Revealing Intermolecular Hydrogen Bonding Structure and Dynamics in a Deep Eutectic Pharmaceutical by Magic-Angle Spinning NMR Spectroscopy.

Liquid forms of pharmaceuticals (ionic liquids and deep eutectic solvents) offer a number of potential advantages over solid-state drugs; a key question is the role of intermolecular hydrogen bonding interactions in enabling membrane transport. Characterization is challenging since high sample viscosities, typical of liquid pharmaceutical formulations, hamper the use of conventional solution NMR at ambient temperature. Here, we report the application of magic-angle spinning (MAS) NMR spectroscopy to the deep eutectic pharmaceutical, lidocaine ibuprofen. Using variable temperature MAS NMR, the neat system, at a fixed molar ratio, can be studied over a wide range of temperatures, characterized by changing mobility, using a single experimental setup. Specific intermolecular hydrogen bonding interactions are identified by two-dimensional 1H-1H NOESY and ROESY MAS NMR experiments. Hydrogen-bonding dynamics are quantitatively determined by following the chemical exchange process between the labile protons by means of line-width analysis of variable temperature 1H MAS NMR spectra.

Local Structural Effects Due to Micronization and Amorphization on an HIV Treatment Active Pharmaceutical Ingredient.

Processing procedures for inducing domain size reduction and/or amorphous phase generation can be crucial for enhancing the bioavailability of active pharmaceutical ingredients (APIs). It is important to quantify these reduced coherence phases and to detect and characterize associated structural changes, to ensure that no deleterious effects on safety, function, or stability occur. Here, X-ray powder diffraction (XRPD), total scattering pair distribution function (TSPDF) analysis, and solid-state nuclear magnetic resonance spectroscopy (SSNMR) have been performed on samples of GSK2838232B, an investigational drug for the treatment of human immunodeficiency virus (HIV). Preparations were obtained through different mechanical treatments resulting in varying extents of domain size reduction and amorphous phase generation. Completely amorphous formulations could be prepared by milling and microfluidic injection processes. Microfluidic injection was shown to result in a different local structure due to dispersion with dichloromethane (DCM). Implications of combined TSPDF and SSNMR studies to characterize molecular compounds are also discussed, in particular, the possibility to obtain a thorough structural understanding of disordered samples from different processes.

Managing emerging mutagenicity risks: Late stage mutagenic impurity control within the atovaquone second generation synthesis.

The mutagenic-impurity control strategy for a second generation manufacturing route to the non-mutagenic antipneumocystic agent atovaquone (2-((1R,4R)-4-(4-chlorophenyl)cyclohexyl)-3-hydroxynaphthalene-1,4-dione) 1 is described. Preliminary assessment highlighted multiple materials of concern which were largely discharged either through returning a negative bacterial mutagenicity assay or through confidence that the impurity would be purged during the downstream processing from when it was first introduced. Additional genotoxicity testing highlighted two materials of concern where initial assessment suggested that testing for these impurities at trace levels within the drug substance would be required. Following a thorough review of process purging detail, spiking and purging experimentation, and an understanding of the process parameters to which they were exposed an ICH M7 Option 4 approach could be justified for their control. The development of two 1H NMR spectroscopy methods for measurement of these impurities is also described as well as a proposed summary table for describing the underlying rationale for ICH M7 control rationales to regulators. This manuscript demonstrates that process purging of potential mutagenic impurities can be realised even when they are introduced in the later stages of a process and highlights the importance of scientific understanding rather than relying on a stage-counting approach.

Probing hydrogen bonding in cocrystals and amorphous dispersions using (14)N-(1)H HMQC solid-state NMR.

