Pan FGFR inhibitors with p
potent antitumour activity in mouse models of
bladder cancer
Hatfield, April 25th 2014
Agenda
 Introduction to Evotec FGFR inhibitor programme
 Biological Background
 Getting Started
 Preparing for a due diligence
 Aftermath
 Lessons Learned
E t FGFR Programme
Evotec
P
Overview
 Ambitions
 Generate assets in the context of a broader R&D effort aimed primarily at TI/TV (i.e. “CureX”), selecting targets we believe
have value for patients, clinicians & commercial partners
 Leverage Evotec’s infrastructure and in particular the intellectual capabilities of key Evotec employees in the oncology /
kinase arena
 Avoid HTS screening approaches
 Rapidly generate proprietary inhibitors against specific targets to showcase Evotec
Evotec’s
s Medchem and CADD design
capabilities
 SPEND WISELY
Th FGFR family
The
f il off receptors
t
in
i cancer
Deregulation of downstream signalling
•
Fibroblast growth factor receptors
(FGFRs) are a small family of four highly
related receptors,
p
, expressed
p
across a
large variety of cell types
•
FGFRs control key bone developmental
processes and are involved in various
processes,
biological processes including wound
healing and phosphate homeostasis
•
Inactivation or deregulation of FGFRs
leads to hyper- or hypophosphatemia,
respectively
•
Cancers display oncogene-addiction to
C
individual FGFRs, hijacking their central
regulatory function
Nature Reviews Cancer, 2010, Turner and Grose, Fibroblast growth factor signalling: from development to cancer
Parke-Davis: FGFR Pioneers
PD- Crystal Structure (pdb 2FGI)
 H-bond between 3-methoxy and N-H of Asp635
 Water mediated H-bond between urea and catalytic lysine
 Orthogonal orientation of phenyl ring relative to hinge binder
Selective FGFR Inhibitors
OMe
Cl
MeO
O
HN
MeO
OMe
NHtBu
OMe
MeO
N
O
N
N
HN
N
H
Parke-Davis
N
N
N
H
Cl
N
NH
AstraZeneca
O
N
N
N
H
N
N
N
N
H
Novartis
CADD pyrrolopyrazine/pyridine FGF inhibitor
Selective FGFR Inhibitors
1st steps: Pyrrolpyrazine/pyridines
FGFR3BAF3 (nM)
KDRBAF3 (nM)
ControlBAF3 (nM)
37
122
3000
2300
15
70
2800
>3160
cLogD
FGFR3 (nM)
2.5
3260
3.7
260
35
3.5
153
28
2.8
3.4
FGFR1,2,4BAF3 (nM)
OMe
O
MeO
N
H
N
OMe
Cl
O
MeO
Cl
N
H
N
OMe
N
MeO
N
N
NMe
N
NMe
N
H
OMe
H
N
M O
MeO
N
N
N
H
OMe
MeO
N
N
N
N
H
NMe
29
7
482
Selective FGFR Inhibitors
Q1 2012 Pyrrolpyrazine/pyridines
FGFR3
(nM)
FGFR3BAF3 (nM)
KDRBAF3 (nM)
ControlBAF3 (nM)
2.8
37
122
3000
2300
29
7
482
3.4
>10000
4.0
113
24
4214
>3160
10
10
4.6
18
13
5000
>4000
10
10
cLogD
FGFR1,2,4BAF3 (nM)
Heps (%QH)
M
R
H
142
62
75
44
139
59
68
38
OMe
MeO
HN
N
N
NMe
N
H
N
OMe
MeO
N
N
N
N
NMe
N
H
OMe
Cl
