Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: Discovery of PF-3084014

Article abstract of DOI:10.1016/j.bmcl.2010.12.118

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aβ in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aβ EC₅₀ lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

Full text of DOI:10.1016/j.bmcl.2010.12.118

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2637–2640
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and in vivo characterization of a novel series of tetralin
amino imidazoles as c-secretase inhibitors: Discovery of PF-3084014
Michael A. Brodney a, , David D. Auperin b, Stacey L. Becker c, Brian S. Bronk a, Tracy M. Brown b,
Karen J. Coffman a, James E. Finley b, Carol D. Hicks b, Michael J. Karmilowicz b, Thomas A. Lanz b,
Dane Liston b, Xingrong Liu c, Barbara-Anne Martin b, Robert B. Nelson b, Charles E. Nolan b,
Christine E. Oborski b, Christine P. Parker b, Karl E. G. Richter b, Nikolay Pozdnyakov b, Barbara G. Sahagan b,
Joel B. Schachter b, Sharon A. Sokolowski b, Barbara Tate b, Douglas E. Wood b, Kathleen M. Wood b,
Jeffrey W. Van Deusen c, Lei Zhang a
⇑
a Neuroscience Medicinal Chemistry, Pfizer PharmaTherapeutics Research and Development, Eastern Point Road, Groton, CT 06340, USA
b Neuroscience Research Unit, Pfizer PharmaTherapeutics Research and Development, Eastern Point Road, Groton, CT 06340, USA
c Pharmacokinetics, Dynamics, and Metabolism, Pfizer PharmaTherapeutics Research and Development, Eastern Point Road, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding
phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent ser-
ies of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Ab in guinea pigs in
the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing
compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations
at the brain Ab EC50 lead to the identification of compound 14f (PF-3084014) which was selected for clin-
ical development.
Received 1 November 2010
Revised 22 December 2010
Accepted 23 December 2010
Available online 30 December 2010
Keywords:
c
-Secretase inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Tetralin-imidazoles
Notch
Ab Reductions
Marginal zone B-cells
Multiple lines of genetic and pathologic evidence have impli-
cated the Ab peptide in the etiology of Alzheimer’s disease (AD).
The last step in production of amyloid-b (Ab) peptide is a C-termi-
We previously described a series of diamide amino imidazole
-secretase inhibitors exemplified by compound 1 ( Fig. 1) that
c
reduce Ab at an in vitro IC50 of 0.4 nM in a whole-cell assay
(WCA) and an IC50 of 1.1 nM in a cell-free assay (CFA).4 In addition,
a single acute dose in vivo (guinea pig) showed a dose dependent
reduction in brain and plasma Ab. To evaluate the potential for
notch-related toxicity over a 24 h time period, B-cell populations
nal proteolytic cleavage by
of the -secretase complex which alter Ab processing induce early-
onset AD. Thus inhibition of -secretase has emerged as a clinically
viable disease-modifying approach to the treatment of AD. One lia-
bility to this approach is substrate specificity; -secretase pro-
cesses additional substrates, most notably Notch. Cleavage of
Notch by -secretase is necessary for differentiation of certain cell
types and toxic effects of
c
-secretase.1 Mutations in components
c
c
c
O
N
H
N
c
N
H
N
F
N
H
c
-secretase inhibitors (GSI) have been ob-
served within the intestine and white blood cell populations.2
Nonetheless, substrate specificity and/or PK/PD relationships may
1
O
CFA IC50 = 1.1 nM
WCA IC50 = 0.4 nM
MDR Er = 8.4
F
provide a possible therapeutic window. Indeed, numerous c-secre-
tase inhibitors such as LY-4501391, BMS-299897, GSI-953, and
BMS-708163, have advanced into human clinical trials and demon-
strated the viability of this approach.3
O
N
NR4R5
N
H
R
N
N
H
R'
2
⇑
Corresponding author. Tel.: +1 860 715 6168.
E-mail address: michael.a.brodney@pfizer.com (M.A. Brodney).
Figure 1. Replacement of the N-terminal amide of imidazole 1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.118

