DESIGN OF A NOVEL ANXIOLYTIC AND ANTIDEPRESSANT AGENT

Target - Serotonin Receptor Notes Important receptor in central nervous system 7 types (5-HT1 - 5HT7) and 14 subtypes G-Protein-coupled receptors (except 5-HT3) Three subtypes of 5-HT2 receptor (5-HT2A 5-HT2B 5-HT2C) 5-HT2C receptor thought to be involved in anxiety mCPP is an agonist with some selectivity for 5-HT2C receptor mCPP causes anxiety in human and animal studies Antagonist with selectivity for 5-HT2C receptor may be useful in treating anxiety meta-Chlorophenylpiperazine (mCPP)

(5-Hydroxytryptamine) Natural Ligand Serotonin (5-Hydroxytryptamine) Notes Serotonin is a neurotransmitter Abnormal levels of serotonin are related to various disorders (e.g. anxiety, depression, migraine) Indole ring system is present

Aims of Drug Design 5-HT2A and 5-HT2C receptors predominate in CNS Selectivity for 5-HT2C receptor Selectivity over 5-HT2A is more important than over 5-HT2B 5-HT2B predominates in peripheral nervous system 5-HT2A and 5-HT2C receptors predominate in CNS Resistance to drug metabolism No effect on metabolic enzymes (drug-drug interactions) Aqueous solubility Non-sedating No interaction with alcohol Fast onset of action High response rate No withdrawal effects

Testing Procedures In vitro tests Radioligand binding studies on 5-HT2A, 5-HT2B and 5-HT2C receptor subtypes Tests for activity against cytochrome P450 enzymes In vivo tests Ability to block hypoactivity in rats caused by mCPP Modelling studies Drug design carried out on model binding sites

Lead Compound Produced by Lilly Pharmaceuticals Serotonin antagonist Urea Indole Aromatic Produced by Lilly Pharmaceuticals Serotonin antagonist Insoluble in water

From Lead Compound to SB 200646 Pyridine SB 200646 Notes Substituents removed from indole ring (simplification) Pyridine ring introduced (ring variation) Pyridine ring more polar - increases water solubility Urea link at optimum positions for both ring systems First selective 5-HT2B/2C antagonist 50-fold selectivity over 5-HT2A receptor Modest in vitro activity Some oral activity in vivo

From SB 200646 to SB 206553 SB 206553 Rigidification SB 200646 Tricyclic ring system Extra ring Metabolically labile Notes Rigidification limits number of possible conformations Rigidify structure such that active conformation still allowed Increased chance of active conformation being present 10-fold increase of in vitro affinity 160-fold selectivity over 5-HT2A receptor 4-fold increase of in vivo activity

From SB 200646 to SB 206553 Conformation of SB 206553 Overall structure is roughly planar

7. From SB 200646 to SB 206553 Metabolism of SB 206553 Metabolically labile N-Demethylation Metabolite is active but non-selective

Analogues of SB 206553 Substitution bad Variation permitted Notes Methyl group replacable with Et, Pr or iPr Slightly increased selectivity for 5-HT2C over 5-HT2A Hydrophobic pocket available for N-alkyl group Slightly bigger for 5-HT2C receptor

Analogues of SB 206553 Ring variation strategy No methyl group Pyrrole Ring variation strategy Thipohene No methyl group Loss of selectivity No methyl group Furan Retains Me group More stable to metabolism Good in vitro affinity and selectivity Poor oral activity Poorly absorbed from gut or rapidly metabolized Administered by IV injection

3D QSAR studies of SB 206553 and analogues Notes Structures overlaid using urea group Dark and light blue = areas accessed by compounds having 5-HT2C activity Light blue = disallowed area for 5-HT2A activity Light blue area = region of N-methyl group of SB 206553

Molecular modelling studies of SB 206553 and analogues Notes Model receptor binding site created SB 206553 docked into binding site Carbonyl oxygen of urea group is crucial for activity Positioned to form hydrogen bond to Ser-312 - Ser-312 present in 5-HT2 receptors but not 5-HT1 receptors - binding to Ser-312 thought to be important for selectivity for 5-HT2 receptors over 5-HT1 receptors Carbonyl oxygen interacts with Ser-312 and Ser-315 Aromatic rings positioned into hydrophobic pockets

Molecular modelling studies of SB 206553 and analogues

Molecular modelling studies of SB 206553 and analogues 1 3 8 4 Phe-223 Trp-324 Phe-327 Phe-328 Phe-220 Phe-224 Val-212 V al-608 Hydrophobic pocket

Molecular modelling studies of SB 206553 and analogues Notes Val-608 and Val-212 are present in the hydrophobic pocket occupied by the N-methyl group Bulkier leucine groups are present in the model binding site for the 5-HT2A receptor Hydrophobic pocket is smaller for the 5-HT2A receptor More difficult for N-methyl group of SB 206553 to fit the 5-HT2A receptor May account for selectivity of SB 206553

From SB 206553 to SB 221284 Aim To replace the metabolically labile N-methyl group with a metabolically stable group The new group must bind to the hydrophobic pocket for selectivity SB 206553 Indolines Notes Indole ring is removed - simplification Indoline structures are synthesized with varying substituents Substituent X needs to bind to the hydrophobic pocket Electron-withdrawing substituent at position 6 is preferred Thioether or ether at position 5 is beneficial

From SB 206553 to SB 221284 SB221284 Electron-withdrawing Fits hydrophobic pocket Metabolically stable SB221284 Fits hydrophobic pocket Metabolically stable SB221284 Inhibits cytochrome P450 enzymes Notes Best balance of affinity vs selectivity Potent inhibitor of 5-HT2B and 5-HT2C receptors Good selectivity over 5-HT2A receptor Inhibits cytochrome P450 enzymes Pyridine N is responsible for inhibiting cyt P450 enzymes Selectivity increases for SEt, SnPr or OiPr, but affinity falls

