Optimization of normal-phase chromatographic separation of compounds with primary, secondary and tertiary amino groups.

The retention behavior of primary, secondary and tertiary amines was studied using normal-phase-HPLC on silica, diol, and cyano stationary phases. Several classes of amines, including benzylamines, anilines, ephedrines, tryptamines, and azatryptamines were chromatographed using mixtures of hexane and ethoxynonafluorobutane with methylene chloride and methanol. Peak tailing, diminished selectivity and low plate count were minimized by the addition of volatile amines to the mobile phase. The optimal additive was n-propylamine at 0.1% concentration. On diol columns, the elution order of free primary, N-N-methyl, and N,N-dimethylamines was predictable, while the elution order of primary and secondary amines on cyano columns varied depending on the alcohol modifier concentration. The feasibility of preparative normal-phase chromatography was demonstrated by the separation of a mixture of primary, secondary and tertiary amines obtained by direct methylation of norephedrine. The procedures described may provide a practical alternative to traditional methods of analysis and purification of potential drug candidates.

Evidence for a novel pentyl radical adduct of the cyclic nitrone spin trap MDL 101,002.

3,4-Dihydro-3,3-dimethyl-isoquinoline-2-oxide (MDL 101,002) is a conformationally constrained cyclic analog of the known spin trap alpha-phenyl N-tert-butyl nitrone (PBN). Because of PBN's ability to scavenge free radicals, MDL 101,002 is currently being evaluated in stroke models as a means to ameliorate the oxidative insult associated with reperfusion injury. To augment our understanding of the radical scavenging mechanism of this potential drug, MDL 101,002 was incubated with soybean lipoxygenase in the presence of linoleic acid to study the interaction between MDL 101,002 and free radicals formed during lipid peroxidation. Analysis of the reaction mixture was performed by high performance liquid chromatography using normal phase conditions with detection by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Similar to the work by Iwahashi et al. [Arch. Biochem. Biophys., 1991, 285, 172], who studied the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN), an adduct that suggested the trapping of pentyl radicals by MDL 101,002 was observed. However, the apparent molecular ion for this adduct (246 Da) was 1 Da lower than would be predicted if a pentyl radical had simply added to MDL 101,002. In addition, the adduct exhibited significant absorbance at 304 nm, consistent with the unsaturated nitrone structure of MDL 101,002. To account for these observations, it is postulated that, after the initial capture of a pentyl radical, subsequent abstraction of a hydrogen atom by a neighboring radical occurs to regenerate a nitrone (1-pentyl analog of MDL 101,002). We present evidence for this adduct and offer a mechanism for its formation. These findings indicate that mass spectroscopic analysis of stable nitrone radical adducts may be useful in the identification of radical-dependent damage in vivo and possibly in clinical development of MDL 101,002 as an antioxidant pharmaceutical.

In vitro and in vivo activity of a novel series of radical trapping agents in model systems of CNS oxidative damage.

Many laboratory and clinical studies suggest that oxygen radical formation and resultant cell damage contribute to CNS injury following stroke and neurotrauma. Accordingly, antioxidants represent a viable therapeutic approach for management of CNS oxidative damage. Recently, several investigators have reported that the spin trap PBN protects against stroked-induced damage and reduces aging-associated neurological deficits. We have prepared and tested a cyclic analog of PBN, MDL 101,002, in a number of in vitro and in vivo assays designed to assess its neuroprotective properties. MDL 101,002 was found to be an effective .OH trap, to inhibit lipid peroxidation, and to decrease infarct size in a gerbil model of stroke. These results further indicate that oxidative damage arising from stroke contributes to infarct formation, and that spin traps are effective in ameliorating ischemia and reperfusion-induced CNS injury.

Easy assembly of ligands for glycosidase affinity chromatography.

An improved, high-yield synthesis of the corresponding N-carboxypentyl derivatives of three iminoalditol glycosidase inhibitors has been developed for affinity chromatography enzyme purification. Reductive amination of 1-deoxynojirimycin (or its D-manno or D-galacto analogues) with methyl 5-formylvalerate and NaBH3CN at neutral pH afforted an aminoester which upon hydrolysis with aqueous 5% HCl gave the desired aminoacid in 97% overall yield. These amino acids could then be covalently attached using water-soluble carbodi-imide to 6-aminohexyl Sepharose 4B.

