Solid-Phase Synthesis and Combinatorial Technologies

can be developed in-house together with high-throughput analytical techniques (78– 82). Usually, these programs detect the presence of the predicted molecular weight for a library component in the sample and may also include minimum threshold purity criteria. The example shown in Fig. 6.7 (83) depicts the analysis of two reaction plates of 96 compounds where the plate on the left contains most of the expected reaction products (green spots, purity higher than the set standard as measured on the UV chromatogram) while the plate on the right is a poor-quality library where a large number of wells do not contain the expected library component or contain it as a mixture with impurities.

Several recent reviews (84–88) cover the characterization of discrete libraries from parallel synthesis with automated systems and using different analytical and/or detection methods; their perspective may help the reader to expand his or her knowledge of this area.

6.2.3 Purification

The SPS of libraries allows the automation of all of the work-up procedures and the elimination of tedious and time-consuming purification steps during the synthesis by simply filtering off the reaction solution and washing the beads repeatedly with fresh solvents. For all SP discrete libraries, the last synthetic step involves the cleavage of resin-bound individuals from the support and their release into solution. Careful selection of linkers and reagents usually allows the elimination of possible by-products from the cleavage, but often the recovered crude materials do not satisfy the purity requirements for screening. The purification of the cleaved compounds from an SP library of discretes is conceptually identical to the purification of the individuals from a solution-phase library of discretes (Chapter 8). However, this latter library format requires the automated parallel work-up/purification of intermediates during most of the synthetic steps. This important topic will be covered more thoroughly in Section 8.3.

Target not found

Target found

Figure 6.7 Visualization of analytical results for a 96-well reaction plate: good-quality (left) and poor-quality (right) libraries.

226 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES

TABLE 6.1 Yields of Selected Tetrahydroquinolines from the SP Discrete Library L1a

aNT = not performed.

bYield from reaction with aldehyde 6.1/aldehyde 6.2. cFailed.

Fig. 6.10. The resin (15 g) was partitioned into 60 aliquots of 250 mg in 60 glass vials, and each was treated with an aniline and an olefin as 0.5 M solutions in acetonitrile in the presence of 2% TFA at room temperature for 24 h. The resin in each vial was filtered, washed, and dried and the product cleaved from the support with 15% TFA–DCM. The library individuals were obtained as solids (>80% by HPLC) after evaporation and trituration of the crude residue with Et2O, with the yields reported in Table 6.1. The reactions proceeded well for alkenes 6.3–6.7 (60–84% yields), the only detectable impurities being the corresponding Schiff bases (5–10%). Alkene 6.8 gave poor yields of the desired product or failed to react completely in some cases (see Table 6.1). All of the tetrahydroquinolines were obtained as single diastereomers, as shown for compounds 6.14 and 6.15 (Fig. 6.10).

A similar library L2 was prepared from the Wang-resin-bound alkene reagents 6.17 and 6.18 (105 g each, obtained via phosphoranes 6.16) by reacting them with anilines and aldehydes. Compound 6.25, which was formed as a single diastereomer, was prepared as a model in good yield (64%) and purity (Fig. 6.11). The use of four anilines (6.9, 6.10, 6.19, and 6.20; Fig. 6.10) and four aldehydes (6.21–6.24; Fig. 6.12) produced a 32-member SP discrete library on a 250-mg resin scale for each individual using identical conditions to those used for L1. The final compounds were obtained in good yields (61–85%) and purities (>85% by HPLC), and as an improvement with respect to L1, the Schiff base side products remained in solution, even though small quantities of unreacted styrenes could sometimes be found in the final library components. The yields for library L2 are reported in Table 6.2, and the diastereopure outcome of the reaction is shown by the structures of 6.25 and 6.26 (Fig. 6.12).

The parallel synthesis of several tens of compounds with two complementary SP routes employing a multicomponent reaction and producing diverse tetrahydroquinolines as decorated, biologically relevant scaffolds (101–103) was realized using simple laboratory equipment and commercially available reagents as monomers. The assessment work to produce compounds 6.14 and 6.25 with the optimized reaction conditions was deemed to be sufficiently robust to pass immediately to library production. This

6.3 EXAMPLES OF SOLID-PHASE DISCRETE LIBRARY SYNTHESIS 227

L1

60 discretes

H

OMe

Figure 6.10 Monomers used for the synthesis of the tetrahydroquinoline library L1 and selected library individuals.