Modelling Studies on SB 221284 Notes SB 221284 is docked into the model binding site for the 5-HT2C receptor Pyridine and indoline rings are positioned in hydrophobic pockets Hydrogen bonding interactions take place with serine residues S-Methyl group fits the hydrophobic pocket CF3 group orientates the thiomethyl group into the correct conformation CF3 acts as a conformational blocker

Modelling Studies on SB 221284 r- 1 3 8 4 Phe-223 Trp-324 Phe-327 Phe-328 Phe-220 Phe-224 Val-212 V al-608 Hydrophobic pocket Note Vacant areas are available in the hydrophobic pocket accommodating the pyridine ring

3D-QSAR Studies on SB 221284 55 Analogues are synthesized Notes 55 Analogues are synthesized Analogues are docked into the model receptor Receptor-ligand complex is minimized Ligands are removed and subjected to CoMFA analysis Predicted affinity versus actual affinity demonstrates a good relationship

3D-QSAR Studies on SB 221284 Notes Steric fields are more important than electrostatic fields Dark blue represents beneficial areas Light blue represents detrimental areas Large number of detrimental areas round the indoline ring Suggests a tight binding pocket Little scope for modification

From SB 221284 to SB 228357 Inhibits cytochrome P450 enzymes Notes Aromatic ring is best placed at position 5 Increased binding interactions with hydrophobic pocket Slightly increased affinity and selectivity Aromatic ring acts as a steric shield for pyridine 100-fold decrease in cytochrome P450 inhibition Level of cytP450 enzyme inhibition is still unacceptable Low oral activity Electron-rich aromatic ring is possibly susceptible to metabolism

From SB 221284 to SB 228357 Structure II Vary ring and rigidification Structure II Pyridine ring is more polar leading to increased water solubility 10-fold increase in affinity due to additional binding interactions Lower selectivity between 5-HT2C and 5-HT2A receptors Structure III Selectivity is recovered by adding a methyl substituent Slightly increased affinity for the 5-HT2C receptor Moderate oral activity Slight drop in cytochrome P450 inhibition

From SB 221284 to SB 228357 Steric clash Structure III Notes Methyl group acts as a conformational blocker Forces rings to be orthogonal Orthogonal rings favored by 5-HT2C receptor but not 5-HT2A receptor

From SB 221284 to SB 228357 Ring variation 4’ 5 Structure III Notes Pyridine nitrogen is responsible for inhibiting cytochrome P450 enzymes Pyridine is replaced by an aromatic ring (ring variation) Poor water solubility Water solubility is improved by a pyridine substituent (R) Substituent is best at position 3 (variation of substituents)

From SB 221284 to SB 228357 4’ 5 Ring variation Structure III Notes Pyridine nitrogen is responsible for inhibiting cytochrome P450 enzymes Pyridine is replaced by an aromatic ring (ring variation) Poor water solubility Water solubility is improved by a pyridine substituent (R) Substituent is best at position 3 (variation of substituents)

From SB 221284 to SB 228357 Improved selectivity 4’ 5 Ring variation Structure III Improved selectivity Higher 5-HT2C affinity Potent oral activity Still inhibits cytochrome P450 enzymes

From SB 221284 to SB 228357 3 Vary substituents Structure VI Electron rich Notes Methyl substituent moved to the ortho position Acts as a conformational blocker Aromatic and pyridine rings cannot be co-planar Increased selectivity for the 5-HT2C over the 5-HT2A receptor Short duration of action Electron-rich aromatic ring is susceptible to metabolism

From SB 221284 to SB 228357 Bad Electron rich Structure VI Structure VIII Notes Analogues made with electron-withdrawing substituents on the aromatic ring Substitution at 2 and 6 is bad - ring is twisted out of plane with the urea group Substitution at positions 4 and 5 is acceptable

From SB 221284 to SB 228357 Increased duration of action (6 hours) Electron rich Structure VI SB 228357 Increased duration of action (6 hours) Modeling studies suggest extra space available in hydrophobic pocket Further extension possible

From SB 228357 to SB 243213 SB 228357 Structure IX Linker Notes Substitution pattern is altered to para Linker oxygen atom is inserted Pushes pyridine further into the hydrophobic pocket Poor oral activity due possibly to reduced solubility

15. From SB 228357 to SB 243213 Linker SB 228357 Structure IX Structure X Original pyridine restored to increase water solubility

From SB 228357 to SB 243213 Linker SB 228357 Structure IX Structure X Structure XI

From SB 228357 to SB 243213 Structure XI Notes ortho Methyl group acts as a conformational blocker Increases torsion angle between the pyridine rings Increased selectivity and affinity for 5-HT2C receptor 80-fold selectivity over 5-HT2B receptor Low cyctochrome P450 activity Good in vivo activity

From SB 228357 to SB 243213 Structure XI SB 243213 Notes Methoxy group is replaced with a methyl group Less liable to metabolism Good profile of affinity, activity and selectivity Negligible cytochrome P450 activity Better aqueous solubility than SB 228357 Entered phase I clinical trials as a non-sedating antidepressant / anxiolytic

From SB 228357 to SB 243213 Modelling studies for SB 243213 Pyridine ring substituent well inserted into hydrophobic pocket

From SB 228357 to SB 243213 Binding interactions for SB 243213 S e r- Phe-223 Trp-324 Phe-327 Phe-328 Phe-220 Phe-224 Val-212 V al-608