Selective Class I PI3K Inhibitors:Identification of a Clinical Candidate

O t b 3 2012October 3, 2012Adrian L. Smith, Ph.D.

CHI Discovery on Target: 3rd Annual Advances inCHI Discovery on Target: 3rd Annual Advances in Targeting the PI3K Pathway - Boston

OutlineI t d ti• Introduction

• HTS Hit Identification• SAR: Hit-to-Lead and Lead Optimization • Profile of the Clinical Candidate (AMG 511)

– Synthesis

– In vitro selectivity

– Pharmacokinetic (PK) profile

– In vivo efficacy • Tumor pharmacodynamic (PD) model

• Xenograft models (U87, HCT116, and BT474)

• Summary

R t t i ki

PI3K signaling pathwayReceptor tyrosine kinase Insulin, IGF, HGF, VEGF, etc.

p

PI3K

p85 p110α/β(δ, γ)

PIP2 PIP3

3 3P

PTENAKTp

MetabolismGlucose uptake Proliferation/survival

mTor

Translation/cell growth

and glycogen

Inhibitors of the PI3K pathway have potentially broad applications across many tumor types

CancerEstimated cases in

USA

PI3Kα% Gain of Function

PTEN % Loss of Function

Advanced Non Small Cell Lung 198,000 4 24

HRPC Prostate 22,000 5 20

Advanced Glioblastoma 19 000 7 66Advanced Glioblastoma 19,000 7 66

Recurrent Ovarian 11,000 12 27

Recurrent Endometrial 6,500 36 61

Metastatic Colon 47,000 14 17

Advanced Bladder 15,000 13 23

Metastatic Breast 46,000 26 38Metastatic Breast 46,000 26 38

Metastatic Melanoma 13,000 3 15

Metastatic Renal 10,500 NA 25

Mutations in the PI3K pathway are among the most frequent in human cancers

Source: Engelman, et al. Nature Reviews Genetics, 2006, 7, 606.

Program goals

In Vivo Technical objective• Identification and development of

ll bi il bl t t ll PI3K

PI3K

Pharmacokinetics Efficacy

Selectivity

orally bioavailable, potent smallmolecule inhibitors of class I PI3Ks for the treatment of solid tumors

PI3K

PI3K

Cellular Potency

Enzyme

PI3K

PI3K

HTS Hit Identification

Enzyme Activity

Desired profile

PI3K

Target Validation

p• Potent inhibition of class I PI3Ks (α, β, δ, γ)

< 10 nM in PI3K enzyme assay< 50 nM in cellular assays

• Selectivity over type-II (e.g., PI3KC2a), t III ( VPS34) t IV ( DNAPK)Validation type-III (e.g., VPS34), type-IV (e.g.,DNAPK)PI3Ks, and mTOR.

• Selectivity over all protein kinases in both enzyme and cellular assays

PI3K enzyme- and cell-based assays[1] ATP l (HTS)[1] ATP loss assay (HTS)

PIP3PIP2ATP

[3] MSD immunoassay (measuring changes in Akt phosphorylation

[2] AlphaScreen assay

PI3KIC50 = Ki(1+[ATP]/Km, ATP) IGF1

changes in Akt phosphorylationat S473 in U87 cells)

[2] AlphaScreen assayExcitation

680 nm

Streptavidin-t d

Emission520-620 nm

1O2

IGFR IRS

PIP2PI3K

PIP3

coated Donor Beads

Biotin-PIP3anti-GST conjugated

Acceptor Beads

PTEN

AKT

PDK1

pAKT

GST-taggedPIP3 Binding Protein

Free PIP3

S473

PIP3PIP2

PI3K

ATP

HTS Hit

• ProsPros– Low MW (358)– Good ligand efficiency (0.33)

