Total Synthesis of Salinosporamide A

Total Synthesis of Salinosporamide A

H O NH OH O Me O Cl

Salinosporamide A

Joshua Pierce Wipf Group January 21, 2006

Endo, A.; Danishefshy, S. J. JACS 2005, 127, 8298.

Josh Pierce @ Wipf Group 1 1/22/2006 Isolation

HO2C NHAc H O O NH NH O S NH OH OH O Me O O O Me Me O Cl OH OH

Salinosporamide A Omuralide Lactacystin

2,500 strains of bacteria belonging to a new taxon, which has been given the genus name Salinospora, have been isolated from tropical or subtropical ocean systems. Preliminary screening of the organic extracts from cultured Salinospora strains showed antibiotic and anticancer activity, causing an increased interest in exploiting these bacteria for drug discovery.

Salinosporamide was isolated by Fenical and co-workers from the strain CNB-392 and showed potent cancer cell cytotoxicity, apparently through its inhibition of the 20S proteasome.

Salinosporamide shares its core structure with Omuralide, a transformation product of the microbial metabolite Lactacystin originally isolated by Omura and co-workers from terrestrial Streptomyces. In early biological testing it has shown to be significantly more active than either Lactacystin or Omuralide.

Fenical et. al. ACIE, 2003, 42, 355. Omura et. al. J. Antibiot. 1991, 44, 113.

Josh Pierce @ Wipf Group 2 1/22/2006 Proteasome Function

Nobel Prize in Chemistry 2004: Aaron Ciechanover, Avram Hershko and Irwin Rose

The proteins that are to be destructed are marked with a label - the molecule ubiquitin. Proteins so labelled are then rapidly broken down in cellular "waste disposers”called proteasomes.

1.The E1 enzyme activates the ubiquitin molecule. This reaction requires energy in the form of ATP.

2.The ubiquitin molecule is transferred to a different enzyme, E2.

3.The E3 enzyme can recognise the protein target which is to be destroyed. The E2-ubiquitin complex binds so near to the protein target that the actual ubiquitin label can be transferred from E2 to the target.

4.The E3 enzyme now releases the ubiquitin-labelled protein.

5.This last step is repeated until the protein has a short chain of ubiquitin molecules attached to itself.

6.This ubiquitin chain is recognised in the opening of the proteasome. The ubiquitin label is disconnected and the protein is admitted and chopped into small pieces.

http://nobelprize.org/chemistry/laureates/2004/public.html

Josh Pierce @ Wipf Group 3 1/22/2006 Current Proteasome Inhibitors

O O O OH H H N H N N B O N N N OH H H H O O O N

Peptide aldehydes, such as Z-Leu-Leu-Leu-H, inhibit Bortezomib (Velcade) is a peptide boronate that is proteasome activity in a potent but reversible manner. the first proteasome inhibitor to be marketed for the treatment of cancer (multiple myeloma). Produced by Millennium Pharmaceuticals. IC50 ~ 0.03 nM

O N HO2C NHAc HO H N N O H O O S O NH HN O O Me OH OH O

Epoxomicin is a cell permeable, potent, and selective Lactacystin is a natural, irreversible, nonpeptide, cell proteasome inhibitor, more potent than lactacystin. permeable inhibitor that is more selective than peptide aldehydes but less selective than peptide boronates.

Josh Pierce @ Wipf Group 4 1/22/2006 Proteasome Inhibition by !-lactone Inhibitors

26S Proteasome Structure

Lactacystin bound to terminal active site threonine. 20S Core (!5 subunit is the target for !-lactone class of inhibitors) Huber et. al. Nature, 1997, 386, 463. Josh Pierce @ Wipf Group 5 1/22/2006 Initial Synthesis and SAR Studies

First total synthesis of Lactacystin was completed by Corey et. al.. Through this work, as well as subsequent improvements by both the Corey group and groups such as Omura-Smith, Baldwin and Chida allowed for the rapid synthesis of both Lactacystin and Omuralide - while allowing for a variety of analogue projects to study SAR for this class of compounds.

First enantioselective synthesis of Lactacystin by Corey in 1998:

HO2C NHAc Procine liver MeS CO2Me MeS CO2H esterase O S 17 linear steps, Me CO Me NH Me CO2Me 2 O 13.4% yield 62%, 95% ee Me OH OH Corey, E. J.; Li, W.; Nagamitsu, T. ACIE, 1998, 37, 1676.

SAR Studies: Replaceable with Et, i-Pr, n-Bu, Bn but NOT H O Me Essential NH O H Me HO NH 20S Proteasome O OH H O OH Me O H Essential Replaceable with Me O H N 2 CONH(!-subunit) Essential Li, W.; Corey, E. J. Chem. Pharm. Bull. 1999, 47, 1.

Josh Pierce @ Wipf Group 6 1/22/2006 Corey’s Approach to Salinosporamide A MeO CO Me H CO Me 2 N 2 LDA, THF-HMPA N CO2Me p-TsOH N OBn MeO MeO Me O 80% O ClCH2OBn 69% O Me HO Me

MeO PMB O CO2Me NaCNBH3 1. TMSCl N Dess-Martin CO2Me OBn AcOH 90% HN 2. Acrylyl Chloride 96% OBn + HO Me i-Pr2NEt then H HO Me 96%

PMB PMB PMB O CO2Me N 1. Quinuclidine 90% O Bu3SnH, AIBN N CO2Me O N CO2Me OBn 2. BrCH Si(CH ) Cl OBn OBn 89% 2 3 2 + O Me Et N, DMAP 95% OH Me 3 Me O (1:9) Si Br

