Solid-Phase Synthesis and Combinatorial Technologies

7.3 DECONVOLUTION METHODS FOR SOLID-PHASE POOL LIBRARIES 293

rule, but according to the complexity of the SP synthesis and the project needs, more than one pool per iteration may be deconvoluted. Sometimes a more active individual may result from a less active pool, and different families/structures of positives may be found. The deconvolution of many pools, though, would again lead to significant efforts that would probably make another library format (discretes) or structure determination method (bead-based, encoding) more effective.

7.3.2 An Example: Iterative Deconvolution of a 1,4-Dihydropyridine Library

Recently Gordeev et al. reported (140, 141) the synthesis and iterative deconvolution of a 300-member focused library of 1,4-dihydropyridines L5 as potential calcium channel blockers inspired by the structure of nifedipine and other known bioactive compounds. The library was built on SP as 30 pools of 10 individuals, following the synthetic scheme shown in Fig. 7.18. Rink amine resin was deprotected, split into 10 portions (steps a and b), and treated with β-ketoesters (step c, M1, 10 representatives A–J, Fig. 7.19) to give 10 discrete N-supported β-enaminoesters 7.30. The resin aliquots were then mixed (step d) and split again into 30 identical, 10-member pools (step e). Each resin portion was treated with a different combination of 1,3-dicarbonyls (M2, three representatives K–M, Fig. 7.19) and aldehydes (M3, 10 representatives N–W, Fig. 7.19) to give 30 pools of 10 open-chain precursors 7.31 (step f), which were cyclatively cleaved to give the final library L3 (step g, Fig. 7.18). Library QC was performed by off-bead MS, identifying all the expected molecular ions in several pools.

The library was tested using a known competition assay (142), and binding activities for the 30 pools were acquired. While the pool complexity was low, as was therefore the possibility of false positives/artifacts, the extreme similarity of all the library components with known calcium channel blockers (compare the monomers in Fig. 7.19 leading to nifedipine, M1 = A, M2 = K, M3 = T, with all the others) meant a constant level of activity was to be expected for all pools. For such a small focused library, parallel synthesis would probably have been more suitable to acquire a refined SAR, but we will see how iterative deconvolution succeeded anyway in both identifying active individuals and showing significant activity differences for different pools. The screening results are reported in Table 7.1. Five pools showed activity > 1 µM, 12 pools had an activity between 100 nM and 1 µM, and 11 pools were active between 10 and 100 nM. Two pools showed an activity around 7–8 nM: They both contained methyl acetoacetate (M2, K) as well as 2-fluorobenzaldehyde (M3, P) and 2-nitroben- zaldehyde (M3, T), respectively.

Deconvolution of these pools consisted simply of a single iteration, preparing the 20 discrete components of pools KP and KT, which produced the results shown in Fig. 7.20. Nifedipine 7.32 was included in the library as a standard and was obtained by deconvolution of the pools together with other active compounds (7.33–7.36, Fig. 7.20). These results point toward an even more focused library where only o-substituted aromatic and heteroaromatic aldehydes would be included as aldehyde monomers.

Many cases of the iterative deconvolution of SP libraries have been reported, and the reader should refer to some recent reviews (143–145) to access further examples. We will mention only several very recent papers in which iterative deconvolution has

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L5

300-member SP pool library

30 pools

10compounds/pool

a:Fmoc deprotection; b: resin portioning (1 to 10); c: 4A MS, DCM, rt; d: mix in one pool;

e:resin portioning (1 to 30); f: pyridine, 4A MS, 45°C; g: 3% TFA, DCM, rt.

Figure 7.18 SP synthesis of the focused dihydropyridine pool library L5.

been successfully applied. Rohrer and co-workers (146–148) reported the iterative deconvolution of two libraries containing respectively 131,760 compounds as 79 pools of 1330 or 2660 individuals and >200,000 compounds as 147 pools. The deconvolution process identified several selective agonists of the somatostatin receptor which were further progressed by more focused efforts. Heizmann et al. (149) reported the synthesis and deconvolution of a 328,509-member SP pool tripeptoid library, made as 69 pools of 4761 compounds and screened for α-melanotropin (α-MSH) and for gastrin-releasing peptide (GRP)/bombesin affinity; the deconvolution process gave two structurally unrelated, low-micromolar ligands for the two targets. An 125-

7.3 DECONVOLUTION METHODS FOR SOLID-PHASE POOL LIBRARIES 295

O O

R4 R3

M2

K:R3 = OMe, R4 = Me

L:R3 = OBn, R4 = Me

M:R3, R4 = Me

CHO

Ar

M3

NO2

N

S

N S

T U V W

NO2

Figure 7.19 Monomer sets M1–M3 used for the synthesis of the focused dihydropyridine pool library L5.

member SP pool library of azasugar peptide conjugates was made by Lohse et al. (150, 151) as five pools of 25 compounds and screened for β-glucosidase inhibition; the simple deconvolution process yielded a medium–low micromolar enzyme inhibitor. An 144-member library of acylated amidinonaphthols was made by Roussel et al. (152) as 18 pools of eight compounds and tested for the inhibition of tissue factor (TF)/factor VIIa complex, providing a high-nanomolar inhibitor. Szardenings et al.

