Discovery of Novel Imidazolines and Imidazoles as Selective TAAR1 Partial Agonists for the Treatment of Psychiatric DisordersGiuseppe Cecere, pRED, Discovery ChemistryF. Hoffmann-La Roche AG, Basel, Switzerland

Biological RationaleTrace amines are known for four decades

-phenylethylamine(PEA)

p-tyramine p-octopamine tryptamine

Trace Amines

Biogenic Amines

• Structurally related to classical biogenic amine neurotransmitters (DA, NE, 5-HT)

• Co-localised & released with biogenic amines in same cells and vesicles

• Low concentrations in CNS, rapidly catabolized by monoamine oxidase (MAO)

• Dysregulation linked to psychiatric disorders such as schizophrenia & depression

dopamine(DA)

norepinephrine(NE)

serotonin (5-HT)

2

Trace AminesMetabolism

3

Biological RationaleTrace Amine-Associated Receptors (TAARs)

• 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 monoaminergic system in the brain

adenylate

cyclasecAMP

intracellular

extracellular

Gs

TAAR1

p-Tyramine

Discrete family of GPCR’s

Subtypes TAAR1-TAAR9 known

Structural similarity with the rhodopsin and adrenergic receptor superfamily

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.

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

4

Biological RationaleElectrical activity of dopaminergic neurons

Lindemann L, Meyer CA, Hoener CM et al., J Pharmacol Exp Ther 2008, 324, 948.

+ p-tyramine

Increased firing rate of dopaminergic neurons of TAAR1 KO mice

The TAAR1 agonist downregulates dopaminergic neurotransmission 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 ( bipolar disorder)

Roche TAAR1 KO mice

5

Medicinal Chemistry ProgrammeAim: Discovery of selective TAAR1 agonists

- Identify potent and selective TAAR1 agonists & partial agonists

- Drug-like series with suitable phys.-chem. and PK properties

- Use to probe behavioural pharmacology in animal models (POC)

Objectives:

Challenges:- The natural ligands are not well suited as in vivo tool compounds due to their low metabolic stability (MAO substrates)

- Selectivity vs. biogenic amine 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 LibraryNumbers

7

(372 specific hits with EC50 < 50 M)

52’757 cpds (Golden Library) HTS

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

2’672 cpds to be tested

181 specific hits with EC50 < 10 M

Confirmation of primary hits and counter screen

Similarity search looking for clusters of parent molecules

Selecting and eliminating (partly manual)

TAAR1 Agonist PharmacophoreStarting point for screening activities

Screening lead to identification of 2-benzylimidazolines as drug-like TAAR1 ligands

Question: Can we find ligands that are selective vs. adrenergic receptor?

X

Rn

β-phenylethylamine

p- tyramine

basic head group

linker

aromaticendogenous ligandsbasic pKa = 10 pharmacophore model adrenergic

compounds

Tolazoline

Tetryzoline

Naphazoline

HTS

8

Benzylimidazolines and BenzylimidazolesStructure Activity and Selectivity Relationship

Preliminary SAR within 2-Benzylimidazole series showed promising selective versus adrenergic receptors when two substituents were added in ortho-position on the phenyl ring

R1, R2

H

o,o’-diMe

o,o’-diEt

o,o’-diiPr

Ki hTAAR1 Ki hTAAR1/Ki 2

400 nM

36 nM

24 nM

630 nM

4.7

4.5

100

14

R1, R2 R1, R2

NH

N

R1, R2 = H, F, Cl, Br, Me, Et, iPr, tBu, CF3, Ph, OH, MeO, PhO, NH2, AcNH, MeSO2,…

1) 2)

9

Restricted SAR

No selectivity vs. 2A

Brain/Plasma <1

2-Benzyl-imidazolesStructure Activity and Selectivity Relationship

10

o o'

m m'

R

HN

NH

based on Ki on h/rTAAR1 and hTAAR1

In functional assay with h/r TAAR1

only H tolerated!

R: Me ~ Et > cBu ~ iPr > nPr, Ph, OH

2-Imidazole < 4-Imidazole

R = H: at least 2 substituents on Ph ringo,o’: C1-C4 alkyl, C1-C3 alkyl/Halo, CF3, F, Cl, Et/OMe o,m: Cl, Oalkyl2, CF3o,m’: F/Me, Me, Me/F, F/CF3m,m’: CF3monosubstituted derivatives:o: cPr, H, Et, OMe, OCF3m: NO2, NH2, Aryltrisubstituted derivatives:o,o’,m: Et/Et/F, Et/Et/F, Et/Et/Br, Et/F/F, F/F/Me, Et/F/Cl

NH mandatory

green < 0.5 M0.5 M < orange > 5 Mred > 5 M

R ≠ H: 0 or 1 o-substituent allowed:o: H, F, Br, Cl, OMe, CF3, Etm: Br, Cl, F, OMe, CF3

disubstituted derivatives:o,m’: Cl/Cl, F/Fo,m: F/F, Me/Me

NH

N

NH

NN

7.31

7.13 M

Profile of RO4992479a Selective TAAR1 Full AgonistFrontrunner from 4-Imidazole Series N

H

N

TAAR1human mouse rat

EC50 46 nM

87%

51 nM

74%

324 nM

62%Effic.

