Solid-Phase Synthesis and Combinatorial Technologies

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expected reaction product with good yields and purity (typically >75%, 25 monomers for each set M1–M3 in Fig. 7.7) are selected for library production. The rejected monomers are replaced with other candidates in each monomer set (path b, n reactions with the scaffold or standard representatives) until the three 50-member sets are selected. If a single monomer is misjudged, that is, is included in the selected sets but does not produce the expected library components, a total of 1 × 50 × 50 = 2500 compounds will be lost and the library value will be diminished. In the example, a few hundred discrete reactions and their careful analytical characterization are the gateway to a high-quality 125,000-member SP pool library. Both off-bead and on-bead methods should be used, and depending on the nature of the library components, some techniques and/or detection methods will be more suitable, including sophisticated on-bead analytical methods (see Sections 1.3 and 1.4).

The following step is represented by the synthesis of a model SP pool library, which is prepared using the same equipment and strategies as planned for the real library but on a smaller scale (typically from 10 to 50 library individuals in small pools). This allows evaluation of the quality of the synthesis as it passes from discretes to mixtures while being able to fully characterize the small pools from an analytical standpoint. These pools are usually analyzed off-bead, and both the presence and the relative amounts of each pool component are determined. The influence of monomers with different reactivities on the library outcome can be checked by the careful selection of monomers for the model library, that is, if the general optimized reaction conditions are suitable both for reactive and less reactive monomers. Examples have been reported in the literature where HPLC (26, 27), MS (28, 29), HPLC/MS (30–33), GC/MS (34), and HPLC/NMR (35) were used to characterize small SP pool libraries of small organic molecules after cleavage from the beads. Most of these papers actually reported only the synthesis of a model SP pool library. It is probable that larger libraries were prepared in the same laboratories using the same synthetic scheme, but their full disclosure via publication was avoided to protect their proprietary chemical diversity.

When both the monomer rehearsal and the model library studies produce good results, the SPS of the planned pool library takes place. Reaction monitoring is now difficult because a mixture of many compounds is present in each well. Transformations related to specific functional groups can be monitored either on-bead (colorimetric methods such as the Kaiser test—appearance or disappearance of an amino group) or off-bead (UV reading, Fmoc deprotection of amines). Information may be obtained from off-bead methods after the cleavage of a small resin aliquot, but while the total or partial transformation of the starting materials can be determined (disappearance or appearance of diagnostic IR bands, or NMR signals), the absolute quantification of each pool component and of each remaining starting material is at best arduous. The rigorous rehearsal of monomers and a successful model library synthesis are the best guarantees for uneventful, high-quality SPS using the mix-and-split approach.

Structure determination is an extremely complex issue in SP pool libraries. Assuming as a hypothetical example a large library of 100,000 individuals in 1000 pools, each containing 100 compounds, we will know the location of each pool but we will not automatically have precise information on the composition of each pool. An accurate determination of each pool’s content would require the cleavage of aliquots

7.1 SYNTHESIS OF SOLID-PHASE POOL LIBRARIES 275

from each pool and their analytical characterization via multiple methods, but above all a strenuous effort to assign the structure of each individual to a specific set of signals, bands, and so on. Even if today’s trend is to have pool libraries containing a small number of components per pool (typically 10 to 30–50), structure determination of all the library components is not possible in a timely and reasonable manner. Thus, accurate structure determination is performed after, rather than before, the library assay and only the positive compounds are structurally identified and confirmed. Several methods exist to perform this crucial operation, and they will be the subject of the next three sections.

This simplified and more timeand cost-effective strategy necessitates an accurate quality control (QC) for the library. While examples of MS characterization of several large peptide pools have been reported (36, 37), an easier and more significant method for SP pool library QC is needed. An interesting and emerging option is bead-based QC. Since mix and split produces beads loaded with a single compound, and a single bead analysis will determine the structure of the loaded compound and its purity, providing that the method is sensitive enough, this technique effectively allows the parallel analysis of discrete beads, with no interference from mixtures. The only difference from the characterization of SP discrete libraries would be the unknown structure of the supported individual. Off-bead MS analysis of a few hundred beads randomly selected from different library pools should allow the identification of library representatives through their molecular weight, and other on-bead techniques already available (FTIR), or to be expected in the near future (NMR, others), would allow the refinement of the bead-related qualitative (structure, presence of impurities) and quantitative (yields, loading of target and impurities) information. A sound proposal expressed by a leading combinatorial group (24) stipulates the lower limit of confirmed library individuals as at least 80% of the examined beads to define a good-quality SP pool library.

The separation and isolation of individual compounds is not in the spirit of a pool library, which is tested as a mixture using various techniques. A final purification by chromatography or extraction, though, may be useful to remove cleavage reagents or impurities that have extremely different physicochemical properties with respect to the library components (salts, greasy reagents, etc.). More details on final purification procedures for released SP pool libraries are reported in Section 8.3.

