Discovery of Novel Imidazolines and as Selective TAAR1 Partial for the Treatment of Psychiatric Disorders Giuseppe Cecere, pRED, Discovery Chemistry F. Hoffmann-La Roche AG, Basel, Switzerland Biological Rationale Trace are known for four decades

Trace Amines

 - phenylethylamine p- p- (PEA)

Biogenic Amines

( DA) (NE) (5-HT)

• Structurally related to classical biogenic (DA, NE, 5-HT) • Co-localised & released with biogenic amines in same cells and vesicles • Low concentrations in CNS, rapidly catabolized by (MAO) • Dysregulation linked to psychiatric disorders such as & 2 Trace Amines

3 Biological Rationale -Associated Receptors

(TAARs) p-Tyramine

extracellular TAAR1  Discrete family of GPCR’s  Subtypes TAAR1-TAAR9 known intracellular Gs     Structural similarity with the and receptor superfamily

adenylate Activation of the TAAR1 cyclase receptor leads to cAMP elevation of intracellular cAMP levels

• First discovered in 2001 (Borowsky & Bunzow); characterised and classified at Roche in 2004 • Trace amines are endogenous ligands of TAAR1 • TAAR1 is expressed throughout the limbic and system in the Borowsky, B. et al., PNAS 2001, 98, 8966; Bunzow, J. R. et al., Mol. Pharmacol. 2001, 60, 1181. Lindemann L, Hoener MC, Trends Pharmacol Sci 2005, 26, 274. 4 Biological Rationale Electrical activity of + p-tyramine

Roche TAAR1 KO mice Increased firing rate of The TAAR1 dopaminergic neurons of TAAR1 downregulates dopaminergic KO mice in neurons from WT but not from KO mice

Working hypothesis based on electrophysiology and behavioral effects of cmpds in rodents -TAAR1 is a negative modulator of dopaminergic neurotransmission -TAAR1 full agonist lowers DA if is too high ( schizophrenia) -TAAR1 partial agonist / antagonist raises DA if too low ( depression) -TAAR1 partial agonist might normalize DA if too low ( )

Lindemann L, Meyer CA, Hoener CM et al., J Pharmacol Exp Ther 2008, 324, 948. 5 Medicinal Chemistry Programme Aim: Discovery of selective TAAR1 agonists Objectives: - Identify potent and selective TAAR1 agonists & partial agonists - -like series with suitable phys.-chem. and PK properties - Use to probe behavioural in animal models (POC)

Challenges:- The natural ligands are not well suited as in vivo tool compounds due to their low metabolic stability (MAO substrates) - Selectivity vs. targets may be difficult to achieve Strategy: Screening a subset of the Roche library to find suitable starting points and optimise these hits in a medicinal chemistry programme

6 HTS of Roche Golden Library Numbers

52’757 cpds (Golden Library)  HTS

Confirmation of primary hits and counter screen

181 specific hits with EC50 < 10 M (372 specific hits with EC50 < 50 M) Similarity search  looking for clusters of parent molecules

2’672 cpds to be tested

Selecting and eliminating (partly manual)

191 new hits with EC50 < 10 M; 78 with EC50 < 1 M

7 TAAR1 Agonist Pharmacophore Starting point for screening activities

basic head group

β- phenylethylamine linker X HTS

Rn

p-tyramine aromatic endogenous ligands pharmacophore model adrenergic basic pK = 10 a compounds

Screening lead to identification of 2-benzylimidazolines as drug-like TAAR1 ligands Question: Can we find ligands that are selective vs. ? 8 Benzylimidazolines and Benzylimidazoles Structure Activity and Selectivity Relationship 1) 2) N N H

R1, R2 R1, R2

R1, R2 = H, F, Cl, Br, Me, Et, i t Pr, Bu, CF3, Ph, OH, MeO, PhO, NH , AcNH, MeSO ,… 1 2 K hTAAR1/Ki 2 2 2 R , R Ki hTAAR1 i

Restricted SAR H 400 nM 4.7 No selectivity vs. o,o’-diMe 36 nM 4.5 2A Brain/Plasma <1 o,o’-diEt 24 nM 100

o,o’-diiPr 630 nM 14

Preliminary SAR within 2-Benzylimidazole series showed promising selective versus adrenergic receptors when two substituents were added in ortho- position on the phenyl ring 9 2-Benzyl-imidazoles Structure Activity and Selectivity

RelationshipR: Me ~ Et > cBu ~ iPr > nPr, Ph, OH N N N R ≠ H: 0 or 1 o-substituent allowed: N o: H, F, Br, Cl, OMe, CF , Et N N 3 H H H m: Br, Cl, F, OMe, CF3 R N H disubstituted derivatives: NH mandatory o,m’: Cl/Cl, F/F oo' o,m: F/F, Me/Me mm' 2- < 4-Imidazole

R = H: at least 2 substituents on Ph ring o,o’: C1-C4 alkyl, C1-C3 alkyl/Halo, CF3, F, Cl, Et/OMe In functional assay with h/r o,m: Cl, Oalkyl2, CF3 TAAR1 o,m’: F/Me, Me, Me/F, F/CF 3 only H tolerated! m,m’: CF3 monosubstituted derivatives: based on Ki on h/rTAAR1 and hTAAR1 o: cPr, H, Et, OMe, OCF3 m: NO2, NH2, Aryl green < 0.5 M trisubstituted derivatives: 0.5 M < orange > 5 M o,o’,m: Et/Et/F, Et/Et/F, Et/Et/Br, Et/F/F, F/F/Me, Et/F/Cl red > 5 M

10 Profile of RO4992479 a Selective TAAR1 Full Agonist N

N Frontrunner. Good selectivity vs. fromother GPCRs 4-Imidazole Series H . Good physicochemical properties . Low oral in both &

TAAR1 Phys.-chem. Properties mouse rat logD 2.62 MW 214 Sol. 410 g/ml EC50 46 nM 51 nM 324 nM pKa 7.31 PAMPA 3.42 x 10-6 cm/s H Effic. 87% 74% 62%

 2a EC 7.13 M Effic. 17% SDPK 50 mouse rat CEREP (panel of 60 targets) clean i.v. dose 5 mg/kg 5 mg/kg

hERGIC50 >10 M I1 IC50 0.66 CL 64 ml/min/kg 66 ml/min/kg Br./Pl. 2.4 1.6 Microsomal Stability human mouse rat p.o. dose 10 mg/kg 10 mg/kg 13 ClMic 59 -- cmax 303 ng/ml 97 ng/ml

MAB 68% 37% -- t1/2 1.0 h 0.3 h Class MM-- Bioavail. F 21% 14% 11 Profile of RO4992479 a Selective TAAR1 Full Agonist N

N Frontrunner. Active in antagonizing from -induced 4-Imidazole Series H hyperlocomotor activity in mouse . Anxiolytic-like effect in stress-induced hyperthermia (SIH) in mouse

8000 mTAAR1 7000 K 28 nM 6000 i EC 60 nM 5000 50 73% efficacy 4000 3000 2000 1000

Horizontal Activity (cm) Activity Horizontal 0 Tw 3 10 Tw 3 10 mg/kg RO

No effect in TAAR1 KO mouse !!

12 Synthesis of 2-Benzyl Imidazoles o,o’-disubstituted required an ad-hoc synthetic route

ethylenediamine Swern [O]

S8, 200°C, W (43-84%) (17-80%)

i. EtLi (2.6 eq.) Ph3P, CBr4 THF, -10 °C CH2Cl2, 0°C to rt

p-TsOH, toluene ii. 4 M aq. HCl (60% ) reflux, 40 min THF, reflux (99%) (93% )

ethylenediamine Swern [O]

CH Cl , rt, 18 h 2 2 (75% ) (60%)

Galley G, Stalder, H, Goergler A, Hoener CM, Norcross RD, BMCL 2012, 22, 5244. 13 Benzylimidazolines and Benzylimidazoles Structure Activity and Selectivity Relationship 1) 2) N 3) N NH R N H