2638
M. A. Brodney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2637–2640
Table 1
were analyzed by fluorescence activated cell sorting (FACS) in
whole-blood and spleen preparations. At a 5 mg/kg dose, com-
pound 1 significantly reduced brain Ab1ÀX (58%) and B-cells popu-
lations (45–70%) suggesting no separation existed between Ab
lowering in the brain and notch mediated side-effects in the plas-
ma. We speculated that the high plasma concentrations required to
observe Ab lowering effects in the brain were negatively impacting
the therapeutic index. In part this could be explained by the signif-
icant P-gp mediated efflux (MDR efflux ratio = 8.4) of compound 1.
Evaluation of wide range of analogs related to compound 1 in the
over expressing P-gp cell line (MDCK) indicated that efflux ratios
(BA/AB) were generally greater than 4 indicating efflux was limit-
ing central penetration.5 Based on this observation, we began a
systematic replacement of the N-terminal amide, a common P-gp
recognition element. This report describes efforts to identify novel
GSIs with increased therapeutic index with respect to Notch
related side-effects.
Whole cell assay (WCA), cell free assay (CFA), and MDR efflux ratios (MDR Er) for
representative gamma secretase inhibitors 7a–k
Compound
R3
WCA
IC50 (nm)
CFA
IC50 (nm)
MDR Erd
a,b
b,c
7a
7b
3100
>10,000
4.4
2.3
310
754
6120
250
F
F
7c
1.9
1.8
F
F
7d
480
499
A general synthetic strategy for the preparation of substituted
amino imidazoles is illustrated in Scheme 1. Starting with nitroim-
idazole 3, reduction of the ester group to its corresponding
aldehyde followed by reduction amination gave nitroimidazole 4
in good overall yield.4 Hydrogenation of the nitroimidazole over
Pd/C provided an intermediate aminoimidazole that was directly
acylated with the activated acid of Boc-norvaline to furnish com-
pound 5. Acid catalyzed deprotection and subsequent reductive
amination with an appropriate aldehyde or ketone gave analogs
7a–k. The synthesis of the corresponding tetralin ketones (9g–k)
was readily accomplished by Friedel-Crafts acylation of the phenyl
acetyl chloride derivatives (8g–k) using ethylene.
7e
7f
272
148
1.8
2.2
H
H
53.9
85.7
7g
7h
7i
155
93.3
8.9
2.9
1.3
3.3
1.8
O
H
12.4
46.8
19.5
Cl
Compounds 7a–d were identified from this initial SAR studies
and the cell free and whole cell potency was evaluated
(Table 1).6 When comparing isopentyl 7a to 3-pentyl analog 7b
there was 10-fold potency improvement in the whole cell assay.
Furthermore, 3,5-difluorobenzyl (7d) and 3,5-difluro phenethyl
(7c) analogs gave modest potency improvements, but were signif-
icantly less active than compound 1. Despite the loss in potency,
we were gratified that analogs 7b–d showed a reduced liability
toward P-gp mediated efflux as measured by MDR efflux ratios
Cl
H
31.0
19.3
H
7j
F
F
F
H
7k
5.4
4.1
1.5
(Er).5 Next, we postulated that incorporating
a-branching would
restrict the conformation of 7c or 7d, reduce the number of
rotatable bonds, and lock the aryl group in an active binding
a
IC50 values in the whole cell assay (WCA) were obtained from H4 APPSw cells by
measuring Ab1ÀX
6
.
b
Values are geometric mean of at least two experiments; compounds were
typically dosed at log intervals from 0.1 nM to 10 M.
l
c
IC50 values in the cell free assay (CFA) were obtained from human HeLa cells by
measuring Ab1À40 by DELFIA-based immunoassay.6
N
d
MDR1-MDCK assay utilizes MDCK cells transfected with the gene that encodes
O
N
CO2Me
a, b
N
N
H
N
for human P-glycoprotein.5
N
N
c,d
N
Boc
N
H
O2N
N
O2N
3
5
4
conformation. Thus, analogs 7e and 7f were prepared to validate
this design concept. Previous SAR in the diamide imidazole series
demonstrated that incorporation of mono or di-fluorines on the
aryl ring of the phenyl acetic acid derivative improved potency.4
Toward this end, substituted tetralins 7h–k were prepared as race-
mic mixtures. We were gratified to find that cell free and whole
cell potency was similar for 7k compared to the diamide 1 while
reducing P-gp mediated efflux.
To facilitate analog synthesis, a new route was established that
incorporated the difluorotetralin in the early stages of the synthe-
sis, avoiding the need for a late stage chiral separation (Scheme 2).
Reductive amination of 10 with (S)-Boc Norvaline t-butyl ester pro-
vided a mixture of diastereomers which was separated using chiral
HPLC to provide 11. Acid catalyzed hydrolysis of the t-butyl ester
11 provided acid 12, whose stereochemistry was unequivocally
confirmed by single-crystal X-ray analysis. With carboxylic acid
12 in hand, we sought coupling conditions with the intermediate
N
N
O
N
O
N
H
N
e
N
N
H2N
f
R3
N
N
H
6
H
7a-k
R1
R1
Cl
O
g
O
R2
R2
8g-k
9g-k
Scheme 1. Reagents and conditions: (a) DIBAL, CH2Cl2, À30 °C; (b) pyrollidine,
4AoMS, CH2Cl2, then Na(OAc)3BH, 60% over 2 steps; (c) Pd/C (10%), H2 (40 psi),
MeOH, rt; (d) BocNorCO2H, TPTU, iPr2EtN, DMF, 80% for 2 steps; (e) TFA, CH2Cl2; (f)
R3CHO or 9g–k, CH2Cl2, Na(OAc)3BH then separate, 30–45%, for 2 steps; (g) C2H4,
AlCl3, CH2Cl2.