7.1.4 An Example: Synthesis of an SP Pool Library of Hexahydroisoindoles

A recent communication by Heerding et al. (38) reported the synthesis of a 3200-mem- ber SP pool library of isoindolines L2 by means of a complex reaction scheme, including an intramolecular metathesis reaction and a Diels–Alder cycloaddition. The target bicyclic nucleus and the selected precursors to obtain the library are depicted in Fig. 7.8. The authors began by assessing a possible reaction scheme in solution using 4-benzyloxybenzoic acid 7.1 as a linker-mimicking synthon (Fig. 7.9). Reaction with allyl amine 7.2a (step a), N-alkylation with the propargyl mesylate 7.4 (steps b,c), intramolecular metathesis (step d), and cycloaddition with maleimide (step e) produced the target cycloadduct 7.7 as a single diastereomer in reasonable yield and purity.

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O

Figure 7.8 Retrosynthetic analysis for the isoindoline SP pool library L2.

7.7

a: EDC, HOBt, TEA, DMF, rt, 18 hrs; b: NaH, DMF, rt, 30'; c: 7.4, rt, 5 hrs;

d:RuCl2(=CHPh)(PCy3)2 cat., benzene, reflux, 18 hrs; e: toluene, reflux, 18 hrs.

Figure 7.9 Validation in solution for the synthetic scheme to the isoindoline SP pool library L2: synthesis of 7.7.

a: NaH, DMF; b: (Ph3P)4Pd, MeNHPh, DMSO/DMF 1/1, 55°C, 18 hrs; c: 7.2a,b, EDC, HOBt, TEA, DMF, rt, 18 hrs; d: tBuOLi, THF, rt, 1.5 hrs; e: 7.4, DMSO, rt, 5 hrs; f: RuCl2(=CHPh)(PCy3)2 cat., benzene, reflux, 18 hrs; g: maleimide, toluene, reflux, 18 hrs.

Figure 7.10 SP chemistry assessment leading to the isoindoline pool library L2.

Some reactions did not go to completion, but the use of excess reagents on SP and the application of multiple reaction cycles were forecasted to drive the conversions to completion.

The same reaction scheme (Fig. 7.10) was followed on SP starting from the chloro derivative of Wang resin and coupling it with the sodium salt of protected phenolcarboxylate 7.8 to give the supported ester 7.9 (classical Mitsunobu reaction of Wang resin with the protected phenol was less successful). After deprotection with Pd (allyl performed better than simple methyl as a protecting group), the carboxylic function was coupled with two allyl amines (7.2a,b) to give the resin-bound secondary amides 7.10a,b. These were N-alkylated by the mesylate 7.4 using two reaction cycles to convert all the starting material; lithium t-butoxide was used as a base, as other bases were either not soluble in the reaction medium or caused instability and degradation of 7.11a,b. These intermediates were cyclized via ruthenium-catalyzed intramolecular metathesis without affecting the linker stability, and maleimide was condensed to give the final resin-bound adducts 7.13a,b as single diastereomers with good yields and purity (Fig. 7.10). All the intermediates of the SP assessment were characterized either on-bead (1H MAS NMR) or off-bead (MS, HPLC, NMR) after cleavage (TFA–water 95/5).

The next step was the synthesis of the pool library L2, made from 10 carboxylic acids (R1), 4 allyl amines (R2), 5 propargyl mesylates (R3), and 16 dienophiles (R4, R5), as 16 pools of 200 individuals where the dienophile position is defined (Fig. 7.11).

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substituted on the aryl ring.

R2

: 4 R2 substituents including H, alkyl or arylalkyl groups.

H2N

Figure 7.11 General structure and composition of the 3200-member isoindoline SP pool library L2.

The jump from two discretes to a 3200-member pool library looks rather ambitious, but it is possible that more discretes and maybe even a small model library were prepared but not reported in the communication. The authors were, as usual, protecting the chemical diversity of the prepared library. In fact, the structures of the 10 + 4 + 5 + 16 = 35 monomers selected were not given in the paper. The library was QCed by randomly sampling 24 beads from each pool, cleaving them individually, and submitting the 384 solutions to LC/MS analysis; 14 out of the 16 pools produced >70% beads with >70% HPLC/MS purity, while the other 2 pools did not give any identifiable product and were discarded. It is safe to assume that 14 × 200 = 2800 individuals possessed a sufficient quality to be tested in various assays, and surely the major pharmaceutical company responsible for this communication had this SP pool library (and maybe expansions by using other monomers) tested on biologically relevant targets.

