Current Literature. Development of Highly Potent and Selective Steroidal Inhibitors and Degraders of CDK8

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1 Current Literature Development of ighly Potent and Selective Steroidal Inhibitors and Degraders of CDK8 ACS Med. Chem. Lett. 2018, ASAP Rational Drug Development simplification Cortistatin A steps; % CDK8 Enzyme IC 50 : 15 nm J-VIII-49 8 steps; 33% CDK8 Enzyme IC 50 : 16 nm Evan Carder Wipf Group Current Literature May 12,

2 Cyclin-dependent kinase 8 (CDK8) Serine and threonine protein kinase Component of the mediator complex multiprotein assembly compromising up to 30 subunits that regulates cell cycle and gene transcription enhancer factor Genomic DA Demonstrates both positive and negative regulation of transcription Elevated expression is associated with enhanced mortality colorectal, breast, and ovarian cancer Mediator complex CDK8 Knock down of CDK8 in cancer cells reduced cell proliferation, induced cell cycle arrest, and promoted apoptosis RA pol II gene expression Inhibiting CDK8 may be a promising approach for cancer therapy. promoter gene Genome Biol. 2014, 15, 122. ature 2008,455,

3 Target modulation - where to begin? Small-molecule anticancer drugs ( ; n = 136) R Cortistatin A 12% 17% S 2 CF 3 9% S* 16 S S* Unaltered natural product semi or total synthetic Synthetic found by random screening Synthetic natural product pharmacophore J-VII-49 (this work) J. at. Prod. 2016, 79, 629; Bioorganic & Medicinal Chem. Lett. 2017, 27, 4488; ACS Med. Chem. 2018, ASAP 3

4 Cortistatin natural products R R R cortistatin C: R = cortistatin D: R = cortistatin E: R = S 1 cortistatin G: R = S 2 cortistatin : R = S 3 cortistatin K: R = S 4 cortistatin F: R = S 1 cortistatin J: R = S 4 R cortistatin A: R = cortistatin B: R = Corticium simplex cortistatin L S 1 S 2 S 3 S 4 J. Am. Chem. Soc. 2006, 128, 3148; Tetrahedron 2007, 63, 4074; Tetrahedron Lett. 2007, 48,

5 Cortistatin A Family of 11 steroidal alkaloid 9-(10,19)-abeo-androstane type igh affinity binder to CDK8 (Kd = 17 nm) Active against human umbilical vein endothelial cells (UVEC) Attractive lead compounds for drug discovery as an anti-angiogenic cancer chemotherapy 17 cortistatin A Semi, formal, and total synthesis: Baran et. al. J. Am. Chem. Soc. 2008, 130, Shair et. al. J. Am. Chem. Soc. 2008, 130, icolaou et. al. Angew. Chem. Int. Ed. 2008, 47, irama et. al. Tet. Lett. 2009, 50, Myers et. al. at. Chem. 2010, 2,

6 Baran et. al. semi-synthesis prednisone Cortistatin A Key step: TMS Br SmI 2, DMPU TF, rt; TBCD TMS Br [Br ] axial attack TMS Br SmI 2 -Br TMS SmI 2 J. Am. Chem. Soc. 2008, 130,

7 Shair et. al. synthesis ajos-parrish ketone Cortistatin A Key step: Ac Ac R TBS Me 2, ZnBr 2 MeC, 50 o C 65% (three steps) Ac Ac TBS Ac Me + Me Ac Me TBS R Aza-Prins/transannular cyclization J. Am. Chem. Soc. 2008, 130,

8 icolaou et. al. synthesis TBS Cortistatin A Key step: TBS K 2C 3, dioxane 125 o C, 52% TBS -[ 2 ] - TBS 1,4-addition / Adol / dehydration Angew. Chem. 2008, 120,

9 TBS cortistatin A steps; over yields: % Current Work Initiate a campaign to design simple, more easily prepared CDK8 inhibitors based on a steroid scaffold that would be more convenient for large-scale synthesis. 9

10 Structural Analysis Structure-activity relationship Cortistatin A-CDK8 co-crystal structure hydroxyls not required important required 18 C17 stereochemistry required C17 & C18 substitution not tolerated W D103 Y32 L158 A100 PDB: 4CRL Baran et. al. Angew. Chem. Int. Ed. 2009, 48, 4328; Corey et. al. J. Am. Chem. Soc. 2009, 131, 9014; icolaou et. al. J. Am. Chem. Soc. 2009, 131,

11 Alternative steroid scaffold Cortistatin A core DEA core Dehydroepiandosterone (DEA) Important endogenous steroid hormone Precursor in the synthesis of steroidal drugs; e.g. abiraterone acetate (Zytiga) treatment of CRPC Commercially available 100 g/$ (ArkPharm) functional handle functional handle DEA 11

12 Rational drug design Design of DEA derivative 3D structure overlay of cortistatin A and DEA derivative 12

13 Binding mode comparison Cortistatin A-CDK8 co-crystal structure W105 DEA derivative-cdk8 model D103 L A100 Y32 05/12/18 Evan Wipf Group 13

14 ypothesis The replacement cortistatin A s core with dehydroepiandrosterone s (DEA) carbocyclic framework will lead to a potent, selective, and scalable CDK8 inhibitor. Cortistatin A core DEA core 14

15 Representative synthesis DEA ethylene glycol, Ts PhMe, 100 o C, 98% DPPA, DEAD PPh 3, TF, 87% 3 P(Me) 3, 2 TF, 85% C, ab 3 C Ts KMDS, TF; 2 EtAc, Me, 92% acetone, 98% Ph(S 2 CF 3 ) 2, 95% Tf () 2 B Pd(Ph 3 ) 4, a 2 C 3 1,4-dioxane, 2 90 o C, 76% KC=CK Ac, DMS TF, 70% 15

16 R X X R CDK8 IC 50 (nm) X R CDK8 IC 50 (nm) 48 10, ,000 10,

17 R X X R CDK8 IC 50 (nm) X R CDK8 IC 50 (nm)

18 Kinase selectivity J-VIII um Kinase % Control at 10 um CDK CDK8 2.9 PIKFVE 8.5 EK1 8.7 RIK

19 Proteolysis Targeting Chimeras - PRTACs PI E3 Ligase PI Ligand E3 Ligase Ligand PRTAC Small-molecule-mediated protein degradation: Ub PRTAC Ub Ub E2 Ub Ub Ub PI PI E3 Ligase PI Proteasomal degradation atl. Acad, Sci. USA 2001, 98,

20 Design of PRTAC PRTAC Allosteric CDK8 ABL Ligand Linker E3 Ligase Ligand J-VIII-49 CDK8 Ligand ethylene glycol linker 2 Pomalidomide Cereblon (CRB) ligand 20

21 05/12/18 21 Evan Wipf Group R CDK8 IC 50 (nm) R /A R CDK8 IC 50 (nm) PRTAC

22 PRTAC biological evaluation n = 4 29 Jurkat cells 6 h 24 h 22

23 Summary cortistatin A steps; % CDK8 Enzyme IC 50 : 15 nm J-VIII-49 8 steps; 33% CDK8 Enzyme IC 50 : 16 nm Rationally designed and developed a potent and selective CDK8 kinase inhibitor 8 steps, 33% overall yield, and amenable for large-scale preparation Further developed a heterobifunctional CDK8 protein degrader Continued work: Demonstrate synthesis on large scale Expand biological evaluation in both in-vitro and in-vivo models 23