MeO
Cl
HN
N
N
N
NMe
N
H
OMe
Cl
MeO
Cl
O
N
N
N
H
NM
NMe
Pyrrolopyrazines
Synthesis
R
R
R
Br
c)
a)
N
b))
Br
N
NH2
OMe
Cl
Br
d)
MeO
N
f)
R
e)
N
N
Cl
HN
N
g)
R
SEM
OMe
N
H
N
OM
OMe
h)
MeO
N
i)
R
N
N
MeO
N
j)
R
N
N
H
SEM
Reagents & Conditions:
a) TMS acetylene/ Pd(PPh3)4 / CuI/ TEA/ Toluene100oC, b) TBAF / THF c) 2-amino 3,5-dibromopyrazine/Pd(PPh3)4/ CuI/ TEA / ACN, 70oC d)
KOtBu/DMF, 100oC e) SEM-Cl / NaH/ THF f) 2,6-dichloro-3,5-Dimethoxy benzylamine / Cs2CO3 / Pd(OAc)2 / X-Phos/ Tol:t-BuOH, 120oC g)
TBAF/ THF h) 3,5-dimethoxy
3 5 di th
styrene,
t
Pd(OA )2 i) H2/Pd,
Pd(OAc)
/Pd j) TBAF/THF
Selective FGFR Inhibitors
Juggling Nitrogens: (Q4 2011)
NHtBu
OMe
O
HN
MeO
OMe
N
MeO
N
N
PD
N
H
N
N
Isoquinoline
N
H
R
Selective FGFR Inhibitors
1st steps: Isoquinolines Q1 2012
cLogD
FGFR3
(nM)
FGFR3BAF3 (nM)
KDRBAF3 (nM)
ControlBAF3 (nM)
FGFR1,2,4BAF3 (nM)
OMe
Heps
(%QH)
Mo
Hu
N
MeO
N
N
4.4
1100
4.0
195
5.2
17
25
1500
3095
14
12
316
21
19
5.4
15
24
1800
1400
15
11
331
27
18
N
H
OMe
N
MeO
N
N
N
N
H
OMe
Cl
N
MeO
Cl
N
N
N
N
H
OMe
Cl
N
MeO
Cl
N
N
N
H
N
FGFR Inhibitor:
I hibit
Isoquinoline
I
i li Synthesis
S th i
Outline reaction scheme
OMe
OMe
Cl
Cl
OMe
Cl
Cl
Br
N
MeO
MeO
N
MeO
N
N
Cl
N
Cl
N
H
+
Cl
N
N
N
N
N
H2N
• Cheap starting materials and many (hetero)aryl amines commercially available
• Short, convergent synthesis facilitates rapid optimization and scale up
Isoquinolines
Initial Rat PK (i.v. leg as cassette)
EOAI3369778 mean plasma levels following IV and PO administration to male Han Wistar
rats at 0.5 mg/kg and 3.8 mg/kg respectively
OMe
Cl
Concentration in plasma (ng/mL)
N
MeO
1000
Cl
N
N
N
N
H
100
intravenous
oral
CL
Vdss
t1/2
F
10
1
0
4
8
12
Time (h)
Ti
16
20
24
30.5 mL/min/kg
19.4 L/kg
8 9 hours
8.9
36%
FGFR Inhibitor:
I hibit
Initial
I iti l Tolerability
T l bilit
Mouse Tolerability - EOAI3369778 (3 mice per group)
OMe
Cl
N
MeO
Cl
N
N
N
H
N
FGFR Inhibitor:
I hibit
Series
S i 2 in
i vivo
i profile
fil
Vehicle
Vehicle
EOAI3369778
EOAI3369778
EOAI3366024 1
Competitor
Competitor
EOAI3378671 2
0.4
0.3
0.2
0.1
%T/C = 44
71
EO
AI
33
78
6
EO
AI
33
66
0
24
78
00
0.0
Ve
hi
cle
Vehicle p.o. q.d.
EOAI3369778 10mpk p.o. q.d.
Competitor 1
p
2
Competitor
0.5
EO
AI
33
69
7
Terminal tumou
ur weight (g)
Mouse efficacy s.c. RT-112 xenograft (10 mice per group)
40
Vehicle
p.o. q.d.
Vehicle
*P-value <0.001
• Efficacy study benchmarking EI3369778 against clinical
competitors @ 10mpk
• RT-112 bladder cancer s.c. xenografts
g
carryy a FGFR3–
TACC3 fusion and amplification
Body w
weight (g)
EOAI3369778
10mg/kg p.o. q.d.
EOAI3369778
EOAI3366024
p.o. q.d.
Competitor10mg/kg
1
EOAI3378671
p.o. q.d.
