Jiyong Hong

Total Synthesis and Molecular Target of Largazole, a Histone Deacetylase Inhibitor

Full details of the concise and convergent synthesis (eight steps, 19% overall yield), its extension to the preparation of a series of key analogues, and the molecular target and pharmacophore of largazole are described. Central to the synthesis of largazole is a macrocyclization reaction for formation of the strained 16-membered depsipeptide core followed by an olefin cross-metathesis reaction for installation of the thioester. The biological evaluation of largazole and its key analogues, including an acetyl analogue, a thiol analogue, and a hydroxyl analogue, suggested that histone deacetylases (HDACs) are molecular targets of largazole and largazole is a class I HDAC inhibitor. In addition, structure-activity relationship (SAR) studies revealed that the thiol group is the pharmacophore of the natural product. Largazoles HDAC inhibitory activity correlates with its antiproliferative activity.

Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.

Moving Bricks with MraY Peptidoglycan, the building brick of bacterial cell walls, is synthesized in the cytoplasm and must be transported across the cell membrane. To achieve this, it is attached to a carrier lipid by the integral membrane protein MraY. MraY is targeted by natural antibacterials and is a promising antibiotic target. Chung et al. (p. 1012) report the crystal structure of MraY at 3.3 Å resolution. The structure, together with mutational mapping, outlines the location of the active site and provides interesting hints for how the enzyme binds the substrate and catalyzes attachment to the carrier lipid. The cell wall component peptidoglycan is attached to a carrier lipid for transfer across the cell membrane. MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg2+ within the active site, and provide a structural basis of catalysis for this class of enzyme.

Largazole: From Discovery to Broad-Spectrum Therapy

The cyclic depsipeptide largazole from a cyanobacterium of the genus Symploca is a marine natural product with a novel chemical scaffold and potently inhibits class I histone deacetylases (HDACs). Largazole possesses highly differential growth-inhibitory activity, preferentially targeting transformed over non-transformed cells. The intriguing structure and biological activity of largazole have attracted strong interest from the synthetic chemistry community to establish synthetic routes to largazole and to investigate its potential as a cancer therapeutic. This Highlight surveys recent advances in this area with a focus on the discovery, synthesis, target identification, structure-activity relationships, HDAC8-largazole thiol crystal structure, and biological studies, including in vivo anticancer and osteogenic activities.

Stereoselective Synthesis of 2,6‐cis‐Tetrahydropyrans through a Tandem Allylic Oxidation/Oxa‐Michael Reaction Promoted by the gem‐Disubstituent Effect: Synthesis of (+)‐Neopeltolide Macrolactone

Structurally complex tetrahydropyrans (THPs) are found in a wide range of biologically interesting natural products. Although considerable effort has been devoted to the development of synthetic routes to THPs, there still exists a great need for a synthetic approach to these classes of molecules that enables rapid and easy access to substrates, proceeds with excellent stereoselectivity in excellent yield, and requires mild reaction conditions compatible with various functional groups. Surprisingly, despite considerable progress in the Michael reaction of carbon nucleophiles, there has been far less interest in the analogous Michael reaction of oxygen nucleophiles (oxa-Michael reaction). The slow development of the oxa-Michael reaction is mainly due to major drawbacks such as the low reactivity of oxygen nucleophiles and the reversibility issue, as well as a lack of control over stereoselectivity. Owing to the poor nucleophilicity of oxygen atoms in the oxaMichael reaction, alcohols must be deprotonated by strong bases to enhance their nucleophilicity, or the conjugate acceptor must be activated by Lewis or Brønsted acids, amines, or transition-metal complexes. These harsh reaction conditions are often incompatible with other functional groups of the substrate. In particular, as a result of competitive acetal formation, instability, and the enolizability of aldehydes, the oxa-Michael reaction of alcohols to a,b-unsaturated aldehydes has been elusive. Herein, we report the stereoselective and efficient synthesis of 2,6-cis-THPs through an unprecedented tandem allylic oxidation/oxa-Michael addition of alcohols to a,bunsaturated aldehydes promoted by the gem-disubstituent effect and its application to the concise synthesis of (+)-neopeltolide macrolactone. To overcome the low reactivity of oxygen nucleophiles, we envisioned the introduction of a structural element that would promote preorganization of the conformation of a substrate for an intramolecular oxa-Michael reaction. We also anticipated that this structural element could help decrease the reversibility of the reaction to form the oxa-Michael product. We hypothesized that a 1,3-dithiane group at the C4 position of an alcohol nucleophile could satisfy these requirements on the basis of the gem-disubstituent effect. Furthermore, the 1,3-dithiane group would serve as a latent functional group for a carbonyl, hydroxy, or olefinic group, or a hydrogen atom. To test this hypothesis, we prepared substrate 4 for the oxa-Michael reaction as follows (Scheme 1): The 1,3-dithiane coupling of 1 with allyl bromide 2, followed by THP deprotection, provided 3. The coupling of 3 with ( )-glycidyl benzyl ether then proceeded smoothly to afford the allylic alcohol 4. As expected, the chemoselective oxidation of 4 with MnO2 and subsequent intramolecular oxa-Michael reaction of the resulting a,b-unsaturated aldehyde 5 provided the desired 2,6-cis-THP 6a with excellent stereoselectivity (d.r.>20:1, 93 %; tandem allylic oxidation/oxa-Michael reaction).