PMB PMB N CO2Me N CO2Me O 1. H , Pd/C 95% O OBn 2 CHO H Me H Me O 2. Dess-Martin 95% O Si Si Me Me Me Me Reddy, L. R.; Saravanan, P.; Corey, E. J JACS, 2004, 126, 6230. Josh Pierce @ Wipf Group 7 1/22/2006 Completion of the Synthesis

ZnCl H H PMB PMB N CO2Me 1. KF, KHCO3 H O O N OH O N OH CHO H2O2 92% CO2Me H Me H CO2Me Me H O 88%, 20:1 dr 2. CAN 83% Me O OH Si Si Me Me Me Me HO

H 1. 3N LiOH-THF O 2. BOPCl, Py NH OH 3. Ph3PCl2, MeCN O 65% (three steps) Me O Cl

17 linear steps, 13.4% yield

Josh Pierce @ Wipf Group 8 1/22/2006 2nd Generation Approach

PMB PMB PMB 1. Ti(Oi-Pr)4, c-C5H9MgCl O N CO2Me O N CO Me O CO2Me t-BuOMe, 30 min 2 N OBn OBn OBn 2. I2, 4 h Me Me HO HO O Me 3. NEt3, 30 min 83% yield > 99:1 dr

PMB 1. [MeTeAlMe2], 12 h O NH O N CO Me 2. Ph3PCl2, Py 89% 2 OH 20 steps, O 3. NEt3•3HF 92% 17.1% yield ODMIPS Me O HO Me Cl HO

Reddy, L. J.; Fournier, J.-F.; Subba Reddy, B. V.; Precedent for Cyclization: Corey, E.J. OL, 2005, 13, 2699. O X Me O Me Cp2Ti(PMe3)2 Cp2Ti - 2 PMe3 X

Me OSi(H)Ph2 Me + 2 PMe3 X H Me Cp Ti Me OSi(H)Ph2 2 O Cp Ti X Ph SiH 2 X 2 2 H Kablaoui, N. M.; Buchwald, S. L. JACS, 1996, 118, 3182.

Josh Pierce @ Wipf Group 9 1/22/2006 Danishefsky’s Approach

Ph Ph Ph 1. Vinylmagnesium Bromide, TMSCl, O O O CuI 75% O N O3, then NaBH4 O N O N 2. LDA 77%, 14:1 dr 86% I OH OBn BnO BnO

H OH 1. Jones Oxidation 1. ClCO2Et, Py O N EtO N CO t-Bu 96% 2. Me2NCH(Ot-Bu)2 2

72% (over 2 steps) 2. TfOH quant OCO2Et OCO2Et 3. Et3OBF4, K2CO3 88% BnO BnO

PMB CO2t-Bu CO2t-Bu EtO N O 1. 1 M HCl 90% O N O LHMDS 82% 2. PMBCl, NaH 60% O O 3. Pd(OH)2-C, H2 quant

BnO HO

PMB 1. PhSeSePh, NaBH4 EtOH O N CO2Bn

CO2t-Bu 2. BnBr, K2CO3 65% (over 2 steps) SePh

HO

Endo, A.; Danishefshy, S. J. JACS 2005, 127, 8298.

Josh Pierce @ Wipf Group 10 1/22/2006 Key Reaction and Completion of the Synthesis

PMB PMB PMB 1. H2O2 1. PhSeBr, AgBF4 O N CO2Bn O N CO Bn O N CO Bn 2. toluene (reflux) 2 BnOH 74% 2 CO2t-Bu CO2t-Bu CO2t-Bu 94% (over 2 steps) SePh (12:1 mixture) 3. Dess-Martin O SePh HO O BnO

ZnCl H PMB 1. n-Bu SnH, AIBN PMB 3 O N CHO 98% O N OH 1. CAN 90% CO2t-Bu CO2t-Bu Me 88% 20:1 dr 2. Na/NH3 2. NaBH4 85% Me O 3. NaBH 97% 3. Dess-Martin 95% O 4 (over 2 steps) BnO BnO

H H H 1. BCl3 O N OH 2. BOPCl, TEA O NH CO2t-Bu 3. Ph PCl , Py OH OH 3 2 O Me 51% (over 3 steps) Me O HO Cl

28 linear steps, 1.8% yield

Josh Pierce @ Wipf Group 11 1/22/2006 Inherent Problems - Future Directions

While !-lactone inhibitors are selective and potent, their half-life in solution at pH 7 is estimated at 5 - 10 min. This, along with other factors, has led to lackluster in-vivo activity. To circumvent this problem, Corey et. al. has synthesized a !-lactam that greatly increases the aqueous stability while maintaining considerable proteasome inhibition (although at a much slower rate).

O NH OH Hogan, P. C.; Corey, E. J. JACS, 2005, 127, 15386. O N Cl H

Although the Nobel Prize in Chemistry in 2004 emphasized the importance of the proteasome pathway, the development of novel treatments that target this pathway have been lagging. While Velcade has shown promise, its toxicity has been a major drawback. Even though proteasome inhibitors of this type are very selective for the proteasome over other cellular targets, their toxicity stems from complete shutdown of the cells waste disposal system.

Since their discovery, E3 ubiqutin ligases have been viewed as ideal drug targets. “Because each E3 is responsible for the destruction of a small number of proteins, specific inhibitors or E3s should be highly effective drugs with few side effects.” -- Dr. J. Wade Harper, Harvard University, 2001

The success of this strategy lies in the ability to interrupt protein-protein interactions with small molecules - something that most companies are not aggressively pursuing. (Roche’s Nutlins are key exception)

Garber, K. J. Nat. Cancer Inst. 2005, 97, 166. Adams, J. DDT, 2003, 8, 307.

Josh Pierce @ Wipf Group 12 1/22/2006