Cocrystals and amorphous solid dispersions have generated interest in the pharmaceutical industry as an alternative to more established solid delivery forms. The identification of intermolecular hydrogen bonding interactions in a nicotinamide palmitic acid cocrystal and a 50% w/w acetaminophen-polyvinylpyrrolidone solid dispersion are reported using advanced solid-state magic-angle spinning (MAS) NMR methods. The application of a novel (14)N-(1)H HMQC experiment, where coherence transfer is achieved via through-space couplings, is shown to identify specific hydrogen bonding motifs. Additionally, (1)H isotropic chemical shifts and (14)N electric field gradient (EFG) parameters, both accessible from (14)N-(1)H HMQC experiments, are shown to be sensitive to changes in hydrogen bonding geometry. Numerous indicators of molecular association are accessible from this experiment, including NH cross-peaks occurring from intermolecular hydrogen bonds and changes in proton chemical shifts or electric field gradient parameters. First-principles calculations using the GIPAW approach that yield accurate estimates of isotropic chemical shifts, and EFG parameters were used to assist in assignment. It is envisaged that (14)N-(1)H HMQC solid state NMR experiments could become a valuable screening technique of solid delivery forms in the pharmaceutical industry.

Identifying guanosine self assembly at natural isotopic abundance by high-resolution 1H and 13C solid-state NMR spectroscopy.

By means of the (1)H chemical shifts and the proton-proton proximities as identified in (1)H double-quantum (DQ) combined rotation and multiple-pulse spectroscopy (CRAMPS) solid-state NMR correlation spectra, ribbon-like and quartet-like self-assembly can be identified for guanosine derivatives without isotopic labeling for which it was not possible to obtain single crystals suitable for diffraction. Specifically, characteristic spectral fingerprints are observed for dG(C10)(2) and dG(C3)(2) derivatives, for which quartet-like and ribbon-like self-assembly has been unambiguously identified by (15)N refocused INADEQUATE spectra in a previous study of (15)N-labeled derivatives (Pham, T. N.; et al. J. Am. Chem. Soc.2005, 127, 16018). The NH (1)H chemical shift is observed to be higher (13-15 ppm) for ribbon-like self-assembly as compared to 10-11 ppm for a quartet-like arrangement, corresponding to a change from NH···N to NH···O intermolecular hydrogen bonding. The order of the two NH(2)(1)H chemical shifts is also inverted, with the NH(2) proton closest in space to the NH proton having a higher or lower (1)H chemical shift than that of the other NH(2) proton for ribbon-like as opposed to quartet-like self-assembly. For the dG(C3)(2) derivative for which a single-crystal diffraction structure is available, the distinct resonances and DQ peaks are assigned by means of gauge-including projector-augmented wave (GIPAW) chemical shift calculations. In addition, (14)N-(1)H correlation spectra obtained at 850 MHz under fast (60 kHz) magic-angle spinning (MAS) confirm the assignment of the NH and NH(2) chemical shifts for the dG(C3)(2) derivative and allow longer range through-space N···H proximities to be identified, notably to the N7 nitrogens on the opposite hydrogen-bonding face.

Analysis of amorphous solid dispersions using 2D solid-state NMR and (1)H T(1) relaxation measurements.

Solid-state NMR (SSNMR) can provide detailed structural information about amorphous solid dispersions of pharmaceutical small molecules. In this study, the ability of SSNMR experiments based on dipolar correlation, spin diffusion, and relaxation measurements to characterize the structure of solid dispersions is explored. Observation of spin diffusion effects using the 2D (1)H-(13)C cross-polarization heteronuclear correlation (CP-HETCOR) experiment is shown to be a useful probe of association between the amorphous drug and polymer that is capable of directly proving glass solution formation. Dispersions of acetaminophen and indomethacin in different polymers are examined using this approach, as well as (1)H double-quantum correlation experiments to probe additional structural features. (1)H-(19)F CP-HETCOR serves a similar role for fluorinated drug molecules such as diflunisal in dispersions, providing a rapid means to prove the formation of a glass solution. Phase separation is detected using (13)C, (19)F, and (23)Na-detected (1)H T(1) experiments in crystalline and amorphous solid dispersions that contain small domains. (1)H T(1) measurements of amorphous nanosuspensions of trehalose and dextran illustrate the ability of SSNMR to detect domain size effects in dispersions that are not glass solutions via spin diffusion effects. Two previously unreported amorphous solid dispersions involving up to three components and containing voriconazole and telithromycin are analyzed using these experiments to demonstrate the general applicability of the approach.

