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TRANSCRIPT
Potent and selective, orally active GPBAR1
agonists as chemical biology probes
Patrizio Mattei, F. Hoffmann-La Roche AG,
Pharma Research & Early Development
Acknowledgements
Discovery Chemistry
Kenneth Atz
Caterina Bissantz
Anne-Sophie Cuvier
Henrietta Dehmlow
Patrick Di Giorgio
Olivier Gavelle
Selina Hodel
Marie-Paule Imhoff
Kersten Klar
Michelle Kovacic
Rainer Martin
Patrizio Mattei
Ulrike Obst
Sherrie Pietranico
Noemi Raschetti
Flore Reggiani
Hans Richter
Martin Ritter
Marianne Rueher
Joana Salta
Petra Schmitz
Jitka Weber
Daniel Zimmerli
Cardiovascular/Metabolic DTA
Andreas Christ
Sabine Grüner
Maria Chiara Magnone
Tanja Minz
Anja Osterwald
Susanne Raab
Wolfgang Rapp
Astride Schnoebelen
Sabine Sewing
Urs Sprecher
Christoph Ullmer
Elisabeth Zirwes
Discovery Technologies
Thilo Enderle
Martin Graf
Arne Rufer
Non-clinical Safety
Rubén Alvarez Sánchez
Andreas Brink
Nicole Clemann
Jürgen Funk
Tobias Manigold
Axel Pähler
Formulation
Jochem Alsenz
Process Research & Synthesis
Stephan Bachmann
Manuela Baumann
Fritz Bliss
Christian Jenny
Jacky Joerger
Fritz Koch
Ernst Kupfer
Michel Lindemer
Karina Müller
Dieter Muri
Kurt Schönbächler
Markus Steiner
Literature
Werner Klaus
Patents
Dörte Klostermeyer
Management
Karin Conde-Knape
Silvia Pomposiello
Martin Stahl
René Wyler
Type 2 diabetes
• Type 2 diabetes
– a disease resulting from a combination of defective insulin secretion and
defective responsiveness to insulin.
– 285 M people (6.4% of world adult population) affected in 2010. (source: International Diabetes Federation, http://www.diabetesatlas.org)
– a manageable chronic condition but risk of long-term complications.
• Initial therapy of type 2 diabetes starts with metformin (in addition, lifestyle changes
are strongly recommended: diet and exercise)
• If sufficient glucose control is not achieved within 3 months, add one of the following
to metformin (ADA/EASD consensus report: Diabetes Care 2012, 35, 1364):
– sulfonylurea (e. g., glibenclamide)
– PPARγ agonist (pioglitazone)
– DPP4 inhibitor (e. g., sitagliptin)
– GLP–1 receptor agonist (e. g., liraglutide)
– insulin
GLP–1 is a potent inducer of
glucose-dependent stimulation of glucose secretion
Bile acids as incretin secretagogues
PYY
• Discovery of the target, GPBAR1 (TGR5, M-BAR) independently at Takeda and Banyu (2002),
leading to intracellular increase of cAMP. Bile acids are endogenous ligands:
• deoxycholic acid EC50 = 1 µM
• lithocholic acid EC50 = 0.53 µM
• Bile acids promote GLP-1 secretion through GPBAR1 (BBRC 2005, 329, 386)
in STC1 enteroendocrine cell line
• Deoxycholic acid administered through intracolonic infusion in humans stimulates
PYY and GLP-1 release through activation of L-cells, to extent similar to that of a
normal meal (Gut 1993, 34, 1219).pl
asm
a P
YY
[pM
]
enteroglucagon
plas
ma
ente
rogl
ucag
on [p
M]
GPBAR1 action on L-cells and macrophages
Both anti-diabetic and anti-inflammatory effects
Kupffer cells
Monocytes
cytokine release:
TNFαααα ����
IL6 ����
• Anti-inflammatory actions
– Improved insulin sensitivity
– Reduced liver inflammation
– Reduced liver fibrosis
cAMP����
Secretagogue:
PYY ����
GLP-1 ����
• Glucose-dependent glycemic
control
– low risk of hypoglycemia
– synergistic action of peptides
– parallels to bariatric surgery
• Islet preservation
• Body weight loss
L-cells
Reference compounds (1)
Synthetic bile acids
• Mixed FXR/GPBAR1 agonist.