More examples of SP pool libraries will be presented in the next sections in relation to structure identification of positives from a library. Other references can be found in

7.2 DIRECT STRUCTURE DETERMINATION OF POSITIVES 279

recent reviews (39–42), and we will mention here some very recent communications of the synthesis of SP pool libraries. Pei et al. (43) reported a 4140-member dihydroquinoline library prepared as 30 pools of 138 compounds from α-amino acids, o-nitrobenzaldehydes, and acyl chlorides; Krchnak and Weichsel (44) reported a 2720-member diazepine library from diamines, amino alcohols, aldehydes, and primary amines; Cao et al. (45) reported a 4608-member library of cyclopentanes prepared as 768 pools of six compounds from diamines, styrene derivatives, and amines using ring-opening cross-metathesis; Nefzi (46) reported a 38,800-member library of hydantoins and thiohydantoins from α-amino acids and alkylating agents; Neustadt et al. (47) reported a 6859-member biphenyl library prepared as 19 pools of 361 compounds from α-amino acids, including exotic amino acids; a 40-member library of 1,3,5-trisubstituted pyridinium salts prepared as pools of compounds from bromonicotinic acid performing a Suzuki coupling and an N-alkylation of pyridines was reported by Amparo Lago et al. (32); Zhu and Boons (33) reported a two-direc- tional strategy to prepare a small library of trisaccharides as pools of compounds using thioglycoside building blocks; Gennari et al. (48) reported the synthesis of three SP pool libraries of N-alkylated vinylogous sulfonamido peptides as pools of compounds using mild N-alkylation conditions.

7.2 DIRECT STRUCTURE DETERMINATION OF POSITIVES FROM SOLID-PHASE POOL LIBRARIES

7.2.1 Structure Determination of Positives

A medium–large SP pool library is typically prepared in large quantities (up to 500 library equivalents), allowing it to be tested on many assays, and novel, relevant, identifiable structures should originate from each of these screening campaigns. While the library QC is performed only once after its synthesis and before screening, as seen in the previous section, fast and reliable methods to identify the active compound(s) from a pool and match activities with structures are needed after every screening.

Direct structure determination methods, where positives are characterized directly via off-bead or on-bead identification of their chemical structure, will be described in detail in this section. Indirect methods that determine the structure of positives from the library architecture will be covered later: they use either deconvolutive methods (Section 7.3), where the iterative synthesis of library pools with decreasing complexity via sequential determination of the best monomers leads to the identification of a positive structure, or encoding methods (Section 7.4), where, during the library synthesis, the structure of each component is coupled to a tag that can be read from a single bead after the library screening.

We will describe these methods illustrating their complementarity with respect to the skills and the equipment available in the laboratory where the SP pool library must be prepared, but most of all with respect to the specific synthetic scheme, format, and size that are planned for each library.

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7.2.2 Off-Bead Direct Structure Determination of Positives

A high-quality SP pool library is usually partitioned into a few aliquots, each containing several library equivalents, after its synthesis and successful QC; then one or more of these portions are cleaved and immediately submitted for screening. Often the screening campaign measures the affinity of the library for the target, which is added to the assay medium as a purified soluble reagent. An off-bead, target-assisted structure determination of library positives is feasible if the assay functions as a separation method dividing the active compounds from the others and if a suitable analytical method can determine the structure of the positives in situ. Alternatively, an analytical technique may be able in determined experimental protocols to detect only the signal related to compounds binding to the target suppressing the signal of any unbound library individual, thus functioning as a screening technique in itself. We will describe the main features of both these methods and will comment on their usefulness in this section, considering that this strategy can be applied both to SP pool libraries, after their cleavage, and to solution-phase pool libraries.

The target may assist in structure determination via the formation of a noncovalent complex with the active library components. These high-MW complexes are then separated from the rest of the low-MW library individuals, and finally the complex is destroyed and the active library components are structurally characterized via a suitable analytical technique. The target acts as an affinity reagent, and care must be taken both in preventing nonspecific associations between the target and some library components and in finding a robust separation protocol to isolate the target-positive complexes, even when their binding constant and their affinity is relatively weak. The analytical technique must be extremely sensitive and soft, as it must be able to detect minimal quantities of the selected compounds without creating artifacts (fragmentation, degradation, etc.).

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft nature of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53–58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66).

As a case example we will consider the approach of van Breemen and co-workers (67–69), who applied pulsed ultrafiltration ESI-MS (70) to the screening for inhibitors of adenosine deaminase (67, 68) and dehydrofolate reductase (69). A schematic representation of the whole process is reported in Fig. 7.12. The authors used an HPLC/MS apparatus where the column was substituted with an ultrafiltration chamber (around 100 L volume) equipped with a 10,000-MW cutoff membrane. Aqueous buffer solutions of the library components and the receptor were incubated, then loaded

a: incubation; b: injection into the ultrafiltration chamber; c: elution of unbound library individuals with aqueous buffer; d: to waste; e: disruption of target-ligand complexes by elution with MeOH/H2O/AcOH;

f: detection of positives by ESI-MS spectrometry.