Competitor10mg/kg
2
30
20
10
12
16
20
Days post implantation
24
FGFR Inhibitor:
I hibit
Isoquinoline
I
i li in
i vivo
i profile
fil
End of efficacy bioanalytics: Plasma, tumor and brain exposure
Plasma Levels
Plasm
ma Concentration
n (nM)
10000.0
Tumor Levels
100000.0
Concentration (nM)
1000.0
100.0
10.0
1.0
10000.0
1000.0
100.0
10.0
1.0
0.1
8
Time (h)
EOAI3369778 (Evotec)
competitor 1
competitor 2
2
24
8
B i Levels
Brain
L l
Concentration (nM)
2
24
time ((hours))
10000.0
1000.0
100.0
10.0
1.0
2
8
Time (h)
24
OMe
Cl
MeO
Cl
Selective FGFR Inhibitors
N
N
R
H
Preparing for due diligence
FGFR3
(nM)
FGFR3BAF3 (nM)
KDRBAF3 (nM)
ControlBAF3 (nM)
5.4
15
24
1800
1800
15
11
4.6
4
12
1300
1600
33
4.3
21
4
1535
10000
54
5.4
23
34
1000
3.2
6
32
2658
cLogD
FGFR1,2,4BAF3 (nM)
Heps (%QH)
hERG
(nM)
Mo
Hu
PC
331
27
18
886
14
345
46
32
3890
6
3
200
41
22
120
10000
40
20
574
36
14
3160
28
22
581
56
26
N
N
N
N
N
N
N
O
N
NH
N
N
O
NH
N
256
OMe
Cl
MeO
Cl
Selective FGFR Inhibitors
N
H
Preparing for due diligence
FGFR3
(nM)
FGFR3BAF3 (nM)
KDRBAF3 (nM)
ControlBAF3 (nM)
5.2
4
5
635
1351
4.3
22
15
2705
2513
N
5.8
87
29
3160
10000
NM
NMe
32
3.2
21
39
731
1305
6.1
5000
36
3000
3000
cLogD
hERG
(nM)
PC
N
N
N
N
N
N
N
N
N
1466
N
N
N
N
N
2280
R
OMe
Cl
MeO
Cl
Selective FGFR Inhibitors
N
N
R
H
hERG binding (due diligence -1)
cLogD
FGFR3
(nM)
FGFR3BAF3
(nM)
KDRBAF3
(nM)
ControlBAF3
(nM)
FGFR1,2,4BAF3 (nM)
Heps
(%QH)
hERG (nM)
Mo
Hu
patch
binding
N
N
N
N
N
N
N
15
24
1800
1400
15
11
331
27
18
886
3
4.6
4
12
1300
1600
33
14
345
46
32
3890
3
4.3
21
4
1535
10000
6
3
200
41
22
120
4
54
5.4
23
34
1000
10000
40
20
574
36
14
3.2
6
32
2658
3160
28
22
581
56
26
256
3
O
N
NH
N
5.4
N
O
NH
N
FGFR Inhibitor:
I hibit
Isoquinolines
I
i li
Effect of 2,6 dihalo
H
N
O
R
N
Cl
N
N
R
H
OMe
F
MeO
Hepatocytes %QH
R
Rat PK
CL
(mL/min/kg)
t1/2
H
N
N
N
N
3369777
3369778
3379459
3379458
4
25
24
12
35
1535
1500
1795
1300
>1000
>10000
3095
1868
1600
>10000
IC50 [nM] BaF3 control
Cl
N
N
3380208
IC50 [nM] BaF3-KDR
MeO
N
N
N
N
IC50 [nM] BaF3-FGFR3
OMe
O
N
N
36
22
63
<19
26
18
34
32
22
<14
2
7.2
6
31.9
8
30.5
9
82
5
5.4
3383105
3383104
3383103
3379365
3379364
IC50 [nM] BaF3-FGFR3
<3
18
20
50
10
IC50 [nM] BaF3-KDR
264
400
658
419
238
>3160
2459
>1000
2608
>10000
F
N
N
H
R
IC50 [nM] BaF3 control
Hepatoc tes %QH
Hepatocytes
R
Rat PK
CL
(mL/min/kg)
t1/2
H
55
22
89
66
11
35.9
1
111
Selective FGFR Inhibitors
Identification of stabilised pyrollopyrazines
cLogD
FGFR3
(nM)
FGFR3
-BAF3
((nM))
KDRBAF3
((nM))
ControlBAF3
