Optimization of -Secretase Modulators
through Variation of Scaffolds
Thomas Lübbers
Proteinase 2013 - 8th RSC-SCI Symposium on Proteinase Inhibitor Design - Basel, 15th-16th April 2013
"No man is rich enough to buy back his past."
- Oscar Wilde -
Acknowledgement
Chemistry
Alexander Flohr
Guido Galley
Erwin Götschi
Luke Green
Synese Jolidon
Henner Knust
Anja Limberg
Thomas Lübbers
Robert Narquizian
Andrew Thomas
Biology
Karlheinz Baumann
Helmut Jacobsen
Laurence Ozmen
Edilio Boroni
Theresa Ballard
Modeling (HTS)
Harald Mauser
Safety
Franziska Boes
Susanne Mohr
Stephan Kirchner
HTS
Joergen Nielsen
Process Research
Fabienne Hoffmann-Emery
Hydrogenation
Shaoning Wang
DMPK
Pascale David-Pierson
Angele Fleury
Monique Schmidt
Formulation
Kerstin Gräser
Patent
Regina Poppe
And many more …
LMU München
Amelie Gutsmiedl
Harald Steiner
Christian Haass
Benedikt Kretner
Richard Page
Akio Fukumori
Keiro Shirotani
Alzheimer Disease
Progression of Neurodegeneration
ca. 10 years
Mild
Cognitive
Impairment
Death
(caused by pneumonia,
infections a. o. reasons
due to bed-ridden condition)
Mild
- loss of recent memory
- personality changes
Moderate
- aggression
- difficulties in daily living
Severe
- loss of body control
Genetics of Alzheimer`s Disease
95% sporadic
onset >65 years
5% genetic
Genetics of familial AD
early onset
39% PS1
0.5%
PS2
60%
other
0.5%
APP
all mutations lead to an increase of the neurotoxic A42
Hallmarks in Alzheimer`s Disease
Neuropathology
Tau pathology
Tangles
Hyperphosphorylated tau
Plaque pathology
•
•
•
•
Decrease of synaptic density
Loss of neurons
Shrinkage of brain tissue
Enlargement of ventricles
-Amyloid plaques
mainly A 42also A 40
Amyloid Cascade Hypothesis
APP
Notch
NH2
NH2
TACE
extracellular
APPs

A

TM


APP-CTF C99
AICD
NICD
CO2H
CO2H
cytosol
Nucleus
Gene regulation
cell differentiation
Properties of -Secretase
 High molecular weight complex:
Presenilin-1 or 2 (PS1/2), Anterior pharynx defective-1 (APH-1),
Presenilin enhancer-2 (PEN-2), Nicastrin
 Located within the membrane
 Atypical Asp-protease (GXGD motif)
 catalytic Aspartates in TMD 6 and 7 of PS
 Low (or unknown?) substrate specificity
-Secretase
Substrates
APP
Notch1
APLP1/2
Notch2-4
ErbB-4
LRP
E-cadherin
CD44
DELTA1
Many others .....
Amyloid Cascade Hypothesis
APP Processing
APP
membrane
membrane
Ratio: 10 : 80 : 10
-secretase
Aspartyl Protease
42
38
D257 TM6
40
D386 TM7
APP model in the TM:
helix has 3.6 residues per turn:
38 and 42 on the same site
GxxxG motif
-secretase
~
~
N …ISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK… C
NL
APPsw

mutation 
A   

AICD
-Secretase Inhibitor Semagacestat
Lack of Efficacy/Worsening of Cognition in Phase III
Trials
•
Worsening of clinical measures of cognition and
the ability to perform activities of daily living
•
Increased risk of skin cancer
•
Gastrointestinal and immune system side effects
(interference with maturation of B- and T-lymphocytes)
Probably related to inhibition of Notch processing.
