A solution-phase screening method for libraries of pharmaceutically relevant molecules is presented. The technique is applicable to screening combinatorial libraries of 20-30 closely related molecules. In this report, individual benzodiazepines are selected from a multicomponent library mixture by formation in solution of noncovalent immunoaffinity complexes with antibodies raised to therapeutically proven drugs such as nitrazepam, temazepam, or oxazepam. Captured compounds are separated from nonspecifically bound library components by centrifugal ultrafiltration. The specifically selected molecules retained on the filter are subsequently liberated from the antibodies by acidification and analyzed by HPLC coupled with pneumatically assisted electrospray (ion spray) ionization mass spectrometric detection. Competition by the benzodiazepines for limited antibody binding sites is controlled by varying the stoichiometry of the complexation mixture. This procedure selects library components with the greatest affinity for a particular antibody. Specific capture of benzodiazepines is demonstrated by screening both a pool of structurally similar benzodiazepines and a more complex mixture of benzodiazepines with an additional set of unrelated compounds. Affinity ultrafiltration and electrospray mass spectrometry complement each other to enhance screening and identification of pooled drug candidates and potentially can be extended to other small-molecule combinatorial libraries and macromolecular receptor preparations.

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N.m.r. spectroscopy demonstrates that N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) exists as a mixture of four isomers, A, B, C and D, the equilibrium ratios of which are 57:8:16:19, respectively, at 25 degrees C. Two of these isomers, A and B, are rotomers of the open chain conformer, while the other two, C and D, are rotomers of the ring tautomer of HPOP and are derived from A and B, respectively, via an intramolecular cyclization reaction. A syn orientation of the nitroso and carbonyl groups favors an open chain configuration (isomer A), while an anti orientation favors cyclization of the molecule (isomer D). Two forms of HPOP (I and II) which are mixtures of isomers A and C, and D and B, respectively were separated chromatographically. These two forms interconvert to each other. The first rate order constants for the interconversion reactions were determined to be 4.7 X 10(-3) and 12.8 X 10(-3)/min, respectively. During these reactions isomers A and D interconvert via the intermediate formation of isomer C. This suggests that rotomerization of C and D is thermodynamically more favorable than rotomerization of their open-chain tautomers A and B, and suggests an intramolecular interaction between the carbonyl and nitroso groups. Isomers A and D are formed during the metabolism of N-nitrosobis(2-oxopropyl)amine (BOP) and cis N-nitroso-2,6-dimethylmorpholine (NNDM), respectively, by hamster liver microsomes and NADH or NADPH. The stereo-specificity of reduction of BOP and the hydroxylation of cis NNDM results in the formation of two slowly interconvertible isomers of HPOP. This, in combination with a possible different metabolic fate of the cyclic and open tautomers of this compound, may have a significant impact on the mechanism of activation of pancreatropic nitrosamines which share HPOP as a common metabolite.