Cellular activity

PI3Kα Ki = 350 nM

– Cellular activity– Good kinase selectivityHTS Hit

PI3Kα Ki 350 nMpAkt (U87) IC50 = 190 nM

SelectivityB-Raf IC50 = 3 nMA375 ERK IC 25 M

• Cons– Poor pharmacokinetics

A375 pERK IC50 = 25 nM

mTor IC50 = 97 nMIC50 >1 μM against 43 / 47 other protein kinases

• phenol (glucuronidation)• benzimidazole

X-ray structure of the HTS Hit co-crystallized with PI3Kγco crystallized with PI3Kγ

Ala805Ribose Pocket

Central Core

Lys833

Asp836

Asp841Lys802

Affinity PocketTyr867

Affinity Pocket

Val882Hinge Region

SAR strategy

N

N

NH

HORibosePocket

N

N

N

NH

CN

CentralCore

PocketAffinityPocket

• Replace phenol and benzimidazole

Hinge Binder

Replace phenol and benzimidazole

• Optimize hinge binder and ribose pocket interactionsp

Hinge bindersLys833Asp836

Ala805

Asp841Lys802

Tyr867

Val882

Heterocycle N

N

N

NH2Et

N

N

N

NH2

PI3KαKi (nM) 350 130 68 2 170 1800

2

mTorIC50 (nM) 97 250 21 7 390 4700

U87(pAkt) 190 180 34 9 410 2600

Peterson, E.A. et al. Bioorg. Med. Chem. Lett. 2011, 21, 2064–2070.

U87(pAkt)IC50 (nM) 190 180 34 9 410 2600

Affinity pocketLys833Asp836

Ala805

Asp841

Lys802

Tyr867

Val882

Ar

PI3KαKi (nM) 2 130 63 31 13 900

mTorIC50 (nM) 7 420 290 98 130 >10 000

U87(pAkt)IC (nM) 9 190 710 110 47 >25 000IC50 (nM)

Central coreLys833Asp836 Ala805

Asp841

Lys802

Core

Tyr867

Val882

CoreN

N

N

N

PI3KαKi (nM) 13 640 110 620 >1000 520 120 >1000

NH

NH

mTorIC50 (nM) 130 >10 000 7800 >10 000 >10 000 >10 000 >10 000 >10 000

U87(pAkt)IC50 (nM) 47 >1000 360 >1000 >1000 >1000 >1 000 >1000

Ribose pocketLys833

Asp836 Ala805

Asp841

Lys802

Tyr867

Val882

R-groupN

N S

PI3KαKi (nM) 110 240 12 39 260 94 9 30

SO O

mTorIC50 (nM) 7800 >10000 >10000 1400 >10000 3700 4800 >10000

U87(pAkt)IC50 (nM) 360 310 83 80 230 62 16 65

Initial SAR milestones

Replacement of phenol (PK liability) and improved

kinase selectivity

Benzimidazolereplacement

(PK liability) andimproved selectivity

N

N

NH

HO

N

N

NH

N

MeO

HTS hit

p yN

N

N

NH

CNN

N

N

NH2

NH

PI3Kα Ki = 350 nMmTor IC50 = 97 nMpAkt (U87) IC50 = 190 nM

PI3Kα Ki = 13 nMmTor IC50 = 130 nMpAkt (U87) IC50 = 47 nM

Improved potencyand selectivity

PI3Kα Ki = 9 nMPI3Kα Ki = 110 nMmTor IC = 7800 nM mTor IC50 = 4800 nM

pAkt (U87) IC50 = 16 nM

mTor IC50 = 7800 nMpAkt (U87) IC50 = 360 nM

Smith, A.L. et al. J. Med. Chem. 2012, 55, 5188–5219.

