Diversity Oriented Synthesis

Synthetic chemists have generally strived to prepare predesigned molecules, such as in total chemical synthesis of complex natural products. Similarly, medicinal chemists are initially designing target molecules, which subsequently are prepared in the laboratory. Such approaches can be classified as target-oriented synthesis (TAS, Figure 4.2). This mindset was modified with the advent of combinatorial chemistry, where compounds were designed and prepared in either parallel or combinatorial manner, instead of preparing target molecules in a linear fashion. Thus combinatorial chemistry could provide a significantly increased number of compounds and it was believed that this increased number eventually would lead to an increased number of drugs on the market. However, it appears as if combinatorial chemistry have not led to the expected revolution in the number of new drug leads, and it is believed that one of the primary issues is the lack of "diversity,"* that is, that in the pursuit of generating large number of compounds, the design and diversity of the compounds might have been compromised.

As a consequence, a fundamental different way of preparing compounds have been proposed, where rather than producing many compounds with limited diversity, compounds should be instead generated with a focus on maximizing diversity of the compounds generated (Figure 4.2). These compounds are not directed toward a single biological target, but can be used to identify new ligands for a number of targets (see Section 4.2.2). The concept of DOS furthermore strives to apply a relatively limited number of chemical reactions; however, each of these reactions should generate maximal diversity. When applying DOS, a number of principally different strategies are being used, in particular strategies that distinguish between building block diversity and skeleton diversity. In the former, a variation of building blocks are attached to the same scaffold, whereas performing DOS with skeleton diversity leads to generation of compounds with fundamental changes in the skeleton.

* It is beyond the scope of this chapter to discuss the philosophical aspects of the term "diversity." Here it is used to describe the extent of variation (of predefined properties) within a set of chemical compounds.

Target-oriented synthesis

Diversity-oriented synthesis (b)

FIGURE 4.2 (a) Illustration of the principles of TAS and DOS. In TAS the synthesis of a single-target molecule is convergent, and the synthetic strategy is devised by a retrosynthetic analysis. The synthesis of multiple target molecules in DOS is divergent and the synthetic strategy planned by forward synthetic analysis. (b) Example of DOS, where different pairings of the functional groups (ester, alkyne/alkene, and nitro group) lead to three different scaffolds.

A number of successful examples for the application of DOS for the synthesis of diverse combinatorial libraries, phenotypic screening, and subsequent elucidation of the biological target have been carried out and will be discussed in Section 4.2.2. In general, the application of DOS in chemical and biological studies is mainly carried out by academic laboratories and further generalization of DOS principles is required to broaden the applicability of DOS in drug design and development.

4.2.1.2 Fragment-Based Approaches

The application of fragments of chemical compounds has been used for some time to simplify computational analysis of ligand binding and in the analysis of pharmacophore elements. However, only recently a similar approach has been used in drug screening, the concept being that by screening low-molecular-weight compounds or fragments, low-affinity binders are found, and, ideally, combining or evolving fragments should then lead to chemical compounds with much-improved affinity. One of the advantages compared to high-throughput screening (HTS) is that hits from fragment-based screening generally have lower molecular weight and more efficient binding affinity and provides better starting points for lead generation.

A requirement for fragment-based screening is that a sufficiently sensitive technology for screening is available, that is, a technology that allows identification of low-affinity (in the mM range) binders. In addition, structural information is required to move from binding fragments to high-potent lead compounds. Therefore screening is carried out using bioassays such as surface plasmon resonance (SPR) in combination with biophysical techniques such as nuclear magnetic resonance (NMR) technologies or x-ray crystallography, the latter providing structural guidance for further development.

Once one or more fragments have been identified, iterative cycles of medicinal chemistry, ideally guided by three-dimensional structural data, are performed to optimize these weak binding fragment hits into potent and selective lead compounds. There are principally, two different approaches for optimization: (1) fragment linking or (2) fragment evolution (Figure 4.3). If two fragments have been identified that bind in separate binding sites that are sufficiently close to each other, those fragments can be chemically linked and provide a lead, this is called fragment linking (Figure 4.3).

HO-NH

HO-NH

(a) MMP3 inhibitors

(a) MMP3 inhibitors

N COOMe -JO

MeO y OMe

MeO y OMe

MeO y OMe

OMe FKBP inhibitors owO

HO O COOH

Thymidylate synthase inhibitors

FIGURE 4.3 Illustration of the two principles applied in fragment-based screening. (a) Fragment linking: two fragments were identified as very weak inhibitors of matrix metalloproteinase 3 (MMP3), but by linking a highly potent lead compound was identified. Similarly, a potent inhibitor of the FKBP was achieved by linking two weaker inhibitors. (b) Fragment evolution: a very potent inhibitor of thymidylate synthase was identified.

COOH

HO O COOH

Thymidylate synthase inhibitors o o os"

O COOH

COOH

COOH

O COOH

COOH

FIGURE 4.3 Illustration of the two principles applied in fragment-based screening. (a) Fragment linking: two fragments were identified as very weak inhibitors of matrix metalloproteinase 3 (MMP3), but by linking a highly potent lead compound was identified. Similarly, a potent inhibitor of the FKBP was achieved by linking two weaker inhibitors. (b) Fragment evolution: a very potent inhibitor of thymidylate synthase was identified.

Alternatively, the knowledge of the identified fragments is used to develop hypotheses on how to expand the fragment by additional interactions in the active site of the protein. This "evolution" then leads to compounds with improved binding, which can be further optimized (Figure 4.3). The fragment "evolution" principle is probably most often applicable and is an elegant demonstration of structure-based drug design (see Chapter 2).

Fragment-based drug design is a highly promising strategy that is widely applied both in industry and at academic institutions. It is still limited to studies of soluble proteins, as easy access to detailed structural information is required. Moreover, due to the novelty of the approach, there are still no drugs on the market, which are developed by fragment-based screening.

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