Figure 7.12 Off-bead structure determination: pulsed ultrafiltration/ESI mass spectrometry.

into the ultrafiltration chamber, which was flushed with water for several minutes to elute all the unbound, low-MW library components. The eluting mixture was then switched to MeOH–water–AcOH to disrupt the receptor–ligand complexes and the free ligands were identified on-line by ESI-MS (Fig. 7.12).

Identification of the noncovalent bound complex between the target and the ligands from the library, without the need to free the active library individual, would be an improvement for off-bead direct structure determination. The experimental protocol would be shortened, but more importantly the method would provide direct information on the strength of each ligand–target interaction and would thus be able to rank the library positives according to their potency. This principle was successfully applied by Bruce and co-workers (71–73), who used Fourier transform ion cyclotron resonance MS (FT-ICR-MS) to identify intact noncovalent complex ions between carbonic anhydrase II and derivatized peptide libraries and to rank them in terms of affinity; Hofstadler et al. (74) screened an artificial mixture of five aminoglycosides against several mass-tagged RNA targets using ESI-FTICR-MS, detecting several interactions between the ligands and prokaryotic RNA targets.

NMR has gained a lot of importance as a screening technique in off-bead targetassisted structure determination lately; several NMR-assisted screening methods providing either a qualitative or a quantitative estimation of the ligand-target binding strength using simple ligand mixtures will be briefly described here.

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Fesik and co-workers reported the so-called SAR by NMR method (75, 76) which monitors the chemical shift perturbation of 15N-1H heteronuclear single quantum correlation (HSQC) data for small proteins in presence of small ligands. The method requires the use of uniformly labeled 15N receptors, but can screen via sophisticated NMR CryoProbes (77) even 10,000 library components per day. Successful applications to identify inhibitors of Human Papillomavirus E2 protein (78), of stromelysin (79), of (80) and of Erm methyltransferases (81) have been reported by the same group. While the method is extremely reliable and accurate, often leading to relevant lead compounds, several bottlenecks must be highlighted: the need of significant quantities of uniformly 15N labelled, purified receptors/proteins/enzymes; the restriction to low MW targets (only up to 30 kDa); the need of complete structure determination and NMR signal assignment for the target; the need of complex and expensive instrumentation which as of today confines this method to a few specialized laboratories. Its usefulness, thus, may be higher for later rather than early discovery phases via HTS.

A method named pulsed field gradient NMR (PFG-NMR) was recently reported by Shapiro and co-workers (82–86) as being able to discriminate between bound and unbound library individuals, which had different diffusion coefficients in solution when complexed with macromolecules. This property allowed the editing of the NMR spectrum to see only the signals of bound molecules. The authors applied this technique to detect interactions between small molecules (82–84), small molecules and vancomycin (85), and small molecules and medium-length oligonucleotides (86). A somewhat different application of PFG-NMR by Hajduk et al. (87) obtained the spectra of bound library components by subtraction of the spectra of the mixtures in the presence and absence of two different proteins, FKBP and stromelysin, and confirmed the detection of two known ligands in each NMR diffusion screening, thus validating this target-assisted screening protocol. The validation studies up to now reported have only hinted towards the usefulness of PFG-NMR, and the publication of a “real” library screening would reinforce the confidence in this method; as of today its low throughput (several hours per sample) and the risk of experimental error (up to 100%) limit its scope.

Meyer et al. recently reported transfer NOE (nuclear overhauser effect) (trNOE) (88), which detects the strong negative trNOE effect of receptor-bound molecules compared to the weak, positive trNOE of unbound compounds without the need of receptor labeling; the method was used to identify an E-selectin antagonist from an artificially assembled 10-member library of saccharides (89).

The same approach has been cleverly exploited by Fejzo et al. (90) by using the so-called SHAPES strategy. The authors selected among the CDC database (91) the meaningful frameworks which are most frequent in the structures of known drugs, i.e., the cyclic arrays of atoms constituting several rings and the connecting atoms working as linkers. A careful computational search and selection (92) limited to 41 the number of frameworks representatives of a large part of CDC. These core scaffolds were used to screen the ACD database (93) and to select commercially available decorated/modified scaffolds without toxic or unstable moieties; with side-chains conferring aqueous solubility; with at least one N or O atom. Several novel compounds obtainable with simple routes were finally added, leading to a SHAPES library of 132 compounds with MWs from 68 to 341 Da and with logPs from –2.2 to 5.5 (see refs. 90 and 92 for more