Chemical modification of pre-formed asymmetric polyazaphane scaffolds by simultaneous addition of functionality (letters) in solution has been developed for the preparation of tertiary nitrogen-based combinatorial chemistry libraries. This approach has some significant advantages over the more commonly employed solid phase bead splitting/reaction/mixing procedures for the preparation of libraries. Three novel, asymmetric polyazaphanes 32, 33, and 37 have been synthesized in high yields by an efficient cyclization of 2,6-bis(bromomethyl)pyridine (31) with new orthogonally protected triamines 29, 30, and 35, respectively. Selective deprotection of 32, 33, and 37 provided mono-t-Boc-protected scaffolds 1−3 suitable for solution phase, simultaneous addition of functionalities. Model studies of small libraries of scaffold 2 using CZE analyses indicated that simultaneous addition of 10 benzylic bromide alkylating functionalities would result in libraries containing approximately equimolar amounts of all possible compounds. Sixteen purified tertiary amine libraries 4−19 (total complexity of 1600 compounds) were generated by this procedure from scaffold 2. A “fix-last” combinatorial method was devised to minimize chemical reactions. Several first-round sublibraries of scaffold 2, containing a mixture of 100 compounds, exhibited potent antimicrobial activities. Twenty single compounds 63−82 with uniform functionalities at the combinatorialized sites were synthesized. Some of these pure compounds were more active, while others were less active, compared with the parent mixtures 5 and 10.

Novel linear pyridinopolyamine derivatives 1−3, 7, and 8 have been synthesized as scaffolds for combinatorial drug discovery. The mono-t-Boc- and monotosyl-protected linear scaffolds 1 and 2 were obtained by a Schiff base type cyclization of 2,6-pyridinedicarboxaldehyde (24) with monoprotected triamines 22 and 23 using Ni2+ as a metal template, followed by reductive cleavage and decomplexation in a one-pot procedure. The unprotected linear scaffold 3 was obtained by treating 1 with TFA. Scaffold 1 was also synthesized from the orthogonally protected pyridinopolyamine 7 which was constructed from 2,6-bis(bromomethyl)pyridine (29) in four steps. Selective deprotection of the key intermediate 7 afforded 8, which was further selectively deprotected to give scaffold 1. A combinatorial chemistry strategy involving solution phase simultaneous addition of functionalities (SPSAF) is described. Thirteen high-purity tertiary amine libraries (9−21) (total 1638 compounds) were synthesized by the SPSAF and fix last methodologies from linear polyamine scaffolds 1 and 2. All libraries were examined by TLC, purified by chromatographic techniques, and characterized by 1H NMR and ESI MS spectral data. A fix last methodology was utilized to minimize chemical reactions and perform SAR studies directly on libraries. Several first-round sublibraries of scaffold 1, containing 126 compounds each, exhibited potent antibacterial activity with MICs of 1−12 μM against Streptococcus pyogenes and Escherichia coli imp -.

A 1638-member pyridinopolyamine library, consisting of 13 sublibraries of 126 members prepared by a solution-phase approach, was completely deconvoluted from orthogonally protected intermediates by a combination of iterative and positional scanning procedures. Antibacterial assays against Streptococcus pyogenes and Escherichia coli imp- and a Candida albicans yeast specificity assay were employed to follow the activity of sublibraries. Screening of the 13 sublibraries, which were prepared by a synthetic method that places the differentiating functionality in a selected position A (secondary amine), at the end of the synthesis (fix last), provided several first-round activities. Subsequently, six single pyridinopolyamines (2-7) were prepared where the first-round winner, a hydrogen atom, is in the first deconvoluted position and the remaining three positions contained the same functionalities. The range of antibacterial and yeast activities of these single compounds suggested that a more active and selective compound may be discovered by completely deconvoluting the first-round active sublibraries. Pyridinopolyamine positions B (secondary benzylamine) and C (primary benzylamine) were then sequentially positionally scanned with a set of six meta-substituted benzyl functionalities to generate two sets of second/third-round sublibraries, containing 21 or 36 compounds in each sublibrary, respectively. High-throughput screening yielded sublibraries 15, 18, and 21 with MICs of 1-5 microM against S. pyogenes and E. coli imp-. Using rounds 1 and 2/3 screening data, two sets of single compounds (22-27) and (28-32) with the combination of m-(trifluoromethyl)-benzyl group at position C and m-(trifluoromethyl)benzyl or m-methylbenzyl group at position B with position D (primary benzylamine) fixed were synthesized in the fourth round deconvolution. Subsequently, broader screening of deconvoluted compounds against a tier II panel of wild-type bacteria identified eight compounds (5, 7, 27, and 29-32) with approximately 100-fold greater selectivity for Gram-positive than Gram-negative bacteria. Thus, S. pyogenes, S. pyogenes (wild-type), Streptomyces aureus, and Enterococcus faecalis were inhibited at MICs of 1-12 microM, whereas MICs for E. coli, Klebsiella pneumoniae, Proteus vulgaris, and Pseudomonas aeruginosa were > 100 microM. These eight compounds were not active (> 100 microM) against fungus C. albicans.

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