230 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES

The generic structure of the library is reported in Fig. 6.13; a retrosynthetic analysis arrived at three acids (e.g., 6.27; only the α-dimethyl was reported as a structure in the original article) as suitable precursors prepared from commercially available materials according to a reported procedure (Fig. 6.13) (112). The cleavage of an ester bond with amines was used to generate diversity and simultaneously release the compounds into solution, and a suitable linker was the 4-hydroxythiophenol linker 6.28 (Fig. 6.13) (113, 114).

The chemistry assessment is reported in Fig. 6.14. The linker 6.28 was coupled with the carboxybenzopyranone 6.27a using standard SP esterification conditions, and the supported benzopyranone 6.29 was reductively aminated. At first, classical SP reductive amination conditions were used, but either incomplete reduction was obtained or extensive cleavage of the benzopyrans from the resin was observed through the use of forcing conditions to drive the reaction to completion. The authors then examined the Ti(OiPr)4/Na(OAc)3H as reducing agent and obtained excellent yields of supported amine 6.30 using benzylamine and anhydrous toluene in an inert atmosphere after 2 h at room temperature with Ti(OiPr)4 and subsequent addition of the borohydride with further stirring at room temperature. The outcome of the reaction was studied by single-bead IR spectroscopy, and the absence of cleaved intermediates from the solid support (<10%) was confirmed by HPLC analysis of the supernatant. An important finding was the negligible amount of insoluble titanium salts formed; the insoluble

Figure 6.13 General structure of the SP benzopyran library L3 and retrosynthetic studies for its synthesis.

6.3 EXAMPLES OF SOLID-PHASE DISCRETE LIBRARY SYNTHESIS 231

a

O

O

O O

S

6.29

b,c

O

O

O HN

S

6.30

d

O

H

N

O HN

6.31

a:DIC, DMAP, NMM, rt; b: Ti(OiPr)4, BnNH2, dry toluene, rt, 2 hrs;

c:Na(OAc)3BH, AcOH, dry toluene/dry THF, rt, 24 hrs; d: n-BuNH2, Py, rt, 24 hrs.

Figure 6.14 SP chemical assessment for the benzopyran library L3 from resin-bound benzopyranone 6.29.

salts could have significantly complicated the washing and filtration of the resin by using anhydrous reaction conditions. Resin-bound 6.30 was finally washed and cleaved with n-butylamine in pyridine at room temperature for 24 h to give compound 6.31 (Fig. 6.14).

The design of the library L3 and the synthetic scheme are reported in Fig. 6.15; filtration of the solutions and washing of the resin aliquots were performed after each step. The three supported benzopyranones 6.32 (step a, from the monomer set M1, three ketones) were obtained in a similar manner to 6.29 on a 300-g scale. Each resin-supported benzopyranone was split into 8 separate peptide synthesis flasks containing 35 g of resin and all of the 24 samples were reductively aminated (steps b and c, monomer set M2, eight amines) to produce 24 resin-bound intermediates 6.33. Each intermediate was then divided into 352 portions of 100 mg of resin that were distributed into four filter-bottom 96-well microtiter plates (2.0 mL capacity per well).

232 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES

a:DIC, DMAP, NMM, rt; b: Ti(OiPr)4, M2, dry toluene, rt, 2 hrs;

c:Na(OAc)3BH, AcOH, dry toluene/dry THF, rt, 24 hrs; d: M3, DIPEA, rt;

e:M4, Py, rt, 48 hrs; f: Supported Liquid Extraction (SLE).

Figure 6.15 SP synthesis of the benzopyran library L3.

These plates allowed the reaction of resin aliquots with reagents in solution by sealing the filter bottom and to discard the solution and wash the resin by filtering the solutions after removal of the seal. The samples 6.33 were then acylated (step d, monomer set M3, 16 acylating agents, including isocyanates, isothiocyanates, and acid chlorides) to produce 384 resin-bound intermediates 6.34, each as a discrete distributed into 22 different wells. Final cleavage from the support was performed (step e, monomer set