Spirocycles and the Development of a MDM2...
Transcript of Spirocycles and the Development of a MDM2...
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Spirocycles and the Development
of a MDM2 antagonist
1
Presenter: Brent Billett
Advisor: Prof. Guangbin Dong
University of Texas at Austin
March 2, 2016
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Outline
• Introduction
• Examples of spirocycle formation/synthesis
• Examples of thiazolidine formation/synthesis; a reoccurring and potent moiety
• Biological Aside– western blot, cell cycle, and P53-MDM2 complex
• Specific thiazolidine drug example
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Spirocycle Examples
cyclopropanes oxiranes oxetanes
spiroazetidinesspirooxindol spirooxindol-3,3’-
thiazolidine
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Spirocycle features
• Rigidify ligand conformation upon ligation to target
• Conformation restriction within molecule• Ligand binding entropy• Greater 3-dimensionality
– more physical properties when compared to planar/aromatic derivatives
• Novelty for patentability
Zheng, Y.; Tice, C. M.; Singh, S. B. Bioorg. Med. Chem. Lett. 2014, 24, 3673.
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Spirocycle feature:
constraint/rigidity
1a is twice as potent and more selective than early lead
1b due to 3 membered spiro constraint.
Zheng, Y.; Tice, C. M.; Singh, S. B. Bioorg. Med. Chem. Lett. 2014, 24, 3673.
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Synthesis of spirocycles
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
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NHC-catalyzed
cycloaddition/Annulation
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Wang, X.; Zhang, Y.; Ye, S. Adv. Synth. Catal. 2010, 352, 1892
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NHC-Catalyzed [3+2]alcohol directed
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Sun, L. H.; Shen, L.T.; Ye, S. Chem. Commun. 2011, 47, 10136
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Cooperative NHC/Lewis Acid
Strategy
Question: Please provide a stereochemical model for the observed
stereochemistry in the NHC/Lewis Acid strategy case. (the conversion of
benzoquinone 38 to spirolactone 47)
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Scheidt, K.A. Chem. Sci. 2012, 3, 53 and Angew. Chem. Int. Ed. 2012, 51, 4963
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Michael Reaction Strategycomplementary spirolactone formation
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Bergonzini, G.; Melchiorre, P. Angew. Chem. 2012, 51, 995
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Zn-ProPhenol Spirolactone Synthesis
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Trost, B. M.: Hirano, K. Org. Lett. 2012, 14, 2446
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Zn-ProPhenol Spirolactone Synthesis
racemic racemic 11% ee
Trost, B. M.: Hirano, K. Org. Lett. 2012, 14, 2446
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Asymmetric [3+2] allylsilane annulation
Takes advantage of
the inherent
enantioselectivity of
allylsilanes
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Franz, A.K. Angew. Chem. Int. Ed. 2012, 51, 989
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Pd catalyzed spirocyclic-pentanes
Using achiral ligand
affords 2:1 trans/cis
diastereomers
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Trost, B. M.; J. Am. Chem Soc. 2007, 129, 12396
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Wall and Flap Model
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Trost, B. M.; Angew. Chem., Int. Ed. 2011, 50, 6167
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Trienamine/DA approach
“remarkably broad strategy for the enantioselective
synthesis of complex spirocycles”
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
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Trienamine/DA approach Strategy
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Jorgensen, K. A.; J. Am. Chem. Soc. 2011, 133, 5053
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Ring Expansion Approach
Franz, A. K.; Hanhan, N. V.; Ball-Jones, N.R. ACS Catal. 2013, 3, 540
Chai, Z.; Rainey, T.J. J. Am. Chem. Soc. 2012, 134, 3615
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Proposed reaction mechanism
Chai, Z.; Rainey, T.J. J. Am. Chem. Soc. 2012, 134, 3615
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Thiazolidines• Indoles and its analogous (such as isatin) are
good pharmacophores for designing chemotherapeutic
• Spiro[indole-thiazolidines] have broad spectrum of pharmacological properties
R. Sakhuja et al. Bioorg. Med. Chem. Lett. 2011, 21, 5465
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Example synthesis of a thiazolidine
R. Sakhuja et al. Bioorg. Med. Chem. Lett. 2011, 21, 5465
racemate of one
ntantiomeric pair
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Another example of thiazolidine
(a) NaH, DMF, 0oC then ArCH2Br or Alk-X; (b) (1) Ar’NH2, EtOH, reflux, 6 h; (2) mercaptoacetic
(aka thioglycolic acid) acid, toluene, reflux, 16 h; (c) mCPBA (5 equiv), CHCl3, rt, 24h (d)
mCPBA (1.1 equiv), CHCl3, 0oC, 1 h
V.V. Vintonyak et al. Tetrahedron 2011, 67, 6713
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Thiazolidine synthesis w/ sulfactant
R. Jain et al. Tetrahedron Letters 2012, 53, 6236
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Biological AsideBefore specific case study
“I try to show the public that chemistry, biology, physics, astrophysics is life. It is not some separate subject that you have to be pulled into a corner to be taught about.” -Neil deGrasse Tyson
• 1) cell cycle
• 2) p53 and MDM2
• 3) Western Blot
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Cell Cycle
Phase Abbreviation Description
Interphase I Includes Go, S, and G1
Gap 1 G1 cell growth, protein and organelles
accumulation
Synthesis S DNAreplication
Gap 2 G2 Cell growth
Mitosis M Cell division
Gap 0 G0 Quiescent/senescent
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P53-MDM2
Crystal structure of the p53-binding domain of MDM2
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Western Blotting1) Cell lysis and separation to extract protein
2) Gel Electrophoresis
- Charged molecules are separated according to physical properties
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Transfer Method: Blotting
3) Blotting• proteins are transferred to a membrane via