M. A. Brodney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2637–2640
2639
F
F
O
in Ab1ÀX. For a more comprehensive analysis of the time course of
Ab changes in brain, CSF, and plasma, guinea pigs were dosed
acutely at 10 mg/kg, sc and tissues were taken at time intervals
from 3 to 24 h (Fig. 2B). Significant reductions in Ab1ÀX were de-
tected from 3.2 h to 10 h with maximal response occurring from
3 to 10 h. At 24 h, the Ab levels in plasma and CSF had returned
to baseline while brain Ab showed a longer duration of action. To
understand the effects of Notch processing, analog 14a was
evaluated in fetal thymic organ culture (FTOC) for B- and T-cell
populations (Table 2).8 Compound 14a had a mean EC50 of
H
N
O
a, b
OH
c
OtBu
F
F
10
11
F
O
H
N
N
NR4R5
CO2Me
N
d-f
N
N
+
H2N
O2N
F
12
15
13
g
3.26 lM which represents >500-fold separation from the APP
F
O
N
whole-cell IC50. To translate this selectivity in vivo, compound
14a was dosed at 7 and 32 mg/kg three times over a 24 h period
(time 0, 12, and 24 h) and tissues were collected at 3 h after the
final dose (Fig. 2C). Under this dosing paradigm, brain and plasma
Ab levels were significantly reduced. At the 7 mg/kg dose, there
was not a significant reduction in blood or marginal zone B cell
populations as measured by FACS. This suggested a potential ther-
apeutic index between the doses needed for a 50% reduction in
brain Ab and doses that impacted B-cell reductions. These results
indicate more potent inhibition of APP versus early and sensitive
biomarkers of notch cleavage, as the latter event is a prerequisite
for B cell maturation.
Previous SAR in the diamide imidazoles suggested that replace-
ment of the pyrrolidine group in the imidazole side chain led to a
potency improvement in vitro and in vivo. Modification using a
range of amines (14b–g) yielded several analogs with comparable
whole cell potency and in vivo efficacy in guinea pig compared to
14a (Table 2). To directly compare analogs, an ED50 in brain was
generated at a 3 h time point. Despite the comparable whole cell
potency for all analogs, the in vivo efficacy was superior for mor-
pholine 14e and neopentyl 14f compared to the other analogs. In
order to fully understand the impact of the FTOC assay compared
with the Ab lowering effects, we decided to normalize the FTOC
EC50 to the plasma concentration (Cp) observed at the brain ED50
NR4R5
H
N
N
N
H
F
14a-g
Scheme 2. Reagents and conditions: (a) Na(OAc)3BH, NH2NorCO2tBu, CH2Cl2; (b)
chiral separation, 20–45% over 2 steps; (c) HCl, 90%; (d) DIBAL, CH2Cl2, À30 °C; (e)
HNR4R5, 4AoMS, CH2Cl2, then Na(OAc)3BH, 30–73% over 2 steps; (f) Pd/C (10%), H2
(40 psi), MeOH, rt; (g) TPTU, iPr2EtN, DMF, 60–80% for 2 steps.
aminoimidazole 13 that avoided protection of the secondary amine
of 12. Coupling attempts with CDI or EDC/HBTU failed to provide
the desired amide but TPTU activation followed by treatment with
the amino-imidazole derived from 15 cleanly provided the desired
analogs 14a–g.
With an improved synthetic route to the single diastereomer of
racemic tetralin 7k, we studied compound 14a (Table 2) in Ab effi-
cacy studies. Guinea pigs were dosed acutely at doses ranging from
3.2 to 32 mg/kg, sc, and tissues were collected at 3 h for Ab mea-
surement in brain, CSF, and plasma by DELFIA (Fig. 2A).7 A clear
relationship existed between inhibition of Ab in the brain, CSF,
and plasma. At the 3.2 mg/kg dose, plasma exposure was 100 ng/
mL (211 nM) and brain exposure was 587 ng/mL (1239 nM), which
produced a significant reduction (33% brain, 31% CSF, 30% plasma)
Table 2
Whole cell potency, FTOC selectivity, and in vivo Ab lowering data for representative
c-secretase inhibitors
NR4R5
WCA IC50 (nM)a
ED50 for Ab1ÀX in brainb
FTOC EC50
3.26
(l
m)c
FTOC/Cp @brain ED50
9.8
e
Compound
14a
2.9
9.9 mg/kg, sc
N
14b
3.3
4.1 mg/kg, sc
2.56
46.1
N
14c
14d
19.3
4.6
41.2 mg/kg, sc
16.1 mg/kg, sc
6.1
3.5
NH
N
3.01
NDd
O
14e
14f
14g
6.9
1.2
4.9
2.9 mg/kg, sc
2.3 mg/kg, sc
14.6 mg/kg, sc
1.09
1.83
5.1
16.3
228.8
37.7
N
NH
NH
CF3
a
6
IC50 values in the whole cell assay (WCA) were obtained from H4 APPSw cells by measuring Ab1ÀX
313 nM. Values are geometric mean of at least two experiments.
. Compounds were dosed at concentrations ranging from 0.01 to
b
In vivo activity was determined by measuring Ab1ÀX, Ab1À40, and Ab1À42 in guinea pig brain, CSF, and plasma by Delfia ELISA.7 Significant differences between groups were
detected by one-way ANOVA followed by Dunnett’s post-hoc in GraphPad Prism v5. Treatment effects were considered statistically significant following p <0.05 at the level of
the ANOVA and post-hoc versus vehicle.
c
Fetal thymic organ cultures (FTOC) were prepared for assessment of compound effects on Notch processing.8
d
ND: not determined.
e
Plasma concentration values were determined using a linear least squares regression model with dose versus measured plasma concentrations.

