1. Ben Munoz, Ph.D. High-Affinity Ligands of  2  1 Subunit of Voltage Gated Calcium Channels

3. What is    ? Increases current amplitude Alters biophysics Alters pharmacology inconsistent effects of gabapentin on calcium currents N SS SS C        C C N C N N R217 N SS SS    C out in  

7. High-Throughput Screening Campaign N -acridin-9-yl-butane-1,4-diamine 6-aryl- 6H -pyrrolo[3,4- d ]-pyridazine *  2  -1 binding assay utilizing 3 H-gabapentin as the radioligand [1,2,4]triazolo[3,4- a ]pthalazine IC 50 (nM)* 180 220 220 MW 295.4 321.5 491.6 LogD c 3.6 0.6 2.9 PSA 45 27 107

8. 6-Aryl-6H-Pyrrolo[3,4- d ]Pyridazine: Strategy 6-aryl- 6H -pyrrolo[3,4- d ]-pyridazine Heterocyclic replacements Scope Improve potency and efficacy Establish strong IP position

9. 6-Aryl-6H-Pyrrolo[3,4- d ]Pyridazine: Rat PK Dose/Route 2 mg/kg i.v. 10 mg/kg p.o. 20 mg/kg i.p. %F n/a 81 n/a AUC (  M.hr) 6.4 27 141 C max (  M) n/a 3.6 101 T max (h) n/a 2.8 0.3 T 1/2 (h) 0.91 4.5 1.2 Clp (mL/min/kg) 18.5 n/a n/a Vdss (L/kg) 1.3 n/a n/a

10. 6-Aryl-6H-Pyrrolo[3,4- d ]Pyridazine Versus Gabapentin: Rat Chung Model

11. Initial SAR of Ethoxyphenyl Ring Tight SAR revealed in the ethoxyphenyl ring R 1  2  -1 IC 50 (nM) 4-OCH 2 CH 3 180 3-OCH 2 CH 3 >10,000 2-OCH 2 CH 3 1,850 4-OCH 3 >10,000 4-O- n Pr 5,180

12. General Synthetic Approach of Pyrrolopyridazine Analogues Removal of one or both pyridazine ring methyl groups with abolished activity

13. Replacement of the Ethoxyphenyl Ring with Heterocycles Heterocyclic replacement not tolerated R 1  2  -1 IC 50 (nM) 180 2,100 5,500 6,100 7,600 >10,000

14. 4-Ethoxyphenyl Analogues Electron donating groups preferred, and 2-substituents 45-fold increase in potency relative to the initial lead (R = H) R 1  2  -1 R 1  2  -1 IC 50 (nM) IC 50 (nM) CH 3 180 2,6-diCH 3 54 2-OCH 3 4 3,6-diCH 3 94 2-CH 2 CH 3 13 3-Cl 100 2-CHCH 2 25 5,6-diCH 3 282 2-CH 3 27 3-CH 2 OH 292 2-SCH 3 28 3-CH 3 298 2-OCH 2 CH 3 33 2-Fl 313

15. Effects of 2-Methyl Substitution on Phenyl Ring

16. General Synthetic Approach of Phenyl Analogues

17. General Synthetic Approach of Pyrrolopyridazine Analogues

18. Pyrrolopyridazine Analogues R 1  2  -1 R 1  2  -1 IC 50 (nM) IC 50 (nM) Methyl 180 Ethyl 250 Phenyl 382 2-Cyclopentylethyl 148 4-Methylphenyl 86 n -Pentyl 360 4-Methoxyphenyl 154 3-Methylbutyl 263 3-Methoxyphenyl 335 2-Methylpropyl 171 2-Methoxyphenyl 737 Cyclopentylmethyl 211 4-Chlorophenyl 701 Cyclopentyl 185 3-Chlorophenyl 1,597 Cyclohexyl 235 2-Chlorophenyl 4,367 Cyclopropyl 170