((nM))
FGFR1,2,4BAF3 (nM)
Heps (%QH)
hERG (nM)
M
R
H
patch
binding
62
75
44
61
69
14
36
45
29
1768
1411
7776
2156
OMe
Cl
MeO
Cl
HN
N
N
N
4.0
113
24
4214
3160
10
10
142
3.5
49
530
10000
10000
2.7
10
20
>3160
>3160
8
6
316
2.9
11
76
3878
3091
67
62
263
NMe
N
H
OMe
Cl
O
MeO
Cl
HN
N
N
N
N
H
N
OMe
Cl
N
MeO
Cl
HN
N
N
N
H
N
N
OMe
Cl
N
MeO
Cl
HN
N
N
N
N
H
N
Pyrrolopyrazines
Initial Rat PK (cassette)
EOAI3381414 mean plasma levels following IV and PO administration to male Han Wistar
rats at 0.5 mg/kg and 1.5 mg/kg respectively
OMe
Cl
1000
N
Pla
asma concentrration (ng/mL)
MeO
Cl
HN
N
N
Intravenous
100
N
N
H
N
Oral
CL
Vdss
t1/2
F
10
1
0
4
8
12
Time (h)
Ti
16
20
24
30.7 mL/min/kg
13.4 L/kg
6 2 hours
6.2
21%
FGFR Inhibitor: Isoquinoline + Pyrrolopyrazine
i vivo
in
i profile
fil
Pyrro
olopyrazines
Isoquinolines
Mouse tolerability s.c. RT-112 xenograft (3 mice per group)
• Preliminary tolerability screen of 12
compounds select compounds in both series
are able to induce regressions at a low
tolerated dose of 10 mg/kg (or 5 mg/kg)
FGFR Inhibitor:
I hibit
I vivo
In
i MTD
Kyphosis phenotype indicates on-target toxicity at MTD
• The majority of compounds tested at MTD
showed associated kyphosis
kyphosis, or prominent
spine morphology, following 10-14 days
dosing
• The same phenotype was observed with
clinical FGF inhibitors
• Phenotype consistently correlates with drug
potency/tumor
t
/t
regressions
i
• On-target toxicity for FGFR inhibitors is
directly correlated with tissue calcification and
hyperphosphatemia
FGFR Inhibitor:
I hibit
I vivo
In
i profile
fil off preferred
f
d candidates
did t
Mouse efficacy s.c. RT-112 xenograft (10 mice per group)
*
*3
3 mice from groups 3 given a 3 day dosing holiday mid study
• Both compounds showed modest regressions at doses as low as 5mpk
• On
On-target
target toxicities (hypophosphataemia) validated drug MoA
FGFR Inhibitor:
I hibit
I vivo
In
i profile
fil off preferred
f
d candidates
did t
End of efficacy bioanalytics: Plasma and tumour exposure
Ki
Kinase
S l ti it
Selectivity
Threats and opportunities
Kinase Tested
ABL1
ACVR1B (ALK4)
AKT1 (PKB alpha)
AMPK A1/B1/G1
AURKA (Aurora
(A
A)
AURKB (Aurora B)
AXL
BTK
CDK1/cyclin B
CDK2/cyclin A
CHEK1 (CHK1)
CHEK2 (CHK2)
CLK2
CSF1R (FMS)
CSNK1G2 (CK1 gamma 2)
CSNK2A1 (CK2 alpha 1)
DYRK3
EGFR (ErbB1)
EPHA2
EPHB4
ERBB2 (HER2)
FGFR1
FLT3
FRAP1 (mTOR)
FRK (PTK5)
FYN
GSK3B (GSK3 beta)
HIPK2
IGF1R
IKBKB (IKK beta)
INSR
IRAK4
JAK1
JAK2
JAK3
EOAI3369777
EOAI3401967
EOAI3401985