2640
M. A. Brodney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2637–2640
obtained from guinea pigs. This would serve as a means to rank or-
100
80
60
40
20
0
A
der compounds with respect to in vivo efficacy and early biomark-
ers related to Notch processing. Compound 14f (PF-3084014)
showed the largest ratio of the tetralin amino-imidazoles and
was therefore selected for additional efficacy studies and long term
safety evaluation.8
In conclusion, a series of tetralin amino imidazoles were de-
signed and synthesized based on SAR from a diamide series that
suffered from significant P-gp mediated efflux. To improve brain
penetration, variation of the C-terminal phenyl acetic acid ana-
logs with non-peptidic groups resulted in the tetralin variations.
Incorporation of fluorines on the aryl ring resulted in a signifi-
cant improvement in whole cell potency. Further in vivo profil-
ing demonstrated that compound 14a reduced brain, CSF, and
plasma Ab in a dose responsive manner over time with a sepa-
ration between Ab and Notch related side-effects. Optimization
of the FTOC/plasma concentrations obtained at the brain ED50
led to the discovery of PF-3084014 (14f) which was selected
for clinical development.
Brain Aβ1-X
CSF Aβ1-X
Plasma Aβ1-X
3.2
10
32
Dose (mg/kg)
160
140
120
100
80
B
Brain Aβ1-X
CSF Aβ1-X
Plasma Aβ1-X
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K.E.; Pozdnyakov, N.; Sahagan, B.G.; Schachter, J.B.; Sokolowski, S.A.; Tate, B.;
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20
0
3
5
10
24
Time (h)
120
100
80
60
40
20
0
C
B Cell
MZB
Plasma Aβ
Brain Aβ
vehicle
7 mg/kg
32 mg/kg
Treatment
Figure 2. In vivo characterization of GSI 14a; dose responsive of brain, CSF and
plasma Ab2A; time course of brain, plasma, and CSF Ab (2B); reduction of marginal
and blood B-cell populations (2C). (A and B) In vivo activity was determined by
measuring Ab1ÀX, Ab1À40, and Ab1À42 were measured in guinea pig brain and plasma
by Delfia ELISA. Extracts were analyzed for changes in Ab1ÀX using an IGEN assay.7
Mean S.E.M. exposure or percentage of vehicle Ab are represented. (C) Spleen and
whole blood B-cells (relative numbers or percentage) were evaluated by flow
cytometry.8
7. Lanz, T. A.; Salatto, C. T.; Semproni, A. R.; Marconi, M.; Brown, T. M.; Richter, K.
E.; Schmidt, K.; Nelson, F. R.; Schachter, J. B. Biochem. Pharmacol. 2008, 75, 1093.
8. Lanz, T. A.; Wood, K. M.; Becker, S. L.; Pozdnyakov, N.; Martin, B. A.; Du, P.;
Oborski, C. E.; Wood, D. E.; Brown, T. M.; Nolan, C. E.; Richter, K. E.; Finley, J. E.;
Sokolowski, S. A.; Hicks, C. C.; Coffman, K. J.; Tate, B.; Geoghegan, K. F.; Brodney,
M. A.; Liston, D. J. Pharmacol. Exp. Ther. 2010, 334, 269.