OH
O
H
N
O
N
H
N
O
-Secretase Modulation
NSAIDs and Alzheimer’s
 Epidemiological studies show reduced prevalence of AD in chronic NSAID users
 Clinical trials in AD with NSAIDs
 Indomethacin (1993) showed significant benefits in a 6 month trial as
compared to placebo Rogers et a., 1993 Neurology
 Diclofenac (1999) showed a reduced change in ADAS scores as
compared to a placebo estimate
-Secretase Modulation
NSAIDs and Alzheimer’s
Effect of a subset of NSAIDs on APP processing
Weggen et al, 2001, Nature, Eriksen et al. 2003 J Clin Invest.
 subset of NSAIDs reduce selectively the toxic A42 peptide in vitro and in
vivo
 in vitro:
reduction of A42
increase of A38
no effect on A40
 effective in COX knock-out cells
Weggen et al, 2001, Nature
 active in cell-free -secretase
assay indicating direct modulation of enzyme activity
Sulindac Sulfide
-Secretase Modulation
H
N
O
N
N
N
N
Cl
RO5434400
OH
% change from vehicle-treated
HEK293/Sw Cells
A38
A40
A42
600
500
control IC50
5xIC50
400
300
A37
A38
200
100
A40
0
-10
-9
-8
-7
-6
A42
RO-02 [log(M)]
RO5434400 [log(M)]
IC50 (A42): 15 nM
Maldi-ToF
A peptides
Production of major A species.
Modulation takes place as expected.
Shift to smaller non-toxic A peptides;
larger peptides not ovserved.
Gutsmiedl et al.
-Secretase Modulation
Photoaffinity Label
H
N
O
O
O
N
N
N
N
O
N
N
O
H
N
N
O
O
O
N
H
N
HN
H
O
3
N
H
NH
H
S
O
RO-57-BpB
IC50 (A42): 185 nM
350
A38
A40
A42
300
250
200
150
100
50
0
-10
-9
-8
-7
RO-57 [log(M)]
-6
% change from vehicle-treated
% change from vehicle-treated
RO-57
IC50 (A42): 47 nM
400
A38
A40
A42
350
300
250
200
150
100
50
0
-8
-7
-6
-5
-4
RO-57-BpB [log(M)]
Gutsmiedl et al.
-Secretase Modulation
% Input +
kDa 0.3 1 105
+
+
+
+
+
+
+
UV
RO-57-BpB
NCT
ve
hi
R cle
O
-5
7
ve
hi
R cle
O
-5
R 7
O
54
34
4
00
Roche GSMs target the Presenilin NTF
Input + kDa 1% - +
+
+
+ UV
+ RO-57-BpB
34
PS1NTF
PS1CTF
34
PS2NTF
34
PS2NTF
16
7
17
PS2CTF
17
APH-1aL/S
34
PS1NTF
17
7
APPCTF
APPCTF
PEN-2
Gutsmiedl et al.
-Secretase Modulation
ve
hi
cl
R e
O
L- 57
68
54
58
D
A
P
LY T
-4
1
be 157
ga 5
ce
st
at
Differential Labeling Competition by GSIs
Input kDa 1 % +
+
+
+
+
+
+
+
+
+
+
+
+
UV
RO-57-BpB
34
PS1NTF
34
PS2NTF
•
GSM compete with RO-57-BpB (data not shown)
•
Only slight competition with transition-state analogue L-685458
•
Other GSIs do not compete
Gutsmiedl et al.
