Post on 16-Mar-2020
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
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