19. General Synthetic Approach for Installation of Heteroatoms Adjacent to Pyridazine

20. Heteroatoms Adjacent to Pyridazine R 1  2  -1 IC 50 (nM) 40 170 75 >10,000 440 240 >10,000

22. Representative Results from HTOS Campaign Results suggest the existence of a large pocket adjacent to the pyridazine nitrogen on the  2  subunit R 1  2  -1 R 1  2  -1 R 1  2  -1 IC 50 (nM) IC 50 (nM) IC 50 (nM) 30 18 84 69 94 221 32 67 1,692 50 54 2,926

23. N -Acridin-9-yl-Butane-1,4-Diamine: Strategy Metabolic liability Stereochemical preference & scope Disrupt planarity of molecule Optimal distance between nitrogen

24. Stereochemical Preference Branched amine important for activity with stereochemical preference for S R 1  2  -1 R 1  2  -1 R 1  2  -1 IC 50 (nM) IC 50 (nM) IC 50 (nM) 220 38 >10,000 110 18 6,000 11,000 9 13,000 52 270 1,100

25. Terminal Amine Substitution Analogues having aromatic heterocyclic groups such as imidazole, triazole and tetrazole were also inactive in the binding assay R 1  2  -1 R 1  2  -1 IC 50 (nM) IC 50 (nM) 110 9,900 5,100 >10,000 >10,000 >10,000 5,900

26. Different Ring Structures R 1 R 2  2  -1 IC 50 (nM) rac-CH 3 960 rac -CH 3 >10,000 rac -CH 3 420 rac -CH 3 160 ( S )-CH 3 43 ( R )-CH 3 760

27. General Synthesis of N -Acridin-9-yl-Butane-1,4-Diamines

28. General Synthesis of Partially Saturated Analogues

29. Specific Binding to  2  -1 Subunit Radio labeled material showed high specific affinity to A710 membranes and soluble human  2  -1 [ 3 H]-Compound Bound (dpm) (Compound) 10/100  M GBP

30. [1,2,4]Triazolo[3,4- a ]Pthalazine: Strategy Improve Solubility Potency Metabolic Stability

31. Isoxazole Replacement Introduction of CF 3 not only improved potency, but more importantly solubility R 1  2  -1 R 1  2  -1 IC 50 (nM) IC 50 (nM) 220 CH 2 OCH 3 131 H 230 iPr 272 CH 3 145 342 CF 3 72 145 SCH 3 194 195 CH 2 CH 3 241

32. Substitution on Phenyl Ring 3-Substituted phenyl ring consistently more potent R 1  2  -1 IC 50 (nM) 2-OCH 3 3,163 3-OCH 3 116 4-OCH 3 829 2-F 291 3-F 161 4-F 630 2-CH 3 448 3-CH 3 15 4-CH 3 260

33. Metabolic Pathway Block dealkylation and/or hydroxylation

34. Modification to Linker Tether Stereochemical preference for S R 1  -1 R 1  2  -1 R 1  2  -1 IC 50 (nM) IC 50 (nM) IC 50 (nM) 36 1,369 >10,000 30 700 29 10,000 10,000 28 183 198 174 229

35. General Synthesis of [1,2,4]Traizole[3,4- a ]Phthalazine

36. Synthesis of Enantioenriched [1,2,4]Traizole[3,4- a ]Phthalazine Derivative

37. Specific Binding to  2  -1 Subunit Radio labeled material showed high specific affinity to A710 membranes and soluble human  2  -1 [ 3 H]-Compound Bound (dpm) [ 3 H]-Compound Bound (dpm) (Compound) 10/100  M GBP

40. Back-Up

41. Spinal Nerve Ligation Model of Neuropathic Pain: Chung Model - tight ligation of L5/L6 spinal nerves - tactile allodynia, thermal hyperalgesia develops 3 days post-op; persists 5-10 weeks L5 DRG DRG DRG Spinal Cord Sciatic Nerve L6