Solid-Phase Synthesis and Combinatorial Technologies

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allowed the preparation of a large SP discrete library that was tested “in a variety of high throughput biological screens” (104). All the monomers used were either commercially available or easily prepared from commercial sources. As often happens in published papers reporting the synthesis of medium–large library, only a few structures of the monomers used and even fewer structures of the library individuals are provided. This keeps the diversity generated in the library as proprietary knowledge and little or no data regarding the activity are usually provided for the libraries. This means that suitably active compounds can be tested further and eventually patented with a more defined property profile. We will cover this topic, which is related to the pharmaceutical applications of chemical libraries, in Section 9.1.

Many other reports of manual parallel SP synthesis leading to significant arrays of small organic molecules have appeared in the last five years, and most of them may be found by consulting the excellent reviews cited in references 1–5. Nevertheless, some recent papers in this area are worth mentioning. For example, Kiselyov et al. have reported the synthesis of two libraries of 14-membered macrocycles, the first (116) made by 12 individuals and prepared from allyl bromide, bromoacetic acid, and aliphatic primary amines with 54–74% yields and >90% purity, the second (117) made by 30 representatives and prepared from functionalized, primary amines and bromoacetic acid. A library of over 1000 thiazolidinones prepared from diamines, aldehydes, mercaptosuccinic acid, and amines with >65% average yields and high purity has been described by Munson et al. (118). Albert et al. (119) have published the synthesis of a 24-membered library of isoxazolylthioamides prepared from phenylacetic acid derivatives and p-substituted aryl isothiocyanates. A 96-membered hydantoin library, which was further expanded to a library of more than 10,000 members, was prepared from α-amino acids with >60% yields and >65% purity (120). Wong et al. (121) have described the synthesis of a 45-membered library of oligosaccharides from orthogonally protected monosaccharides with varying yields (5–90%) and purity. The Ugi four-component coupling (4CC) has been used by Li et al. for the preparation of an 108-membered library of α,α,α-difluoromethylene phosphonic acids from isonitriles, aldehydes, Rink amine resin, and a phosphonic acid with yields ranging from 10 to 95% and >70% purity (122). The same reaction has also been employed by Kim et al. (123), who used it to prepare a library of 96 peptidomimetics from aldehydes, carboxylic acids, Rink amine resin, and a single isonitrile with varying yields and >90% purity. Peptidomimetic-based libraries have been described by Souers et al. (124) and Ogbu et al. (125). The former highlighted the preparation of a 5500-member library of β-turn mimetics from primary amines, α-amino acids, and α-halo acids, whereas Ogbu et al. used the Diels–Alder cycloaddition of 1,2,4-tria- zolinediones and dienoic esters or amides to construct a 1500-member library of constrained β-strand mimetics. The venerable Pictet–Spengler cyclization of tryptophan, an aldehyde, and a primary amine was used by Fantauzzi and Yager to prepare a library of 345 tetrahydro-β-carbolines in high yield and purity (126). A 24-member aminopyrazoline library was prepared in unspecified yield from α,β-unsaturated nitriles and hydrazines with >80% purity (127). Mohan et al. (128) has shown that nucleophilic aromatic substitution can be useful for the synthesis of an amidinophenoxypyridine library. A large library of benzimidazoles was prepared from fluoroben-

6.3 EXAMPLES OF SOLID-PHASE DISCRETE LIBRARY SYNTHESIS 235

zoic acid, aldehydes, and amines with >80% yields and >70% purity by Mayer et al. (129). Lin and Ganesan (130) have described the preparation of a 1000-member library of carbamoylguanidines with moderate to good yields and >85% purity. Van Loevezijn et al. (131) have reported the synthesis of a 42-member library of polycondensed heterocyclic analogues of indolyl diketopiperazine alkaloids with 50–100% yields and 70–99% NMR purity after recovery. A robust SP and solution-phase protocol for parallel synthesis of >100 member libraries of substituted ureas with high purity (>80%, MS) has been reported by Nieuwenhuijzen et al. (132). Romoff et al. (133) published an efficient protocol for SP parallel synthesis of polyfunctionalized 3- acyltetramic acids using a cyclative cleavage approach with average good yield and excellent HPLC/MS purity. Another cyclative cleavage approach was reported by Villalgordo et al. (134) to prepare SP discrete libraries of 3H-quinazolin-4-ones with average high yield (>70%, recovered material) and >95% HPLC purity. Nefzi (135) reported the synthesis of two 97-member libraries of diethylenetriamines using Houghten’s tea-bag synthetic protocol (136) and obtaining good average yield and purity for the library individuals. The protocol to obtain parallel aldehyde libraries was reported, with good yield and purity, by Salvino et al. (137); the further use of these cleaved libraries as reagents for a “library from library” approach in a Horner–Em- mons protocol using a supported phosphonate was also reported. Hennequin and Piva-Le Blanc (138) reported the procedure to prepare a discrete library of oxindole quinazolines, providing several examples with moderate to good yield and excellent purity. Roussel et al. (139) presented a 43-member discrete library of amidinonaphthols with an average purity of 60% (HPLC) that was used as a model library for mix-and-split synthesis of SP pool libraries. A small 30-member library of 2,5-dihydrofurans and 1,3-dihydroisobenzofurans was obtained with average 80% yield and >90% purity via iodine–magnesium exchange and further Grignard reaction by Rottlander and Knochel (140). Paio et al. (141) reported the SP protocol for the synthesis of polysubstituted amine libraries with moderate to good yield and good purity using supported benzotriazoles as key reaction intermediates.

