1. BACE-1 Inhibitors Alzheimer’s Disease Application of Fragment-Based NMR Screening, X-ray Crystallography, Structure-Based Design, and Focused Chemical Library Design to Identify Novel μM Leads for the Development of nM BACE-1 ( β-Site APP Cleaving Enzyme 1) Inhibitors: JMC 2010 (Schering-Plough) Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010 (Evotec) Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I;Inhibitor Design and Validation: JMC 2010 (Schering-Plough) Design and Synthesis of 5,5’-Disubstituted Aminohydantoins as Potent and Selective Human β-Secretase (BACE1) Inhibitors: JMC 2010 (Wyeth) 4/13/111CHEM E-120

2. Amyloid Plaque Formation 2 Insoluble, neurotoxic, protein clusters formed from Amyloid Precursor Protein (APP) 770 AAAA669 Asp BACE β-secretase AA669 AA770 Β-CFT γ-secretase Aβ – β-amyloid 1-40(42) AChE fibril peripheral site of AChE N terminus extracellular C terminus intracellular 4/13/11CHEM E-120

3. BACE β-secretase Transmembrane protein : NH 2 -terminal protease domain, connecting region, transmembrane region, cytosol domain Aspartic acid protease with a catalytic diad of 2 aspartic acid amino acids BACE-1 cleaves APP in the interior of the cell at a pH of ~ 5 BACE patents filed 1997 – 2010 (C&EN 2010, April 5) Amgen5 BMS25 Elan65 Eli Lilly3 GSK15 Merck45 Schering-Plough25 Pfizer50 Wyeth18 4/13/113CHEM E-120

4. BACE β-secretase Science 2000, 290, 150 OM-99-2 4/13/114CHEM E-120

5. BACE β-secretase Science 2000, 290, 150 OM-99-2 4/13/115CHEM E-120

6. BACE β-secretase Application of Fragment-Based NMR Screening, X-ray Crystallography, Structure-Based Design, and Focused Chemical Library Design to Identify Novel μM Leads for the Development of nM BACE-1 ( β-Site APP Cleaving Enzyme 1) Inhibitors JMC 2010, 53, 942-950 from Schering-Plough Strategy: Discover low molecular weight compounds by Nuclear Magnetic Resonance – binding affinity Validate hits by X-ray cystallography Determine IC 50 of enzyme inhibition Optimize hits by small focused libraries 4/13/116CHEM E-120

7. 7 Fragment-Based Screening Discover of a “lead” compound – a compound that binds to the target, has functional activity, and possesses “drug-like” properties. Modern methods: 1. High-throughput screening of large numbers of compounds: 10,000’s to 100,00’s 2. Fragment-Based lead discovery: screen “components” of drugs, <5000 compounds Screen small compounds of molecular weights 120-300 Rule of 3mw < 300HBA < 3rotatable bonds < 3 HBD < 3clogP < 3 Thought is these compounds are more likely to fit into a binding site, the binding affinity is usually quite low – K D of 100 μM to 10 mM “hit-rate” of 2-10% Target structure known either by X-ray crystallography or NMR Start to combine this fragment with others to fit better into the binding site with greater affinity. Structure-guided modification of the lead fragment. 4/13/11CHEM E-120

8. 8 Fragment-Based Screening Chemical Fragments that Hydrogen Bond to Asp, Glu, Arg, and His Side Chains in Protein Binding Sites: J Med. Chem 2010, 533086 4/13/11CHEM E-120

9. 9 Fragment-Based Screening 4/13/11CHEM E-120

10. 10 Fragment-Based NMR Screening 2D 15 N-HSQC NMR Measure the changes in chemical shifts of 15 N-labled BACE-1 catalytic domain in the presence of a fragment. Express BACE-1 with 15 NH 4 Cl as sole nitrogen source Purify Assign resonances - Identify the amino acids in the catalytic domain in the NMR spectrum Crystal structure helps Add a mixture of 10 compounds and measure the spectrum, compare with spectrum of the active site without compounds Hit – measure each compound separately to identify hit Quantify by K d 1/τ = k off + k on [S] y = b + mx k obs ( s -1 ) [S] k off (K d ) Δδ (ppm) vs [drug] 4/13/11CHEM E-120

11. 11 Heteronuclear Two-Dimensional Coupling HMQC (heteronuclear multiple quantum correlation) HSQC (heteronuclear single quantum correlation) Correlation between the carbon atoms and the hydrogens attached DIRECTLY to the carbon, 1 H vs 13 C 4/13/11CHEM E-120

