Jason T. Lowe

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

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.

Synthesis and Profiling of a Diverse Collection of Azetidine-Based Scaffolds for the Development of CNS-Focused Lead-like Libraries

The synthesis and diversification of a densely functionalized azetidine ring system to gain access to a wide variety of fused, bridged, and spirocyclic ring systems is described. The in vitro physicochemical and pharmacokinetic properties of representative library members are measured in order to evaluate the use of these scaffolds for the generation of lead-like molecules to be used in targeting the central nervous system. The solid-phase synthesis of a 1976-membered library of spirocyclic azetidines is also described.

Synthesis of a 35-member stereoisomer library of bistramide A: evaluation of effects on actin state, cell cycle and tumor cell growth.

Synthesis and preliminary biological evaluation of a 35-member library of bistramide A stereoisomers are reported. All eight stereoisomers of the C1-C13 tetrahydropyran fragment of the molecule were prepared utilizing crotylsilane reagents 9 and 10 in our [4+2]-annulation methodology. In addition, the four isomers of the C14-C18 gamma-amino acid unit were accessed via a Lewis acid mediated crotylation reaction with use of both enantiomers of organosilane 11. The spiroketal subunit of bistramide A was modified at the C39-alcohol to give another point of stereochemical diversification. The fragments were coupled by using a standard peptide coupling protocol to provide 35 stereoisomers of the natural product. These stereochemical analogues were screened for their effects on cellular actin and cytotoxicity against cancer cell lines (UO-31 renal and SF-295 CNS). The results of these assays identified one analogue, 1.21, with enhanced potency relative to the natural product, bistramide A.

Total Synthesis of (−)-Kendomycin

An enantioselective synthesis of (-)-kendomycin is described and is based on the application of the organosilane-based [4 + 2]-annulation strategy for the assembly of the C1a-C10 fragment. An underutilized samarium(II) iodide-assisted cyclization (intramolecular Barbier-type reaction) is employed to afford the protected macrocycle.

Regioselective Intramolecular Dipolar Cycloaddition of Azides and Unsymmetrical Alkynes

Enantioenriched allenylsilanes are used in three-component propargylation reactions with aldehydes and silyl ethers to form syn-homopropargylic ethers that contain an imbedded azide. These materials then undergo thermally induced intramolecular 1,3-dipolar cycloaddition reactions, resulting in unique fused ring systems containing 1,2,3-triazoles. The ability to modify all three components of the reaction allows for expedient access to compounds containing significant structural and stereochemical variation.

Diversity-oriented synthesis of 13- to 18-membered macrolactams via ring-closing metathesis.

An efficient build/couple/pair approach to diversity-oriented synthesis was employed to access several structurally complex macrolactams. In this paper, we describe the successful evaluation of ring-closing metathesis toward the systematic generation of skeletal diversity. By appropriately varying the nature and chain length of the alkenol fragment, a diverse collection of 13- to 18-membered macrolactams were obtained.

Splicing modulators act at the branch point adenosine binding pocket defined by the PHF5A–SF3b complex

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A–SF3B1 forms a central node for binding to these splicing modulators.

Total Synthesis of 6-Deoxypladienolide D and Assessment of Splicing Inhibitory Activity in a Mutant SF3B1 Cancer Cell Line

A total synthesis of the natural product 6-deoxypladienolide D (1) has been achieved. Two noteworthy attributes of the synthesis are (1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and (2) the development of a scalable and cost-effective synthetic route to support drug discovery efforts. 6-Deoxypladienolide D (1) demonstrates potent growth inhibition in a mutant SF3B1 cancer cell line, high binding affinity to the SF3b complex, and inhibition of pre-mRNA splicing.

Synthesis of Stereochemically and Skeletally Diverse Fused Ring Systems from Functionalized C-Glycosides

A diversity-oriented synthesis (DOS) strategy was developed for the synthesis of stereochemically diverse fused-ring systems containing a pyran moiety. Each scaffold contains an amine and methyl ester for further diversification via amine capping and amide coupling. Scaffold diversity was evaluated in comparison to previously prepared scaffolds by a shape-based principal moments of inertia (PMI) analysis.

Abstract 5564: Total synthesis of 6-deoxypladienolide D and assessment of splicing inhibitory activity in a mutant SF3B1 cancer cell line

Hotspot mutations in several components of the spliceosome have been reported in various hematological (CLL, MDS, etc.) and solid tumor (melanoma, pancreatic, etc.) malignancies. SF3B1 is a component of the U2 snRNP complex of the spliceosome and is involved in the recognition of 3′-splice sites during early spliceosomal assembly. We and others have demonstrated that mutations in SF3B1 result in neomorphic activity and trigger the production of aberrantly spliced transcripts. Thus, the discovery of small molecule modulators of SF3B1 splicing activity may have therapeutic potential in cancers harboring SF3B1 mutations. Members of the pladienolide family of natural products have been shown to affect RNA splicing through interaction with SF3B1. We have found that one particular natural product in this family, 6-deoxypladienolide D, demonstrates potent growth inhibition and cellular lethality in Panc 05.04 cells (a hotspot mutant SF3B1 cancer cell line). Due to the limited natural supply of 6-deoxypladienolide D and our interest in identifying chemical matter able to modulate splicing in these newly-identified mutant SF3B1 cancers, a total synthesis of 6-deoxypladienolide D using versatile and modular fragments was initiated. We will describe the first total synthesis of the natural product 6-deoxypladienolide D. Two noteworthy synthetic attributes are: 1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and 2) the use of cost-effective starting materials and reagents to enable access to 6-deoxypladienolide D and its analogs for biological evaluation. We will show that 6-deoxypladienolide D demonstrates: 1) high binding affinity to the SF3b complex, 2) ability to modulate canonical pre-mRNA splicing, and 3) modulation of aberrant splicing induced by mutant SF3B1. Citation Format: Kenzo Arai, Silvia Buonamici, Betty Chan, Laura Corson, Atsushi Endo, Baudouin Gerard, Ming-Hong Hao, Craig Karr, Kazunobu Kira, Linda Lee, Xiang Liu, Jason T. Lowe, Tuoping Luo, Lisa A. Marcaurelle, Yoshiharu Mizui, Marta Nevalainen, Morgan Welzel O9Shea, Eun Sun Park, Samantha A. Perino, Sudeep Prajapati, Mingde Shan, Peter G. Smith, Parcharee Tivitmahaisoon, John Yuan Wang, Markus Warmuth, Kuo-Ming Wu, Lihua Yu, Huiming Zhang, Guo Zhu Zheng, Gregg F. Keaney. Total synthesis of 6-deoxypladienolide D and assessment of splicing inhibitory activity in a mutant SF3B1 cancer cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5564. doi:10.1158/1538-7445.AM2015-5564