% Inhibition
% Inhibition
% Inhibition
mean
mean
mean
64
85
99
-1
-12
9
-2
3
-2
-5
-1
-3
23
17
34
-10
11
23
22
39
15
-1
-6
31
-5
-6
-4
-3
-5
-4
-2
-12
1
-2
2
-4
4
2
-5
-3
-6
98
98
96
-4
-21
-15
-8
-14
-5
-7
-13
-7
-6
-7
2
-8
-2
48
9
5
33
-21
-13
-22
99
98
98
18
19
37
-5
-12
-1
18
5
87
37
36
79
-4
4
-7
7
-4
4
-11
-10
-11
-1
-4
13
15
7
-3
29
3
29
-8
-10
1
7
-2
-2
6
15
1
14
37
3
EOAI3369777
EOAI3401967
EOAI3401985
Kinase Tested
% Inhibition
mean
% Inhibition
mean
% Inhibition
mean
KDR (VEGFR2)
KIT
LCK
MAP2K1 (MEK1)
MAP3K8 ((COT))
MAP4K4 (HGK)
MAPK1 (ERK2)
MAPK14 (p38 alpha)
MAPK3 (ERK1)
MAPK8 (JNK1)
MAPKAPK2
MARK2
MET (cMet)
MKNK1 (MNK1)
NEK1
NTRK1 (TRKA)
PAK4
PDGFRA (PDGFR alpha)
PDGFRB (PDGFR beta)
PHKG2
PIM1
PLK1
PRKACA (PKA)
PRKCB1 (PKC beta I)
PTK2 (FAK)
RET
ROCK1
RPS6KA3 (RSK2)
RPS6KB1 (p70S6K)
SNF1LK2
SRC
SYK
TBK1
TEK (Tie2)
ZAP70
PIK3CA/PIK3R1 (p110 alpha/p85 alpha)
(p110 delta/p85
p alpha)
p )
PIK3CD/PIK3R1 (p
PIK3CG (p110 gamma)
88
37
68
3
-11
17
1
39
-7
-11
0
0
10
-6
1
47
-13
15
24
2
6
-16
2
0
-9
63
-8
9
0
-3
37
-6
-9
71
-6
0
14
1
91
34
88
8
-4
41
-6
23
-16
-10
-1
-6
18
-5
17
40
-7
24
21
5
10
-15
-3
3
-10
68
-14
7
1
5
37
-19
-14
89
-7
-7
11
7
93
65
92
-3
10
4
-2
13
-9
3
-3
0
5
-7
-2
78
-22
29
34
-11
1
-12
-3
1
-5
73
-9
-4
-1
47
89
-12
-7
79
-5
-9
-28
-2
OMe
Cl
N
MeO
N
Cl
N
3369777
N
N
H
OMe
Cl
N
MeO
Cl
N
N
N
3401967
N
H
OMe
Cl
N
MeO
Cl
HN
N
N
N
N
H
N
3401985
Ki
Kinase
S l ti it
Selectivity
Interaction with threonine gatekeeper
S
Summary
and
d Lessons
L
L
Learned
d
Overview
 2 series of selective FGFR inhibitors identified using low/moderate resources
 In vivo
i o efficac
efficacy demonstrated with
ith both series
 Potency at least comparable to clinical FGF inhibitors
 Excellent pharmacokinetic profile attained
 Progressing compounds in parallel has it’s downsides
 Compounds were progressed that would have been stopped earlier using sequential profiling
 Progressing compounds in parallel has it’s upsides
 Some of the efficacy data was surprising in light of the in vitro data
 Compounds disclosed are being investigated further, together with related analogues
A k
Acknowledgments
l d
t
 Chemistry





Matthew Mills
Manojkumar Prabhu
Uday Joshi
Thane team
Andrew Phillips




Michael Garrigou
Patricia Amouzegh
James Jenkins
Tim Jones
 Reformatting team
 Adam Grey
 Sally Davies
 Richard Dunn
 CADD
 Mike Mazanetz
 Michelle Southey
 DMPK
 Chris Chantler
 Joanne Sproston
 Pharmacology
 Joanna Lisztwan
 Saretius
 ACD/Carna
 Invitrogen
 Precos