R1, R2 R1, R2 R1, R2

R1, R2 = H, F, Cl, Br, Me, Et, i t Pr, Bu, CF3, Ph, OH, MeO, Restricted SAR 1 2 PhO, NH2, AcNH, MeSO2,… R , R K hTAAR1 Ki hTAAR1/Ki 2 i Low selectivity vs. 2 Restricted SAR H 400 nM 4.7 Often high clearance in PK No selectivity vs. 2 Submicromolar inhibition o,o’-diMe 36 nM 4.5 of CYP (expecially at 2C9) Brain/Plasma <1 o,o’-diEt 24 nM 100

o,o’-diiPr 630 nM 14

• All three series show a very restricted structure-selectivity-relationship • Metabolite ID studies on RO4992479 showed extensive oxidation of o,o’- ethyl residues and GSH adduct formation

14 Discovery of the 2-atom linker 4-Imidazole series Successful Application of a SOSA N ApproachNH NH

1 65 nM vary 11 nM 48 nM (5.3) (1.6) sidechain (1.6) h 2a Ki = 18 nM

N N NH NH

N N 32 nM (3.9) 2 22 nM 55 nM (24) (5.9) hTAAR1 Ki h 2a Ki = 40 nM (2a Ki/hTAAR1 Ki)

Aminomethyl imidazoles are known adrenergic ligands – SOSA approach* Selectivity for TAAR1 could be achieved by variation of substituents* Wermuth CG: Selective Optimisation of Side Activities. DDT 2006, 11, 160. 15 4-Aminomethyl-Imidazoles Structure Activity and Selectivity EC hTAAR1 Relationship 50 R 5 nM m-Cl N NH 10 nM H m,m'-di-F m-Cl,p-F m,p-di-F p-NHC(O)Aryl m-Me N p-Cl m-F m-Cl-m'-F m-OCF CF H R m-OMe m-Cl,o'-F p-F,m-Me 2 2 m,p-di-Cl m-Cl,p-F m-[1-pyrrole]

Broader SAR allowed 50 nM p-Br, m-F m-F,o'-F m-OBn m,m'-di-Cl Good selectivity vs alpha m-[-naphthyl] p-F m-OCHF2 m-OPh 2 100 nM p-Cl,m-CF Good PK possible p-OBn p-Cl,m-OMe 3 selectivity vs. 2a o-F m-OEt m-NH 2 p-OPh m-c-Pr (based on EC50 and IC50) i m-CF3 m-O Pr m-OCF3 p-F,m-OMe m-O-[3-pyridine] green > 200 200 > orange > 50 m-CHOH-Ph p-F,m-OPh p-Cl,m-F red < 50 300 nM p-Cl,o-F p-OMe,m-F m-Ph m-Br m-OH 16 RO5073012 – a TAAR1 partial agonist Frontrunner from 4-Imidazole Series . Balanced TAAR1 efficacy profile across . Good selectivity vs. other GPCRs . Good physicochemical properties . Good oral PK profile in both mouse & rat TAAR1 Phys.-chem. Properties human mouse rat logD 3.3 MW 250 Sol. 97 g/ml EC50 23 nM 33 nM 25 nM pKa 3.5 / 6.7 PAMPA 2.7 x 10-6 cm/s H Effic. 35% 25% 24%

SDPK  2a EC50 > 50 M IC50 = 11 M mouse rat

CEREP (panel of 60 targets) clean i.v. dose 5 mg/kg 4.8 mg/kg

hERGIC50 >10 M IC20 7.8 M CL 24 ml/min/kg 40 ml/min/kg Br./Pl. 0.6 6.6 Microsomal Stability human mouse rat p.o. dose 10 mg/kg 3.8 mg/kg

ClMic 18 18 70 l/min/mg cmax 1740 ng/ml 519 ng/ml

MAB 46% 64% 30% t1/2 1.8 h 1 h Class MMH Bioavail. F 86% 67% 17 RO5073012 – a TAAR1 partial agonist N NH Active in key behavioural assays rTAAR1 N Ki 1 nM

EC50 25 nM 24% efficacy PrimaryPrimary inin vivovivoscreeningscreening assayassay 240 Cl 18000 220

15000 200

12000 180 * 9000 * 160 6000 140 Immobility time (s)

Horizontal Activity 3000 120 0 100 Tw 30 Tw 3 10 30 mg/kg Vehicle 100 3 10 30 Cocaine (20 mg/kg ip) DMI RO5073012 (mg/kg p.o.)

