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Solid-Phase Synthesis and Combinatorial Technologies. Pierfausto Seneci Copyright © 2000 John Wiley & Sons, Inc.

ISBNs: 0-471-33195-3 (Hardback); 0-471-22039-6 (Electronic)

5Synthetic Organic Libraries: Library Design and Properties

From now until Chapter 9 our attention will focus on synthetic organic libraries, which are by far the most relevant libraries produced by combinatorial technologies. Their basic properties and their design by means of computational techniques will be described in this chapter as an introduction to the different synthetic organic library formats, which will be covered in the next chapters.

So-called primary, or unbiased, libraries will be described first, then focused, or biased or targeted, libraries will be presented and thoroughly discussed. A somewhat intermediate class of synthetic organic libraries, which we call biased-targeted, will also be introduced, and their properties and their usefulness will be highlighted. For each of these three classes there will be a discussion of their rationale, their synthesis, and the selection of the scaffolds and the monomer sets used to build the libraries.

The fourth section of this chapter will address a fundamental topic, the use of computational chemistry methods to help and to drive the design of each library class. A section for each library class will briefly illustrate all the relevant methodologies and their applications through the discussion of specific examples.

5.1 PRIMARY LIBRARIES: SHOOTING IN THE DARK?

5.1.1 Properties

We call primary, or unbiased, or diversity based, the libraries that are not designed on the basis of structural information, but rather those intended as diverse collections of molecules as sources of active compounds for different targets. Examples of such libraries have been reported frequently in the literature (1–3), and their main properties are shown in Fig. 5.1.

These libraries contain a large number of individuals (typically thousands or more) and are often prepared as pool libraries, even though the recent progress of automated parallel synthesis allows the preparation of thousands of discretes in a timely manner. Primary libraries are predominantly prepared on SP because of the easier preparation of high-quality pools on SP and the negligible work-up required for each SP sample. The library components are prepared in small quantities, often significantly less than a milligram. They are diversity-based (see Section 5.4.1) and are usually tested on many different targets: if their design is successful, they represent a source of positives for most of the screenable targets. The positives obtained from these libraries, called

165

166 SYNTHETIC ORGANIC LIBRARIES: LIBRARY DESIGN AND PROPERTIES

PRIMARY LIBRARIES

-LARGE (thousands to millions)

-FREQUENTLY ON SOLID PHASE

-FREQUENTLY IN POOLS

-<<1mg PER LIBRARY INDIVIDUAL

-DIVERSITY-BASED

-TESTED ON MANY TARGETS

-DESIRED OUTCOME: HIT

Figure 5.1 Primary libraries: main features.

hits, typically have a moderate or even weak activity and represent the starting points for further structure optimization.

5.1.2 Rationale

Whenever a library is designed in a particular format, some basic questions should be addressed to justify its synthesis. We will address them first in theory; then we will apply the same principles to a published example of a primary library.

•Why? A primary library can be planned for many purposes. Sometimes the project goal is purely academic, that is, simply to open a new combinatorial synthetic route and to report it. This respectable option allows complete freedom to prepare any primary library, with no constraints dictated by parameters such as application, cost, or resources. We will not comment further upon this approach in this section. More often, though, a primary library is prepared as a source of relevant active molecules on various biological targets. This obviously necessitates the availability of several robust and reliable HTS assays for these targets, as well as the synthetic resources, the analytical resources, and the instrumentation to ensure the successful synthesis and analytical characterization of the library.

•When? The combinatorialization of the planned synthesis is often long and arduous for primary libraries, so several applications must be available for the library to compensate for this major undertaking: A single target would not justify the efforts required, and hence another library format would be preferable.

•How? The large number of components of primary libraries is more suitable for SP pool libraries, but each library has to be designed keeping in mind the available equipment and the expertise of the chemical resource(s). The same is true for the analytical methods used to check every step of the library synthesis,

5.1 PRIMARY LIBRARIES: SHOOTING IN THE DARK? 167

which must produce a reliable quality profile for all or most of the library components. It is important to have a certain flexibility, that is, to be able to use different library formats for different applications without negative consequences on the quality of the prepared primary libraries.