-Secretase Modulator
High Througput Screen
HTS Hit
O
S
H
N
O
N
O
O
N
H
N
Cl
Eisai
N
S
N
N
TorryPines
N
N
A42 IC50 = 800 nM
IC50 = 140 nM
O
N
N
H
N
A42 IC50 = 214 nM
F
IC50 = 671 nM (H4 cells)
42 nM (rat primary neurons)
S
N
N
N
Cl
Me-Imidazol improves
activity by factor of 4
Aminothiazoles
Profile of RO5188492
A38, 40, 42
H
N
O
RO5188492-000-001
S
%
350
viability
%
300
N
N
N
150
250
Cl
200
120
150
90
100
60
50
30
0
0.1
A42 IC50 = 214 nM (H4 cells)
130 nM (rat primary neurons)
1
10
0
0.1
µM
permeability
Low
Cmax/dose [ng/mL]
181
logD
>3
T1/2 [h]
2.6
clogP
6.5
Vss [l/kg]
2.7
Solubility [mg/L]
<1
Cl [mL/min/kg]
15
IC50 CYP 3A4 / 2D6 / 2C9 [M]
Microsomes
1.2, 0.7, 0.7
F [%]
64
MAB [%]
h
17
Br/pl
0.61
m
40
Fu [%]
<0.1
flag
10
µM
Mouse PK Profile po
GSH adduct h
1
Aminothiazoles
in vivo Profile of RO5188492
Transgenic mice model
APP Sw mice po 4h
brain A levels
Non-Transgenic rat model
Wistar rats canjulated in the Cisterna Magna
Brain CSF A levels
Ve
hi
cl
e
MR
10 K 5
m 60
g/
kg
RO
15 518
0 m 84
g/ 92
kg
150 mg/kg
F
F
F
O
O
S
O
Cl
N
H
F
F
S
O
Aminothiazoles
Profile of RO5220778
A38, 40, 42
H
N
O
RO5220778-000-001
%
640
S
560
N
N
N
viability
%
150
480
120
400
90
320
240
60
160
30
80
A42 IC50 = 44 nM (H4 cells)
64 nM (rat primary neurons)
0
0.01
0.1
1
µM
Similar physicochemical and MDO profile as RO5188492
Microsomes
MAB [%]: h
52
m
49
GSH adduct h
flag
0
0.01
0.1
1
µM
Transgenic mice model
APP Sw mice po, 4h
brain A levels
Aminothiazoles
in vivo Profile of RO5220778
A42
A
Atotal
Aof ctrl ± SEM)
A40
le
ic
h
Ve
10
10
MRK
560
30
100 mg/kg
RO5220778
le
ic
h
Ve
10
MRK
560
10
30
100 mg/kg
RO5220778
le
ic
h
Ve
10
MRK
560
• In vitro profile translates into in vivo profile
10
30
100 mg/kg
RO5220778
le
ic
h
Ve
10
MRK
560
10
30
100 mg/kg
RO5220778
-Secretase Modulator
Head Group SAR
important to avoid
CYP inhibition
R
IC50 A42 [nM]
MeO
44
F
53
NC
120
H
130
Me
730
Cl
680
CF3
inactive
H
N
Annelated Aminothiazoles
S
Head Group SAR
40 nM
240 nM
N
IC50 A 42
inactive
5547 nM
86 nM
127 nM
252 nM
N
651 nM
3431 nM
1000 nM
320 nM
483 nM
2867 nM
•
2-Substitution is sometimes allowed - sometime not, 3-Me beneficial
•
Nitrogen atom needed in 3-/4-position of 5-/6-membered heterocyclic head group
•
No correlation of in vitro potentcy to basicity or H-bond strength
N
376 nM
Annelated Aminothiazoles
Head Group SAR
H
N
N
N
S
N
N
N
N
2180 nM
IC50 A 42
862 nM
N
S
N
N
N
S
N
N
221 nM
N
N
N
N
414 nM
O
270 nM
•
A piperidyl ring is a good replacement for the phenyl ring.
•
The head group determines the exposure of the compound in vivo
and thus its in vivo activity
•
The SAR of the piperidyl and the phenyl head groups are not transferrable
N
N
N
S
1820 nM
-Secretase Modulator
Evolution of the Aminothiazoles
O
N
N
H
N
H
N
O
S
R
H
N
N
N
N
N
N
N
N
N
MeO 477 nM
F
129 nM
A42 IC50 = 44 nM
N
154 nM
Lipophilic
promiscous
O
Position identified, which
permits the introduction of
a polar group
N
N
H
N
N
N
OH
N
CF3
100 nM
O
N
H
N
N
N
R
H 322 nM
CF3 317 nM
Aminopyrimidine
Profile of RO5321402
O
N
N
H
N
N
OH
A38, 40, 42
RO5321402-000-001
N
%
240
200
160
120
CF3
80
40
A42 IC50 = 100 nM (H4 cells)
31 nM (rat primary neurons)
Notch IC50 >10000 nM
0
0.1
1
10
µM
Physicochemical properties not improved
Ames
2.5 M
clean
76
MNT
borderline
95
TDI
clean
GSH adduct h
clean
phototox
clean
IC50 CYP 3A4 / 2D6 / 2C9 [M]
Microsomes
9, 16, 12
MAB [%]
h
m
hERG IC20
Aminopyrimidines
in vivo Profile of RO5321402
Transgenic mice model
APP Sw mice po, 4h
brain A levels
brain A42
100
*
75
**
**
50
25
RO5321402
m
g/
kg
10
0
kg
30
m
g/
kg
g/
m
10
MRO5
R 02
K 8
5661
07
0
Ve
hi
cl
e
A (% of ctrl  SEM)
125
Aminopyrimidines
MNT Issue – Screening Strategy
• The onset of the clastogenic effect was accompanied by a strong cytotoxic activity.
• Changes observed in the nuclei of treated cells indicate an interference in the
replication phase of the cell cycle leading to cell arrest possibly due to DNA
intercalation.
• No particular kinase inhibitory effect was found (ambit screen).
Screening Strategy:
• LD-cells (replicating):
cytotoxicity will indicate cell cycle arrest and general cytotoxicity
• HD-cells (non-replicating):
captures general cytotoxicity e.g. by interference with metabolism, cell integrity, etc.
• Ratio HD/LD will indicate a specific contribution of the cytotoxicity due to cell cylce
arrest
Aminopyrimidines
MNT Issue – Screening Results
Cytotoxicity on cT3 cultures
O
N
N
H
N
N
R1
R1
R2
LD-IC50
[M]
HD-IC50
[M]
HD/LD
MNT
H
H
51
>100
>1.9
neg
Me
Me
0.2
197
1214
pos
H
6.8
>100
>17
pos
i-Pr
n
n
n
pos
3.8
46
12
pos
6.1
12
1.9
neg
4.1
14
3.4
neg
N
R2
MNT due to
Small size of the modulators
OH
OH
Pyrimidine core
Imidazole head group
OH
OH
N
OH
F
F
O
OH
F
F
Good correlation between specific cytotoxic effect on low-density cells with simplified
MNT assessment.
Aminopyrimidines
Profile of RO5434400
H
N
O
N
N
A38, 40, 42
OH
RO5434400-000-001
N
N
%
360
300
240
180
Cl
120
60
A42 IC50 = 180 nM (H4 cells)
13 nM (rat primary neurons)
Notch IC50 >10000 nM
0
0.01
0.1
µM
1
Physicochemical properties not improved
Ames
1.5 M
negative
22
MNT
negative
60
TDI
positive
GSH adduct h
borderline
IC50 CYP 3A4 / 2D6 / 2C9 [M]
Microsomes
1.4, 1.1, 1.5
MAB [%]: h
m
hERG IC20
Aminopyrimidines
in vivo Profile of RO5434400
Transgenic mice model
APP Sw mice po, 4h
brain A levels
Non-Transgenic rat model
Wistar rats canjulated in the Cisterna Magna
Brain CSF A levels
A42
100
100
**
75
***
***
50
***
25
CSF A42 (% of ctrl)
120
80
60
40
RO5434400 10 mg/kg
RO5434400 30 mg/kg
vehicle
-1
00
m
g/
kg
kg
-3
0m
g/
0
O
54
34
40
0
0
20
0
0.0
2.5
5.0
7.5
time (h)
R
R
O
54
34
40
R
O
54
34
40
00
-3
-1
0m
g/
m
g/
kg
kg
kg
30
m
g/
R
O
54
34
4
Ve
h
ic
le
0
Ei
sa
ir
ef
.-
A (% of ctrl  SEM)
A 4 2
125
•
RO5434400 shows comparable in vivo efficacy to E2012 in mice after 4 hs po.
•
Dose dependent and prolonged (up 8h) decrease of CSF A42 at 10 and 30 mg/kg.
24.0
Aminopyrimidines
in vivo Profile of RO5434400
Transgenic mice model
APP Sw mice po 4h
brain A levels
Reduction of brain A42 [%]
Dose by shape
H
N
O
N
N
OH
N
N
Cl
RO5434400
O
ED50 ≤ 30 mg/kg
O
N
F
N
N
Chiral
RO5272554
E2012
Exposure plasma [ng/ml]
•
Excellent correlation between exposure and in vivo effect.
