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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