Solid-Phase Synthesis and Combinatorial Technologies

3.5 SOLID-PHASE SYNTHETIC STRATEGIES: SELECTED EXAMPLES 133

2.Depending on biological results on the prostaglandin EP3 receptor a larger set of compounds could easily be obtained using other amines or N-nucleophiles (route a) or terminal alkenes (route b) or cuprates (both routes);

				TMTtO
				Et
			P	Si
				O
				Et
		route a		3.117
				O
	O			R3
				N
	Et			R4
P	Si	R2
	EtO	3.124
			O
O			R3
			N
			R4

R2

HO

3.125

20 amides overall yields

from PS-DES: 18-56%

Br

route b

	O	R1
	Et
P	Si	R2
	EtO	3.126
	O	R1

R2

HO

3.127

6 alkyl derivatives overall yields

from PS-DES: 32-41%

R1 = H, Me, Et; R2 = A or B; HNR3R4 = 8 primary and 2 secondary alkyl amines

O
OH A	B OH

Figure 3.47.

134SOLID-PHASE SYNTHESIS: SMALL ORGANIC MOLECULES

3.The SP route should be of general use, and different classes of monomer sets could focus either towards other prostaglandin receptors or even generate large libraries of diverse prostaglandins.

REFERENCES

1.Bolton, G. L., Hodges, J. C. and Rubin, J. R., Tetrahedron 53, 6611–6634 (1997).

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3.March, J., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure , fourth edition. John Wiley & Sons, New York, 1992.

4.Bruice, P. Y., Organic Chemistry, second edition. Prentice Hall Press, Englewood Cliffs, NJ, 1998.

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9.Schore, N. E. and Najdi, S. D., J Am. Chem. Soc. 112, 441–442 (1990).

10.Spitzer, J. L., Kurth, M. J., Schore, N. D. and Najdi, S. D., Tetrahedron 53, 6791–6808 (1997).

11.Sieber, P., Tetrahedron Lett. 28, 6147–6150 (1987).

12.Fruechtel, J. S. and Jung, G., Angew. Chem., Int. Ed. Engl. 35, 17–42 (1996).

13.Hermkens, P. H. H., Ottenheijm, H. C. J. and Rees, D. C., Tetrahedron 52, 4527–4554 (1996).

14.Hermkens, P. H. H., Ottenheijm, H. C. J. and Rees, D. C., Tetrahedron 53, 5643–5678 (1997).

15.Booth, S., Hermkens, P. H. H., Ottenheijm, H. C. J. and Rees, D. C., Tetrahedron 54, 15385–15443 (1998).

16.Hall, S. E., Annu. Rep. Combi. Chem. Mol. Div. 2, 15–26 (1999).

17.Andres, C. J., Denhart, D. J., Desphande, M. S. and Gillman, Comb. Chem. High Throughput Screen. 2, 191–210 (1999).

18.Kingsbury, C. L., Mehrman, S. J. and Takacs, J. M.,Curr. Org. Chem. 3, 497–555 (1999).

19.Burgess, K., Solid-Phase Organic Synthesis. Wiley Interscience, New York, 2000.

20.Nicolaou, K. C., Pfefferkom, J. A., Cao, G.-Q., Kim, S. and Kessabi, J., Org. Lett. 1, 807–810 (1999).

21.Nicolaou, K. C., Pastor, J., Barluenga, S. and Wissinger, N.,Chem. Commun. 1947–1948 (1999).

22.Jackson, W. P., Ley, S. V. and Morton, J. A., Tetrahedron Lett. 22, 2601–2604 (1981).

23.Verhe’, R., Schamp, N. and De Buyck, L., Synthesis 392–393 (1975).

24.Nicolaou, K. C., Pfefferkom, J. A., Kim, S. and Wei, H. X., J. Am. Chem. Soc. 121, 4724–4725 (1999).

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25.Meseguer, B., Alonso.Diaz, D., Griebenow, N., Herget, T. and Waldmann, H., Angew. Chem. Int. Ed. 38, 2902–2906 (1999).

26.Kogan, T. P., Somers, T. C. and Venuti, M. C., Tetrahedron 46, 6623–6632 (1990).

27.Thompson, L. A. and Eliman, J. A., Tetrahedron Lett. 35, 6333–6336 (1994).