Profile of lead

Plasma - 2422813#1 - IV Bolus 1 mg/kg

1000

1

10

100

Conc

(ng/

mL)

123

PI3Kα Ki (nM) 9

PI3Kβ Ki (nM) 5

0.1

1

0 5 10 15 20

Time (h)

PI3Kγ Ki (nM) 2

PI3Kδ Ki (nM) 2

mTor IC50 (nM) 4800

hVPS34 IC (nM) >9000 Plasma - 2422813#1 - PO 2 mg/kg

100

1000

mL)

4

hVPS34 IC50 (nM) >9000

pAKT IC50 (U87) (nM) 16

HLM / RLM (μL/min/mg) <14 / 26

0.1

1

10C

onc

(ng/

m 456

Rat PK

CL (L/h/kg) 1.7

MRT (h) 1.50 5 10 15 20 25 30

Time (h)Vss (L/kg) 2.6

%F (PO) 77

Metabolite ID studies

MetaSite Metabolism Predications*

2229

Incubation with rat and human

li i

Sco

res

liver microsomesor hepatocytes

Atom ID

*Cruciani, G. et al. J. Med. Chem. 2005, 48, 6970–6979.

Affinity pocket – SAR refinements

Lys833Asp836

3.5 Å4.3 Å

refinements

T 867

Asp841

Ar

Tyr867

N

Cl

N

NMeO

PI3KαKi (nM) 9 5300 24 110 17 18 37

N N

mTorIC50 (nM) 4800 >10 000 >10 000 >10 000 1400 >10 000 >10 000

U87(pAkt)IC50 (nM) 16 >10 000 35 240 62 6 23

Ribose pocket – SAR refinements

Ala805Lys802

NN

MeON

RF

Thr887Asp950

Ile963

Met953

N

N

N

NH2

NH

R

PI3Kα 18 4 4 3 2

NN

SO O

NN

SO O

NN SO O

NN

SO O

PI3KαKi (nM) 18 4 4 3 2

U87(pAkt)IC50 (nM) 6 7 4 1 7

R

PI3Kα 4 2 1 13 4

NN SO O

NN

SO O

NN SO O

NN

SO O

3 αKi (nM) 4 2 1 13 4

U87(pAkt)IC50 (nM) 3 31 1 12 3

In Vivo Rat PK

Pharmacokinetic profiles of lead compounds

R

In Vivo Rat PKIV PO

CL (L/kg/h)

Vdss(L/kg)

MRT (h) %F AUC (mM*h)

N

MeON

RF

2.0 3.3 1.7 45 0.95N

N

N

NH2

NH

N

2.9 6.4 2.2 57 0.35

0 4 1 7 3 9 60 5 0AMG 511 0.4 1.7 3.9 60 5.0

1.0 3.2 3.2 60 2.3

AMG 511

1.3 4.0 3.1 49 1.4

0.7 3.5 4.9 24 1.3NN SO O

Lys833Ala805

Asp836

3 5 Å3.6 Å Lys833

Ala805

Lys802A C

Lys802

Asp964

3.5 Å

Asp841

Lys833

Val882 Asp836

Tyr867

Asp841

Tyr867

p

Asp964

Asp950Ile963Met953

Tyr867Val882

Thr887

Asp950

Lys833

Asp836Lys802

B DLys833

Ala805

Asp964

Ala805

Asp841Lys802

Tyr867Val882

Thr887

Asp950Ile963

Synthesis of AMG 511OH Br HN NBoc

N

F

CHON

F

N

F

MeMgBr, THF,

0 25 °C, 5 min

HN NBocN

F

NNBoc

SOBr2, CH2Cl2,

25 °C, 3 h96%

KI, K2CO3, CH3CN,80 °C, 1 h

73%, 2 steps

1. n-BuLi, THF, –78 °C, 1 h

2. B(Oi-Pr)3

3. NaOH / aqueous workup

N

F

NNBoc

B(OH)2

PdCl2(AmPhos)2, KOAc,81%

chiral separation N

F

NNBoc>99% ee

dioxane–H2O, 100 °C, 15 h

N

N

N

N(PMB)2

Cl

N

N

N

NHPMB

Cl

N

N

N

Cl

Cl

N

N

N

Cl Cl

Cl

79% 95% 93%

MeMgBr, Et20,0°C, 30 min

PMBNH2, EtNHiPr,<20°C, 30 min

NaH, DMF, PMBCl,0 °C, 1.5 h

N

F

NNBoc

N N

N

F

NN

N N

1. TFA, CH2Cl22. MsCl, Et3N, DCM, 0 °C NH2

N

MeO

NaHMDS, THF, 0 °C

F

SO O

93%N

N

N

N(PMB)2

N

N

N

N(PMB)2

NN

MeO NN TFA TfOH (10%)

