patrizio mattei, f. hoffmann-la roche ag, pharma research & … mattei_tcm18-223170.pdf ·...

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


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


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[%]


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[%]


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[%]


67 3.3 2.2 29 40

Cl[mL/min/kg]

Vss[L/kg]

t1/2[h]

F[%]


5 0.5 0.7 87 600

Cl[mL/min/kg]

Vss[L/kg]

t1/2[h]

F[%]


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.

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patrizio mattei, f. hoffmann-la roche ag, pharma research & … mattei_tcm18-223170.pdf ·...

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