2a

MW 214

EC50 Effic. 17%

Microsomal Stabilityhuman mouse rat

ClMic 13

68%

59

37%

--

--MAB

Class M M --

Phys.-chem. Properties

logD 2.62 410 g/mlSol.

pKa PAMPA 3.42 x 10-6 cm/s H

SDPKmouse rat

p.o. dose 10 mg/kg 10 mg/kgcmax 303 ng/ml 97 ng/ml

Bioavail. F 21% 14%

t1/2 1.0 h 0.3 h

i.v. dose 5 mg/kg 5 mg/kg

CL 64 ml/min/kg 66 ml/min/kgBr./Pl. 2.4 1.6

CEREP (panel of 60 targets) clean

11

Good selectivity vs. other GPCRs

Good physicochemical properties

Low oral bioavailability in both mouse & rat

>10 MhERGIC50 I1 IC50 0.66

Profile of RO4992479a Selective TAAR1 Full AgonistFrontrunner from 4-Imidazole Series N

H

N

12

Active in antagonizing cocaine-induced hyperlocomotor activity in mouse

Anxiolytic-like effect in stress-induced hyperthermia (SIH) in mouse

Tw 3 10 Tw 3 100

10002000300040005000600070008000

Hor

izon

tal A

ctiv

ity (c

m)

Ki 28 nMEC50 60 nM

73% efficacy

mTAAR1

No effect in TAAR1 KO mouse !!

mg/kg RO

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

Galley G, Stalder, H, Goergler A, Hoener CM, Norcross RD, BMCL 2012, 22, 5244. 13

(43-84%)

ethylenediamine

S8, 200°C, W

Swern [O]

(17-80%)

ethylenediamine

(75%)

Ph3P, CBr4CH2Cl2, 0°C to rt

Swern [O]

ii. 4 M aq. HCl THF, reflux

CH2Cl2, rt, 18 h

(60%)

(60%)

(93%)

p-TsOH, toluenereflux, 40 min

i. EtLi (2.6 eq.) THF, -10 °C

(99%)

Benzylimidazolines and BenzylimidazolesStructure Activity and Selectivity Relationship

• 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

R1, R2

H

o,o’-diMe

o,o’-diEt

o,o’-diiPr

Ki hTAAR1 Ki hTAAR1/Ki 2

400 nM

36 nM

24 nM

630 nM

4.7

4.5

100

14

R1, R2 R1, R2

NH

N

R1, R2 = H, F, Cl, Br, Me, Et, iPr, tBu, CF3, Ph, OH, MeO, PhO, NH2, AcNH, MeSO2,…

1) 2)

14

Restricted SAR

No selectivity vs. 2

Brain/Plasma <1

R1, R2

RN

N H3)

Restricted SAR

Low selectivity vs. 2

Often high clearance in PK

Submicromolar inhibition of CYP (expecially at 2C9)

Discovery of the 2-atom linker 4-Imidazole seriesSuccessful Application of a SOSA Approach

1

2

N

NHN

NHN

NH

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

hTAAR1 Ki

(2a Ki/hTAAR1 Ki)

h 2a Ki = 18 nM

h 2a Ki = 40 nM

11 nM 48 nM65 nM

(1.6)(5.3) (1.6)

22 nM(24)

varysidechain

55 nM(5.9)

32 nM(3.9)

NHN

N

4-Aminomethyl-ImidazolesStructure Activity and Selectivity Relationship

16

selectivity vs. 2a(based on EC50 and IC50)

green > 200200 > orange > 50red < 50

R

N

NNH

m-OCF3m-CF3

p-F,m-OPhm-CHOH-Ph

m-O-[3-pyridine]p-F,m-OMe

p-OPh

p-Cl,m-F

m-OPh

p-Cl,m-CF3

m-c-Pr

m-F,o'-F

p-OBn

m-[1-pyrrole]

m-OCF2CF2Hp-F,m-Me

p-NHC(O)Aryl

10 nM

m-Cl,p-Fm,p-di-Cl

EC50 hTAAR1

5 nM

R

300 nM

100 nM

50 nM

m-Cl

p-Cl

H

m,m'-di-Cl

m-Cl,p-F

m-Cl-m'-F

m,m'-di-F

m-Me

m-OBn

m-Cl,o'-F

m,p-di-F

m-OMe

m-OiPr

p-Fm-[-naphthyl] m-OCHF2

p-Br, m-F

m-F

o-F

p-Cl,o-F

p-Cl,m-OMe

m-NH2m-OEt

m-Ph m-Br m-OHp-OMe,m-F

Broader SAR allowed

Good selectivity vs alpha 2

Good PK possible

3.5 / 6.7

> 50 M

RO5073012 – a TAAR1 partial agonistFrontrunner from 4-Imidazole Series Balanced TAAR1 efficacy profile across species Good selectivity vs. other GPCRs Good physicochemical properties Good oral PK profile in both mouse & rat

TAAR1human mouse rat

EC50 23 nM

35%

33 nM

25%

25 nM

24%Effic.