Complete (1)H resonance assignment of beta-maltose from (1)H-(1)H DQ-SQ CRAMPS and (1)H (DQ-DUMBO)-(13)C SQ refocused INEPT 2D solid-state NMR spectra and first principles GIPAW calculations.

A disaccharide is a challenging case for high-resolution (1)H solid-state NMR because of the 24 distinct protons (14 aliphatic and 10 OH) having (1)H chemical shifts that all fall within a narrow range of approximately 3 to 7 ppm. High-resolution (1)H (500 MHz) double-quantum (DQ) combined rotation and multiple pulse sequence (CRAMPS) solid-state NMR spectra of beta-maltose monohydrate are presented. (1)H-(1)H DQ-SQ CRAMPS spectra are presented together with (1)H (DQ)-(13)C correlation spectra obtained with a new pulse sequence that correlates a high-resolution (1)H DQ dimension with a (13)C single quantum (SQ) dimension using the refocused INEPT pulse-sequence element to transfer magnetization via one-bond (13)C-(1)H J couplings. Compared to the observation of only a single broad peak in a (1)H DQ spectrum recorded at 30 kHz magic-angle spinning (MAS), the use of DUMBO (1)H homonuclear decoupling in the (1)H DQ CRAMPS experiment allows the resolution of distinct DQ correlation peaks which, in combination with first-principles chemical shift calculations based on the GIPAW (Gauge Including Projector Augmented Waves) plane-wave pseudopotential approach, enables the assignment of the (1)H resonances to the 24 distinct protons. We believe this to be the first experimental solid-state NMR determination of the hydroxyl OH (1)H chemical shifts for a simple sugar. Variable-temperature (1)H-(1)H DQ CRAMPS spectra reveal small increases in the (1)H chemical shifts of the OH resonances upon decreasing the temperature from 348 K to 248 K.

MAS NMR Investigation of Molecular Order in an Ionic Liquid Crystal.

The structure and molecular order in the thermotropic ionic liquid crystal (ILC), [choline][geranate(H)octanoate], an analogue of Choline And GEranate (CAGE), which has potential for use as a broad-spectrum antimicrobial and transdermal and oral delivery agent, were investigated by magic-angle spinning (MAS) nuclear magnetic resonance (NMR), polarizing optical microscopy, small-angle X-ray scattering (SAXS), and mass spectrometry. Mass spectrometry and the 1H NMR chemical shift reveal that CAGE-oct is a dynamic system, with metathesis (the exchange of interacting ions) and hydrogen exchange occurring between hydrogen-bonded/ionic complexes such as [(choline)(geranate)(H)(octanoate)], [(choline)(octanoate)2(H)], and [(choline)(geranate)2(H)]. These clusters, which are shown by mass spectrometry to be significantly more stable than expected for typical electrostatic ion clusters, involve hydrogen bonding between the carboxylic acid, carboxylate, and hydroxyl groups, with rapid hydrogen bond breaking and re-formation observed to average the 1H chemical shifts. The formation of a partial bilayer liquid crystal (LC) phase was identified by SAXS and polarizing optical microscopy at temperatures below ∼293 K. The occurrence of this transition close to room temperature could be utilized as a potential temperature-induced "switch" of the anisotropic properties for particular applications. The presence of an isotropic component of approximately 23% was observed to coexist with the LC phase, as detected by polarizing optical microscopy and quantified by both 1H-13C dipolar-chemical shift correlation (DIPSHIFT) and 1H double-quantum (DQ) MAS NMR experiments. At temperatures above the LC-to-isotropic transition, intermediate-range order (clustering of polar and nonpolar domains), a feature of many ILs, persists. Site-specific order parameters for the LC phase of CAGE-oct were obtained from the MAS NMR measurement of the partially averaged 13C-1H dipolar couplings (DCH) by cross-polarization (CP) build-up curves and DIPSHIFT experiments, and 1H-1H dipolar couplings (DHH) by double-quantum (DQ) build-up curves. The corresponding order parameters, SCH and SHH, are in the range 0-0.2 and are lower compared to those for smectic (i.e., layered) phases of conventional nonionic liquid crystals, resembling those of lamellar phases formed by lyotropic surfactant-solvent systems.

