The Importance of Fluorine in the Design of BACE Inhibitors

1. KaoHsiung Medical School KaoHsiung, Taiwan December 14, 2009 The Importance of Fluorine in the Design of BACE Inhibitors James R. McCarthy

2. 14,000,000 12,000,000 10,000,000 8,000,000 NUMBER OF VICTIMS 6,000,000 4,000,000 2,000,000 0 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 YEARS Age 65-74 Years Age 75-84 Years Age 85+ Years Prevalence of Alzheimer’s Disease (AD)(By decades in U.S.A. from 1900-2050)

3. Contrast Between the Healthy Brain and the Advanced AD Brain Ventricle Hippocampus

4. Two main pathological hallmarks of AD: neuritic plaques and neurofibrillary tangles Neurtic plaques Neurofibrillary tangles Largely Ab peptide Hyperphosphorylated tau More specific to AD Found in many neurological disorders

5. BACE (b-SECRETASE) HYPOTHESIS Blocking the first cleavage of APP by b-secretase will limit the substrate for g-secretase and diminish Ab peptide production. sAPPb Amyloid Precursor Protein KPI OX NH2+ NH2+ b-amyloid (Ab38-49) (b-secretase) First step + CLEARANCE Diffuse Primitive (Immature) g-secretase COO- COO- Classic (Typical) C99 (b-CTF) Compact (Burnt-out) - Lilly Confidential BACE Clark and Trojanowski, 2000, Neurodegenerative Dementias, p151.

6. Common isosteres that mimic the tetrahedral intermediate of amide bond hydrolysis as catalyzed by aspartyl proteases (TSAs): N-terminal Central core C-terminal P1’ P2 P1 P3 P2’ Ethanolamines Hydroxyethylenes BACE (b-APP Cleaving Enzyme) is a membrane bound aspartyl protease Aspartyl protease mechanism of action

7. BACE1, an Aspartyl Protease, co-crystallizes with a substrate mimetic inhibitor OM99-2 (Tang, Hong et. al., Science, 2000, 290, 150-153) The active site of BACE is defined by OM99-2, containing the hydroxyethylene core (Leu*Ala) Ki= 1.6 nM

8. OM99-2/BACE1 Interactions (Ki = 1.6 nM)

9. Hippocampus Vehicle 0.5 treated 0.4 67% 67% 60% 60% 0.3 *** *** Hippocampus Ab (x-40) (ng/gm) ** ** 0.2 0.1 0.0 mdr_KO PO mdr_KO SQ A Novel Sulfone That Is Efficacious in Pgp Transporter Deficient Mice mcaFRET IC50 = 0.4 nM Ab IC50 = 1 nM Mouse microsome metabolism = 90% Rat microsome metabolisn = 87% Caco2 = 3%

10. Polar P2 Binding Groups Give Potent Inhibitors That Do Not Penetrate the Brain of Wild Type Mice

11. Physical properties of BACE inhibitors are key for brain penetration For a reasonable chance of compound crossing Blood-Brain Barrier (BBB), following physical properties are important: • cLog P > 2 and < 5 • PSA < 85 Å2 • MW < 500 • Caco-2 > 200 nM/sec • cpKa < 8 Van der Waterbeemd, H.; Camenisch, G.; Folkers, G.; Chretien, J.R.; Raevsky, O.A. Estimation of blood-brain barrier crossing of drugs Using molecular size and shape and H-bonding descriptors. J. Drug Targeting, 1998, 6(2), 151-165 Kelder, J.; Grootenhuis, P.D.J.; Bayada, D.M.; Delbressine, L.P.C.; Ploemen, J.-P. Polar molecular surface as a dominating determinant For oral absorption and brain penetration of drugs. Pharm. Res., 1999, 16(10), 1514-1519

12. Designing Efficacious BACE Inhibitors • Which pockets are important to occupy? • Utilize directed SAR to define binding opportunities.

