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A microtube screening approach affords simple and convenient assessment of the selective adsorption of metal impurities by a variety of different process adsorbents. This approach is helpful in identifying rapid solutions to metal impurity problems in pharmaceutical process research. Several examples illustrating the utility of the approach are presented.

Chromatography plays a vital role in supporting preclinical pharmaceutical development, whether in providing assays for purity determinations, preparative separation of small amounts of intermediates for route selection studies, or purification of bulk drug substances on multikilogram scale. All three approaches are illustrated in the recent development of a candidate HIV protease inhibitor in these laboratories. Chiral supercritical fluid chromatography (SFC) on the hundreds-of-milligrams scale afforded an enantiopure intermediate to facilitate early synthetic studies, HPLC on the tens-of-grams scale provided purified material for use in salt form investigations, and HPLC using a 30 cm column was used to purify 5.6 kg of a key intermediate to provide material for early preclinical evaluations.

An improved method for rapid LC/MS screening of chiral stationary phases based on the use of isotopically labeled enantiomers is reported.

An 15N-NMR study at natural abundance of 04/N3-substituted pyrimidine and C6-substituted purine ribonucleosides has shown that the exact location of the protecting group (substituent) on either 04 or N3 in pyrimidines has a strong influence on the electronic properties of the resultant pyrimidine system, mainly because of the change of state of hybridization of the N3-nitrogen. The basicity of N3 in some C4-substituted pyrimidines has been studied by following the 15N chemical shifts of protonated species in the presence of CF3COOH both in DMSO and in CH2Cl2 solution. A comparison of the basic character of N3 in C4-substituted pyrimidine and of N1 in C6-substituted purine nucleosides has shown that the magnitude of the 15N shift of N3 (or N1) upon protonation is governed mainly by the electronic properties of the heteroatom linked to C4 (or C6). It also clearly emerged in this study that there is very litle difference in basicities of N3 of pyrimidine and N1 of purine nucleosides despite the presence of the fused imidazole moiety in the latter.

Dilute aqueous solutions of ethyl(hydroxyethyl)cellulose (EHEC) and sodium dodecyl sulfate (SDS) have been investigated by ¹H NMR shift and relaxation measurements. The shift measurements indicate no presence of EHEC at the core of the SDS micelles but at and just inside the micellar surface. Both the spin−lattice relaxation time (T1) and the spin−spin relaxation time (T2) imply that molecular motions slow as the system aggregates. T1 measured at both 270 and 400 MHz indicates nonextreme narrowing conditions with molecular motions on the nanosecond time scale. T2 monitors slower dynamics of the EHEC/SDS/water as compared to both of the systems polyethylene oxide (PEO)/SDS/water and SDS/water, attributed to the rigid EHEC backbone as compared to the more flexible PEO chain. An important observation is that the NMR relaxation data correlate well with the microviscosity in the EHEC/SDS/water system as measured by intramolecular excimer formation of the fluorescent probe 1,3-di(1-pyrenyl)propane (P3P).

Disulfide cyclization is a powerful method for reducing the conformational space of a peptide. This in turn may enable the study of its bioactive conformation. Several analogues of angiotensin II (Ang II) containing a disulfide bridge between amino acids 3 and 5 have been reported. Among these the cyclic octapeptides c[Hcy^3,5]-Ang II, c[Cys^3,5]-Ang II, and c[Pen^3,5]-Ang II showed significant activity at Ang II receptors. We have performed conformational analysis studies using theoretical calculations and ¹H-NMR spectroscopy on tripeptide model compounds of these cyclic octapeptides which show that the cyclic moieties of c[Cys^3,5]-Ang II and c[Pen^3,5]-Ang II preferentially assume an inverse γ-turn conformation. On the basis of these results, we substituted amino acid residues 3−5 in Ang II with two different γ-turn mimetics giving four diastereomeric Ang II analogues. Interestingly, two of these are equipotent to Ang II in binding to AT1 receptors. In the contractile test using rabbit aorta rings, one of the analogues is an agonist with full contractile activity approximately equipotent to c[Pen^3,5]-Ang II but 300-fold less potent than Ang II. This low potency may suggest that Ang II does not adopt a γ-turn in the 3−5 region when interacting with the receptor.

A series of extended length heterobifunctional coupling agents is described. The successive aminocaproic acid homologation of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, a known 9-atom long maleimide active ester linker, yielded 16-, 23-, and 30-atom long maleimide active ester homologues. The performance study of these coupling agents in automated microparticle enzyme immunoassays showed that, in the α fetoprotein assay, in which the linkers were employed in the construction of the alkaline phosphatase−antibody conjugates, the signal increased 64% when the length of the linker was incremented from 9 atoms to 23 atoms and 82% for the 30-atom long linker as compared with the 9-atom homologue. Similar improvements were observed in the performance of carbohydrate antigen, marker of ovarian cancer (CA-125), immunoassay where the linkers were used for conjugation of the capture antibody anti-CA-125 to the microparticle. Thus, a 300% signal improvement resulted when a 30-atom linker was used instead of the 9-atom homologue. The observed differences in the performance of the conjugates are interpreted as resulting from improved antibody binding and lowering of the steric hindrance of the complementarity-determined region of the antibody when longer coupling agents were used.

Human glutathione transferases (GSTs) were shown to catalyze the reductive glutathione conjugation of aminochrome (2, 3-dihydroindole-5,6-dione). The class Mu enzyme GST M2-2 displayed the highest specific activity (148 micromol/min/mg), whereas GSTs A1-1, A2-2, M1-1, M3-3, and P1-1 had markedly lower activities (<1 micromol/min/mg). The product of the conjugation, with a UV spectrum exhibiting absorption peaks at 277 and 295 nm, was 4-S-glutathionyl-5,6-dihydroxyindoline as determined by NMR spectroscopy. In contrast to reduced forms of aminochrome (leucoaminochrome and o-semiquinone), 4-S-glutathionyl-5, 6-dihydroxyindoline was stable in the presence of molecular oxygen, superoxide radicals, and hydrogen peroxide. However, the strongly oxidizing complex of Mn3+ and pyrophosphate oxidizes 4-S-glutathionyl-5,6-dihydroxyindoline to 4-S-glutathionylaminochrome, a new quinone derivative with an absorption peak at 620 nm. GST M2-2 (and to a lower degree, GST M1-1) prevents the formation of reactive oxygen species linked to one-electron reduction of aminochrome catalyzed by NADPH-cytochrome P450 reductase. The results suggest that the reductive conjugation of aminochrome catalyzed by GSTs, in particular GST M2-2, is an important cellular antioxidant activity preventing the formation of o-semiquinone and thereby the generation of reactive oxygen species.