Reversal of cocaine-induced Reduction of immobility time hyperlocomotion in in Forced Swim Test (FST) in rats  indicative for  indicative for depression schizophrenia 18 Bioorg. Med. Chem. Lett. 2012, 22, 5244-5248 RO5073012 – a TAAR1 partial agonist Frontrunner from 4-Imidazole Series . Balanced TAAR1 efficacy profile across species . Good selectivity vs. other GPCRs . Good physicochemical properties . Some key safety flags need to be resolved ! TAAR1 Phys.-chem. Properties human mouse rat logD 3.3 MW 250 Sol. 97 g/ml EC50 23 nM 33 nM 25 nM pKa 3.5 / 6.7 PAMPA 2.7 x 10-6 cm/s H Effic. 35% 25% 24%

CYP P450 Inhibition  2a EC50 > 50 M IC50 = 11 M CEREP (panel of 60 targets) clean 3A4 2D6 2C9 1A2 2C19

IC50 1.4 M 8.1 M 1.0 M 2.3 M 1.3 M hERGIC50 >10 M IC20 7.8 M

Microsomal Stability GSH Adducts In Vitro Tox human mouse rat human rat Ames MNT ClMic 18 18 70 l/min/mg FLAG FLAG NEG NEG MAB 46% 64% 30% Class MMH 19 Reactive Metabolite Formation Strong propensity to form GSH adducts

N NH

N GSH [O] R

N-C-linked Imidazoles

aromatic amino-quinone In line with mass peaks found: para- reactive or ortho- metabolite Aryl-NH2 + GSH - 2H + O oxidation Aryl-NH-alkyl + GSH - 2H + O (M-alkyl) + GSH - 2H + O

• Most selective imidazole compounds bear N-C linker (embedded aniline) • Nearly all aniline-containing cmpds showed pronounced formation of GSH adducts upon metabolic activation in both rat and human microsomes

20 Imidazole Series – conclusions at mid- LO stage In vivo-activity seen in several behavioral models Attributes . Selectivity and PK profile suitable for use as POC compounds . Active in behavioural models in rodents . Provided first insights into behavioural pharmacology of TAAR1

Liabilities . High DDI risk: strong CYP inhibition due to unsubstituted imidazole . Only N-C linker affords sufficient selectivity . N-C linker associated with high reactive metabolite risk (GSH adducts) . Mainly partial agonists at TAAR1: full agonists rare . Judged unlikely to deliver a clinical candidate. SERIES TERMINATED 21 Acknowledgements

Discovery Chemistry In vitro Pharmacology Isotopic Labelling A Beurier Molecular & Biology T Hartung G Galley M-C Höner A Goergler L Lindemann Patenting R Hutter V Metzler R Poppe D Krüsi D Buchy R Norcross A Nilly P Schmid S Chaboz DMPK H Stalder G Hoffmann D Zimmerli F Schuler In vivo Pharmacology A Pähler J-L Moreau Molecular Modelling R Mory Safety N Kratochwil E Prinssen S Mohr W Guba W Spooren L Polunchek M Ebeling S Durkin G Schmitt

Transgenic Mice High Throughput Screening Microdialysis Electrophysiology T Enderle E. Borroni & lab F Knoflach M Dietz L Lindemann G Trube S Jensen-Zoffmann C-A Meyer P Pflimlin M Graf L Ozmen & lab

Preclinical Team - Sept 2007 Doing now what patients need next

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