electroelution. • Nonspecific sites of the remaining membrane
surface are blocked• Proteins are labeled for detection
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Specific Case StudyDevelopment of a MDM2 antagonist
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
1
Inhibits 30% of p53-MDM2 interaction
by mimicking p53 residues Phe19 and
Trp23 that bind to MDM2
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Outline of MDM2 antagonist Discovery
Structural-based design
Library construction
and in vitro
cytotoxicity
(paper 1) Library construction and
in vitro cytotoxicity
(paper 2)
1 2
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
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Synthesis of derivatives
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
Cysteine ethyl ester
(i) Cys-OEt, NaHCO3 in MeOH, MW; (ii) triphosgene TEA, THF, rt, 10 min, then R2-
NH2; (iii) MeOH, TEA, reflux, 1-3 hr
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Cytotoxic activity of derivatives
Cell Lines: HEK, transformed human embryonic kidney cell line; M14, human
melanoma cell line; U937, Human leukemia monocyte lymphoma
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
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Cytotoxic activity of derivatives
Cell Lines: HEK, transformed human embryonic kidney cell line; M14, human
melanoma cell line; U937, Human leukemia monocyte lymphoma
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
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Hit compounds 9c and 10d
To undergo additional in vitro
testing and derivatization
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
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Derivatives 9c and 10d exhibit
good cell selectivity a) Human papillary
thyroid carcinoma
TPC1
b) Normal thyroid TAD-2
cell
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
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Derivatives of 9c and 10d
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
acylated series
less potent
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Video Time Lapse Microscopy
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
treated cells experienced reduced cell division, but
what was the cause of the reduced cell division?
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Determining mode of Cytotoxicity
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
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Determining mode of Cytotoxicity
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
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Both treated cells exhibit
and increase in p53
concentration
Determining mode of Cytotoxicity
a) p53-MDM2 complex and b)
9c c) 10d d) nutlin-3 treated
Gomez-Monterrey. et al. J. Med. Chem., 2010, 53, 8319
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Further Development of 9c
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Derivatization
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Synthesis of Series 4
What about stereoselectivity? => lets look at a similar system
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
(i) Cys-OEt, NaHCO3 in EtOH; (ii) R2-COCl, TEA, THF, 2 h, rt
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Facile Ring Opening of Spirooxindolesa look at a similar system
Yujun Zhao et al. J. Am. Chem. Soc. 2013, 135, 7223 and J. Med. Chem. 2013, 56, 5553
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Stable cis-cis isomer
Open ring intermediate
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Isomerization Mechanisma look at a similar system
Yujun Zhao et al. J. Am. Chem. Soc. 2013, 135, 7223 and J. Med. Chem. 2013, 56, 5553
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Stereochemistry
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
Question: Please provide a mechanistic rational for the
observed isomerization.
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Synthesis of Series 5
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
(i) 4-Cl-C6H4COCl, TEA, THF, 2h, rt; (ii) Cys-OEt, NaHCO3, EtOH
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Biological effects
MCF-7: Human breast adenocarcinoma cell. HT29: Human colon carcinoma cell
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Biological effects
MCF-7: Human breast adenocarcinoma cell. HT29: Human colon carcinoma cell
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Hit compound 4n
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Binding Model
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Determination of p53 levels
Quantification of immunoblotsWestern blot
Total cell lysates
After
immunoprecipitation
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Cell cycle progression
a) 24 hr b) 48hrs c)72 hrs d) 24 hr and varying concentration
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Apoptotic cell death
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Mechanism of apoptosis
Bertamino, A. et al. J. Med. Chem., 2013, 56, 5407
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Summary• Examples of spirocycle formation/synthesis
• Examples of thiazolidine formation/synthesis; a reoccurring and potent moiety
• Biological Aside– western blot, cell cycle, and P53-MDM2 complex
• Specific thiazolidine drug example– 4n high efficiency in breast, colon, lung, and leukemia
cancer cell lines
– Docking studies allowed to predict bind mode
– 4n induces apoptosis
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THANK YOU
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Question 1
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Question 2: Western Blotting1) Cell lysis and separation to extract protein
2) Gel Electrophoresis
- Charged molecules are separated according to physical properties
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Question 2: Transfer Method: Blotting
3) Blotting• proteins are transferred to a membrane via
electroelution. • Nonspecific sites of the remaining membrane
surface are blocked• Proteins are labeled for detection
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Question 3
“Base-catalyzed
isomerization of
thiazolidines through
Schiff Base
intermediates (I) is a
well-known process”
Trost, B.M. et al. Angew. Chem. Int. Ed., 2011, 50, 6167
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Wall and Flap Model
Trost, B.M. et al. Angew. Chem. Int. Ed., 2011, 50, 6167