28.Peng, G., Sohn, A. and Gallop, M. A., J. Org. Chem. 64, 8342–8349 (1999).

29.Armstrong, P., Grigg, R., Jordan, M. W. and Malone, J. F., Tetrahedron 41, 3547–3558 (1985).

30.Dragoli, D. R., Thompson, L. A., O’Brien, J. and Ellman, J. A.,J. Comb. Chem. 1, 534–539 (1999).

31.Thompson, L. A., Moore, F. L., Moon, Y. C. and Ellman, J. A., J. Org. Chem. 63, 2066–2067 (1998).

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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)

4Combinatorial Technologies: Basic Principles

This and the following seven chapters will cover the main topic of this book, combinatorial technologies. The field of combinatorial technology was born only very recently; however, it has rapidly captured the imagination of an enormous number of research workers generating a vast literature in the process. A discipline that was primarily confined to applications in the pharmaceutical industry is now being widely applied in other fields and has attracted the attention of world renowned scientists such as Nicolaou, Ley, Boger, Weinberg, Burgess, Schultz, Curran, and Geysen, to name but a few of those who have contributed substantially to the expansion of combinatorial technologies.

This introductory chapter will be devoted to three main topics, presenting the reader with the most useful basic principles of the most relevant aspects of combinatorial technologies. To begin with, a glossary will be provided, in which the most relevant concepts of combinatorial technologies will be briefly defined to familiarize the reader with the terms and with their meaning used in the area. This will be followed by a historical background, which will describe the main motives leading to the birth of combinatorial chemistry and its attendant technologies through the milestone contributions presented in chronological order. The different classes of combinatorial libraries will then be briefly presented and discussed, highlighting their relative merits and usefulness for various applications.

4.1 COMBINATORIAL TECHNOLOGIES

4.1.1 Glossary

The purpose of this section is to present the reader with basic definitions of some of the most commonly encountered terms in the area of combinatorial technologies rather than attempting to provide an exhaustive list. Many concepts introduced here will appear often in the following chapters.

Combinatorial chemistry refers to the synthetic chemical process that generates a set or sets (combinatorial libraries) of compounds in a simultaneous rather than a sequential manner. The broader concept of combinatorial technologies includes all of the disciplines involved in the synthesis and the applications of synthetic chemical libraries, such as their attendant analytical and computational chemistry methods and high-throughput screening (HTS, vide infra), as well as the disciplines related to other

136

4.1 COMBINATORIAL TECHNOLOGIES 137

libraries that have found applications in the biological disciplines and also in materials science.

A combinatorial library is a single entity composed of many individuals (typically hundreds to millions) that can be prepared in a variety of formats through the use of different techniques. A synthetic organic library is prepared using standard organic chemistry, either in solution or on SP. A biosynthetic library is usually composed of natural oligomers and is prepared by natural means (e.g., phage display libraries) or through the use of biological or biochemical reagents (e.g., enzymes and oligonucleotide amplification systems). A materials science library is made from inorganic compounds and is prepared using techniques peculiar to this field, such as sputtering deposition of thin films, electron beam evaporation, and moving-masks techniques, among others.

A primary, or unbiased, library is a large set of compounds (typically thousands to millions) based on diversity and aimed at the discovery of samples of interest for targets for which little, if any, information is available. Diversity is a concept unrelated to the library size that attempts to evaluate the representation of chemical space by a chemical library using computational methods: If this space is sampled evenly by the components of a library, then this library is considered to be diversity based (Fig. 4.1, left). A focused, or biased, library is a similarity-based set of compounds (typically hundreds to thousands) aimed at the discovery and optimization of lead structures for a target for which a structural model on which to design the library is available. Similarity is a concept unrelated to the library size that is opposite to diversity: if the library components are clustered around the model structure A, the library is similarity based (see Fig. 4.1, right).

A discrete library is a set of compounds that are obtained as individuals at the end of the library synthesis. Parallel synthesis leads to a discrete library by simultaneous addition of reactants in different reaction vessels and parallel handling of each library sample (see the example in Fig. 4.2, where 15 discretes are prepared from the common intermediate A with two parallel reaction steps). Conversely, a pool library is a set of compounds that are obtained as mixtures, or library pools, at the end of the synthesis. Mix and split (or divide and recombine) is the process leading to an SP pool library

x	x	x		x	x		x
	x	x	x	x		x
x	x	x		x	x		x CHEMICAL
								SPACE
	x	x	x	x		x		X = library
x	x	x		x	x		x	individual

DIVERSITY - BASED

PRIMARY LIBRARY

xx xx x xxx x xxxxxx x x xxxAxxx xxxxxxx x

x x x

SIMILARITY - BASED FOCUSED LIBRARY

Figure 4.1 Chemical diversity and similarity: primary and focused libraries in the chemical space.