Aminopyrimidines
Metabolic Stability – human versus mouse
H
N
O
OH
N
N
N
N
N
N
OH
N
OH
N
N
N
N
OH
N
O
Cl
MetID
confirms main
metabolic site
IC50 A42
MAB [%] h
•
Cl
Cl
Cl
Cl
180 nM
320 nM
230 nM
420 nM
540 nM
RO5434400
RO5458087
RO5461303
RO5462431
RO5464157
22
48
55
76
100
Stability in human microsomes can be achieved.
OH
Aminopyrimidines
Introduction of Polar Groups
H
N
O
N
N
N
OH
N
N
N
N
N
N
N
OH
N
OH
N
N
OH
Cl
OH
Cl
OH
OH
F
IC50 A42
180 nM
130 nM
logD
solubility [mg/L]
3.7
<1
3.9
<1
RO5434400
RO5456508
22
no
59
no to weak
MAB [%] h
PgP substrate
N
170 nM
RO5454686
good
Cl
OH
Cl
OH
F
400 nM
450 nM
258 nM
3.8
<1
3.3
5.0
3.1
27
RO5465930
RO5465931
RO5468879
90
good
58
80
strong
•
Introduction of hydroxy group in several positions allowed.
•
Lower plasma exposure and lower brain/plasma ratio – PgP
substrates.
How to improve in vitro Potency?
Scaffold Hopping
F
O
N
H
N
N
S
N
H
N
O
N
N
OH
O
H
N
N
N
N
N
N
N N
Cl
IC50 A42
Thiazoles
Pyrimidines
Triazoles
RO5220778
RO5324400
RO5506339
40 nM
180 nM
92 nM
Eight Combinations of Triazolopyridines
F
N
N N
H
N
O
N
N
H
N
N
S
N
N
F
F
F
N
N
N
N
N
N
N N
N
IC50 A42 [nM]
Solubility [g/ml]
MAB h/m
Pgp substrate
In vivo, 30mpk”
br/pl
RO5505096
330
<1
65/ 51
good
80%
0.1
RO5503981
324
<1
72/ 46
good
inactive
0.08
RO5506339
92
<1
63/ 39
RO5502119
53
<1
too scatt.
65%
0.9
inactive
-
IC50 A42 [nM]
Solubility [g/ml]
MAB h/m
Pgp substrate
In vivo, 30mpk”
br/pl
RO5507303
367
430
100/ 60
weak/ good
88%
0.1
RO5505101
547
265
70/ 22
weak/ good
inactive
0.2
RO5506284
219
<1
55/ 29
RO5505290
260
8
31/ 14
52%
0.4
81%
0.4
*APPSw mice, 4h, po, 30 mg/kg, brain %A42
Aminotriazolopyridines
in vivo Profile of RO5514194
F
O
N
N
H
N
N
A42 IC50 = 283 nM (H4 cells)
N N
brain
Exposure brain/plasma [ng/g]:
460/ 4640
0.10
2740/ 4560
0.60
Transgenic mice model
APP Sw mice po, 4h
brain A levels
Pgp transport is responsible
for low brain/plasma ratio
Aminotriazolopyridines
Profile of RO5519402
F
O
H
N
N
Cl
N
N
N N
RO5519402
in vivo brain A 42 56% (30 mg/kg)
62% (10 mg/kg)
APPSw mice 4h po
GSH adduct h positive
A42 IC50 = 30 nM (H4 cells)
 6-Me substituent improves CYPs and exposure
[O]
GSH adduct:
Working hypothesis
H
N
O
Human/ rat liver
microsomes
N
N
O
N
O
O
SG
M
+GSH
-Me-imidazole
+O
-2Da
Aminotriazolopyridines
Profile of RO5506284
F
H
N
N
S
N
N N
N
N
A42 IC50 = 219 nM (H4 cells)
Met ID
permeability
Med to high
logD
n.o.