NN

MeO NN

F

93%

F

NH

N

N

N

N

N(PMB)2

N

75 °C, 1.5 h

TFA, TfOH (10%), NH

N

N

N

N

NH2

N

AMG 511

SO O

SO O

52%

442 Ambit

AMG 511 is highly selectivePIK3CA(C420R)

PIK3CA(E542K)

PIK3CB

442 Ambit Kinases POC < 50

PI3K α (Ki) = 4 nMPI3K β (Ki) = 6 nMPI3K δ (Ki) = 2 nMN

N

MeON N

NS

F

PIK3CG

PIK3CA(E545A)

PIK3CA(Q546K)

PIK3CA(E545K)

PIK3CA(I800L)

( i)PI3K γ (Ki) = 1 nM

mTOR (IC50) > 10 μMhVPS34 (IC50) > 9 μM

N

N

N

NH2

NH

SO O

EPHA3 – POC 15

PIK3C2G

PIK3CA

PIK3CD

PIK3CA(H1047Y)

DNAPK (IC50) = 1.2 μM

PIK3C2B

PIK3CA(H1047L)

EPHA3 – POC 15

PIK3CA(M1043I)

CDK7 – POC 42

Class I PI3K’sClass II PI3K’s

POC @ 1 uM from Ambit Kinase Panel(442 kinases)

0 10050

AMG 511 @ 1000 nM

Pharmacokinetic profile of AMG 511PO PK of AMG 511 in Male Sprague-Dawley RatsMicrosomal Clearance

(μL/min/mg)

MsLM = 21MsLM = 21 RLM = 28DLM = 9 MkLM = 93HLM = 27 C

onc

(ng/

mL)

CL Vd MRT

Plasma Protein Binding(% bound)

Ms = 90

Time (h)

Species CL(L/h/kg)

Vdss(L/kg)

MRT(h) %F

Mouse 0.90 3.4 3.8 80

Ms = 90 R = 92D = 96 Mk = 93H = 92

Rat 0.45 1.7 3.9 60

Dog (Beagle) 0.047 1.6 25 65

Monkey (Cyno) 2 0 3 2 1 6 49Monkey (Cyno) 2.0 3.2 1.6 49

Human (projected) 0.34 2.6 5.3 –

Treatment with AMG 511 results in a dose-dependent decrease of pAKT in tumorsdependent decrease of pAKT in tumors

U87 MG Glioblastoma Xenograft Model

350

400

140000

160000

U87 MG Glioblastoma Xenograft Model

250

300

100000

120000

(n

g/m

L)

AK

T *

100

150

200

40000

60000

80000

asm

a C

on

c.

mal

ized

pA

*

*

0

50

0

20000

Veh 0.3 mpk 1 mpk 3 mpk

Pla

No

rm

AMG 511 * = p<0.05= Plasma Conc.

AMG 511 inhibits pAKT in tumors up to 16 hours16 hours

U87 MG Glioblastoma Xenograft ModelA

KT

mL)

mal

ized

pA

Con

c. (n

g/

Nor

m

Plas

ma

C

* = p<0.05AMG 511 (1 k)AMG 511 (1 mpk) AMG 511 (10 mpk) = Plasma Conc.