•What? The chemistry required for a primary library synthesis must be extremely robust and assessed to accommodate a wide range of monomers/scaffolds in order to produce the large numbers of diverse individuals needed. A compromise between “easy” reaction schemes, which readily produce a reliable library but may not lead to novel positives, and “hard” ones, where the library components are novel but the optimization of reaction conditions is difficult and requires major efforts, should be found for each specific project.

•How Much? Every synthesis has its costs, both in terms of resources and reagents: A primary library synthesis employs a large number of chemicals, and its total cost will always be significant. The introduction of expensive reagents, monomers, or scaffolds should be avoided as a general rule, using commercially available chemicals whenever possible and utilizing computational tools to improve selection among them to help prepare a diversity-based primary library (see Section 5.4.1). The same is true for monomers/scaffolds requiring a long synthesis, which are better suited for more focused libraries, as we will describe in the next section.

The primary library shown in Fig. 5.2, which was reported by Baldwin (1), was clearly designed as a source of biologically active molecules on several targets (why), thus compensating the efforts required for the chemical assessment and for a satisfactory characterization (when). The SP library was prepared in pools using chemical encoding (4, 5) to produce a population of around 62,000 individuals (how). The synthetic scheme was composed of both simple and more complex SP steps, and several monomer sets (A–F, Fig. 5.2) were used (what). These monomers were either commercially available or easily prepared from commercial precursors, while the library benzopyranic core was formed during the synthesis (how much).

5.1.3 Synthesis and Characterization

After the design of a library in the most suitable format, the synthetic process starts with the chemical assessment. Usually the designed synthetic route is first tested in solution on one or a few representative structures, then its transfer onto SP is accomplished while optimizing the reaction conditions for model individuals. This process already has been described in detail in Chapter 3.

The preparation of a large primary library requires the use of some monomer sets containing a considerable number of monomers, and before embarking on the library synthesis, a so-called monomer rehearsal (6) is necessary. The monomer set is split into homogeneous subsets, and a representative of each subset is used in the relevant SP reaction step. A theoretical example is shown in Fig. 5.3, where eight representative aldehydes (M1-CHO–M8-CHO) are examined for a reductive amination with R–NH2. If even after significant efforts to adapt the poorly performing monomers (e.g., change

168 SYNTHETIC ORGANIC LIBRARIES: LIBRARY DESIGN AND PROPERTIES

			NO2
	P			N	R1
		H
		N	O			R7 N R6
			O			O
						0-1	R4
						O
		61,740 compounds					NH
						R3
						R2	R5
	A					HOOC
	NO2							O
		R1
	N						O
HO	O	O					0-1
								OH
O				O	C		B
	7 monomers							R2
			R3		R4
						6 monomers
				NHBoc
			3 monomers
R5	X D		R6	NH2 E		R7	X F
7 monomers			7 monomers			10 monomers

Figure 5.2 Example of a benzopyran primary library.

of protecting groups) their reactivity remains poor, they are removed from the whole set: In our example only six aldehydes pass the rehearsal, producing the expected reaction product with good yields and purities (Fig. 5.3). During the monomer rehearsal an extensive analytical characterization is essential, using on-bead and off-bead methods, to detect the side products formed, to assess the purity of the resin-bound intermediates, and to understand the influence of each modification of the reaction conditions on the outcome. Rather often this process is not extensively reported in published works, as for (1), but the unequivocal basis of a good-quality pool library lies in careful monomer rehearsal.