The refocused INADEQUATE MAS NMR experiment in multiple spin-systems: interpreting observed correlation peaks and optimising lineshapes.

The robustness of the refocused INADEQUATE MAS NMR pulse sequence for probing through-bond connectivities has been demonstrated in a large range of solid-state applications. This pulse sequence nevertheless suffers from artifacts when applied to multispin systems, e.g. uniformly labeled (13)C solids, which distort the lineshapes and can potentially result in misleading correlation peaks. In this paper, we present a detailed account that combines product-operator analysis, numerical simulations and experiments of the behavior of a three-spin system during the refocused INADEQUATE pulse sequence. The origin of undesired anti-phase contributions to the spectral lineshapes are described, and we show that they do not interfere with the observation of long-range correlations (e.g. two-bond (13)C-(13)C correlations). The suppression of undesired contributions to the refocused INADEQUATE spectra is shown to require the removal of zero-quantum coherences within a z-filter. A method is proposed to eliminate zero-quantum coherences through dephasing by heteronuclear dipolar couplings, which leads to pure in-phase spectra.

1H-detected double-J-modulated INEPT-INADEQUATE for simultaneous determination of one-bond and long-range carbon-carbon connectivities and the measurement of all carbon-carbon coupling constants.

We describe a new NMR experiment, (1)H-detected double-J-modulated (DJM)-INEPT-INADEQUATE, for tracing out the carbon skeleton of molecules. This experiment allows simultaneous correlation of directly bonded carbon atoms and those separated by multiple bonds, while at the same time also providing the values of all J(CC) coupling constants. This is achieved by replacing both fixed carbon-carbon coupling evolution intervals of the INEPT-INADEQUATE experiment by variable time intervals, which are incremented in concert with the DQ evolution period (t(1)). We show that the analysis of the fine structure of cross-peaks in DJM-INEPT-INADEQUATE spectra leads to accurate values of coupling constants and give guidelines for the proper usage of the method. The proposed experiment is two times less sensitive that the original INEPT-INADEQUATE experiment. We show that, using a 600-MHz cryoprobe and 20 mg of a monosaccharide, spectra that are suitable for the analysis of coupling constants as small as 2 Hz can be obtained within 24 h. Instead of performing multiple experiments, a single DJM-INEPT-INADEQUATE experiment can thus provide a wealth of information for the structural analysis of small molecules.

A multicenter, randomized, phase III study of docetaxel plus best supportive care versus best supportive care in chemotherapy-naive patients with metastatic or non-resectable localized non-small cell lung cancer (NSCLC).

This was an open-label randomized Phase III study of 207 patients with either unresectable or metastatic non-small cell lung cancer (NSCLC) who were treated with docetaxel plus best supportive care (BSC) or best supportive care alone. Patients in the chemotherapy arm of the study received docetaxel 100 mg/m(2) as a 1 h intravenous infusion every 21 days until they showed evidence of progressive disease, or estimated maximum benefit obtained or unacceptable side effects. Patients who received docetaxel were pretreated with oral dexamethasone. Patients in the BSC arm should not receive chemotherapy or anticancer therapy except for palliative radiotherapy. Overall survival obtained in the docetaxel arm was significantly longer than in the BSC arm (P=0.026). Two-year survival in the docetaxel arm was 12%, whereas none of the BSC patients survived after 20 months. The response rate was 13.1% (95% CI, 7.5-18.8%). There was a significantly longer time to progression in the docetaxel versus the BSC arm (P<0.001), and statistically significant improvement of clinical symptoms with docetaxel compared to BSC. The quality-of-life descriptors were in favor of docetaxel, and the difference was significant for pain, dyspnea and emotional functioning. The safety profile of docetaxel for this study was similar to that already reported in this patient population.