clogP
3.8
Solubility [mg/L]
<1
IC50 CYP 3A4 / 2D6 / 2C9 [M]
Microsomes
>50, 1.0, 15
MAB [%]: h
55
m
29
TDI
positive
GSH adduct h
positive
Ames
negative
MNT
negative
Desulfuration of the thiadiazole
Human liver
microsomes
RO5429560
H
N
HO
HN
N
M-14:
M-S+O+2Da
N
H
N
G
S
GSH adduct: M-S+GSH-2Da
N
HN
N
Aminotriazolopyridines
in vivo Profile of RO5506284
F
H
N
N
S
N
N N
N
N
20
93
60
52
 Dose dependent reduction of brain A42
Transgenic mice model
APP Sw mice po, 4h
brain A levels
-Secretase Modulator
Summary
 -Secretase modulator bind to the N-terminus of PS
 Nanomolar -secretase modulators identified starting form an HTS hits
 Good Pk properties
 Excellent in vivo activity in transgenic mouse model
 Several safety issues solved
 Reactive metabolites (TDI and GSH) are still an issue to be solved
Doing now what patients need next
-Secretase Inhibitors
Representative Examples
OH
H
N
O
O
H
N
O
NH 2
N
H
O
O
H
N
F
O
OH
N
H
O
O
O
O
L-685458
F
S
O
Cl
MRK 560
O
O
F
F
N
H
F
F
S
Cl
N
H
N
O
LY-411575
DAPT
O
O
F
F
O
H
N
F
S
S
N
H
O
Begacestat
OH
CF3
CF3
OH
O
H
N
O
N
H
N
O
Semagacestat
-Secretase Modulation
GSMs Display Differential Labeling Competition
O
O
O
N
OH
OH
F
O
N
N
F
N
F
F
Cl
S
Input - +
1% + +
+
+
+
+
GSM-1
180 nM
weak PS1, strong PS2
ve
hi
c
R le
O
-5
su 7
l.
s
G u lfi
SM d
-1 e
Sulindac Sulfide
70 M
strong
ve
hi
cl
R e
O
-5
ES 7
IC50 (A42):
competition
Eisai (ES)
40 nM
strong
F
Input 1% +
+
+
+
+
+
+
+
+
UV
RO-57-BpB
PS1NTF
PS2NTF
Gutsmiedl et al.
Aminothiazoles
Synthesis
Meerwein Reaction
DBU
CuCl2
tBuONO
NH 2
R
+
Cl
R
Cl
R
R
O
+
R
+
Cl
R
O
O
EtOH,
reflux
O
N
O
Cl
NO2
N
H
N
S
N
O
1. KOH, DMSO
H2O, 80°C
2. Pd/C H2
R
R
N
N
NH2
1. EtOCONCS
2. NaOH
O
N
N
H
N
NH2
S
Benzylaminopyrimidine
Synthesis
O
COOEt
+
COOEt
Cl
HO
N
KOtBu
Et2O
r.t.
COOH
H
N
O
N
O
+
EtOH
K2CO3
heat
H
N
O
N
COOEt
N
N
N
2 HCl
N
orotic acid
NH2
COOEt
NH
N
OH
O
Cl
Cl
MeMgCl/THF
HO
N
COOMe
N
OH
Cl
O
NH2
N
OH
N
+
N
Cl
N
N
N
Cl
Cl
N
RO5434400-000
Cl
ZnCl
Cl
Cl
dioxane, K2CO3
N
N
N
COOMe
H
N
O
Pd(0)
N
N
Cl
Cl
OH
N
Cl
Pd(0)
N
O Si
N
Cl
N
Cl
O Si
OH
Aminopyrimidines
MNT – Conformational Aspects
MNT due to
Small size of the modulators
Pyrimidine core
F
N
N
N
N
N
Imidazole head group
1 MNT: neg. (0-50 uM)
X-ray structure of 2
X-ray structure (MeO)
Cl
MeO
N
N
N
N
N
HO
Overlay of 1 (blue) and 14 (red). Minimized
conformations de-rived from x-ray structure of 2.
14 MNT: neg. (0-35 uM)
Benzyl substituents abolish the MNT effect