138 COMBINATORIAL TECHNOLOGIES: BASIC PRINCIPLES

A A A A A

	AB			AC			AD
A	A	A	A	A	A	A	A	A
B	B	B	C	C	C	D	D	D
G	H	I	G	H	I	G	H	I

	AE			AF
A	A	A	A	A	A
E	E	E	F	F	F
G	H	I	G	H	I

A = common starting intermediate reagents: B-F (step 1), G-I (step 2)

Figure 4.2 Parallel synthesis of a 15-member discrete library ABG-AFI.

by simultaneous addition of reactants in different reaction vessels followed by mixing and splitting the resin portions to eventually produce the final library pools in which each resin bead carries a single library component (see Section 7.1.1).

A scaffold is the common structural element contained in all of the individuals of the library: It can be an iterated chemical bond (e.g., in oligomeric libraries such as peptides it is a repeating amide backbone), a functional group (e.g., in libraries of substituted guanidines), or a ring motif (e.g., in libraries of substituted benzodiazepines as in Fig. 4.3). A building block is a reagent used during the synthesis of a library (e.g., the lithium salt of acetanilide; Fig. 4.3). A monomer set is a class of reagents with a common functional group that is used either to produce substituted scaffolds or to decorate preexisting scaffolds (e.g., substituted anthranilic acids, alkyl halides, and α-amino acids; Fig. 4.3). A monomer is a reagent that is part of a monomer set (e.g., 5-chloroanthranilic acid, 2-methoxybenzyl bromide, and 2-thienylalanine methyl ester; Fig. 4.3). A randomization point (R1, R2, and R3 in Fig. 4.3) is a position where a monomer can be inserted into a library during the construction of the scaffold or where it can be coupled to a preexisting scaffold to produce library components containing all of the possible combinations of the selected monomers.

The term chemical assessment describes the process through which the reaction scheme to arrive at a target molecule is combinatorialized. This process may include the transfer of a reaction from solution onto SP and/or the adaptation of the reaction conditions to the use of many monomers with different reactivities and stabilities for library synthesis. Monomer rehearsal is an accurate check of the reactivity of a monomer set in the synthetic scheme for the buildup of the library so that the unreactive/difficult monomers are removed from the set. A model library is a small set of discretes, or a small pool, that is prepared using the planned synthetic route for the library and is fully characterized by the appropriate analytical tools: Only if the results are satisfactory is the library synthesis carried out. Quality control determines the analytical profile of a library as a single entity, but data from each library individual, or a significant percentage of library individuals, are acquired. A library with 80% confirmed pure compounds is a good-quality library, but the 20% of samples that are

benzodiazepine R1 scaffold

R1: from

N Me		R2: from
O-Li+
building block
		R3: from
		randomization
		points

4.1 COMBINATORIAL TECHNOLOGIES 139

R2	O
N
	R3
	N
O	H
	NH		NH2
	2
R1
	COOH	Cl	COOH
	X		Br
	R2
			OMe
	R3		S
H2N	COOMe	H2N	COOMe

	monomer	monomers
	set

Figure 4.3 Structural elements of a library: scaffolds, building blocks, monomers, monomer sets, and randomization points.

not confirmed, or are impure, are known and discarded before the library screening (vide infra).

Structure determination links a desired activity observed during the screening of the library with the structure of the active library component and is relatively straightforward for discrete libraries, while several techniques can be applied for the structure determination of compounds in pooled libraries. Deconvolution is the process whereby the complexity of an active SP pool is unraveled through iterative synthesis of smaller pools while tracking the new location of the desired activity. The final iteration is made of individual compounds, and the structure of the active component can be determined. Chemical encoding is a process in which the structure of every library component is unambiguously encoded by another chemical structure, called a tag or code, that is easily identified by automated procedures. This allows the indirect structure determination of the active library component after the library screening. The principle of nonchemical encoding is identical, but the tag is not a chemical entity; examples include radiofrequency tags or optical tags.