296

TABLE 7.1 Biological Activity of the SP Peptidomimetic Pool Library L3

aA: >1000 nM; B: 100–1000 nM; C: 10–100 nM; D: 1–10 nM.

H

7.32 (nifedipine) IC50: 18 nM

Figure 7.20 Iterative deconvolution of the focused dihydropyridine pool library L5: deconvoluted active structures 7.32–7.36.

(153) reported the synthesis and the deconvolution of a 1225-member diketopiperazine library as 35 pools of 35 compounds; the pools were screened and deconvoluted for their activity on two matrix metalloproteinases (MMPs), gelatinase-B and collagenase- 1. The process yielded several target-selective, low-nanomolar inhibitor.

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Ng et al. (154) reported the identification of several antimicrobial peptoids from an 845-member SP pool library organized as 65 pools of 13 compounds inspired by previous, larger combinatorial efforts (155). A 400-member SP library based on a 1,3-disubstituted cyclohexanone scaffold, prepared as 20 pools of 20 compounds, was reported by Abato et al. (156); a selective inhibitor of a serine protease, plasmin, was identified from iterative deconvolution of the library.

Rather often, examples of the iterative deconvolution of large libraries do not contain all the details about the library structure, the monomer sets, and the positives obtained in order to protect the proprietary information extracted from the library. This makes it much more difficult to judge the real potential of iterative deconvolution in the structure determination of positives, and it remains only to accept the method as it is, with the practical considerations made in the previous section.

7.3.3 Other Deconvolutive Methods

Another popular deconvolution method is based on the synthesis and screening of multiple library copies, where different positions are determined to identify the best monomer for each library randomization point (positional scanning) (157). We can examine positional scanning with the same hypothetical 625-member library used to illustrate iterative deconvolution (monomers A–T, see Figs. 7.16 and 7.17). Rather than performing iterative cycles on one original pool library, positional scanning implies the up-front synthesis of four complete copies of the library (step a, Fig. 7.21). The corresponding 20 pools P–T (sublibrary 1), K–O (sublibrary 2), F–J (sublibrary 3), and A–E (sublibrary 4) are assayed (step b). While some pools are weakly active (M, R, T), the four most active pools of each library copy are respectively D, J, L, and S. Simply by combining the information derived from the screening and selecting the most active monomer representatives (step c), an active library individual, DJLS, is obtained (Fig. 7.21).

The up-front synthesis of multiple library copies, and the deconvolution of large pools to derive directly the active structure are the main features of positional scanning. The synthetic scheme is also more demanding with respect to typical mix-and-split methods: for example, sublibrary 1, or P–T, requires 5 + 25 + 25 + 25 = 80 reactions (Fig. 7.22), while iterative deconvolution that corresponds to sublibrary 4, or A–E (Fig. 7.16), requires only 5 + 5 + 5 + 5 = 20 reactions. Multiple-pool deconvolution is, however, much easier using positional scanning: Referring to the example (pools M, R, and T as less active than the selected most active pools), compounds DJMS, DJLR, DJLT, DJMR, and DJMT can be prepared as discretes together with DJLS with no additional deconvolution efforts. The observations made for iterative deconvolution on pool size (<50) and concentration of library individuals (depending on the screening sensitivity) are also valid for positional scanning.

This approach has often been applied to peptide libraries (158–161), but its use with small organic molecules has been, to date, restricted to two examples of 72- (162) and 54- (163) member solution libraries, which proved the validity of the technique with simple test cases; to a 1600-member SP amide library (164); to a set of large peptide, peptoid, and polyamine libraries (more than 70,000,000 total individuals) (165); and

300 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

e

625-member SP pool library

M1 determined, M2-M4 randomized P Q R S T 5 pools, 125 components/pool

80 reactions to prepare it

a:resin portioning (1 to 5); b: coupling with M1, five discrete reactions; c: split in 25 aliquots;

d:coupling with Mx, 25 discrete reactions; e: mix in five pools.

Figure 7.22 Positional scanning: synthesis of the four hypothetical sublibraries 1–4.

Unrandomization of Random Oligomer Fragments) (173) have been applied either on SP or in solution to peptide or oligonucleotide library deconvolution, but none have found relevant applications in small organic molecule libraries. Several reviews dealing with deconvolution methods for SP libraries were mentioned in the previous

7.4 ENCODING METHODS FOR SOLID-PHASE POOL LIBRARIES 301

section, and the interested reader may refer to them to expand this subject. A few papers (174–176) have tried to compare the efficacy of various deconvolution methods with other structure determination methods for pool libraries. Iterative deconvolution resulted as the method of choice, and positional scanning was similarly, if somewhat less, successful. All the other methods mentioned in this section were significantly less successful in finding the most active structures when the activity of library individuals (oligonucleotides) was known and virtual pools were constructed to make the identification of positives difficult.

Deconvolution was very popular in the early 1990s in combinatorial technologies. It allowed the screening of a significantly lower number of samples with a consequent reduction in costs when compared with the same library of discrete individuals, which were also more demanding in terms of synthesis, while obtaining the activity information on the whole library. The advent of medium–high throughput parallel synthesis and of bead-based SP pool libraries, however, has largely oriented the major active groups toward these libraries and has made the use of deconvolution methods to identify positives from medium–large primary libraries less appealing. These new library formats can be easily tested due to the general diffusion of high-throughput screening (HTS) methods, in pharmaceutical research and in other fields, which allow the rapid testing of large numbers of discrete or bead-based samples.

False positives have always hampered the deconvolution approaches, and often good positives were lost in the iterative/scanning process. These are, in fact, the only structure determination methods that do not identify all but only one or a few positives (sometimes not the best ones). Nevertheless, groups that enter this field and do not have adequate analytical instrumentation to prepare bead-based pool libraries, or HTS throughput screening methods to test them, but do have the need to prepare large SP pool libraries, and testing them for a specific application may still benefit largely from these low-cost methods.

7.4 ENCODING METHODS FOR SOLID-PHASE POOL LIBRARIES

7.4.1 Chemical Encoding

Structure determination of positives from SP pool libraries can also be made through the coupling of a tag, or code, that will unequivocally encode the structure of the library component to each library individual. These tags may be divided in two classes: chemical tags, which are compounds with a completely different chemical nature from the library compounds, and nonchemical tags, which use nonchemical entities to code for all the library structures.

Encoding methods are typically used for bead-based libraries, with a single exception (vide infra), and possess some attractive features. We will start with chemical encoding, which appeared early in the history of combinatorial chemistry and is still widely used by various research groups. The chemical tags are coupled to the resin beads in parallel with the library synthesis using the mix-and-split method, which ensures the presence of a single code and a single chemical structure on each resin

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bead. This variation of classical mix and split is shown in Fig. 7.23 for a hypothetical example where three monomer sets M1–M3 each composed of 10 representatives are used. The synthesis starts from an orthogonally protected solid support where most of the sites (loading/library sites) are protected by P1 and a small portion (tag sites) are protected by P2. The monomer sets M1(P1)–M3(P1) are coupled (step c), after deprotection of loading sites (P1, step a) and splitting into 10 aliquots (step b), while the tags T1(P2)–T3(P2) are coupled after their respective monomer sets (step e), following deprotection of tag sites (P2, step d). Each monomer set–tag coupling cycle is followed by mixing the resin aliquots into a single pool (step f, Fig. 7.23). The coupling of each monomer–tag pair may be inverted [M1(P1), then T1(P2) or T1(P2), then M1(P1)] depending on the stability of resin-bound library intermediates. Sometimes the differentiation of loading and tag sites with orthogonal protecting groups may not be necessary (electrophoric tags, vide infra). After its synthesis and QC characterization, the library is tested using a bead-based assay, either off-bead or on-bead, and the positive beads are determined. Finally, the tags are cleaved, their structure is determined, and from this information the structure of the active library individual is obtained directly.

The tags are often used as binary-coded mixtures, as shown in Fig. 7.24 for a hypothetical example where six tags are available. Using tags to code individually for

1 pool containing

1000 encoded compounds

a:P1 deprotection; b: resin portioning (1 to 10); c: coupling with Mx(P1), ten discrete reactions;

d:P2 deprotection; e: coupling with Tx(P2), ten discrete reactions; f: mix in one pool.

Figure 7.23 Hypothetical SP synthesis of a 1000-member pool library using chemical encoding.