Johnson et al. (142) reported the SP synthesis of 346 discrete benzothiophenes as potential thrombin inhibitors from the decoration of a core structure with alcohols and acylating agents with good yields and purities after parallel silica gel chromatography on 96-well plates. Tremblay et al. (143) presented the synthesis of a 20-member SP model library of 7-modified estradiols as potential estrogen receptor antagonists, using standard peptide SPS protocols and obtaining moderate yields (around 30%) and high purities (typically >85% by HPLC) for the five step protocol. Haskell-Luevano et al. (144) prepared a medium, >900-member SP library based on the known β-turn motif using a final cyclative cleavage and obtaining novel melanocortin-1 receptor activators with good yields and purities.

6.3.3 Semiautomated Parallel Synthesis: A Library of 1,4-Benzodiazepine-2,5-Diones

A recent paper by Boojamra et al. (145) reported the synthesis of a 2508-membered SP discrete library L4 of 1,4-benzodiazepine-2,5-diones, further exploiting the first

236 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES

reported SP synthesis of a library of small organic molecules (146, 147) based on a final cyclization step. The generic structure of the library is shown in Fig. 6.17, together with the three monomer classes used and their components. Nine of 10 chosen α-amino acid esters were coupled as racemic mixtures or were racemized during the synthesis, so that the library was prepared as 1320 samples containing 132 discretes and 1188 enantiomeric pairs.

An extensive assessment of the chemistry was performed, and several individual compounds were prepared to demonstrate the chemistry. The optimized synthetic route and reaction conditions are reported in Fig. 6.18 for a specific example. The acid-labile dimethoxybenzaldehyde 6.41-based linker was chosen and linked to a Merrifield resin to give 6.42 (step a), which was reductively aminated to 6.43 under racemizing conditions to obtain the two enantiomers from L-leucine methyl ester (representative of monomer set M1, step b). The resin-bound secondary amine was then acylated with unprotected 4-chloro anthranilic acids (representative of monomer set M2, step c) to give 6.44 using EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride) as the only effective coupling agent. Cyclization occurred using lithium acetanilide as the base with an optimal pKa to give the resin-bound cyclic anion 6.45 (step d). This was subsequently quenched with ethyl iodide (representative of monomer set M3, step e) to give the resin-bound benzodiazepinedione 6.46, which was then released under acidic conditions (step f) to provide 6.47 in a 75% overall yield based on the amino acid ester loading (Fig. 6.18). The assessment allowed satisfactory reaction conditions for the whole library synthesis to be determined (vide infra) but also served to discard potential monomers on the basis of their reactivity. In this case, serine and valine were rejected from M1 due to α-elimination of the hydroxyl group and incomplete acylation of the secondary amine in the following step, respectively. p-Substituted anthranilic acids bearing electron-donating groups were rejected from M2 because they led to significant acylation of the unprotected aromatic amine function and formation of higher order anthranilic oligomers. The racemizing conditions used for the reductive amination step (preequilibration of the amino ester hydrochloride, DIEA (diisopropyl ethyl amine), and the resin for 6 h in DMF, then addition of the reducing agent and acetic acid) were easily changed for nonracemizing conditions (sequential addition of acetic acid, reducing agent, and amino ester to a stirred slurry of resin in DMF), which allowed the synthesis of enantiomerically pure benzodiazepinediones. This allowed the “chiral deconvolution” of each enantiomeric mixture showing through the preparation of the two single enantiomers. A total of 23 compounds were prepared as discretes in yields varying from 40 to 92% for the purified material; some of them are shown in Fig. 6.18. These results encouraged the authors to pursue synthesis of a medium–large library L4. These compounds were fully characterized by means of HPLC, MS, and NMR and served as standards for the quality control of the library as a whole (vide infra).

The first steps in the synthesis of the library were performed manually, and the usual filtration/washing cycles were carried out after each step. The aldehyde linker was coupled to 50 g of Merrifield resin in a 2 L-flask; then ten 3.5-g portions of resin were placed into ten separate 100-mL flasks and four batches reductively aminated under nonracemizing conditions using racemic mixtures of Ala, 2-naphthylAla and

6.3 EXAMPLES OF SOLID-PHASE DISCRETE LIBRARY SYNTHESIS 237

O

Figure 6.17 General structure of the benzodiazepine libraryL4 and of the used monomer sets

M1–M3.

238 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES

6.41OMe

6.47

yield from 6.43: 75%

Other discretes from chemical assessment (α-Amino Ester, Anthranilic Acid (AA), Alkylating Agent, Yield of purified material):

Figure 6.18 SP chemical assessment for the benzodiazepine library L4 from resin-bound α-amino ester 6.43.