Library screening is the process where the activity of each library individual is measured and reliably determined; it may consist of a biological assay (for pharmaceutical purposes) or an analytical measurement (for materials science, for catalysis,

140 COMBINATORIAL TECHNOLOGIES: BASIC PRINCIPLES

or for polymer applications among others). High-throughput screening (HTS) is the fast, automated version of classical screening protocols and is the ideal counterpart of combinatorial chemistry: Large libraries made by combinatorial chemistry are screened by HTS methods and the whole process is fast and reliable. A positive is a library component that shows a significant activity during the library screening and whose structure is unequivocally determined via the above-mentioned structure determination methods.

4.1.2 Historical Background

Historically, the use of compounds derived from natural sources satisfied many of the medical and technological needs of society, but the advent of rational scientific disciplines and of chemistry in particular has culminated in the development of synthetic chemical methods that can supply compounds designed to possess specific attributes suited to a wide range of applications. The classical synthetic process, which is still used in many areas of chemistry, implies the synthesis of a compound, its purification to determine structure and quality, and its characterization in the test system for which it was prepared to determine its properties. This latter step is performed through reliable, low-throughput assays that match the low throughput of classical synthetic methods. An example related to pharmaceutical research would be the synthesis and thorough evaluation of around 100 druglike molecules per year by a small group of chemists that could be considered successful using classical synthetic and pharmacological methods.

The gradual shift to the so-called HTS methods, driven in part by the needs of pharmaceutical research, has revolutionized the concept of the drug discovery process. Implementation of assays aimed at molecular targets, requiring minimal quantities of compound, and that are easy to automate became common in the late 1980s. The consequence of this was to significantly increase the demand for chemical entities to be tested in the high-throughput biological assays. Every major pharmaceutical company possesses a large compound collection containing many proprietary compounds (typically hundreds of thousands) that were previously only tested for a specific mechanism of action in a specific assay. With the advent of this new screening philosophy, these collections were tested randomly on many different assays in the hope of producing novel and relevant biologically activity hits. An interesting experimental study has recently analyzed the usefulness of compound collections in drug discovery in providing novel, unpredictable active structures on specific targets (1).

While useful, these collections were generally biased by the previous activities of each company, which largely depend on the historical involvement in certain markets to the exclusion of others. The repeated use of these collections for HTS eventually brought about the consumption of many of their components. Methods to produce large sets of structurally related compounds in a high-throughput manner therefore became extremely important because of the need to replenish and extend available compound collections for testing for potential pharmaceutical applications. These methods, which aimed at providing a compound flow that could match the expectations of HTS, produced libraries of compounds with defined structures either to be used for

4.1 COMBINATORIAL TECHNOLOGIES 141

generic applications or that were designed to include specific structural features related to selected biological targets. Both industrial and academic groups started to become active in this field around the mid-1980s.

Initially attention was focused on natural oligomeric molecules such as peptides and oligonucleotides because the preparation of libraries based on these structures, either by chemical synthesis or by biosynthetic methods, was easily carried out while large sets of other classes of compounds required more effort and/or more complex procedures.

The first attempts to create peptide synthetic libraries were reported by Furka (2) in an obscure communication in 1982, but not having been published in a major journal, their importance was completely underestimated. In 1984 Geysen et al. (3) reported the synthesis and the biological characterization of a peptide library made up of several hundred discrete compounds using a rack of plastic polystyrene pins as supports (Section 1.1.4). A year later, Houghten (4) disclosed the use of the so-called tea bags, each containing 10–20 mg of resin beads, to prepare and biologically characterize as discretes a few hundred peptide 13-mers. After several other reports by Furka et al. (5), Frank et al. (6), and Houghten et al. (7), an important breakthrough was achieved by Lam et al. (8) in 1991, who described the synthesis of around 2,500,000 peptides using the so-called mix-and-split method and introduced the “one bead, one peptide” concept. In the same year, Fodor et al. (9) reported the synthesis of thousands of discrete peptides in 10 steps using the so-called spatially addressable parallel synthesis on glass support. In the following years the synthesis of large peptide libraries containing up to several million compounds has become a somewhat routine task using automated SPS.