A risk-based statistical investigation of the quantification of polymorphic purity of a pharmaceutical candidate by solid-state 19F NMR.

The DMAIC (Define, Measure, Analyse, Improve and Control) framework and associated statistical tools have been applied to both identify and reduce variability observed in a quantitative (19)F solid-state NMR (SSNMR) analytical method. The method had been developed to quantify levels of an additional polymorph (Form 3) in batches of an active pharmaceutical ingredient (API), where Form 1 is the predominant polymorph. In order to validate analyses of the polymorphic form, a single batch of API was used as a standard each time the method was used. The level of Form 3 in this standard was observed to gradually increase over time, the effect not being immediately apparent due to method variability. In order to determine the cause of this unexpected increase and to reduce method variability, a risk-based statistical investigation was performed to identify potential factors which could be responsible for these effects. Factors identified by the risk assessment were investigated using a series of designed experiments to gain a greater understanding of the method. The increase of the level of Form 3 in the standard was primarily found to correlate with the number of repeat analyses, an effect not previously reported in SSNMR literature. Differences in data processing (phasing and linewidth) were found to be responsible for the variability in the method. After implementing corrective actions the variability was reduced such that the level of Form 3 was within an acceptable range of ±1% ww(-1) in fresh samples of API.

Detection of low-level PTFE contamination: An application of solid-state NMR to structure elucidation in the pharmaceutical industry.

We report a novel use of solid-state ¹9F nuclear magnetic resonance to detect and quantify polytetrafluoroethylene contamination from laboratory equipment, which due to low quantity (up to 1% w/w) and insolubility remained undetected by standard analytical techniques. Solid-state ¹9F NMR is shown to be highly sensitive to such fluoropolymers (detection limit 0.02% w/w), and is demonstrated as a useful analytical tool for structure elucidation of unknown solid materials.

Measurement of 1H-1H residual dipolar coupling constants for structural studies of medium size molecules.

Residual dipolar coupling constants (RDCs) are being increasingly applied to elucidate the configuration and conformation of small organic molecules, peptides and oligosaccharides. In this paper we describe a set of robust 1D NMR methods for accurate and precise measurement of proton-proton RDCs of small and medium size molecules. The performance of these techniques is not impeded by the presence of overlapping and broad (1)H multiplets that are typically observed for such molecules in weakly aligned media. The use of these techniques provides access to a large pool of proton-proton RDCs opening new avenues for the solution structure elucidation of medium size molecules by NMR. The techniques are illustrated on the determination of the alignment tensor of the reducing monosaccharide ring of cellobiose and the determination of the relative configuration of sodium cholate.

Quantifying hydrogen-bonding strength: the measurement of 2hJNN couplings in self-assembled guanosines by solid-state 15N spin-echo MAS NMR.

(2h)J(NN) hydrogen-bond mediated J couplings are measured in the solid state for two synthetic deoxyguanosine derivatives by (15)N MAS NMR spin-echo experiments. The use of rotor-synchronised Hahn-echo pulse train (RS-HEPT) (1)H decoupling, with a duty cycle of 6%, allows spin-echo durations out to 200 ms, hence enabling the accurate determination of J couplings as small as 3.8 Hz. A single-crystal X-ray diffraction structure exists for the shorter alkyl chain derivative dG(C(3))(2): the observation of significantly different (2h)J(NN) couplings, 6.2 +/- 0.4 and 7.4 +/- 0.4 Hz, for the two resolved N7 resonances is to be expected given the NH...N hydrogen-bonding distances of 2.91 and 2.83 A for the two distinct molecules in the asymmetric unit cell. For the longer alkyl chain derivative, dG(C(10))(2), for which there is no single-crystal diffraction structure, a (15)N refocused INADEQUATE spectrum (Pham et al., J. Am. Chem. Soc., 2005, 127, 16018-16019) has demonstrated the presence of N2-H...N7 intermolecular hydrogen-bonds indicative of a quartet-like structure. The (2h)J(NN) hydrogen-bond mediated J coupling of 5.9 +/- 0.2 Hz is at the lower end of the range (5.9-8.2 Hz) of (2h)J(NN) couplings determined from solution-state NMR of guanosine quartets in quadruplex DNA. A full discussion of the determination of error bars on the fitted parameters is given; specifically, error bars determined by a non-linear fitting (using the covariance matrix) or in a Monte-Carlo fashion are found to give effectively identical results.