AMG 511 inhibits tumor growth in a PTEN-null xenograft modelPTEN-null xenograft model

U87 MG Glioblastoma Xenograft Model

1000

1100

1200

)

vehicle, po, qd

AMG 511 - 1 mpk, po, qdED 0 6 mg/kg

700

800

900

ume

(mm

3 AMG 511 - 3 mpk, po, qd

AMG 511 - 10 mpk, po, qd

ED50 = 0.6 mg/kg

AUC = 3.6 μg.h/mL

300

400

500

600

Tum

or V

olu

p < 0.0001

10 15 20 25

100

200

T

Rxp < 0.0001

p < 0.0001

10 15 20 25

Time (days)

AMG 511 Inhibits Growth of KRAS Mutant and of HER2 Amplified Xenograft Modelsof HER2 Amplified Xenograft Models

1500

m3 )

AMG 511 - 3 mpk po qd

vehicle, po, qd

AMG 511 - 1 mpk, po, qd

HCT-116 Colorectal Cancer Xenograft Model

600

700

) AMG 511 3 mpk po qd

AMG 511 - 1 mpk, po, qdvehicle, po, qd

BT-474 Breast Cancer Xenograft Model

500

1000

or

Vol

ume

(mm AMG 511 3 mpk, po, qd

AMG 511 - 10 mpk, po, qd

p = 0.0318

p=N.S.

300

400

500

r V

olu

me

(m

m3

AMG 511 - 10 mpk, po, qdAMG 511 - 3 mpk, po, qd

p=0.0165

p < 0 0001

10 15 20 25 30 350

500

Tum

o

Rx

p < 0.0001

Rx

10 15 20 25 30 350

100

200

Tum

or

p<0.0001

p < 0.0001

10 15 20 25 30 35

Time (days)

10 15 20 25 30 35

Time (days)

Summary• A novel series of pyridyl-triazine PI3K inhibitors was

identified

• SAR studies from a HTS hit led to the discovery of a clinical candidate, AMG 511

• AMG 511 is a potent class I PI3K inhibitor with excellent selectivity over mTOR, related phosphatidylinositoly , p p ykinases, and a broad panel of protein kinases

• AMG 511 inhibited pAKT in tumor tissue in a dose- andAMG 511 inhibited pAKT in tumor tissue in a dose and time-dependent manner

• AMG 511 inhibited tumor growth in PTEN-null KRASAMG 511 inhibited tumor growth in PTEN null, KRASmutant, and HER2 amplified xenograft models

Medicinal ChemistryY i B

OncologyPaul Hughes

CBSSHerve Lebrec

PI3K TeamProcess ChemistryJason TedrowYunxin Bo

Shon Booker Vic CeeNoel D’Angelo

Paul HughesSean CaenepeelNancy ZhangDan FreemanLing Wang

Herve LebrecIrene SotoKeith BaileyDavid HollenbackNancy Everds

Jason TedrowEric BercotAnil GuramMattew Bio

Brad HerberichFang-Tsao HongClaire JacksonBrian Lanman

Ti CaiBarbara GrubinskaXiaoling ZhangJulie LofgrenShiwen Zhang

PKDMRaju SubramanianJian JiangGary Skiles

DSUDavid BauerMichele KubrykJie Yan

Lillian Liao Longbin LiuNobuko Nishimura Mark Norman

Shiwen ZhangTerri Burgess

HTSTisha San Miguel

y

PharmaceuticsTian Wu

Early Development

Molecular StructureKristin AndrewsDoug Whittington

T M d Cha o aLiping PettusTony ReedAdrian SmithMark Stec

Erin MulladyLeeanne Zalameda John McCarterPaul Andrews

y pGreg Friberg

Molecular SciencesGary Means

mTor Med ChemEmily PetersonDaniel LaRuss GraceffaKatrina CopelandMark Stec

Seifu Tadesse Nuria TamayoAndrew TaskerKevin Yang

Rob WahlRenee Emkey

OutsourcingErich Wohlhieter

LegalTodd Crissey

pAlessandro Boezio

Kevin YangRyan WurzBin Wu

y