2-thienylAla, and Gly. The remaining six batches were reductively aminated under racemizing conditions. Each portion of resin was further divided into 12 aliquots and transferred to 120 filter cartridges. According to the recovered mass, after the reductive amination the resin aliquots varied in weight between 266 mg (3.55-g portion, Leu) and 316 mg (4.21-g portion, Gln). Each chosen acylating agent was reacted with the cartridges containing the 10 different resin-bound amino esters to give 120 intermedi-

240 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES

well. After 8 h, the resin aliquots were rapidly washed, and the final products were cleaved by soaking the 17 multiplates into seventeen 96-well 2-mL microtiter plates containing the cleavage cocktail (1 mL/well) for 40 h. The multitubes wereremovedslowly to allow all of the liquid to drain into the corresponding well below and then submerged into a second set of 17 plates containing a rinse cocktail (DCE–DMF 1/1, 1 mL/well). The solutions were concentrated in a plate concentrator, the rinse cocktails were also added by using semiautomated multipipetters, and the residues were finally obtained and statistically characterized for yields and purity by HPLC in the presence of an external standard (multiple randomly selected wells) or NMR (36 randomly selected wells).

The use of such a synthetic strategy in which a semiautomated device allowed a significant improvement in the speed of the synthesis and ease of handling of the 1320 library samples, while using common glassware or laboratory equipment for the early steps, proved to be a good choice for the synthesis of this library.

Other groups have recently reported the preparation of discrete libraries in which part of the synthesis has involved the use of semiautomated or fully automated devices. Examples include the work of MacDonald et al. (148), who described the preparation of an eight-member quinolone library using the so-called Diversomer kit (15) and that of Shankar et al. (149), who reported an eight-member isoxazoline library using the same technology. An 80-member N-(alkoxyacyl)amino alcohol library using a 96- deep-well microtiter plate, a multichannel automated pipetter to deliver reagents and resin slurries, and a multichannel automated washing station has been reported by Krchnak et al. (150), while Wilson et al. (151) have demonstrated the use of a sophisticated liquid-handling robot modified to handle and deliver corrosive solutions and reagents for the synthesis of several 3-thio-1,2,4-triazole libraries. Boeijen et al. (152) reported the preparation of a 42-member hydantoin library using a semiautomated multiblock device (153). Krchnak (154) has recently reported the use of the Domino reaction block (20) to prepare several hundred amino acid–derived library individuals. The semiautomated synthesis of around 700 diketopiperazines from multicomponent reaction protocols using in-house developed instrumentation was reported by Szardenings et al. (155). Finally, Souers et al. (156) presented an 176member β-turn mimetic library prepared using a semiautomated apparatus very similar to the multitube device described in the above-mentioned example. A 400-member phenyl stilbene library (157) was prepared using a semi-automated commercially available synthesizer (158) employing Wittig and Suzuki couplings. Han et al. (159) reported a small library of highly substituted thiophenes prepared via the prototype of a popular SP synthesizer (160).

6.3.4 Automated Parallel Synthesis: A Library of Tricyclic Compounds from the Tsuge Reaction

Bicknell et al. (161) has recently reported the successful transfer onto SP of a modified version of the Tsuge reaction (162) to produce a 96-member discrete library L5 of tricyclic compounds by means of a commercially available automated SP multiple organic synthesizer (163). In this synthetic adaptation, the Tsuge reaction, that is, cycloaddition of pyridinium methilides with olefinic dipolarophiles, as shown in

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Other recent reports of the automated synthesis of SP libraries of discretes include those of Wilson et al. (166), who prepared a library of more than 1000 aminohydantoins from α-hydrazino amino acids, amines, and aldehydes; Perumattam et al. (167), who reported a 200-member library based on an anhydride template using anhydrides, primary amines, and α-amino acids; Smith et al. (168), who described the synthesis of a library of more than 1000 piperazinediones from α-amino acids; Crawshaw et al. (169), who presented a >200-member library of cyclohexanones from maleimides, nitrostyrenes, and aminobutadienes; Shao et al. (170), who described a 96-member library of quinazolinediones from anhydrides and amines; Lebl et al. (21), presenting a 30,816-member

a:ClCH2COCl, DCM, 0°C, 2 hrs; b: Py, DCM, rt, 24 hrs; c: N-subst. maleimide, dry THF, rt, 1', then TEA, rt, 1 hr; d: R2CClNOH, dry THF, rt, 1', then TEA, rt, 2 hrs; e: TFA/DCM 1/1, rt.

Discretes prepared during the chemical assessment (R1, R2, yield, HPLC purity):

Me, Ph, 76%, 63%

Me, 3-PhOPh, 80%, 90% Me, 4-tBuPh, 81%, 96% Ph, 4-tBuPh, 83%, 79% 4-AcPh, 4-tBuPh, 92%, 83% PhCH2, 4-tBuPh, 88%, 76%

Figure 6.21 SP chemistry assessment for the tricyclic libraryL5 from resin-bound pyridinium methilide 6.48.