Identification by 15N refocused INADEQUATE MAS NMR of intermolecular hydrogen bonding that directs the self-assembly of modified DNA bases.

15N solid-state NMR refocused INADEQUATE spectra of two lipophilic deoxyguanosine derivatives unambiguously identify different intermolecular hydrogen-bonding arrangements that are indicative of either guanine ribbon or quartet self-assembly. The observation of guanine quartet formation in the absence of metal ions is a further example that challenges the accepted dogma that quartet formation requires metal ions.

An investigation of weak CH...O hydrogen bonds in maltose anomers by a combination of calculation and experimental solid-state NMR spectroscopy.

Two-dimensional (1)H-(13)C MAS-J-HMQC solid-state NMR spectra of the two anomeric forms of maltose at natural abundance are presented. The experimental (1)H chemical shifts of the CH and CH(2) protons are assigned using first-principles chemical shift calculations that employ a plane-wave pseudopotential approach. Further calculations show that the calculated change in the (1)H chemical shift when comparing the full crystal and an isolated molecule is a quantitative measure of intermolecular C-H...O weak hydrogen bonding. Notably, a clear correlation between a large chemical shift change (up to 2 ppm) and both a short H...O distance (<2.7 A) and a CHO bond angle greater than 130 degrees is observed, thus showing that directionality is important in C-H...O hydrogen bonding.

Determination of sugar structures in solution from residual dipolar coupling constants: methodology and application to methyl beta-D-xylopyranoside.

We have developed methodology for the determination of solution structures of small molecules from residual dipolar coupling constants measured in dilute liquid crystals. The power of the new technique is demonstrated by the determination of the structure of methyl beta-d-xylopyranoside (I) in solution. An oriented sample of I was prepared using a mixture of C(12)E(5) and hexanol in D(2)O. Thirty residual dipolar coupling constants, ranging from -6.44 to 4.99 Hz, were measured using intensity-based J-modulated NMR techniques. These include 15 D(HH), 4 (1)D(CH), and 11 (n)D(CH) coupling constants. The accuracy of the dipolar coupling constants is estimated to be < +/- 0.02 Hz. New constant-time HMBC NMR experiments were developed for the measurement of (n)D(CH) coupling constants, the use of which was crucial for the successful structure determination of I, as they allowed us to increase the number of fitted parameters. The structure of I was refined using a model in which the directly bonded interatom distances were fixed at their ab initio values, while 16 geometrical and 5 order parameters were optimized. These included 2 CCC and 6 CCH angles, and 2 CCCC and 6 CCCH dihedral angles. Vibrationally averaged dipolar coupling constants were used during the refinement. The refined solution structure of I is very similar to that obtained by ab initio calculations, with 11 bond and dihedral angles differing by 0.8 degrees or less and the remaining 5 parameters differing by up to 3.3 degrees . Comparison with the neutron diffraction structure showed larger differences attributable to crystal packing effects. Reducing the degree of order by using dilute liquid crystalline media in combination with precise measurement of small residual dipolar coupling constants, as shown here, is a way of overcoming the limitation of strongly orienting liquid crystals associated with the complexity of (1)H NMR spectra for molecules with more than 12 protons.