Castanospermine analogues: their inhibition of glycoprotein processing alpha-glucosidases from porcine kidney and B16F10 cells.

We have used a simple and efficient procedure for the synthesis of N-5-carboxypentyl-1-deoxynojirimycin, an affinity ligand for alpha-glucosidase I (Bernotas, R. C. and Ganem, B., Biochem. J., 270, 539-540, 1990). The affinity gel was used to purify alpha-glucosidase I in one step from crude extract. In subsequent steps, partially purified alpha-glucosidase II was obtained. We have synthesized several analogues of castanospermine and studied their inhibition of alpha-glucosidase I in vitro using purified alpha-glucosidase I and in vivo in cultured B16F10 cells. Although the castanospermine analogues were significantly less active against the purified enzyme (IC50 approximately 1-23 micrograms/ml) as compared to castanospermine (IC50 = 0.02 microgram/ml), several compounds had up to 30-fold higher activity than castanospermine against alpha-glucosidase I in B16F10 cells, based on the accumulation of G3M7-9N2 oligosaccharide-containing glycoproteins. These results suggest that these analogues with lipophilic side chains cross the membrane barrier more efficiently than castanospermine. Once inside the cell, they may be converted to their active metabolite, castanospermine, by cellular esterases to give enzyme inhibition.

Characterization of the radical trapping activity of a novel series of cyclic nitrone spin traps.

alpha-Phenyl-tert-butyl nitrone (PBN) is a nitrone spin trap, which has shown efficacy in animal models of oxidative stress, including stroke, aging, sepsis, and myocardial ischemia/reperfusion injury. We have prepared a series of novel cyclic variants of PBN and evaluated them for radical trapping activity in vitro. Specifically, their ability to inhibit iron-induced lipid peroxidation in liposomes was assessed, as well as superoxide anion (O2(-.)) and hydroxyl radical ((.)OH) trapping activity as determined biochemically and using electron spin resonance (ESR) spectroscopy. All cyclic nitrones tested were much more potent as inhibitors of lipid peroxidation than was PBN. The unsubstituted cyclic variant MDL 101,002 was approximately 8-fold more potent than PBN. An analysis of the analogs of MDL 101,002 revealed a direct correlation of activity with lipophilicity. However, lipophilicity does not solely account for the difference between MDL 101,002 and PBN, inasmuch as the calculated octanol/water partition coefficient for MDL 101,002 is 1.01 as compared to 1.23 for PBN. This indicated the cyclic nitrones are inherently more effective radical traps than PBN in a membrane system. The most active compound was a dichloro analog in the seven-membered ring series (MDL 104,342), which had an IC50 of 26 mum, which was 550-fold better than that of PBN. The cyclic nitrones were shown to trap (.)OH with MDL 101,002 being 20 25 times more active than PBN as assessed using 2-deoxyribose and p-nitrosodimethylaniline as substrates, respectively. Trapping of (.)OH by MDL 101,002 was also examined by using ESR spectroscopy. When Fenton's reagent was used, the (.)OH adduct of MDL 101,002 yielded a six-line spectrum with hyperfine coupling constants distinct from that of PBN. Importantly, the half-life of the adduct was nearly 5 min, while that of PBN is less than 1 min at physiologic pH. MDL 101,002 also trapped the O2(-.) radical to yield a six-line spectrum with coupling constants very distinct from that of the (.)OH adduct. In mice, the cyclic nitrones ameliorated the damaging effects of oxidative stress induced by ferrous iron injection into brain tissue. Similar protection was not afforded by the lipid peroxidation inhibitor U74006F, thus implicating radical trapping as a unique feature in the prevention of cell injury. Together, the in vivo activity, the stability of the nitroxide adducts, and the ability to distinguish between trapping of (.)OH and O2(-.) suggest the cyclic nitrones to be ideal reagents for the study of oxidative cell injury.