Large sets of peptides or oligonucleotides have also been obtained from biological sources. The so-called phage display technique was introduced by Smith in 1985 (10), who then described its potential application to library generation in 1988 (11), which was eventually exploited in 1990 (12, 13). The expression of large, random assortments of peptides on the surface of gram-negative bacteria using fusion protein strategies and their application as peptide libraries, vaccines, or antibodies was reported. There have been many accounts of the creation of phage display libraries in the literature since these first reports (Section 10.1).

While complementary DNA (cDNA) and genomic libraries have long been used in molecular biology, the breakthrough for oligonucleotide libraries came when Tuerk and Gold (14) introduced SELEX (systematic evolution of ligands by exponential) enrichment to prepare large, amplifiable oligonucleotide libraries and to select from their populations sequences of nucleic acids capable of binding to specific targets (aptamers). This methodology, which was inspired by previous reports in the late 1980s (15–17), has become very popular. A similar selection–amplification process was reported by Pan and Uhlenbeck (18) and Bartel and Szostak (19) to find catalytic nucleic acid sequences called ribozymes, which are able to catalyze the cleavage or formation of various chemical bonds. Many reports of aptamer or ribozyme libraries appeared in the literature in the last 10 years (Section 10.2).

The use of oligonucleotide and especially peptide libraries was instrumental in uncovering to find new sequences able to interact with molecular targets and also in finding oligomeric structures suitable for use as a variety of biological tools. These

142 COMBINATORIAL TECHNOLOGIES: BASIC PRINCIPLES

molecules, though, suffer from many serious drawbacks when considered as potential drugs and the need for high-throughput synthesis of more “druglike” molecules for pharmaceutical applications became more pressing. The so-called small organic molecule (SOM) combinatorial libraries first appeared in 1992 (20) in a paper by Bunin and Ellman in which the preparation of a few 1,4-benzodiazepines on SP was described. This SPS was extended in 1994 (21) to give a discrete library of 192 1,4-benzodiazepines on plastic pins. The last few years have witnessed an enormous growth in interest for SOM libraries with the production of many molecules of interest for a wide range of pharmaceutical applications. The SOM libraries offer a more diverse range of structures when compared to the oligomeric libraries and can be tailored more precisely to specific needs. These libraries have gradually become the main target for combinatorial chemists for pharmaceutical applications, and many applications in other fields have also been reported. The accurate description of these libraries and of the related combinatorial methodologies will span five full chapters (5–9) of this book.

Finally, the use of libraries in the field of materials science libraries is also growing steadily after the first report by Xiang et al. in 1995 (22), which described the parallel synthesis of spatially addressable libraries of solid-state materials and their screening to produce novel materials with superconducting properties. This and other groups have reported the discovery of magnetoresistive, photoluminescent, and dielectric materials among others (Section 11.1 and 11.2). A significant number of polymer libraries were also reported in the last five years; they will be extensively covered in Section 11.3.

4.2 COMBINATORIAL LIBRARIES

4.2.1 Synthetic Organic Libraries: Natural and Unnatural Oligomers

The synthesis of oligomeric libraries, and especially peptide libraries, has been described in a large number of reports. We will discuss the main properties and applications of such libraries using the four examples reported in Figs. 4.4–4.8.

A linear hexapeptide amide library (L1, Fig. 4.4) was prepared on SP by Dooley et al. (23) by randomizing the 20 natural L-amino acids in positions X1 and X2 and 19 of them in positions X3–X6 (Cys omitted). The library consisted of 20 × 20 × 19 × 19 × 19 × 19 = 52,128,400 different hexapeptides and was prepared as a pool library made up of 400 subsets, or library pools, of 19 × 19 × 19 × 19 = 130,321 individuals. Among thesepools,YPX3X4X5X6 (L2, Fig. 4.4) was the most active as inhibitor of the binding of an enkephalin-related peptide to rat brain homogenates and was further deconvoluted to find a few active sequences (Fig. 4.4) among which YPFGFR–CONH2 was the most active inhibitory concentration (IC50 = 13 nM). The huge number of possible library components that can be made from the 20 commercially available precursors, together with the robust and readily available automated SPS of peptides (see Section 2.1), make synthetic peptide libraries with up to eight randomized positions a useful reservoir for “ligand fishing,” that is, to identify peptidic sequences that are able to interact with a selected target. Biosynthetic peptide libraries (Section 11.1) are more