Stereoselective Synthesis of over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assays

Understanding the cellular function of a protein usually requires a means to alter the function. This is commonly done by mutating the gene encoding the protein (genetic approach). It can also be done by binding the protein directly with a small molecule ligand (chemical genetic approach). Such ligands, primarily natural products or synthetic variants of them, can either inactivate1 or activate2 protein function. For the chemical genetic approach to have its maximal impact, efficient methods of ligand discovery will be required to provide, in the limit, a small molecule partner for every gene product. Our laboratory has recently described miniaturized cell culture assays for screening large numbers of small molecule ligands, potentially on a genome-wide basis.3 However, despite its success in natural products synthesis, synthetic chemistry has not yet been applied to the synthesis of vast numbers of compounds with structures both reminiscent of natural products and compatible with miniaturized assays. These features of a synthesis will likely be required in order to discover nonnatural compounds having the binding affinities and specificities characteristic of natural products. The synthetic strategy we have undertaken is to develop highly efficient multistep syntheses of natural product-like compounds that include several coupling steps and to use split-pool techniques4 at these steps in order to generate diverse outcomes. This technique requires that one of the coupling substrates be immobilized on a solid support. Our syntheses are further constrained by the water-compatible photolabile supports required when carrying out large numbers (>105) of miniaturized “nanodroplet” cell culture assays aimed at identifying cell permeable ligands.3 This presents a considerable synthetic challenge since the structural elements that provide these properties (e.g., poly(ethylene glycol), nitrobenzyl moieties) are incompatible with numerous reagents used in conventional synthetic chemistry. With these considerations in mind, we first converted shikimic acid, 1, into both enantiomers of epoxycyclohexenol carboxylic acid 2,5 which were then coupled to a photocleavable linker on solid support by standard methods (Figure 1).6 Treatment of the resin-bound epoxycyclohexenol, 3, with various nitrone carboxylic acids,7 7a-f and 9b-d, under esterification conditions yielded tetracyclic compounds 4a-f and 5b-d with complete regioand stereoselectivity, presumably via tandem acylation/1,3-dipolar cycloaddition.8 No intermediate nitrone ester or carboxy isoxazolidine structures were observed. The iodoaryl tetracycles 4b-d and 5b-d were selected for further study since the supportbound aryl iodides could be modified with commercially available reagents, avoiding the need for solution-phase syntheses of numerous nitrone acids. Tetracycles 4b-d and 5b-d are rigid, densely functionalized compounds that can undergo further reactions to introduce a variety of functional groups around the central octahydrobenzisoxazole structure, notably, without the use of protecting groups. The iodoaryl groups can serve as substrates for palladium cross-coupling reactions. The electrophilic lactone and epoxide can react with nucleophiles while simultaneously unmasking alcohols for subsequent reactions. Furthermore, reductive N-O bond cleavage would provide two additional handles for functionalization. We developed several such reactions (Figure 2) with product purity ranging from ∼50 to g98% following photocleavage.9 Since purification is not practical in a large split-pool synthesis, only reactions generating products in g90% purity were acceptable. The most promising reactions were studied in detail to define the scope, limitations, and optimal conditions for each (Figure 3). Iodobenzyl tetracycles 4b-d were selected as scaffolds since (1) See http://www-schreiber.chem.harvard.edu on the Web. (2) Crabtree, G. R.; Schreiber, S. L. Trends in Biochem. Sci. 1996, 21, 418-422. (3) (a) Borchardt, A.; Liberles, S. D.; Biggar, S. R.; Crabtree, G. R.; Schreiber, S. L. Chem. Biol. 1997, 4, 961-968. (b) You, A. J.; Jackman, R. J.; Whitesides, G. M.; Schreiber, S. L. Chem. Biol. 1997, 4, 969-975. (4) (a) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487-493. (b) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84. (5) (a) Wood, H. B.; Ganem, B. J. Am. Chem. Soc. 1990, 112, 89078909. (b) McGowan, D. A.; Berchtold, G. A. J. Org. Chem. 1981, 46, 23812383. (c) Mitsunobu, O. Synthesis 1981, 1-28. (6) The resin structure shown represents Tentagel S NH2, a poly(ethylene glycol)-polystyrene copolymer, loaded with a photocleavable 3-amino-3-onitrophenylpropionic acid linker. (Brown, B. B.; Wagner, D. S.; Geysen, H. M. Mol. DiV. 1995, 1, 4-12.). (7) Keirs, D.; Overton, K. Heterocycles 1989, 28, 841-848. (8) Tamura, O.; Okabe, T.; Yamaguchi, T.; Gotanda, K.; Noe, K.; Sakamoto, M. Tetrahedron 1995, 51, 107-118. (9) Photocleavage products were analyzed by 1H NMR, FAB-MS, HPLC, and TLC. Figure 1. Synthesis of tetracycles and nitrones. a: X ) H, b: X ) 2-I, c: X ) 3-I, d: X ) 4-I, e: X ) 4-CF3, f: X ) 3,4-(OMe)2.