Polymorphs of Theophylline Characterized by DNP Enhanced Solid-State NMR

Abstract

We show how dynamic nuclear polarization (DNP) enhanced solid-state NMR spectroscopy can be used to characterize polymorphs and solvates of organic solids. We applied DNP to three polymorphs and one hydrated form of the asthma drug molecule theophylline. For some forms of theophylline, sample grinding and impregnation with the radical-containing solution, which are necessary to prepare the samples for DNP, were found to induce polymorphic transitions or desolvation between some forms. We present protocols for sample preparation for solid-state magic-angle spinning (MAS) DNP experiments that avoid the polymorphic phase transitions in theophylline. These protocols include cryogrinding, grinding under inert atmosphere, and the appropriate choice of the impregnating liquid. By applying these procedures, we subsequently demonstrate that two-dimensional correlation experiments, such as (1)H-(13)C and (1)H-(15)N HETCOR or (13)C-(13)C INADEQUATE, can be obtained at natural isotopic abundance in reasonable times, thus enabling more advanced structural characterization of polymorphs.

Keywords: dynamic nuclear polarization; polymorphs; solid-state NMR; theophylline.

Scheme 1. Molecular Structure of Theophylline

Red numbers correspond to carbon peak labeling associated with Figure 2.

Figure 1

Schemes for the preparation and interconversion of the different theophylline-containing crystal structures considered herein. Form II of anhydrous theophylline was obtained commercially. Form IV is an anhydrous polymorph of theophylline obtained by stirring Form II in methanol for 15 days. Form I is an anhydrous polymorph of theophylline obtained by heating Form II at 270 °C for 2 h. Form M is a theophylline-containing hydrate obtained by recrystallizing Form II in water. Dashed arrows illustrate the conversions that are induced by grinding or impregnation.

Figure 2

¹³C solid-state NMR spectra recorded at 105 K and B₀ = 9.4 T without microwave irradiation and with 64 scans for all nonimpregnated polymorphic forms of theophylline studied here: Form II (red), Form I (blue), Form IV (green), and Form M (purple). The tick marks show the predicted shifts from GIPAW DFT calculations. ¹³C, ¹H, and ¹⁵N solid-state NMR spectra can be found in Figures S1–S3. Additionally, comparison between 298 and 105 K ¹³C solid-state NMR spectra is presented in Figure S1.

Figure 3

¹³C DNP enhanced solid-state NMR spectra of Form II of theophylline obtained at 105 K and B₀ = 9.4 T (i.e., ¹H resonance frequency of 400 MHz) with 16 scans for microwave on, 64 scans for microwave off, and 20 s recycle delay. The powder was finely ground in a mortar and pestle and impregnated with a solution of TCE:methanol-d₄ (95:5) containing TEKPol as biradical.

Figure 4

¹³C CPMAS NMR spectra recorded at 105 K and B₀ = 9.4 T of what was initially powdered theophylline, Form I, subjected to different preparation procedures. From top to bottom: (a) pure powdered Form I before any procedure, (b) a mixture of Forms I and II obtained after manual grinding of pure Form I in air at room temperature, (c) Form I manually ground cold at −55 °C in a glovebox, (d) mixture of Forms II and I after impregnation with a 16 mM TCE:methanol-d₄ (95:5) solution, (e) Form I manually ground cold at −55 °C in a glovebox and impregnated with a toluene-d₈:decalin (i.e., Tol:dec) (90:10) solution, (f) pure Form II.

Figure 5

DNP enhanced ¹³C CPMAS NMR spectrum recorded at 105 K and B₀ = 9.4 T of Form I of theophylline impregnated with a toluene-d₈:decalin (90:10) solution containing 14 mM TEKPol with 4 scans for microwave on, 64 scans for microwave off, and 20 s recycle delay.

Figure 6

DNP enhanced ¹³C CPMAS NMR spectra recorded at 105 K and B₀ = 9.4 T of Form IV of theophylline impregnated with a TCE:methanol-d₄ (95:5) solution containing 16 mM TEKPol, with 4 scans for microwave on, 16 scans for microwave off, and 20 s recycle delay.

Figure 7

Comparison of ¹³C CPMAS solid-state NMR spectra as a function of the grinding procedure and the solvent used for impregnation of Form M. All spectra were recorded with a 16.4 T spectrometer at ca. 300 K. (a) Pure Form M before any procedure. (b) Mixture of Forms M and II obtained after grinding at room temperature. (c) Form M after grinding with a drop of water. (d) Mixture of Forms M and II after grinding with a drop of water and impregnation with TCE:methanol-d₄ (95:5) solution. (e) Form M after grinding with a drop of water and impregnated with a glycerol-d₈:D₂O:H₂O (60:30:10) solution. (f) Pure Form II.

Figure 8

DNP enhanced ¹³C CPMAS NMR spectra of theophylline/water Form M ground with a drop of water, and impregnated with a glycerol-d₈:D₂O:H₂O (60:30:10) solution containing AMUPol. Spectra were recorded at 105 K and B₀ = 9.4 T with 4 scans for microwave on, 16 scans for microwave off, and 20 s recycle delay.