13. Strategy: Design BACE inhibitors with clogP > 2 and < 5 PSA < 85 MW < 500 cpKa < 8 • Replace polar N-terminal groups with smaller groups • Retain potency through novel C-terminal rings to increase • rigidity R = Me BACE1 IC50 = 80 mM BACE1 IC50 > 100 mM BACE1 IC50=2 nM

14. Difluorophenyl at P1 is 5 times more potent vs. phenyl Increase in potency consistent with van der Waals Forces between the fluorines and the surface of the protein. (Will be illustrated with a crystal structure)

15. BACE1 IC50 = 20 nM 9.2 Hz 9.5Hz 1.8Hz 8 9 7 6 unsubstituted 9.2 Hz 6.5Hz 3.05 Hz 7 9 8 6 Extreme coupling constants for compound 1 indicate a more rigid solution structure 7 8 1 BACE1 IC50 = 926 nM 9 2

16. Cyclization Strategies BACE1 IC50 = 20 nM • 5 and 6-membered rings exhibit optimal binding to BACE1 (by DOCKing experiments) BACE1 IC50 = 0.35nM Ab IC50 = 5.7nM

17. Elimination of polar group at P2 & decrease in MW have a small impact on BBB penetration P2 P2 P2’ P3 P1 P3 P1 P1 2 3 1 BACE1 IC50 = 0.4 nM Ab IC50 = 5.7 nM B/P = 0.02 MW = 553 PSA = 132 BACE1 IC50 = 0.5 nM Ab IC50 = 1.5 nM B/P = 0.02 MW = 580 PSA = 127 BACE1 IC50 =80000 nM B/P = 0.06 MW = 312 PSA = 61 Hypothesis: Basicity of nitrogen on compound 3 (cpKa = 9.5) hinders blood brain barrier penetration

18. Optimization strategy • Di-axial groups are required to fill S1’ and S2’ pockets • Addition of hydrophobic P1’ & P2’ groups led to the design of smaller less polar inhibitors S1’ pocket BACE1 IC50 = 0.35 nM S2’ pocket

19. Introduce P2’ group to improve potency Both forms exist in solution

20. Enhance inhibitor preorganization and rigidity via stereoelectronic effects

21. Synthesis of 3-Alkyl-2-Alkoxy morpholine BACE inhibitors X-ray crystal structure unequivocally established the stereochemistry at all three chiral centers

22. Synthesis of 3-Alkyl-2-Alkoxy Morpholines (cont)

23. Alternative Synthesis of 3-Alkyl-2-Alkoxy morpholine BACE inhibitors

24. BACE IC50 = 1320 nM BACE IC50 = 222 nM BACE IC50 = 306 nM B/P 0.07 (Brain to Plasma exposure ratio) B/P 0.42 B/P 0.48 Progression of the SAR: P2’/P1’ on morpholine 2434074 BACE IC50 = 77 nM Ab IC50 = 91 nM MW =414 BACE IC50 = 2800 nM Ab IC50 = 2160 nM B/P 0.38 Morpholine ring decreased pKa & provided anomeric effect Introduction of P1’ methyl increased potency

25. P1’ axial P2’ cLogP = 2.4 PSA = 70 A2 MW = 414 cpKa = 7 Caco-2 perm ~ 300 nM/sec Anomeric effect favors axial P2’ O-neopentyl 2434074 exhibits desired physical properties for a CNS drug o

26. Pharmacokinetics of 2434074 in wild type mice shows minimal exposure after oral administration (10 mg/kg) s.c 2434074

27. rat A P+O B O-dealkylated B A control parent Rat hepatocyte metabolism for 2434074 • Metabolism predominantly on P2’ neopentyl group

28. Approach to decrease metabolism at P2’ • Mouse hepatic microsomal experiments show that most metabolism • is concentrated on the P2’ position Introduction of fluorine at P2’

29. Fluorinated alcohols designed for attachment at the P2’ site on the morpholine inhibitors

30. Routes to Fluoro-Neopentyl Alcohols Represented by the Syntheses of 3-Fluoro-2-fluoromethyl-2-methyl-propan-1-ol (3)

31. Synthesis of (R) and (S)-3,3-difluorocyclohexanemethanol

32. Representative synthesis of P2’ fluorinated neopentyl BACE inhibitor

33. Approach to decrease metabolism at P2’ 2434074 The bis (difluoroMe) analog of 2434074 maintains potency with decreased metabolism

