An enantioselective total synthesis of GEX1Q1 has been accomplished in a convergent manner. The C-5 asymmetric center has now been assigned through synthesis. GEX1Q1 displayed slightly better spliceosome inhibitory activity over its C-5 epimer. The salient features of this synthesis include an asymmetric hetero-Diels-Alder reaction to construct the tetrahydropyran ring and a Suzuki cross-coupling to assemble the key segments.

Spliceostatins and thailanstatins are intriguing natural products due to their structural features as well as their biological significance. This family of natural products has been the subject of immense synthetic interest because they exhibit very potent cytotoxicity in representative human cancer cell lines. The cytotoxic properties of these natural products are related to their ability to inhibit spliceosomes. FR901564 and spliceostatins have been shown to inhibit spliceosomes by binding to their SF3B component. Structurally, these natural products contain two highly functionalized tetrahydropyran rings with multiple stereogenic centers joined by a diene moiety and an acyclic side chain linked with an amide bond. Total syntheses of this family of natural products led to the development of useful synthetic strategies, which enabled the synthesis of potent derivatives. The spliceosome modulating properties of spliceostatins and synthetic derivatives opened the door for understanding the underlying spliceosome mechanism as well as the development of new therapies based upon small-molecule splicing modulators. This review outlines the total synthesis of spliceostatins, synthetic studies of structural derivatives, and their bioactivity.

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP's association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5' end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

FR901464 (1) and spliceostatin A (2) are potent inhibitors of spliceosomes. These compounds have shown remarkable anticancer activity against multiple human cancer cell lines. Herein, we describe efficient, enantioselective syntheses of FR901464, spliceostatin A, six corresponding diastereomers and an evaluation of their splicing activity. Syntheses of spliceostatin A and FR901464 were carried out in the longest linear sequence of 9 and 10 steps, respectively. To construct the highly functionalized tetrahydropyran A-ring, we utilized CBS reduction, Achmatowicz rearrangement, Michael addition, and reductive amination as key steps. The remarkable diastereoselectivity of the Michael addition was specifically demonstrated with different substrates under various reaction conditions. The side chain B was prepared from an optically active alcohol, followed by acetylation and hydrogenation over Lindlar's catalyst. The other densely functionalized tetrahydropyran C-ring was derived from readily available (R)-isopropylidene glyceraldehyde through a route featuring 1,2-addition, cyclic ketalization, and regioselective epoxidation. These fragments were coupled together at a late stage through amidation and cross-metathesis in a convergent manner. Six key diastereomers were then synthesized to probe the importance of specific stereochemical features of FR901464 and spliceostatin A, with respect to their in vitro splicing activity.

Thailanstatin A has been isolated recently from the fermentation broth of B. thailandensis MSMB43. We describe here an enantioselective convergent synthesis of thailanstatin A methyl ester and evaluation of its splicing activity. Synthesis of both highly functionalized tetrahydropyran rings were carried out from commercially available tri- O-acetyl-d-glucal as the key starting material. Our convergent synthesis involved the synthesis of both tetrahydropyran fragments in a highly stereoselective manner. The fragments were then coupled using cross-metathesis as the key step. The synthesis of the diene subunit included a highly stereoselective Claisen rearrangement, a Cu(I)-mediated conjugate addition of MeLi to set the C-14 methyl stereochemistry, a reductive amination reaction to install the C16-amine functionality, and a Wittig olefination reaction to incorporate the diene unit. The epoxy alcohol subunit was synthesized by a highly selective anomeric allylation, a Peterson olefination, and a vanadium catalyzed epoxidation that installed the epoxide stereoselectively. Cross-metathesis of the olefins provided the methyl ester derivative of thailanstatin A. We have carried out in vitro splicing studies of the methyl ester derivative, which proved to be a potent inhibitor of the spliceosome.

An enantioselective total synthesis of spliceostatin G has been accomplished. The synthesis involved a Suzuki cross-coupling reaction as a key step. The functionalized tetrahydropyran ring was constructed from commercially available optically active tri-O-acetyl-d-glucal. Other key reactions include a highly stereoselective Claisen rearrangement, a Cu(I)-mediated 1,4 addition of MeLi to install the C8 methyl group, and a reductive amination to incorporate the C10 amine functionality of spliceostatin G. Biological evaluation of synthetic spliceostatin G and its methyl ester revealed that it does not inhibit splicing in vitro.

The protein SF3B1 is a core component of the spliceosome, the large ribonucleoprotein complex responsible for pre-mRNA splicing. Interest in SF3B1 intensified when tumor exome sequencing revealed frequent specific SF3B1 mutations in a variety of neoplasia and when SF3B1 was identified as the target of three different cancer cell growth inhibitors. A better mechanistic understanding of SF3B1's role in splicing is required to capitalize on these discoveries. Using the inhibitor compounds, we probed SF3B1 function in the spliceosome in an in vitro splicing system. Formerly, the inhibitors were shown to block early steps of spliceosome assembly, consistent with a previously determined role of SF3B1 in intron recognition. We now report that SF3B1 inhibitors also interfere with later events in the spliceosome cycle, including exon ligation. These observations are consistent with a requirement for SF3B1 throughout the splicing process. Additional experiments aimed at understanding how three structurally distinct molecules produce nearly identical effects on splicing revealed that inactive analogs of each compound interchangeably compete with the active inhibitors to restore splicing. The competition indicates that all three types of compounds interact with the same site on SF3B1 and likely interfere with its function by the same mechanism, supporting a shared pharmacophore model. It also suggests that SF3B1 inhibition does not result from binding alone, but is consistent with a model in which the compounds affect a conformational change in the protein. Together, our studies reveal new mechanistic insight into SF3B1 as a principal player in the spliceosome and as a target of inhibitor compounds.

Herboxidiene is a potent inhibitor of spliceosomes. It exhibits excellent anticancer activity against multiple human cancer cell lines. Herein, we describe an enantioselective synthesis of a desmethyl derivative and the corresponding carba-derivatives of herboxidiene. The synthesis involved Suzuki coupling of a vinyl iodide with boronate as the key reaction. For the synthesis of carba-derivatives, the corresponding optically active cyclohexane-1,3-dicarbonyl derivatives were synthesized using an enantioselective desymmetrization of meso-anhydride. The biological properties of these derivatives were evaluated in an in vitro splicing assay.

Spliceostatin A is a potent inhibitor of spliceosomes and exhibits excellent anticancer activity against multiple human cancer cell lines. We describe here the design and synthesis of a stable cyclopropane derivative of spliceostatin A. The synthesis involved a cross-metathesis or a Suzuki cross-coupling reaction as the key step. The functionalized epoxy alcohol ring was constructed from commercially available optically active tri- O-acetyl-d-glucal. The biological properties of the cyclopropyl derivative revealed that it is active in human cells and inhibits splicing in vitro comparable to spliceostatin A.

An enantioselective total synthesis of spliceostatin E has been accomplished. The δ-lactone unit A was constructed from readily available (R)-glycidyl alcohol using a ring-closing olefin metathesis as the key reaction. A cross-metathesis of ring A containing δ-lactone and the functionalized tetrahydropyran B-ring provided spliceostatin E. Our biological evaluation of synthetic spliceostatin E revealed that it does not inhibit splicing in vitro and does not impact speckle morphology in cells. Spliceostatin E was reported to possess potent antitumor activity.