2a

MW 250

EC50 IC50 = 11 M

Microsomal Stabilityhuman mouse rat

ClMic 18

46%

18

64%

70 l/min/mg

30%MAB

Class M M H

Phys.-chem. Properties

logD 3.3 97 g/mlSol.

pKa PAMPA 2.7 x 10-6 cm/s H

SDPKmouse rat

p.o. dose 10 mg/kg 3.8 mg/kgcmax 1740 ng/ml 519 ng/ml

Bioavail. F 86% 67%

t1/2 1.8 h 1 h

i.v. dose 5 mg/kg 4.8 mg/kg

CL 24 ml/min/kg 40 ml/min/kg

Br./Pl. 0.6 6.6

CEREP (panel of 60 targets) clean

17

>10 MhERGIC50 7.8 MIC20

RO5073012 – a TAAR1 partial agonistActive in key behavioural assays

NNH

Cl

N Ki 1 nMEC50 25 nM

24% efficacy

rTAAR1

Reduction of immobility timein Forced Swim Test (FST) in rats indicative for depression

mg/kg

Cocaine (20 mg/kg ip)

*

Tw 30 Tw 3 10 300

3000

6000

9000

12000

15000

18000

Hor

izon

tal A

ctiv

ity

Vehicle 100 3 10 30100

120

140

160

180

200

220

240

Imm

obili

ty ti

me

(s)

RO5073012 (mg/kg p.o.)DMI

*

Reversal of cocaine-induced hyperlocomotion in rats indicative for schizophrenia

Bioorg. Med. Chem. Lett. 2012, 22, 5244-5248

Primary in vivo screening assayPrimary in vivo screening assay

18

3.5 / 6.7

> 50 M

RO5073012 – a TAAR1 partial agonistFrontrunner 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 !

TAAR1human mouse rat

EC50 23 nM

35%

33 nM

25%

25 nM

24%Effic.

2a

MW 250

EC50 IC50 = 11 M

Microsomal Stabilityhuman mouse rat

ClMic 18

46%

18

64%

70 l/min/mg

30%MAB

Class M M H

Phys.-chem. Properties

logD 3.3 97 g/mlSol.

pKa PAMPA 2.7 x 10-6 cm/s H

CEREP (panel of 60 targets) clean

19

>10 MhERGIC50 7.8 MIC20

CYP P450 Inhibition

3A4 2D6 2C9IC50

1A2 2C191.4 M 8.1 M 1.0 M 2.3 M 1.3 M

GSH Adductshuman rat

FLAG FLAG

In Vitro ToxAmes MNT

NEG NEG

GSH

amino-quinonereactive

metabolite

aromaticpara-

or ortho-oxidation

In line with mass peaks found:

Aryl-NH2 + GSH - 2H + OAryl-NH-alkyl + GSH - 2H + O(M-alkyl) + GSH - 2H + O

[O]

Reactive Metabolite Formation Strong propensity to form GSH adducts

NHN

N

R

N-C-linked Imidazoles

• 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 liver microsomes

20

Imidazole Series – conclusions at mid- LO stageIn vivo-activity seen in several behavioral models

21

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

Acknowledgements

Molecular ModellingN KratochwilW GubaM Ebeling

Transgenic MiceMicrodialysisE. Borroni & labL LindemannC-A MeyerL Ozmen & lab

ElectrophysiologyF KnoflachG TrubeP Pflimlin

PatentingR Poppe

Discovery ChemistryA BeurierG GalleyA GoerglerR HutterD KrüsiR NorcrossP SchmidH StalderD Zimmerli

In vitro PharmacologyMolecular & Cell BiologyM-C HönerL LindemannV MetzlerD BuchyA NillyS Chaboz

In vivo PharmacologyJ-L Moreau R MoryE PrinssenW SpoorenS Durkin

SafetyS MohrL PolunchekG Schmitt

DMPKG HoffmannF SchulerA Pähler

Isotopic LabellingT Hartung

Preclinical Team - Sept 2007

High Throughput ScreeningT EnderleM DietzS Jensen-ZoffmannM Graf

Doing now what patients need next

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