34. Approach to decrease metabolism at P2’ • Introduction of oxygen or fluorine on cyclic P2´substituents lead to decreased metabolism but also decreased activity

35. S2’ Iterative Docking and X-ray Studies for introduction of fluorine on P2’ Cyclohexyl • SAR showed polar groups not tolerated in S2’ pocket • Docking suggested 3-cyclohexyl position may allow addition of metabolic stabilizing difluoro substitution • Subsequent x-ray structure (yellow) verified this finding {

36. Significant difference in activity of the two enantiomers mcaFRET IC50 = 45 nM Ab IC50 = 67 nM Rat surrogate metabolism: 49% PSA = 72.9 cLogP = 2.6 MW = 475 Caco-2 = 24.2 mcaFRET IC50 = 280 nM Ab IC50 = 473 nM Rat surrogate metabolism: 50%

37. X-ray crystal structure of 1 co-crystallized with BACE 1

38. X-ray crystal structure of 1 co-crystallized with BACE 1

39. Summary • Structure-based drug design was utilized to obtain drugable BACE • inhibitors • The importance of fluorine in the design of the BACE inhibitors was • shown to be two-fold: • - Fluorines substituted on the meta-positions of P1 phenyl group resulted • in an increase in potency via van der Waals forces between the surface • of the protein and the fluorines. • - The addition of the aliphatic fluorines on the 3-position of the P2’ • cyclohexyl group resulted in decreased metabolism. The crystal • structure shows that the fluorines are situated at the edge of the • pocket and are not interfering with interactions of the cyclohexyl group • with amino acids in the S2’ pocket.

40. Neuroscience Yuan Su Binhui Ni Len Boggs Zhixiang Yang Patty Gonzalez-Dewhitt Patrick May Bruce Gitter Dan Czilli Sheila Little Ed Johnstone Ginnie Yang Jiangqing Huang Xiyun Chai Tingui Yin Yuan Tu Kelly Bales Steven Paul Suizhen Lin Carrie Jones Beth Hoffman Lijun Yang Harlan Shannon Chemistry Dave Bender Patrick Hahn Todd Kohn Dawn Brooks Chris Rito Allie Tripp Shui-Hui Chen Yvonne Yip Deqi Guo Jason Lamar Jingdan Hu Cindy Cwi Dave Timm Jim Copp Michael Wiley Timothy Shepherd Rob Dally Bob Murff Dave Mitchell Mike Shapiro Teddy Zartler Richard Harper Fuyao Zhang Fred Bruns Jon Erickson Kenn Henry Jayana Lineswala Brian Warson Scott Sheehan Tim Durham Jim Toth Chemistry Howard Broughton Jose Alfredo Martin Alicia Marcos Isabel Rojo Charo Gonzalez Ana Belen Bueno Fatima Iradier Alicia Torrado Javier Agejas Gema Sanz ADME/Toxicology /Biopharm Terry Lindstrom Mike Clay Kate Hillgren Vasu Vasudevan Tim Ryan Yu-hua Hui Helen Huang Tom Raub Statistician Viswanath Devanarayan DCSG José Francisco Soriano Juan Ramón Rodríguez José Miguel Mínguez (MPC) Chin Liu Gema Ruano PPM Stan Sorgen Scott Lindsey Medical Eric Siemers AT Juan F. Espinosa Paloma Vidal Maria Luz de la Puente Alfonso Espada Amelia González Pilar López Leticia Cano Aranzazu Marín Cristina Anta María Sánchez Manuel Molina Enabling Biology Ami Boodhoo Don McClure Aimin Lin Joseph Brunson Haihong Guo LOB Rick Brier Deb Laigle Jim McGee Raquel Torres Enrique Jambrina Juan Valesco Legal Janet Gongola Robert Titus Nelsen Lentz Charles Cohen Lynn Gossett