12. 12 Fragment-Based NMR Screening 4/13/11CHEM E-120

13. 13 Fragment-Based NMR Screening Screened 10,000 compound fragment library, ~ 50% obeyed Rule-of-3 4/13/11CHEM E-120

14. 14 Fragment-Based NMR Screening Selected 204 isothioureas from fragment library and IC 50 for BACE-1 inhibition measured 15 compounds active at 50 μg/mL which where characterized by NMR to generate 3 36 fold increase binding affinity got X-Ray structure Ligand Efficiency (LE) free binding energy per non-hydrogen atom (kcal/mole/heavy atom) LE > 0.3 4/13/11CHEM E-120

15. Fragment-Based NMR Screening 4/13/1115CHEM E-120

16. 16 Fragment-Based NMR Screening Isothioureas will have hydrolytic stability problems - bioisosters 4/13/11CHEM E-120

17. 17 Fragment-Based NMR Screening Analyzed 32 2-aminopyridines by NMR giving compound 4 K d = 32 μM LE = 0.38 similar to 3 but no inhibition of BACE-1 up to 1 mM but pK a ~ 7.2 good for BBB penetration 3 4/13/11CHEM E-120

18. Hit-to-Lead Optimization Small-focused libraries by parallel synthesis step e most likely 4/13/1118CHEM E-120

19. Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010, 20, 5329 (Evotec) Screened a 20,000 compound fragment library, average mw = 250 screened at 1 mM in typical enzyme assay hits confirmed using surface plasmon resonance (SPR) and X-ray crystallography 4/13/1119CHEM E-120

20. Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010, 20, 5329 (Evotec) Able to cocrystallize 3 with BACE1 Asp32&228 catalytic diad 4/13/1120CHEM E-120

21. Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010, 20, 5329 (Evotec) 4/13/1121CHEM E-120

22. Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010, 20, 5329 (Evotec) 4/13/1122CHEM E-120

23. 23 Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I;Inhibitor Design and Validation: JMC 2010, 53, 951-965 Problem – need to cross BBB (pK a ’s of ~ 7 are best) but the enzyme exists at a pH ~ 5 They focused on the second heterocycle – bioisotere of thioisourea with pK a > 6-10 4 HBD in protonated state 4/13/11CHEM E-120

24. Test the idea Computational ligand binding studies suggested the chlorophenyl group will fit into S1 Parallel Synthesis CH 3 NH 2 PhCH 2 NH 2 XPhCH 2 NH 2 RCH 2 NH 2 P1A P1B P1C P1DP4D ROW A 4/13/1124CHEM E-120

25. Type 2 did not bind by NMR and did not inhibit BACE-1 Type 1 hits Discovered two modes of binding 4/13/1125CHEM E-120

26. Problem! Have two binding modes that are not obvious to control. Know want to extend into S1, S3 and S1’. With what?? Prepare a library 500 compounds using high throughput organic synthesis. 3 sites or points of diversity. Fix R 1 and R 2 = CH 3 and isobutyl steroechemistry?? Use racemic R 3 = 500 amines 4/13/1126CHEM E-120

27. A-site needs 2 HBD 4/13/1127CHEM E-120

28. IC 50 = 27 nM 3 with IC 50 = 0.2 mM to 39 with IC 50 = 27 nM 7407 fold enhancment clog p = 7.5 mw = 545 LE = 0.25 modest bioavailability 4/13/1128CHEM E-120

29. Can extend from S1 into S3? 40 IC 50 = 605 nM BUT brain penetration IC 50 = 27 nM 4/13/1129CHEM E-120

30. 4/13/1130CHEM E-120

31. Design and Synthesis of 5,5’-Disubstituted Aminohydantoins as Potent and Selective Human β-Secretase (BACE1) Inhibitors: JMC 2010, 53, 1146 (Wyeth) Discovered through FRET assay 4/13/1131CHEM E-120

32. Types of Compounds Tested to Develop SAR 4/13/1132CHEM E-120

33. IC 50 (nM) BACE140 BACE2780 cathepsin D12,500 IC 50 (nM) BACE110 BACE2810 cathepsin D820 100 mg/kg po 69% reduction of plasma Aβ 40 limited brain exposure!!! 4/13/1133CHEM E-120