Optimal flip angle for robust practical quantitative NMR measurement using a fixed pulse length.

NMR quantification has been traditionally performed by using internal standards. Although methods using external reference in NMR quantification have been developed, the major obstacles in using external referencing method are the measurement deviations associated with changing sample conditions and the requirement of pulse width calibration for every sample in order to compensate these errors. The calibration process is time consuming and in some cases impossible. We developed a quantitative NMR method fixed pulse length (FIXPUL) for all measurements without sample-by-sample calibration. The method is based on the use of an optimal flip angle calibrated for an external standard so that the quantitative errors associated with the pulse width variations are minimized. FIXPUL can be implemented on most basic NMR spectrometers and is robust and easily automated. The method is applicable to a wide range of solution NMR samples in chemistry, biology, and drug research and discovery.

In-tube derivatization for determination of absolute configuration and enantiomeric purity of chiral compounds by NMR spectroscopy.

We have developed an in-tube derivatization method using commercially available polymer-supported coupling agents to prepare derivatives of chiral compounds directly in NMR tube with high yield and purity. Because the method does not require any workup or purification, the configuration and enatiopurity can be quickly determined by NMR analysis for a small amount of chiral compounds, which is critical for today's fast-paced medicinal chemistry efforts in drug discovery. The application of the method was demonstrated for the derivatization of chiral amines, alcohols, diols, amino alcohols, thiols, and carboxylic acids using various chiral derivatizing agents and coupling agents. This article also serves as a practical guide for in-tube derivatization and selection of suitable chiral derivatizing agents and coupling agents for various types of chiral compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Real time measurement of PEG shedding from lipid nanoparticles in serum via NMR spectroscopy.

Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle mediated delivery. The most clinically advanced siRNA-containing nanoparticles are polymer-coated supramolecular assemblies of siRNA and lipids (lipid nanoparticles or LNPs), which protect the siRNA from nucleases, modulate pharmacokinetics of the siRNA, and enable selective delivery of siRNA to target cells. Understanding the mechanisms of assembly and delivery of such systems is complicated by the complexity of the dynamic supramolecular assembly as well as by its subsequent interactions with the biological milieu. We have developed an ex vivo method that provides insight into how LNPs behave when contacted with biological fluids. Pulsed gradient spin echo (PGSE) NMR was used to directly measure the kinetics of poly(ethylene) glycol (PEG) shedding from siRNA encapsulated LNPs in rat serum. The method represents a molecularly specific, real-time, quantitative, and label-free way to monitor the behavior of a nanoparticle surface coating. We believe that this method has broad implications in gaining mechanistic insights into how nanoparticle-based drug delivery vehicles behave in biofluids and is versatile enough to be applied to a diversity of systems.

Stable Fatty Acid Solvates of Dasatinib, a Tyrosine Kinase Inhibitor: Prediction, Process, and Physicochemical Properties.

Exploration of alternate solid forms for dasatinib, a potent oncogene tyrosine kinase inhibitor classified under Biopharmaceutics Classification System (BCS) class II drugs with low water solubility and high permeability, has been performed using COSMO-RS excess enthalpy (Hex) to increase dissolution. The theoretical prediction resulted in the potential for the formation of C6-C8 fatty acid solvates with dasatinib. A crystallization process has been identified for the preparation of the predicted solvates and successfully scaled up till the 100 g level. The fatty acid solvates are completely characterized using powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform infrared (FT-IR) spectroscopy, and proton nuclear magnetic resonance (1H NMR) spectroscopy. Unique powder X-ray diffraction patterns and powder indexing of C6-C8 fatty acid solvates indicate the purity of the solid phase. The red shift in the acid carbonyl stretching frequency of C6-C8 fatty acids in FT-IR spectra and the intactness of the fatty acid proton in 1H-NMR spectra provide evidence for solvate formation. The stoichiometry of active pharmaceutical ingredients (APIs) with solvent in solvates is measured using TGA and 1H-NMR spectroscopy. Dasatinib C6-C8 fatty acid solvates were found to retain their solid form under various stress and pharmaceutical processing conditions. In addition, they exhibited improved powder dissolution over dasatinib Form H1-7 by 2.2-fold. They also showed stability at 40 °C and 75% RH for 3 months. C8 fatty acid is a USFDA GRAS listed solvent, and hence may be a viable option for drug product development.

The use of MPA amide for the assignment of absolute configuration of a sterically hindered cyclic secondary amine by 'mix and shake' NMR method.

We present here a new method using methoxyphenylacetic acid (MPA) as the chiral derivatizing agent (CDA) for the assignment of absolute configuration of cyclic secondary amines. The MPA amides were prepared using the purification-free 'mix and shake' method. A detailed conformational analysis for the two diastereomeric amides was conducted by 2D NMR experiments and molecular mechanics calculations. We have established that, in the most stable conformation of each syn rotamer of MPA amides, the H-alpha in the MPA moiety is oriented toward the bulky substituent group at the asymmetric carbon in the chiral amine, presumably to avoid steric and/or electrostatic interactions. The observed NMR data were correlated with the conformational model to allow unambiguous assignment of absolute configuration of secondary amines. The results demonstrate that the MPA can be used as a useful CDA in the case of sterically crowded cyclic secondary amines from which the MTPA amides are usually difficult to make.

Aging and sympathetic modulation of immune function in Fischer 344 rats: effects of chemical sympathectomy on primary antibody response.

In aged Fischer 344 (F334) rats, sympathetic innervation of the spleen is markedly diminished compared with young rats. To determine if this diminished noradrenergic (NA) innervation maintains a functional connection with the immune system, 3- and 17-month-old male F344 rats were treated with the NA-selective neurotoxin, 6-hydroxydopamine (6-OHDA), to ablate peripheral NA nerve fibers. In sympathectomized rats immunized with keyhole limpet hemocyanin (KLH), a T-dependent protein antigen, anti-KLH IgM, IgG, IgG1, IgG2b antibody titers were increased in young and old rats 14 days after immunization compared to vehicle controls. Furthermore, the number of IgM and IgG anti-KLH antibody-secreting spleen cells was elevated 7 and 14 days post-immunization. These effects were prevented by pretreatment with desipramine, a catecholamine uptake blocker that blocks 6-OHDA uptake and subsequent sympathectomy. Chemical sympathectomy also increased KLH-induced proliferation in vitro by spleen cells from old, but not young animals. Isoproterenol (ISO), a beta-adrenergic receptor agonist, elicited a rise in cAMP in spleen cells from NA-intact young and old rats, but the increase was attenuated in spleen cells from old rats. These results demonstrate that, although NA innervation in the F344 rat spleen is diminished with age, sympathetic signaling of the immune system remains intact. Thus, the SNS can inhibit antibody produced in response to a protein antigen in both young and old F344 rats.

Conformational refinement of hydroxamate-based histone deacetylase inhibitors and exploration of 3-piperidin-3-ylindole analogues of dacinostat (LAQ824).

Inspired by natural product HDAC inhibitors, we prepared a series of conformationally restrained HDAC inhibitors based on the hydroxamic acid dacinostat (LAQ824, 7). Several scaffolds with improved biochemical and cellular potency, as well as attenuated hERG inhibition, were identified, suggesting that the introduction of molecular rigidity is a viable strategy to enhance HDAC binding and mitigate hERG liability. Further SAR studies around a 3-piperidin-3-ylindole moiety resulted in the discovery of compound 30, for which a unique conformation was speculated to contribute to overcoming increased lipophilicity and attenuating hERG binding. Separation of racemate 30 afforded 32, the R enantiomer, which demonstrated improved potency in both enzyme and cellular assays compared to dacinostat.