The next step in primary library synthesis is to prepare the so-called model library, which contains a small number of compounds (typically from 5 to 50) prepared using the very same instrumentation, techniques, and format planned for the library synthesis (6, 7). Simply moving from single compounds to simultaneous multiple synthesis requires adaptation of the technical operations (addition of solids or liquids, evaporation of solvents, stirring, reaction times, etc.), and the impact of these modifications is sometimes significant: A complete analytical characterization of the model library using the most appropriate techniques allows selection of the best operating conditions to be used for the library synthesis. The direct synthesis of a large library after monomer rehearsal may sometimes produce poor-quality results for the above-mentioned rea-

5.1 PRIMARY LIBRARIES: SHOOTING IN THE DARK? 169

						O					+
					X			H
					X = M1-8, 8
				monomer candidates
O				A
					S
M1			H
O				A				O
					F
M2			H			M					H
O						2
				A
					S
M3			H					O
O				A
					F
M4			H			M4					H
O				A
M5			H		S			O
O				A
M			H		F	M6					H
6
O				A				O
M			H		F	M7					H
7
O				A
					S
M8			H

R NH2

B F

B S

B S

B F

reductive

amination

R

X N H

O					O				O
M1			H	M3			H	M4			H
O					O				O
M5			H	M6			H	M8			H

ACCEPTED AS MONOMERS experimental conditions as in B

O O

M2 H M7 H

REJECTED

A= first experimental conditions tried B= experimental condition adjustment S= successful

F= failed

Figure 5.3 Monomer rehearsal: A hypothetical example for reductive amination.

sons, wasting substantial amounts of precious materials and the significant efforts of both synthetic and analytical chemists. It is highly recommendable to follow this gradual scale-up process via high-quality model libraries to minimize the risk of synthesis failures.

The primary library synthesis is then carried out using any of the techniques that will be illustrated in detail in the next three chapters for solutionor solid-phase, pool or discrete libraries. Many choices in terms of instrumentation or automation are available, and each of them may afford high-quality results. The same is true for analytical quality control, which determines the synthesis outcome and decides if the library is suitable for screening against desired targets.

5.1.4 Scaffolds and Monomers

A synthetic organic library is characterized by a common core structure for all its components: This motif may be used to start a library synthesis that consists of the

170 SYNTHETIC ORGANIC LIBRARIES: LIBRARY DESIGN AND PROPERTIES

decoration of this scaffold or it may be assembled from the various synthons to give the scaffold. For both options monomer sets will be used, either to decorate the scaffold through its randomization points (decoration libraries) or to build the scaffold and simultaneously introduce diversity in the library components (assembly libraries).

The assessment of a solid and reliable synthetic route for a primary library is essential, but it is not the only requirement to fulfill. A large library requires the availability of large monomer sets that should, if possible, be composed of commercially available chemicals. The costly and time-intensive synthesis of several monomers can be justified only when the compounds produced using them have a high probability of being useful, not when diversity is the only criteria driving the library synthesis. Examples of monomer classes that are suitable for large library synthesis include alcohols, amines, aldehydes, carboxylic acids, α-amino acids, and other common functionalities. The appetite for new proprietary monomers and for new monomer sets is increasing with the interest for combinatorial technologies; when a serious and continuous involvement in this new discipline is planned, even the synthesis of precious proprietary monomers (e.g., bifunctional monomers with orthogonal protection and poorly represented functionalities) in large amounts to be used repeatedly for high-value primary libraries becomes convenient and even desirable due to the novelty they ensure to library components. We will discuss the computational methods available for the selection of monomers for a primary library within Section 5.4.

A different scenario must be considered for a scaffold, especially when the library is made by its decoration. A highly functionalized proprietary scaffold is a very desirable asset, and a difficult synthesis may be accepted because even easily accessible commercial monomers will produce original library individuals by its decoration. Significant efforts to design hindered and constrained polyfunctional scaffolds, often taking advantage of the structures of natural products, are commonly accepted and may represent the key factor to prepare a high-quality meaningful primary library.

5.2 FOCUSED LIBRARIES: HIGH-THROUGHPUT STRUCTURE–ACTIVITY RELATIONSHIPS

5.2.1 Properties

We call focused, or biased, or similarity based the libraries designed using structural information (a compound active on the target, or on a similar target) or a rational hypothesis and allowing the library structure to focus on very specific features. The main properties of such libraries are shown in Fig. 5.4.

