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Enantioselective total syntheses of FR901464 and spliceostatin A and evaluation of splicing activity of key derivatives.

Ghosh AK, Chen ZH, Effenberger KA, Jurica MS - J. Org. Chem. (2014)

Bottom Line: To construct the highly functionalized tetrahydropyran A-ring, we utilized CBS reduction, Achmatowicz rearrangement, Michael addition, and reductive amination as key steps.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Department of Medicinal Chemistry, Purdue University , 560 Oval Drive, West Lafayette, Indiana 4790, United States.

ABSTRACT
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.

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Impact of analogues on in vitro splicing. A and B: (1) Top panels: Denaturing gel analysisof radiolabeled RNA isolated from splicing reactions. The first fivelanes include a time course of splicing reactions in 1% DMSO followedby 30 min time points of splicing reactions incubated with indicatedconcentration. Identities of bands are schematized to the left as(from top to bottom) lariat intermediate, pre-mRNA, mRNA, 5′exon intermediate. (2) Bottom panels: Native gel analysis of spliceosomeassembly. Aliquots of the splicing reactions described above wereseparated under native conditions. The identity of splicing complexesis denoted with assembly occurring in the following order: H/E →A → B → C. C: Quantification of normalizedsplicing efficiency vs inhibitor concentration for the splicing reactionsshown in (A) and (B), respectively. D: Summary of splicing inhibition data. IC50 refersto the concentration required to reduce in vitro splicingefficiency by half compared to DMSO control.
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fig2: Impact of analogues on in vitro splicing. A and B: (1) Top panels: Denaturing gel analysisof radiolabeled RNA isolated from splicing reactions. The first fivelanes include a time course of splicing reactions in 1% DMSO followedby 30 min time points of splicing reactions incubated with indicatedconcentration. Identities of bands are schematized to the left as(from top to bottom) lariat intermediate, pre-mRNA, mRNA, 5′exon intermediate. (2) Bottom panels: Native gel analysis of spliceosomeassembly. Aliquots of the splicing reactions described above wereseparated under native conditions. The identity of splicing complexesis denoted with assembly occurring in the following order: H/E →A → B → C. C: Quantification of normalizedsplicing efficiency vs inhibitor concentration for the splicing reactionsshown in (A) and (B), respectively. D: Summary of splicing inhibition data. IC50 refersto the concentration required to reduce in vitro splicingefficiency by half compared to DMSO control.

Mentions: Thebiological properties of FR901464 (1), spliceostatinA (2), along with their six diasteromers (47–52) were evaluated in an in vitro splicing system (Figure 2).44 We added the compounds to splicing reactions containinga synthetic pre-mRNA substrate, ATP, and nuclear extract from HeLacells. Splicing chemistry was examined by denaturing PAGE to separatethe substrate and product mRNA, while splicing efficiency was quantifiedas the percent of pre-mRNA converted to mRNA. In this system, DMSOalone has no effect on splicing efficiency, while spliceostatin A(2) and FR901464 (1) both inhibit splicingwith an IC50 of 0.01 and 0.05 μM, respectively (Figure 2, A, C, and D). Surprisingly, compounds 47, 49, and 50 showed an approximately 100-foldreduction in potency relative to spliceostatin A, with IC50 values between 1 and 1.5 μM (Figure 2, A–D). Additionally, compounds 48, 51, and 52 were the least potent splicing inhibitors withIC50 between 10 and 35 μM.


Enantioselective total syntheses of FR901464 and spliceostatin A and evaluation of splicing activity of key derivatives.

Ghosh AK, Chen ZH, Effenberger KA, Jurica MS - J. Org. Chem. (2014)

Impact of analogues on in vitro splicing. A and B: (1) Top panels: Denaturing gel analysisof radiolabeled RNA isolated from splicing reactions. The first fivelanes include a time course of splicing reactions in 1% DMSO followedby 30 min time points of splicing reactions incubated with indicatedconcentration. Identities of bands are schematized to the left as(from top to bottom) lariat intermediate, pre-mRNA, mRNA, 5′exon intermediate. (2) Bottom panels: Native gel analysis of spliceosomeassembly. Aliquots of the splicing reactions described above wereseparated under native conditions. The identity of splicing complexesis denoted with assembly occurring in the following order: H/E →A → B → C. C: Quantification of normalizedsplicing efficiency vs inhibitor concentration for the splicing reactionsshown in (A) and (B), respectively. D: Summary of splicing inhibition data. IC50 refersto the concentration required to reduce in vitro splicingefficiency by half compared to DMSO control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4066912&req=5

fig2: Impact of analogues on in vitro splicing. A and B: (1) Top panels: Denaturing gel analysisof radiolabeled RNA isolated from splicing reactions. The first fivelanes include a time course of splicing reactions in 1% DMSO followedby 30 min time points of splicing reactions incubated with indicatedconcentration. Identities of bands are schematized to the left as(from top to bottom) lariat intermediate, pre-mRNA, mRNA, 5′exon intermediate. (2) Bottom panels: Native gel analysis of spliceosomeassembly. Aliquots of the splicing reactions described above wereseparated under native conditions. The identity of splicing complexesis denoted with assembly occurring in the following order: H/E →A → B → C. C: Quantification of normalizedsplicing efficiency vs inhibitor concentration for the splicing reactionsshown in (A) and (B), respectively. D: Summary of splicing inhibition data. IC50 refersto the concentration required to reduce in vitro splicingefficiency by half compared to DMSO control.
Mentions: Thebiological properties of FR901464 (1), spliceostatinA (2), along with their six diasteromers (47–52) were evaluated in an in vitro splicing system (Figure 2).44 We added the compounds to splicing reactions containinga synthetic pre-mRNA substrate, ATP, and nuclear extract from HeLacells. Splicing chemistry was examined by denaturing PAGE to separatethe substrate and product mRNA, while splicing efficiency was quantifiedas the percent of pre-mRNA converted to mRNA. In this system, DMSOalone has no effect on splicing efficiency, while spliceostatin A(2) and FR901464 (1) both inhibit splicingwith an IC50 of 0.01 and 0.05 μM, respectively (Figure 2, A, C, and D). Surprisingly, compounds 47, 49, and 50 showed an approximately 100-foldreduction in potency relative to spliceostatin A, with IC50 values between 1 and 1.5 μM (Figure 2, A–D). Additionally, compounds 48, 51, and 52 were the least potent splicing inhibitors withIC50 between 10 and 35 μM.

Bottom Line: To construct the highly functionalized tetrahydropyran A-ring, we utilized CBS reduction, Achmatowicz rearrangement, Michael addition, and reductive amination as key steps.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Department of Medicinal Chemistry, Purdue University , 560 Oval Drive, West Lafayette, Indiana 4790, United States.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus