An aldol-based build/couple/pair strategy for the synthesis of medium- and large-sized rings: discovery of macrocyclic histone deacetylase inhibitors

Abstract

An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti-aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (S(N)Ar), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.

Figure 1

An aldol-based B/C/P strategy for generating macrocycles and medium-sized rings.

Figure 2

Scatter plot representing molecular shape diversity of the bare library scaffolds. Normalized PMI ratios are plotted in a triangular graph to compare shape space covered by skeletons derived from the three pairing reactions: S_NAr (8- and 9-membered rings), Click (1,4- and 1,5-triazoles) and RCM. The triangle is defined by its three corners, which are represented as rod-, disc- and sphere-like shapes. Representative examples of molecules exhibiting these extremes are shown. Minimum energy conformers ≤3 kcal/mol from the global minimum are plotted on the graph for all diastereomers.

Scheme 1

Syn- and Anti-Aldol Reaction to Provide Four Stereoisomers of γ-amino Acid 1

Scheme 2

^a Protecting Group Modifications to Yield Final Library Scaffolds

Scheme 3

Solid-Phase Synthesis of RCM Library

Figure 3

Mean Fsp³ values for various compound collections where Fsp³ = (number of sp³ hybridized carbons/total carbon count). DOS-RCM = RCM library compounds (14,400 compounds); DOS-all = All DOS compounds derived from the S_NAr, Click and RCM pathways (30,099 compounds); NPs = Natural products and natural product derivatives retrieved from GVK BIO database (2,265 compounds); Drugs = Small-molecule drugs retrieved from DrugBank (4,239 compounds); MLSMR = Compounds contained in NIH's Molecular Libraries Small-Molecule Repository (307,983 compounds). Mean Fsp³ values were determined according to the method of Lovering et al.

Figure 4

Primary screening data displayed as percent inhibition for the HDAC2 biochemical screen. A total of 1,604 compounds derived from two diastereomeric RCM scaffolds, (2R,5S,6R,12R)-30a and (2R,5S,6S,12R)-30e, were tested. The assay was conducted in a kinetic mode using 4.6 μM substrate, 2.2 nM HDAC2, 150 nM trypsin, and 17 μM compound. The full matrix of building blocks for R₁ (y-axis) and R₂ (x-axis) is shown. Piperonaldehyde (AL11) was the preferred building block at R₂. Empty cells represent compounds that were not tested due to low purity or unavailability.

Figure 5

Stereo/structure-activity relationships for HDAC inhibition. IC₅₀ values below 50 M are provided. A more potent stereoisomer with low micromolar activity against HDACs 1, 2 and 3 (2S,5R,6R,12R)-32 (BRD-4805) was identified. (See also Table S-1).

Scheme 4

Solution-phase synthesis of BRD-4805

Figure 6

Lineweaver-Burk plot of the effect of BRD-4805 on the HDAC2-catalyzed deacetylation (Ac)Leu-Gly-Lys-acetyl-AMC peptide substrate. The experiment was performed at pH 7.0, room temperature. Inhibitor concentrations were 0, 1.56, 3.1, 6.2, 12.5, 25 and 50 μM and substrate concentrations were 2.5, 5, 10, 20, 40 and 80 μM.

Figure 7

Induction of histone acetylation increase in primary mouse forebrain neurons. Neurons were treated for 24 hours with the indicated compounds (95 μM), then fixed and stained for AcH4K12 or AcH3K9. Data are shown normalized to the level of acetylation in the presence of DMSO. Error bars represent +/− the standard deviation of the measurement.

Figure 8

Imaging of histone acetylation in primary mouse forebrain neurons. (A) Compound treated primary neuronal cultures were stained for acetylation of histone H3 lysine 9 (AcH3K9; shown in green) and MAP2B (neuronal marker; shown in red). To quantify cellular activity of compounds, the mean nuclear intensity of histone signal was measured and the percentage of cells with a value above a predefined threshold was measured. Scale bar = 50 μm. (B) Neuronal nuclei with representative levels of acetylation for each treatment (DMSO, ent-BRD-4805, BRD-4805, BRD-8172). Nuclei of cells treated with BRD-4805 and analog BRD-8172 scored as “bright green” in our analysis. Scale bar = 10 μm.