These libraries contain a relatively small number of individuals (typically tens to hundreds) and are almost always prepared as discrete libraries using parallel synthesis and automated or semiautomated devices. Focused libraries are predominantly prepared in solution because of the easier shift from classical organic synthesis to solution-phase combinatorial chemistry, while automated purification procedures for relatively small arrays of discrete compounds in solution are common nowadays. The

5.2 FOCUSED LIBRARIES 171

FOCUSED LIBRARIES

-MEDIUM/SMALL (tens to thousands)

-FREQUENTLY IN SOLUTION

-FREQUENTLY AS DISCRETES

->1mg PER LIBRARY INDIVIDUAL

-SIMILARITY-BASED

-TESTED ON A SPECIFIC TARGET

-DESIRED OUTCOME: LEAD

Figure 5.4 Focused libraries: main features.

quantities of single library components are usually a few milligrams but may even reach a few tens of milligrams. They are similarity based (see Section 5.4.2), and they are usually tested only on the target/assay for which the library was prepared. If their design is successful, they provide a relatively accurate, even if preliminary, structure– activity relationship for the specific target allowing the selection of a compound, or a few compounds, called lead(s), with significant activity on the target.

5.2.2 Rationale

•Why? A focused library is used as a source of lead compounds for a specific target, with a better activity profiling than for hits from primary libraries: smaller numbers allow testing of the library components in mediumor even lowthroughput assays. Other properties besides the potency on the target are evaluated for active library components to determine their usefulness (solubility, toxicity, aspecificity, and physicochemical properties, among others). Measurement and evaluation of these parameters must be fast to allow rapid project progression. The synthetic and analytical instruments required to synthesize and analytically characterize the library are significantly less complex than for a primary library; even the normal equipment of an organic synthesis laboratory is often appropriate.

•When? The combinatorialization, usually in small solutionor solid-phase arrays, of the planned synthesis is not particularly demanding for focused libraries. Moreover, an active compound from a primary library is often the structural information around which the focused library has been designed so that a detailed chemical assessment has already been performed. The use of focused libraries should become more and more frequent given the simplicity of their synthesis and the significant impact they may have on the progression of many projects.

172SYNTHETIC ORGANIC LIBRARIES: LIBRARY DESIGN AND PROPERTIES

•How? Each library has to be designed according to the equipment and the expertise of the combinatorial chemist(s) but also, if possible, using the knowledge accumulated during the synthesis of related primary libraries. The same is true for the analytical methods used to check every step of the library synthesis; the components of a focused library must be thoroughly evaluated, their purity requirements are high, and their analytical characterization may be slower but more accurate than for a primary library. The flexibility of using different library formats for different applications is especially important for the automated purification procedures that are becoming popular and allow isolation of significant amounts of every library component with a high degree of purity.

•What? The chemistry required for a focused library synthesis may be extremely robust and assessed, but even less robust synthetic schemes can be adapted to accommodate the needs of a small focused library synthesis. All the library components are inspired by the structural information available, but nevertheless they must provide a detailed exploration of the structure–activity relationship for analogues of the parent structure.

•How Much? A focused library is by definition composed of a manageable number of entities, and its total cost will not be significant most of the time. The introduction of meaningful, expensive reagents, monomers, or scaffolds must be accepted and encouraged, even when they require significant synthetic efforts, providing that less expensive or commercial replacements are not available.

A small focused library, shown in Fig. 5.5, which was reported by Wipf and co-workers (8, 9), was designed to exploit the information provided by natural inhibitors of serine/threonine protein phosphatases (why) and to gain additional

				H	O
	R4						R3
				N	N
		O COOH
				O	NH
	18 compounds
	as discretes				R2
				N
					O
				R1
	COOH
	N			H				O
					R3
R1	O R2			N			R4
		Alloc			N					Cl
					H
2 monomers				3 monomers			3 monomers
	A			B				C

Figure 5.5 Example of an oxazole focused library.