• Phase 3 (primary biliary cirrhosis);
Phase 2/3 (non-alcoholic steatohepatitis)
Intercept/Università di Perugia:
INT-777 [6-EMCA]
HOH
OHH
OH
O
H
H
H
Intercept/Università di Perugia:
Obeticholic acid (INT-747)
HOH
OHH
OH
O
H
H
H
OH
hGPBAR EC50 0.5 µMFXR: EC50 0.03 µM
hGPBAR EC50 0.82 µMFXR: inactive
• Selective GPBAR1 agonist.
• Anti-diabetic effects:
Cell Metabolism 2009, 10, 167.
• Reduction of atherosclerosis by reducing
macrophage inflammation and lipid
loading: Cell Metabolism 2011, 14, 747.
• "lead candidate to advance into clinical
studies" (J. Med. Chem. 2009, 52, 7958).
cholic acid
hydroxyl
chenodeoxycholic acid
core structure
Reference compounds (2)
Non-bile acids
Exelixis/Bristol-Myers Squibb: XL-475
• Orally administered GPBAR1 agonist. Designed
to selectively target the GPBAR1 receptors
in the intestine without significant systemic exposure to enhance the therapeutic index for
potential chronic administration.
• Effective in lowering blood glucose, improving
glucose tolerance, improving plasma and
hepatic lipid levels, and reducing hepatic
steatosis.
GlaxoSmithKline: SB-050
• Compound SB-050 progressed to the clinic but was stopped due to inconsistent
PD effects/large inter-individual variations
(e.g. GLP-1 release) across doses.
• Not clear whether systemic exposure is
required for PD effect.
• 241st ACS National Meeting & Exposition,
Anaheim 2011, MEDI-335.
N N SSO
O
O
OO
O
O
O
hGPBAR1 EC50 1.2 µM
structure not disclosed
Today's topics
• A new class of non-bile acid derived, orally bioavailable GPBAR1 agonists
� Start from scratch, by way of high-throughput screening
• Characterisation of the new GPBAR1 agonists as antidiabetic agents
� Glucose, PYY, GLP–1
N
ClN
O
FF
F
FFF
NN
SO O
N O
N
O
F3C
NO
Cl
Cl Cl NN
NOH
N
GPBAR1 – HTS
• High-throughput screening of the whole Roche library (n = 940000)
• 5147 validated hits, of these 847 at hEC50 < 3 µM
• Examples (other than bile acids, steroids):
hEC50 [µM] 1.21 0.49 0.58 0.051
mEC50 [µM] >20 2.7 >20 0.28
NK1 antagonists(n = 43)
nicotinamides(n = 1)
"Novartis-type"(n = 4)
oximes(n = 8)
OHH
NH
O
H H
H
OH
SO
OO N
Cl
N
OF
F
F
F FF
NNSOO
N
O
F FF
NO
NO
Cl
Cl Cl N
N
NOH
N
anionic/polar
aromatic/hydrophobic:
essential feature
aromatic
HBA
hydrophobic
aromatic/hydrophobic
Ligand alignment: HTS hits/synthetic bile acid
hEC50 = 0.095 µM
J. Med. Chem.
2007, 50, 4266.hEC50 = 1.21 µM hEC50 = 0.58 µM
hEC50 = 0.48 µM hEC50 = 0.051 µM
Should we consider oximes at all?
Marketed drugs
Antibiotics
N
S
O
NH
OHO
O
NN
OH
S
NH2
H
O
O
NOH
H
H
H
H
Hormonal contraceptives
INN cefdinir norgestimate
marketed by Astellas/Abbott + generics Johnson & Johnson + generics
peak sales (year) ca. USD 900 M (2006) n/a
dose, administration route oral, 300 -600 mg/day Oral, 0.18 – 0.25 mg per tablet, predominantly
in combination with ethynylestradiol
half life 1.7 h n/a
clogP -0.5 5.1
metabolism none (renal excretion of parent) Complete first-pass metabolism (intestine,
liver) to active metabolites norelgestromine
(deacetylation), and norgestrel (deacetylation
+ ketone formation)
Resynthesis of the HTS hit
O
NN
O
NN
N
N
OH
N
N
NN
OH
O
NN
O
H
PhB(OH)2
Et2Zntoluene, 60°C
NH2OH.HCl,NaOAc,
EtOH/H2O+
E/Z ca. 6:1
+NaOH, RT (neat)
Perkin 1 2001, 3258
N
NH2
+
N
O
HCl, 1,4-dioxane
Profile of the oxime hits
N
NOH
N N
NOH
Br
human/mouse EC50 [nM] 45 19
mouse EC50 [nM] 2000 760
solubility [mg/L] (LYSA, pH 6.5) <1 <1
logD (pH 7.4) 3.4 >3
aqueous stability (pH 1, 4, 6, 8, 10) n. d. stable
permeability (prediction) medium/high medium/high
Clmic [mL/min/mg protein] (h/m) 169 / 460 242 / 388
CYPs [μM] (3A4, 2D6, 2C9) <0.2 / 3.1 / <0.2 <0.2 / 2.2 / <0.2
hERG IC20 [μM] n.d. 5.5
Cl [mL/min/kg] 77
Vss [L/kg] 1.6
F [%] 32
t1/2 [h] 0.2 – 0.5
protein binding fu [%] (h/m) 0.2 / 0.5
Mo
use
PK
Initial SAR: Oxime, pyridine
X
NN
0.045 µM 0.26 µM >10 µM >10 µM
NOH
NOH O N
O
N
NN
OH
hEC50 0.045 µM >10 µM >10 µM >10 µM >10 µM
F NN
N
(–)-Enantiomer: 0.028 µM
(+)-Enantiomer: 0.105 µM
hEC50
Optimisation of "northern" vector
N
N
OH
N
hEC50 = 0.045 µM
NH
hEC50 0.025 µM 0.077 µM 0.1 µM 0.38 µM >10 µM >10 µM
Lipophilic unsubstituted 4-pyridyl derivatives
3A4/2C9 pharmacophore
0.1
1
10
100
-2 -1 0 1 2 3 4 5
0.1
1
10
100
-2 -1 0 1 2 3 4 5
0.1
1
10
100
-2 -1 0 1 2 3 4 5
N
Br
OH
N
IC5
0[µ
M]
logD7.4
hEC50 0.012 µM
CYP3A4, IC50 <0.2 µM
CYP2D6, IC50 4.3 µM
CYP2C9, IC50 <0.2 µM
logD >4
logD7.4 logD7.4
CYP3A4 (n = 539) CYP2D6 (n = 520) CYP2C9 (n = 497)
3-Methyl-4-pyridyl is not a CYP450 pharmacophore
CYP3A4 (n = 554) CYP2D6 (n = 540) CYP2C9 (n = 538)
IC5
0[µ
M]
logD7.4 logD7.4 logD7.4
0.1
1
10
100
-2 -1 0 1 2 3 4 5
0.1
1
10
100
-2 -1 0 1 2 3 4 5
N
R
3-Methyl-4-pyridyl is not a CYP450 pharmacophore
N
Br
OH
N
CYP3A4 (n = 554) CYP2D6 (n = 540) CYP2C9 (n = 538)
IC5
0[µ
M]
logD7.4
hEC50 0.028 µM
CYP3A4, IC50 5 µM
CYP2D6, IC50 19 µM
CYP2C9, IC50 1.6 µM
logD >4
logD7.4 logD7.4
0.1
1
10
100
-2 -1 0 1 2 3 4 5
0.1
1
10
100
-2 -1 0 1 2 3 4 5
Other head groups with reduced CYP interaction
hEC50 0.012 µM 0.028 µM 0.008 µM >10 µM 0.179 µM 0.87 µM
CYP3A4, IC50 <0.2 µM 5 µM >50 µM 15 µM n. a. >50 µM
CYP2D6, IC50 4.3 µM 19 µM 24 µM 25 µM 50 µM >50 µM
CYP2C9, IC50 <0.2 µM 1.6 µM 5.1 µM 4.4 µM 32 µM 6.6 µM
logD >4 >4 3.8 3.8 2.9 1.0
R
N
Br
OH
N NNO
N
O
NO
CONH2
NO
COOH
Introduction of polarity at "south eastern" exit vector
N
Br
OH
N
hEC50 = 0.028 µM 0.26 µM 0.7 µM 0.02 µM 0.29 µM 0.01 µM
mEC50 = 0.74 µM 3.7 µM 6.1 µM 0.093 µM 0.86 µM 0.07 µM
logD = >4 1.1 0.9 2.5 2.4 1.0
permeatility = medium/high medium/high medium/high medium/high medium/high LOW
COOH
COOH
COOHCOOH
N OH
N
COOH
hEC50 0.011 µM 0.057 µM
N OH
N
COOH
O
NH
COOH
Comparison of in vitro profiles
human EC50 [nM] 19 11
mouse EC50 [nM] 760 130
FXR transactivation inactive inactive
off-target activity: PPARs, LXR, PXR, RXR n.d. inactive
solubility [mg/L] (LYSA, pH 6.5) <1 9
logD (pH 7.4) >3 2.7
aqueous stability (pH 1, 4, 6, 8, 10) stable stable
permeability (prediction) medium/high medium/high
Clhep [mL/min/mg protein] (h/m) n.d. 16 / 53
CYPs [μM] (3A4, 2D6, 2C9) <0.2 / 2.2 / <0.2 45 / >50 / 14
hERG IC20 [μM] 5.5 >10
protein binding fu [%] (h/m) 0.2 / 0.5 0.04 / 0.2
BrN
N
OH N
N
OHOH
O
HTS hit benzoic acid lead compound
Improved properties translate into an improved PK
1
10
100
1000
10000
0 2 4 6 8Time (h)
p.o. 8.1 mg/kg
i.v. 2.2 mg/kg
N
N
OH
OH
OBr
N
N
OH
1
10
100
1000
10000
100000
0 2 4 6 8Time (h)
pla
sma c
onc. (
ng/m
L) p.o. 23 mg/kg
i.v. 1.0 mg/kg
Cl[mL/min/kg]
Vss[L/kg]
t1/2[h]
F[%]
cmax norm.[ng/mL]/[mg/kg]
77 1.6 0.2-0.5 32 65
Cl[mL/min/kg]
Vss[L/kg]
t1/2[h]
F[%]
cmax norm.[ng/mL]/[mg/kg]
15 0.5 2.5 80 800
HTS hit benzoic acid lead compound
pla
sma c
onc. (
ng/m
L)
mouse
PK/PD for benzoic acid lead compound
PYY as mechanistic readout
• No indirect or delayed effect
• Plasma exposure appears to be a good surrogate for GPBAR1 action.
100
1000
10000
100000
0 5 10 15 20 25
time [h]
concentr
ation
exposure [ng/mL]
PYY [pg/mL]
0
500
1000
1500
2000
2500
3000
10 100 1000 10000 100000
exposure [ng/mL]Tota
l PY
Y [
pg/m
L]
p.o. 100 mg/kg
p.o. 50 mg/kg
i.v. 2 mg/kg
PK/PD relationshipconcentration vs. timedose: 100 mg/kg p.o.
N
N
OH
OH
O
C57BL6 mice
Oral glucose tolerance test of lead compound
Dose dependent improvement of glucose tolerance∆ G
lucose
AU
C0-1
20m
in[%
]
100705020
Dose [mg/kg]
§§ -22.8%
-18.9%
+5%
-10.4%
10
0
-10
-20
-30
§§ p<0.01 Jonckheere's trend test supports
high probability of dose dependent effects
N
N
OH
OH
O
compound administration 1 h
before glucose challenge
C57BL6 mice
Second generation: Reduce logD/protein binding
N
N
OH
OH
O
N
N
OHOH
O
O
SN
N
OH
O
OO
NN
N
OH
OH
O
hEC50[nM]
mEC50[nM]
solubility[mg/L]
logD fu (h/m) [%]
4 28 273 1.5 0.4/1.9
Cl[mL/min/kg]
Vss[L/kg]
t1/2[h]
F[%]
cmax norm.[ng/mL]/[mg/kg]
15 0.5 2.5 80 800
hEC50[nM]
mEC50[nM]
solubility[mg/L]
logD fu (h/m) [%]
60 450 250 1.3 0.4/0.4
hEC50[nM]
mEC50[nM]
solubility[mg/L]
logD fu (h/m) [%]
26 1300 300 1.7 2.1/11
hEC50[nM]
mEC50[nM]
solubility[mg/L]
logD fu (h/m) [%]
11 130 9 2.7 0.04/0.2
Cl[mL/min/kg]
Vss[L/kg]
t1/2[h]
F[%]
cmax norm.[ng/mL]/[mg/kg]
67 3.3 2.2 29 40
Cl[mL/min/kg]
Vss[L/kg]
t1/2[h]
F[%]
cmax norm.[ng/mL]/[mg/kg]
5 0.5 0.7 87 600
Cl[mL/min/kg]
Vss[L/kg]
t1/2[h]
F[%]
cmax norm.[ng/mL]/[mg/kg]
5 0.4 1.7 100 650
RO5527239
in vitro:
mouse PK:
H
O
Br
CO2EtNC ClMg
HNMe(OMe)
N
O
BrOMe
O
Br
OH
O
Br
EtO
CN
O
Br
EtO
CN
N
O
BrOMe
O
BrN
O
NN
OH
O
N
NN
OH
O
OH
N
Br
NH COOEt
Morpholine, ∆∆∆∆, 2h
87% 2-Me-THF, RT, 1hquant.
