (marked with asterisks). The signal-to-noise ratios (SNR) are indicated next to each trace. The spectra were acquired at 11.7 T using a 1.3 mm HFX MAS probe, averaging 32 scans.

Figure 3.

a) ¹H-¹³C and ¹⁹F-¹³C CPMAS spectra of mefloquine. The spectra were acquired at 20.0 T using a 1.9 mm HX MAS probe, averaging 512 scans and 1920 scans and CP contact time of 1.4 and 6 ms, respectively. The ¹⁹F-¹³C spectrum was acquired without decoupling. b) ¹⁹F-¹³C CPMAS spectra of mefloquine without decoupling acquired (top trace), with ¹⁹F decoupling (middle trace), and with ¹H and ¹⁹F decoupling (bottom trace). The spectra were acquired at 16.4 T using a 1.6 mm HFXY MAS probe, averaging 512 scans; the CP contact time was 7 ms. The MAS frequency was 40 kHz in every case. Assignments for individual carbon signals are shown in the different spectra.

Figure 4.

a) 2D ¹H-¹³C HETCOR spectra acquired with CP contact times of 0.5 ms (yellow) and 1.1 ms (blue). b)-d) 2D ¹⁹F-¹³C HETCOR spectra acquired with CP contact times of 1ms and 7 ms (b), 7 ms and 10 ms with ¹⁹F decoupling (c), and with a CP contact time of 7 ms with ¹H and ¹⁹F decoupling (d). The rf fields for ¹⁹F and ¹³C were 15 kHz and 25 kHz (b) or 50 kHz (c), respectively. The spectrum shown in d) was acquired at 16.4 T with 8 scans. All other spectra were acquired at 20.0 T with 256 scans. The MAS frequency was 40 kHz. Carbon assignments are indicated. e) Short- and long-range intramolecular ¹⁹F-¹³C distances in the mefloquine crystal structure consistent with correlations in the HETCOR spectra for 1ms and 7 ms contact times. f) Intermolecular ¹⁹F-¹³C distances < 6 Å in the crystal lattice.

Figure 5.

a)-d) Pulse sequence (a), 1D ¹⁹F MAS NMR spectrum (b) with ¹⁹F DANTE selective excitation of the 8CF₃ resonance at 15.9 ppm (c) and 2CF₃ resonance at 8.8 ppm (d). Spectra were acquired at 20.0 T, with a MAS frequency of 40 kHz, averaging 16 scans and a DANTE interpulse delay of 4 rotor periods. e) 2D ¹⁹F-¹⁹F RFDR spectra with increased mixing times from 1.6 ms to 30.4 ms. Intramolecular and intermolecular correlations are shown in black and orange, respectively. f) The intra- and intermolecular interfluorine distances in crystal structure of mefloquine. The notation for each ¹⁹F-¹⁹F distance is identical to the corresponding correlation 2D ¹⁹F-¹⁹F RFDR spectra. The fluorine atoms in CF₃ groups are shown in light cyan. g) Left panels: 2D ¹⁹F-¹⁹F RFDR spectra acquired at the MAS frequencies of 40 kHz (left), 50 kHz (middle), and 60 kHz (right). The RFDR mixing times were 3.2 ms (gray) and 8.0 ms (dark purple). Right panel: the 1D traces of the 2D spectra with 8 ms RFDR mixing extracted at 8.61 ppm for MAS frequencies of 40 kHz (cyan), 50 kHz (black) and 60 kHz (orange). The peak widths are indicated in the slices.

Figure 6.

a) Pulse sequence for the 1D RFDR experiment with ¹⁹F DANTE-excitation. b),c) 1D ¹⁹F-¹⁹F DANTE-RFDR spectra with DANTE 90° selective excitation applied to the ¹⁹F resonances of 2CF₃ (b) and 8CF₃ (c), respectively. Spectra acquired with RFDR mixing times of 1.6 ms and 8.0 ms are shown in black and blue, respectively. The position of the DANTE excitation is shown with arrows and the resonances to which the magnetization was transferred by asterisks. d),e) Left: Experimental and simulated ¹⁹F-¹⁹F DANTE-RFDR magnetization exchange curves for the 8CF₃ (d) and 2CF₃ (e) resonances. The experimental data points are shown as black circles, the simulated curves, as dashed lines. In d), the 2CF₃ spins were excited by DANTE pulses and magnetization was transferred to the 8CF₃ spins during RFDR mixing period. In e), DANTE excitation was applied to the 8CF₃ resonances and magnetization was transferred to the 2CF₃ groups. Errors in the data points as defined by the standard deviation of the noise in a region of over 10 ppm are smaller than the size of the circles. The RMSDs of the simulated DANTE-RFDR magnetization exchange curves (dashed lines) are 0.008 (orange, the 2CF₃ — 2CF₃ distance is 7.2 Å) and 0.014 (black, the 2CF₃ — 2CF₃ distance is 7.4 Å) for 8CF₃ (d) and 0.014 for 2CF₃ (e). Right: Sets of interfluorine distances used in the 5-spin simulations, see also Table 3.