Anticolon Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor

Histone deacetylases (HDACs) are validated targets for anticancer therapy as attested by the approval of suberoylanilide hydroxamic acid (SAHA) and romidepsin (FK228) for treating cutaneous T cell lymphoma. We recently described the bioassay-guided isolation, structure determination, synthesis, and target identification of largazole, a marine-derived antiproliferative natural product that is a prodrug that releases a potent HDAC inhibitor, largazole thiol. Here, we characterize the anticancer activity of largazole by using in vitro and in vivo cancer models. Screening against the National Cancer Institutes 60 cell lines revealed that largazole is particularly active against several colon cancer cell types. Consequently, we tested largazole, along with several synthetic analogs, for HDAC inhibition in human HCT116 colon cancer cells. Enzyme inhibition strongly correlated with the growth inhibitory effects, and differential activity of largazole analogs was rationalized by molecular docking to an HDAC1 homology model. Comparative genomewide transcript profiling revealed a close overlap of genes that are regulated by largazole, FK228, and SAHA. Several of these genes can be related to largazoles ability to induce cell cycle arrest and apoptosis. Stability studies suggested reasonable bioavailability of the active species, largazole thiol. We established that largazole inhibits HDACs in tumor tissue in vivo by using a human HCT116 xenograft mouse model. Largazole strongly stimulated histone hyperacetylation in the tumor, showed efficacy in inhibiting tumor growth, and induced apoptosis in the tumor. This effect probably is mediated by the modulation of levels of cell cycle regulators, antagonism of the AKT pathway through insulin receptor substrate 1 down-regulation, and reduction of epidermal growth factor receptor levels.

Synthesis and activity of largazole analogues with linker and macrocycle modification.

To characterize largazoles structural requirements for histone deacetylase (HDAC) inhibitory and antiproliferative activities, a series of analogues with modifications to the side chain or 16-membered macrocycle were prepared and biologically evaluated. Structure-activity relationships suggested that the four-atom linker between the macrocycle and octanoyl group in the side chain and the (S)-configuration at the C17 position are critical to repression of HDAC activity. However, the valine residue in the macrocycle can be replaced with alanine without significant loss of activity.

A Small‐Molecule Antagonist of the Hedgehog Signaling Pathway

The Hedgehog (Hh) signaling pathway plays an important role in embryonic pattern formation and adult tissue maintenance by directing cell differentiation and proliferation. In mammals, three Hh genes, Sonic (Shh), Indian (Ihh), and Desert (Dhh), have been identified. Binding of Hh protein to the membrane receptor Patched (Ptc) releases its inhibitory effect on Smoothened (Smo). Activated Smo further transduces downstream signals to activate the Gli family of transcription factors, which promote the expression of Hh signaling target genes. Hh signaling has recently attracted considerable interest based on the discovery that aberrant activation of Shh signaling leads to the formation of various tumors, which include pancreatic cancer, medulloblastoma, basal cell carcinoma, small cell lung cancer, and prostate cancer. 5] Cell-based phenotypic assays and, more recently, pathway screens of natural products and synthetic small molecules have provided useful chemical tools for modulating and/or studying complex cellular processes, both in vitro and in vivo. Several Hh antagonists, including cyclopamine, CUR61414, and SANT1-4 have been reported. Some of these antagonists exert antiproliferative effects by binding directly to Smo. However, cancer cells with mutations downstream of Smo are resistant to these antagonists. Therefore, Hh antagonists that interrupt downstream Hh signaling could lead to antiproliferative agents with a broader spectrum of activity. Here, we report the identification and characterization of the Hh signaling antagonist, JK184, and initial studies to characterize its biological mechanism of action. To screen small molecule libraries for compounds that antagonize Hh signaling, we developed a protocol using mesenchymal progenitor (C3H10T1/2) cells derived from the mouse embryonic mesoderm. These cells were stably transfected with a reporter construct that encoded luciferase, which was driven by Gli responsive elements, along with a neomycin resistanceconferring gene. Stably transfected C3H10T1/2 cells were plated into 384-well plates, and treated with a library of approximately 20000 heterocycles (2 mm, final concentration). After treatment with compound for 36 h in the presence of recombinant peptide that corresponded to the N terminus of Shh (100 ngmL ), luciferase activity was assayed, and a number of active 2,4-disubstituted thiazole compounds were identified. One of these compounds, JK184 (Figure 1A), inhibited Gli-dependent transcriptional activity in a dose-dependent manner with an IC50 value of 30 nm. This effect was fur-

Role of natural product diversity in chemical biology.

Through the natural selection process, natural products possess a unique and vast chemical diversity and have been evolved for optimal interactions with biological macromolecules. Owing to their diversity, target affinity, and specificity, natural products have demonstrated enormous potential as modulators of biomolecular function, been an essential source for drug discovery, and provided design principles for combinatorial library development.

Total synthesis of cyanolide A and confirmation of its absolute configuration.

The tandem allylic oxidation/oxa-Michael reaction promoted by the gem-disubstituent effect and the 2-methyl-6-nitrobenzoic anhydride (MNBA)-mediated dimerization were explored for the efficient and facile synthesis of cyanolide A.

Stereoselective Synthesis of 2,6-trans-Tetrahydropyran via Primary Diamine-Catalyzed Oxa-Conjugate Addition Reaction of α,β-Unsaturated Ketone: Total Synthesis of Psymberin

The total synthesis of psymberin was achieved employing a readily available chiral epoxide to prepare two of the three subunits in the natural product. The key reaction was a highly stereoselective organocatalytic oxa-conjugate addition reaction of α,β-unsaturated ketone catalyzed by primary diamine for the synthesis of the 2,6-trans-tetrahydropyran embedded in psymberin.