CH3CO2H, H2SO4
15 h, 110°C
HCl
TBTU, NEt3, DMF
(rac)
46% (R)42% (S)
Reprosil Chiral NR
n-Heptane/i-PrOH 80:20
72%
91%
n-BuLi,2-Me-THF,
-100°C
Pd2(dba)3, X-PhosNaOtBu, toluene
1. NH2OH.HCl, NaOAc, EtOH, reflux2. HCl, 1,4-dioxane
1.
2. NaOH, EtOH, H2O
76%
73%
76%
RO5527239
Synthesis of RO5527239
RO5527239 binds specifically to GPBAR1
Same binding site as lithocholic acid
NN
N
OHOH
O
T
free radioligand (nM)
specific
bin
din
g [
pm
ol/
mg]
0 40 80 100
0
1
2
3
4
5
6
7
20 60
Kd = 70 nM
Bmax = 10.2 pmol/mg protein
[3H]-RO5527239
No meaningful off-target activity identified
• radioligand binding panel (Cerep, n = 97)
• transactivation assays:
• FXR
• LXRα, LXRβ
• PPARα, PPARγ, PPARδ
• RXRα
Cellular activity of RO5527239
GPBAR1 expressing enteroendocrine STC-1 cells
GLP-1
(%
of
PM
A)
-4
0
4
8
12
16
20
24
28
concentration (µM)
0.1 1 10
NN
N
OHOH
O
EC50 = 1.59 µMRO5527239
EC50 > 10 µM deoxycholic acid
EC50 = 0.32 µM
EC50 = 11 µM
concentration (µM)
0.1 1 10
cA
MP (
nM
)
0
40
80
120
160
cAMP GLP–1
60 min
tota
l GLP
-1 [p
g/m
l]
0
20
40
60
80
100
60 min
tota
l PY
Y [p
g/m
l]
0
400
800
1200
RO5527239 in humanised DIO (C57/Bl6) mice
GLP-1/PYY secretion and glucose tolerance
min0 30 60 120
Pla
sma G
lucose
[m
M]
4
6
8
10
12
14
16
18
20
-37%-28-31
∆ ∆ ∆ ∆ AUC Glucose
[mM
×m
in]×
100
0
2
4
6
8
10
Vehicle
Glucose
• Higher in vivo efficacy of RO5527239 in GLP-1/PYY release translates into
superior glucose tolerance.
10 mg/kg
30 mg/kg
NN
N
OHOH
O
100 mg/kg
N
N
OHOH
O
GLP–1PYY
benzoic acid lead compoundRO5527239
Acute improvement in glucose tolerance in db/db mice
Synergistic effects with metformin and sitagliptin
fold
incre
ase
10
20
40
50
− + + − RO5527239
− − + + metformin
0
30
Total GLP-1
time [min]
-120 0 15 30 60 120
4
8
12
16
20
glu
cose
[m
M]
-24%
AUC Glucose
[mM
×m
in]
×1000
0
1
2
3 NN
N
OHOH
O
a) with metformin
time [min]-60 0 15 30 60 120
0
5
10
15
20
25
vehicle
RO5527239 (30 mg/kg)
sitagliptin (10 mg/kg)
sitagliptin (10 mg/kg)
+ RO5527239 (30 mg/kg)
glu
cose
[m
M]
b) with sitagliptin
(DPP4 inhibitor)
RO5527239
0
5
10
15
20
25
30
Active GLP-1
− + + − RO5527239
− − + + sitagliptinfo
ld incre
ase
vehicle
RO5527239 (30 mg/kg)
metformin (100 mg/kg)
metformin (100 mg/kg)
+ RO5527239 (30 mg/kg)
Summary
• We have identified a new class of orally available GPBAR1 agonists starting
from high-throughput screening hits.
• An unsubstituted oxime group is required for this class of compounds.
• Optimised GPBAR1 agonists bind to the target in the same manner as bile
acids, resulting in secretion of PYY and GLP-1.
• RO5527239, the most potent compound of this class, improves glucose
tolerance in rodents and shows synergistic effects with sitagliptin and
metformin.