Figure 1.23. Fluoroquinolone libraries.

quences. The product illustrated in Fig. 1.24 has the best combination of in vitro and in vivo properties in this grouping (131). Reports of related studies have also appeared (132, 133).

A library of cephalosporin antibiotic analogs was made on a solid support (basic alumina) without requiring protection-deprotec-tion. The compounds were prepared in high yield (82-93%)and purity in about 2 min with the aid of microwave irradiation (Fig. 1.25) (134). Microwave acceleration in combinatorial chemistry is a powerful technology for enhancing reactions on solid phases.

The small molecule libraries just exemplified belong to the class called focused. That is, in each case a discrete molecular target was available at the outset and chemical routes were generally available. After some adaptation to the needs of the method and rehearsal cf the chemistry, libraries could be generated relatively quickly. Many analogs were then available by comparatively simple variations in the reactants employed. Clearly, in drug seeking, one can operate in much the same manner after identification of a suitable hit molecule.

The strategy required in hit seeking, however, is rather different. Here the initial libraries are usually bigger and more diverse. After the library is screened, and useful molecules are uncovered, subsequent refining libraries are employed that are progressively smaller and more focused. Each succeeding library benefits from the information gained in the previous work so this can be considered the chemist's equivalent of biological evolution. As the work progresses, the needs for quantities of material for evaluation become more and more so the work usually proceeds back into the larger scale one at a time mode resembling the BC (before combichem) era.

A couple of examples represent the very large amount of work carried out in this manner. First, consider the discovery and progression of OC 144-093, an orally active modulator of P-glycoprotein-mediated multiple drug resistance that has entered clinical studies. First, a 500-membered library of variously substituted imidazoles was prepared on a mixture of aldehyde and amine beads (Fig. 1.26). The choice of materials was based on prior knowledge of the structures of other P-glyco-protein modulators. Screening this library in whole cells led to the identification of two main leads, A, possessing an IC50 of 600 nM, and B, possessing an IC50 of 80 nM. In addition, B possessed an oral bioavailability in dogs of about 35%. These results were very encouraging.

The third stage involved making a solution-based library based on the structures of A and B. Screening produced leads C, possessing an IC50 of 300 nM, and D, with an IC50 of 150 nM. Interestingly, D was an unexpected by-product. The chemistry in libraries does not always go as intended. In addition to reasonable potency, D showed enhanced metabolic stability, so it was chosen as the lead for the next phase. Analoging around structure D lead ultimately to OC 144-093, with an IC50 of 50 nM and an


Strong gram +ve Moderate gram -ve Potent in vivo

Strong gram +ve Moderate gram -ve Potent in vivo


ii. Chromat.


ii. Chromat.

Figure 1.24. Oxazolidinone libraries.

estimated 60% bioavailability after oral administration in humans (135, 136).

Later biological studies in vitro and in vivo have shown that the agent enhances the activity of paclitaxel by interfering with its export by P-glycoprotein. It is not a substrate for and interferes with paclitaxel metabolism only at comparatively high doses. After IV administration, OC 144-093 does not interfere with paclitaxel's pharmacokinetic profile but elevates its area under the curve when given orally. The results are interpreted as meaning that OC 144-093 interferes with gut P-glycoprotein, enhancing oral bioavailability. Further studies are in progress and it is hoped that a marketed anticancer adjunct will emerge in due course as a result of combinatorial chemistry (137).

In a different study, a search through a company compound collection was made in an attempt to find an inhibitor of the Erm family of methyltransferases. These bacterial enzymes produce resistance to the widely used macrolide-lincosaminide-streptogramin B an-

Figure 1.25. Cephalosporin libraries.

tibiotics by catalyzing S-adenosylmethionine-based methylation of a specific adenine residue in 23S bacterial ribosomes. This interferes with the binding of the antibiotics and conveys resistance to them. Using NMR (SAR by NMR) screening, a series of compounds including 1,3-diamino-5-thiomethyltriazine were found to bind to the active site of the enzyme, albeit weakly (1.0 mM for the triazine named) (Fig. 1.27). Analogs were retrieved from the collection, and analogs A, B, and C identified as promising for further work. A solution phase parallel synthesis study was performed from which compound D emerged as being significantly potent. Next a 232-compound library was prepared to discover the best R group on the left side of compound D. From this, compounds E and F emerged. These were now potent in the low micromolar range. The left side cf analog E was fixed and the right side was investigated through a 411-membered library. From this, E emerged as the best substance with a K{ of 4 yM against Erm-AM and 10 jaM against ErmC. Thus, starting with a very weak lead with a malleable structure, successive libraries produced analogs with quite significant potency for further exploration (138).

It is iust a decade after this field became generally active, yet already most of the common drug series and hundreds of different heterocyclic classes have been prepared in library form. Originally the emphasis was on bead-

based chemistry, and this actually slowed general acceptance of the method because few organic and medicinal chemists were familiar with the techniques needed to make small molecules on beads through non-iterative methods, and indeed, much of the needed technology had yet to be developed and disseminated. These problems have largely been overcome, and today the choice of beads or no beads is partly a matter of taste, the size of the libraries being made, and the length of the reaction sequences required.

The remainder of this chapter deals with selected examples that illustrates particular concepts and methodologies.

4.1 Purification

In communicating their results, chemists explicate the route with formulae and often discuss the relative strengths and weaknesses of key reagents but almost never devote time to workup. Even so, the details of the workup require attention to detail in the performance and are sometimes quite challenging. This factor becomes even more demanding in combinatorial work where the need for rapid, effective workup is intensified. Little is gained if one saves much time in construction only to have to give this back by tedious and repetitious purification schemes. Performing chemistry on beads addresses this in that simple filtration and washing often suffices. This is not as useful if the reactions do not go to completion, so considerable excess of reagents and more lengthy times are often employed to drive the reactions further to completion. Separation from solution in solid form or simple evaporation is very convenient, and manifolds for filtration and for solvent removal are commercially available. From a drugability standpoint, there is a danger in this. Compounds that separate readily from polar solvents are often of very low water solubility and present difficulties in testing. A number of commercially available combinatorial screening libraries are peppered with such substances.

Column chromatography is powerful but often labor intensive and solvent consuming. Separation of hundreds of analogs by column chromatography would be a nightmare. With smaller, focused libraries, this is often more manageable.

Phase 1.

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